Download - Katherine Gillette-Browne Thesis

β-ADRENOCEPTOR DETERMINANTS OF CONTRACTILITY IN THE HUMAN HEART: THE

ROLE OF PHOSPHODIESTERASE ENZYMES

Katherine Tegan Gillette-Browne BPharm BSci (Hons)

06126979

Submitted in [partial] fulfilment of the requirements for the degree of

Doctor of Philosophy

School of Biomedical Sciences

Faculty of Health

Queensland University of Technology

[May 2015]

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes i

Keywords

adrenaline, affinity, arrhythmia, β-adrenoceptor, β-blocker, cardiac, carvedilol,

cilostamide, contractility, enantiomer, esmolol, heart failure, inotropy, lusitropy,

metoprolol, noradrenaline, PDE, PDE3, PDE4, pharmacology, phosphodiesterase

enzyme, phosphodiesterase inhibitor, rolipram, ryanodine, RyR2

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes ii

Abstract

The β-blockers carvedilol and metoprolol provide important therapeutic strategies for

the management of heart failure. While short-term treatment with phosphodiesterase

(PDE) inhibitors has proven beneficial in acute settings, long-term therapy in heart

failure patients results in increased mortality. It is unknown whether PDE3 and PDE4

mediate their inotropic and lusitropic effects through β1- and/or β2-adrenoceptors in

human myocardium. These studies investigated whether the PDE3-selective

inhibitor cilostamide (0.3 µM) or PDE4-selective inhibitor rolipram (1-10 µM)

modified the positive inotropic and lusitropic effects of catecholamines in ventricular

myocardium from heart failure patients treated with or without metoprolol or

carvedilol. Ventricular trabeculae from freshly explanted hearts of 5 non-β-blocked

patients, 15 metoprolol-treated patients, and 9 carvedilol-treated patients with

terminal heart failure were paced to contract at 1 Hz. The effects of (-)-noradrenaline,

mediated through β1-adrenoceptors (β2-adrenoceptors blocked with ICI 118,551), and

(-)-adrenaline, mediated through β2-adrenoceptors (β1-adrenoceptors blocked with

CGP 20712A), were assessed in the absence and presence of the PDE inhibitors.

The inotropic potency, estimated from shifts of concentration-effect curves,

increased 4-fold for (-)-noradrenaline and 5-fold for (-)-adrenaline in metoprolol-

treated but did not change for (-)-noradrenaline and decreased 16-fold for (-)-

adrenaline in right ventricular trabeculae from carvedilol-treated, compared to non-

β-blocked patients. The positive inotropic and lusitropic effects of (-)-noradrenaline

were potentiated (2-3-fold) by cilostamide in metoprolol-treated but not in non-β-

blocker-treated and carvedilol-treated patients. Cilostamide caused marginal, 3-5-

fold and 19-35 fold potentiation of the inotropic and lusitropic effects of (-)-

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes iii

adrenaline, in non-β-blocker-treated, metoprolol-treated and carvedilol-treated

patients respectively. Results from left ventricle were similar to those of right

ventricle. Rolipram did not affect the inotropic and lusitropic potencies of (-)-

noradrenaline or (-)-adrenaline, suggesting negligible control by PDE4.

Carvedilol, and to a lesser extent metoprolol, induce control by PDE3 of the

inotropic and lusitropic effects mediated through β2-adrenoceptors, plausibly by

changing the feedback between receptor activity and PDE3. Treatment of heart

failure patients with carvedilol induces PDE3 to selectively control the positive

inotropic and lusitropic effects mediated through ventricular β2-adrenoceptors

compared to β1-adrenoceptors. This property of carvedilol may provide protection

against β2-adrenoceptor-mediated ventricular overstimulation in PDE3 inhibitor

treated patients. PDE4 does not control β1- or β2-adrenoceptor-mediated inotropic

and lusitropic effects in metoprolol or carvedilol -treated patients.

β-blockers all share a common racemic structure and in general, the antagonistic

efficacies of the S-enantiomers are much more potent (up to 500-fold) than those of

the R-enantiomers. The pharmacodynamics and pharmacokinetics of the racemates

also differ from one another, which may lead to adverse effects from one racemate

while the other provides the majority of the clinical benefits. With very few

exceptions, the clinically used β-blockers are only available as racemic mixtures,

despite the fact that enantiomerically pure preparations may increase clinical efficacy

while reducing the incidence of adverse effects. In this work, the effects of R-(+) and

S-(-) esmolol on human right atrial contractility, β1L- and β3-adrenoceptors, and their

affinities at human β1- and β2-adrenoceptors was investigated. S-(-) esmolol was a

competitive antagonist at β1-adrenoceptors with a β1/β2-adrenoceptor selectivity ratio

of ~ 4.3. The enantiomers of esmolol showed stereoselectivity for blockade of β1-

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes iv

and β2-adrenoceptors. S-(-) esmolol had ~80 fold higher affinity for β1-adrenoceptors

than R-(+) esmolol. The stereoselectivity requirements at β2-adrenoceptors were

lower where S-(-) esmolol had ~13-fold higher affinity for β2-adrenoceptors than R-

(+) esmolol.

As for the majority of β-blockers, the S-(-) enantiomer of esmolol has higher affinity

for blocking both β1- and β2-adrenoceptors than the R-(+) enantiomer. However,

because the β2-adrenoceptor has a much lower stereoselectivity, this could indicate a

role of R-(+) esmolol in the mediation of inotropic and/or lusitropic effects via the

β2-adrenoceptor in vivo and in the clinical setting.

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes v

Table of Contents

Keywords ..................................................................................................................... i Abstract ...................................................................................................................... ii Table of Contents ........................................................................................................ v List of Figures .......................................................................................................... viii List of Tables ............................................................................................................. xi List of Abbreviations ............................................................................................... xiii Statement of Original Authorship ............................................................................. xv Publications .............................................................................................................. xvi Acknowledgements ................................................................................................. xvii CHAPTER 1: LITERATURE REVIEW ............................................................... 1 1.1 Background ........................................................................................................... 1 1.2 The β-adrenoceptor System: Healthy vs. Failing Heart ........................................ 2 1.2.1 The β-adrenoceptor System in the Healthy Heart .................................. 2 1.2.2 The β-adrenoceptor System in Heart Failure ......................................... 5 1.3 The Ryanodine Receptor ....................................................................................... 6 1.3.1 Ryanodine Receptor Structure: The Healthy Heart ............................... 6 1.3.2 Mediation of ECC in the Healthy Heart ................................................ 8 1.3.3 The Ryanodine Receptor in the Failing Heart ....................................... 9 1.4 The Clinical Use of β-Blockers for the Treatment of Heart Failure ................... 11 1.4.1 The Development of β-Blockers .......................................................... 11 1.4.2 The Evolution of the β-Blocker ........................................................... 12 1.4.3 Efficacy of β-Blockers Clinically Used for Heart Failure ................... 13 1.4.4 Signaling Bias of β-Blockers ............................................................... 18 1.4.5 Dynamic Bias ....................................................................................... 21 1.5 The Enantiomers of β-Blockers .......................................................................... 22 1.5.1 Racemic Mixtures ................................................................................ 22 1.5.2 Esmolol ................................................................................................ 28 1.5.3 The Clinical Relevance of Enantiomerically Pure β-Blockers ............ 32 1.6 Phosphodiesterase Enzymes: Healthy vs. Failing Hearts ................................... 33 1.6.1 Phosphodiesterases in the Healthy Heart ............................................. 33 1.6.2 Phosphodiesterase Compartmentalization ........................................... 36 1.6.3 Roles of the PDE Subfamilies in Healthy and Failing Hearts ............. 36 1.7 Hypothesis, Summary, and Implications ............................................................ 44 CHAPTER 2: MATERIALS AND METHODS ...................................................47 2.1 Ventricular Experiments ..................................................................................... 47 2.1.1 Research Design Summary ...................................................................47

2.1.2 Materials and Methods ......................................................................... 47 2.1.3 Analysis of Contractile Data ................................................................ 52 2.1.4 Drugs ................................................................................................... 52 2.1.5 Participants ........................................................................................... 53 2.2 Right Atrial Experiments .................................................................................... 55

2.2.1 Research Design Summary .................................................................. 55 2.2.2 Explantation and Transport ................................................................. 55 2.2.3 Dissection and Set-Up of Trabeculae .................................................. 55

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes vi

2.2.4 Specific Activation of β1- and β2-Adrenoceptors ................................ 56 2.2.5 Analysis of Contractile Data ................................................................ 57 2.2.6 Drugs .................................................................................................... 57 2.2.7 Participants ........................................................................................... 58 CHAPTER 3: THE EFFECTS OF METOPROLOL ON PDE3 AND PDE4 CONTROL OF β1- AND β2-ADRENOCEPTOR MEDIATED INOTROPY AND LUSITROPY IN HUMAN FAILING VENTRICLE ................................. 59 3.1 Background and Purpose .................................................................................... 59 3.2 Methodology and Research Design .................................................................... 59

3.2.1 Research Design ................................................................................ 59 3.3 Materials and Methods ........................................................................................ 60

3.3.1 Participants ........................................................................................... 60 3.3.2 In vitro Human Heart Trabeculae Experiments ................................. 61 3.3.3 Isolated Ventricular Trabeculae from Heart Transplant Patients ........ 62 3.3.4 Specific Activation of β1- and β2-Adrenoceptors ................................ 62 3.3.5 Analysis of Contractile Data ................................................................ 63 3.3.6 Drugs .................................................................................................... 64

3.4 Results ................................................................................................................ 64 3.4.1 Chronic Metoprolol Treatment Increases the Inotropic Potencies of Catecholamines ....................................................................................... 64 3.4.2 Cilostamide Fails to Potentiate the Inotropic Effects of

Catecholamines in Right Ventricular Trabeculae from Non- β-Blocker-Treated Patients ..................................................................... 68

3.4.3 Cilostamide Potentiates the Effects Mediated Through β2- Adrenoceptors More than β1-Adrenoceptors in Ventricular Trabeculae from Metoprolol-Treated Patients .............................................................70

3.4.4 Rolipram Does Not Modify Inotropic or Lusitropic Potencies of (-)-Noradrenaline or (-)-Adrenaline ..........,..............................................77

3.5 Discussion ........................................................................................................... 80 3.5.1 Control by PDE3 of β1- and β2-Adrenoceptors in Heart Failure

Patients Treated with Metoprolol .............................................................. 80 3.5.2 PDE4 Inhibition Does Not Affect the Inotropic or Lusitropic Effects

of Catecholamines ..................................................................................... 83 3.6 Clinical Implications ........................................................................................... 86 3.7 Conclusions ......................................................................................................... 87 CHAPTER 4: THE EFFECTS OF CARVEDILOL ON PDE3 AND PDE4 CONTROL OF β1- AND β2-ADRENOCEPTOR MEDIATED INOTROPY AND LUSITROPY IN HUMAN FAILING VENTRICLE ................................. 88 4.1 Background and Purpose .................................................................................... 88 4.2 Methodology and Research Design .................................................................... 88 4.2.1 Heart Transplant Patients ........................................................................... 88 4.2.2 Isolated Right Ventricular Trabeculae from Heart Transplant Patients ..... 89 4.2.3 Specific Activation of β1- and β2-Adrenoceptors ...................................... 90 4.2.4 Analysis and Statistics ............................................................................... 91 4.3 Results ................................................................................................................. 92 4.3.1 Decrease of Inotropic and Lusitropic Potencies for (-)-Adrenaline by Chronic Treatment with Carvedilol ........................................................... 92 4.3.2 Cilostamide Potentiates the Effects of (-)-Adrenaline More Than

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes vii

(-)-Noradrenaline in Trabeculae From Carvedilol-Treated Patients ......... 99 4.3.3 Rolipram Does Not Modify Inotropic or Lusitropic Potencies of (-)-Noradrenaline or (-)-Adrenaline in Trabeculae From Patients Treated With Carvedilol ....................................................................................... 102 4.4 Discussion ..........................................................................................................102 4.4.1 Carvedilol Treatment Reduces Responses Through β2-Adrenoceptors

But Not β1-Adrenoceptors ...........................................................................103 4.4.2 Carvedilol Facilitates the Control by PDE3 of β2- More Than β1- Adrenoceptor-Mediated Effects ...............................................................104 4.4.3 Quantitative Comparison of β1- and β2-Adrenoceptor Blockade by Carvedilol in Combination with PDE3 Inhibition ................................... 107 4.4.4 PDE4 Does Not Control the Inotropic or Lusitropic Effects of Catecholamines in Carvedilol-Treated Patients with Heart Failure .........108 4.5 Conclusions ....................................................................................................... 109 CHAPTER 5: ENANTIOMERS OF ESMOLOL .............................................. 112 5.1 Background and Purpose ................................................................................. 112 5.2 Methods ............................................................................................................. 112

5.2.1 Participants ..........................................................................................112 5.2.2 Preparation of Right Atrial Trabeculae .............................................. 113 5.2.3 Acute Effects of S-(-) Esmolol on Human Right Atrium .................. 114 5.2.4 Effect of S-(-) Esmolol in the Presence of 3-isobutyl-1-

methylxanthine and Nadolol .............................................................. 114 5.2.5 Determination of the Affinity of S-(-) and R-(+) Esmolol at

Human Atrial β1- and β2-Adrenoceptors ............................................ 114 5.3 Results ................................................................................................................115

5.3.1 Acute Effects of S-(-) Esmolol on Human Right Atrium .................. 115 5.3.2 Effect of S-(-) Esmolol in the Presence of 3-isobutyl-1-

methylxanthine and Nadolol .............................................................. 117 5.3.3 Determination of the Affinity of S-(-) and R-(+) Esmolol at Human

Atrial β1- and β2-Adrenoceptors ........................................................ 118 5.4 Discussion ......................................................................................................... 122

5.4.1 Patient Population ..........,................................................................... 122 5.4.2 Acute Effects of S-(-) Esmolol on Human Right Atrium .................. 122 5.4.3 Lack of Agonist Activity of S-(-) Esmolol on β1L-Adrenoceptors .... 122 5.4.4 Affinity of S-(-) and R-(+) Esmolol at Human Atrial β1- and β2-Adrenoceptors ............................................................................... 123

5.5 Conclusions and Future Directions ................................................................... 123 CHAPTER 6: CONCLUSIONS AND FUTURE DIRECTIONS ..................... 125 6.1 Role of PDE3 and PDE4 on Contractility of the Human Heart ....................... 125 6.2 The Future of β-Blockers .................................................................................. 129 6.2.1 Biased β-Blockers .............................................................................. 129 6.2.2 Enantiomerically Pure β-Blocker Preparations .................................. 132 6.3 The Future of Phosphodiesterase Inhibitors ..................................................... 133 BIBLIOGRAPHY ................................................................................................. 136 APPENDICES ....................................................................................................... 177 Appendix A: British Journal of Pharmacology Publication ................................... 177 Appendix B: Naunyn-Schmeideberg's Archives of Pharmacology Publication ..... 188

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes viii

List of Figures

Figure 1.1. The ryanodine receptor macromolecular signaling complex ......... 7 Figure 1.2. RYR2 mediates excitation-contraction coupling in the healthy heart ....................................................................................... 9

Figure 1.3. Example of ligand-biased signaling for the β2-adrenoceptor ....... 20 Figure 1.4. Chemical structures of β-blockers ................................................ 25 Figure 1.5. Catecholamine-induced activation of β-adrenoceptors leads to a cAMP-PKA-mediated phosphorylation of the RyR2 .................... 34 Figure 3.1. Effects of chronic administration of metoprolol compared with no- β-blocker on inotropic effects of (-)-noradrenaline and (-)-adrenaline in right ventricular trabeculae from failing hearts ................................................................................................. 65 Figure 3.2. Effects of chronic administration of metoprolol compared with no β-blocker on lusitropic effects of (-)- noradrenaline and (-)-adrenaline in right ventricular trabeculae from failing hearts ..... 67 Figure 3.3. Lack of effect of cilostamide on the inotropic responses of (-)- noradrenaline and (-)-adrenaline in right ventricular trabeculae from patients with heart failure not treated with a β-blocker ........... 68 Figure 3.4. Effect of cilostamide on the lusitropic responses of (-)- noradrenaline and (-)-adrenaline in right ventricular trabeculae from patients with heart failure not treated with a β-blocker ........... 69 Figure 3.5. Representative experiment carried out on right ventricular trabeculae obtained from a 48-year-old male patient with IHD, chronically administered metoprolol ....................................... 72 Figure 3.6. Potentiation of the inotropic effects of (-)-adrenaline by cilostamide in right ventricular trabeculae from seven patients from Brisbane/Dresden with heart failure chronically administered metoprolol ................................................................... 73 Figure 3.7. Cilostamide, but not rolipram, potentiates the lusitropic effects of (-)-adrenaline and (-)-noradrenaline in right ventricular trabeculae from seven patients with heart failure chronically administered metoprolol ................................................................... 74 Figure 3.8. Cilostamide potentiates the inotropic effects of (-)-adrenaline in left ventricular trabeculae from patients with heart failure chronically administered metoprolol ................................................ 75

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes ix

Figure 3.9. Cilostamide, but not rolipram, potentiates the lusitropic effects of (-)-noradrenaline and (-)-adrenaline in left ventricular trabeculae from patients with heart failure chronically administered metoprolol ................................................................... 76 Figure 3.10. Effects of the combination of cilostamide and rolipram on the inotropic responses of (-)-noradrenaline and (-)-adrenaline in right ventricular trabeculae from three patients with heart failure chronically administered metoprolol ................................................ 78 Figure 3.11. Effects of the combination of cilostamide and rolipram on the lusitropic responses of (-)-noradrenaline and (-)-adrenaline in right ventricular trabeculae from 3 patients with heart failure chronically administered metoprolol ................................................ 79

Figure 4.1. Marked reductions in potency for inotropic effects of (-)- adrenaline but not for (-)-noradrenaline in right ventricular trabeculae .......................................................................................... 93 Figure 4.2. Effects of chronic administration of carvedilol compared to no β-blocker on lusitropic effects of (-)-noradrenaline and (-)- adrenaline in right ventricular trabeculae from failing hearts .......... 95 Figure 4.3. Potentiation of inotropic effects of both (-)-noradrenaline and (-)- adrenaline in the presence of cilostamide but not rolipram ....... 97 Figure 4.4. Marked potentiation of the inotropic effects of (-)-adrenaline vs. (-)-noradrenaline by cilostamide in right ventricular trabeculae from three hearts chronically treated with carvedilol ................................. 98 Figure 4.5. Marked potentiation of lusitropic effects of (-)-noradrenaline and (-)-adrenaline by cilostamide in right ventricular trabeculae from three hearts chronically treated with carvedilol ....................... 99 Figure 4.6. Cilostamide potentiates the inotropic effects of (-)-adrenaline more than (-)-noradrenaline in right ventricular trabeculae from heart failure patients chronically administered carvedilol .............. 100 Figure 4.7. Cilostamide, but not rolipram, potentiates lusitropic effects of (-)-adrenaline and (-)-noradrenaline in right ventricular trabeculae from patients with heart failure chronically administered carvedilol ........................................................................................ 101

Figure 5.1. Effect of S-(-) esmolol on force of contraction and relaxation, TPF, and t50 ....................................................................................... 116 Figure 5.2. Lack of agonist activity of S-(-) esmolol at β1L-adrenoceptors .. 117

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes x

Figure 5.3. Determination of the affinity of S-(-) and R-(+) esmolol at β1- adrenoceptors in human right atrium ................................................. 119 Figure 5.4. Determination of the affinity of S-(-) and R-(+) esmolol at β2- adrenoceptors in human right atrium ................................................. 121

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes xi

List of Tables

Table 1.1. Generations of β-blockers .........................................................................13 Table 1.2. Multi-center, double-blind, placebo-controlled studies on survival benefits of β-blockers ....................................................................................14 Table 1.3. β-adrenoceptor ligands and their efficacy patterns towards adenylyl cyclase and the β-arrestin/ERK1/2 pathways of the β1-and β2-adrenoceptors ..........................................................................................21 Table 1.4 Families, subfamilies, substrates, and tissue expression of

phosphodiesterase enzymes ...........................................................................35 Table 3.1. Summary of Brisbane/Dresden patients that were not taking a β–blocker or chronically administered metoprolol prior to heart transplantation ...............................................................................................61 Table 3.2. Summary of Oslo patients chronically administered metoprolol prior to heart transplantation .........................................................................61 Table 3.3. Inotropic potencies of (-)-noradrenaline and (-)-adrenaline acting through ventricular β1- and β2-adrenoceptors, respectively ..........................66 Table 3.4. Lusitropic potencies of (-)-noradrenaline and (-)-adrenaline, acting through right ventricular β1- and β2-adrenoceptors, respectively .....70 Table 3.5. Lusitropic potencies of (-)-noradrenaline and (-)-adrenaline, acting through left ventricular β1- and β2-adrenoceptors of metoprolol-treated patients ..........................................................................................................77 Table 3.6. Reduction of inotropic and lusitropic responses as well as protection against arrhythmias, mediated through myocardial β1- and β2- adrenoceptors, by PDE3 and PDE4 in different species ...............................85 Table 4.1.A. Patients that were not taking a β–blocker prior to heart

transplantation ................................................................................................89 Table 4.1.B Summary of patients chronically administered carvedilol prior to heart transplantation ......................................................................................89 Table 4.2. ΔpEC50 obtained in the presence or absence of PDE inhibitor and calculated from mean values for each patient ..............................................94 Table 4.3. Lusitropic potencies of (-)-noradrenaline and (-)-adrenaline, acting through right ventricular β1- and β2-adrenoceptors, respectively, from patients chronically treated with carvedilol ..................................................96

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes xii

Table 5.1. Summary of patient details .....................................................................113 Table 5.2. pKB values of S-(-) esmolol and R-(+) esmolol determined at human β1- and β2-adrenoceptors .............................................................................118

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes xiii

List of Abbreviations

AC adenylyl cyclase ADR adrenaline AF atrial fibrillation AKAP A kinase anchoring proteins AMP adenosine 5’-monophosphate ATP adenosine 5’-triphosphate β1L-adrenoceptor low affinity β1-adrenoceptor β1H-adrenoceptor high affinity β1-adrenoceptor β-blocker β-adrenoceptor blocking agent Ca2+ calcium CAMKII calmodulin-dependent protein kinase cAMP cyclic 3’-5’ adenosine monophosphate CGP 12177 (±)-CGP-12177 hydrochloride, 4-[3-[(1,1-

Dimethylethyl)amino]-2-hydroxypropoxy]-1, 3-dihydro-2H-benzimidazol-2-one hydrochloride

CGP20712A (±)-2-Hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4

(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl] amino]ethoxy]-benzamide

CICR calcium-induced calcium release GPCR G-protein coupled receptor GTP guanosine 5-triphosphate Gi inhibitory G protein Gs stimulatory G protein IBMX 3-isobutyl-1-methylxanthine ICaL L-type Ca2+ channel

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes xiv

ICI 118,551 (±)-1-[2,3-(Dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride

KB dissociation constant at equilibrium Lmax length/tension required to produce a maximal contraction mAKAP muscle-selective A-kinase anchoring protein PBZ phenoxybenzamine PDE phosphodiesterase enzyme pEC50 -log molar concentration of agonist required to produce half

maximum response PKA cAMP-dependent protein kinase A PKB protein kinase B pKB -log molar concentration of antagonist which occupies 50% of

receptors PO open probability NA noradrenaline RA right atrium/ right atrial RV right ventricle/ right ventricular RyR2 ryanodine receptor/channel S.E.M standard error of the mean SERCA sarcoplasmic reticulum Ca2+-ATPase SR sarcoplasmic reticulum t ½ half-life TPF time to peak force t50 time to 50% relaxation

QUT Verified Signature

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes xvi

Publications and Communications

Molenaar, P.M., Christ, T., Hussain, R.I., Engel, A., Berk, E., Gillette, K.T., Chen,

L., Galindo-Tovar, A., Krobert, K.A., Ravens, U., Levy, F.A., & Kaumann, A.J.

(2013). PDE3, but not PDE4, reduces β1- and β2- adrenoceptor-mediated inotropic

and lusitropic effects in failing ventricle from metoprolol-treated patients. British

Journal of Pharmacology, 169, 528-538.

Molenaar, P.M., Christ, T., Berk, E., Engel, A., Gillette, K.T., Galindo-Tovar, A.,

Ravens, U., & Kaumann, A.J. (2014). Carvedilol induces greater control of β2- than

β1-adrenoceptor-mediated inotropic and lusitropic effects by PDE3, while PDE4 has

no effect in human failing myocardium. Naunyn-Schmeideberg’s Archives of

Pharmacology, 387, 629-640.

Molenaar, P., Christ, T., Hussain, R.I., Engel, A., Berk, E., Gillette, K.T., Chen, L.,

Galindo-Tovar, A., Krobert, K.A., Ravens, U., Levy, F.O., & Kaumann, A.J. (2012).

Carvedilol induces greater control by PDE3 of β2- than β1-adrenoceptor-mediated

inotropic effects in human failing myocardium while PDE4 has no effect. ASCEPT-

APSA Sydney.

Molenaar, P., Christ, T., Gillette, K.T., Chen, L., & Kaumann, A.J. (2012).

Carvedilol induces greater control by PDE3 of β2- than β1-adrenoceptor-mediated

inotropic effects in human failing myocardium while PDE4 has no effect. The Prince

Charles Hospital Research Forum Brisbane.

Molenaar, P., & Gillette, K.T. (2013). β1- and β2-adrenoceptor – phosphodiesterase

control over human heart contractility. ASCEPT-SEAWP Melbourne.

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes xvii

Acknowledgements

Firstly, I would like to acknowledge that these experiments are the result of a

collaboration between the groups here in Brisbane, Australia: Peter Molenaar and

myself, in Dresden, Germany: Alberto Kaumann, Alejandro Galindo-Tovar, Torsten

Christ, Andreas Engle, Emanuel Berk, and Ursula Ravens, and in Oslo, Norway:

Rizwan Hussain, Kurt Krobert, and Finn Olav Levy. The members of these groups

have all collaborated for the experiments, the data analysis, and the writing of results

for publication. I myself contributed to all three stages, including tissue retrieval,

experiment completion, data compilation, statistical analysis, writing, table

formulation, and proofreading. This work has already resulted in two publications,

which are part of the appendix of this thesis.

I would like to thank QUT for allowing me this amazing opportunity and for their

financial support, which made completion of this endeavour possible. I would also

like to thank Professor Malcolm West and Associate Professor Ian Yang of the

University of Queensland School of Medicine for allowing me to work in the in vitro

Human Heart Laboratory. Special thanks also goes to the surgeons of The Prince

Charles Hospital, Brisbane, and the Gustav Carus Hospital, Dresden, for providing

the tissues for experiments and to the Transplant Coordinators for gaining patient

consents.

I would thank my supervisors Associate Professor Peter Molenaar and Dr. Annalese

Semmler for their guidance and support. A very heartfelt and humbled thank you

goes to Peter Molenaar, who has helped and encouraged me through every step of

this challenging adventure, and whose patience with tired PhD candidates and

unflagging dedication to his field deserve special commendation.

Lastly but certainly not least, I would like to thank my family for their constant

support and encouragement; my Husband Lucas for being my wailing wall and

β-adrenoceptor determinants of contractility in the human heart: the role of phosphodiesterase enzymes xviii

biggest cheerleader, and my Mom for supplying her little science nerd with

everything from microscopes to buckets for adopted salamanders.

CHAPTER 1 Literature Review 1

1. Literature Review

1.1 BACKGROUND

Heart failure and arrhythmic disease are major causes of death in Australia, ranked 8

in females and 11 in males in 2012 (Australian Bureau of Statistics, 2012). Ischemic

heart disease, which commonly progresses to heart failure, is ranked as the leading

cause of death in both males and females. In terms of rankings, no progress appears

to have been made since 1998 (Australian Bureau of Statistics, 2012). The

sympathetic nerve- β-adrenoceptor system contributes to the progression of heart

failure, morbidity and mortality (Molenaar & Parsonage, 2005).

Heart failure can clinically be defined as a syndrome “in which patients have typical

symptoms and signs resulting from an abnormality of cardiac structure or function”

(ESC Guidelines, 2012), and 50% of patients who are diagnosed with heart failure

will die within five years (Nguyen et al., 2014). Sudden cardiac death accounts for

approximately 30-50% of these deaths (Zipes et al., 2009; Adamson & Gilbert,

2006). Patients who have died suddenly whilst having a Holter monitor recording to

detect the presence of arrhythmias have primarily shown complex ventricular activity

and ventricular tachyarrhythmias most often degenerating into ventricular

fibrillation. Structural changes and remodeling associated with heart failure

combined with increased sympathetic nerve β-adrenoceptor activity is largely

responsible for ventricular arrhythmias leading to sudden death (Adamson & Gilbert,

2006).


1.2 THE β-ADRENOCEPTOR SYSTEM: HEALTHY VS. FAILING HEART

1.2.1 The β-adrenoceptor system in the Healthy Heart

There are two stimulatory β-adrenoceptors in the human heart, the β1-adrenoceptor

and the β2-adrenoceptor (Brodde, 1991; Molenaar et al., 2007). Noradrenaline,

released from sympathetic nerves in response to physiological demands including

exercise or low blood pressure, or pathologic stimuli including heart failure, activates

β1-adrenoceptors on heart muscle cells to cause an increase in heart rate (positive

chronotropy) and an increase in force of contraction (positive inotropy). This causes

an increase in heart function with consequent increase in cardiac output to meet

physiological demand. Adrenaline, released from the adrenal medulla in response to

stress, circulates in the blood and upon reaching the heart, activates both β1- and β2-

adrenoceptors on heart muscle cells to increase heart rate and force of contraction.

Human heart β1-adrenoceptors are associated with heart failure, coronary artery

disease, and arrhythmias. The role of β2-adrenoceptors in pathology is less clear. The

harmful effects of the sympathetic nerve β-adrenoceptor system have largely been

attributed to the β1-adrenoceptor. In contrast a ‘protective’ role of β2-adrenoceptors

has been advocated leading to proposals to administer β2-adrenoceptor agonists for

the management and treatment of human heart failure (Zheng, Zhu, Han, & Xiao,

2005). Based on observations of differences between β1- and β2-adrenoceptors made

in animal species a ‘current consensus’ has been described in which “β1-adrenoceptor

stimulation serves as a causal factor of congestive heart failure, while β2-

adrenoceptor activation may actually be protective for the failing heart” (Zheng et

al., 2005). Furthermore a rationale for a “combination of β1-adrenoceptor blockade

with β2-adrenoceptor activation as a new prevention and intervention strategy for the


treatment of congestive heart failure” has been proposed (Zheng et al., 2005).

However, based on other work (Kaumann et al., 1999; Molenaar et al., 2000;

Molenaar et al., 2007) the proposal for implementation of β2-adrenoceptor activation

for the management of human heart failure is likely to contribute to fatal ventricular

arrhythmias and sudden death.

There are fundamental differences between β2-adrenoceptor signalling in rodent and

human hearts. Release of noradrenaline and adrenaline from sympathetic nerves and

the adrenal medulla causes cardiostimulation through both β1- and β2-adrenoceptors

in the human heart. In the human heart, but not necessarily in other mammals,

activation of β1- and β2-adrenoceptors causes similar increases of cAMP, cAMP-

dependent protein kinase (PKA), and comparable PKA-catalyzed phosphorylation of

target proteins including phospholamban, troponin I, and myosin binding protein C

(Kaumann et al., 1999; Molenaar et al., 2000; Molenaar et al., 2007). As a result of

these biochemical effects, β1- and β2-adrenoceptors also mediate similar positive

inotropic and lusitropic effects (Kaumann et al., 1999; Molenaar et al., 2000;

Molenaar et al., 2007). Therefore, activation of β2-adrenoceptors could also be

responsible for the occurrence of arrhythmias, including fatal ventricular

arrhythmias.

In the human atrium and ventricle, activation of β1- and β2-adrenoceptors causes an

increase in contractile force and a hastening of relaxation, (positive inotropy and

lusitropy, respectively) (Kaumann et al., 1999; Molenaar et al., 2000; Molenaar et al.,

2002; Sarsero et al., 2003; Molenaar et al., 2006; Molenaar et al., 2007). Activation

of β1-adrenoceptors in the human heart occurs as a physiological response to heart

failure, enabling the heart to maintain adequate haemodynamic function and


perfusion pressure in organs. The β1-adrenoceptor has at least two binding sites,

which can both be activated to cause cardiostimulation. The first, termed the β1H-

adrenoceptor (high affinity site of β1-adrenoceptor) is activated by noradrenaline and

adrenaline and blocked by relatively low concentrations of β-blockers including

metoprolol, bisoprolol and carvedilol (Kaumann & Molenaar, 2008). The other,

termed the β1L-adrenoceptor (low affinity site of β1-adrenoceptor) has lower affinity

for noradrenaline and adrenaline and is activated by some β-blockers including CGP

12177 and pindolol, both at higher concentrations than those required to block the

receptor (Kaumann & Molenaar, 2008). Activation of β2-adrenoceptors located on

pre-junctional sympathetic nerve terminals facilitates the release of noradrenaline to

increase contractility, while activation of β2-adrenoceptors located directly on human

heart muscle causes maximal or near maximal increases in contractile force in the

human atrium (Molenaar et al., 2007) and ventricle (Kaumann et al., 1999; Molenaar

et al., 2000).

Human atrial and ventricular β1-adrenoceptors (β1H- and β1L-adrenoceptors) and β2-

adrenoceptors couple to the stimulatory Gsα-protein → adenylyl cyclase → cyclic

AMP → cyclic AMP dependent protein kinase (PKA) pathway. Evidence of

activation of PKA includes PKA dependent phosphorylation of Ser16-

phospholamban, C-protein and troponin I for both the β1H-adrenoceptor and the β2-

adrenoceptor (Kaumann et al., 1999; Molenaar et al., 2000; Molenaar et al., 2007),

which is consistent with increases in contractile force and hastening of relaxation.

Activation of the β1L-adrenoceptor, like that of β1H-adrenoceptor, also causes an

increase in contractile force and hastening of relaxation. β1L-adrenoceptor responses

are enhanced in the presence of non-selective blockade of phosphodiesterase


enzymes with isobutylmethylxanthine (IBMX). In the presence of IBMX,

accumulation of cyclic AMP and activation of PKA can be demonstrated in human

right atrium (Kaumann & Molenaar 2008; Sarsero et al., 2003).

1.2.2 The β-adrenoceptor System in Heart Failure

As the human heart fails, the body uses a variety of mechanisms to compensate for

decreased cardiac output and organ perfusion (Lehnart et al., 2005). One such

compensatory mechanism is an increase in sympathetic signaling, primarily through

the activation of the β1-adrenoceptor (Marks, 2001; Molenaar & Parsonage, 2005).

Acute sympathetic activation (activation that lasts for minutes to days) serves an

important function in the healthy heart in response to events such as trauma, blood

loss, or exercise; it allows the heart to increase its hemodynamic output significantly

within a matter of seconds (Port & Bristow, 2001). In contrast, chronic sympathetic

activation in the failing heart leads to adverse changes mediated through α1- β1- and

β2-adrenoceptors (Packer et al., 1998), and sympathetic activation is inversely

proportional to survival rate (Cohn, 1984). Sustained increases in circulating

noradrenaline leads to oxidative stress and apoptosis through both α1- and β1-

adrenoceptors (Packer et al., 1998; Port & Bristow, 2001). Activation of α1-

adrenoceptors impairs the ability of the kidneys to excrete both salt and water; this

leads to ventricular hypertrophy and increased intravascular volume, and ultimately

in increased oxygen demand by cardiomyocytes (Packer et al., 1998). Over time, the

chronic adrenergic activation of the failing heart leads to cardiac malfunction, and

more specifically to altered Ca2+ mediated excitation-contraction coupling

(Andersson & Marks, 2010; Blayney & Lai, 2009). The desensitization of β1- and β2-

adrenoceptors by chronic stimulation also leads to the uncoupling of the β-


adrenoceptors to Gsα, ultimately leading to a reduction in the amount of cAMP

normally accumulated by their activation; the higher the increase in adrenergic

activation, the higher the level of desensitization and consequent cardiomyocyte

damage (Port & Bristow, 2001).

In the healthy human heart, the ratio of β1- to β2-adrenoceptors is approximately

70:30. In heart failure, where the β1-adrenoceptor is down-regulated, this ratio is

closer to 50:50 (Port & Bristow, 2001; Barrese & Taglialatela, 2013). Both β1- and

β2-adrenoceptors are decoupled from their signaling pathways, in large part due to an

upregulation of the phosphorylating G-protein coupled receptor kinase GRK2, or

βARK1 (Lohse et al., 2003; Port & Bristow, 2001). Giα subunits, which antagonize

β-adrenoceptor signaling, are up-regulated (Neumann et al., 1988). Taken together,

these alterations in cardiac signaling effectively limit the heart’s ability to contract

normally and retain adequate systemic organ perfusion (Lohse et al., 2003).

1.3 THE RYANODINE RECEPTOR

1.3.1 Ryanodine Receptor Structure: The Healthy Heart

The ryanodine receptor, or RyR2, is a large homotetrameric Ca2+ channel within the

lipid bilayer of the sarcoplasmic reticulum (SR) in cardiac and skeletal muscle cells

(Marks, 1996; Dulhunty, Beard, Pouliquin, & Casarotto, 2007). On its cytoplasmic

terminus, the RyR2 associates with Protein Kinase A (PKA) and PDE4D3 (Blainey

& Lai, 2009). The phosphatases PP1 and PP2A also associate with the cytoplasmic

portion of the RyR2, and all of these cytosolic accessory compounds help to

physically and temporally regulate phosphorylation and dephosphorylation of the

RyR2 (Blayney & Lai 2009; Dulhunty et al., 2007; Marx et al., 2000). (Figure 1.1)


Phosphorylation of the RyR2 activates the channel by increasing its open probability

(PO) and dephosphorylation results in channel stabilisation and closing (Fill &

Copello, 2002; Wehrens & Marks, 2003). (Figure 1.1)

Figure 1.1. The ryanodine receptor macromolecular signaling complex. Four RyR2 monomers contribute to the tetrameric Ca2+ release channel macromolecular complex. Regulatory proteins and enzymes associate with the large cytoplasmic RyR2 domains protruding into the cytosolic space. Calmodulin (CaM), and FKBP12.6 (calstabin2) are thought to bind directly to the RyR2 monomers, whereas binding of other subunits is mediated by specific targeting proteins. Triadin and junctin interact with RyR2 on the luminal side of the channel complex. PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; CAMKII, calmodulin-dependent kinase II; PDE, phosphodiesterase enzyme; PKA, protein kinase A; Ser, serine; mAKAP, muscle-A kinase anchoring protein. (Figure from Mohler & Wehrens, 2007).


1.3.2 Mediation of ECC in the Healthy Heart

The RyR2 channels serve to mediate excitation–contraction coupling (ECC) in the

heart by controlling Ca2+ release from the luminal sarcoplasmic reticulum (SR)

stores. Upon depolarisation, the L-Type Calcium Channel (ICaL) allows an influx of

Ca2+ into the cytosolic space between the sarcolemma and the SR known as the

dyadic cleft. This Ca2+ influx results in the RyR2 channel opening, releasing a much

larger amount of Ca2+ into the cleft in a process called calcium-induced calcium

release (CICR) (Wehrens & Marks, 2003; Marks, 1996). Activation of β1- and β2-

adrenoceptors causes dissociation of the Gsα subunit, which in turn activates

adenylate cyclase to make cAMP. cAMP then activates Protein Kinase A (PKA)

which phosphorylates the RyR2, further regulating the opening of the RyR2 (Marx et

al., 2000). The Ca2+ released by the RyR2 into the cytosol then binds to troponin C, a

regulatory protein of the myofilaments; once this occurs, the myofilaments actin and

myosin are able to interact. The activation of actin and myosin leads to a shortening

of the sarcomere, causing contraction of the muscle, or systole (Andersson & Marks,

2010). (Figure 1.2)

Phosphorylation of the RyR2 by PKA increases the PO of the channel by dissociating

calstabin2 from the macromolecular complex. The removal of calstabin2 causes

increased sensitivity of the channel to Ca2+ influx via the ICaL (Wehrens & Marks,

2003; Marx et al., 2000; Dulhunty et al., 2007; Brillantes et al., 1994). During

diastole the RyR2 closes, which stops the flow of Ca2+ into the dyadic cleft; Ca2+

diffuses away from troponin C and is pumped into the SR by the sarcoplasmic

reticulum Ca2+ ATPase pump, SERCA2A. This allows the sarcomeres to lengthen,


the muscle to relax, and ultimately allows the heart to fill. (Figure 1.2) SERCA2A is

under the control of phospholamban, and phosphorylation of phospholamban by

PKA at Ser16 results in decreased inhibition of SERCA2A (Simmerman et al., 1986;

Koss & Kranias, 1996; Kaumann et al., 1999).

Figure 1.2. The ryanodine receptor (RyR2) mediates excitation-contraction coupling in the healthy heart. CICR, calcium-induced calcium release; SR, sarcoplasmic reticulum; PLB, phospholamban; SERCA, sarcoplasmic reticulum Ca2+-ATPase; NCX, Na+/Ca2+ exchange pump; SL, sarcolemma; AP, action potential; LCC, L-type Ca2+ channel. Figure from Blayney & Lai (2009).

1.3.3 The Ryanodine Receptor in the Failing Heart

The increased adrenergic activation during heart failure results in increased cAMP

levels in cardiac myocytes, higher localised PKA levels, hyperphosphorylation of

RyR2, dissociation of calstabin2, and an increase in the channel’s PO (Marks, 2001;

Lehnart et al., 2005). Without calstabin2, the RyR becomes unstable during diastole,

allowing Ca2+ to leak into the dyadic cleft. Dissociation of calstabin2 has been shown


to cause a four-fold increase in transient Ca2+ releases from the RyR2, termed Ca2+

sparks (Marks 2001; Marx et al., 2000; Brillantes et al., 1994).

An arrhythmic contraction is orchestrated through the untimely release of calcium

from the RyR2. Delayed after depolarizations, or DADs, can be caused by an

inappropriately timed, spontaneous efflux of Ca2+ escaping from the sarcoplasmic

reticulum via the RyR2; DADs are a characteristic of heart failure (Blayney & Lai,

2009). RyR2 is phosphorylated by PKA (Witcher, Kovacs, Schulman, & Cefali,

1991; Marx et al., 2000; Rodriguez, Bfhogal, & Colyer, 2003), and by

Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Witcher et al., 1991;

Wehrens, Lehnart, Reiken, & Marks, 2004). PKA phosphorylation of RyR2 causes

increased sensitivity to Ca2+ induced activation, increases the probability of channel

opening and destabilizes RyR2, which may lead to arrhythmias (Blayney & Lai,

2009; Marx et al., 2000).

A further consequence of hyperphosphorylation of the RyR2 is depletion of luminal

SR Ca2+ stores; as more Ca2+ is leaked from the lumen, the less is available for the

next contraction, resulting in smaller contractions and a longer time to decay (Marks,

2001; Dulhunty et al., 2007; Wehrens & Marks, 2003). The RyR2 has also been

shown to be down-regulated along with SERCA2a in failing human hearts, which

leads to further disruption of effective ECC (Wehrens & Marks, 2003; Marks, 1996;

Blayney & Lai, 2009).


1.4 THE CLINICAL USE OF β-BLOCKERS FOR THE TREATMENT OF HEART FAILURE

1.4.1 Development of the β-blockers

Noradrenaline and adrenaline first bind to key amino acids of β1- and β2-

adrenoceptors to enable them to activate the receptors. The binding of noradrenaline

and adrenaline to the active site of β1- and β2-adrenoceptors is highly specific; β-

blockers competitively reduce the ability of noradrenaline and adrenaline to bind to

the active site of both of the β-adrenoceptors (Bristow, 1993). Treating heart failure

requires a complex approach to target the signaling pathways which lead to both

functional and structural remodeling of cardiomyocytes (Javed & Deedwania, 2009).

Considering that heart failure causes a reduction in cardiac output, inotropy, and

lusitropy, using an agent which blocks the positive effects of catecholamines that

normally serve to increase cardiac output may seem counter-intuitive; however,

multiple studies after their discovery in the 1960’s showed a clear survival benefit of

β-blockers in both coronary artery disease and heart failure (Black et al., 1965;

Packer et al., 1998; Waagstein, Hjalmarson, Varnauskas, & Wallentin, 1975;

Barrrese & Taglialatela, 2013).

The survival benefit of the β-blockers was put down to two different mechanisms of

action: their ability to block the harmful chronic effects of catecholamines, and their

ability to re-sensitize down-regulated β1-adrenoceptors to acute increases in

catecholamines (Nguyen et al., 2014). When first administered, β-blockers do in fact

reduce cardiac output; however, low-dose initiation with subsequent titration to

target dosing does minimize adverse effects, and long-term use of bisoprolol (CIBIS-

II, 1997; Lechat et al., 1997), metoprolol (Waagstein et al., 1993; MERIT-HF, 1993)

and carvedilol (Colucci et al., 1996; Packer et al., 1996; Packer et al., 2001) have all


shown improvements in left ventricular ejection fraction (Packer et al., 1998; ESC

Guidelines, 2012) and reductions in hospitalizations and overall mortality (ESC

Guidelines, 2012). These long-term clinical improvements indicate that the chronic

administration of β-blockers is able to restore cardiac function sufficiently enough to

overcome any short-term cardiodepressant effects (Packer et al., 1998).

1.4.2 The Evolution of the β-blocker

There are three generations of β-blockers, each distinguished by their affinity for

adrenergic receptors (Bristow, 2000). The first generation included the non-β-

adrenoceptor-selective propranolol, timolol, and pindolol; second-generation were

designed to be β1-adrenoceptor selective in order to avoid complications in patients

with concomitant respiratory diseases such as COPD (Black et al., 2005), and include

atenolol, metoprolol, and bisoprolol. This generation does, however, block β2-

adrenoceptors at higher doses (Javed & Deedwania, 2009). Third generation β-

blockers are also non-selective, but mediate vasodilation in addition to β-blockade;

this class includes carvedilol and bucindolol (Javed & Deedwania, 2009; Mansoor &

Kaul, 2009). (Table 1.1) Both bucindolol and carvedilol mediate their vasodilatory

actions via the α1-adrenoceptor, and improve insulin sensitivity versus the first

generation β-blockers (Javed & Deedwania, 2009). Interestingly, many of the first

generation β-blockers, which were developed to be β1-adrenoceptor selective,

actually have poor β1-adrenoceptor selectivity in intact cells (Baker, 2005).

Furthermore, many of the β-blockers are not merely antagonists, but at differing

concentrations can also act as partial agonists (such as pindolol) and inverse agonists

(such as bucindolol) at β-adrenoceptors (Baker, 2005; Barrese & Taglialatela, 2013).

_______________________________________________________________


Generation Selectivity Drugs _________________________________________________________________ 1st non-selective propranolol, timolol, pindolol, no vasodilation nadolol, sotalol 2nd β1 –selctive atenolol, bisoprolol, metoprolol no vasodilation β1-selective acebutolol with vasodilation 3rd non-selective carvedilol, bucindolol, nebivolol with vasodilation __________________________________________________________________

Table 1.1: Generations of β-blockers, adapted from Mansoor & Kaul (2009), and Bristow (2000b).

1.4.3 Efficacy of β-Blockers Clinically Used for Heart Failure

There have been many well-designed, multi-center, double blind, placebo-controlled

trials that have shown clear survival benefits for bisoprolol (CIBIS-II, 1999),

metoprolol (MERIT-HF Study Group, 1999; COMET, 2003), carvedilol

(COPERNICUS, 2002; COMET, 2003), and nebivolol (SENIORS, 2005) in heart

failure patients. In fact, most of the trials were stopped after one year due to vast

improvements in morbidity and mortality in the treatment groups versus the placebo

groups (Nguyen et al., 2014; Barrese & Taglialatela, 2013). (Table 1.2) The survival

benefit of β-blockers has been demonstrated so effectively that they are now

recommended to be initiated as soon as signs and symptoms of heart failure appear

(Nguyen et al., 2014); moreover, of the four oral medications currently used (the

other three being ACE inhibitors, anti-platelet therapy, and statins) to ameliorate

cardiovascular morbidity and mortality, β-blockers have been shown to have the


highest survival benefit (Mansoor & Kaul, 2009; Javed & Deedwania, 2009; ESC

guidelines, 2012).

TRIAL n DOSE LVEF RESULTS

US Carvedilol HF Study; 1996

1094 6.25-‐50mg/bd 28%

Carvedilol lowered risk of death by 65%

(p<0.001)

CIBIS II; 1999 2647 1.25-‐10mg/ day

27.5% Bisoprolol significantly improved survival

(p<0.0001)

MERIT-‐HF (Metoprolol CR/XL Randomized Intervention Trial in Congestive Heart Failure); 1999

3991 200mg/ day 28%

Metoprolol CR/XL significantly improved survival (p=0.00009)

COPERNICUS (Carvedilol Prospective

Randomized Cumulative Survival

Trial); 2002

2289 3.15-‐25mg/ day

19.9% Carvedilol significantly improved survival

(p=0.0014)

COMET (Carvedilol or Metoprolol European

Trial); 2003 3029

Carv: 25mg/bd,

Met: 50mg/bd

26%

Carvedilol significantly improved survival vs. metoprolol TARTRATE

(p=0.0017)

SENIORS; 2005 2128 1.25-‐10mg/ day

36% Nebivolol significantly reduced morbitity and mortality (p=0.039)

Table 1.2: Multi-center, double-blind, placebo-controlled studies on survival benefits of β-blockers; modified from Nguyen et al. (2014).

Metoprolol was one of the first β-blockers to be studied extensively for the treatment

of systolic heart failure, and in 1999 the MERIT-HF trial found a 34% reduction in

overall mortality, with a 39% reduction in myocardial infarction of metoprolol versus

placebo (MERIT-HF, 1999). Bisoprolol was studied around the same time, and a

similar survival benefit was shown in a large double-blind trial (CIBIS-II, 1999). The


fact that α1-, β1- and β2-adrenoceptors all mediate harmful effects of chronic

adrenergic stimulation in the heart, together with the fact that β-blockers are not all in

fact solely antagonists at β-adrenergic receptors (Barrese & Taglialatela, 2013), led

researchers to question the possibility of a non-selective β-blocker improving

morbidity and mortality in patients with heart failure versus a selective β1-

adrenoceptor blocker (Packer 1998). In addition, the intolerability of some patients

with severe heart failure to the initial cardiodepressant effects of the first-generation

β-blockers makes an agent that blocks both β1- and β2-adrenoceptors, as well as

having vasodilatory effects, in theory, a better therapeutic alternative (Packer, 1998;

Book & Hott, 2003). Carvedilol is the obvious stand-out for these additional

properties, as it is orally administered, blocks α1-, β1-, and β2-adrenoceptors,

ameliorates insulin sensitivity, and decreases smooth muscle cell proliferation

(Barrese & Taglialatela, 2013).

In 1999, three separate studies reported a higher increase in left ventricular ejection

fraction in carvedilol versus metoprolol-treated patients. However, the studies were

small, some not blinded, and only showed significant benefits of carvedilol when

patients were followed for more than six months (Metra et al., 2000; Di Lenarda et

al., 1999; Kukin et al., 1999; Sanderson et al., 1999). In 2000, Metra and colleagues

studied the effects of metoprolol tartrate in a larger, randomized, double blind, head-

to-head trial versus carvedilol. They found that carvedilol had a greater benefit in left

ventricular ejection fraction and stroke volume during exercise, but that the two

drugs had similar benefits in overall symptom improvement and quality of life.

Unfortunately, they did not use survival as a primary endpoint, and still only had a

small cohort of patients. The first large, (n=3029) multi-center, head-to-head trial of


carvedilol versus immediate release metoprolol tartrate with survival as a primary

endpoint found a significant survival benefit of carvedilol over metoprolol, although

the researchers could not positively identify any of carvedilol’s additional adrenergic

effects as the reason for the reported increased survival benefit (COMET, 2003).

Despite the significant benefit of carvedilol versus metoprolol in the 2003 COMET

trial, questions emerged regarding the suitability of both the dose and dosage form of

metoprolol used in the study (Bristow et al., 2003). Metoprolol is orally available in

two formulations: as an immediate release metoprolol tartrate, which is dosed twice

daily, and as a controlled release metoprolol succinate (metoprolol XL/CR), which

only requires once daily dosing. Critics of the COMET study argued that the

metoprolol dosing used during the trial was unable to achieve adequate β1-

adrenoceptor blockade when compared to the carvedilol dosing; this was due to both

the use of the wrong formulation of metoprolol and administration of sub-therapeutic

maintenance doses. Immediate release metoprolol was not approved for the treatment

of heart failure due to wide fluctuations in plasma concentrations, leading to both

loss of β1-adrenoceptor selectivity at high concentrations and loss of β1-adrenoceptor

blockade at low concentrations (Wikstrand, 2000). In addition, the average

maintenance dose of metoprolol for the COMET trial was only 85 mg daily, which is

lower than the dose of metoprolol used in the Metoprolol in Dilated Cardiomyopathy

Trial, upon which COMET based its dosing (Waagstein et al., 1993; Bristow, 2003).

Critics of the findings from COMET argue that these oversights in the metoprolol

dosing are what led to the significant survival benefit of carvedilol (Bristow et al.,

2003).

Several groups have sought to answer the carvedilol-versus-metoprolol question

since the COMET results were published in 2003, with either original research or


meta-analyses of existing trials. Unfortunately, results have remained conflicting

(Barrese & Taglialatela, 2013). In 2012, Shore and colleagues released a 10-year

study including over 3,700 patients with either ischemic or non-ischemic heart

failure treated with either carvedilol or the extended release metoprolol succinate.

They found that the type of heart failure significantly modified patient response; the

ischemic heart failure group showed greater survival when treated with metoprolol

succinate, whereas the non-ischemic group had greater survival when treated with

carvedilol. However, both extended release metoprolol and carvedilol showed similar

reductions in hospitalizations (Shore et al., 2012).

In 2013, a meta-analysis of trials studying atenolol, bisoprolol, bucindolol,

carvedilol, metoprolol succinate, or nebivolol was conducted to see if any of the

included β-blockers had a clearly significant benefit in survival endpoints over the

others (Chatterjee et al., 2013). While the investigators declared that survival

benefits and improvements in left ventricular ejection fraction appeared to be a class

effect and not significantly different between β-blockers, they did suggest that a

definitive answer as to any β-blocker’s superiority was not possible with their

available data, and that more head-to-head trials would need to be conducted, as

carvedilol did demonstrate the lowest (albeit not significantly lower) mortality

(Chatterjee et al., 2013). Another study was put forth by Pasternak et al. (2014)

which followed 11,664 Danish heart failure patients who were treated with either

carvedilol or metoprolol succinate over 3 years. Similar to the Chatterjee group, the

Pasternak study did not find any significant difference in mortality of carvedilol

versus slow-release metoprolol (Pasternak et al., 2014). In contrast, a meta-analysis

of 11 randomized trials spanning more than 5,000 patients found a significant benefit


of carvedilol on overall survival versus atenolol, bisoprolol, metoprolol, and

nebivolol (DiNicolantonio et al., 2013). Again, the group was unable to pinpoint the

reasons for carvedilol’s improved survival rates; they speculated the benefits were

due to carvedilol’s additional α1-mediated vasodilation and β2-adrenoceptor

blockade, as well as its anti-arrhythmic effects (DiNicolantonio et al., 2013). These

studies demonstrate a clear need for more head-to-head trials not only investigating

the survival benefits of the β-blockers, but also how the β-blockers ameliorate their

beneficial effects in heart failure patients.

1.4.4 Signaling Bias of β-blockers

The studies conducted on the clinically available β-blockers have indicated that these

drugs should not just be grouped as a single class, or even into distinct generations.

β-blockers can be full, partial, or inverse agonists/antagonists of either the β1-and/or

the β2-adrenoceptor, and their differing effects on disease states such as heart failure

and asthma indicate that there is no “class effect” of β-blockers, and that in fact β-

blockers are not just simply β-blockers at all (Thanawala et al., 2014). Recent studies

have found a variety of ligands, including the β-blockers, which intrinsically activate

specific downstream signaling cascades of GPCRs rather than activating the

receptor’s full repertoires, by directly stabilizing a distinct receptor conformation;

this ability has been termed ‘ligand bias’ (Galandrin & Bouvier, 2006; DeWire &

Violin, 2011).


β1-Adrenoceptor Signaling Bias

In addition to activating the cAMP pathway, β1-adrenoceptors have also been shown

to activate the EKR1/2 pathway via the scaffolding protein β-arrestin; in mice,

studies found this pathway to protect against catecholamine-induced apoptosis

(DeWire & Violin, 2011). Theoretically, biased ligands which could selectively

activate the ERK1/2 pathway may prove more cardioprotective than those which

block both the cAMP and ERK1/2 pathways. In a study which investigated the

potential ligand bias of 20 β-blockers, carvedilol was the only one approved for

clinical use in heart failure which activated this non-canonical ERK1/2 pathway,

which could be one explanation for why carvedilol has demonstrated an increase of

survival over the other β-blockers in certain studies (Kim et al., 2008; Barrese &

Taglialatela, 2013).

β2-Adrenoceptor Signaling Bias

The β2-adrenoceptor has at least two distinct signaling pathways: the adenylyl

cyclase pathway which ultimately results in the accumulation of cAMP, and the

mitogen-activated protein kinase (MAPK) pathway that activates ERK1 and ERK2

via the scaffolding protein β-arrestin (Audet & Bouvier, 2008; Thanawala et al.,

2014). (Figure 1.3)


Figure 1.3. Example of ligand-biased signaling for the β2-adrenoceptor. Three of the signaling effectors (Gs, Gi and β-arrestin) that mediate β2-adrenoceptor–promoted activation of AC and MAPK are illustrated. From Audet & Bouvier, 2008.

Each β-blocker has shown distinct affinities and efficacies for each pathway; this

selective activation of distinct ligand-directed signaling is termed ‘biased signaling’

(Thanawala et al., 2014). Carvedilol and propranolol are both inverse agonists of the

cAMP signaling pathway, but partial agonists of the ERK1/2 pathway. Adrenaline

and formoterol are both full agonists of the β2-adrenoceptor for both pathways,

whereas nadolol and ICI 118,551 are inverse agonists for both pathways (Galandrin

& Bouvier, 2006; Thanawala et al., 2014). For the efficacy profiles of common

ligands of β-adrenoceptors, please refer to Table 1.3, below.


Ligand β1-Adrenoceptor β2-Adrenoceptor AC ERK1/2 AC ERK1/2 Isoproterenol AGO AGO AGO AGO Labetalol AGO NEUT AGO AGO Bucindolol AGO AGO NEUT AGO Carvedilol AGO AGO NEUT AGO Propranolol INV AGO INV AGO Metoprolol INV NEUT INV INV Bisoprolol INV NEUT INV INV Atenolol INV NEUT INV INV

Table 1.3. β-adrenoceptor ligands and their efficacy patterns towards adenylyl cyclase (AC) and the β-arrestin/ERK1/2 pathways of the β1- and β2-adrenoceptors. AGO, partial or full agonist; NEUT, neutral agonist; INV, inverse agonist. Modified from Galandrin & Bouvier (2006). 1.4.5 Dynamic Bias

Building on the theory of ligand and signaling bias is the theory of dynamic bias; the

idea that both progressive disease states and chronic therapy with certain drugs can

lead to a dynamic shift in signaling bias in vivo, which is not seen in static, in vitro

studies (Michel, Seifert, & Bond, 2014). Michel and colleagues suggest that this

dynamic shifting in signaling bias may be what accounts for the seemingly

paradoxical benefit of β-blockers reducing mortality in heart failure, and may also

explain why despite acute worsening of cardiac function and output, long-term


administration of β-blockers demonstrates a clear therapeutic benefit (Michel et al.,

2014).

As stated previously, the pharmacology of the clinically used β-blockers is

quite varied in terms of β1- versus β2-adrenoceptor affinities from generation to

generation. As even un-occupied β-adrenoceptors have intrinsic activity, β-blockers

are termed not just as antagonists but also as inverse agonists, due to their ability to

go beyond simply blocking the β-adrenoceptors and further reduce their resting

intrinsic activity (Maack et al., 2003). Pharmacodynamics also vary widely between

the β-blockers; for example, carvedilol is able to bind to the α1- and β1/ β2-

adrenoceptors for a longer duration than the other β-blockers, and actually exerts its

antagonistic effects even at concentrations too low to be detected in plasma (Maack

et al., 2003). In contrast, the β-blocker esmolol has such a rapid onset and short

duration of action that it is used peri-and post-operatively when rapid titration of

cardiac output is required (Zangrillo et al., 2009; Wiest, 1995; Byrd et al., 1984).

1.5 THE ENANTIOMERS OF β-BLOCKERS

1.5.1 Racemic Mixtures

For a drug to exist as an enantiomer, it must have a chiral center, around which the

molecules can rotate so as to create two distinct structural isoforms of the compound,

which are mirror images of each other (Mehvar & Brocks, 2010; Patil & Kothekar,

2006). The isomers are then classed as being “R” or “S” depending on the actual

arrangement of atoms, as well as being either dextro (+) or levo (-), which indicates

how polarized light is rotated around them (clockwise or anti-clockwise,

respectively) (Stoschitzky, Zernig, & Linder, 1998; Patil & Kothekar, 2006).


Compounds that do not have a chiral centre and therefore do not exist as racemates

are termed ‘enantiomerically’ or ‘optically’ pure (Patil & Kothekar, 2006).

In order for a drug to interact and bind with its receptor(s), its molecular structure

must “fit,” or meet a stereospecific conformation unique to that receptor. Therefore,

different enantiomers of the same drug may not possess the same affinity or efficacy

within the human body, due to different binding conformations for their target

receptor(s) (Mehvar & Brocks, 2001). The pharmacokinetics of enantiomeric

compounds can be markedly different between racemates, especially in regards to

their protein binding profiles and metabolism, where receptor stereoselectivity is

more common (Caldwell, 1995; Lowenthal et al., 1985). The presence of racemates

within a compound can lead to clinical implications such as differences in efficacy,

bioavailability, volume of distribution, and rate of elimination (or subsequent

accumulation of one racemate) (Patil & Kothekar, 2006). This is especially true for

racemic mixtures in which one racemate either binds to receptors other than the

target receptor (such as R-amlodipine resulting in peripheral edema), has different

activity at the target receptor, (for example, (-)-pindolol activates β1L-adrenoceptors

at higher concentrations required to block the receptor (Kaumann & Lobnig, 1986),

or is metabolized into a molecule which accumulates or causes toxicity, such as R-

omeprazole leading to gastric carcinoids in poor CYP2C19 and CYP3A4

metabolizers (Patil & Kothekar, 2006).

It is estimated that as many as 60% of therapeutic agents contain chiral centers

(Tucker & Lennard, 1985) and that up to 20% of these agents are only clinically

available as racemic mixtures (Caldwell, 1995; Caner, Groner, & Levy, 2004). The


administration of racemic mixtures may increase the incidence of toxicity or adverse

effects, especially when those unwanted effects are a product of the “inactive”

racemate (Caldwell, 1995; Mehvar & Brocks, 2001). By performing a “chiral

switch,” or removing the inactive or less-active racemate completely, many

compounds can be made more efficacious and better tolerated, especially for those

compounds in which the inactive form has different absorption and elimination rates

than its active isomer (Mehvar & Brocks, 2001; Patil & Kuthekar, 2006).

As β-blockers are synthetically derived from isoprenaline, a β-adrenoceptor agonist,

they all share a common racemic structure, the pharmacophore of which is an

aryloxyamino alcohol (Peter & Gyeresi, 2011; Davies, 1990; Stoschitzky et al.,

1998). (Figure 1.4) Common features of noradrenaline, adrenaline and all clinically

used β-blockers is the presence of a protonated nitrogen atom, which forms a strong

hydrogen bond with the receptor, and a chiral carbon atom (Kaumann & Molenaar,

2008). The presence of a chiral carbon in β-blockers means that they exist as

enantiomers which differ by the arrangement of hydrogen and hydroxyl groups

around the chiral carbon.


Figure 1.4. Chemical structures of β-blockers. The asterisk denotes the chiral carbon. From Mehvar & Brocks. (2001)

With the exception of sotalol, the S-enantiomer of which has very limited

antagonistic effect at both β1-and β2-adrenoceptors (Johnston et al., 1985; Gomoll &

Bartek, 1986; Waldo et al., 1996), the efficacies of the S-enantiomers of the β-

blockers are much more potent (up to 500-fold) than those of the R-enantiomers, and

their pharmacokinetics and pharmacodynamics often differ from one another as well

(Stoschitzky et al., 1998; Agustiana et al., 2010; Peter & Gyeresi, 2011; Bekhradnia

& Ebrahimzadeh, 2012). The differences of the pharmacokinetics of the R- isomers

versus the S-isomers of many β-blockers are especially marked in genetically poor

metabolisers due to polymorphisms of both CYP1A and CYP2D6, and in patients

under exercise-induced stress (Benny & Adithan, 2001; Mehvar & Brocks, 2001).


The differences in pharmacokinetics between racemates of the β-blockers are due

largely to the high stereoselectivity of the β-adrenoceptor (Stoschitzky et al., 1998).

Absorption appears not to differ between the racemates, as they are passively

absorbed through the gastrointestinal tract (Wetterich et al., 1996). The distribution

between racemates depends on their binding to plasma proteins; for example, (-)-

propranolol has a higher unbound fraction in plasma, and reaches higher

concentrations in certain tissues, including the heart (Mehvar & Brocks, 2001).

Metabolism and excretion of the β-blockers occurs both through hepatic and renal

pathways, and each pathway can show stereoselectivity for one racemate over

another; for example, S-metoprolol is favored by α-hydroxylation, whereas R-

metoprolol is favored by O-demethylation (Murthy et al., 1990).

The physicochemical and pharmacological properties of the enantiomers of the β-

blockers can also differ significantly (Kaumann & Molenaar, 2008). For example, (-

)-pindolol blocks both β1- and β2-adrenoceptors at sub-nanomolar concentrations

with moderate selectivity for β2-adrenoceptors (Kaumann & Lobnig, 1986), while

higher concentrations (~ 2½ - 3 log units) activate β1-adrenoceptors though the low

affinity binding site (Kaumann & Lobnig, 1986; Joseph et al., 2003; Kaumann &

Molenaar, 2008). (+)-Pindolol blocks β1- and β2-adrenoceptors at higher (~ 2 log

units) concentrations than those required for (-)-pindolol (Walter, Lemoine, &

Kaumann, 1984; Kaumann & Lobnig, 1986; Joseph et al., 2003). The steric

requirements for pindolol are higher at β1-adrenoceptors than β2-adrenoceptors

(Kaumann & Lobnig, 1986). Finally, (+)-pindolol causes a positive chronotropic

effect in guinea-pig atrium, predominantly through activation of β2-adrenoceptors

(Walter et al., 1984).


Despite demonstrated differences in pharmacology, the clinically available β-

blockers are still, for the most part, only available as racemic mixtures; this has

resulted in an increased demand for enantiomerically pure preparations, as they will

allow for more predictable pharmacokinetic and pharmacodynamic profiles in

patients (Caldwell, 1995; Agustiana et al., 2010; Peter & Gyeresi, 2011; Patil &

Kuthekar, 2006). With more powerful separation technologies available now

compared to when many β-blockers were first marketed, studies investigating each

enantiomer’s distinct pharmacokinetics and pharmacodynamics are becoming more

available and critical (Gorczynski et al., 1984; Lowenthal et al., 1985; Tucker &

Lennard, 1990; Wang et al., 2011; Tang et al., 2012).

In order to study the effects of enantiomerically pure preparations of β-blockers in an

in vitro setting, it is desirable to utilise one which rapidly diffuses through tissue and

therefore displays a rapid onset of effect, so that contractility is not affected by long

incubation times. Esmolol, a parenterally available β-blocker, has a 60-second onset

and a very short half-life (Zuppa, Shi, & Adamson, 2003; Iskandrian et al., 1986).


1.5.2 Esmolol

ASK-8052, or esmolol, is an ultra-short acting, β1-adrenoceptor-selective antagonist

with a half-life reported from 2 minutes to 19 minutes (Zangrillo et al., 2009),

although it is generally reported to have a 9 minute half-life in human plasma (Zuppa

et al., 1986; Kirshenbaum, Kloner, McGowan, & Antman, 1988; Spahn et al., 1993).

It has a rapid onset of 60 seconds, 90% β-blockade within 5 minutes, a maximum

effect at 6-10 minutes, and a duration of action ranging from 10-20 minutes,

(Zangrillo et al., 2009; Marik & Varon, 2009; Wiest, 1995). Esmolol levels drop to

undetectable concentrations in as little as 20 minutes after infusion, and complete β-

adrenoceptor recovery occurs in approximately 18 minutes (Wiest, 1995). While

esmolol has a similar structure to the other second-generation, β1-selective blockers

such as atenolol and metoprolol, the presence of an ester on its phenyl ring allows

esmolol to bind more selectively to the β1-adrenoceptors in the heart versus the β2-

adrenoceptors located in the bronchial tissue and peripheral blood vessels

(Lowenthal, Porter, Saris, Bies, Slegowski, & Stuadacher, 1985; Wiest, 1995; Pringle

& Riddel, 1989; Mansoor & Kaur, 2009). The selectivity of the β-blockers for

cardiac β1- or bronchial/peripheral β2-adrenoceptors depends largely on the

configuration of its aryloxypropanolamine group, and more specifically on the

placement of the nitrogen and oxygen atoms within this aryloxypropanolamine

group; different formations allow for stronger affinity to distinct β-adrenoceptors

(Bekhradnia & Ebrahimzadeh, 2012).

Esmolol is rapidly hydrolyzed in vivo; its acid metabolite only has 0.3% the activity

of the active metabolite and its metabolism by erythrocyte esterases within the

cytosol of red blood cells rather than by plasma cholinesterases give esmolol its

ultra-short duration of action (Gorczynski, 1985; Lowenthal et al., 1985; Zuppa et al.,


2003; Tang, He, Yao, & Zeng, 2004, Tang, Wang, Hu, Yao, & Zeng, 2012; Marik &

Varon, 2009). The acid metabolite is almost entirely eliminated through the urine,

with only 2% being excreted unchanged (Lowenthal et al., 1985).

Esmolol was synthesized and first studied in canine models in 1982 when it was

suggested that a shorter-acting, rapidly titratable β-blocker would have improved

safety over the longer-acting preparations for acute peri-and post-operative

hypertensive and arrhythmic complications, which often precipitated symptoms of

CHF (Erhardt, Woo, Anderson, & Gorczynski, 1982a, 1982b; Zaroslinksi et al.,

1982; Turlapaty et al., 1987). Studies in human patients with supraventricular

arrhythmias soon followed in 1984, and esmolol demonstrated cardioselectivity and

efficacy at controlling ventricular rates in these patients, due to its ability to slow

signal conduction through the AV node (Byrd, Sung, Mark, & Parmley, 1984;

Gorczynski, 1985). Left untreated, tachyarrhythmias and hypertension can lead to

mortality or major morbidities, including myocardial infarction, stroke, and

irreversible organ damage (Turlapaty et al., 1987; Garnock-Jones, 2012). Esmolol

has been shown to reduce heart rate, ejection fraction, and cardiac output by reducing

oxygen demand, contractility, and shear stress by increasing the sinus and AV node

conduction and refractory times (Bakker et al., 2011; Wiest, 1995) in patients with

preserved left ventricular function (Iskandrian et al., 1986). These effects have also

been demonstrated in patients with unstable angina and history of myocardial

infarction and in patients with moderate left ventricular dysfunction or acute

myocardial infarction (Kirshenbaum et al., 1988). In canine reperfusion models,

esmolol showed an improvement in regional myocardial function and a reduction in

infarct size, although a total cardioprotective effect was not demonstrated (Spahn et


al., 1993). A reduction in myocardial infarct size and oedema has also been

demonstrated in humans (Geissler et al., 2000).

Due to its cardioselectivity, rapid onset and washout times regardless of hepatic or

renal function (Marik & Varon, 2009), esmolol is used clinically when rapid titration

is required (Byrd et al., 1984; Kirshenbaum et al., 1988). It is used perioperatively

(Lowenthal et al., 1985; Kirshenbaum et al., 1988) and postoperatively (Marik &

Varon, 2009) to prevent and control hypertension, ventricular fibrillation and

tachycardia (Kirshenbaum et al., 1988). The short t ½ also allows for rapid reversal if

hypotension or bradycardia develops (Zuppa et al., 2003). The specific

pharmacokinetic profile of esmolol allows for rapid titration in vivo simply by

controlling infusion rate (Fang, Bykowski-Jurkiewicz, Sarver, & Erhardt, 2010).

Orally administered β-blockers half-lives which range from 2-14 hours, which makes

them much more difficult to titrate should adverse effects occur (Esmolol Research

Group, 1986).

Atrial fibrillation and sinus tachycardia occur in 25% of coronary artery bypass graft

and valve replacement patients, and is yet more common in patients who have had β-

blocker therapy ceased pre-operatively (Gray, Bateman, Czer, Conklin, & Matloff,

1985; Garnock-Jones, 2012). In these patients, postoperatively administered esmolol

effectively restores sinus rhythm and controls heart rate (Tang et al., 2012; Gray et

al., 1985). Esmolol is used both peri- and postoperatively in patients with acute

ischaemic heart disease (Iskandrian et al., 1986; Bakker et al., 2011; Garnock-Jones,

2012), atrial fibrillation/flutter (Tang et al., 2004; 2012), and for supraventricular

tachyarrythmias in patients with mild CHF, COPD, and valvular disease (Geissler et

al., 2011; Gray et al., 1985; Byrd et al., 1984; Yamakage, Iwasaki, Jeong, Sato, &


Namiki, 2009) and in low doses in those with severe left ventricular dysfunction

(Iskandrian et al., 1986) and severe postoperative hypertension (Marik & Varon,

2009). Esmolol has demonstrated a reduction in perioperative mortality, (Bakker et

al., 2011) and in postoperative AF/MI following CABG surgery (Zangrillo et al.,

2009). However, esmolol can increase the incidence of peri-and post-operative

bradycardia and hypotension, and its cardioprotective efficacy has yet to be

effectively demonstrated (Bakker et al., 2011, Zangrillo et al., 2009; Garnock-Jones,

2012).

The most common adverse effect of esmolol is hypotension (incidence up to 50%),

due to decreased inotropy and chronotropy, and possibly due to a decrease in

peripheral vascular resistance (Byrd et al., 1984; Wiest, 1995), however it is usually

rapidly reversible (Garnock-Jones, 2012). Its tolerability is greatly due to its

cardioselectivity; esmolol is 18 times more selective for cardiac β1-adrenoceptors

versus bronchial β2-adrenoceptors than metoprolol, and 50 times more β1-selective

than propranolol (Gorczynski et al., 1983; Garnock-Jones, 2012).

Esmolol, like most β-blockers, is currently only marketed as a racemic mixture,

although its antagonistic effects are believed to reside solely in the S-(-) enantiomer

(Zuppa et al., 2003; Fang et al., 2010; Tang et al., 2012), with the R-(+) enantiomer

having been demonstrated as being inactive (Lowenthal et al., 1985). The red blood

cell esterases responsible for the majority of its metabolism have not shown

stereoselectivity in previous human studies (Zuppa et al., 2003; Tang et al., 2012).

However, plasma esterases demonstrate a high level of stereoselectivity for the R-(+)

enantiomer, due to a significant difference in protein bound fractions between the

enantiomers (Tang et al., 2012).


1.5.3 The Clinical Relevance Of Enantiomerically Pure β-Blockers

Enantiomerically pure compounds are in greater demand, with the FDA now calling

for the justification of racemic mixtures being marketed for clinical use (Agustiana et

al., 2010). In both the United States and the European Union, all drug development

studies must now include a thorough investigation of any chiral structure, how the

enantiomers may be separated, the relative contributions of the separate enantiomers

to the compound’s overall efficacy, and how the racemates will be marketed; these

guidelines, introduced in the early 1990’s, have led to a world-wide reduction in the

marketing of racemic mixtures (Caner et al., 2004). This is not true for the β-

blockers, however, as timolol is the only one of its class which is available world-

wide as an enantiomerically pure preparation; S-atenolol and S-metoprolol were

introduced in India in 2003, and are still being trialled (Mehvar & Brocks, 2001;

Dasbiswas & Dasbiswas, 2010). The S-isomers of both atenolol and metoprolol have

been isolated with the intent of improving cardioselectivity at higher doses; the

racemic mixtures lose β1-selectivity as dose increases, and this is believed to be due

to the R-isomers relative β2-selectivity (Mehvar & Brocks, 2001; Patil & Kuthekar,

2006). R-metoprolol also causes interactions with commonly prescribed drugs such

as cimetidine, paroxetine, verapamil, and some antibiotics, due to competitive

binding for metabolising enzymes such as CYP2D6 (Stout et al., 2011); its removal

should also eliminate potentiation by co-administration with these compounds (Patil

& Kuthekar, 2006).


1.6 PHOSPHODIESTERASE ENZYMES: HEALTHY VS. FAILING HEARTS

1.6.1 Phosphodiesterase Enzymes in the Healthy Heart

Phosphodiesterase (PDE) enzymes are 3’, 5’ cyclic nucleotide phosphatases that are

responsible for degrading the second messengers cAMP and cGMP by hydrolyzing

their phosphodiester bond (Bischoff, 2004). PDEs accomplish this degradation in

both spatially and temporally specific patterns due to highly regulated

compartmentation (Omori & Kotera, 2007; Lenhart & Marks, 2006). cAMP is a

second messenger of the β1- and β2-adrenoceptor pathways, and activates cAMP-

dependent protein kinase a (PKA) which is responsible for the phosphorylation of

many proteins orchestrating the ECC, including troponin I, PLB, and the L-type Ca2+

channel and the RyR2 complex (Guellich et al., 2014). Over-stimulation of these

proteins by chronic β-adrenoceptor activation leads to hypertrophy, fibrosis, altered

Ca2+ handling and arrhythmias, and eventually leads to ventricular dysfunction

(Guellich et al., 2014). The degradation of cAMP reduces the phosphorylation of

target proteins by PKA, and protects the heart against overstimulation by Ca2+ via

RyR2-mediated ECC (Omori & Kotera, 2007; Lenhart & Marks, 2006; Levy, 2103).

(Figure 1.5)


Figure 1.5. Catecholamine-induced activation of β1 and β2-adrenoceptors leads to a cAMP-PKA-mediated phosphorylation of the RyR2.

There are 11 PDE families that are separated into 60 isoforms based on sequence,

structure, substrate specificity, and sensitivity to both physiological and

pharmacological inhibitors (Bischoff, 2004; Lenhart & Marks, 2006; Mika, Leroy,

Vandecasteele, & Fischmeister, 2011; Omori & Kotera, 2007). PDEs 1-5, 8 and 9

have been found in the mammalian heart, although PDE9 does not appear to regulate

cardiac physiology or pathology (Knight & Yan, 2012). All of the PDEs regulate

cAMP and/or cGMP signaling by hydrolyzing their phosphodiester bond, which

effectively reduces both cyclic nucleotides to their inactive forms. However, each

family is localized to different tissues in the human body (Bischoff, 2004; De

Courcelles, De Loore, Freyne, & Janssen, 1992), and consequently, selectively

inhibiting PDE enzymes leads to tissue-specific responses (De Courcelles et al.,

1992). (Table 1.4)

RyR2


FAMILY ISOFORM(S) SUBSTRATE(S)/ REGULATION TISSUE EXPRESSION

PDE1 PDE1A,C cAMP/cGMP: Ca2+/CaM-stimulated

cardiac myocytes, vascular myocytes, neurons, lymphoid cells, myeloid cells, testes

PDE2 PDE2A cAMP/cGMP: cGMP-stimulated

brain, cardiac myocytes, liver, adrenal cortex, endothelium, platelets

PDE3 PDE3A,B cAMP: cGMP-inhibited

cardiac myocytes, vascular myocytes, brain, liver, adipose, pancreas, endothelium, epithelium, oocytes, platelets

PDE4 PDE4A-D cAMP cardiovascular myocytes, neurons, immune system, inflammatory system

PDE5 PDE5A cGMP vascular myocytes, diseased cardiac myocytes, lung, brain, platelets, kidney, gastrointestinal tissues, penis

PDE6 PDE6A,B,C cGMP photoreceptors, pineal gland

PDE7 PDE7A,B cAMP spleen, brain, lung, kidney, lymphoid cells, myeloid cells

PDE8 PDE8A,B cAMP testes, thyroid

PDE9 PDE9A cAMP spleen, brain, intestinal cells

PDE10 PDE10A cAMP brain, testes

PDE11 PDE11 cGMP prostate, testes, salivary gland, pituitary gland

Table 1.4. Families, subfamilies, substrates, and tissue expression of phosphodiesterase enzymes. Table adapted from Maurice et al., 2014 and Miller & Yan, 2010.


1.6.2 Phosphodiesterase Compartmentalization

PDEs have very specific spatial localizations, not just within specific tissue types but

also within subcellular compartments, which allows their effects on signaling

cascades to be very tightly regulated (Ahmad et al., 2012). The effects of PDEs are

not only spatially regulated, but also functionally and temporally regulated by means

of their targeting other effectors such as anchoring proteins, kinases, cyclases, PKA,

PKG, ion channels, and Epacs, (Beca et al., 2013; Ahmad et al., 2012; Knight &

Yan, 2012). They are incorporated into a wide variety of macromolecular complexes,

which allows them even further specificity and compartmentation, and any change in

their activation or degradation can upset this delicate balance of regulation, leading

to a disruption of homeostasis of cyclic nucleotides and, eventually, to progression of

disease (Guellich et al., 2014; Knight & Yan, 2012).

1.6.3 Roles of the PDE Subfamilies in Healthy and Failing Hearts

PDE expression and activity can be either up-regulated or down-regulated in patients

with heart failure, depending on the PDE family (Mehel et al., 2013; Moltzau et al.,

2014; Lee & Kass, 2012; Lenhart & Marks, 2006). The failing heart has decreased

levels of cAMP, and this decrease is thought to be a protective mechanism against

the harmful effects of chronic β-adrenergic stimulation. The aim of the

administration of PDE inhibitors is to restore cAMP levels by blocking the enzyme

responsible for its degradation (Amsallem et al., 2013). In doing so, PDE inhibitors

increase the uptake of Ca2+ into the sarcoplasmic reticulum, which results in positive

inotropy (Arnold et al., 1993). Unfortunately, along with positive inotropy, broad

PDE inhibition also leads to increased energy expenditure, the development of

arrhythmias, and cardiomyocyte apoptosis (Eschenhagen, 2013).


Role of Phosphodiesterase 1

PDE1 is encoded by three different genes (A, B, and C), is activated by CAMK-II,

and is found in the hearts of many species, including humans (Conti & Beavo, 2007).

While PDE1C was known to be the dominant isoform expressed in the human heart

(Conti & Beavo, 2007; Lee & Kass, 2012; Omori & Kotera, 2007), it was originally

thought not to be present in myocytes; however, PDE1C has since been found in the

cytosol of cardiomyocytes (Guellich et al., 2014), particularly along the Z- and M-

lines (Vandeput et al., 2007). PDE1 hydrolyses both cAMP and cGMP and is

activated by Ca2+, calmodulin, and PDE1A. It is up-regulated in heart failure, and

may contribute to hypertrophy and maladaptive remodeling (Guellich et al., 2104).

However, without a readily available PDE1-selective inhibitor, its possible functions

in the human heart remain unknown (Fischmeister et al., 2006; Guellich et al., 2014).


PDE2 has also been identified in human myocytes, where it hydrolyzes both cAMP

and cGMP. It is activated by cGMP in a negative-feedback mechanism, which

protects the heart when cGMP is activated by increased levels of nitric oxide (Mehel

et al., 2013; Guellich et al., 2014). When cGMP levels are increased, PDE2 is

activated and stimulates the hydrolysis of cAMP in the vicinity of the L-type Ca2+

channel by up to 30-fold, attenuating the effects of β-adrenoceptor stimulation

(Hartzell & Fischmeister, 1986; Mehel et al., 2013). In heart failure, PDE2 is up-

regulated approximately 2-fold; this up-regulation leads to desensitisation of the

myocytes to acute β-adrenoceptor activation, and may be a protective mechanism of

the failing heart to stress and noradrenaline-induced hypertrophy (Mehel et al., 2013;

Moltzau et al., 2014). The inhibition of PDE2 may also restore human cardiac


function, however this has not yet been established in clinical studies (Mehel et al.,

2013).


PDE3 has been identified in the cytosol of both healthy and failing human hearts, and

has similar affinity for both cAMP and cGMP; high cGMP levels inhibits its ability

to bind to and hydrolyze cAMP (Knight & Yan, 2012). Cyclic AMP in the human

heart is mainly hydrolyzed by PDE3 (Bender & Beavo, 2006; Movsesian et al.

1991), thereby controlling atrial (Christ et al., 2006; Kaumann, Semmler, &

Molenaar, 2007) and ventricular (Omori & Kotera, 2007) β1- and β2-adrenoceptor

mediated positive inotropic and lusitropic effects.

PDE3 has two subfamilies, PDE3A and PDE3B. Both hydrolyze cAMP and cGMP;

PDE3B is expressed in hepatocytes, adipocytes, and the β-cells of the pancreas,

whereas PDE3A is highly expressed in vascular smooth muscle cells and the heart

(Ahmed et al., 2012). In both rat and human studies, the PDE3A subfamily regulates

cardiac contractility by associating with SERCA and PLB to regulate uptake of Ca2+

into the sarcoplasmic reticulum, whereas PDE3B appears to have minimal effects on

basal cardiac contractility (Ahmad et al., 2012; Beca et al., 2013). PDE3A knockout

mice have altered expressions of both SERCA and RyR2, which leads to impaired

contractility (Beca et al., 2013). In addition, in vitro studies show that chronic

inhibition of PDE3A leads to cardiomyocyte apoptosis (Guellich et al., 2014). In

transgenic mice with myocardial overexpression of the enzyme, PDE3A1 has been

shown to negatively regulate β-adrenoceptor signaling, attenuating catecholamine-

induced increases of inotropy, chronotropy, and lusitropy, and to prevent apoptosis


and ischemic reperfusion injury (Oikawa et al., 2103). In contrast, it has been

suggested that PDE3B may play a role in cardioprotection in times of cardiac stress,

such as during exercise, surgery, or in chronic heart failure (Beca et al., 2013).

PDE3B is activated by PKA, PKB, and PKC-mediated phosphorylation, whereas

PDE3A is only activated by PKA and PKB-mediated phosphorylation (Omori &

Kotera, 2007). Both the PDE3A and PDE3B families have multiple and distinct

phosphorylation sites in their N-terminus domains, which preferentially interact with

PKA, PKB, or PKC (Movsesian et al., 2011).

PDE3 inhibitors compete with cAMP to bind to PDE3, competitively inhibiting the

enzyme’s ability to phosphorylate cAMP and allowing it to accumulate within the

cell (Amsallem et al., 2013). The increased intracellular concentration of cAMP

results in increased ECC, and therefore positive inotropy and chronotropy. Acute

administration of PDE3 inhibitors has been shown to be helpful in restoring the ECC

in heart failure, however chronic administration of PDE3 inhibitors leads to

dysregulation of Ca2+ signaling and an increase in mortality (Packer et al., 1991; De

Courcelles et al., 1992; Lenhart & Marks, 2006; Levy, 2013). The role of PDE3

remains unclear in human heart failure, with downregulation reported by some

studies, (Silver et al., 1990; Ding et al., 2005a, 2005b), but normal function reported

in others (Movsesian et al., 1991; Von Der Leyen et al., 1991).

PDE3 inhibitors have been used clinically to restore hemodynamics in patients with

heart failure in an acute setting (De Courcelles et al., 1992), although several large-

scale studies investigating the chronic administration of milrinone, a PDE3 inhibitor,

found that its clinical use leads to worsening of heart failure and an increase in


overall mortality (Packer et al., 1993; Shakur et al., 2002; Lenhart & Marks, 2006;

Yan, Miller, & Abe, 2007). The reason for the increase in mortality is still unknown,

with researchers indicating excessive accumulation of cAMP, a direct increase in

arrhythmias, and possible interaction with concomitant administration with

vasodilators as possible mechanisms (Shakur et al., 2002; Amsallem et al., 2013).


PDE4 is the largest of the PDE families (Guellich et al., 2014), is activated by PKA

(Conti & Beavo, 2007), and is cAMP specific (Fischmeister et al., 2006). PDE4 is

encoded by four genes (A-D) and has more than 25 isotypes; however PDE4A,

PDE4B, and PDE4D are the only ones expressed in the human heart, with PDE4D

being the dominant gene expressed (Richter et al., 2011). β1-adrenoceptors interact

preferentially with PDE4D8, while β2-adrenoceptors and the RyR2 interact

preferentially with PDE4D5 and PDE4D3, respectively (Richter et al., 2011). PDE4

is also responsible for controlling the PKA-mediated regulation of ICaL, troponin I,

and the PLB/SERCA complex associated with the RyR2 (Richter et al., 2011).

PDE4 is a well-known regulator of chronotropic and inotropic effects in animal

hearts (Heaslip, Buckley, Sickels, & Grimes, 1991; Herzer, Thomas, Carcillo,

Tofovic, & Jackson, 1998; Shakur et al., 2002; Kaumann et al., 2009; Richter et al.,

2011). PDE4B is highly localized to the sarcolemma in mouse cardiomyocytes, and

modulates β1, but not β2-adrenoceptor-mediated excitation-contraction coupling with

the RyR2 and the L-type Ca2+ channel (Mika et al., 2014). The reason for the β1-

adrenoceptor specific activation may be due to the fact that β1-adrenoceptors are

widely distributed throughout cardiomyocytes, whereas β2-adrenoceptors are


spatially restricted to very specific subcellular domains (Nikolaev et al., 2006; Mika

et al., 2014).

In the rat heart, PDE4D3 mediates cardiac hypertrophy resulting from chronic β-

adrenoceptor activation, which leads to progressive cardiomyopathy and arrhythmias

in PDE4D3-knockout mice (Kaumann, 2011; Ahmad et al., 2012). PDE4 protects

against β1-adrenoceptor mediated arrhythmias in rodent ventricle (Lenhart et al.,

2005; Galindo-Tovar & Kaumann, 2008) and human atrium (Molina et al., 2012).

In rat myocytes, agonist stimulation of the β2-adrenoceptor by isoprenaline recruits

PDE4D5, along with β-arrestin, which results in a desensitization of the β2-

adrenoceptor to both the cAMP-PKA and ERK1 signaling cascades by competitively

inhibiting Gα-activation of adenylyl cyclase and increasing local cAMP hydrolysis

(Houslay et al., 2007).

PDE4D9 associates with the β2-adrenoceptor in mice at rest, and becomes

dissociated upon β-adrenoceptor stimulation, when PDE4D5 and PDE4D8 are

recruited to the β2-adrenoceptor, likely via binding to β2-adrenoceptor/β-arrestin

complexes (De Arcangelis et al., 2009). This highly localized and specific

recruitment of PDE4 isoforms by the β2-adrenoceptor suggests a very tightly

controlled spatial and temporal regulation of cAMP signaling in response to

catecholamine-induced β2-adrenoceptor activation, which suggests an important role

of the β2-adrenoceptor in the mediation of cardiac contractility.

The effects of PDE4 in both healthy and failing human hearts are largely unknown

(Shakur et al., 2002; Richter et al., 2011), and there is still controversy regarding the


role of PDE4 in human heart, with some studies showing no effect on inotropy and

chronotropy in human ventricle (Movsesian et al., 1991; Christ et al., 2006) while

another study showed a significant potentiation of both β1 and β2 – mediated

arrhythmias in human atrium (Molina et al., 2012; Eschenhagen, 2013). Due to its

presence in the RyR2 complex, PDE4 may in theory mediate catecholamine-induced

arrhythmias via the β-adrenoceptors, however PDE4 does not seem to limit the

positive inotropic effects of (-)-noradrenaline and (-)-adrenaline through β1- and β2-

adrenoceptors respectively, in non-failing human atrium obtained from patients

treated with or without β-blockers (Christ et al., 2006; Kaumann et al., 2007). The

disparity between these studies could be due to the fact that PDE4 is activated at

higher localized cAMP levels than PDE3; thus, PDE4 inhibitors may only mediate

inotropy and lusitropy when cAMP levels are increased by either PDE3 inhibition or

the administration of isoprenaline, or in tissues where resting adenylyl cyclase

activity is higher, such as the human atrium (Molina et al., 2012; Guellich et al.,

2014).

PDE4 activity is reduced in heart failure (Levy, 2013), specifically PDE4D3 which

helps to regulate Ca2+ release from the RyR2 (Lenhart et al., 2005; Lenhart & Marks,

2006). PDE4D3 is an integral part of the RyR2 complex, and is also reduced in

failing human hearts (Lenhart et al., 2005), but its role in adrenergic stimulation of

contractility via β1- and β2-adrenoceptors in the human ventricle is not currently

known (Christ, Galindo-Tovar, Thoms, Ravens, & Kaumann, 2008; Galido-Tovar &

Kaumann, 2008; Nikolaev, Bunemann, Schmitteckert, Lohse, & Engelhardt, 2006;

Rochais et al., 2006).



PDE5A is found in human heart, lung, and smooth muscle, and PDE5A inhibitors

were first trialed in humans with the aim of treating coronary artery disease and

hypertension (Omori & Kotera, 2007; Lee & Kass, 2012). In mice, PDE5 has proven

to be an integral component of cardiac remodeling after myocardial infarction, with

its inhibition resulting in reduced hypertrophy and cardiomyocyte apoptosis

(Guellich et al., 2014). Although there appears to be minimal expression of PDE5A

in the healthy human heart, its expression is increased in hypertrophic and failing

hearts, which has led to new trials showing significant effects of PDE5A inhibition in

the failing heart (Lee & Kass, 2012). In the acute setting, PDE5 inhibition may offer

ischemic preconditioning (Kukreja et al., 2005; Das, Salloum, Xi, Rao, & Kukreja,

2009), and chronic administration may reverse hypertrophy (Fisher, Salloum, Das,

Hyder, & Kukreja, 2005) and reduce the morbidity and mortality of cardiomyopathy

due to doxorubicin toxicity (Koka & Kukreja, 2010). Despite all these findings,

PDE5 inhibition in the human failing heart has not been shown to improve clinical

outcomes (Redfield et al., 2013).


Western blots have shown that PDE8 is expressed in human cardiac tissue, and a

single study in knockout mice has shown that PDE8A controls at least some

localization of cAMP in mouse cardiac myocytes, as the removal of the gene leads to

a higher incidents of Ca2+ sparks through the RyR2 and increased Ca2+ transients

upon β-adrenoceptor activation (Petrucco et al., 2010). However, the role of PDE8 in

the human heart has yet to be elucidated.


1.7 HYPOTHESIS, SUMMARY, AND IMPLICATIONS While PDE3 and PDE4 subtypes have been extensively studied in knockout mice,

their roles in the mediation of human heart contractility have not yet been clarified.

Murine and human hearts have stark differences: where PDE4 is the dominant

isoform controlling catecholamine-induced inotropy and chronotropy in murine

hearts, accounting for approximately 30% of overall cardiac PDE activity, it only

accounts for approximately 10% of PDE activity in the human heart, and its

localization to areas of the heart associated with the mediation of contractility has

been conflicting between human heart studies (Eschenhagen, 2013). In murine,

rabbit, dog, and human heart failure studies, both PDE3 and PDE4 are down-

regulated, whereas PDE1, PDE2, and PDE5 are up-regulated (Guellich et al., 2014).

The main question that still remains to be answered is whether downregulation of

PDE3 and PDE4 is simply a product of the failing heart, or if they are an integral part

of the maladaptive remodeling which leads to heart failure - and if so, which

subfamilies need to be targeted in order to improve mortality in these patients?

While PDE3 and/or PDE4 appear to control ventricular effects of catecholamines

through the β1- and the β2-adrenoceptor in several species, their relative effects in

failing human ventricle are still unknown. These studies investigate whether the

PDE3-selective inhibitor cilostamide (300 nM–1 µM) or PDE4 inhibitor rolipram (1–

10 µM) modify the positive inotropic and lusitropic effects of catecholamines in

human failing myocardium of patients chronically treated with either metoprolol or

carvedilol versus no β-blocker therapy. This study seeks to fill the current literature

gap in the roles of PDE3 and PDE4 in human heart failure, specifically their effects

on β-adrenoceptor mediated inotropy and lusitropy in failing human ventricles.


The aim of Chapter 3 is to investigate whether PDE3 and/or PDE4 enzymes play a

role in the mediation of positive inotropy and lusitropy through ventricular β1- or β2-

adrenoceptors in patients with terminal heart failure in response to the administration

of catecholamines, and whether or not chronic administration of metoprolol modifies

the effects of PDE inhibition. The aim of Chapter 4 is to investigate whether the

inotropic and lusitropic effects of catecholamines, mediated through ventricular β1-

or β2-adrenoceptors from patients with terminal heart failure, are enhanced by

selective PDE3 and/or PDE4 inhibition, and whether or not chronic administration of

carvedilol modifies the effects of PDE inhibition. I predict that both PDE3 and PDE4

control catecholamine-induced effects of β1- and β2-adrenoceptor mediated positive

inotropy and lusitropy in human failing ventricle.

The aim of Chapter 5 is to investigate the affinities of R-(+) and S-(-) esmolol at

human β1- and β2-adrenoceptors and their effects on human right atrial contractility

and β1L- and β3-adrenoceptors, in order to determine whether or not an

enantiomerically pure preparation of S-(-) esmolol would be beneficial in a clinical

setting. I predict that S-(-) esmolol will have higher affinity at β1H,- β1L,- and β2-

adrenoceptors, and that R-(+) esmolol will have no significant affinity or efficacy in

mediating contractility through any of the investigated β-adrenoceptors.

CHAPTER 2 Methods & Research Design 47

2. Methods & Research Design

2.1 VENTRICULAR EXPERIMENTS

2.1.1 Research Design Summary

Ventricular trabeculae from freshly explanted hearts of 5 non-β-blocked, 15

metoprolol-treated, and 9 carvedilol-treated patients with terminal heart failure were

paced to contract at 1 Hz. The inotropic and lusitropic effects of (-)-noradrenaline,

mediated through β1-adrenoceptors and (-)-adrenaline, mediated through β2-

adrenoceptors, were assessed in the absence and presence of the PDE3-selective

inhibitor cilostamide (0.3 nM – 1 µM) or PDE4 inhibitor rolipram (1-10 µM). The

inotropic potencies of (-)-noradrenaline and (-)-adrenaline were then determined by

fitting a Hill function with variable slopes to concentration-effect curves from

individual experiments, and then estimating the catecholamine concentrations

producing a half maximum response, –LogEC50M (pEC50).

2.1.2 MATERIALS AND METHODS

Explantation and Transport

Trabeculae from right or left ventricle from explanted hearts with terminal heart

failure were obtained immediately upon explantation and handed directly from the

surgeon to the researcher. The time between full removal of the heart and handover

to the experimenters was carefully monitored (< 1 minute) in order to ensure a

minimum of tissue damage due to lapse in oxygenation. Upon its receipt, the entire

organ was placed directly into ice-cold Krebs solution containing (mM; Na+ 125, K+

Chapter 2: Methods & Research Design 48

5, Ca2+ 2.25, Mg2+ 0.5, Cl- 98.5, SO42- 0.5, HCO3

- 32, HPO42- 1, EDTA 0.04) for

Brisbane experiments and into Tyrode’s solution (mM): Na+ 149, K+ 5.4, Ca2+ 1.8,

Mg2+ 1.05, Cl- 137.8, HCO3- 22, HPO4

2- 0.42, EDTA 0.04, ascorbate 0.2, glucose 5.5,

and equilibrated with 95% O2/5% CO2 for Dresden experiments (Kaumann et al.,

1996; 1999). Krebs and Tyrode’s solutions were oxygenated in advance, so as to

further minimize the possibility of damage due to loss of tissue oxygenation. Once

the organs were in the solutions, they were immediately placed back onto ice for the

brief (less than two minute) transport to the laboratory.

Dissection and Set-Up of Trabeculae

Trabeculae were dissected in ice-cold Krebs or Tyrode’s solution which were

continuously oxygenated with 95% O2/5% CO2. Trabeculae chosen for dissection

were approximately 1 mm or smaller in thickness, in order to maximize the diffusion

of gases, nutrients, and drugs across the tissue throughout the experiment. Each was

carefully removed using fine surgical scissors in order to avoid any damage to the

endocardium, as it can lead to a reduction in contractility. A fine silk thread was

attached to one end of each trabeculum and knotted around the smallest amount of

tissue possible, again to minimize any interference with basal contractility. The other

ends of the sutures were tied into a loop, which were then attached to Swema SG4-45

strain gauge transducers. The non-looped ends of the trabeculae were then clamped

to Blinks tissue electrode blocks (Blinks, 1965), care being taken to clamp the

smallest portion of the trabeculae as possible, while still ensuring a secure enough

attachment to prevent dislodgement during either contraction or length-tension

testing. The design of the electrode blocks described by Blinks (1965) allow

trabeculae to be securely clamped without sacrificing oxygen flow; rather than


oxygen being bubbled directly at tissues, the solution itself (Krebs or Tyrode’s) is

briskly oxygenated and flowed past tissues. The thinness of the trabeculum

preparations and means of clamping allow for high oxygen perfusion and access to

all surfaces (with the exception of the small clamped portion) of the tissues.

The tissue electrode blocks were housed within 50 ml organ baths, large enough to

accommodate 2 trabeculae on their respective clamps in the oxygenated solution

(above) in each bath. Within the organ baths, the Krebs or Tyrode’s solution was

supplemented with (mM): Na+ 15, fumarate 5, pyruvate 5, L-glutamate 5, and

glucose 10. This solution provided metabolic support to the tissues throughout the

duration of the experiment (Kaumann et al., 1999). Up to 10 separate organ baths (20

trabeculae) were then lowered into a water bath held at 37°C by heaters which

continuously cycled and monitored water temperature, ensuring that temperatures

were held as closely to physiological conditions as possible (Kaumann et al., 1996;

1999).

Once dissected, clamped, and brought to 37°C, trabeculae were driven with

squarewave pulses of 5 ms duration to contract at 1 Hz. Currents were individually

adjusted for each trabeculum in order to establish a rhythmic contraction, and were

then held just above threshold current for the duration of the experiment (Kaumann

et al., 1999; Molenaar et al., 2006). Once contracting, each tissue was stretched

slowly and the resulting change in force of contractions monitored, in order to

establish a length tension curve for each individual trabeculum; the Lmax, or stretched

length producing maximal contractions, was established and maintained for the

duration of the experiment (Gille et al., 1985; Kaumann et al., 1996; 1999; Molenaar


et al., 2006).

Specific Activation of β1- and β2-Adrenoceptors

Optimized conditions were used to selectively activate β1- or β2-adrenoceptors. For

both conditions, α-adrenoceptors, neuronal and extraneuronal uptake were

irreversibly blocked by 5 µM phenoxybenzamine, which was incubated with the

tissues for 90 minutes prior to the addition of catecholamines. After 90 minutes, the

incubation solution was exchanged to remove unbound phenoxybenzamine and

supplemented with ascorbic acid (0.2 mM) to prevent catecholamine oxidation. (Kaumann et al., 1987; Molenaar et al., 2000; Christ et al., 2006).

ICI 118,551 has up to 300-fold higher affinity for β2- than β1-adrenoceptors (Bilski et

al., 1983; Lemoine et al., 1985a; Kaumann & Lemoine, 1987). ICI 118,551 at a

concentration of 50 nM antagonizes adrenaline-induced increases in contractile force

(Kaumann & Lemoine, 1987), and the KD of ICI, 118,551 at the β2-adrenoceptor is

0.5-1.5 nM (Lemoine et al., 1985a; Kaumann & Lemoine, 1987; Hall, Kaumann, &

Brown, 1990). Therefore, to selectively activate β1-adrenoceptors, (-)-noradrenaline

was used in the presence of the β2-adrenoceptor selective blocker ICI 118,551 50 nM

(Gille et al., 1985; Kaumann et al., 1999; Molenaar et al., 2000; 2006; Christ et al.,

2006).

CGP20712A has a 10,000-fold higher affinity for β1- versus β2-adrenoceptors

(Kaumann & Lemoine, 1987; Dooley & Bittiger, 1984; Lemoine et al., 1985b). The

KD of CGP 20712A at β1-adrenoceptors is 0.3-0.1 nM (Kaumann & Lemoine, 1987;

Hall et al., 1990; Lemoine et al., 1985a). Therefore, to selectively activate β2-


adrenoceptors, (-)-adrenaline was used in the presence of 300 nM CGP 20712A

(Kaumann et al., 1989; 1999; Molenaar et al., 2000; 2006; Christ et al., 2006).

To determine the effect of PDE enzymes on effects mediated through activation of

β1- or β2-adrenoceptors, PDE3-selective inhibition was obtained with cilostamide

(300 nM) and PDE4-selective inhibition obtained with rolipram (1 µM) pre-

incubated with trabeculae for 30-45 minutes prior to incubation with catecholamines.

The IC50 of cilostamide at PDE3 is approximately 50 nM (Beavo, 1995; Sudo et al.,

2000; Vargas et al., 2006); therefore, 300 nM of cilostamide blocks 86% of PDE3

but less than 0.4% of PDE4 (Vargas et al., 2006; Galindo-Tovar et al., 2009). The

IC50 of rolipram at PDE4 is approximately 1 µM; therefore 1 µM of rolipram will

block approximately 50% of PDE4 and only 0.4% of PDE3 (Vargas et al., 2006).

At the completion of concentration-effect curves to catecholamines on ventricular

trabeculae, the maximal response was recorded for each trabeculae by incubating

tissues with 200 µM (-)-isoprenaline, which surmounts blockade by both ICI

188,551 and CGP 20712A, therefore activating both β1- and β2-adrenoceptors

(Kaumann et al., 1987). This allowed all force readings of the concentration-effect

curves to be calculated as a % maximal response for each tissue, as all were different

sizes and had individual basal contraction forces. Since up to 20 contracting

trabeculae were obtained from the same heart, (upper limit of 20 was due to total

number of available clamps within organ baths, not due to limit of suitable trabeculae

per heart) it was often possible to compare the influence of the PDE inhibitors on

responses mediated through both β1 and β2-adrenoceptors. Experiments were

concluded with the addition of Ca2+ to raise the final bath concentration to 9.25 mM


(Molenaar et al., 2000).

2.1.3 Analysis of Contractile Data

A complete single concentration-effect curve for (-)-noradrenaline or (-)-adrenaline

was obtained in the absence or presence of cilostamide (300 nM) or rolipram (1 µM).

Responses to the catecholamines were expressed as a percentage of each tissue’s

maximal response to (-)-isoprenaline (200 µM). The catecholamine concentrations

producing a half maximum response, or –LogEC50M (pEC50), were estimated from

fitting a Hill function with variable slopes to concentration-effect curves from

individual experiments. The data are expressed as mean ± S.E.M. of n = number of

patients or trabeculae (as indicated).

Contractile force (F), time to peak force (TPF) and time to half-relaxation (t0.5) were

recorded through PowerLab amplifiers on a Chart for Windows, Version 5.0

recording program (ADInstruments Pty Ltd., Castle Hill, Australia) or on Graphtec 8

and 12 channel recorders. Measurements taken from the Graphtec channel recorders

were then measured by hand.

Significance of differences between means were assessed with the use of either

Student’s t test or ANOVA followed by Tukey-Kramer Multiple comparisons ad hoc

test at P<0.05 using Instat software (GraphPad Software Inc., San Diego, CA USA).

2.1.4 Drugs

(-)-Adrenaline (+)-bitartrate salt and (-)-noradrenaline bitartrate salt (hydrate) were

purchased from Sigma-Aldrich (St. Louis, MO, USA or Castle Hill, Australia).


Rolipram, cilostamide, CGP 20712A (2- hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-

4-(trifluorometyl)-1H-imidazol-2yl] phenoxy]propyl]amino]ethoxy]-benzamide) and

ICI 118,551 (1-[2,3-dihydro-7-methyl-1H-inden-4-yl]oxy- 3-[(1 methylethyl)amino]-

2-butanol) were purchased from Tocris Bioscience (Bristol, UK) or Sigma-Aldrich

(St. Louis, MO, USA or Castle Hill, Australia). Stock solutions were prepared in

purified water and kept at -20°C to avoid oxidation. Further dilutions of the drugs

were made fresh daily and kept cool (0–4°C) and dark. Repetitive experiments

showed that drug solutions treated in these ways are stable.

2.1.5 Participants

All participants had terminal heart failure and underwent cardiac transplant surgery

at The Prince Charles Hospital, Brisbane, ethics approval numbers EC9876,

HREC10/QPCH/184 or Gustav Carus Hospital, Dresden Technological University

ethics committee (Document EK 1140 82202). The studies conformed with the

World Medical Association Declaration of Helsinki Ethical Principles for Medical

Research Involving Human Subjects (Rickham, 1964).

Written informed consent was obtained from all patients. Each patient was

approached by the Transplant Coordinator of their hospital and provided with

layman-appropriate written information about the study being performed, the

reasoning and purpose behind it, and how their information, tissue, and the results

from their tissue would be utilised both during and after the study, included

experiments, and any communication or publication regarding the study. Participants

were invited to ask further questions of both experimenters and surgeons, and care

was taken to ensure that all patients had adequate time to make their decision, were


comfortable with the parameters of the study, and were able to provide the

coordinator with informed and confident decisions to participate. Any patient who

was either not confident with their agreement or uncomfortable with any aspect of

the study was not included. Participants were also informed they were able to

withdraw at any time, for any reason.

Patients were administered with metoprolol, carvedilol, or no β-blocker prior to

transplantation, up to and including the day of transplantation and tissue collection.

Other medications administered included aspirin, warfarin, clopidogrel, digoxin,

amiodarone, nitrates, thyroid hormones, ACE inhibitors, (lisinopril, perindopril,

ramipril, enalapril) diuretics, (amiloride, frusemide) L-type calcium channel

blockers, (amlodipine, diltiazem, nifedipine) angiotensin-II receptor antagonists,

(candesartan, telmisartan, irbesartan, losartan) proton pump inhibitors (omeprazole,

esomeprazole, lansoprazole), statins (atorvastatin, rosuvastatin) and others, such as

antibiotics or nicotine replacement therapy. All medications were administered up to

transplantation, with the exception of aspirin, clopidogrel, and warfarin, which were

ceased up to 7 days prior to surgery. Reasons for transplantation included

cardiomyopathy, dilated cardiomyopathy, idiopathic dilated cardiomyopathy,

congestive heart failure, ischemic heart disease, and sick sinus syndrome. For

quantitative patient details and statistics, please see patient tables in Chapters 3 and

4.


2.2 RIGHT ATRIAL EXPERIMENTS

2.2.1 Research Design Summary

Right atrial appendages from 39 patients were obtained from patients undergoing

coronary artery bypass surgery were paced to contract at 1 Hz. The affinities of S-(-)

and R-(+) esmolol at β1-adrenoceptors were assessed in the presence of S-(-) esmolol

(1-100 µM) or R-(+) esmolol (100 µM) and concentration-effect curves to

noradrenaline established; The affinities of S-(-) and R-(+) esmolol at β2-

adrenoceptors were assessed in the presence of S-(-) esmolol (10 –100 µM) or R-(+)

esmolol (100 µM) and concentration-effect curves to adrenaline established.

2.2.2 Explantation and Transport

The right atrial appendages from non-failing hearts of patients undergoing coronary

artery bypass graft surgery were obtained at time of aortic cannulation and placed

immediately into ice-cold pre-oxygenated Krebs solution, as described above.

Appendages were then placed immediately back onto ice and were transported (less

than two minutes) to the laboratory.

2.2.3 Dissection and Set-Up of Trabeculae

Right atrial trabeculae were dissected under continuous oxygenation with 95%

O2/5% CO2, and set up in pairs onto Blinks tissue electrode blocks, as described

above. Trabeculae were driven with squarewave pulses of 5 ms duration to contract

at 1 Hz, and currents adjusted for individual trabeculae, as above. After establishing

a length-tension curve for each trabeculum, the length was set to obtain 50% of the

resting tension associated with maximum developed force.


2.2.4 Specific Activation of β1- and β2-Adrenoceptors

Optimized conditions were used to selectively activate β1- or β2-adrenoceptors, as

described above. To determine the acute effects of S-(-) esmolol, S-(-) esmolol (1 -

100 µM) was added to the tissue bath after stabilization of trabeculae and effects on

contractility observed for 30 min. To determine whether S-(-) esmolol activates β1L-

adrenoceptors (Sarsero et al 2003; Christ et al 2011), trabeculae were incubated with

nadolol (200 nM) for 30 min followed by 3-isobutyl-1- methylxanthine (IBMX, 10

µM) for approximately 20 min until equilibrium was obtained. S-(-) esmolol (100

µM) was then added and observed for 30 min.

For the determination of the equilibrium dissociation constant (KB) of S-(-) and R-(+)

esmolol at β1-adrenoceptors, trabeculae were incubated with S-(-) esmolol (1 - 100

µM) or R-(+) esmolol (100 µM) and 50 nM ICI 118,551 for at least 90 minutes and

then concentration-effect curves to (-)-noradrenaline established. For the

determination of the equilibrium dissociation constant (KB) of S-(-) and R-(+)

esmolol at β2-adrenoceptors, trabeculae were incubated with S-(-) esmolol (10 – 100

µM) or R-(+) esmolol (100 µM) and 300 nM CGP 20712A for at least 90 minutes

and then concentration-effect curves to (-)-adrenaline established. Concentration-

effect curves to (-)-noradrenaline and (-)-adrenaline were followed by a single

concentration of (-)-isoprenaline (200 µM) and calcium (7 mM, to final bath

concentration 9.25 mM).


2.2.5Analysis of Contractile Data


was obtained in the absence or presence of S-(-) esmolol (1 – 100 µM) or R-(+)

esmolol (100 µM). Responses to the catecholamines were expressed as a percentage

of each tissue’s maximal response to (-)-isoprenaline (200 µM). The catecholamine

concentrations producing a half maximum response, or –LogEC50M (pEC50), were

estimated from fitting a Hill function with variable slopes to concentration-effect

curves from individual experiments. The data are expressed as mean ± S.E.M. of n =

number of patients or trabeculae (as indicated).

Contractile force (F), time to peak force (TPF) and time to half-relaxation (t0.5) were

recorded and measured as described above.



test at P < 0.05 using Instat software (GraphPad Software Inc., San Diego, CA USA).

2.2.6 Drugs

(-)-Adrenaline (+)-bitartrate salt and (-)-noradrenaline bitartrate salt (hydrate) were

purchased from Sigma-Aldrich (St. Louis, MO, USA or Castle Hill, Australia). S-(-)

and R-(+) enantiomers of esmolol were provided by Baxter Healthcare, Old

Toongabbie, NSW, Australia.


2.2.7 Participants

Ethics approval was obtained from the University of Queensland Human Research

Ethics Committee (Ethics approval 2011001285) and The Prince Charles Hospital,

Metro North Health Service District (Ethics Approval HREC/11/QPCH/148). The

studies conformed with the World Medical Association Declaration of Helsinki

Ethical Principles for Medical Research Involving Human Subjects (Rickham, 1964).

Written informed consent was obtained from all patients prior to surgery, as above,

with information provided to the patient directly from the researcher. Participants

were informed they were able to withdraw at any time, for any reason. All patients

had been treated with a β-blocker prior to surgery. Other medications administered

included aspirin, clopidogrel, nitrates, ACE inhibitors, (lisinopril, perindopril,

ramipril, enalapril) diuretics, (amiloride, frusemide) L-type calcium channel

blockers, (amlodipine, diltiazem, nifedipine) angiotensin-II receptor antagonists,

(candesartan, telmisartan, irbesartan, losartan) statins (atorvastatin, rosuvastatin), and

hypoglycemic agents (metformin, gliclazide, insulin). All medications were

administered up to transplantation, with the exception of aspirin and clopidogrel,

which were ceased up to 7 days prior to surgery.

CHAPTER 3: The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle 59

3. The Effects of Metoprolol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor-Mediated Inotropy and Lusitropy in Human Failing Ventricle

3.1 BACKGROUND AND PURPOSE

Phosphodiesterases PDE3 and/or PDE4 control ventricular effects of catecholamines

in several animal species but their relative effects in failing human ventricle are as

yet unknown. The aim of this study is to investigate whether or not the PDE3-

selective inhibitor cilostamide (0.3-1µM) or the PDE4 inhibitor rolipram (1-10µM)

modifies the positive inotropic and lusitropic effects of catecholamines in human

failing myocardium in patients chronically administered metoprolol versus non non-

β-blocker-treated patients.

3.2 METHODOLOGY AND RESEARCH DESIGN

3.2.1 Research Design Ventricular trabeculae from freshly explanted hearts of 5 non-β-blocked and 15

metoprolol-treated (7 from Brisbane/Dresden group, 8 from Oslo group) patients

with terminal heart failure were paced to contract at 1Hz. The inotropic and

lusitropic effects of (-)-noradrenaline, mediated through β1-adrenoceptors and (-)-

adrenaline, mediated through β2-adrenoceptors, were assessed in the absence and

presence of the PDE3-selective inhibitor cilostamide (300 nM-1 µM) or PDE4

inhibitor rolipram (1-10 µM). The inotropic and lusitropic potencies of (-)-

noradrenaline and (-)-adrenaline were then estimated from the half maximal effect,

or pEC50 from non-linear curve fitting.


3.3 MATERIALS AND METHODS

3.3.1 Participants

Written informed consent was obtained from all patients. All patients had terminal

heart failure and underwent cardiac transplant surgery at The Prince Charles

Hospital, Brisbane, ethics approval numbers EC9876, HREC10/QPCH/184 and

Gustav Carus Hospital, Dresden Technological University ethics committee

(Document EK 1140 82202). The study conformed with the World Medical

Association Declaration of Helsinki Ethical Principles for Medical Research

Involving Human Subjects. Clinical data from metoprolol and non-β-blocker treated

Brisbane & Dresden patients are shown on Table 3.1. Clinical data from metoprolol-

treated Oslo patients are shown on Table 3.2.


n Aetiology n Age Sex n EF Medication n ________ 5 CHF 3 47 ± 3.2 M 5 26 ± 1.8 A5,B0,C4,D1,E4,F1,G0,H4,I2,J0,M4 DCM 1 F 0 DCM/SSS 1 Table 3.1.A. Summary of Brisbane/Dresden patients that were not taking a β–blocker prior to heart transplantation n Aetiology n Age Sex n EF Dose Medication n_______ 7 ICM 3 53 ± 1.1 M 6 23 ± 1.8 115 ± 26 A4,B1,C4,D2,E3, DCM 3 F 1 F1,G2,H1,I0,J0,M2 IHD 1 Table 3.1.B. Summary of Brisbane/Dresden patients chronically administered metoprolol prior to heart transplantation. n Aetiology n Age Sex n EF Dose Medication n_______ 8 CAD 6 52 ± 4 M 7 21.5 ± 1. 92 ± 26 A4,B3,C7,D0,E6,F0 CMP 2 F 1 G3,H5,I1,J1,K7,L1,M8 Table 3.2. Summary of Oslo patients chronically administered metoprolol prior to heart transplantation n, number of patients; Aetiology, CMP (cardiomyopathy), DCM (dilated cardiomyopathy), ICM (idiopathic cardiomyopathy), CHF (congestive heart failure), IHD (ischemic heart disease), SSS (sick sinus syndrome); Age (years); Sex, Male (M), Female (F); Left ventricular ejection fraction (EF); Dose (mg per day); Medication: A angiotensin converting enzyme inhibitor, B angiotensin receptor blocker, C diuretic, D digoxin, E warfarin, F nitrate, G statin, H amiodarone, I thyroid hormone, J clopidogrel, K aspirin, L aldosterone antagonist, M other.

3.3.2 In vitro human heart trabeculae experiments

Trabeculae from right ventricle from explanted hearts with terminal heart failure

were obtained immediately upon explantation, and placed directly into ice-cold pre-

oxygenated modified Krebs solution containing (mM; Na+ 125, K+ 5, Ca2+ 2.25,

Mg2+ 0.5, Cl- 98.5, SO42- 0.5, HCO3

- 32, HPO42- 1, EDTA 0.04), sealed and

transported to the laboratory.


3.3.3 Isolated Ventricular Trabeculae From Heart Transplant Patients

Trabeculae were dissected in ice-cold Kreb’s solution with 95% O2/5% CO2 and

prepared for electrical stimulation by attaching a fine silk thread loop to one end of

each trabeculum. Trabeculae were then clamped to an electrode block and the thread-

looped ends attached to Swema SG4-45 strain gauge transducers. Blinks tissue

electrode blocks were housed within 50 ml organ baths, to accommodate 2 trabeculae

in the oxygenated solution (above) supplemented with (mM): Na+ 15, fumarate 5,

pyruvate 5, L-glutamate 5, glucose 10 at 37°C (Kaumann et al., 1999). Trabeculae

were driven with squarewave pulses of 5 ms duration just above threshold current to

contract at 1 Hz. A length tension curve was established to determine Lmax, the

length producing maximal contractions, and maintained (Kaumann et al., 1999;

Molenaar et al., 2006).


Optimized conditions were used to selectively activate β1- or β2-adrenoceptors. For

both conditions, α-adrenoceptors, neuronal and extraneuronal uptake were

irreversibly blocked by 5 µM phenoxybenzamine (Christ et al., 2006). To selectively

activate β1-adrenoceptor, (-)-noradrenaline was used in the presence of the β2-

adrenoceptor selective blocker ICI 118,551 50 nM (Kaumann et al., 1999; Molenaar

et al., 2000; 2006; Christ et al., 2006). To selectively activate β2-adrenoceptors, (-)-

adrenaline was used in the presence of the β1-adrenoceptor selective blocker 300 nM

CGP 20712A (Kaumann et al., 1999; Molenaar et al., 2000; 2006; Christ et al., 2006;

Alexander et al., 2011). To determine the effect of PDE enzymes on effects mediated

through activation of β1- or β2-adrenoceptors, PDE3-selective inhibition was

obtained with cilostamide (300 nM) and PDE4-selective inhibition obtained with


rolipram (1-10 µM) pre-incubated with trabeculae for 30 minutes prior to incubation

with catecholamines. At the completion of concentration-effect curves to

catecholamines on right ventricular trabeculae, the effects of a maximal

concentration of (-)-isoprenaline (200 µM) were determined. Since up to 20

contracting trabeculae were obtained from the same heart, it was often possible to

compare the influence of the PDE inhibitors on responses mediated through both β1

and β2-adrenoceptors.

3.3.5 Analysis Of Contractile Data


was obtained in the absence or presence of cilostamide (300 nM) or rolipram (1-10

µM). Responses to the catecholamines were expressed as a percentage of the

response to (-)-isoprenaline (200 µM). The catecholamine concentrations producing a

half maximum response, –LogEC50M (pEC50), were estimated from fitting a Hill

function with variable slopes to concentration-effect curves from individual

experiments. The data are expressed as mean ± S.E.M. of n = number of patients or

trabeculae (as indicated).

Contractile force, time to peak force (TPF) and time to half-relaxation (t0.5) were


recording program (ADInstruments Pty Ltd., Castle Hill, Australia) or on Graphtec 8

and 12 channel recorders.




test at P<0.05 using Instat software (GraphPad Software Inc., San Diego, CA USA).

3.3.6 Drugs

(-)-Adrenaline (+)-bitartrate salt, (-)-noradrenaline bitartrate salt (hydrate), prazosin

hydrochloride, and atropine sulphate were purchased from Sigma-Aldrich (St. Louis,

MO, USA or Castle Hill, Australia). Rolipram, cilostamide, CGP 20712A (2-

hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluorometyl)-1H-imidazol-2yl]

phenoxy]propyl]amino]ethoxy]-benzamide) and ICI 118,551 (1-[2,3-dihydro-7-

methyl-1H-inden-4-yl]oxy- 3-[(1 methylethyl)amino]-2-butanol) were from Tocris

Bioscience (Bristol, UK) or Sigma-Aldrich (St. Louis, MO, USA or Castle Hill,

Australia). Stock solutions were prepared in purified water and kept at -20°C to

avoid oxidation. Further dilutions of the drugs were made fresh daily and kept cool

(0–4°C) and dark. Repetitive experiments showed that drug solutions treated in these

ways are stable.

3.4 RESULTS

3.4.1 Chronic Metoprolol Treatment Increases the Inotropic Potencies of Catecholamines

Chronic treatment of patients with metoprolol sensitized right ventricular trabeculae

to the inotropic effects of (-)- noradrenaline and (-)-adrenaline. The inotropic

potencies of (-)-noradrenaline and (-)-adrenaline were increased fourfold and

fivefold, respectively, in metoprolol-treated (P < 0.05) compared with non-β-blocker

treated patients (Figure 3.1.A and B, Table 3.3.). The lusitropic effects (TPF, t50) of

(-) noradrenaline, mediated through β1-adrenoceptors, were not significantly


enhanced but the t50-abbreviating potency of (-)-adrenaline increased sevenfold (P <

0.001) by treatment of patients with metoprolol (Figure 3.2). These results are

consistent with the up-regulation of the β1-adrenoceptor density and enhanced

inotropic responses through these receptors in metoprolol treated patients (Heilbrunn,

Shah, Bristow, Valantine, Ginsburg, Fowler, 1989; Sigmund, Jacob, Becker,

Hanrath, Schumacher, & Eschenhagen, 1996).

Figure 3.1. Effects of chronic administration of metoprolol compared with no β-blocker on inotropic effects of (-)-noradrenaline through activation of β1-adrenoceptors (a) and (-)-adrenaline through activation of β2-adrenoceptors (b) in right ventricular trabeculae from failing hearts. Note the increased potency of (-)-noradrenaline and (-)-adrenaline for inotropic effects in metoprolol-treated patients. β-adrenoceptor blockade did not significantly increase basal force [P = 0.07 for (-)-noradrenaline, P = 0.095 for (-)-adrenaline] and maximum force [P = 0.10 for (-)-noradrenaline, P = 0.054 for (-)-adrenaline]. Data is from four [(-)-noradrenaline experiments] or five [(-)-adrenaline experiments] patients with heart failure not treated with a β-blocker and seven patients with heart failure treated with metoprolol. See text and Table 3.3 for further detail.


(-)-Noradrenaline (-)-Adrenaline ____________________________________________________________________ Non-βB Metoprolol Non-βB Metoprolol pEC50 (n) pEC50 (n) pEC50 (n) pEC50 (n) RV Control 5.65 ± 0.15 (11/4) 6.25 ± 0.13 (18/7)* 5.70 ± 0.27 (14/5) 6.40 ± 0.11 (18/7)* Cilostamide 5.89 ± 0.24 (10/4) 6.75 ± 0.17 (17/7) 6.19 ± 0.27 (12/5) 7.11 ± 0.16 (13/7)† Rolipram - 6.19 ± 0.15 (15/7) - 6.50 ± 0.17 (12/7) LV Control - 6.34 ± 0.16 (7/6) - 6.26 ± 0.16 (10/7) Cilostamide - 6.77 ± 0.19 (7/6) - 6.93 ± 0.12 (8/7)†† Rolipram - 6.25 ± 0.10 (7/6) - 6.29 ± 0.17 (8/7)

Table 3.3. Inotropic potencies of (-)-noradrenaline and (-)-adrenaline acting through ventricular β1- and β2-adrenoceptors, respectively. Effects of cilostamide (300 nM right ventricle, 1 µM left ventricle) and rolipram (1 µM right ventricle, 10 µM left ventricle) and chronic metoprolol treatment. Non-βB: not treated with β-blockers; LV, left ventricle; RV, right ventricle; *P < 0.05 versus non- βB; †P < 0.001 paired Student’s t-test for comparison between cilostamide and control (no PDE inhibitor); ††P < 0.05 versus control, one-way ANOVA with Bonferroni adjustment for multiple a priori comparisons for comparison between cilostamide, rolipram and control. Numbers in parentheses are (trabeculae/patients).


Figure 3.2. Effects of chronic administration of metoprolol compared with no β-blocker on lusitropic effects [time to peak force and time to 50% relaxation (t50)] of (-)-noradrenaline through activation of β1-adrenoceptors (a,c) and (-)-adrenaline through activation of β2-adrenoceptors (b,d) in right ventricular trabeculae from failing hearts. Data from four [(-)-noradrenaline experiments] or five [(-)- adrenaline experiments] patients with heart failure not treated with a β-blocker and seven patients with heart failure treated with metoprolol. See text and Table 3.4 for further detail.


3.4.2 Cilostamide Fails to Potentiate the Inotropic Effects of

Catecholamines in Right Ventricular Trabeculae from Non-β-Blocker-Treated Patients

Cilostamide (300 nM) did not significantly increase contractile force or hasten

relaxation in the presence of ICI 118,551 or CGP 20712A in trabeculae from non β-

blocker-treated patients. Cilostamide did not potentiate the positive inotropic

effects of (-)-noradrenaline or (-)-adrenaline (Figure 3.3, Table 3.3). Cilostamide did

not affect the lusitropic effects of (-)-noradrenaline (Figure 3.4 A and C, Table 3.4)

but potentiated the (-)-adrenaline-evoked shortening of t50 (Figure 3.4 D, Table 3.4).

Figure 3.3. Lack of effect of cilostamide on the inotropic responses of (-)-noradrenaline and (-)-adrenaline in right ventricular trabeculae from four [(-)-noradrenaline experiments] or five [(-)-adrenaline experiments] patients with heart failure not treated with a β-blocker. Shown are concentration–effect curves to (-)-noradrenaline (a) and (-)-adrenaline (b) in the absence or presence of cilostamide (300 nM). Cilostamide did not significantly increase basal force (P = 0.36 for the noradrenaline group, P = 0.46 for the adrenaline group) or enhance the maximum force caused by (-)-noradrenaline (P = 0.41) or (-)-adrenaline (P = 0.13). See text and Table 3.3 for further detail.


Figure 3.4. Effect of cilostamide on the lusitropic responses of (-)-noradrenaline and (-)-adrenaline in right ventricular trabeculae from four [(-)-noradrenaline experiments] or five [(-)-adrenaline experiments] patients with heart failure not treated with a β-blocker. Shown are concentration–effect curves to (-)-noradrenaline (a,c) and (-)-adrenaline (b,d) in the absence or presence of cilostamide (300 nM). Cilostamide potentiated the (-)-adrenaline-evoked hastening of relaxation (shortening of t50). See text and Table 3.4 for further detail.


(-)-Noradrenaline (-)-Adrenaline ____________________________________________________________________ Non-βB Metoprolol Non-βB Metoprolol pEC50 (n) pEC50 (n) pEC50 (n) pEC50 (n) TPF Control 6.45 ± 0.20 (11/4) 6.41 ± 0.13 (17/7) 6.33 ± 0.28 (13/5) 6.62 ± 0.10 (13/7) Cilostamide 6.49 ± 0.26 (9/4) 6.80 ± 0.12 (13/7) 6.80 ± 0.28 (12/5) 7.06 ± 0.12 (13/7)† Rolipram - 6.67 ± 0.21 (11/7) T50 Control 6.21 ± 0.19 (11/4) 6.61 ± 0.13 (16/7) 5.96 ± 0.26 (13/5) 6.79 ± 0.16 (15/7)* Cilostamide 6.30 ± 0.24 (9/4) 7.12 ± 0.12 (16/7)† 6.84 ± 0.28 (12/5) 7.41 ± 0.14 (13/7)† Rolipram - 6.64 ± 0.11 (15/7) - 6.59 ± 0.22 (12/7)

Table 3.4. Lusitropic potencies of (-)-noradrenaline and (-)-adrenaline, acting through right ventricular β1- and β2-adrenoceptors, respectively. Effects of cilostamide (300 nM) and rolipram (1µM) in non- β-blocker (Non-βB) treated and chronic metoprolol-treated patients. * P < 0.01, vs. non-βB †P < 0.05 Paired Student’s t-test for comparison between Cilostamide and Control (no phosphodiesterase inhibitor) Numbers in parentheses are (trabeculae/patients).

3.4.3 Cilostamide Potentiates the Effects Mediated Through β2- Adrenoceptors More Than β1-Adrenoceptors in Ventricular Trabeculae from Metoprolol-Treated Patients

Cilostamide (300 nM) alone did not significantly change contractile force in the

presence of ICI 118,551 or CGP 20712A on right ventricular trabeculae. Cilostamide

caused leftward shifts of the inotropic concentration–effect curves of (-)-

noradrenaline and (-)-adrenaline as shown in the representative experiment in Figure

3.5. Inotropic results from right ventricular trabeculae of seven patients are shown in

Figure 3.6. Cilostamide almost significantly increased the inotropic potency of (-)-

noradrenaline (P = 0.06) (Figure 3.6 A, Table 3.3). When data from right ventricular

trabeculae of two additional metoprolol-treated Oslo patients (results not shown)

were pooled with the data from seven Brisbane–Dresden patients, cilostamide

significantly (P < 0.02, n = 9) potentiated the inotropic effects of (-)-noradrenaline.


Cilostamide (300 nM) potentiated the effects of (-)-adrenaline on force (fivefold, P <

0.05, Figure 3.6 B, Table 3.3). Cilostamide potentiated threefold the effects of (-)-

noradrenaline on t50 (P < 0.05) but not time to peak force (TPF) (Figure 3.7 A and C,

Table 3.4). Cilostamide potentiated the effects of (-)-adrenaline on TPF (threefold)

and t50 (fourfold) respectively (both P < 0.05, Figure 3.7 B and D, Table 3.4).

Cilostamide (300 nM) caused a non-significant (P < 0.07) leftward shift of the

concentration–effect curve for the inotropic effects of (-)-noradrenaline on left

ventricular trabeculae (P < 0.07, Figure 3.8 A, Table 3.3), but potentiated the

inotropic effects of (-)-adrenaline fivefold (P < 0.05, Figure 3.8 B, Table 3.3).

Cilostamide did not potentiate the TPF effects of (-)- noradrenaline or (-)-adrenaline

on left ventricular trabeculae but potentiated the effects on time to reach 80%

relaxation fourfold and fivefold respectively (both P < 0.05, Figure 3.9, Table 3.5).

Cilostamide caused a non-significant trend of greater potentiation of the inotropic

effects of (-)-adrenaline through β2-adrenoceptors (0.80 ± 0.11 log units, n = 9) than

(-)-noradrenaline through β1-adrenoceptors in right ventricular trabeculae from

metoprolol-treated patients (0.48 ± 0.18 log units, n = 9, Brisbane/Dresden/Oslo

hearts) (P = 0.14, paired Student’s t-test). However, when all right and left

ventricular inotropic data from metoprolol-treated patients were pooled, cilostamide

(0.3–1 µM) potentiated significantly more the β2-adrenoceptor-mediated effects of (-

)-adrenaline (0.78 ± 0.12 log units, n = 15) than the β1-adrenoceptor-mediated effects

of (-)-noradrenaline (0.47 ± 0.12 log units, n = 15) (P = 0.037). These results suggest

that PDE3 limits the inotropic responses through β2-adrenoceptors more than β1-

adrenoceptors.


Figure 3.5. Representative experiment carried out on right ventricular trabeculae obtained from a 48-year-old male patient with ischaemic heart disease, left ventricular ejection fraction 25 %, chronically administered metoprolol 142.5 mg daily. Shown are original traces for (-)-noradrenaline and (-)-adrenaline in the absence or presence of cilostamide (Cil, 300 nM), rolipram (Rol, 1 µM), or Cil + Rol, followed by (-)-isoprenaline (ISO, 200 µM). The bottom panels show the corresponding graphical representation with non-linear fits. Note the clear potentiation of inotropic effects of both (-)-noradrenaline and (-)-adrenaline in the presence of cilostamide but the lack of potentiation by rolipram.


Figure 3.6. Potentiation of the inotropic effects of (-)-adrenaline by cilostamide (P < 0.05) in right ventricular trabeculae from seven patients from Brisbane/Dresden with heart failure chronically administered metoprolol (b). In the same hearts, cilostamide caused a leftward shift of the inotropic effects of (-)-noradrenaline (a), which was not quite significant (P = 0.06). Rolipram had no effect on the inotropic effects of (-)-noradrenaline or (-)-adrenaline. See text for further explanation. Shown are concentration–effect curves to (-)-noradrenaline (a) and (-)-adrenaline (b) in the absence or presence of cilostamide (300 nM) or rolipram (1 µM).


Figure 3.7. Cilostamide, but not rolipram, potentiates the lusitropic effects of (-)-adrenaline and (-)-noradrenaline in right ventricular trabeculae from seven patients with heart failure chronically administered metoprolol. Shown are concentration-effect curves to (-)-noradrenaline (a,c) and (-)-adrenaline (b,d) in the absence or presence of cilostamide (300 nM) or rolipram (1 µM).


Figure 3.8. Cilostamide potentiates the inotropic effects of (-)-adrenaline in left ventricular trabeculae from seven [(-)-noradrenaline experiments] or eight Oslo patients [(-)-adrenaline experiments] with heart failure chronically administered metoprolol. Shown are concentration–effect curves to (-)-noradrenaline (a) and (-)-adrenaline (b) in the absence or presence of cilostamide (1 µM) or rolipram (10 µM). Inotropic data are normalized as a percentage of the maximal response to (-)-noradrenaline or (-)-adrenaline.


Figure 3.9. Cilostamide, but not rolipram, potentiates the relaxant effects of (-)-noradrenaline and (-)-adrenaline in left ventricular trabeculae from seven ((-)-noradrenaline experiments), or eight Oslo patients ((-)-adrenaline experiments) with heart failure chronically administered metoprolol. Shown are concentration-effect curves to (-)-noradrenaline (a,c) and (-)-adrenaline (b,d) in the absence or presence of cilostamide (1 µM) or rolipram (10 µM). See Table 3.5 for analysis.


(-)-Noradrenaline (-)-Adrenaline pEC50 (n) pEC50 (n) TPF Control 6.54 ± 0.16 (7/6) 6.56 ± 0.31 (8/7) Cilostamide 7.00 ± 0.20 (7/6) 7.13 ± 0.17 (8/7) Rolipram 6.30 ± 0.11 (7/6) 6.76 ± 0.19 (8/7) t50 Control 6.64 ± 0.15 (7/6) 6.59 ± 0.19 (10/7) Cilostamide 7.41 ± 0.16 (7/6)† 7.33 ± 0.13 (8/7)† Rolipram 6.57 ± 0.13 (7/6) 6.58 ± 0.17 (8/7)

Table 3.5. Lusitropic potencies of (-)-noradrenaline and (-)-adrenaline, acting through left ventricular β1- and β2-adrenoceptors of metoprolol-treated patients. Effects of cilostamide (1 µM) and rolipram (10 µM). Ventricular trabeculae were treated with rolipram (10 µM), cilostamide (1 µM) or untreated (control) for ~15-30 min prior to administration of increasing concentrations of either (-)-noradrenaline or (-)-adrenaline as described in Methods. Values in parentheses are (trabeculae/patients). Data are mean ± S.E.M. † P < 0.05 vs. control, Oneway ANOVA with Bonferroni adjustment for multiple a-priori comparisons. TPF, time to peak force; t50, ime to 50% relaxation

3.4.4 Rolipram Does Not Modify Inotropic or Lusitropic Potencies of (-)-Noradrenaline and (-)-Adrenaline

Rolipram did not significantly modify force, TPF, t50 or t80 in right and left

ventricular trabeculae incubated with ICI 118,551 or CGP 20712A. The inotropic

and lusitropic effects of (-)-noradrenaline and (-)-adrenaline were not significantly

changed by rolipram (1 µM) in right ventricular trabeculae (inotropic: Figures 3.5

and 3.6, Table 3.3; lusitropic: Figure 3.7, Table 3.4) or rolipram (10 µM) in left

ventricular trabeculae (inotropic: Figure 3.8, Table 3.3; lusitropic: Figure 3.9, Table

3.5). The effects of the combination of cilostamide (300 nM) and rolipram (10 µM)

on the inotropic and lusitropic potencies of (-)-noradrenaline and (-)-adrenaline were

investigated in three metoprolol-treated patients. Cilostamide + rolipram potentiated


the inotropic [(-)-noradrenaline P < 0.05; (-)- adrenaline P < 0.05] and lusitropic [(-)-

noradrenaline TPF, t50 both P < 0.05; (-)-adrenaline TPF, t50 both P < 0.05] effects of

both (-)-noradrenaline and (-)-adrenaline, but the degree of potentiation did not

significantly (P > 0.05) differ from the potentiation caused by cilostamide alone

(inotropic: Figure 3.10; lusitropic: Figure 3.11).

Figure 3.10. Effects of the combination of cilostamide and rolipram on the inotropic responses of (-)-noradrenaline (a) and (-)-adrenaline (b) in right ventricular trabeculae from three patients with heart failure chronically administered metoprolol. While the combination of cilostamide and rolipram potentiated the inotropic responses of (-)-noradrenaline and (-)-adrenaline, the degree of potentiation did not differ from that caused by cilostamide alone.


Figure 3.11. Effects of the combination of cilostamide (300 nM) and rolipram (10 µM) on the lusitropic responses of (-)-noradrenaline and (-)-adrenaline in right ventricular trabeculae from 3 patients with heart failure chronically administered metoprolol. Whilst the combination of cilostamide and rolipram potentiated the lusitropic responses of (-)-noradrenaline and (-)-adrenaline, the degree of potentiation did not differ from that caused by cilostamide alone.


3.5 DISCUSSION

This work revealed two important aspects of the control by PDEs of the inotropic

effects of catecholamines. Chronic treatment of heart failure patients with metoprolol

induced PDE3 to reduce the inotropic responses more through β2-adrenoceptors than

β1-adrenoceptors. PDE4 appears not to be involved in the inotropic and lusitropic

through either β1- or β2-adrenoceptors.

3.5.1 Control by PDE3 of the Function of β1- and β2-Adrenoceptors in Heart Failure Patients Treated with Metoprolol

PDE3 activity is stimulated by activation of β1-adrenoceptors and β2-adrenoceptors,

which in turn causes a negative feedback by hydrolyzing cAMP and thereby

reducing inotropic and lusitropic effects. Atonic receptor activation by endogenous

catecholamines increases cAMP and PKA activity in a compartment that allows the

latter to phosphorylate and activate PDE3 (Gettys, Blackmore, Redmon, Beebe, &

Corbin, 1987), which in turn hydrolyses cAMP. This effect is likely to be more

important for β2-adrenoceptors, at least in human heart, because these receptors are

more efficient than β1-adrenoceptors at activating Gs and stimulating ventricular

adenylyl cyclase (Kaumann & Lemoine, 1987), as verified with recombinant

receptors (Levy, Zhu, Kaumann, & Birnbaumer, 1993). Therefore, inhibition of

PDE3 may potentiate β2-adrenoceptor-mediated responses more than β1-

adrenoceptor-mediated responses, as shown here for human failing ventricle and

previously for non-failing atrial myocardium from patients without heart failure

(Christ et al., 2006).


A reduction in the expression and activity of PDE3 has been reported in heart failure

patients (Silver et al., 1990; Ding et al., 2005a, 2005b). Treatment with isoprenaline

causes sustained down-regulation of PDE3A (Ding et al., 2005b), as also observed in

human heart failure and animal heart failure models (Ya & Abe, 2007), presumably

due to the high catecholamine plasma levels. A down-regulation of PDE3A would be

expected to increase cAMP levels in heart failure so that inhibition of the enzyme

would conceivably affect the effects of catecholamines less. The lack of significant

potentiation by cilostamide of the inotropic and lusitropic effects of both (-)-

noradrenaline through β1-adrenoceptors and marginal potentiation of the effects of

adrenaline through β2-adrenoceptors in this study’s five non-β-blocked patients is

consistent with this expectation. In contrast, chronic treatment of heart failure

patients with metoprolol revealed robust potentiation of the inotropic and lusitropic

effects of the catecholamines through β1- and β2-adrenoceptors. The ability of

chronically administrated metoprolol to potentiate positive inotropy and lusitropy of

catecholamines may be due to chronic β-adrenoceptor blockade. In a failing heart,

high circulating levels of catecholamines would normally suppress PDE3 activity.

When the β-adrenoceptors are blocked, these higher levels of catecholamines are

prevented from suppressing PDE3 activity; this allows cAMP concentrations to

increase and more PKA phosphorylation and subsequent activation of RyR2 to

occur, ultimately resulting in increased inotropic and lusitropic responses to

circulating catecholamines. The increased β2-adrenoceptor-mediated ventricular

inotropic and lusitropic effects in ventricular trabeculae caused by metoprolol

treatment of heart failure patients agree with a similar (six-fold) enhancement of the

β2-adrenoceptor mediated inotropic potency of (-)-adrenaline in human atria obtained

from patients without heart failure chronically treated with atenolol (Hall, Kaumann,


& Brown, 1990). The increased cardiac responsiveness to adrenaline through β2-

adrenoceptors appears to be the result of chronic β1-adrenoceptor blockade.

Experimental long-lasting exposure to catecholamines elicits up-regulation of Gia

(Eschenhagen et al., 1992). A similar situation occurs in heart failure in which the

sympathetic nervous system is hyperactive (Cohn, 1989), plasma noradrenaline

levels are increased, (Thomas & Marks, 1978) and ventricular Giα is increased

(Neumann, Schmitz, Scholz, von Meyerinck, Döring, & Kalmar, 1988). Human β2-

adrenoceptors can couple to and activate Giα, in addition to Gsα, when they are

stimulated by a very high isoprenaline concentration in human atrium (Kilts et al.,

2000). Through chronic β1-adrenoceptor blockade of patients by treatment with

metoprolol or possibly atenolol, the noradrenaline-induced elevation of Giα ceases,

Giα levels are reduced (Sigmund et al., 1996), conceivably thereby favoring coupling

of the β2-adrenoceptor to Gsα to allow enhanced inotropic and lusitropic effects of

adrenaline through β2-adrenoceptors. This hypothesis requires future research.

The results presented here suggest that chronic β-adrenoceptor blockade facilitates

the control by PDE3s of catecholamine effects, particularly through β2-

adrenoceptors. However, the generality of this argument has to be restricted to heart

failure, because in atrial myocardium obtained from patients without heart failure it

has been reported that cilostamide potentiated the effects of adrenaline, mediated

through β2-adrenoceptors, more than the effects of noradrenaline, mediated through

β1-adrenoceptors, regardless of whether or not patients had been treated with β1-

adrenoceptor-selective blockers (Christ et al., 2006). Changes of ventricular β2-

adrenoceptor function in heart failure (Nikolaev et al., 2010) and profound

anatomical differences between ventricle and atrium (Bootman, Smyrnias, Thul,


Coombes, & Roderick, 2011) may be relevant to account for the different

consequences of PDE3 control in the two tissues with respect to β1-adrenoceptor and

β2-adrenoceptor function after chronic β-adrenoceptor blockade.

The lusitropic (Figures 3.7, 3.9, 3.11, Tables 3.4 and 3.5) effects mediated through

β1- and β2-adrenoceptors were usually potentiated by cilostamide to a similar extent

as the corresponding inotropic effects in trabeculae from β-blocker treated patients

(Figures 3.6, 3.8, 3.10, Table 3.3). PDE3 activity in human ventricle is associated

with membrane vesicle-derived t-tubules and junctional sarcoplasmic reticulum (SR),

causing hydrolysis of cAMP in the vicinity of phospholamban (PLB) (Movsesian et

al., 1991; Lugnier, Muller, Le Bec, Beaudry, & Rousseau, 1993). In ventricular

myocardium from failing hearts, noradrenaline and adrenaline produce similar

increases in PKA-catalyzed phosphorylation of the proteins mediating myocardial

relaxation, PLB (at Ser16), troponin-I (TnI) and cardiac myosin-binding protein-C

(Kaumann et al., 1999). These lusitropic results are consistent with an increased

phosphorylation of PLB, TnI and myosin-binding protein-C by isoprenaline in the

presence of the PDE3 inhibitor pimobendan in human failing myocardium (Bartel et

al., 1996).

3.5.2 PDE4 Inhibition Does not Affect the Inotropic or Lusitropic Effects of Catecholamines

PDE4 isoenzymes, their subtypes and splicing variants, are equally expressed in

rodent and human ventricle but murine hearts have a considerably higher PDE4

activity than human hearts (Richter et al., 2011). Inhibition of PDE4 causes

potentiation of the positive inotropic effects mediated through rodent β1-

adrenoceptors (Kaumann, 2011). In contrast, these results from human failing


ventricle demonstrate that inhibition of PDE4 with rolipram did not potentiate the

positive inotropic and lusitropic effects of (-)-noradrenaline or (-)-adrenaline,

mediated through β1-and β2-adrenoceptors, respectively. It could be argued that this

study was unable to demonstrate a potentiating effect of rolipram because PDEs,

including PDE4s, are down-regulated in heart failure (Ding et al., 2005a, 2005b;

Lehnart et al., 2005). However, studies have also reported for human atrial

myocardium, obtained from non-failing hearts, that rolipram failed to potentiate the

positive inotropic effects of (-)-noradrenaline or (-)-adrenaline mediated through β1-

and β2-adrenoceptors (Christ et al., 2006; Kaumann et al., 2007). These findings are

consistent with an early report demonstrating that cilostamide but not rolipram

inhibited SR-associated PDE activity in human ventricle from heart failure patients

(Movsesian et al., 1991).

Taken together, the present results and a critical appraisal of the literature make it

unlikely that PDE4s modulate human inotropic and lusitropic effects of

catecholamines, mediated through either β1- or β2-adrenoceptors in non-failing and

failing hearts. Moreover, extrapolation of results from the PDE4 function in mouse

and rat hearts to human inotropic and lusitropic effects of physiological

catecholamines can actually be misleading. However, PDE4s can reduce the

occurrence of catecholamine-evoked arrhythmias in murine ventricle (Galindo-Tovar

& Kaumann, 2008; Lehnart et al., 2005) and apparently in human atrium (Molina et

al., 2012). However, clinical trials with a PDE4 inhibitor, roflumilast, have not

provided evidence for cardiovascular side effects in approximately 1500 roflumilast-

treated patients compared with 1500 placebo patients (Calverley, Rabe, Goehring,

Kristiansen, Fabri, & Martinez, 2009). A comparison between human and other


species of the control of β1-adrenoceptor and β2-adrenoceptor-mediated inotropy and

lusitropy by PDE3 and PDE4, as well as protection against arrhythmias, is

summarised in Table 3.6.

________________________________________________________________________

Function Species PDE βAR References

Inotropism Human PDE3 β1&β2 Christ et al., 2006; Kaumann et al., 2007; This study Lusitropism PDE3 β1&β2 This study

Arrhythmias PDE3 β1&β2 Molina et al., 2012 &PDE4

Inotropism Mouse PDE4 β1 Galindo-Tovar & Kaumann, 2008

Lusitropism PDE4 β1 Beca et al., 2011

Arrhythmias PDE4 β1 Galindo-Tovar and Kaumann, 2008; Leroy et al., 2011

Inotropism Rat PDE4 β1&β2 Christ et al., 2009 ; Afzal et al., 2011

Lusitropism PDE4 β1&β2 Christ et al., 2009 ; Afzal et al., 2011

Arrhythmias PDE4 β1 Boer et al., 2011

Inotropism Rabbit PDE3 β1 Kaumann et al., 2009 &PDE4 Arrhythmias PDE4 β1 Holbrook & Coker, 1991 ________________________________________________________________________ Table 3.6. Reduction of inotropic and lusitropic responses as well as protection against arrhythmias, mediated through myocardial β1-adrenoceptor and β2-adrenoceptor, by PDE3 and PDE4 in different species.


3.6 CLINICAL IMPLICATIONS Although this study did not detect a direct inotropic change with cilostamide, this

PDE3 inhibitor potentiated the inotropic effects of the endogenous catecholamines

mediated through ventricular β1-and β2-adrenoceptors of metoprolol-treated patients,

consistent with PDE3 inhibition. The induction of PDE3 activity in metoprolol-

treated patients could further reduce cardiostimulation by endogenous

catecholamines. In human atrium, metoprolol blocks the effects of catecholamines

through β1-adrenoceptors only by 2.5-fold more than β2-adrenoceptors (Figure 3.12).

It is therefore logical to predict that heart failure patients under therapy with a PDE3

inhibitor + metoprolol could be at risk of not being protected against adverse stress-

induced surges of adrenaline, acting through β2-adrenoceptors. From simple

competitive inhibition, the concentration ratio (CR) of a catecholamine in the

presence and absence of metoprolol can be calculated from CR = 1 + ([metoprolol] x

KB-1). The therapeutic plasma level of 100 ng·mL-1 (310 nM) metoprolol

(Kindermann et al., 2004) which hardly binds to plasma proteins, using KB values of

40 nM for β1-adrenoceptors and 98 nM for β2-adrenoceptors (Figure 3.12), would

produce CR values of 8.8 for β1-adrenoceptors and 4.2 for β2-adrenoceptors. The

fivefold potentiation of the inotropic effects of (-)-adrenaline by cilostamide suggests

that endogenous increases in plasma (-)-adrenaline could conceivably surmount the

β2-adrenoceptor blockade caused by metoprolol in patients also treated with a PDE3

inhibitor.


3.7 CONCLUSIONS Treatment with metoprolol induces the control by PDE3 of the ventricular inotropic

and lusitropic effects of (-)-noradrenaline and (-)-adrenaline through β1-

adrenoceptors and β2-adrenoceptors, respectively, plausibly by restoring the

decreased activity and expression of PDE3 in heart failure. Quantitative

considerations, based on differences in the affinity profile of metoprolol for β1- and

β2-adrenoceptors, suggest that treatment with a PDE3-selective inhibitor could

potentially facilitate adverse stress-induced adrenaline effects through β2-

adrenoceptors in patients treated with metoprolol. PDE4 does not control the

inotropic and lusitropic effects mediated through β1- and β2-adrenoceptors in human

heart.

Chapter 4: The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle 88

4. The Effects of Carvedilol on PDE3 and PDE4 Control of β1-and β2-Adrenoceptor Mediated Inotropy and Lusitropy in Human Failing Ventricle

4.1 BACKGROUND AND PURPOSE

Both metoprolol and carvedilol provide important therapeutic benefits for heart

failure patients. The previous chapter concluded that chronic administration of

metoprolol facilitates the control by phosphodiesterase PDE3, but not by PDE4, of

inotropic effects of catecholamines in failing human ventricle. However, it is not

known whether carvedilol has the same effect. This chapter investigates whether the

PDE3-selective inhibitor cilostamide (0.3 µM) or PDE4-selective inhibitor rolipram

(1 µM) modifies the positive inotropic and lusitropic effects of catecholamines in

ventricular myocardium of heart failure patients chronically administered carvedilol.

4.2 METHODOLOGY AND RESEARCH DESIGN

4.2.1 Heart Transplant Patients Written informed consent was obtained from all patients. Patients with terminal heart

failure underwent heart transplant surgery at The Prince Charles Hospital, Brisbane,

ethics approval EC9876, HREC10/QPCH/184 and Gustav Carus Hospital, Dresden

Technological University ethics committee (Document EK 1140 82202). Clinical

data for patients are is shown in Table 4.1. The use of human myocardium conforms

with the principles outlined in the Declaration of Helsinki (Rickham, 1964).


n Aetiology n Age Sex n EF Medication n ________ 5 CHF 3 47 ± 3.2 M 5 26 ± 1.8 A5,B0,C4,D1,E4,F1,G0,H4,I2,J0,M4 DCM 1 F 0 DCM/SSS 1 Table 4.1.A. patients that were not taking a β–blocker prior to heart transplantation. (Please note: this is the same control group utilised in Chapter 3).

n Aetiology n Age (year) Sex n LVEF Dose (mg/day) Medication 9 IDCM 3 44±2.9 M5 20±0.5 43±9 A7, B1, C8, D4, DCM 4 F4 E4, F2, G6, H3, IHD 2 I2, J1

Table 4.1.B Summary of patients chronically administered with carvedilol prior to heart transplantation. n, number of patients; DCM, dilated cardiomyopathy; IDCM, idiopathic dilated cardiomyopathy; IHD, ischaemic heart disease; M, male; F, female, LVEF, left ventricular ejection fraction; A, angiotensin-converting enzyme inhibitor; B, angiotensin-receptor blocker; C, diuretic; D, digoxin; E, warfarin; F, nitrate; G, statin; H, amiodarone; I, thyroid hormone; J, clopidogrel. M other.

4.2.2 Isolated Right Ventricular Trabeculae from Heart Transplant Patients

Hearts from heart transplantation surgery were placed immediately into ice-cold pre-


Mg2+ 0.5, Cl- 98.5, SO4 2- 0.5, HCO3 - 32, HPO4 2- 1, EDTA 0.04), sealed and

transported to the laboratory where right ventricular trabeculae were dissected under

continuous oxygenation with 95% O2/5% CO2. Dresden experiments were carried

out in Tyrode’s solution (mM): Na+ 149, K+ 5.4, Ca2+ 1.8, Mg2+ 1.05, Cl- 137.8,

HCO3- 22, HPO4

2- 0.42, EDTA 0.04, ascorbate 0.2, glucose 5.5, and equilibrated

with 95% O2/5% CO2. Trabeculae were clamped to an electrode block and the other

end attached to Swema SG4-45 strain gauge transducers. Electrode blocks were


housed within 50 ml organ baths, to accommodate 2 trabeculae in the oxygenated

solution (above) supplemented with (mM): Na+ 15, fumarate 5, pyruvate 5, L-

glutamate 5, glucose 10 at 37°C (Kaumann et al., 1999). Trabeculae were driven with

squarewave pulses of 5ms duration just above threshold current to contract at 1 Hz.

Contractile force, time to peak force (TPF) and time to half-relaxation (t0.5) were


recording programme (ADInstruments Pty Ltd., Castle Hill, Australia) or on

Graphtec 8 and 12 channel recorders (Kaumann et al., 1999; Molenaar et al., 2006).

After determination of a length-tension curve, the length of each trabeculum was set

to obtain a resting tension associated with maximum developed force. To irreversibly

block tissue uptake of catecholamines and α-adrenoceptors, trabeculae were

incubated for 90 minutes with phenoxybenzamine (5 µM) followed by washout

(Kaumann et al., 1999; Molenaar et al., 2006).


To determine the effects of β1-adrenoceptor-selective activation, concentration-effect

curves for (-)-noradrenaline were obtained in the presence of ICI 118,551 (50 nM) to

selectively block β2-adrenoceptors (Kaumann et al., 1999; Molenaar et al., 2006). To

determine the effects of β2-adrenoceptor-selective activation, concentration-effect

curves for (-)-adrenaline were determined in the presence of CGP 20712A (300 nM)

to selectively block β1-adrenoceptors (Kaumann et al., 1999; Molenaar et al., 2006).

To assess the influence of the PDE3- selective inhibitor cilostamide (300 nM) and

the PDE4-specific inhibitor rolipram (1 µM) on the effects of the catecholamines, a

single concentration-effect curve for a catecholamine was obtained in the absence or


presence of a PDE inhibitor. Trabeculae were incubated with PDE inhibitors for 30–

45 minutes prior to commencement of catecholamine concentration-effect curves. At

the completion of concentration-effect curves to catecholamines on right ventricular

trabeculae, the effects of a maximal concentration of (-)-isoprenaline (200 µM) were

determined. Since up to 20 contracting trabeculae were obtained from the same heart,

it was often possible to compare the influence of the PDE inhibitors on responses

mediated through both β1 and β2-adrenoceptors.

4.2.4 Analysis and Statistics A single concentration-effect curve for (-)-noradrenaline or (-)-adrenaline was

obtained in the absence or presence of cilostamide (300 nM) or rolipram (1 µM).

Responses to the catecholamines were expressed as a percentage of the response to

(-)-isoprenaline (200 µM). The catecholamine concentrations producing a half

maximum response, –LogEC50M (pEC50), were estimated from fitting a Hill function

with variable slopes to concentration-effect curves from individual experiments. The

data are expressed as mean ± S.E.M. of n = number of patients or trabeculae as

indicated. Significance of differences between means were assessed with the use of

either Student’s t test or ANOVA followed by Tukey-Kramer Multiple comparisons

ad hoc test at P<0.05 using Instat software (GraphPad Software Inc., San Diego,

CA).


4.3 RESULTS

4.3.1 Decrease of Inotropic and Lusitropic Potencies for (-)-Adrenaline by Chronic Treatment with Carvedilol

The potencies of (-)-adrenaline through activation of β2-adrenoceptors were reduced

(inotropic 16-49– fold, P < 0.0001), (lusitropic 13-35– fold, P < 0.0001) in carvedilol

treated patients compared to non-β-blocker treated patients from previous studies

(Kaumann et al., 1999; Molenaar et al., 2006; previous chapter) (inotropic, Figure

4.1, Table 4.2; lusitropic Figure 4.2, Table 4.3). In contrast, the inotropic and

lusitropic potencies of (-)-noradrenaline for activation of β1-adrenoceptors in

carvedilol treated patients were only slightly (less than 4-fold) or not reduced

compared to non-β-blocker treated patients (Kaumann et al. 1999; Molenaar et al.

2006; previous chapter). (Figure 4.1, Table 4.2, Figure 4.2, Table 4.3). In trabeculae

obtained from 3 carvedilol-treated patients, the responses to (-)-adrenaline were

greatly depressed and almost absent, preventing the estimation of meaningful pEC50

values. Inotropic and lusitropic results from these 3 patients are therefore shown

separately (Figures 4.3, 4.4, and 4.5). There was no difference with the daily dose of

carvedilol administered to the two groups of patients (Figure 4.1, 47.9 ± 12.3 mg, n =

6; Figure 4.4, 33.3 ±1 1.0 mg, n = 3, P = 0.5).


Figure 4.1. Marked reductions in potency for inotropic effects of (-)-adrenaline (b) through β2-adrenoceptors, but not for (-)-noradrenaline (a) through β1-adrenoceptors, in right ventricular trabeculae from six ((-)-adrenaline experiments) or nine carvedilol-treated patients ((-)-noradrenaline experiments), compared to five non-β-blocker-treated patients from the previous chapter.


Carvedilol-treated patients (-)-Noradrenaline (-)-Adrenaline pEC50 (n) pEC50 (n) Control 5.68±0.10 (24/9) 4.50±0.10 (18/6) Cilostamide 5.89±0.14 (23/9) 5.51±0.25 (18/6) ΔpEC50 0.285±0.127 (9) 1.462±0.408 (6) P1 0.0533 0.0491 P2 0.006 Rolipram 5.72±0.16 (13/7) 4.34±0.13 (10/5) ΔpEC50 0.021±0.080 (7) 0.4995±0.25 (5) P1 0.9374 0.3039 Historical data from carvedilol and non-β-blocker treated patients (-)-Noradrenaline (-)-Adrenaline Non-β-blocker Carvedilol Non-β-blocker Carvedilol pEC50 (n) pEC50 (n) pEC50 (n) pEC50 (n) Historical Dataa 5.93±0.13 (18/11) 5.68±0.16 (19/11) Historical Datab 6.21±0.11 (18/9) 5.62±0.12 (31/15) 6.19±0.13 (21/10) 4.63±0.12 (41/15) Historical Datac 5.65±0.15 (11/4) 5.70±0.27 (14/5) Table 4.2. ΔpEC50 of (-)-noradrenaline and (-)-adrenaline in the absence or presence of PDE3 and PDE4 inhibitor. ΔpEC50is the difference in pEC50 values for (-)-noradrenaline or (-)-adrenaline obtained in the presence of phosphodiesterase inhibitor (cilostamide or rolipram) and without phosphodiesterase inhibitor (control) calculated from mean values for each patient. Values in parentheses are (trabeculae/patients) or (patients). P1 are P values obtained from paired Student’s t test for comparisons between phosphodiesterase inhibitor (cilostamide or rolipram) and control (no phosphodiesterase inhibitor). P2 are comparisons of the effects of (-)-noradrenaline and (-)-adrenaline. P2 is the value obtained from Student’s t test for comparisons of ΔpEC50 between (-)-noradrenaline and (-)-adrenaline. Values calculated for effects of cilostamide only. aKaumann et al., 1999; bMolenaar et al., 2006; cPrevious chapter.


Figure 4.2. Effects of chronic administration of carvedilol compared to no β-blocker on lusitropic effects (time to peak force and time to 50 % relaxation (t50)) of (-)-noradrenaline through activation of β1-adrenoceptors (a, c) and (-)-adrenaline through activation of β2-adrenoceptors (b, d) in right ventricular trabeculae from failing hearts. Data from four ((-)-noradrenaline experiments) or five ((-)-adrenaline experiments) patients with heart failure not treated with a β-blocker (from previous chapter) and nine ((-)-noradrenaline experiments) or six ((-)- adrenaline experiments) patients with heart failure treated with carvedilol. Carvedilol-treated patients showed marked reductions in potency for lusitropic effects of (-)-adrenaline (b, d). See Table 4.3 for more details.


(-)-Noradrenaline (-)-Adrenaline pEC50 (n) pEC50 (n) TPF Control 6.16±0.11 (24/9) 4.96±0.20 (18/6) Cilostamide 6.49±0.12 (23/9) 6.40±0.17 (16/6) ΔpEC50 0.423±0.126 (9) 1.27±0.18 (6) P1 0.0086 0.0294 P2 0.0016 Rolipram 6.26±0.20 (12/7) 5.07±0.16 (10/5) ΔpEC50 0.396±0.226 (7) 0.301±0.166 (5) P1 0.1614 0.2051 t50

Control 6.18±0.10 (24/9) 4.84±0.14 (16/6) Cilostamide 6.52±0.17 (22/9) 6.38±0.18 (16/6) ΔpEC50 0.418±0.24 (9) 1.54±0.277 (6) P1 0.1181 0.0051 P2 0.0098 Rolipram 6.31±0.08 (12/7) 4.90±0.11 (10/5) ΔpEC50 0.216±0.11 (7) 0.0879±0.304 (5) P1 0.22518 0.7905 Historical Data from non-β-blocker-treated patients (-)-Noradrenaline (-)-Adrenaline Non-β-blocker Non-β-blocker pEC50 (n) pEC50 (n) TPF Historical Dataa 6.18±0.19 (18/11) 6.51±0.11 (19/11) Historical Datab 6.45±0.20 (11/4) 6.33±0.28 (13/5) t50 Historical Dataa 6.43±0.17 (18/11) 6.24±0.15 (19/11) Historical Datab 6.21±0.19 (11/4) 5.96±0.26 (13/5) Table 4.3. Lusitropic potencies of (-)-noradrenaline and (-)-adrenaline, acting through right ventricular β1- and β2-adrenoceptors, respectively, from patients chronically treated with carvedilol. Effects of cilostamide (300 nM) and rolipram (1 µM). Historical data is given for non-β-blocked patients. ΔpEC50 is the difference in pEC50 values for (-)-noradrenaline or (-)-adrenaline obtained in the presence of phosphodiesterase inhibitor (cilostamide or rolipram) and without phosphodiesterase inhibitor (control) calculated from mean values for each patient. Values in parentheses are (trabeculae/patients) or (patients). P1 are P values obtained from paired Student’s t test for comparisons between phosphodiesterase inhibitor (cilostamide or rolipram) and control (no phosphodiesterase inhibitor). P2 is the value obtained from Student’s t test for comparisons of ΔpEC50 between (-)-noradrenaline and (-)-adrenaline. Values calculated for effects of cilostamide only aKaumann et al., 1999; bPrevious chapter.


Figure 4.3. Potentiation of inotropic effects of both (-)-noradrenaline and (-)- adrenaline in the presence of cilostamide (Cil, 300 nM) but not rolipram (Rol, 1 µM). Representative experiment carried out on right ventricular trabeculae obtained from a 38-year-old male patient with DCM, left ventricular ejection fraction of 20 %, and chronically administered carvedilol 37.5 mg daily. Shown are original traces for (-)-noradrenaline and (-)-adrenaline followed by (-)-isoprenaline (ISO, 200 µM). The bottom panels show the corresponding graphical representation of the concentration-effect curves to the catecholamines with non-linear fits.


Figure 4.4. Marked potentiation of the inotropic effects of (-)-adrenaline (b) versus (-)-noradrenaline (a) by cilostamide in right ventricular trabeculae from three hearts chronically treated with carvedilol. The hearts were notable for poor responsiveness to (-)-adrenaline in the absence of cilostamide. (Note: S.E.M. are not displayed for rolipram data in order to allow better clarity of the graph)


Figure 4.5. Marked potentiation of lusitropic effects of (-)-noradrenaline (a) and (-)-adrenaline (b) by cilostamide in right ventricular trabeculae from three hearts chronically treated with carvedilol. The hearts were notable for poor responsiveness to (-)-adrenaline in the absence of cilostamide; shown are the concentration-effect curves to (-)-noradrenaline and (-)-adrenaline in the absence or presence of cilostamide (300nM) or a combination of both cilostamide (300 nM) and rolipram (1 µM). Rolipram on its own (not shown) did not have an effect on either (-)-adrenaline or (-)-noradrenaline-mediated lusitropic effects.

4.3.2 Cilostamide Potentiates the Effects of (-)-Adrenaline More Than (-)-Noradrenaline in Trabeculae from Carvedilol-Treated Patients

Cilostamide (300 nM) caused a small, not quite significant 1.9-fold increase of the

inotropic potency of (-)-noradrenaline (P = 0.053) (Figure 4.6, Table 4.2) but

significantly increased the potency of (-)-noradrenaline to shorten TPF by 2.6-fold

(P<0.01) (Figure 4.7, Table 4.3). Cilostamide did not significantly alter (-)-

noradrenaline-evoked shortening of t0.5 (P = 0.12) in trabeculae from 9 carvedilol-

treated patients (Figure 4.7, Table 4.3). Cilostamide significantly potentiated the


inotropic effects of (-)-adrenaline, 10.2-fold in trabeculae from patients treated with

carvedilol (P<0.05) (Figure 4.6, Table 4.2). The lusitropic effects of (-)-adrenaline

were potentiated by cilostamide (28-fold for TPF, P<0.03; 35-fold for t0.5, P<0.01)

(Figure 4.7, Table 4.3). The effects of cilostamide (300 nM) were also investigated

on additional trabeculae from the 3 carvedilol-treated patients that were poor

responders to (-)-adrenaline (Figures 4.3, 4.5). In these patients, cilostamide partially

restored the inotropic responses to (-)-adrenaline, allowing estimates of inotropic

potentiation. The average log concentration-ratios of (-)-adrenaline in the presence

and absence of cilostamide at the 10% and 20% of maximum response levels to

isoprenaline were approximately 2.0 and 1.7 log units, i.e. equivalent to a 100-fold

and 50-fold potentiation, respectively. The responses to (-)-noradrenaline of this

group tended to be potentiated and were included in the group displayed in Figure

4.6. Cilostamide also potentiated the lusitropic responses of (-)-adrenaline by 0.93 ±

0.12 log units (TPF) and 1.47 ± 0.64 log units (t50) in these 3 patients (Figure 4.5).

Figure 4.6. Cilostamide (300 nM) potentiates the inotropic effects of (-)-adrenaline (b, n = 6) more than (-)-noradrenaline (a, n = 9) in right ventricular trabeculae from heart failure patients chronically administered carvedilol. Rolipram did not potentiate the effects from either (-)-noradrenaline or (-)-adrenaline.


Figure 4.7. Cilostamide, but not rolipram, potentiates lusitropic effects of (-)-adrenaline (TPF P<0.03; t50 P<0.01) and (-)-noradrenaline (TPF P<0.01, but not t50

P=0.12) in right ventricular trabeculae from 6 (-)-adrenaline experiments and nine (-)-noradrenaline experiments of patients with heart failure chronically administered carvedilol. Shown are the concentration-effect curves to (-)-noradrenaline (a, c) and (-)-adrenaline (b, d) in the absence or presence of cilostamide (300 nM) or rolipram (1 µM). (Note: S.E.M. are not displayed for rolipram experiments in order to allow better clarity of the graph)


4.3.3 Rolipram Does Not Modify Inotropic or Lusitropic Potencies of (-)-Noradrenaline and (-)- Adrenaline in Trabeculae from Patients Treated with Carvedilol

Rolipram (1 µM) did not significantly modify force, TPF or t50, in trabeculae from

carvedilol treated patients. The inotropic (Figures 4.6, 4.3, 4.4, 4.5, Table 4.2) and

lusitropic potencies (Figures 4.7, 4.5, Table 4.3) of (-)-noradrenaline and (-)-

adrenaline were not significantly changed by rolipram (1 µM) in trabeculae from

carvedilol-treated patients.

The effects of the combination of cilostamide (300 nM) and rolipram (1 µM) on the

inotropic and lusitropic potencies of (-)-noradrenaline and (-)-adrenaline were

investigated in 3 carvedilol-treated patients of Figures 4.3 and 4.4. Cilostamide +

rolipram potentiated the inotropic and lusitropic effects of both (-)-noradrenaline

(inotropic and TPF P < 0.003, but not t50, P = 0.07) and (-)-adrenaline (P<0.01), but

the degree of potentiation did not significantly (P>0.07) differ from the potentiation

caused by cilostamide alone (Figures 4.3, 4.4, and 4.5).

4.4 DISCUSSION This work confirms that chronic treatment of heart failure patients with carvedilol

reduces the inotropic and lusitropic effects and potencies of (-)-adrenaline.

Carvedilol therapy induced marked potentiation by cilostamide of the inotropic and

lusitropic effects of (-)-adrenaline mediated through β2-adrenoceptors. In contrast,

cilostamide had markedly less ability to increase the potency of (-)-noradrenaline for

β1-adrenoceptor mediated inotropic and lusitropic effects. PDE4 did not affect the

inotropic and lusitropic responses through β1- or β2-adrenoceptors.


4.4.1 Carvedilol-treatment Reduces Responses Through β2- Adrenoceptors but Not β1-Adrenoceptors

The β-adrenoceptor blocking effects of carvedilol partially persist after removal of

tissues from the heart in isolated atrial and ventricular trabeculae (Kindermann et al.,

2004; Molenaar et al., 2006). Studies have previously shown that persistent β2-

adrenoceptor blockade is considerably greater than the residual β1-adrenoceptor

blockade after removal of tissue from the failing hearts of carvedilol-treated patients

(Molenaar et al., 2006). Furthermore, this work demonstrates with exogenously

administered antagonist, that carvedilol has a 13-fold higher affinity for human atrial

β2- than β1-adrenoceptors (Molenaar et al., 2006). The 16-fold reduction of the

inotropic potency of (-)-adrenaline, mediated through β2-adrenoceptors, found in

right ventricular trabeculae of carvedilol-treated patients compared to non β-blocker-

treated patients, is consistent with a 25-fold potency reduction reported previously

(Molenaar et al., 2006). In contrast, the inotropic and lusitropic potency of (-)-

noradrenaline was not changed by the chronic treatment of patients with carvedilol,

consistent with a mere 1.8-fold decrease of (-)-noradrenaline inotropic potency

(Molenaar et al., 2006).

Therapy of heart failure patients with the β-blocker metoprolol increases β1-

adrenoceptor density and function (Heilbrunn et al., 1989; Gilbert et al., 1996;

Sigmund et al., 1996). The unchanged noradrenaline potency in trabeculae from

carvedilol-treated patients could be the result of an actual upregulation of β1-

adrenoceptor density combined with partial β1-adrenoceptor occupancy with

carvedilol firmly bound to the receptors. Residual binding of carvedilol to β1-

adrenoceptors would also explain the lack of increase of β1-adrenoceptor receptor

density reported in heart failure patients treated with carvedilol (Gilbert et al., 1996).


4.4.2 Carvedilol Facilitates the Control by PDE3 of β2- More than β1- Adrenoceptor-Mediated Effects

Cilostamide produced a small potentiation of the positive inotropic and lusitropic

effects of (-)-noradrenaline, mediated through β1-adrenoceptors, but marked

potentiation of the effects of (-)-adrenaline mediated through β2-adrenoceptors in

ventricular trabeculae from carvedilol treated patients.

These data indicate that chronic administration of carvedilol to patients with severe

heart failure differentially modifies the control of PDE3 metabolism of cAMP

accumulated by activation of β1- and β2-adrenoceptors. The existence of spatial

microdomains incorporating β1- or β2-adrenoceptors, G proteins, cAMP, protein

kinases, PDEs and AKAP scaffolding proteins has been described in mouse and rat

hearts (Nikolaev et al., 2006, 2010; Perera & Nikolaev, 2013). In rat and mouse non-

failing ventricular cardiomyocytes, β2-adrenoceptor signalling was reported to be

localized to deep transverse tubules, whereas β1-adrenoceptor signalling was

distributed across the whole cell surface (Nikolaev et al., 2010). However, in a rat

model of chronic heart failure induced by myocardial infarction, β2-adrenoceptor

signalling in ventricular cardiomyocytes became diffuse and was distributed across

the whole cell surface (Nikolaev et al., 2010). The demonstration of organized

signalling structures and their ability, at least for β2-adrenoceptor signalling, to re-

organize in rat failing heart may shed light on the current findings. It may be possible

that the chronic administration of carvedilol in patients with human heart failure

adjusts the spatial alignment of PDE3 with β2- more than β1-adrenoceptor signalling

so that cAMP accumulated by activation of β2-adrenoceptors is more efficiently

metabolized by PDE3. This remains to be verified by high resolution imaging


techniques (Nikolaev et al., 2010), particularly in human myocardium.

Following β-adrenoceptor-mediated increases in cAMP levels and PKA activity,

PDE3 is phosphorylated (Gettys et al., 1987) and in turn hydrolyzes cAMP, thereby

reducing the positive inotropic and lusitropic effects of endogenous catecholamines.

This effect is likely to be more important for β2-adrenoceptors, at least in human

heart, because these receptors are more efficient than β1-adrenoceptors at activating

Gs protein and stimulating atrial and ventricular adenylyl cyclase from non-failing

human hearts (Gille, Lemoine, Ehle, & Kaumann, 1985; Kaumann & Lemoine,

1987), as later verified with recombinant receptors (Green, Holt, & Liggett, 1992;

Levy et al., 1993). Therefore, inhibition of PDE3 may potentiate responses more

through β2- than β1-adrenoceptors. β2-adrenoceptor selective potentiation of the

inotropic and lusitropic effects of (-)-adrenaline was also observed in human atrial

myocardium obtained from patients without heart failure, treated or not treated with

β-blockers (Christ et al., 2006) and in the previous chapter with ventricular

trabeculae from metoprolol-treated patients with heart failure. Interestingly, however,

these results from carvedilol-treated patients with heart failure reveal an even more

pronounced control of PDE3 of β2-adrenoceptor-mediated effects than in the

previous studies of Christ et al. (2006). Although β2-adrenoceptors of non-failing

human ventricle couple tightly to Gs (Kaumann & Lemoine, 1987) they appear

partially uncoupled from the Gs-cAMP pathway in chronic heart failure (Bristow et

al., 1989). Chronic treatment with carvedilol may improve the coupling of β2-

adrenoceptors to the Gs-cAMP pathway, thereby facilitating PKA-catalyzed

phosphorylation and activation of PDE3.


The α subunits of Gi protein, Giα, are up-regulated in heart failure (Neumann et al.,

1988) and β2-adrenoceptors can couple to and activate Gi, in addition to Gs, when

stimulated by isoprenaline in human atrium (Kilts et al., 2000). A 3-month treatment

of heart failure patients with metoprolol causes a 74% decrease in Giα (Sigmund et

al., 1996). This reduction of Giα would allow a greater coupling of β2-adrenoceptors

to Gs, thereby facilitating inotropic and lusitropic effects mediated through these

receptors. I speculate that a decrease of Gi activity may also have occurred in chronic

carvedilol-treated patients, as observed with metoprolol (Sigmund et al., 1996),

thereby facilitating coupling of β2-adrenoceptors to Gs.

When the persistent β2-adrenoceptor blockade by carvedilol was surmounted with (-

)- adrenaline, improved coupling to Gs protein may occur and lead therefore to

greater PKA-catalyzed phosphorylation and activation of PDE3. Inhibition of PDE3

by cilostamide may then cause potentiation of the effects of (-)-adrenaline through

two plausible mechanisms, selective coupling of β2-adrenoceptors to Gs and

decreased Gi coupling. The potentiation by PDE3 inhibition of (-)-noradrenaline

effects is smaller than corresponding effects of (-)-adrenaline through β2-

adrenoceptors, apparently because β1-adrenoceptors couple less tightly to Gs than β2-

adrenoceptors and they do not couple to Gi.

As found with the inotropic effects, cilostamide potentiated the lusitropic effects

mediated through β2- more than β1-adrenoceptors in trabeculae from carvedilol-

treated patients (Figures 4.7 and 4.5, Table 4.3). This was expected from similar

increases in PKA-catalyzed phosphorylation of the proteins mediating myocardial

relaxation, phospholamban (at Ser16), troponin-I and cardiac myosin-binding protein


C through β1- and β2-adrenoceptors in ventricular myocardium from failing hearts

(Kaumann et al., 1999). These lusitropic results are consistent with the localization of

PDE3 in the vicinity of phospholamban (Movsesian et al., 1991; Lugnier et al.,

1993).

4.4.3 Quantitative Comparison of β1- and β2-Adrenoceptor Blockade by Carvedilol in Combination with PDE3 Inhibition

Following the oral administration of the therapeutically used concentration of 25 mg

carvedilol, peak plasma levels of 100 ng.ml-1 (246 nM) carvedilol have been

measured in heart failure patients (Tenero et al., 2000). Since approximately 98% of

carvedilol is bound to plasma protein (Martindale, 2002) the β-adrenoceptors would

be expected to be in contact with approximately only 4.9 nM of plasma carvedilol.

The concentrations of endogenous noradrenaline and adrenaline needed to surmount

the carvedilol-induced blockade of β1- and β2-adrenoceptors can be estimated from

the surmountable antagonism against noradrenaline and adrenaline respectively, and

the corresponding dissociation equilibrium constants KB (Molenaar et al., 2006).

From simple competitive inhibition, the concentration-ratio (CR) of a catecholamine

in the presence and absence of carvedilol can be calculated from CR = 1 +

([carvedilol] x KB-1). Using KB values of carvedilol of 0.95 nM for the β1-

adrenoceptor and 0.074 nM for the β2-adrenoceptor (Molenaar et al. 2006), 4.9 nM

carvedilol would produce CR values of 6.2 and 67 for β1- and β2-adrenoceptors,

respectively. Therefore, due to the β2-adrenoceptor-selective affinity of carvedilol,

endogenous adrenaline would hardly be expected to cause cardiostimulation through

β2-adrenoceptors in carvedilol-treated patients treated with a PDE3 inhibitor, despite

the 10-35-fold potentiation of the adrenaline effects.


The estimates of blocking potency from plasma concentrations of carvedilol may

actually not represent the concentration of carvedilol at the β1- and β2-adrenoceptors

because carvedilol still causes substantial β-adrenoceptor blockade at plasma levels

below the carvedilol detection level of 1 ng.ml-1 (Kindermann et al., 2004). It is

likely that under these conditions carvedilol stored in cardiac tissues is slowly

released and diffuses across the cardiomyocytes thereby causing some occupancy of

β1-adrenoceptors and greater occupancy of β2-adrenoceptors due to its higher affinity

for the latter (Molenaar et al., 2006). The cardiac capture and storage of carvedilol is

supported by evidence demonstrating that the cardiac concentrations of active S-

enantiomer of carvedilol exceed the plasma concentrations by 7-fold (Stahl,

Mutschler, Baumgartner, & Spahn-Langguth, 1993). The ex-vivo experiments,

demonstrating consistently greater persisting blockade of β2- than β1-adrenoceptors

in atrial and ventricular tissues from heart failure patients treated with carvedilol

(Molenaar et al., 2006 confirmed in this work), are consistent with these

mechanisms.

4.4.4 PDE4 Does Not Control the Inotropic or Lusitropic Effects of Catecholamines in Carvedilol Treated Patients with Heart Failure

Rolipram did not potentiate the positive inotropic and lusitropic effects mediated

through β1- or β2-adrenoceptors in trabeculae from carvedilol-treated patients with

heart failure. Furthermore, rolipram did not enhance the potentiation caused by

cilostamide (Figure 4.4). A similar failure of rolipram to potentiate catecholamine

effects was reported previously for human non-failing atrium (Christ et al., 2006;

Kaumann et al., 2007) and failing ventricle from metoprolol-treated patients

(Previous Chapter). This evidence, taken together, allows the conclusion that under

these conditions PDE4 does not control human atrial and ventricular inotropic and


lusitropic effects, mediated through β1- and β2-adrenoceptors. However, it has been

reported that PDE4 reduces the incidence of arrhythmias mediated through human

atrial β1- and β2-adrenoceptors (Molina et al., 2012). Furthermore, although PDE4

protects against β1-adrenoceptor-mediated ventricular arrhythmias in mice (Lehnhart

et al., 2005; Galindo-Tovar & Kaumann, 2008), it is unknown whether this happens

in failing human ventricle.

4.5 CONCLUSIONS By preventing cAMP hydrolysis, PDE3 inhibitors (e.g. milrinone and enoximone)

enhance cardiac contractility through activation of cAMP-dependent pathways.

Short-lasting infusions or low-dose oral treatment with PDE3 inhibitors have been

shown to improve systolic function in chronic heart failure (Anderson, 1991; Van

Tassel, Radwanski, Movsesian, & Munger, 2008). It is plausible that the potentiation

of the effects of catecholamines contributes to the positive inotropic effects of PDE3

inhibitors in the clinic (Previous chapter and this study). It could be argued that the

consequences of the suppression by cilostamide of the PDE3-induced control of the

inotropic and lusitropic responses mediated through β1- and β2-adrenoceptors are

transient because they were only observed in a time frame of less than 2 hours.

Conceivably, at a later stage compensatory PDEs could be activated through PKA-

catalyzed phosphorylation by the cAMP accumulated during PDE3 inhibition.

Preliminary results reveal, however, that cilostamide reduces the fade of the inotropic

responses to noradrenaline up to 21 hours after cilostamide administration

(Unpublished experiments of Galindo-Tovar & Kaumann), inconsistent with

intervention of other PDEs.


High-dose chronic treatment with PDE3 inhibitors worsens heart failure and

increases mortality, particularly through sudden death (Packer et al., 1991;

Amsallem, Kasparian, Haddour, Boissel, & Nony, 2005), presumably due to

ventricular fibrillation. Arrhythmias, elicited by endogenous catecholamines, may be

facilitated by PDE3 inhibitors. (-)-Noradrenaline and (-)-adrenaline produce a similar

incidence of arrhythmic contractions, mediated through β1- and β2-adrenoceptors

respectively, in human atrial myocardium from patients without heart failure

(Kaumann & Sanders, 1993), despite the lower density of β2-adrenoceptors

(Kaumann, Lynham, Sanders, Brown, & Molenaar, 1995), likely related to the tighter

coupling of human β2-adrenoceptors to Gs protein. Elevations of endogenous plasma

adrenaline levels during stress (Wortsman, 2002), stress cardiomyopathy (Wittstein

et al., 2005) and cardiopulmonary bypass surgery (Reves et al., 1981) are

considerably higher than increases in noradrenaline. Human ventricle from failing

hearts also shows pro-arrhythmic β2-adrenoceptor-mediated after-contractions and

after-transients and increases in Ca2+ transient amplitude, SR load, and twitch [Ca2+]

decline rate (De Santiago et al., 2008).

This study indicates that the joint treatment of heart failure patients with a PDE3

inhibitor and the slightly β1-adrenoceptor-selective blocker metoprolol could

facilitate adverse stress-induced adrenaline effects such as arrhythmias mediated

through β2-adrenoceptors (Previous Chapter). In contrast, due to the selective

blockage of β2-adrenoceptors, the quantitative assessment (see above) allows the

prediction that carvedilol also protects the heart against adverse effects of adrenaline

in heart failure patients treated together with a PDE3 inhibitor.


Carvedilol appears to produce more benefit than metoprolol in heart failure as shown

in the COMET trial (Poole-Wilson et al., 2003). The greater benefit could be

partially related to the β2-adrenoceptor-selective (Molenaar et al., 2006) blockage.

Carvedilol, but not metoprolol, would prevent the appearance of β2-adrenoceptor-

mediated arrhythmias (Remme et al., 2007; Di Lenarda et al., 1999) by endogenous

adrenaline, which was even shown to decrease the incidence of arrhythmias in heart

failure patients carrying antibodies with agonist activity through β2-adrenoceptors

(Chiale et al., 1995; Li, Wang, Zhao, Zhou, & Zhu, 2010). However, experimental

evidence demonstrating β2-adrenoceptor-mediated arrhythmias, facilitated by PDE3

inhibition, is still pending for human myocardium.

CHAPTER 5: Enantiomers of Esmolol 112

5. Enantiomers of Esmolol

5.1. BACKGROUND AND PURPOSE

All β-blockers share a common racemic core structure, and the antagonistic

efficacies of the S-enantiomers are generally more potent than those of the R-

enantiomers. As both the pharmacodynamics and pharmacokinetics of the racemates

also commonly differ, it is desirable to know which racemates produce either clinical

benefits or unwanted side effects. Most β-blockers used clinically are only available

as racemic mixtures, including esmolol, which is commonly used in acute and

surgical settings.

The β1-adrenoceptor has two separate affinity states: the β1H-adrenoceptor, which is

called the “high affinity” receptor due to the high affinity of β-blockers to block its

activity, is activated by noradrenaline. Conversely, the β-blockers have low affinity

for the blockade of the β1L-adrenoceptor, which is activated by (–)-CGP 12177 and

(–)-pindolol (Molenaar et al., 1997; Kaumann, 1989; Sarsero et al., 1999). As β–

blockers can have differing affinities for these two physiological states of the β-

adrenoceptor, it is therefore possible that the separate racemates of β-blockers may

be responsible for the disparity in activity at these two sites. This chapter will

investigate the effects of R-(+) and S-(-) esmolol on human right atrial contractility,

β1L- and β3-adrenoceptors, and their affinities at human β1- and β2-adrenoceptors.

5.2 METHODS

5.2.1 Participants

Right atrial appendages were obtained from patients undergoing coronary artery

bypass surgery at The Prince Charles Hospital. Ethics approval was obtained from

Chapter 5: Enantiomers of Esmolol 113

the University of Queensland Human Research Ethics Committee (Ethics approval

2011001285) and The Prince Charles Hospital, Metro North Health Service District

(Ethics Approval HREC/11/QPCH/148). Written informed consent was obtained

from all patients prior to surgery. Patient characteristics are given in Table 5.1.

____________________________________________________________________ n Age Sex EF β-blocker, daily dose, mg (n) Other medication ____________________________________________________________________ 39 61.7±1.3 35M/4F 56.2±1.6 Aten 43.9±4.9 (9) Bisop 8.3±1.7(3) A34,B23,C5 D37 Carv 50 (1) Met 62.4±7.9 (26) E11,F32,J8 ____________________________________________________________________ Table 5.1. Summary of patient details. A, Angiotensin converting enzyme inhibitor/AT1 receptor antagonist; B, nitrate; C, diuretic, D, hypolipidemic; E, L-type Ca2+channel antagonist; F, Aspirin/Clopidogrel; J, hypoglycemic agent.

5.2.2 Preparation of Right Atrial Trabeculae

After surgical removal, right atrial tissues were placed immediately into ice-cold pre-


Mg2+ 0.5, Cl- 98.5, SO42- 0.5, HCO3- 32, HPO42- 1, EDTA 0.04) and transported

to the laboratory (within ~5 min of surgical removal) where atrial strips containing

intact trabeculae were dissected under continuous oxygenation with 95% O2/5%

CO2, set up, on occasion in pairs, and driven with square-wave pulses of 5 ms

duration just above threshold voltage to contract at 1 Hz in an apparatus with a 50 ml

organ bath in the solution above supplemented with (mM): Na+ 15, fumarate 5,

pyruvate 5, L-glutamate 5, glucose 10 at 37°C (Molenaar et al., 2007). The tissues

were attached to Swema SG4-45 strain gauge transducers and force recorded through

PowerLab amplifiers on a Chart for Windows, Version 7.0 recording programme

(ADInstruments Pty Ltd., Castle Hill, Australia) or on Graphtec 8 and 12 channel

recorders (Molenaar et al., 2007). After determination of a length-tension curve, the


length of each trabeculum was set to obtain 50% of the resting tension associated

with maximum developed force.

5.2.3 Acute effects of S-(-) Esmolol on Human Right Atrium

Upon stabilization of trabeculae, S-(-) esmolol (1 - 100 µM) was added to the tissue

bath and effects on contractility observed for 30 min.

5.2.4 Effect of S-(-) Esmolol in the Presence of 3-isobutyl-1- methylxanthine and Nadolol

All clinically used β-blockers block β1-adrenoceptors and some, including pindolol,

also activate β1L-adrenoceptors (Kaumann & Molenaar, 2008). To determine whether

S-(-) esmolol activates β1L-adrenoceptors (Sarsero et al., 2003; Christ et al., 2011),

trabeculae were incubated with nadolol (200 nM) for 30 min followed by 3-isobutyl-

1- methylxanthine (IBMX, 10 µM) for approximately 20 min until equilibrium was

obtained. S-(-) esmolol (100 µM) was then added and observed for 30 min.

5.2.5 Determination of the Affinity of S-(-) and R-(+) Esmolol at Human Atrial β1- and β2-Adrenoceptors

Tissues were incubated with 5 µM phenoxybenzamine to block α-adrenoceptors and

neuronal and extraneuronal uptake of catecholamines and either 50 nM ICI 118,551

to block β2-adrenoceptors and/or 300 nM CGP 20712A to block β1-adrenoceptors

(Gille et al., 1985; Kaumann et al., 1999). For the determination of the equilibrium

dissociation constant (KB) of S-(-) and R-(+) esmolol at β1-adrenoceptors, trabeculae

were incubated with S-(-) esmolol (1 - 100 µM) or R-(+) esmolol (100 µM) and 50

nM ICI 118,551 for at least 90 minutes and then concentration-effect curves to (-)-

noradrenaline established. For the determination of the equilibrium dissociation


constant (KB) of S-(-) and R-(+) esmolol at β2-adrenoceptors, trabeculae were

incubated with S-(-) esmolol (10 – 100 µM) or R-(+) esmolol (100 µM) and 300 nM

CGP 20712A for at least 90 minutes and then concentration-effect curves to (-)-

adrenaline established. Concentration-effect curves to (-)-noradrenaline and (-)-

adrenaline were followed by a single concentration of (-)-isoprenaline (200 µM) and

calcium (7 mM, final concentration 9.25 mM).

5.3 RESULTS

5.3.1 Acute Effects of S-(-) Esmolol on Human Right Atrium

S-(-) Esmolol caused time and concentration (1 – 100 µM) dependent reductions in

contractile force of electrically stimulated human right atrial trabeculae (Figure 5.1

a), most likely due in part to blockade of (-)-noradrenaline. There was no effect on

the duration of contraction, time to peak force (Figure 5.1 b) or time to 50%

relaxation (Figure 5.1 c).


Figure 5.1. Effect of S-(-) esmolol on force of contraction (a) and relaxation, time to peak force (b) and time to 50% relaxation. S-(-) esmolol caused a concentration and time dependent reduction in force of contraction, but did not affect the duration of the contraction. Force is expressed as a percentage of force at time 0 min.


5.3.2 Effect of S-(-) esmolol in the Presence of 3-isobutyl-1-methylxanthine (IBMX) and Nadolol

The addition of S-(-) esmolol (100 µM) to trabeculae equilibrated with nadolol (200

nM) and IBMX (10 µM), conditions designed to detect agonist activity at β1L-

adrenoceptors (Christ, Molenaar, Klenowski, Ravens, & Kaumann, 2011), caused a

time dependent reduction in contractile force (Figure 5.2). No agonist activity at β1L-

adrenoceptors was detected.

Figure 5.2. Lack of agonist activity of S-(-) esmolol at β1L-adrenoceptors. Right atrial trabeculae were equilibrated with nadolol (200 nM) and 3-isobutyl-1- methylxanthine (IBMX, 10 µM) and incubated with or without S-(-) esmolol (100 µM). Force is expressed as a percentage of initial force immediately before addition of nadolol or IBMX. Numbers in parentheses are (trabeculae/patients).


5.3.3 Determination of the Affinity of S-(-) and R-(+) Esmolol at Human Atrial β1- and β2-Adrenoceptors

β1-adrenoceptors

(-)-Noradrenaline caused concentration dependent increases in contractile force in

the absence and presence of S-(-) and R-(+) esmolol through activation of β1-

adrenoceptors (Figure 5.3 a). The pKB (-logKB) for S-(-) esmolol was 6.48 ± 0.12, n

= 33. The slope of the Schild plot was not different to unity (Figure 5.3 c). The

affinity of R-(+) esmolol, determined with one concentration of R-(+) esmolol (100

µM) at β1-adrenoceptors (pKB 4.57 ± 0.31, n = 7, Figure 7b) was ~ 80-fold less than

S-(-) esmolol.

__________________________________________________________ β1-adrenoceptors β2-adrenoceptors ________________________________________ S-(-) Esmolol 6.48 ± 0.12 (33/10) 5.85 ± 0.13 (20/9) R-(+) Esmolol 4.57 ± 0.31 (7/4) 4.74 ± 0.42 (5/3) __________________________________________________________

Table 5.2. pKB values of S-(-) Esmolol and R-(+) Esmolol determined at human atrial β1- and β2-adrenoceptors. S-(-) Esmolol, the slope of the Schild plot for experiments at β1-adrenoceptors was not different to unity. The slope of the Schild plot for experiments at β2-adrenoceptors was different to unity and therefore the pKB value is an estimate. R-(+) Esmolol pKB values determined with the use of a 100 µM concentration only. Numbers in parentheses are (trabeculae-patients).


Figure 5.3. Determination of the affinity of S-(-) and R-(+) esmolol at β1-adrenoceptors in human right atrium. Shown are cumulative concentration effect curves for (-)-noradrenaline at β1-adrenoceptors in the absence or presence of S-(-) (a) or R- (+) esmolol (b). The corresponding Schild plots are shown in (c). Numbers in parentheses are (trabeculae/patients).

!


β2-adrenoceptors

(-)-Adrenaline caused concentration dependent increases in contractile force in the

absence and presence of S-(-) and R-(+) esmolol through activation of β2-

adrenoceptors (Figure 5.4 a). The pKB (-logKB) for S-(-) esmolol was 5.85 ± 0.11, n

= 20. The slope of the Schild plot was different to unity (Figure 5.4 c) and therefore

the affinity value, pKB 5.85 is an estimate. The affinity of R-(+) esmolol, determined

with one concentration of R-(+) esmolol (100 µM) at β2-adrenoceptors (pKB 4.74 ±

0.94, n = 5, Figure 5.4 b) was ~ 13-fold less than S-(-) esmolol.


Figure 5.4. Determination of the affinity of S-(-) and R-(+) esmolol at β2-adrenoceptors in human right atrium. Shown are cumulative concentration effect curves for (-)-adrenaline at β2-adrenoceptors in the absence or presence of S-(-) (a) or R-(+) esmolol (b). The corresponding Schild plots are shown in (c). Numbers in parentheses are (trabeculae/patients).

A β2AR


5.4. DISCUSSION

5.4.1 Patient Population

This study was carried out in a relatively homogenous population of patients

undergoing open chested heart surgery at The Prince Charles Hospital. Right atrium

was obtained from patients, less than 75 years of age undergoing coronary artery

bypass surgery and who were chronically administered a β-blocker. Responses to (-)-

noradrenaline and (-)-adrenaline through activation of β1- and β2-adrenoceptors

respectively, differ in patients chronically administered a β-blocker or not taking a β-

blocker (Molenaar et al., 2007). Some patients that present for coronary artery bypass

surgery do not receive chronic administration of β-blockers.

5.4.2 Acute Effects of S-(-) Esmolol on Human Right Atrium

S-(-) esmolol caused a concentration and time dependent reduction in contractile

force of electrically stimulated right atrial trabeculae. This is most likely due to

blockade of β1-adrenoceptors activated by small amounts of (-)-noradrenaline

released upon nerve stimulation. There was no difference in the duration of

contraction, suggesting no alteration in phosphorylation of phospholamban or

troponin I, key proteins associated with relaxation in human atrium (Molenaar et al.,

2007).

5.4.3 Lack of Agonist Activity of S-Esmolol on β1L-Adrenoceptors

The β1-adrenoceptor is activated by (-)-noradrenaline and inhibited by all clinically

used β-blockers. Some β-blockers, for example (-)-pindolol, also activate β1-

adrenoceptors at higher concentrations (~2-3 orders of magnitude) than those

required to block it (Kaumann & Molenaar, 2008) through activation of a low


affinity binding site, the β1L-adrenoceptor. Using conditions optimized to detect

agonist activity of β-blockers at β1L-adrenoceptors, in the presence of nadolol and

IBMX (Christ et al., 2011), S-(-) esmolol 100 µM had no agonist activity in human

atrial trabeculae. On the other hand, S-(-) esmolol 100 µM caused cardiodepression.

5.4.4 Affinity of S-(-) and R-(+) Esmolol at Human Atrial β1- and β2- Adrenoceptors

S-(-) esmolol was a competitive antagonist at β1-adrenoceptors with a pKB of 6.48.

An estimated pKB value at β2-adrenoceptors was determined to be 5.84. This

represents a β1/β2-adrenoceptor selectivity ratio of ~ 4.3. The enantiomers of esmolol

showed stereoselectivity for blockade of β1- and β2-adrenoceptors. S-(-) esmolol had

~80 fold higher affinity for β1-adrenoceptors than R-(+) esmolol. The

stereoselectivity requirements at β2-adrenoceptors were lower where S-(-) esmolol

had ~13-fold higher affinity for β2-adrenoceptors than R-(+) esmolol.

5.5 CONLCUSIONS AND FUTURE DIRECTIONS

These results agree with previous findings that the main activity of β-blockers resides

mostly in the activity of the S-(-) enantiomer versus the R-(+) enantiomer (Kaumann

& Lobnig, 1986; Joseph et al., 2003; Kaumann & Molenaar, 2008). Interestingly, and

consistent with previous studies on the racemates of pindolol (Walter et al., 1984),

the β2-adrenoceptor displayed much lower stereoselectivity requirements, which may

indicate a role of R-(+) esmolol in the mediation of inotropic and/or lusitropic effects

via the β2-adrenoceptor in vivo. This may be another reason behind adverse effects

seen from racemic mixtures β-blockers; rather than just a product of altered

pharmacokinetics such as metabolism or excretion, the R-(+) enantiomers may


actually be mediating unwanted effects through the β2-adrenoceptor due to its

apparently less rigid stereoselectivity.

Timolol is already available in certain countries as an enantiomerically pure mixture,

however all of the other clinically available β-blockers are still only marketed as

racemic mixtures (Mehvar & Brocks, 2001). The affinities and efficacies of the

individual racemates of clinically important β-blockers such as metoprolol, atenolol,

bisoprolol, bucindolol, and carvedilol should be investigated in order to determine

the possible clinical benefit of their enantiomerically pure preparations. The

determination of the affinity values for both S- and R- enantiomers of β-blockers at

cloned human β1- and β2-adrenoceptors is recommended for the confirmation of in

vitro efficacy findings, and is also recommended for the confirmation of the findings

of this study (Kaumann et al., 2007; Kaumann & Molenaar, 2008).

Chapter 6: Conclusions and Future Directions 125

6. Conclusions and Future Directions

6.1 ROLE OF PDE3 AND PDE4 ON CONTRACTILITY OF THE HUMAN HEART

Ischemic heart disease, which leads to heart failure, is still the largest killer of both

men and women in all first-world countries, despite decades of research and

corresponding clinical advancements (Australian Bureau of Statistics, 2012).

Approximately 30-50% of the deaths from patients with heart failure are due to

sudden death, most likely due to the development ventricular arrhythmias (Adamson

& Gilbert, 2006; Zipes et al., 2009), which arise as a result of remodeling, apoptosis,

and altered Ca2+ signaling from chronic β1- and β2-adrenoceptor activation (Packer et

al., 1998; Bristow, 2001). PDE3 and PDE4 inactivate cAMP, effectively inhibiting

its accumulation and the subsequent increases in Ca2+ release and positive inotropy

and lusitropy that result from local increases in cAMP levels (Omori & Kotera, 2007;

Lenhart & Marks, 2006; Levy, 2103).

This work was carried out in order to determine whether PDE3 and/or PDE4 mediate

inotropic and lusitropic effects through β1- and/or β2-adrenoceptors in the failing

heart, and whether or not chronic administration of metoprolol or carvedilol alters

their effects on contractility. Previous studies have investigated the roles of PDE3

and PDE4 in animal models, however their roles in the human heart was largely

unknown. While PDE3 inhibition has been shown previously to increase the

inotropic potency of isoprenaline in failing and non-failing (Von Der Leyen et al.,

1991) and dobutamine (Metra et al., 2002) in failing human hearts. However, the role

of β1- and/or β2-adrenoceptors was not investigated. Metra and colleagues (2002)


investigated the effect of chronic administration of metoprolol and carvedilol on the

inotropic responses to the PDE3 inhibitor enoximone in vivo, and found that neither

β-blocker had any effect on the inotropic response to its administration (Metra et al.,

2002).

Even less is known about the possible role of PDE4 in human ventricular

myocardium, despite the downregulation of certain subfamilies of PDE4 in human

heart failure (Lehnart et al., 2005). This work was able to directly investigate the

roles of PDE3 and PDE4 in the failing human ventricle, utilising a long-standing

model of live in vitro trabecular setup to compare not only β-blocked to non-β-

blocked human tissues, but also to compare β1-selective to non β-selective blockade

on inotropic and lusitropic responses to catecholamines.

These findings revealed that PDE3, but not PDE4, helps to control β1-and β2-

adrenoceptor-mediated increases in inotropy and lusitropy in the failing human

ventricle. Metoprolol, a β1-adrenoceptor selective antagonist, moderately induced (3-

5-fold) control by PDE3 of the positive inotropy and lusitropy mediated by β2-

adrenoceptors. In contrast, cilostamide had markedly less ability to increase the

potency of (-)-noradrenaline for β1-adrenoceptor mediated inotropic and lusitropic

effects. Chronic administration of metoprolol increased the inotropic potency 4-fold

for (-)-noradrenaline via β1-adrenoceptors and 5-fold for (-)-adrenaline via β2-

adrenoceptors in the absence of PDE inhibition. The ability of metoprolol

administration to potentiate the effects of PDE3 inhibition may be attributed to a

variety of mechanisms: firstly, by blocking β1-adrenoceptors, metoprolol also

reduces the negative feedback mechanism that circulating catecholamines would

normally exert on PDE3 enzymes. With the suppression of PDE3 lifted, cAMP is


able to accumulate, increasing PKA phosphorylation and ultimately increasing

inotropic and lusitropic responses to noradrenaline via β1-adrenoceptors, and

adrenaline via β2-adrenoceptors. Secondly, metoprolol may restore the activity and

expression of PDE3 in failing hearts, in which PDE3 expression is down-regulated.

Thirdly, metoprolol administration may stop the catecholamine-induced increased

Giα levels, allowing β2-adrenoceptors to bind more tightly to the stimulatory Gsα

pathway, again increasing inotropic and lusitropic responses to adrenaline. This is

likely why metoprolol induces the effects of adrenaline, acting through β2-

adrenoceptors, more than it does the effects of noradrenaline via β1-adrenoceptors.

Carvedilol, a non-selective β-adrenoceptor and α-adrenoceptor antagonist, strongly

induced (19-35 fold) control by PDE3 of the positive inotropic and lusitropic effects

mediated by β2-adrenoceptors, likely due to its higher affinity for β2-adrenoceptors

over β1-adrenoceptors. Carvedilol may have induced positive inotropy and lusitropy

through a variety of mechanisms. Firstly, carvedilol administration may reduce the

activity of Giα which is normally seen in the failing heart, allowing improved

coupling of the β2-adrenoceptors to the stimulatory Gsα pathway. In addition,

carvedilol may help to restore the spatial localization of the β2-adrenoceptors, which

has been shown to be de-localised from stimulatory pathway proteins in failing

murine hearts. This re-localization would allow more efficient cAMP metabolism by

PDE3, and therefore a greater control over inotropic and lusitropic responses to

catecholamines. Chronic administration of carvedilol did not alter the inotropic

potency of (-)-noradrenaline via β1-adrenoceptors, but decreased the inotropic

potency of (-)-adrenaline via β2-adrenoceptors 16-fold in the absence of PDE

inhibition.


PDE4 did not control β1- or β2-adrenoceptor-mediated inotropic and lusitropic effects

in metoprolol- or carvedilol-treated patients, in contrast with previous animal heart

failure model studies.

Role of Carvedilol in the Prevention of Arrhythmias

Carvedilol has a 13-fold higher affinity for β2-adrenoceptors in human atrium, and

the residual blockade of β2-adrenoceptors after removal of tissue from patients

chronically administered carvedilol continues for longer than the residual β1-

adrenoceptor blockade (Molenaar et al., 2006). Carvedilol strongly induced PDE3’s

control of the inotropic and lusitropic effects mediated through β2-adrenoceptors,

plausibly by changing the feedback between PDE3 and β-adrenoceptor activity.

Chronic administration of carvedilol in heart failure patients induces PDE3 to

selectively control the positive inotropic and lusitropic effects mediated through

ventricular β2-adrenoceptors more than β1-adrenoceptors. This selective potentiation

of the inotropic and lusitropic responses of β2-adrenoceptors by PDE3 may have

occurred due to the ability of the β2-adrenoceptors to activate the Gs protein pathway,

which leads to local increases of cAMP levels, more efficiently than the β1-

adrenoceptors (Gille et al., 1985; Kaumann & Lemoine, 1987).

High-dose, chronic treatment with PDE3 inhibitors such as milrinone leads to

worsening of heart failure and increased mortality, particularly through sudden death

(Packer et al., 1991; Amsallem et al., 2005). As both β1- and β2-adrenoceptors

activate the stimulatory Gs pathway (Kaumann & Lemoine, 1987), they may also

both mediate arrhythmias in the human heart. Therefore, chronic administration with


the β1-adrenoceptor selective metoprolol may actually facilitate the development of

arrhythmias in response to stress- or surgery-induced increases of adrenaline. The

ability of carvedilol to selectively block β2-adrenoceptor-mediated arrhythmias

(Molenaar et al., 2006) may be the reason behind its apparent survival benefit versus

metoprolol (Poole-Wilson et al., 2003), and may provide protection against β2-

adrenoceptor-mediated ventricular overstimulation in PDE3 inhibitor treated

patients. However, experimental evidence demonstrating β2-adrenoceptor-mediated

arrhythmias, facilitated by PDE3 inhibition, is still pending for human myocardium;

determining the role of PDE3 inhibition on the development of β2-adrenoceptor-

mediated arrhythmias in failing human hearts is the next step for these experiments.

Limitations of this Work

The data for these studies was collected from three different research sites: Oslo,

Dresden, and Brisbane lab teams have collaborated and pooled data in order to obtain

tissue numbers necessary for statistical power. While the Oslo team worked

primarily with left ventricular trabeculae (and was therefore not pooled with the

other lab’s data), the Dresden and Brisbane labs data were pooled together. While

patient statistics were similar across groups and collection/experimental/analytical

protocol was kept as identical as possible, the presence of multiple researchers may

always introduce an element of inconsistency.

This work also did not utilize chemical assays to investigate the expression of PDE3

or PDE4 in the trabeculae that were included. As certain subfamilies become up or

down regulated as a result of human heart failure, each heart may have had unique

levels of PDE 3 and 4 expression, which may also have had an effect on inotropic


and lusitropic responses.

6.2 THE FUTURE OF β-BLOCKERS

6.2.1 Biased β-blockers

Biased ligands, or those with the ability to selectively activate a variety of signaling

pathways opens up an entirely new field of possible GPCR-based targets, where the

drug does not merely act as an ‘agonist’ or ‘antagonist’, simply activating or

blocking all of the targeted pathways of a specific GPCR, but is designed to be

selectively biased towards activating or inactivating a specific pathway in its

particular GPCR target’s repertoire (DeWire & Violin, 2011). Ligand-biased

targeting would, in theory, minimize unwanted side-effects. For example, having a

“β-blocker” which could block the effects of chronic β1-and β2-adrenoceptor

activation in the heart without having any risk of worsening concomitant disease

states such as asthma would be extremely useful.

Recent studies in mice have found another signaling pathway of the β1-adrenoceptor

through the β-arrestin–ERK1/2 pathway, which has anti-apoptotic and

cardioprotective properties in mice when selectively activated (DeWire & Violin,

2011). Biased ligands which selectively activate the ERK1/2 pathway of the β1-

adrenoceptor may prove to have more cardioprotective benefits than classical, non-

biased β1-adrenoceptor antagonists. The β2-adrenoceptor is also linked to this ‘non-

canonical’ ERK1/2 signaling pathway (Audet & Bouvier, 2008), and could also

potentially mediate cardioprotective effects in response to chronic adrenergic

activation in heart failure. Interestingly, carvedilol is the only clinically approved β-

blocker for heart failure that can activate the β-arrestin-ERK1/2 pathway via both β1-


and β2-adrenoceptors (DeWire & Violin, 2011; Thanawala et al., 2014). This biased

activation of theoretically cardioprotective signaling pathways may explain the

increase in survival in carvedilol-treated patients seen in multiple studies (COMET,

2003; DiNicolantonio et al., 2013).

Yet another reason behind carvedilol’s apparent survival benefits, and furthermore a

possible explanation behind the ostensibly paradoxical benefits of adding a β-blocker

to an already β-adrenergic down-regulated and desensitized heart in a failing state, is

the theory of dynamic signaling put forth by Michel et al. (2014). They propose that

both chronic administration of drugs and progressive disease states further regulate

the efficacy profiles and signaling bias of ligands, which would explain why chronic

administration of β-blockers demonstrates clear therapeutic benefits, despite an

initial worsening of heart function at the beginning of therapy. It may also explain

why, over time, PDE3 inhibitors show a worsening of mortality, in contrast to their

clinical benefits upon acute administration (Packer et al., 1993).

The ligand-biased efficacy of the β-blockers is certainly a mechanism of action

worth investigating further, for both refining the use of the existing β-blockers, and

in the development of newer, possibly more strongly biased β-adrenoceptor targeting

ligands. Investigating the efficacy profiles of the existing β-blockers will help create

a link between signaling bias and the possible therapeutic indications for drugs with

pluridimensional signaling efficacies (Galandrin & Bouvier, 2006). In addition to

investigating the existing β-adrenoceptor modifiers, more strongly biased ligands

need to be designed so that signaling bias and its translation to clinical effects can be

further elucidated.


6.2.2 Enantiomerically Pure β-Blocker Preparations

Yet another possible avenue for capitalizing on the β-blocker’s ability to increase

survival while potentially minimizing current unwanted side effects is to use

enantiomerically pure preparations. Previous studies have shown that the both the

affinities and the efficacies of the racemates of β-blockers can differ markedly in

vitro, (Kaumann & Lobnig, 1986; Mehvar & Brocks, 2001; Kaumann & Molenaar,

2008) and the findings with esmolol presented here confirm these pharmacodynamic

differences.

With new separation techniques available, it is now easier and more cost-effective to

prepare single racemate solutions than when β-blockers first became clinically

available (Wang et al., 2011; Tang et al., 2012). β-blockers commonly used for the

treatment of heart failure such as metoprolol, bisoprolol, and carvedilol should be

separated and tested in vitro to assess the potential for possible clinical benefits over

their mixed counterparts. In fact, S-metoprolol has already been released in India and

is still undergoing clinical trials; as the R- enantiomer displays higher β2-

adrenoceptor selectivity, investigators are hoping to see fewer adverse effects

associated with β2-adrenoceptor activation with the new S-metoprolol (Mehvar &

Brocks, 2001; Patil & Kuthekar, 2006). The findings from S-metoprolol along with

the FDA’s push for enantiomerically pure preparations will likely prompt many

researchers to investigate the potential benefits of purified β-blocker preparations

(Agustiana et al., 2010).


6.3 THE FUTURE OF PHOSPHODIESTERASE INHIBITORS

Clinically, PDE inhibitors are already mainstays in the treatment of pulmonary

hypertension, erectile dysfunction, and chronic obstructive pulmonary disease

(Francis et al., 2011). However, their role in the treatment of heart failure remains

controversial. Despite the previously demonstrated increases in mortality rates, the

use of PDE3 inhibitors may still have a place in the future of long-term heart failure

therapy. Rather than chronic administration, PDE3 inhibitors may be used repeatedly

in patients, but as a discontinuation therapy where the PDE inhibitor is used only

during acute worsening of cardiac function, and, once restored, removed once more

from therapy (Amsallem et al., 2005). This treatment strategy has not yet been

assessed in clinical trials, and would be a logical next step in the research of heart

failure combination therapies.

The level of intricacy and specificity of the structures, functions, expression,

distribution, (on the tissue, cellular, and sub-cellular levels) signaling, and regulation

of each of the PDE3/4 subfamilies is one of the reasons they are still being

investigated; despite more than 50 years of information gleaned from hundreds of

studies, not much is known regarding how PDE3 and PDE4 mediate contractility in

the human heart. Each new discovery leads to yet more important questions, and

there is a vast area of knowledge yet to fill; in future investigations, these questions

need to be answered one subfamily at a time (Guellich et al., 2014). As total

inhibition of PDE3 and/or PDE4 has initially proven harmful, it is possible that

inhibiting a specific subfamily or a specific subcellular localization of PDEs may

prove beneficial in the treatment of heart failure.


While PDE3A seems to control basal heart function in rat studies, PDE3B has been

suggested as a cardioprotective subtype against stress (Beca et al., 2013). The

worsening of heart failure in patients chronically treated with PDE3 inhibitors (such

as milrinone in Packer et al., 1993) might not have been a consequence of PDE3

inhibition in general, but of inhibition of PDE3B specifically; trials investigating the

effects of targeted PDE3A inhibition will be required to determine whether this is the

case. Firstly, however, the roles of PDE3A and PDE3B will need to be determined in

both healthy and failing human hearts, as Molenaar et al. (2000; 2007) have shown

that what is demonstrated in murine hearts is not necessarily able to be extrapolated

to the human heart. In addition, the “PDE3 inhibitor” milrinone actually has similar

selectivity for both PDE3 and PDE4 (Eschenhagen, 2013). Therefore, the increase in

mortality in patients who received long-term therapy with milrinone, demonstrated in

multiple studies including the large Packer et al. study in 1993, may actually be the

result of a combination of the inhibition of certain PDE4 subtypes in addition to

PDE3.

The highly specific recruitment of distinct PDE4D isoforms in mouse heart by the β2-

adreoceptor demonstrated by De Arcangelis et al. (2009) may be a further

explanation as to why PDE4 inhibition in this study did not result in changes of

inotropic or lusitropic response: certain subtypes may be recruited upon stimulation,

whereas others may control basal cAMP levels. In future studies, the specific

inhibition of distinct PDE4 subfamilies and/or isoforms may uncover a role of PDE4,

and therefore subtype-specific PDE4 inhibition, in the treatment of heart failure.


While the results of the studies researching the effects of PDE3 and PDE4 in both

murine and human hearts may be contradictory, a common theme does emerge from

their collective data: firstly, that different subfamilies and isoforms of the PDE3 and

PDE4 enzymes have highly specific roles in the regulation of β-adrenoceptor-

mediated inotropic and lusitropic responses to catecholamines, which demonstrates

the need for subtype-specific PDE inhibition in order to effectively determine the

roles of these enzymes. Secondly, that results from animal hearts cannot simply be

extrapolated to the human heart, as the human myocardium has striking differences

in both the expression of PDE families and the roles they play in basic cardiac

function and response. Moving forward, the inhibition of specific subtypes of both

PDE3 and PDE4 need to be carried out in both healthy and failing human ventricle

and atria to determine their role (or lack thereof) in human cardiac contractility; once

this has been achieved, the place of PDE3 inhibitors in the management of heart

failure may be determined.

136

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