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  • -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]

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

    Keywords

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

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

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

    enzyme, phosphodiesterase inhibitor, rolipram, ryanodine, RyR2

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

    Abstract

    The -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 1- and/or 2-adrenoceptors in

    human myocardium. These studies investigated whether the PDE3-selective

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

    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--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 1-adrenoceptors (2-adrenoceptors blocked with ICI 118,551), and

    (-)-adrenaline, mediated through 2-adrenoceptors (1-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-

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

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

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

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

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

    adrenaline, in non--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 2-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 2-adrenoceptors

    compared to 1-adrenoceptors. This property of carvedilol may provide protection

    against 2-adrenoceptor-mediated ventricular overstimulation in PDE3 inhibitor

    treated patients. PDE4 does not control 1- or 2-adrenoceptor-mediated inotropic

    and lusitropic effects in metoprolol or carvedilol -treated patients.

    -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 -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, 1L- and 3-adrenoceptors, and their

    affinities at human 1- and 2-adrenoceptors was investigated. S-(-) esmolol was a

    competitive antagonist at 1-adrenoceptors with a 1/2-adrenoceptor selectivity ratio

    of ~ 4.3. The enantiomers of esmolol showed stereoselectivity for blockade of 1-

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

    and 2-adrenoceptors. S-(-) esmolol had ~80 fold higher affinity for 1-adrenoceptors

    than R-(+) esmolol. The stereoselectivity requirements at 2-adrenoceptors were

    lower where S-(-) esmolol had ~13-fold higher affinity for 2-adrenoceptors than R-

    (+) esmolol.

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

    for blocking both 1- and 2-adrenoceptors than the R-(+) enantiomer. However,

    because the 2-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

    2-adrenoceptor in vivo and in the clinical setting.

  • -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 -adrenoceptor System: Healthy vs. Failing Heart ........................................ 2 1.2.1 The -adrenoceptor System in the Healthy Heart .................................. 2 1.2.2 The -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 -Blockers for the Treatment of Heart Failure ................... 11 1.4.1 The Development of -Blockers .......................................................... 11 1.4.2 The Evolution of the -Blocker ........................................................... 12 1.4.3 Efficacy of -Blockers Clinically Used for Heart Failure ................... 13 1.4.4 Signaling Bias of -Blockers ............................................................... 18 1.4.5 Dynamic Bias ....................................................................................... 21 1.5 The Enantiomers of -Blockers .......................................................................... 22 1.5.1 Racemic Mixtures ................................................................................ 22 1.5.2 Esmolol ................................................................................................ 28 1.5.3 The Clinical Relevance of Enantiomerically Pure -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 ...................................................4