aldehyde dehydrogenase activation prevents reperfusion arrhythmias by inhibiting local renin release...

Racha Estephan, Randi B. Silver, Daria Mochly-Rosen and Roberto LeviKenichiro Koda, Mariselis Salazar-Rodriguez, Federico Corti, Noel Yan-Ki Chan,

Inhibiting Local Renin Release From Cardiac Mast CellsAldehyde Dehydrogenase Activation Prevents Reperfusion Arrhythmias by

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Aldehyde Dehydrogenase Activation Prevents ReperfusionArrhythmias by Inhibiting Local Renin Release From

Cardiac Mast CellsKenichiro Koda, MD*; Mariselis Salazar-Rodriguez, MPharm*; Federico Corti, MPharm;

Noel Yan-Ki Chan, BS; Racha Estephan, PhD; Randi B. Silver, PhD;Daria Mochly-Rosen, PhD; Roberto Levi, MD, DSc

Background—Renin released by ischemia/reperfusion from cardiac mast cells activates a local renin-angiotensin system(RAS). This exacerbates norepinephrine release and reperfusion arrhythmias (ventricular tachycardia and fibrillation),making RAS a new therapeutic target in myocardial ischemia.

Methods and Results—We investigated whether ischemic preconditioning (IPC) prevents cardiac RAS activation in guineapig hearts ex vivo. When ischemia/reperfusion (20 minutes of ischemia/30 minutes of reperfusion) was preceded by IPC(two 5-minute ischemia/reperfusion cycles), renin and norepinephrine release and ventricular tachycardia and fibrillationduration were markedly decreased, a cardioprotective anti-RAS effect. Activation and blockade of adenosine A2b/A3

receptors and activation and inhibition of protein kinase C� (PKC�) mimicked and prevented, respectively, the anti-RASeffects of IPC. Moreover, activation of A2b/A3 receptors or activation of PKC� prevented degranulation and reninrelease elicited by peroxide in cultured mast cells (HMC-1). Activation and inhibition of mitochondrial aldehydedehydrogenase type-2 (ALDH2) also mimicked and prevented, respectively, the cardioprotective anti-RAS effects ofIPC. Furthermore, ALDH2 activation inhibited degranulation and renin release by reactive aldehydes in HMC-1.Notably, PKC� and ALDH2 were both activated by A2b/A3 receptor stimulation in HMC-1, and PKC� inhibitionprevented ALDH2 activation.

Conclusions—The results uncover a signaling cascade initiated by A2b/A3 receptors, which triggers PKC�-mediatedALDH2 activation in cardiac mast cells, contributing to IPC-induced cardioprotection by preventing mast cell reninrelease and the dysfunctional consequences of local RAS activation. Thus, unlike classic IPC in which cardiacmyocytes are the main target, cardiac mast cells are the critical site at which the cardioprotective anti-RAS effectsof IPC develop. (Circulation. 2010;122:771-781.)

Key Words: arrhythmia � ischemia � norepinephrine � renin � reperfusion

How critical mast cells are in cardiac pathophysiology isnot well understood. Numerous mast cells are present in

the mammalian heart (�50 000 mast cells per 1 g humanheart tissue) in close proximity to vessels and nerves, andtheir density markedly increases in heart failure, ischemiccardiomyopathy, and experimental infarct models.1 Mastcells synthesize, store, and release a variety of mediators. Werecently reported that cardiac mast cells are also an importantsource of the aspartyl protease renin.2 When released byischemia/reperfusion (I/R), this renin initiates the activationof a local renin-angiotensin system (RAS); the locally formedangiotensin II (Ang II) exacerbates norepinephrine releasefrom cardiac sympathetic nerves and elicits reperfusion ar-rhythmias.3 When mast cells are depleted or pharmacologi-

cally stabilized, renin and norepinephrine release and reper-fusion arrhythmias are markedly reduced.3 Thus, the releaseof mast cell–derived renin represents a new target in theprevention and treatment of ischemic cardiac dysfunction.

Editorial see p 761Clinical Perspective on p 781

Brief ischemic exposures have repeatedly been shown toprotect the heart both structurally and functionally from asubsequent prolonged exposure to I/R. Ischemic precondi-tioning (IPC), as this phenomenon is known, has been theobject of wide attention since its discovery.4–6 Adenosinerelease and activation/translocation of protein kinase C(PKC) have been identified as necessary steps in the cardio-

Received March 9, 2010; accepted June 14, 2010.From the Departments of Pharmacology (K.K., M.S.-R., F.C., N.Y.-K.C., R.L.) and Physiology and Biophysics (R.E., R.B.S.), Weill Cornell Medical

College, New York, NY, and Department of Chemical and Systems Biology (D.M.-R.), Stanford University School of Medicine, Stanford, Calif.*The first 2 authors contributed equally to this work.The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.110.952481/DC1.Correspondence to Roberto Levi, MD, DSc, Department of Pharmacology, Weill Cornell Medical College, 1300 York Ave, New York, NY 10065.

E-mail [email protected]© 2010 American Heart Association, Inc.

Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.110.952481

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protection afforded by classic IPC (eg, infarct size reduc-tion).6–8 Of the PKC family of serine/threonine kinases, thePKC� isoform has been shown to play a key cardioprotectiverole against I/R9,10 and to exhibit antisecretory activity inmast cells.11

Accordingly, we set out to determine whether IPC mightalso prevent the activation of a local cardiac RAS initiated bythe release of mast cell renin and, if so, whether this novelIPC paradigm involves the activation of PKC� in mast cells.Given that phosphorylation of mitochondrial aldehyde dehy-drogenase type-2 (ALDH2) by PKC� correlates with cardio-protection,12 we hypothesized that IPC could promote PKC�-induced activation of ALDH2, which would then remove thetoxic aldehydes known to degranulate mast cells such asacetaldehyde and 4-hydroxynonenal (4-HNE), which areformed by lipid peroxidation.13–15 Our study outlines a novelprotective anti-RAS effect of IPC; we find that the sequentialactivation of adenosine A2b and A3 receptors, PKC�, andALDH2 in cardiac mast cells diminishes the release of reninelicited by I/R and thus curtails Ang II– and norepinephrine-induced arrhythmias.

MethodsSee the online-only Data Supplement for the completeMethods section.

Ex Vivo HeartsIn total, 132 male Hartley guinea pigs (weight, 300 to 350 g; CharlesRiver Laboratories, Kingston, NY) were anesthetized with CO2 andeuthanized by stunning with approval from the Institutional AnimalCare and Use Committee. Isolated hearts were perfused at constantpressure with oxygenated Ringer at 37°C in a Langendorff apparatus(Radnoti Glass Technology, Monrovia, Calif).

Ischemia/ReperfusionAfter equilibration, all hearts were subjected to 20 minutes of globalischemia followed by 30 minutes of reperfusion. For IPC, two5-minute cycles of ischemia were each followed by 5 minutes ofreperfusion. For pharmacological prevention of IPC, antagonistswere perfused for 20 minutes (glyceryl trinitrate [GTN] for 30minutes) before and during IPC and then washed out for 15 minutesbefore I/R. For pharmacological preconditioning, given agents wereperfused for two 5-minute cycles before I/R except for �V1-1 (PKC�inhibitor), which was administered during the entire 30-minutereperfusion following the 20-minute ischemia. For prevention ofpharmacological preconditioning, antagonists were perfused for 20minutes (GTN for 30 minutes) before and during pharmacologicalpreconditioning and then washed out for 15 minutes before I/R.

Coronary flow was measured every 2 minutes; samples wereassayed for renin, norepinephrine, �-hexosaminidase, and creatinephosphokinase (CPK). Surface ECG was obtained from the leftventricle and right atrium, recorded in digital format, and analyzedwith Power Laboratory/8SP (AdInstrument, Colorado Springs,Colo).

Cell CultureThe human mastocytoma cell line (HMC-1) was a gift of from Dr I.Biaggioni (Vanderbilt University, Nashville, Tenn). Cells weremaintained in suspension culture as previously described.2

�-Hexosaminidase and Renin Assay�-Hexosaminidase and renin coronary overflow was measured aspreviously described.3 HMC-1 cells were suspended in Ringerbuffer, and equal volumes were divided into aliquots in Eppendorftubes and incubated at 37°C with a given agent (ie, Alda-1,

��RACK, or LUF5835 plus IBMECA) for 10 minutes (preceded ornot by a 30-minute incubation with GTN). Acetaldehyde, H2O2, or4-HNE was subsequently added for 20 minutes. All results werenormalized and expressed as percent above control.

Norepinephrine AssayCoronary effluent was assayed for norepinephrine by high-performance liquid chromatography with electrochemical detectionas previously described.3

CPK AssayCoronary effluent was assayed for CPK release with a CPK assay kit(Genzyme Diagnostics, Charlottetown, Prince Edward Island,Canada).

Polymerase Chain Reaction and ImmunostainingFor reverse-transcription polymerase chain reaction (RT-PCR), totalRNA was extracted from HMC-1 cells with TRIzol reagent (Invitro-gen, Carlsbad, Calif), 1 �g total RNA from each sample was reversetranscribed, and complementary DNA was amplified by RT-PCRwith a Qiagen (Valencia, Calif) 1-step RT-PCR kit. PCR productswere analyzed by agarose gel electrophoresis and ethidium bromidestaining. For immunostaining, HMC-1 cells were fixed and perme-abilized on glass slides and stained with the goat anti–A2b receptorantibody (Santa Cruz Biotechnology, Santa Cruz, Calif) conjugatedto Alexa Fluor 488 donkey anti-goat IgG and with rabbit anti–A3

receptor antibody (Santa Cruz) conjugated to Alexa Fluor 488donkey anti-rabbit IgG. Nuclei were stained with DAPI. For immu-nofluorescence, cells were examined with an inverted fluorescencemicroscope (Nikon Eclipse TE 2000-U, Morrell Instruments,Melville, NY) interfaced to an electron multiplying charge-coupleddevice (Hamamatsu Photonics, Bridgewater, NJ) and processedwith Metamorph software (version 6.2, Universal Imaging Corp,Downington, Pa).

Translocation of PKC�Cytosolic and membrane fractions of HMC-1 cells were separated,and Western blot analysis was performed with a PKC�-specificantibody (Santa Cruz).

ALDH2 Enzymatic Activity AssayALDH2 activity in HMC-1 cells was determined spectrophotometri-cally by monitoring the reductive reaction of NAD� to NADH at 340nm as previously described.16

Drugs and ChemicalsAcetaldehyde, H2O2, IBMECA, MRS1754, MRS1523, DPCPX,chelerythrine, 5-hydroxydecanoate, and cyanamide were purchasedfrom Sigma-Aldrich (St Louis, Mo); 4-HNE in ethanol solution waspurchased from Cayman Chemical (Ann Arbor, Mich). LUF5835was a gift from Dr M.W. Beukers (University of Leiden, Leiden, theNetherlands); EXP3174 was a gift from Merck Sharp & Dohme Ltd(Whitehouse Station, NJ); ��RACK, �V1-1, and Alda-1 weresynthesized in Dr Mochly-Rosen’s laboratory (Stanford UniversitySchool of Medicine, Palo Alto, Calif). Phorbol 12-myristate 13-acetate was purchased from LC Laboratories (Woburn, Mass). GTNwas purchased from Hospira Inc (Lake Forest, Ill). Human plasmaangiotensinogen was purchased from Calbiochem (San Diego,Calif).

StatisticsData are presented as mean�SEM. Nonparametric tests were usedthroughout the study. For 2-group comparisons, the Mann–Whitneytest was used (Figures 1 and 2). For comparisons among �2 groups,the Kruskal-Wallis test followed by the posthoc Dunn test was used(Figures 1 through 3, 4D, 4F, and 5 through 7). GraphPad Prismversion 4.03 for Windows (GraphPad Software, San Diego, Calif)was used. Values of P�0.05 were considered statistically significant.

772 Circulation August 24, 2010



ResultsIPC Prevents the Activation of a Cardiac RAS andAlleviates Arrhythmic DysfunctionSpontaneously beating Langendorff-perfused guinea pighearts were subjected to 20 minutes of global ischemiafollowed by 30 minutes of reperfusion. This resulted in mastcell degranulation, demonstrated by a 202�31% (n�5;mean�SEM) increase in �-hexosaminidase overflow into thecoronary effluent. I/R also caused large increases in renin andnorepinephrine overflow (ie, �2.5- and �75-fold, respec-tively) and severe ventricular arrhythmias (tachycardia andfibrillation [VT/VF]) that lasted �12 minutes (Figure 1). We

had previously shown that the enhanced norepinephrineoverflow and arrhythmias result from the activation of a localRAS by renin released from cardiac mast cells.3

When I/R was preceded by IPC (ie, two 5-minute cyclesof ischemia, each followed by 5 minutes of reperfusion),mast cell degranulation was only approximately half thatoccurring with I/R alone, as indicated by a markeddecrease in �-hexosaminidase overflow (ie, the overflowof �-hexosaminidase increased by 202�31% and 109�14%with I/R and I/R preceded by IPC, respectively; n�5 and 5;P�0.05). IPC also greatly reduced the overflow of renin andnorepinephrine and the duration of VT/VF (ie, an overall�70% to 85% decrease; Figure 1), clearly indicating acardioprotective anti-RAS effect of IPC.

Figure 1. IPC reduces renin and norepinephrine (NE) releaseand shortens arrhythmias caused by I/R in guinea pig hearts exvivo. This cardioprotective anti-RAS effect is mimicked or pre-vented by activation or blockade of adenosine A2b and A3receptors in combination but is unaffected by adenosine A1receptor blockade (A), mimicked by PKC� activation, and pre-vented by PKC� inhibition (B). A, Coronary overflow of renin andnorepinephrine and duration of reperfusion arrhythmias (VT/VF)in I/R (n�6) and I/R preceded by IPC (n�8) or I/R preceded byIPC in the presence of DPCPX (adenosine A1 receptor antago-nist, 300 nmol/L) or MRS1754 and MRS1523 (adenosine A2band A3 receptor antagonists; 50 and 100 nmol/L) (n�6 each).Instead of IPC, other hearts were perfused with a combinationof A2b (LUF 5835, 50 nmol/L) and A3 receptor agonists(IBMECA, 50 nmol/L) for two 5-minute cycles plus a 5-minutewashout before I/R (n�6). B, Hearts were pretreated with theselective PKC� inhibitor �V1-2 (1 �mol/L) and then subjected toI/R preceded by IPC (n�5). Additional hearts were perfused withthe selective PKC� activator ��RACK (500 nmol/L) instead ofIPC (two 5-minute cycles plus a 5-minute reperfusion; n�5)before I/R. Bars represent mean�SEM of overflows during thefirst 4 minutes of reperfusion or duration of VT/VF. **P�0.01,***P�0.001 from I/R; †P�0.05, ††P�0.01 from IPC by Mann–Whitney test (for I/R vs LUF5835 plus IBMECA or ��RACK) orKruskal-Wallis with the Dunn multiple comparison test (for allothers).

Figure 2. Selective inhibition of the PKC� isoform prevents thecardioprotective IPC-like anti-RAS effects generated by thecombined activation of adenosine A2b and A3 receptors (A).Selective desensitization of ALDH2 with GTN prevents the car-dioprotective effects of PKC� activation (B). A, Coronary over-flow of renin and norepinephrine (NE) and duration of VT/VF inguinea pig hearts subjected to I/R (n�6). Instead of IPC, otherhearts were perfused with a combination of selective A2b (LUF5835, 50 nmol/L) and A3 receptor agonists (IBMECA, 50 nmol/L)for two 5-minute cycles plus a 5-minute washout before I/R(n�6). Other hearts were perfused with the selective PKC� in-hibitor �V1-2 (1 �mol/L) followed by a combination of A2b (LUF5835) and A3 (IBMECA) receptor agonists for two 5-minutecycles plus a 5-minute washout instead of IPC before I/R (n�6).B, Instead of IPC, hearts were perfused with the selective PKC�activator (��RACK, 500 nmol/L) for two 5-minute cycles plus a5-minute washout before I/R (n�5). Other hearts were perfusedwith the ALDH2 desensitizer GTN (2 �mol/L, perfused for 30minutes), followed by ��RACK for two 5-minute cycles plus a5-minute washout instead of IPC before I/R (n�5). Bars repre-sent mean�SEM of overflows during the first 4 minutes ofreperfusion or duration of VT/VF. **P�0.01, ***P�0.001 from I/R;†P�0.05, ††P�0.01 from the A2b and A3 receptor agonist com-bination (A); †P�0.05 from ��RACK (B) by Mann–Whitney test(for I/R versus IPC) or Kruskal-Wallis with the Dunn multiplecomparison test (for all others).

Koda et al ALDH2 Inhibits Renin Release From Heart Mast Cells 773



Notably, the overflow of renin and norepinephrine duringpreconditioning was only slightly and not significantly higherthan that in nonconditioned hearts (renin overflow, 7.10�0.99pg � h�1 � g�1 Ang I formed during IPC and 6.81�2.12pg � h�1 � g�1 Ang I in control conditions; norepinephrine over-flow, 3.82�1.17 pmol/g during IPC versus 4.02�0.77 pmol/g incontrol conditions; mean�SEM; n�8�6; P�0.95 and P�0.75for renin and norepinephrine, respectively). Thus, the findingthat IPC markedly attenuated the I/R-induced release of reninand norepinephrine was not due to depletion of renin andnorepinephrine pools during preconditioning.

Combined Activation of Adenosine A2b and A3

Receptors Mimics the Cardioprotective Anti-RASEffects of IPCAdenosine, PKC, and mitochondrial ATP-sensitive potassiumchannels (mKATP) have been identified as necessary steps in the

cardioprotection afforded by classic IPC (eg, infarct size reduc-tion).6–8 Furthermore, although Ang II is widely perceived as adeleterious agent, it has also been found to mimic the cardio-protective effects of IPC.17–19 Thus, we investigated whetheradenosine, mKATP channels, Ang II, and PKC play a role in thecardioprotective anti-RAS IPC paradigm.

First, we assessed whether inhibition or activation of adeno-sine receptors prevents or mimics, respectively, the IPC-mediated attenuation of renin release in hearts subjected to I/R.We found that the combined blockade of adenosine A2b and A3

receptors with the MRS1754 (50 nmol/L)20 and MRS1523 (100nmol/L)21 compounds prevented the IPC-induced attenuation ofrenin and norepinephrine release and the alleviation of reperfu-sion arrhythmias (Figure 1A). Conversely, the combined activa-tion of A2b and A3 receptors with the LUF5835 (50 nmol/L)22

and IBMECA (50 nmol/L)23 compounds mimicked the cardio-protective anti-RAS effects of IPC (Figure 1A). In contrast,activation of A2b or A3 receptors alone failed to mimic theeffects of IPC (renin overflow, 26.94�2.80, 19.77�3.9, and24.37�2.58 pg � h�1 � g�1 Ang I formed for I/R, I/R plusLUF5835, and I/R plus IBMECA, respectively; n�6�5�5,P�0.20). Blockade of adenosine A1 receptors with DPCPX(300 nmol/L; IC50�18 nmol/L)24 was also ineffective (Figure1A). Thus, only the combined activation of A2b and A3 receptorsappears to induce the cardioprotective anti-RAS effects of IPC.

Because the opening of mKATP channels is known to beinvolved in the mediation of classic IPC-induced cardioprotec-tion,7,25,26 we questioned whether these channels may also playa role in the anti-RAS effects of IPC. Thus, we induced IPC inthe presence of the mKATP antagonist 5-hydroxydecanoate(100 �mol/L; IC50�30 �mol/L).27 5-Hydroxydecanoate failedto affect the cardioprotective anti-RAS effects of IPC. In fact, theIPC-induced attenuation of renin and norepinephrine release andthe abbreviation of reperfusion arrhythmias were the same in thepresence and absence of 5-hydroxydecanoate (Figure IA in theonline-only Data Supplement). Hence, the cardioprotective anti-RAS effects of IPC do not appear to depend on the opening ofmKATP channels.

According to early reports in rabbits and rats, Ang II mim-icked the cardioprotective effects of classic IPC.17–19 Thus, wedetermined whether Ang II, which is locally produced in theheart subjected to I/R,3 contributes to the anti-RAS effects ofIPC. For this, we induced IPC in hearts perfused with the AT1

receptor antagonist EXP3174. EXP3174 (300 nmol/L; IC50�6nmol/L)28 did not prevent the cardioprotective anti-RAS effectsof IPC (Figure IA in the online-only Data Supplement). In fact,the IPC-induced attenuation of renin and norepinephrine releaseand the abbreviation of reperfusion arrhythmias were the same inthe presence and absence of EXP3174 (Figure IA in theonline-only Data Supplement). Therefore, AT1 receptors areprobably not involved in the mediation of the cardioprotectiveanti-RAS effects of IPC.

Translocation of PKC� Mediates theCardioprotective Anti-RAS Effects of AdenosineA2b and A3 Receptor ActivationBecause PKC activation/translocation is likely to be involvedin the cardioprotective effects of classic IPC,8,29 we nextinvestigated the role of PKC in the cardioprotective anti-RAS

Figure 3. Selective activation of ALDH2 with Alda-1 mimics thecardioprotective anti-RAS effects of IPC. ALDH inhibition withcyanamide or selective ALDH2 desensitization with GTN eachprevents the effects of IPC and Alda-1. A, Coronary overflow ofrenin and norepinephrine (NE) and duration of VT/VF in guineapigs hearts subjected to I/R (n�6) or I/R preceded by IPC eitherin the absence (n�8) or presence (n�5) of the ALDH inhibitorcyanamide (CYA; 5 mmol/L). Other hearts were perfused withthe selective ALDH-2 activator Alda-1 (20 �mol/L) instead ofIPC (two 5-minute cycles plus a 5-minute washout; n�5) beforeI/R. Other hearts were perfused with cyanamide, followed byAlda-1 (two 5-minute cycles plus a 5-minute reperfusion)instead of IPC before I/R (n�5). B, Hearts were pretreated withthe ALDH2 desensitizer GTN (2 �mol/L for 30 minutes) and thensubjected to I/R preceded by IPC (n�5). Some hearts were per-fused with Alda-1 instead of IPC before I/R (n�5). Other heartswere perfused with GTN (2 �mol/L for 30 minutes), followedby Alda-1 instead of IPC before I/R (n�5). Bars representmean�SEM of overflows collected during the first 4 minutes ofreperfusion or duration of VT/VF. *P�0.05, **P�0.01, ***P�0.001from I/R; #P�0.05, ##P�0.01, ###P�0.001 from IPC; †P�0.05,††P�0.01 from Alda-1 by Kruskal-Wallis with the Dunn multiplecomparison test.




effects of IPC. Treatment of hearts with the general, non–isoform-selective PKC activator phorbol 12-myristate 13-acetate (PMA; 0.05 nmol/L, two 5-minute cycles before I/R)mimicked the protective effects of IPC on renin and norepi-nephrine release and reperfusion arrhythmia duration (FigureIB in the online-only Data Supplement). Moreover, inhibitionof PKC with the specific but non–isoform-selective chel-erythrine (2.8 �mol/L) prevented the effects of IPC on thesame parameters (Figure IB in the online-only Data Supple-ment). Thus, general PKC activation appears to mediate thecardioprotective anti-RAS effects of IPC.

Of the PKC family of serine/threonine kinases, the PKC�isoform has been shown to play a key cardioprotective roleagainst I/R.9,10,30,31 Thus, we tested whether ��RACK, aselective activator peptide of PKC�,32 mimics the anti-RASeffects of IPC. ��RACK (500 nmol/L), perfused for two5-minute cycles followed by a 5-minute washout before I/R,mimicked the protective anti-RAS effects of IPC. Indeed, theoverflow of renin and norepinephrine and the duration ofVT/VF were reduced by �55% to 90% compared with I/Rhearts (Figure 1B). Moreover, selective inhibition of PKC�with �V1-2 (1 �mol/L)33 prevented the effects of IPC on thesame parameters (Figure 1B). Thus, PKC� activation appearsto be required and sufficient for the genesis of the cardiopro-tective anti-RAS effects of IPC.

Stimulation of adenosine A2b and A3 receptors mimickedthe anti-RAS effects of IPC (see Figure 1A); selectiveactivation of the PKC� isoform also had anti-RAS effectssimilar to IPC (see Figure 1B). Given that adenosine is knownto activate PKC, thus initiating the traditional preconditioningcascade,6–8 we determined whether the anti-RAS IPC-likeeffects of A2b and A3 receptors rely on the consequentactivation of PKC�. To verify this notion, we assessedwhether PKC� blockade would prevent the IPC-like effects ofA2b and A3 receptor agonists. We found that selectiveinhibition of the PKC� isozyme with �V1-2 (1 �mol/L)33

prevented the anti-RAS IPC-like effects resulting from thecombined activation of A2b and A3 receptors (Figure 2A).Thus, A2b and A3 receptor–mediated activation of PKC�appears to be the first significant step in the anti-RASpreconditioning pathway.

Activation of ALDH2 Is Pivotal for theCardioprotective Anti-RAS Effects of PKC�Because the cardioprotective infarct-sparing effects of PKC�activation30 have been found to depend on phosphorylation ofmitochondrial ALDH2,12,34 we next assessed whether theanti-RAS effects of IPC are also determined by ALDH2activation. For this, we assessed whether inhibition/inactiva-tion of ALDH2 would abolish the anti-RAS effects of IPCand whether activation of ALDH2 would mimic them. Wefound that GTN, perfused for 30 minutes at a concentrationknown to inactivate ALDH2 (2 �mol/L),12 prevented thecardioprotective anti-RAS effects of ��RACK (ie, GTNabolished the ��RACK-induced inhibition of renin and nor-epinephrine release and the alleviation of VT/VF; Figure 2B).We also found that the general ALDH inhibitors cyanamide(5 mmol/L)12 and GTN prevented the anti-RAS effects of IPC(Figure 3A and 3B). Conversely, selective activation of

ALDH2 with Alda-112,34 (20 �mol/L, two 5-minute cycles)reproduced all of the anti-RAS effects of IPC, an action thatwas also prevented by cyanamide (Figure 3A) and bypretreatment with GTN (Figure 3B). Collectively, thesefindings suggest that ALDH2 activation by PKC� is a crucialmechanistic step in the development of the anti-RAS effectsof IPC.

Mast Cells Are the Site of the CardioprotectiveAnti-RAS Action of IPCGiven the pivotal role that mast cells play in the activation ofRAS in the heart,3 cardiac mast cells are likely to be the siteat which the anti-RAS effects of IPC develop. Becausecombined activation of adenosine A2b and A3 receptorsmimics the cardioprotective anti-RAS effects of IPC, whereasthe combined blockade of the same receptors prevents theanti-RAS effects (see Figure 1A), we first ascertained thepresence of A2b and A3 receptors on mast cells. For this, weused human mast cells in culture (HMC-1 cells). Total RNA(1 �g) was extracted from HMC-1 cells, reverse transcribed,and amplified by PCR using sense and antisense primersspecific for human A2b and A3 receptor genes. Figure 4Adepicts an ethidium bromide–stained gel showing that theHMC-1 PCR products for these adenosine receptor subtypesare consistent with those reported by others.35 HMC-1 cellswere also immunopositive for the 2 adenosine receptorsubtypes (Figure 4B).

We next made certain that mast cell PKC� can be activated.Using Western analysis in cytosolic and membrane fractionsof HMC-1 cells, we found that the phorbol ester PMA(positive control) markedly increased the translocation ofPKC� from cytosol to membrane (ie, a hallmark of PKC�activation; Figure 4C through 4F). Incubation of HMC-1 cellswith the PKC� activator ��RACK (500 nmol/L for 30minutes) before a 7-minute incubation with a below-thresholdconcentration of PMA (3 nmol/L)36 also significantly trans-located PKC� from cytosol to membrane (Figure 4C and 4D).Moreover, incubating HMC-1 cells with the adenosine A2b

and A3 receptor agonists in combination (LUF5835 andIBMECA, 50 nmol/L each for 1 hour) also translocated PKC�(Figure 4E and 4F).

Our findings in isolated guinea pig hearts and culturedmast cells suggested that IPC may result from the activationof adenosine A2b and A3 receptors expressed by cardiac mastcells and consequent PKC�-dependent activation of mito-chondrial ALDH2. Thus, we next investigated the role ofALDH2 in mast cell degranulation and renin release elicitedby prototypic toxic compounds formed in I/R. For this, wemeasured mast cell degranulation in response to acetalde-hyde, 4-HNE, another toxic aldehyde that accumulates duringcardiac ischemia, and hydrogen peroxide (H2O2), whichtriggers toxic aldehydes formation by membrane lipid per-oxidation.13,15 Incubation of HMC-1 cells with acetaldehyde(300 to 700 �mol/L), H2O2 (0.1 to 1 mmol/L), or 4-HNE (3to 30 �mol/L) elicited a concentration-dependent increase inthe release of �-hexosaminidase (�15% to 42%, an indica-tion of mast cell degranulation) and renin (�15% to 80%;Figure 5). Notably, preincubation of HMC-1 cells with theALDH2 activator Alda-1 (20 �mol/L)12,34 prevented the




degranulating effects of each acetaldehyde, H2O2, and 4-HNE(Figure 5A through 5F). Moreover, pretreatment of HMC-1cells with the ALDH2 desensitizer GTN (2 �mol/L for 30minutes) prevented the antidegranulating effects of Alda-1(Figure 5). These findings suggested that ALDH2 activationin mast cells prevents their degranulation by toxic aldehydesproduced in the I/R heart and that this represents a crucialmechanistic step in the anti-RAS effects of IPC.

We next measured ALDH2 activity in HMC-1 cells inresponse to the specific ALDH2 activator Alda-1,37 thenon–isoform-specific PKC activator PMA, the PKC�-selective agonist ��RACK, and the adenosine A2b and A3

receptor agonists in combination. We found that the ALDH2enzymatic activity (ie, NADH production) was enhanced byAlda-1 (100 �mol/L), PMA (300 nmol/L), ��RACK (0.5 and1 �mol/L), and LUF5835 (50 nmol/L) and IBMECA (50nmol/L) combined (Figure 6). Notably, selective PKC� inhi-bition with �V1-2 (1 �mol/L) prevented the increase inALDH2 activity elicited by A2b and A3 receptor agonists incombination (Figure 6).

Given that activation of A2b and A3 receptors in HMC-1cells increased ALDH2 activity and that this was inhibited by

the PKC� antagonist (see Figure 6), we next examinedwhether activation of A2b and A3 receptors and PKC� wouldeach protect mast cells from degranulation and renin release.We found that the large concentration-dependent increase in�-hexosaminidase and renin release elicited by incubationwith H2O2 (0.1 to 1 mmol/L) was markedly inhibited byLUF5835 (50 nmol/L) and IBMECA (50 nmol/L) combined(Figure 7A and 7C) and by ��RACK (500 nmol/L; Figure 7Band 7D). These effects were prevented by prior ALDH2desensitization with GTN pretreatment (2 �mol/L for 30minutes; Figure 7A through 7D). Collectively, these findingsindicate that activation of adenosine A2b and A3 receptors onthe mast cell membrane leads to an increase in PKC� activityand thus ALDH2 activation, which prevents the degranulat-ing effects of toxic aldehydes such as those produced in I/R.

Finally, we sought to establish whether the cardioprotec-tive anti-RAS effects of IPC, which most likely result from anaction at the mast cell level, are independent of the IPC-induced reduction of myocyte damage. Because both PKC�activation10 and PKC� inhibition32 protect cardiac myocytesfrom I/R-induced damage, we compared PKC� activationwith PKC� inhibition in terms of CPK release, as well as

Figure 4. Expression of adenosine receptors by HMC-1 cells. ��RACK or adenosine A2b and A3 receptor agonists in combinationselectively activate PKC� in HMC-1 cells. A, Ethidium bromide gel of transcripts of A2b and A3 receptors. B, Immunostaining of HMC-1cells expressing A2b and A3 receptors. Nuclei (blue) are stained with DAPI. Scale bar�10 �m. C and E, Translocation of PKC� (ie, PKC�activation) was determined by Western blot analysis in cytosolic and membrane fractions of HMC-1 cells. C, Control cells were incu-bated with Na Ringer for 30 minutes, demonstrating PKC� basal activity. Incubation of HMC-1 cells with the PKC activator PMA 300nmol/L for 7 minutes (positive control) or the PKC� activator ��RACK 500 nmol/L for 30 minutes, before an additional 7-minute incuba-tion with a below-threshold concentration of PMA (3 nmol/L), elicits an increase in PKC� translocation from cytosol to membrane (ie,PKC� activation). Notably, treatment with 3 nmol/L PMA for 7 minutes alone does not cause PKC� translocation. E, Control cells wereincubated with Na Ringer for 1 hour to demonstrate PKC� basal activity. Incubation of HMC-1 cells with 300 nmol/L PMA for 7 minutes(positive control) or the adenosine A2b and A3 receptor agonists LUF5835 and IB-MECA in combination (both at 50 nmol/L for 1 hour)elicits an increase in PKC� translocation from cytosol to membrane (ie, PKC� activation). D and F, Quantification of PKC translocationfrom cytosolic to membrane fractions, expressed as percent of control. Bars are mean�SEM (D, n�4 to 6; F, n�7) normalized relativeto �-actin. *P�0.05, **P�0.01 from control by Kruskal-Wallis test followed by the posthoc Dunn test.




renin and norepinephrine release and VT/VF duration. Wefound that I/R (ie, 20 minutes of global ischemia followed by30 minutes of reperfusion) caused a characteristic increase inCPK overflow into the coronary effluent of isolated guineapig hearts that peaked between the 4th and 10th minutes ofreperfusion (Figure IIA in the online-only Data Supplement).IPC, ��RACK pretreatment (500 nmol/L, two 5-minutecycles), and reperfusion with the PKC� antagonist �V1-1(500 nmol/L) each and all reduced total CPK overflow (0 to20 minutes of reperfusion) by �50% (Figure IIB in theonline-only Data Supplement). However, whereas ��RACKpretreatment markedly reduced the activation of RAS, thusdisplaying cardioprotective anti-RAS effects, reperfusionwith �V1-1 did not affect renin and norepinephrine release oralleviate VT/VF, thus failing to protect the heart from theconsequences of mast cell degranulation, including induction ofVT/VF (Figure IIC in the online-only Data Supplement). Thesefindings indicate that the cardioprotective anti-RAS effects ofIPC are mediated by a separate pathway that is independent ofcardiac myocyte salvage but directly dependent on the modula-tion of renin release from cardiac mast cells.

DiscussionThe notion of a local tissue-specific RAS, in addition to theclassic circulating RAS, has now gained general recogni-tion.38,39 Renin, the rate-limiting step in RAS activation, hasbeen found in cardiac myocytes,38,40 and renin mRNA hasbeen identified in heart fibroblasts and endothelial and

smooth muscle cells of coronary vessels.38 Our laboratoriesrecently demonstrated that mast cells can synthesize andsecrete renin.2,3 I/R causes the release of renin from cardiacmast cells, activating a local RAS, which results in severearrhythmic dysfunction.3

We have now uncovered a novel cardioprotective anti-RAS paradigm of IPC and delineated its transductionalpathway. IPC prevents I/R-induced renin release from cardiacmast cells via an adenosine-mediated activation of PKC� inthese cells, followed by activation of mitochondrial ALDH2,which effectively prevents mast cell degranulation. Given thepivotal role played by mast cell renin in local RAS activa-tion,1–3 we propose that the cardioprotective anti-RAS effectof IPC, typified by a marked decrease in the overflow of�-hexosaminidase, renin, and norepinephrine, and the curtail-ing of VT/VF are based on an inhibitory action at the mastcell level. This novel IPC paradigm is unlike classic IPC. Inclassic IPC, myocytes are the main target of cardioprotection,and IPC is characterized by infarct-size reduction and im-proved recovery of contractility.4–6

Although the adenosine A1 receptor has often been asso-ciated with IPC-induced protection of cardiac myocytes,6,8

we found here that the anti-RAS effects of IPC do not involveA1 receptors because A1 receptor blockade failed to modifythe anti-RAS effects of IPC. This agrees with the reportedlack of A1 receptors in mast cells41 and with our proposal thatmast cells are the critical site at which the cardioprotectiveanti-RAS effects of IPC develop.

Figure 5. Mast cell degranulation andrenin release elicited by acetaldehyde(A and D), H2O2 (B and E), and 4-HNE(C and F); blockade by Alda-1 andreversal by GTN pretreatment. A20-minute incubation of HMC-1 cellswith acetaldehyde, H2O2 or 4-HNEelicits a concentration-dependentrelease of both �-hexosaminidaseand renin. Preincubation with Alda-1(10 minutes) attenuates the release of�-hexosaminidase and renin. Pretreat-ment with GTN (2 �mol/L for 30 min-utes) prevents the effects of Alda-1.Points are mean (�SEM; n�3 to 17).*P�0.05, **P�0.01, ***P�0.001 from control;#P�0.05, ##P�0.01, ###P�0.001 fromAlda-1 plus GTN by Kruskal-Wallis with theDunn multiple comparison test.




Mast cells are known to express both adenosine A2b and A3

receptors42,43 (see also Figure 4). We found that the combinedactivation of A2b and A3 receptors displayed IPC-like effects:It attenuated the release of renin and norepinephrine andalleviated reperfusion arrhythmias associated with I/R. Thesecardioprotective anti-RAS effects were similar to those af-forded by IPC, which in fact was abolished by blockade ofadenosine A2b and A3 receptors in combination. AlthoughA2b and A3 receptors are known as low-affinity receptors (Ki

�5 and 1 �mol/L for A2b and A3, respectively),44 both werelikely activated by endogenous adenosine during IPC. Indeed,interstitial adenosine was shown to reach a 7-�mol/L levelafter 6 minutes of IPC in the isolated heart.45

Significantly, the combined activation of A2b and A3

receptors in HMC-1 cells in culture also prevented peroxide-induced degranulation and renin release, supporting ourconclusion that these mast cell receptors play a major role inthe anti-RAS effects of IPC. Activation of either A2b or A3

receptor alone failed to mimic the cardioprotective effects ofIPC, demonstrating the necessity that both receptors beactivated for the initiation of IPC. Other actions of adenosinesuch as coronary dilatation have also been shown to require aconcomitant activation of both A2b and A3 receptors.46

That the activation of A2b and A3 receptors in HMC-1 cellsprevents degranulation and renin release concurs with the

protective antisecretory effect of A2b receptors, demonstratedby enhanced mast cell activation when A2b receptors aredeleted in mice.47 Yet, other investigators have shown thatactivation of A2b and A3 receptors promotes the release ofmediators and cytokines from human lung fragments, ratRBL-2H3 cells, HMC-1 cells, and macrophages from A2b

receptor–deleted mice.42,43,48 The discrepancy between thesefindings and ours most likely depends on differences in cellsand animal species and on the different stimuli used todegranulate mast cells.

Opening of KATP channels in the inner membrane ofmitochondria in cardiac myocytes has been found to contrib-ute to the protective effects of classic IPC.6–8Yet, the mKATP

channel antagonist 5-hydroxydecanoate27 failed to modify thecardioprotective anti-RAS effects of IPC, indicating thatthese mitochondrial channels are not involved in the mastcell–dependent anti-RAS effects of IPC.

Having established that activation of A2b and A3 receptorscontributes to the anti-RAS effects of IPC, we asked whether

Figure 6. Activation of adenosine A2b and A3 receptors in combi-nation or PKC� increases ALDH2 activity in HMC-1 cells. Selectiveinhibition of the PKC� isoform prevents the increase in ALDH2activity by the combined activation of adenosine A2b and A3 recep-tors. Incubation of HMC-1 cells with the selective ALDH2 activatorAlda-1 (100 �mol/L for 10 minutes), the PKC activator PMA (300nmol/L for 10 minutes), the PKC�-selective activator ��RACK (0.5and 1 �mol/L for 30 minutes), or the adenosine A2b and A3 recep-tor agonists LUF5835 and IB-MECA in combination (both at 50nmol/L for 1 hour) increases ALDH2 activity (measured by the rateof NADH production at 340 nm). Pretreatment of HMC-1 cells withthe selective PKC� antagonist ��V1-2 (1 �mol/L for 20 minutes)prevents the effects of A2b and A3 receptor activation. Bars aremean percent increases from control (�SEM; n�4 to 8). BasalNADH production was 3.63�0.25 �mol � min�1 � /mg�1 protein.*P�0.05, **P�0.01, ***P�0.001 from control; #P�0.05 from thecombination of A2b and A3 receptor agonists by Kruskal-Wallis testfollowed by the posthoc Dunn test.

Figure 7. Activation of A2b and A3 receptors (A and C) or PKC�(B and D) inhibits mast cell degranulation and renin release elic-ited by H2O2; prevention by GTN pretreatment. Incubation ofHMC-1 cells with H2O2 for 20 minutes elicits a concentration-dependent release of both �-hexosaminidase and renin. Prein-cubation of HMC-1 cells with the A2b and A3 receptor agonistsLUF5835 and IB-MECA (10 minutes) in combination or with��RACK (10 minutes) attenuates the H2O2-induced release of�-hexosaminidase and renin. Pretreatment with GTN (2 �mol/Lfor 30 minutes) prevents the effects of A2b and A3 receptors andPKC�. Points are mean (�SEM; n�3 to 19). *P�0.05, **P�0.01,***P�0.001 from control; #P�0.05, ##P�0.01, ###P�0.001from LUF5835 plus IB-MECA plus GTN or ��RACK plus GTN,respectively, by Kruskal-Wallis with the Dunn multiple compari-son test.




A2b and A3 receptors might signal via PKC�, given that thisisoform has both cardioprotective9,10 and antisecretory prop-erties in mast cell–like RBL-H3 cells.11 Indeed, we found thatselective blockade of PKC� with �V1-233 abolished theanti-RAS effects of IPC, whereas selective activation ofPKC� with ��RACK10 mimicked them. Notably, the IPC-likeanti-RAS effects of A2b and A3 receptor agonists in combi-nation were prevented by selective PKC� inhibition. Collec-tively, at this point, our findings suggested that the initial stepin the anti-RAS signaling sequence of IPC could be anadenosine-induced stimulation of A2b and A3 receptors re-sulting in PKC� translocation in cardiac mast cells. In fact,A2b and A3 receptor activation in HMC-1 cells caused thetranslocation of PKC� from cytosol to membrane, the hall-mark of PKC� activation.

Acetaldehyde and 4-HNE are formed during I/R,14,15 inpart by lipid peroxidation caused by reactive oxygen speciessuch as hydrogen peroxide.13 These toxic aldehydes, knownto elicit mast cell degranulation,49 can be removed bymitochondrial ALDH2, a phosphorylation target of PKC�.12

Indeed, we found that Alda-1, a selective ALDH2-activatingdrug,12,34,37 not only mimicked the cardioprotective anti-RASeffects of IPC in the guinea pig heart ex vivo but alsoprevented the degranulating effects of acetaldehyde, H2O2,and 4-HNE in HMC-1 cells in culture. These effects wereabolished by selective inactivation of ALDH2 with GTN.Similarly, pretreatment of HMC-1 cells with GTN preventedthe antidegranulating effects caused by activation of A2b andA3 receptors or PKC�. Hence, these findings add furthersupport to the proposal that activation of ALDH2 in cardiacmast cells is the final crucial step of the protective anti-RASpathway.

Because A2b and A3 receptors, PKC�, and ALDH2 are alsopresent in cardiac myocytes,37,50 it is conceivable that inaddition to mast cells, activation of ALDH2 in cardiacmyocytes mediates cardioprotection from I/R. To rule out thispossibility, we used the PKC� inhibitor �V1-1, which pro-tects cardiac myocytes from damage by I/R51 but does notaffect mast cell degranulation (Palandiyani SS and Mochly-Rosen D, unpublished results). When we compared thecardioprotective effects resulting from PKC� activation withthose caused by PKC� inhibition,32 we found that the mastcell–dependent anti-RAS effects of IPC were clearly separa-ble from the protective effects on cardiac myocytes. Onewould have expected that the cardioprotective effects ofPKC� inhibition might reduce reperfusion arrhythmias, ifthey were to originate mostly from myocytes. On the con-trary, PKC� inhibition, which reduced myocyte damage byI/R to a similar extent as PKC� activation, did not abolishreperfusion arrhythmias. Thus, mast cell renin release andRAS activation are the predominant cause of arrhythmicdysfunction in I/R, and activation of ALDH2 in mast cells isan important cardioprotective mechanism in ischemicconditions.

Cardiac myocytes have been reported to express all of theRAS components, including renin.38,40 Renin mRNA has alsobeen found in heart fibroblasts and in endothelial and smoothmuscle cells of coronary vessels.38 Thus, it is conceivable thatin addition to cardiac mast cells, myocytes and other cells in

the heart may contribute to renin production and RASactivation in I/R. In fact, the incidence of reperfusion arrhyth-mias was markedly reduced, but not completely abolished, inhearts isolated from mice lacking mast cells or in guinea pighearts perfused with mast cell–stabilizing agents.3 This sug-gests that other cells in the heart could release renin inresponse to I/R.

ConclusionsWe propose that adenosine released from various cells duringI/R and IPC activates A2b and A3 receptors on the surface ofcardiac mast cells and that this is followed by activation/translocation of PKC�, which then increases the catalyticactivity of mitochondrial ALDH2 (see Figure 8). By elimi-nating reactive aldehydes and their mast cell–degranulatingeffects, ALDH2 prevents renin release and RAS activation.This reduces Ang II formation, inhibits excessive norepineph-rine release, and prevents the generation of reperfusionarrhythmias. Notably, the relevance of our findings extendsbeyond the disclosure of a new IPC mechanism. Indeed,although the discovery of IPC has generated a wealth ofstudies, its clinical translation has yet to come to fruition.52

As the search for cardioprotective drugs continues unabated,our findings elucidate novel basic mechanisms of pharmaco-logical cardioprotection (ie, the detoxification of reactivealdehydes and ROS in I/R by increasing the catalytic activityof mitochondrial ALDH2, thus alleviating the dysfunctionalconsequences of RAS activation in the heart). This newfinding suggests that in addition to protecting cardiac myo-cytes from I/R-induced injury, drugs that prevent cardiacmast cell degranulation may prevent the activation of a localRAS, thus providing an additional benefit to patients withmyocardial infarction and perhaps in other cardiac oxidativestress conditions such as those occurring in heart failure.

Sources of FundingThis work was supported by National Institutes of Health grantsHL34215, HL73400 (Dr Koda, Dr Estephan, Dr Silver, Dr Levi, M.Salazar-Rodriguez, F. Corti), HL47073, HL46403 (Dr Levi), andAA11147 (Dr Mochly-Rosen); by CDCH B-09-11-3999-2005 ofCentral University of Venezuela (M. Salazar-Rodriguez); and by aPharmaceutical Research and Manufacturers of America Foundation

Figure 8. Proposed mechanisms of the cardioprotective anti-RAS effects of IPC.




predoctoral fellowship (N. Yan-Ki Chan). We thank Paul J. Christos,DrPH, for statistical help, partially supported by the Clinical Trans-lational Science Center grant UL1-RR024996.

DisclosuresDr Mochly-Rosen is the founder of KAI Pharmaceuticals Inc, acompany that plans to bring PKC regulators to the clinic. However,none of the work in her laboratory is in collaboration with orsupported by the company. The other authors report no disclosures.

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CLINICAL PERSPECTIVEIschemia/reperfusion is a known cause of cardiac dysfunction that is often accompanied by infarction, contractile failure,and severe arrhythmias. The production of reactive oxygen species and reactive aldehydes is considered a prominent causebecause aldehyde detoxification by mitochondrial aldehyde dehydrogenase type-2 displays cardioprotective effects. Werecently reported that ischemia/reperfusion elicits the release of renin from cardiac mast cells, which in turn activates alocal renin-angiotensin system, causing severe arrhythmic dysfunction. Reactive oxygen species and reactive aldehydes arelikely responsible for mast cell degranulation and renin release; hence, means to prevent such effects could be clinicallybeneficial. We describe here a novel pathway that is independent of cardiomyocyte salvage and relies instead on aldehydedehydrogenase type-2 activation in mast cell mitochondria. Aldehyde dehydrogenase type-2 detoxifies reactive aldehydesproduced in ischemia/reperfusion, thus preventing mast cell degranulation and renin release. This avoids the activation ofa local renin-angiotensin system and reperfusion arrhythmias. This new finding suggests that in addition to protectingcardiac myocytes from ischemia/reperfusion-induced injury, drugs that prevent cardiac mast cell degranulation mayprevent the activation of a local renin-angiotensin system, thus providing an additional benefit to patients with myocardialinfarction and perhaps in other cardiac oxidative stress conditions such as those occurring in heart failure.




Supplemental Materials MATERIALS AND METHODS

Animals. 132 male Hartley guinea pigs weighing 300-350 grams (Charles River

Laboratories, Kingston, NY) were used for the study. All experiments were

approved by the Institutional Animal Care and Use Committee of Weill Cornell

Medical College.

Perfusion of guinea-pig hearts ex vivo. Guinea pigs were anesthetized with

CO2 and euthanized by stunning. Hearts were rapidly isolated and perfused at

constant pressure (55 cm H2O) with oxygenated Ringer’s solution (NaCl 154

mmol/L, KCl 5.61 mmol/L, CaCl2 2.16 mmol/L, NaHCO3 5.95 mmol/L and

dextrose 5.55 mmol/L) at 37oC in a Langendorff apparatus (Radnoti Glass

Technology Inc., Monrovia, CA).

Ischemia/reperfusion (I/R): following an equilibration period all hearts were

subjected to 20-min global ischemia induced by complete cessation of coronary

perfusion, followed by 30-min reperfusion (I/R). Ischemic preconditioning (IPC):

hearts were subjected to 2 x 5-min cycles of ischemia, each followed by 5-min

reperfusion. Pharmacological prevention of IPC: hearts were perfused with a

given antagonist for 20 min (30 min for GTN) before and during IPC, and then

washed out for 15 min before I/R. Pharmacological preconditioning: hearts were

perfused for 2 x 5-min cycles with a given agent, except for the selective PKCδ

inhibitor δV1-1 which was administered during the entire 30-min reperfusion

following the 20-min ischemia. Prevention of pharmacological preconditioning:

hearts were perfused with a given antagonist for 20 min (30 min for GTN) before



and during pharmacological preconditioning. All agents were then washed out for

15 min before I/R.

Coronary flow was measured by timed collections of the effluent every 2-

min; all samples were assayed for renin and norepinephrine, some for β-

hexosaminidase and creatine phosphokinase (CPK). Surface ECG was obtained

from leads attached to the left ventricle and the right atrium, recorded in digital

format through each experiment, and analyzed using Power Lab/8SP

(AdInstrument, Colorado Springs, CO). Onset and duration of reperfusion

arrhythmias were recorded and evaluated according to the Lambeth

conventions.1

Cell culture. The human mastocytoma cell line (HMC-1) was kindly provided to

us by Dr. I. Biaggioni (Vanderbilt University, Nashville, TN) and J. H. Butterfield

(Mayo Clinic, Rochester, MN). Cells were maintained in suspension culture at

high density in Iscove’s modified Dulbecco’s medium supplemented with 10%

FBS and 1.2 mmol/L monothioglycerol and kept at 37°C, 5% CO2.

β-Hexosaminidase and renin assay. Coronary effluent from guinea-pig hearts

ex vivo was assayed for β-hexosaminidase using the method of Schwartz et al.2

Samples of coronary effluent (20 µL) were placed in the well of a 96-well plate

and total β-hexosaminidase content was determined. For the measurement of

renin overflow, coronary effluent was immediately concentrated 8-fold by

centrifugal filtration (Millipore, Billerica, MA). Concentrated samples were

incubated overnight with human angiotensinogen and then renin activity

(angiotensin I formed) was determined by GammaCoat Plasma Renin Activity 125I



Radioimmunoassay (DiaSorin, Stillwater, MN). The detection limit was

approximately 0.01 pmol.

For HMC-1 cells, 4 to 6 pooled confluent flasks were pelleted and cells

washed twice with Ringer buffer (pH 7.4). Pelleted HMC-1 were then

resuspended in Ringer buffer and equal volumes of cell suspension were

aliquoted in Eppendorf tubes and incubated at 37°C with gentle agitation. Cell

suspensions were then incubated with a given agent (i.e., Alda-1, ψεRACK or

LUF5835 + IBMECA) for 10 min (preceded or not by a 30-min incubation with

GTN). Acetaldehyde, H2O2 or 4-HNE was subsequently added to the incubation

mixture for 20 minutes. At the end of protocol, samples were transferred to ice

and then centrifuged at 1,000 g for 10 min. Supernatants were collected and kept

in ice until β-hexosaminidase and renin release was evaluated. Cell pellets were

lysed with 0.5% triton X-100 and centrifuged at 13,000 g. Supernatants were

then used for protein and total β-hexosaminidase content determination. All

results were normalized and expressed as percent above control.

NE assay. Coronary effluent was assayed for norepinephrine by HPLC with

electrochemical detection as previously described.3 The detection limit was

approximately 0.2 pmol.

CPK assay. Coronary effluent was assayed for creatine phosphokinase release

using a creatine kinase assay kit (Genzyme Diagnostics, Charlottetown, PE,

Canada). Substrate and buffer reagent were added to the collected coronary

effluent and then incubated at 37oC for 2 min. The change in absorbance at 340



nm at one- min intervals was recorded in the spectrometer until the change in

absorbance was constant.

PCR and immunostaining. RT-PCR: total RNA was extracted from HMC-1 cells

using TRIzol reagent (Invitrogen, Carlsbad, CA), 1 µg of total RNA from each

sample was reverse-transcribed and cDNA amplified by RT-PCR using a

QIAGEN One-step RT-PCR kit. The sense primers specific for adenosine A2b-

and A3-receptors were: 5’-TAAGATCTTCCTGGTGGCCT-3’, and 5-

AGATGCCCAACAACAGCACT-3’, respectively. Antisense primers specific for

adenosine A2b- and A3-receptors were: 5’-GCTTGGCAGAGAAGATAC-3’, and 5’-

ATCTGACGGTAAGCTTGACC-3’, respectively. The amplification profile used

was: 50°C for 30 min, 95°C for 15 min, then 94°C for 30 sec, 55°C for 30 sec,

72°C for 1.5 min (40 cycles), and finally 72°C for 10 min. PCR products

generated were, ~330 and ~354 bp for adenosine A2b- and A3-receptor,

respectively. PCR products were analyzed by agarose gel electrophoresis and

ethidium bromide staining.

Immunostaining: HMC-1 cells were fixed and permeabilized on glass

slides and stained with the goat anti-A2b-receptor Ab (Santa Cruz Biotechnology,

Santa Cruz, CA)(1:200) conjugated to Alexa Fluor 488 donkey anti-goat IgG

(1:500) and with rabbit anti-A3-receptor Ab (Santa Cruz) (1:200) conjugated to

Alexa Fluor 488 donkey anti-rabbit IgG (1:500). Nuclei were stained with DAPI.

For viewing immunofluorescence, cells were examined with an inverted

fluorescence microscope (Nikon Eclipse TE 2000-U) interfaced to an electron



multiplying charge coupled device (Hamamatsu) and processed with Metamorph

software (version 6.2; Universal Imaging Corp.).

Translocation of PKCε. Following incubation of HMC-1 cells with PMA, the

PKCε-selective agonist ψεRACK, or the adenosine A2b- and A3-receptor agonists

LUF5835 and IB-MECA in combination, cells were homogenized in buffer (200

µL) (20 mmol/L Tris-HCl, 2 mmol/L EDTA, 10 mmol/L EGTA, 0.25 M sucrose, β-

mercaptoethanol and 1X protease inhibitors cocktail). Cell homogenates were

then passed through syringes with needle (30 Gauge) 10 times, and cell lysates

were centrifuged at 100,000 g for 30 min to collect cytosolic fractions

(supernatant). The pellets were resuspended in homogenization buffer (50 µL)

with 1% Triton X-100, and then centrifuged at 100,000 g for 30 min to collect

membrane fractions (supernatant). Translocation of PKCε was assessed by

using a PKCε-specific antibody (Santa Cruz; 1:1000 dilution) in Western blot

analysis. Methods for Western blot analysis were as previously described.3 The

ratio of PKCε in membrane to that in cytosol was expressed as PKCε

translocation.

ALDH2 enzymatic activity assay. Enzymatic activity of ALDH2 in HMC-1 cells

was determined spectrophotometrically by monitoring the reductive reaction of

NAD+ to NADH at 340 nm as previously described.4 The assays were carried out

in 50 mmol/L sodium pyrophosphate buffer, pH = 9.0, at 25oC. 300 µg of cell

lysates and 2.5 mmol/L NAD were added to the buffer. To start the reaction, 10

mmol/L acetaldehyde was added and the accumulation of NADH was recorded

for 3 min with measurements being taken every 15 s. ALDH2 reaction rates were



calculated as µmol NADH/min/mg proteins, and compared to cells that were

treated with Na Ringer (control) and expressed as % increase from control.

Drugs and chemicals. Acetaldehyde (17.8 M) and H2O2 (30% w/w, 9.8 M) were

purchased from Sigma-Aldrich (St. Louis, MO); 4-hydroxy Nonenal (4-HNE) in

ethanol solution was purchased from Cayman Chemical and dissolved in DMSO

to 300 mmol/L stock concentration after solvent evaporation. DMSO

concentration during experiments was always below 0.1% and did not affect the

cell response. LUF5825 was a gift from Dr. M.W. Beukers (University of Leiden,

Leiden, Netherlands); EXP3174 was a gift from Merck Sharp & Dohme Ltd

(Whitehouse Station, NJ); ψεRACK, δV1-1 and Alda-1 were synthesized in the

Mochly-Rosen lab (Stanford University School of Medicine, Palo Alto, CA).

IBMECA, MRS1754, MRS1523, DPCPX, chelerythrine, 5-hydroxydecanoate and

cyanamide were purchased from Sigma-Aldrich. Phorbol 12-myristate 13-acetate

was purchased from LC Laboratories (Woburn, MA). Glyceryl trinitrate was

purchased from Hospira Inc. (Lake Forest, IL). Human plasma angiotensinogen

was purchased from Calbiochem (San Diego, CA). Cyanamide and chelerythrine

were dissolved in water; EXP3174, DPCPX and 5-hydroxydecanoate were

dissolved in ethanol; IBMECA, MRS1754, MRS1523, phorbol 12-myristate 13-

acetate, and Alda-1 were dissolved in DMSO.

Figure Legends

Figure 1. IPC reduces renin and NE release, and shortens arrhythmias

caused by I/R in guinea-pig hearts ex vivo. This cardioprotective anti-RAS



effect is mimicked or prevented by activation or blockade of adenosine A2b-

and A3-receptors in combination, but unaffected by adenosine A1-receptor

blockade (Panel A), and mimicked by PKCε activation and prevented by

PKCε inhibition (Panel B). Panel A: Coronary overflow of renin and NE, and

duration of reperfusion arrhythmias (VT/VF) in isolated hearts subjected to 20-

min global ischemia and 30-min reperfusion (n=6). In other hearts, I/R was

preceded by IPC (2 x 5-min ischemia cycles each followed by 5-min reperfusion;

n=8). Other hearts were subjected to I/R preceded by IPC in the presence of

DPCPX (300 nmol/L, adenosine A1-receptor antagonist) or MRS1754 and

MRS1523 (50 and 100 nmol/L, adenosine A2b- and A3-receptor antagonists,

respectively) (n=6 each). Instead of IPC, other hearts were perfused with a

combination of A2b- (LUF 5835; 50 nmol/L) and A3-receptor agonists (IBMECA;

50 nmol/L) for 2 x 5-min cycles each followed by 5-min washout before I/R (n=6).

Panel B: Hearts underwent I/R preceded by IPC either in the absence (n=8) or

presence (n=5) of the selective PKCε inhibitor εV1-2 (1 µmol/L). Additional hearts

were perfused with the selective PKCε activator ψεRACK instead of IPC (500

nmol/L, 2 x 5-min cycles each followed by 5-min reperfusion; n=5) before I/R.

Basal, pre-ischemic overflows of active renin (i.e., ANG I formed) and NE were

6.81 ± 2.12 pg/hr/g and 4.02 ± 0.77 pmol/g, respectively. Bars represent means ±

SEM of overflows during the first 4 min of reperfusion or duration of VT/VF. **

And ***, P<0.01 and P<0.001, respectively, from I/R; † and ††, P<0.05 and P<0.01

from IPC, respectively, by Mann-Whitney test (for I/R vs LUF5835 + IBMECA or

ψεRACK) or Kruskal-Wallis with Dunn’s multiple comparison test (for all others).



Figure 2. Selective inhibition of the PKCε isoform prevents the

cardioprotective IPC-like anti-RAS effects generated by the combined

activation of adenosine A2b- and A3-receptors (Panel A). Selective

desensitization of ALDH2 with glyceryl trinitrate (GTN) prevents the

cardioprotective IPC-like anti-RAS effects generated by PKCε activation

(Panel B). Panel A: Coronary overflow of renin and NE, and duration of VT/VF in

ex vivo guinea-pig hearts subjected to I/R (n=6). Instead of IPC, other hearts

were perfused with a combination of selective A2b- (LUF 5835; 50 nmol/L) and

A3-receptor agonists (IBMECA; 50 nmol/L) for 2 x 5-min cycles each followed by

5-min washout before I/R (n=5). Other hearts were perfused with the selective

PKCε inhibitor εV1-2 (1 µmol/L), followed by a combination of A2b- (LUF 5835; 50

nmol/L) and A3-receptor agonists (IBMECA; 50 nmol/L) for 2 x 5-min cycles each

followed by 5-min washout instead of IPC before I/R (n=6). Panel B: Instead of

IPC, hearts were perfused with the selective PKCε activator (ψεRACK; 500

nmol/L) for 2 x 5-min cycles each followed by 5-min washout before I/R (n=5).

Other hearts were perfused with the ALDH2 desensitizer glyceryl trinitrate (GTN;

2µmol/L, perfused for 30 min), followed by ψεRACK (500 nmol/L) for 2 x 5-min

cycles each followed by 5-min washout instead of IPC before I/R (n=5). Bars

represent means ± SEM of overflows during the first 4 min of reperfusion or

duration of VT/VF. ** And ***, P<0.01 and P<0.001 from I/R, respectively; † and ††,

P<0.05 and P<0.01, respectively from the A2bR and A3R agonist combination



(Panel A), and †, P<0.05 from ψεRACK (Panel B), by Mann-Whitney test (for I/R

vs IPC) or Kruskal-Wallis test with Dunn’s multiple comparison test (for all others).

Figure 3. Selective activation of ALDH2 with Alda-1 mimics the

cardioprotective anti-RAS effects of IPC. General ALDH inhibition with

cyanamide or selective desensitization of ALDH2 with glyceryl trinitrate

(GTN), each prevents the cardioprotective anti-RAS effects of IPC and Alda-

1. Panel A: Coronary overflow of renin and NE, and duration of VT/VF in guinea-

pigs hearts subjected to I/R (n=6) or I/R preceded by IPC either in the absence

(n=8) or presence (n=5) of the ALDH inhibitor cyanamide (CYA; 5 mmol/L). Other

hearts were perfused with the selective ALDH-2 activator Alda-1 instead of IPC

(20 µmol/L, 2 x 5-min cycles each followed by 5-min washout; n=5) before I/R.

Other hearts were perfused with CYA (5 mmol/L), followed by Alda-1 (20 µmol/L,

2 x 5 min cycles each followed by 5-min reperfusion) instead of IPC before I/R

(n=5). Panel B: Hearts were pretreated with the ALDH2 desensitizer glyceryl

trinitrate (nitroglycerin, GTN; 2 µmol/L, perfused for 30 min) and then subjected

to I/R preceded by IPC (n=5). Other hearts were perfused with the selective

ALDH-2 activator Alda-1 instead of IPC (20 µmol/L, 2 x 5-min cycles each

followed by 5-min reperfusion; n=5) before I/R. Other hearts were perfused with

GTN (2 µmol/L) for 30 min, followed by Alda-1 (20 µmol/L, 2 x 5 min cycles each

followed by 5-min reperfusion) instead of IPC before I/R (n=5). Bars represent

means ± SEM of overflows collected during the first 4 min of reperfusion or

duration of VT/VF. *, ** And ***, P<0.05, P<0.01 and P<0.001 from I/R,



respectively; #, ## and ###, P<0.05, P<0.01 and <0.001 from IPC, respectively; †

and ††, P<0.05 and P<0.01 from Alda-1, respectively, by Kruskal-Wallis test with

Dunn’s multiple comparison test.

Figure 4. Expression of adenosine receptors by HMC-1 cells. ψεRACK or

adenosine A2b- and A3-receptor agonists in combination induces selective

activation of PKCε in HMC-1 cells. A, Ethidium bromide gel of transcripts of the

adenosine A2b- and A3-receptor sub-types in HMC-1 cells. B, Immuno-staining of

HMC-1 cells expressing the adenosine A2b- and A3-receptor sub-types. Nuclei

(blue) are stained with DAPI. Scale bar = 10 μm. C and E, Translocation of PKCε

(i.e., activity of PKCε) was determined by Western blot analysis in cytosolic and

membrane fractions of HMC-1 cells. C, Control cells were incubated with Na

Ringer for 30 min demonstrating the basal activity of PKCε. Incubation of HMC-1

cells with the PKC activator phorbol ester myristate (PMA) 300 nmol/L for 7 min

(positive control) or the PKCε activator ψεRACK 500 nmol/L for 30 min, prior to

an additional 7-min incubation with a below-threshold concentration of PMA (3

nmol/L), elicits an increase in PKCε translocation from cytosol to membrane (i.e.,

PKCε activation). Notably, treatment with 3 nmol/L PMA for 7 min alone does not

cause PKCε translocation. E, Control cells were incubated with Na Ringer for 1

hr demonstrating the basal activity of PKCε. Incubation of HMC-1 cells with 300

nmol/L PMA for 7 min (positive control) or the adenosine A2b- and A3-receptor

agonists LUF5835 and IB-MECA in combination (both at 50 nmol/L; 1 hour)

elicits an increase in PKCε translocation from cytosol to membrane (i.e., PKCε



activation). D and F, quantification of PKCε translocation from cytosolic to

membrane fractions, expressed as % of control. Bars are means ± SEM (D, n=4-

6; F, n=7) normalized relative to β-actin. * And **, P<0.05 and P<0.01, from

control respectively, by Kruskal-Wallis test followed by post-hoc Dunn’s test.

Figure 5. Mast cell degranulation and renin release elicited by acetaldehyde

(A and D), H2O2 (B and E) and 4-HNE (C and F): blockade by Alda-1 and its

reversal by pretreatment with GTN. Incubation of HMC-1 cells with

acetaldehyde (300-700 µmol/L, 20 min), H2O2 (0.1-1 mmol/L, 20 min) or 4-HNE

(3-30 µmol/L, 20 min) elicits a concentration-dependent release of both β-HEX

and renin. Pre-incubation of HMC-1 cells with the ALDH2 activator Alda-1 (20

µmol/L, 10 min) attenuates the acetaldehyde- , H2O2- and 4-HNE-induced

release of β-HEX and renin. Pre-treatment of HMC-1 cells with the ALDH2-

desensitizer glyceryl trinitrate (GTN, 2 µmol/L, 30 min) prevents the effects of

Alda-1. Points are means (± SEM; n=3-17). *, ** and ***, P<0.05, P<0.01 and

P<0.001, from control, respectively; #, ## and ###, P<0.05 and P<0.01, from Alda-1

+ GTN respectively, by Kruskal-Wallis with Dunn’s multiple comparison test.

Figure 6. Activation of adenosine A2b- and A3-receptors in combination or

PKCε increases ALDH2 activity in HMC-1 cells. Selective inhibition of the

PKCε isoform prevents the increase in ALDH2 activity by the combined

activation of adenosine A2b- and A3-receptors. Incubation of HMC-1 cells with

the selective ALDH2 activator Alda-1 (100 µmol/L; 10 min), the PKC activator



phorbol ester myristate (PMA) (300 nmol/L; 10 min), the PKCε-selective agonist

ψεRACK (0.5 and 1 µmol/L; 30 min), or the adenosine A2b- and A3-receptor

agonists LUF5835 and IB-MECA in combination (both at 50 nmol/L; 1 hour)

increases ALDH2 activity (measured by the rate of NADH production at 340 nm).

Pre-treatment of HMC-1 cells with the PKCε antagonist εV1-2 (1 µmol/L; 20 min)

prevents the effects of adenosine A2b- and A3-receptor activation. Bars are mean

percent increases from control (±SEM; n=4-8). Basal NADH production was 3.63

± 0.25 µmol/min/mg protein. *, ** and ***, P<0.05, P<0.01 and P<0.001 from

control, respectively, by Kruskal-Wallis test followed by post-hoc Dunn’s test. #,

P<0.05 from the A2b- and A3-receptor agonists combination by Kruskal-Wallis test

followed by post-hoc Dunn’s test.

Figure 7. Activation of adenosine A2b- and A3-receptors (A and C), or PKCε

(B and D), inhibits mast cell degranulation and renin release elicited by

H2O2: prevention by GTN pretreatment. Incubation of HMC-1 cells with H2O2

for 20 min elicits a concentration-dependent release of both β-HEX and renin.

Pre-incubation of HMC-1 cells with the adenosine A2b- and A3-receptor agonists

LUF5835 and IB-MECA (both at 50 nmol/L, 10 min) in combination, or with the

PKCε activator (ψεRACK, 500 nmol/L, 10 min) attenuates the H2O2-induced

release of β-HEX and renin. Pre-treatment of HMC-1 cells with the ALDH2

desensitizer glyceryl trinitrate (GTN, 2 µmol/L, 30 min) prevents the effects

resulting from the activation of A2b- and A3-receptors and PKCε. Points are

means (± SEM; n=3-19). *, ** and ***, P<0.05, P<0.01 and P<0.001, from control,



respectively; #, ## and ###, P<0.05, P<0.01 and P<0.001 from LUF5835 + IB-

MECA + GTN or ψεRACK + GTN, respectively, by Kruskal-Wallis with Dunn’s

multiple comparison test.

Figure 8. Proposed mechanisms of the cardioprotective anti-RAS effects of

ischemic preconditioning.

Legend to Supplemental Figure I. The cardioprotective anti-RAS effects of

IPC are unaffected by mitochondrial KATP channel blockade or angiotensin

AT1-receptor blockade (Panel A), and mimicked by general PKC activation

and prevented by general PKC inhibition (Panel B). Panel A: Coronary

overflow of renin and NE, and duration of VT/VF in ex vivo guinea-pig hearts

subjected to I/R (as in Fig.1; n=6). Other hearts underwent I/R preceded by IPC

in the presence of 5-hydroxydecanoate, a mitochondrial KATP channel blocker (5-

HD; 100 µmol/L; n=6), or EXP3174, an angiotensin AT1-receptor antagonist (300

nmol/L; n=5). Panel B: Hearts were pretreated with the general PKC inhibitor

chelerythrine (2.8 µmol/L; Chel) and then subjected to I/R preceded by IPC (n=6).

Additional hearts were perfused with the general PKC activator phorbol ester

myristate (0.05 nmol/L; PMA) instead of IPC (2 x 5-min cycles + 5-min

reperfusion; n=6) before I/R. Bars are means ± SEM of overflows during the first

4 min of reperfusion or duration of VT/VF. ** And ***, P<0.01 and <0.001 from I/R,

respectively; †† and †††, P<0.01 and P<0.001 from IPC, respectively, by Mann-



Whitney test (for I/R vs PMA) or Kruskal-Wallis with Dunn’s multiple comparison

test (for all others).

Legend to Supplemental Figure II. Selective inhibition of PKCδ during

reperfusion following ischemia prevents CPK release from isolated guinea-

pig hearts (Panel A and B), but fails to affect the cardioprotective anti-RAS

effect of IPC (Panel C). Time course of CPK overflow (Panel A) and total CPK

release during 20 min of reperfusion (Panel B) in ex vivo guinea-pig hearts

subjected to I/R preceded or not by IPC or PKCε activation (with ψεRACK, 500

nmol/L) (n=5-7). Other hearts underwent I/R and received the selective PKCδ

inhibitor δV1-1 (500 nmol/L, n=4) during the 30-min reperfusion. Panel C:

Coronary overflow of renin, NE, and duration of VT/VF, in ex vivo guinea-pig

hearts subjected to I/R, preceded or not by IPC (n=8 and 6, respectively) or the

selective PKCε activator ψεRACK (500 nmol/L) for 2 x 5-min cycles each

followed by 5-min washout (n=5). Other hearts underwent I/R and received the

selective PKCδ inhibitor δV1-1 (500 nmol/L) during the 30-min reperfusion (n=4).

In Panel A and B, bars represent means ± SEM of overflows collected during the

first 20 min of reperfusion. In Panel C, bars represent means ± SEM of overflows

collected during the first 4 min of reperfusion or duration of VT/VF. *, ** And ***,

P<0.05, P<0.01 and P<0.001 from I/R, respectively, by Mann-Whitney test.

Reference List 1. Walker MJ, Curtis MJ, Hearse DJ, Campbell RW, Janse MJ, Yellon DM,

Cobbe SM, Coker SJ, Harness JB, Harron DW, Higgins AJ, Julian DG, Lab



MJ, Manning AS, Northover BJ, Parratt JR, Riemersma RA, Riva E, Russell DC, Sheridan DJ, Winslow E, Woodward B. The Lambeth Conventions: guidelines for the study of arrhythmias in ischaemia infarction, and reperfusion. Cardiovasc Res. 1988;22:447-455.

2. Schwartz LB, Austen KF, Wasserman SI. Immunologic release of beta-hexosaminidase and beta-glucuronidase from purified rat serosal mast cells. J Immunol. 1979;123:1445-1450.

3. Seyedi N, Mackins CJ, Machida T, Reid AC, Silver RB, Levi R. Histamine H3-receptor-induced attenuation of norepinephrine exocytosis: a decreased PKA activity mediates a reduction in intracellular calcium. J Pharmacol Exp Ther. 2005;312:272-280.

4. Churchill EN, Disatnik MH, Mochly-Rosen D. Time-dependent and ethanol-induced cardiac protection from ischemia mediated by mitochondrial translocation of varepsilonPKC and activation of aldehyde dehydrogenase 2. J Mol Cell Cardiol. 2009;46:278-284.



Supplemental Figure I

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δV1-1ψεRACKIPC

Ischemia/Reperfusion

δV1-1ψεRACKIPC



aldehyde dehydrogenase activation prevents reperfusion arrhythmias by inhibiting local renin release...

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