Published online before print July 14, 2003, doi: 10.1161/01.CIR.0000081943.93653.73
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(Circulation. 2003;108:869.)
© 2003 American Heart Association, Inc.
Basic Science Reports |
Additive Protection of the Ischemic Heart Ex Vivo by Combined Treatment With 
-Protein Kinase C Inhibitor and 
-Protein Kinase C Activator
From the Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, Calif (K.I., D.M.-R.), and the Department of Internal Medicine, Division of Cardiology, University of Cincinnati Medical Center, Cincinnati, Ohio (H.S.H., G.W.D.).
Correspondence to Daria Mochly-Rosen, PhD, Department of Molecular Pharmacology, Stanford University School of Medicine, CCSR, Room 3145A, 269 Campus Dr, Stanford, CA 94305-5174. E-mail mochly{at}stanford.edu
Received February 10, 2003; revision received April 17, 2003; accepted April 18, 2003.
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Abstract |
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Background— Protein kinase C (PKC) plays a major role in cardioprotection from ischemia/reperfusion injury. Using an HIV-1 Tat protein–derived peptide to mediate rapid and efficient transmembrane delivery of peptide regulators of PKC translocation and function, we examined the cardioprotective effect of selective
-PKC inhibitor (V1-1) and 
-PKC activator (
RACK) peptides for ischemia/reperfusion damage in isolated perfused rat hearts. Furthermore, we examined the protective effects of these PKC isozymes in isolated perfused hearts subjected to ischemia/reperfusion damage using transgenic mice expressing these peptides specifically in their cardiomyocytes.
Methods and Results— In isolated perfused rat hearts, administration of V1-1 but not RACK during reperfusion improved cardiac function and decreased creatine phosphokinase release. In contrast, pretreatment with RACK but not V1-1, followed by a 10-minute washout before ischemia/reperfusion, also improved cardiac function and decreased creatine phosphokinase release. Furthermore, administration of RACK before ischemia followed by V1-1 during reperfusion only conferred greater cardioprotective effects than that obtained by each peptide treatment alone. Both the -PKC inhibitor and -PKC activator conferred cardioprotection against ischemia/reperfusion injury in transgenic mice expressing these peptides in the heart, and coexpression of both peptides conferred greater cardioprotective effects than that obtained by the expression of each peptide alone.
Conclusions— -PKC inhibitor prevents reperfusion injury, and -PKC activator mimics ischemic preconditioning. Furthermore, treatment with both peptides confers additive cardioprotective effects. Therefore, these peptides mediate cardioprotection by regulating ischemia/reperfusion damage at distinct time points.
Key Words: ischemia • reperfusion • cardioprotection • enzymes • kinases
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Protein kinase C (PKC) is a key enzyme in signal transduction involved in a variety of cellular functions.1 PKC plays a major role in cardioprotection from ischemia/reperfusion injury in various cell culture studies and animal models.2–4 Multiple PKC isozymes are expressed in the heart.5,6 The role of individual isozymes in cardioprotection has been difficult to assess because the pharmacological tools currently available are not isozyme selective.
We previously developed several PKC translocation inhibitor and activator peptides that, when introduced into cells, cause selective regulation of translocation and function of the corresponding PKC isozymes.7 These peptides were designed on the basis of the observation that isozyme-unique functions are mediated by binding of each isozyme to its corresponding selective anchoring protein, receptor for activated C-kinase (RACK), each localized to a selective subcellular site.5,8,9 More recently, we showed that the Tat protein–derived peptide provides a means to deliver the PKC-regulating peptides into cardiomyocytes when infused through the coronary arteries.10 Using this peptide delivery technique, we showed that pretreatment with the -PKC inhibitor peptide V1-1 or the -PKC activator peptide RACK reduced the damage of isolated adult rat cardiomyocytes and of isolated perfused mouse and rat hearts from simulated ischemia.10–12
In the present study, we determined whether the time at which each of these PKC-regulating peptides is delivered affects induced cardioprotection, the effects of these peptides in acute (Tat-conjugated peptide) and chronic (transgenic mice) administration, and the effect of treatment with both peptides on cardioprotection from ischemia/reperfusion damage in isolated hearts.
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Methods |
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Peptide Synthesis
We synthesized
V1-1 (-PKC inhibitor, amino acids 8 to 17 [SFNSYELGSL])10 and RACK (-PKC activator, amino acids 85 to 92 [HDAPIGYD])11 at Stanford’s Protein and Nucleic Acid facility and conjugated them to Tat (carrier peptide, amino acids 47 to 57 [YGRKKRRQRRR])13 via a cysteine–cysteine S-S bond at their N termini, as previously described.14
Isolated Perfused Rat Heart Model
Wistar rats (300 to 350 g) were heparinized (2000 U/kg IP) and then anesthetized with sodium pentobarbital (100 mg/kg IP). The hearts were rapidly excised and then perfused with an oxygenated Krebs-Henseleit solution containing (in mmol/L) NaCl 120, KCl 5.8, NaHCO3 25, NaH2PO4 1.2, MgSO4 1.2, CaCl2 1.0, and dextrose 10, pH 7.4, at 37°C in a Langendorff coronary perfusion system. The coronary flow rate was kept constant during the experiment at 10 mL/min. A thin latex balloon filled with water was inserted into the left ventricular (LV) cavity and connected to a pressure transducer (PowerLab/8SP, AD Instruments) for measurement of the isovolumic LV pressure. The LV volume was adjusted initially at an end-diastolic pressure (EDP) of 3 to 5 mm Hg. Hearts were paced at 3.3 Hz (200 bpm) with a bipolar pacing catheter. Hearts were submerged into a heat-jacketed organ bath set at 37°C. Coronary effluent was collected to determine creatine phosphokinase (CPK) release. After 
10 minutes of equilibration, the experiment began, and the LV pressure and coronary perfusion pressure were monitored throughout the experiment.
Experimental Protocol in Isolated Perfused Rat Hearts
Hearts were subjected to a 20-minute global ischemia and a 30-minute full reperfusion to measure functional recovery and CPK release during reperfusion. (Sets 1 to 3 in Figures 1 to 3
) or a 40-minute global ischemia and a 120-minute full reperfusion to measure infarct size after ischemia/reperfusion (Set 4 in Figure 4). In the first set of experiments, the hearts were perfused with Tat peptide conjugated to V1-1 (V1-1), Tat peptide conjugated to RACK (RACK), Tat peptide alone, or vehicle during the first 10 or 20 minutes of reperfusion at the indicated concentration (
500 nmol/L; Set 1 in Figure 1). In the second set of experiments, the hearts were perfused with V1-1, RACK, scrambled RACK, or vehicle for 10 minutes with or without a peptide-washout period of 10 minutes before ischemia (Set 2 in Figure 2). In the third and fourth sets of experiments, the hearts were perfused with either V1-1 (500 nmol/L), RACK (500 nmol/L), Tat peptide (500 nmol/L), or vehicle for 10 minutes before ischemia and for 20 minutes during reperfusion or with RACK (500 nmol/L) for 10 minutes before ischemia and then with V1-1 (500 nmol/L) for 20 minutes during reperfusion (Sets 3 and 4 in Figures 3 and 4 ).
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P<0.002 vs 50 nmol/L of Tat, ¶P<0.002 vs 500 nmol/L of Tat, 
P<0.002 vs
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Histomorphometry
At the end of the reperfusion period, hearts were sliced into 1-mm-thick transverse sections and incubated in triphenyltetrazolium chloride solution (1% in phosphate buffer, pH 7.4) at 37°C for 15 minutes as reported previously.15 Infarct size was expressed as a percentage of the risk zone (equivalent to total LV muscle mass).
Transgenic Mice
Details of transgenic mice (FVB/N background) expressing the -PKC inhibitor (V1) and -PKC activator (RACK) under control of the 
-myosin heavy chain promoter have been described previously.11,16,17 The V1 transgene expression was under the attenuated promoter TRE-2,16 and the morphometric or hemodynamic characteristics of these transgenic mice have been described previously.16 Compound transgenic mice expressing V1 and RACK (R/V1) were obtained by breeding heterozygous V1 and heterozygous RACK mice. Hemodynamic and morphometric parameters in these transgenic mice as measured by echocardiographic measurements in vivo were not statistically different from those measured in nontransgenic (NTG) control animals (n=12 to 26). Similarly, there was no statistical difference between the R/V1 mice and the NTG mice when ex vivo hemodynamic measurements were used before introduction of ischemia and reperfusion. (These measurements included heart rate, LV systolic and diastolic pressures, and their derivatives.) Transgenes, alone and in combination, were identified by genomic Southern analysis of tail-clip DNA. Isolated hearts from 12- to 20-week-old mice were perfused on Langendorff apparatus for 20 minutes for equilibration, followed by a 40-minute period of no-flow ischemia and a 20-minute reperfusion period as reported previously.10,11,16 Contractile function was measured as the peak rate of increase of pressure (+dP/dt). CPK was assayed in the perfusate during reperfusion.
Statistical Analysis
Data are expressed as mean ± SEM. The main effects of the peptide were tested by 2-factor ANOVA for repeated measures in functional recovery (Figures 1 to 3
, A through C, and Figure 5A), and differences of infarct size, total CPK release, and LV developed pressure (LVDP) at individual time points between the groups were assessed by 1-factor ANOVA with Bonferroni’s multiple-comparison test.
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Results |
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-PKC Inhibitor Mediates Protection From Reperfusion InjuryWhen the
-PKC inhibitor V1-1 was administered immediately after ischemia only during the reperfusion period, rat hearts exhibited improved LVDP, EDP, and coronary vascular resistance (CVR) and decreased CPK release (Figure 1). Even at 1 minute after the administration of this peptide, there was a significantly improved LVDP (33.9±6.4 mm Hg in V1-1, 14.9±3.1 mm Hg in Tat; P<0.006) and CVR (9.6±0.8 in V1-1, 13.3±0.6 in Tat; P<0.006; Figure 1). The effect of V1-1 was specific, because the Tat carrier peptide did not have cardioprotective effects against reperfusion injury. In contrast to V1-1, RACK did not improve LVDP, EDP, or CVR and did not decrease CPK release when administered only during reperfusion (Figure 1). V1-1 conferred cardioprotection at a wide range of concentrations, from 500 pmol/L to 500 nmol/L, as measured by CPK release (Figure 1F). Thus, V1-1 but not RACK protected cardiomyocytes and prevented the decrease of LV function when delivered during reperfusion.
Ischemic Preconditioning–Like Effects of -PKC–Selective Activator
When RACK and V1-1 were administered before ischemia and were not removed by washing, these peptides significantly improved LVDP and CVR and decreased CPK release during reperfusion (Figure 2). To test whether either of these peptides mimicked preconditioning, we followed peptide administration with a 10-minute washout before ischemia. V1-1 exhibited no cardioprotection when administered before ischemia followed by a 10-minute washout period preceding the ischemia (Figure 2, E and F). In contrast, RACK treatment significantly protected cardiomyocytes even when the peptide was washed out before ischemia (Figure 2, E and F). As expected, the inactive form of RACK, scrambled RACK, did not have a significant cardioprotective effect (Figure 2). Thus, RACK but not V1-1 had an ischemic preconditioning–like effect; RACK administration provides cardioprotection when given before the ischemic event.
Enhanced Cardioprotection From Ischemia/Reperfusion Damage by Combined Treatment With the -PKC Activator and the -PKC Inhibitor
Because V1-1 was cardioprotective when delivered during reperfusion and RACK was cardioprotective when delivered before the ischemic period, we hypothesized that combined treatment with V1-1 and RACK would yield an additive benefit. When we administered RACK before ischemia and V1-1 during reperfusion, there was a greater functional recovery after ischemia/reperfusion compared with treatment with each peptide alone (Figure 3). Although treatment with either peptide limits the infarct size to 50% to 60% of the untreated control or Tat carrier peptide, treatment with both peptides reduced infarct size by an additional 20% (Figure 4). Thus, -PKC inhibitor and -PKC activator mediate cardioprotection by regulating ischemia/reperfusion damage at distinct time points of the process, and their therapeutic effect is additive.
Cardioprotective Effects of -PKC Inhibitor and -PKC Activator in Transgenic Mouse Hearts
Treatments with the -PKC inhibitor and -PKC activator improved both cardiac function and CVR in isolated perfused rat hearts, suggesting effects of the PKC regulators on both endothelial cells and cardiac myocyte cells. To determine whether cardioprotection can occur when the PKC regulators are present only in cardiac myocytes, we used transgenic mice that express PKC regulators selectively only in these cells. To this end, we constructed transgenic mice expressing V1, RACK, or both V1 and RACK (R/V1 compound mice) using the cardiac myocyte–specific -myosin heavy chain promoter.11,16,17 The transgenic mice had a low number of copies of the PKC regulators and exhibited normal morphometric and hemodynamic characteristics as measured by echocardiography in vivo and by studies of isolated perfused hearts ex vivo (data not shown). After subjecting these mice to ischemia/reperfusion, we found that the expression of V1, RACK, or R/V1 in the myocytes improved cardiac function significantly during reperfusion compared with NTG (Figure 5A). Although functional recovery of the 3 groups of transgenic mice was similar, when muscle damage as measured by CPK release was assessed, concomitant inhibition of -PKC and activation of -PKC in cardiomyocytes provided additive protection from ischemia and reperfusion compared with regulating each PKC alone (Figure 5B). In addition, because the expression of the PKC regulators was restricted to the cardiac myocytes, we can conclude that substantial cardiac protection from ischemia and reperfusion damage can be obtained by selectively regulating PKC isozymes in the myocytes only.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In this study, we used rationally designed selective PKC isozyme regulator peptides conjugated to Tat carrier peptide to determine the role of PKC isozymes in ischemia/reperfusion injury. We found that (1) the
-PKC inhibitor has cardioprotective effects against reperfusion injury, (2) the -PKC activator has ischemic preconditioning–like effects, (3) combined treatment with an -PKC activator before ischemia and -PKC inhibitor at the onset of reperfusion induces a greater cardioprotective effect against ischemia/reperfusion injury than the treatment with each peptide alone, and (4) the selective expression of -PKC inhibitor or -PKC activator only in cardiomyocytes protects cardiomyocytes directly against ischemia/reperfusion injury and the coexpression of both these PKC regulators in cardiomyocytes yields an additive benefit as determined by reduction in muscle damage. The preconditioning-like effect induced by RACK is predicted from previous studies.10–12 However, the finding that effective cardioprotection is achieved when V1-1 is delivered only during reperfusion suggests that, as predicted by Bolli,18 a substantial amount of cardiac damage occurs during reperfusion only. Our data here indicate that -PKC activity mediates reperfusion damage at least in part because inhibition of -PKC immediately at the onset of reperfusion can decrease cardiac damage by >50%.
The Role of -PKC in Reperfusion Injury
The molecular basis of -PKC in reperfusion injury is not known. Recently, the involvement of apoptosis in ischemia/reperfusion injury has been demonstrated in a number of species, including humans.19–21 In experimental ischemia/reperfusion models, although apoptosis may be initiated during ischemia, the number of apoptotic cells is increased during reperfusion, and apoptosis may actually be accelerated by the process of reperfusion.20,21 These observations suggest that the apoptotic component contributes particularly to the death of the myocardium during reperfusion. In vitro overexpression of -PKC is associated with inhibition of proliferation, cell cycle arrest, enhanced differentiation, and/or accelerated apoptosis in several nonheart cell lines.22 Thus, the cardioprotective effects of a -PKC inhibitor may result from preventing cell death by modifying cellular signaling involving this isozyme in apoptosis. Scarabelli et al23 reported that in the very early stages of reperfusion, apoptosis is first seen in the endothelial cells of small coronary vessels and then spreads in a radial fashion to the surrounding cardiac myocytes in isolated perfused rat hearts. We observed cardioprotection when the peptide was applied through the coronary artery; thus, the peptide can act on all heart cells, including endothelial cells. Cardioprotection was also observed in mice expressing the -PKC inhibitor in their myocytes (previous study16 and this study). Therefore, it appears possible that cardioprotection by V1-1 is a result of its effects both on cardiomyocytes and on endothelial cells.
How -PKC is activated during reperfusion is also unclear. Recent data on the role of endothelin A in this process provide a possible explanation. The level of endothelin increases in plasma and myocardial tissue during ischemia/reperfusion,24 resulting in direct vascular and myocardial damage.25 Moreover, treatment with an endothelin A antagonist significantly limits the progressive decrease in postischemic microvascular reflow and decreases the infarct size when given at the time of reperfusion.26 Endothelin-1 induces the translocation of -PKC from soluble to particulate fractions in neonatal rat ventricular cardiomyocytes.27 Thus, if translocation of -PKC is induced by endothelin during reperfusion, -PKC may mediate the endothelin-induced damage.
PKC and Ischemia/Reperfusion Injury
The hypothesis that the translocation of PKC plays a key role in ischemic preconditioning is supported by many studies. However, which isozyme mediates this effect is still controversial. Ping et al28 reported that -PKC and 
-PKC translocate selectively to the particulate fraction in rabbit myocardium after several cycles of brief coronary occlusion/reperfusion. Translocation of -PKC and -PKC is induced by ischemic preconditioning in neonatal rat cultured cardiomyocytes2 and in rat whole hearts.29 These data correlated translocation of different PKC isozymes with preconditioning and suggested that the role of PKC isozymes may differ in various animal species. However, we found that the -PKC activator, RACK, alone conferred cardioprotective effects against ischemia/reperfusion in rats and mice10–12 and that the -PKC inhibitor inhibited cardiac protective effects induced by ischemic preconditioning in mice, rats, and rabbits.3,10,30 In addition, Ping and coworkers demonstrated cardioprotection from ischemia/reperfusion of transgenic mice overexpressing -PKC.31 Moreover, another report demonstrated loss of ischemic preconditioning in -PKC–knockout mice.32 Our findings that RACK can be washed away before the ischemic event and still confer cardiac protection from ischemia are in agreement with -PKC as a trigger for the preconditioning effect.2 In contrast to our data, some earlier studies suggested that -PKC plays a cardioprotective role in ischemic preconditioning.33,34 Kawamura et al33 showed that ischemic preconditioning under low-Ca2+ perfusion induced translocation of -PKC but not -PKC, which was correlated with improved cardiac function after ischemia/reperfusion in isolated rat heart. They also showed that although the treatment of bisindolylmaleimide (PKC inhibitor) before preconditioning inhibited translocation of -PKC but not -PKC after preconditioning, this inhibitor did not prevent the cardioprotection of preconditioning.33 These data indicate that the effects of preconditioning are mediated by either -PKC or -PKC. Zhao et al34 showed that the protective effect of the constitutively active -PKC can be transmitted to cocultured nontransfected myocytes. They suggest that -PKC activation may be induced by -PKC–mediated increased release of adenosine and activation of PKC via the A1 adenosine receptors.35
The present study showed that pretreatment with an -PKC activator followed by washout before ischemia/reperfusion conferred cardioprotection. We also showed that concomitant treatment with both PKC regulators provides superior protection compared with each regulator alone. Because here, we used isozyme-selective pharmacological tools and regulated the function of the endogenous enzyme, the simplest explanation of our data is that -PKC activation before ischemia protects the heart by mimicking preconditioning, whereas inhibition of -PKC during reperfusion protects the heart from reperfusion damage.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (HL-52141 to Dr Mochly-Rosen and HL-52318 to Drs Dorn and Mochly-Rosen) and from the Yokoyama Foundation for Clinical Pharmacology to Dr Inagaki.
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Footnotes |
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Dr Mochly-Rosen is a founder of a pharmaceutical company (Anchora) that has licensed the rights to commercialize peptide inhibitors and activators of PKC (developed in the author’s laboratory) as human therapeutics.
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References |
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F. Ikeno, K. Inagaki, M. Rezaee, and D. Mochly-Rosen Impaired perfusion after myocardial infarction is due to reperfusion-induced {delta}PKC-mediated myocardial damage Cardiovasc Res, March 1, 2007; 73(4): 699 - 709. [Abstract] [Full Text] [PDF]
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S. V. Pierre, C. Yang, Z. Yuan, J. Seminerio, C. Mouas, K. D. Garlid, P. Dos-Santos, and Z. Xie Ouabain triggers preconditioning through activation of the Na+,K+-ATPase signaling cascade in rat hearts Cardiovasc Res, February 1, 2007; 73(3): 488 - 496. [Abstract] [Full Text] [PDF]
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V. Kheifets, R. Bright, K. Inagaki, D. Schechtman, and D. Mochly-Rosen Protein Kinase C {delta} ({delta}PKC)-Annexin V Interaction: A REQUIRED STEP IN {delta}PKC TRANSLOCATION AND FUNCTION J. Biol. Chem., August 11, 2006; 281(32): 23218 - 23226. [Abstract] [Full Text] [PDF]
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R. Mangat, T. Singal, N. S. Dhalla, and P. S. Tappia Inhibition of phospholipase C-{gamma}1 augments the decrease in cardiomyocyte viability by H2O2 Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H854 - H860. [Abstract] [Full Text] [PDF]
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Z.-Q. Zhao and J. Vinten-Johansen Postconditioning: Reduction of reperfusion-induced injury Cardiovasc Res, May 1, 2006; 70(2): 200 - 211. [Abstract] [Full Text] [PDF]
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K. Inagaki, E. Churchill, and D. Mochly-Rosen Epsilon protein kinase C as a potential therapeutic target for the ischemic heart Cardiovasc Res, May 1, 2006; 70(2): 222 - 230. [Abstract] [Full Text] [PDF]
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D. J. Hausenloy and D. M. Yellon Survival kinases in ischemic preconditioning and postconditioning Cardiovasc Res, May 1, 2006; 70(2): 240 - 253. [Abstract] [Full Text] [PDF]
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S. Philipp, X.-M. Yang, L. Cui, A. M. Davis, J. M. Downey, and M. V. Cohen Postconditioning protects rabbit hearts through a protein kinase C-adenosine A2b receptor cascade Cardiovasc Res, May 1, 2006; 70(2): 308 - 314. [Abstract] [Full Text] [PDF]
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A. J. Zatta, H. Kin, G. Lee, N. Wang, R. Jiang, R. Lust, J. G. Reeves, J. Mykytenko, R. A. Guyton, Z.-Q. Zhao, et al. Infarct-sparing effect of myocardial postconditioning is dependent on protein kinase C signalling Cardiovasc Res, May 1, 2006; 70(2): 315 - 324. [Abstract] [Full Text] [PDF]
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J. C. Kostyak, J. C. Hunter, and D. H. Korzick Acute PKC{delta} inhibition limits ischaemia-reperfusion injury in the aged rat heart: Role of GSK-3{beta} Cardiovasc Res, May 1, 2006; 70(2): 325 - 334. [Abstract] [Full Text] [PDF]
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 A. D.T. Costa, K. D. Garlid, I. C. West, T. M. Lincoln, J. M. Downey, M. V. Cohen, and S. D. Critz Protein Kinase G Transmits the Cardioprotective Signal From Cytosol to Mitochondria Circ. Res., August 19, 2005; 97(4): 329 - 336. [Abstract] [Full Text] [PDF]
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 D. Omiyi, R. J. Brue, P. Taormina II, M. Harvey, N. Atkinson, and L. H. Young Protein Kinase C {beta}II Peptide Inhibitor Exerts Cardioprotective Effects in Rat Cardiac Ischemia/Reperfusion Injury J. Pharmacol. Exp. Ther., August 1, 2005; 314(2): 542 - 551. [Abstract] [Full Text] [PDF]
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A. Phillipson, E. E. Peterman, P. Taormina Jr., M. Harvey, R. J. Brue, N. Atkinson, D. Omiyi, U. Chukwu, and L. H. Young Protein kinase C-{zeta} inhibition exerts cardioprotective effects in ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, August 1, 2005; 289(2): H898 - H907. [Abstract] [Full Text] [PDF]
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E. N. Churchill, C. L. Murriel, C.-H. Chen, D. Mochly-Rosen, and L. I. Szweda Reperfusion-Induced Translocation of {delta}PKC to Cardiac Mitochondria Prevents Pyruvate Dehydrogenase Reactivation Circ. Res., July 8, 2005; 97(1): 78 - 85. [Abstract] [Full Text] [PDF]
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 L. A. Turner, K. Fujimoto, A. Suzuki, A. Stadnicka, Z. J. Bosnjak, and W.-M. Kwok The Interaction of Isoflurane and Protein Kinase C-Activators on Sarcolemmal KATP Channels Anesth. Analg., June 1, 2005; 100(6): 1680 - 1686. [Abstract] [Full Text] [PDF]
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W. Wang, L. Jia, T. Wang, W. Sun, S. Wu, and X. Wang Endogenous Calcitonin Gene-related Peptide Protects Human Alveolar Epithelial Cells through Protein Kinase C{epsilon} and Heat Shock Protein J. Biol. Chem., May 27, 2005; 280(21): 20325 - 20330. [Abstract] [Full Text] [PDF]
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 S. Bae, R. D. Gilbert, C. A. Ducsay, and L. Zhang Prenatal cocaine exposure increases heart susceptibility to ischaemia-reperfusion injury in adult male but not female rats J. Physiol., May 15, 2005; 565(1): 149 - 158. [Abstract] [Full Text] [PDF]
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 M. Tanaka, F. Gunawan, R. D. Terry, K. Inagaki, A. D. Caffarelli, G. Hoyt, P. S. Tsao, D. Mochly-Rosen, and R. C. Robbins Inhibition of heart transplant injury and graft coronary artery disease after prolonged organ ischemia by selective protein kinase C regulators J. Thorac. Cardiovasc. Surg., May 1, 2005; 129(5): 1160 - 1167. [Abstract] [Full Text] [PDF]
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 K. Inagaki, R. Begley, F. Ikeno, and D. Mochly-Rosen Cardioprotection by {epsilon}-Protein Kinase C Activation From Ischemia: Continuous Delivery and Antiarrhythmic Effect of an {epsilon}-Protein Kinase C-Activating Peptide Circulation, January 4, 2005; 111(1): 44 - 50. [Abstract] [Full Text] [PDF]
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 M. E. Jung, M. B. Gatch, and J. W. Simpkins Estrogen Neuroprotection Against the Neurotoxic Effects of Ethanol Withdrawal: Potential Mechanisms Experimental Biology and Medicine, January 1, 2005; 230(1): 8 - 22. [Abstract] [Full Text] [PDF]
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G. Asemu, N. S. Dhalla, and P. S. Tappia Inhibition of PLC improves postischemic recovery in isolated rat heart Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2598 - H2605. [Abstract] [Full Text] [PDF]
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C. L. Murriel, E. Churchill, K. Inagaki, L. I. Szweda, and D. Mochly-Rosen Protein Kinase C{delta} Activation Induces Apoptosis in Response to Cardiac Ischemia and Reperfusion Damage: A MECHANISM INVOLVING BAD AND THE MITOCHONDRIA J. Biol. Chem., November 12, 2004; 279(46): 47985 - 47991. [Abstract] [Full Text] [PDF]
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 D. H. Korzick, J. C. Hunter, M. K. McDowell, M. D. Delp, M. M. Tickerhoof, and L. D. Carson Chronic Exercise Improves Myocardial Inotropic Reserve Capacity Through {alpha}1-Adrenergic and Protein Kinase C-Dependent Effects in Senescent Rats J. Gerontol. A Biol. Sci. Med. Sci., November 1, 2004; 59(11): 1089 - 1098. [Abstract] [Full Text] [PDF]
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 A. Hassouna, B. M. Matata, and M. Galinanes PKC-{epsilon} is upstream and PKC-{alpha} is downstream of mitoKATP channels in the signal transduction pathway of ischemic preconditioning of human myocardium Am J Physiol Cell Physiol, November 1, 2004; 287(5): C1418 - C1425. [Abstract] [Full Text] [PDF]
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M. Tanaka, R. D. Terry, G. K. Mokhtari, K. Inagaki, T. Koyanagi, T. Kofidis, D. Mochly-Rosen, and R. C. Robbins Suppression of Graft Coronary Artery Disease by a Brief Treatment With a Selective {epsilon}PKC Activator and a {delta}PKC Inhibitor in Murine Cardiac Allografts Circulation, September 14, 2004; 110(11_suppl_1): II-194 - II-199. [Abstract] [Full Text] [PDF]
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M. Mayr, Y.-L. Chung, U. Mayr, E. McGregor, H. Troy, G. Baier, M. Leitges, M. J. Dunn, J. R. Griffiths, and Q. Xu Loss of PKC-{delta} alters cardiac metabolism Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H937 - H945. [Abstract] [Full Text] [PDF]
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M. Mayr, B. Metzler, Y.-L. Chung, E. McGregor, U. Mayr, H. Troy, Y. Hu, M. Leitges, O. Pachinger, J. R. Griffiths, et al. Ischemic preconditioning exaggerates cardiac damage in PKC-{delta} null mice Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H946 - H956. [Abstract] [Full Text] [PDF]
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 A. K. Salahudeen Cold ischemic injury of transplanted kidneys: new insights from experimental studies Am J Physiol Renal Physiol, August 1, 2004; 287(2): F181 - F187. [Abstract] [Full Text] [PDF]
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J. A. Shumilla, S. M. Sweitzer, E. I Eger II, M. J. Laster, and J. J. Kendig Inhibition of Spinal Protein Kinase C-{epsilon} or -{gamma} Isozymes Does Not Affect Halothane Minimum Alveolar Anesthetic Concentration in Rats Anesth. Analg., July 1, 2004; 99(1): 82 - 84. [Abstract] [Full Text] [PDF]
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S. C Armstrong Protein kinase activation and myocardial ischemia/reperfusion injury Cardiovasc Res, February 15, 2004; 61(3): 427 - 436. [Abstract] [Full Text] [PDF]
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K. Inagaki, L. Chen, F. Ikeno, F. H. Lee, K.-i. Imahashi, D. M. Bouley, M. Rezaee, P. G. Yock, E. Murphy, and D. Mochly-Rosen Inhibition of {delta}-Protein Kinase C Protects Against Reperfusion Injury of the Ischemic Heart In Vivo Circulation, November 11, 2003; 108(19): 2304 - 2307. [Abstract] [Full Text] [PDF]
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