Published online before print December 20, 2004, doi: 10.1161/01.CIR.0000151614.22282.F1
(Circulation. 2005;111:44-50.)
© 2005 American Heart Association, Inc.
Coronary Heart Disease |
Cardioprotection by 
-Protein Kinase C Activation From Ischemia
Continuous Delivery and Antiarrhythmic Effect of an 
-Protein Kinase C-Activating Peptide
From the Department of Molecular Pharmacology, Stanford University School of Medicine (K.I., R.B., D.M.-R.), and Department of Cardiovascular Medicine, Stanford University (F.I.), Stanford, Calif.
Correspondence to Daria Mochly-Rosen, Department of Molecular Pharmacology, 269 Campus Dr, 3145 CCSR, Stanford University School of Medicine, Stanford CA 94305-5174. E-mail mochly{at}stanford.edu
Received July 24, 2004; revision received September 10, 2004; accepted September 30, 2004.
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Abstract |
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Background— We previously showed that a selective activator peptide of
-protein kinase C (PKC), 
RACK, conferred cardioprotection against ischemia-reperfusion when delivered ex vivo before the ischemic event. Here, we tested whether in vivo continuous systemic delivery of RACK confers sustained cardioprotection against ischemia-reperfusion in isolated mouse hearts and whether RACK treatment reduces infarct size or lethal arrhythmias in porcine hearts in vivo.
Methods and Results— After RACK was systemically administered in mice either acutely or continuously, hearts were subjected to ischemia-reperfusion in an isolated perfused model. Whereas RACK-induced cardioprotection lasted 1 hour after a single intraperitoneal injection, continuous treatment with RACK induced a sustained preconditioned state during the 10 days of delivery. There was no desensitization to the therapeutic effect, no downregulation of PKC, and no adverse effects after sustained RACK delivery. Porcine hearts were subjected to ischemia-reperfusion in vivo, and RACK was administered by intracoronary injection during the first 10 minutes of ischemia. RACK treatment reduced infarct size (34±2% versus 14±1%, control versus RACK) and resulted in fewer cases of ventricular fibrillation during ischemia-reperfusion (87.5% versus 50%, control versus RACK).
Conclusions— The PKC activator RACK induced cardioprotection both in vivo and ex vivo, reduced the incidence of lethal arrhythmia during ischemia-reperfusion, and did not cause desensitization or downregulation of PKC after sustained delivery. Thus, RACK may be useful for patients with ischemic heart disease. In addition, the RACK peptide should be a useful pharmacological agent for animal studies in which systemic and sustained modulation of PKC in vivo is needed.
Key Words: arrhythmia • ischemia • ischemic preconditioning • peptides • protein kinase C
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The hypothesis that the translocation of
-protein kinase C (PKC) plays a key role in ischemic preconditioning is supported by many studies. Translocation of PKC is induced by ischemic preconditioning in neonatal rat cultured cardiomyocytes and in the rat and rabbit myocardium.1–3 A selective PKC antagonist inhibits cardiac protective effects induced by ischemic preconditioning in mice, rats, and rabbits.1,4,5 Conversely, a selective PKC activator peptide, RACK, confers cardioprotection from ischemia-reperfusion injury in various cell culture and isolated perfused heart models.4,6–9 Importantly, RACK acts as a cardiac preconditioning-mimicking agent; it is cardioprotective when delivered before the ischemic event. However, previous reports have not demonstrated the cardioprotective effects of a selective PKC activator after in vivo delivery in an in vivo ischemic heart model. Treatment of patients at high risk for ischemic episodes could include long-term administration of a cardiac preconditioning-mimicking agent. However, desensitization to the agent or to the effect may further limit the use of such an agent. Indeed, long-term delivery of cardioprotective agents like adenosine causes desensitization to the protective effect.10,11
Here, we studied whether in vivo continuous delivery of the preconditioning-mimicking agent, RACK, confers a sustained therapeutic effect without desensitization and whether RACK treatment during ischemia induces cardioprotection and reduces ventricular tachyarrhythmias in a porcine model in vivo.
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Methods |
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Peptide Synthesis
The
PKC selective agonist RACK (-receptor for activated C-kinase) derived from PKC (amino acids 85 to 92 [HDAPIGYD]) and TAT47–57 derived from the transactivator protein (TAT, amino acids 47 to 57 [YGRKKRRQRRR]) were synthesized as previously described,4,12 and RACK was conjugated to TAT47–57 peptide by an S-S conjugation through free cysteines at the C terminus of each peptide.9,12 TAT47–57 carrier peptide was used as a control peptide to study the effect of RACK on ischemia-reperfusion-induced damage and on translocation of PKC.
Isolated Perfused Mouse Heart Model of Myocardial Infarction Ex Vivo
Animal protocols were approved by the Stanford University Institutional Animal Care and Use Committee. TAT47–57-RACK (RACK) or TAT47–57 (control) was administered either by an intraperitoneal injection (in 200 µL of saline) or continuously via the subcutaneous implantation of an osmotic pump (10 mmol/L peptide in saline; 0.5 µL/h, 5.0 nmol/h) in FVB/N mice (15 to 20 g).
Hearts were rapidly excised after mice were injected with heparin (4000 U/kg IP) and anesthetized with sodium pentobarbital (200 mg/kg IP); then, hearts were perfused with an oxygenated Krebs-Henseleit solution at 37°C in a Langendorff coronary perfusion system. Coronary flow rate was kept at 2.5 mL/min. Hearts were subjected to a 30-minute global ischemia, followed by a 120-minute reperfusion. Coronary effluent during reperfusion was collected to determine myocytolysis by measuring creatine phosphokinase (CPK) release with a CPK assay kit (Sigma). At the end of the reperfusion period, hearts were sliced into 1-mm-thick transverse sections and incubated in triphenyltetrazolium chloride solution (TTC) to determine infarct size as previously described.8 Infarcted tissue (white) and live tissue (red) were visualized in hearts subjected to ischemia-reperfusion. Infarct size was calculated by detection of specific colors in digital images with the Adobe Photoshop 7.0 graphical analyzing software.
Organs collected 10 days after long-term administration of RACK were harvested and fixed in 4% phosphate buffered formalin, embedded in paraffin, sliced, and stained with hematoxylin and eosin. All tissues were evaluated by a pathologist blinded to these experimental conditions.
Porcine Heart Model of Myocardial Infarction In Vivo
We applied a balloon catheter into the middle left anterior descending artery of female Yorkshire juvenile pigs (35 to 40 kg) under anesthesia (1% isoflurane) and inflated the balloon to produce total occlusion for 30 minutes as previously described.13 The guidewire was removed, and TAT47–57 (control; n=16) or TAT47–57-RACK (RACK; n=12) was infused via the lumen of the balloon catheter for the first 10 minutes of ischemia (2.5 µg/kg in 10 mL saline, 1 mL/min). Amiodarone (150 mg) was administered to all animals just before ischemia. ECG was monitored from the induction of anesthesia until 30 minutes after reperfusion. When ventricular fibrillation (VF) or sustained ventricular tachycardia (VT) occurred, DC shocks were performed to convert to normal sinus rhythm. Left ventriculograms and echocardiography were performed to determine cardiac function. Left ventriculograms (40° left anterior oblique projection, 30 frames per second) were obtained before ischemia and 30 minutes and 24 hours after reperfusion. Echocardiography (Sonos 5500, Hewlett-Packard) was obtained before ischemia and 5 days after reperfusion (n=16 for control, n=12 for RACK). Ejection fraction and hypokinetic area in the left ventriculograms were calculated with the Plus Plus, Sanders Data System software. Hearts were harvested 5 days after ischemia. Double staining with Evan’s Blue dye and TTC marked areas at risk for ischemia and infarcted areas, respectively.13 Troponin T levels in blood, as an indicator of cardiac cytolysis, were also determined 24 hours after reperfusion. No pigs were excluded from this study, and none died during or after the procedure. Wedge biopsies of liver, spleen, lung, and kidney were fixed in 10% buffered neutral formalin and embedded in paraffin, and 8-µm-thick sections were stained with hematoxylin and eosin. All tissues were evaluated by a pathologist blinded to the experimental conditions.
Quantification and Stability of RACK in Blood Plasma and Saline
After RACK (160 nmol in 200 µL saline) was injected intravenously into the mouse tail vein, blood was drawn from the abdominal vein 1, 15, 30, 60, and 120 minutes after administration. A Quantum triple quadrupolar mass spectrometer (MS) (ThermoFinnigan) was used to quantify the plasma concentrations of RACK (Figure 2D). We measured the concentration of RACK in saline over time when 10 mmol/L RACK was placed in an Eppendorf tube at 37°C or after 2 weeks in a subcutaneously implanted pump (Figure 3A and 3B). The concentration of RACK in saline was determined by HPLC-ESI MS with a surveyor HPLC and an LCQ ion trap MS (ThermoFinnigan).
Levels and Translocation of PKC Isozymes
Whole-tissue lysate and soluble and particulate fractions from mouse hearts were prepared as previously described.14 PKC and 
PKC levels and translocation were determined by SDS-PAGE, followed by Western blot analysis with anti-PKC and anti-PKC antibodies (Santa Cruz). PKC served as control for both protein levels and translocation.
Statistical Analysis
Data are expressed as mean±SEM. Unpaired t test for differences between 2 groups, 1-factor ANOVA with Fisher’s test for differences among >2 groups, and 
2 test or Fisher’s exact test for categorical data were used to assess significance (P<0.05).
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Results |
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Intraperitoneal administration of the
RACK peptide (conjugated to TAT47–57 as an intracellular carrier) renders the heart resistant to ischemic damage.
To determine whether RACK confers cardioprotection when administered systemically, we injected RACK intraperitoneally into mice. Intraperitoneal delivery of 0.5 or 20 but not 0.05 nmol RACK reduced cardiac damage from a subsequent ischemia-reperfusion insult by 
70%, as indicated by decreased infarct size and decreased release of the intracellular cardiac enzyme CPK (Figure 1). The effect of RACK was specific; injection of the control carrier peptide (TAT47–57) resulted in no cardioprotective effects against ischemia-reperfusion injury.
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P<0.05 vs control peptide; ¶P<0.05 vs 0.05 nmol
Duration of Cardioprotection From a Single Administration of RACK
We examined the duration of cardioprotection conferred by a single 20-nmol intraperitoneal injection of RACK. Intraperitoneal administration of RACK rendered the hearts resistant to subsequent ex vivo ischemia-reperfusion-induced injury for up to 60 minutes only (Figure 2A and 2B); cardioprotection was not observed 90 minutes after RACK injection.
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To determine whether the cardioprotection was dependent on enhanced PKC activation, the duration of increased PKC translocation in the heart after a single administration of RACK was also determined. Intraperitoneal delivery of RACK resulted in translocation of PKC in the myocardium 10 and 60 minutes after administration. However, at 90 minutes, when cardioprotection was lost, translocation of PKC returned to baseline (Figure 2C). These data indicate that cardioprotection induced by a single intraperitoneal administration of RACK was transient because peptide-induced PKC translocation also was transient.
RACK (160 nmol per mouse) was also delivered intravenously. The theoretical initial concentration in the blood was 80 µmol/L. After 1 minute, the blood plasma concentration of RACK was 1.1 nmol/L (Figure 2D), indicating a 99.99% reduction in peptide concentration. The concentration of RACK in the plasma rapidly decreased further until no peptide was detected by 120 minutes after delivery. The initial decrease in concentration was likely due to degradation and to uptake of RACK by cells.
Continuous Delivery of RACK Confers Sustained Cardioprotection
To obtain sustained treatment, we first examined the stability of the RACK disulfide conjugate in vitro by incubating it in saline at 37°C, and using HPLC-ESI MS, we found that the half-life of RACK was 2 weeks at 37°C in vitro and in a subcutaneously implanted osmotic pump in vivo (Figure 3A and 3B). We then determined whether continuous delivery of RACK could confer sustained cardioprotection. We found that sustained pump delivery of RACK for 1 hour reduced infarct size and CPK release compared with delivery of TAT47–57 alone (control) (Figure 3C and 3D, left 2 bars). An important point is that sustained delivery of RACK for 10 days also conferred cardioprotection from ischemia equal to that obtained after 1 hour of sustained delivery (Figure 3C and 3D, right 2 bars).
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on left) are provided on same graph for comparison but were not included in the statistical analysis.
Next, mice were implanted with pumps containing a range of concentrations of RACK to investigate the dose dependence of peptide-induced cardioprotection. There was a significant correlation between the concentration of RACK in the pump and infarct size (n=13, r=–0.588, P<0.05; Figure 3E). There was no change in body or heart weight, blood pressure, heart rate, or cardiac contractility by echocardiography or pathological examination in mice treated with RACK for 10 days compared with mice treated with control carrier peptide (Table 1). Thus, sustained systemic delivery of RACK induced a dose-dependent cardioprotection.
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Chronic PKC Activation Does Not Induce Desensitization
To determine whether sustained activation of PKC by RACK causes downregulation of PKC or desensitization in the translocation of PKC, we measured protein level and translocation of PKC in hearts after continuous delivery of RACK. Neither the overall protein levels of PKC nor PKC translocation (Figure 4A and 4B) was decreased after continuous administration of RACK for 10 days compared with treatment for 1 hour. Moreover, protein levels and translocation of PKC were unaffected by the PKC-selective agonist (Figure 4C and 4D). Therefore, continuous activation of PKC in the heart by sustained delivery of the PKC-selective activator RACK resulted in sustained and selective activation of PKC without desensitization or downregulation of the enzyme.
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In vivo RACK delivery during ischemia conferred cardioprotection and reduced ventricular tachyarrhythmias in a porcine model.
We studied the cardioprotective effect of RACK delivery during ischemia in an in vivo model. RACK delivery resulted in no acute changes in blood pressure or heart rate in pigs (Table 2). RACK delivery reduced infarct size (14±1% versus 34±2% in RACK versus control; P<0.05; Figure 5A and 5B) and improved cardiac function (Figure 5C) and troponin T release (0.6±0.12 versus 1.34±0.7 ng/mL in RACK versus control; P<0.05). RACK delivery resulted in fewer cases of VF and VT (50% versus 87.5% in RACK versus control; P<0.05) and fewer VF and VT events per animal (0.6±0.2 versus 1.8±0.3; P<0.05) during ischemia-reperfusion (Table 3). Thus, PKC activation during ischemia reduced infarct size and suppressed incidence of VF and VT during ischemia.
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As in the mouse study, there was no evidence for adverse effects after RACK delivery in pigs on physiological and pathological examination.
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Discussion |
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In this study, we found that continuous administration of
RACK conjugated to TAT47–57 to obtain intracellular delivery conferred a sustained therapeutic effect for the duration of its delivery, as evidenced by continuous cardioprotection from ischemia-reperfusion-induced injury. Short bouts of ischemia (preconditioning) and adenosine agonists are also cardioprotective against ischemia-reperfusion if administered before an ischemic event.7,15 However, cardioprotection is markedly attenuated or diminished when the ischemic insult is preceded by many repeated short bouts of ischemia.16 Similarly, sustained delivery of an adenosine agonist for 72 hours also results in complete desensitization of the myocardium to the protective effect resulting from desensitization and downregulation of adenosine receptor.10,11 In contrast, our findings that sustained PKC activation induced by continuous delivery of RACK did not lead to downregulation of the enzyme or desensitization to the cardioprotective effect demonstrate a unique feature of RACK as a preconditioning-mimicking agent. In contrast, sustained strong activation of PKC by the tumor promoter phorbol ester17–20 or hormones21 causes desensitization or downregulation of PKC as a result of an increased rate of proteolysis. The dogma on PKC downregulation relies on studies using large amounts of PMA (100 nmol/L to 16 µmol/L) that cause 100% activation of PKC.19,22,23 However, prolonged treatment with lower amounts of PMA (1 to 10 nmol/L) in cultured cardiac myocytes does not cause downregulation of PKC even after 48 hour of treatment.23 The lack of desensitization or downregulation of PKC after sustained treatment with RACK (Figure 4) may be due to mild activation of this isozyme by RACK treatment (20% to 30% increase over basal), which is not sufficient to trigger downregulation of PKC.
We previously expressed the RACK peptide in cardiac myocytes of transgenic mice during postnatal development using an 
MHC expression vector. Expression of RACK also resulted in sustained activation of PKC in cardiac myocytes and sustained protection of the myocardium from ischemic insult without desensitization or downregulation of PKC.6,8 However, studies using transgenic mice do not address the following issues: What are the effects of systemic delivery of the peptide as opposed to delivery limited only to cardiomyocytes? Is the lack of desensitization in the transgenic mice expressing the peptide in their hearts due to only increases in the expression of the peptide? The rate of expression of the transgenes could be altered; therefore, it is possible that the levels of RACK increase to compensate for the desensitization. In contrast, the rate of drug delivery via the pump is constant or may be decreasing, so the data unequivocally show lack of desensitization to RACK; there was as much cardiac protection after 10 days as after 1 hour of delivery (Figure 3). Therefore, in the present study, we provide the first report that a peptide regulator of intracellular signaling, delivered as a pharmacological agent in a continuous fashion, yields sustained effects without desensitization or downregulation of target protein. In addition, this is the first report on the lack of desensitization to a cardiac preconditioning agent.
Using porcine hearts, we also found that treatment with RACK as a pharmacological agent during ischemia in vivo reduced infarct size by 60% and improved ejection fraction by 17% as measured 5 days after infarction. Unexpectedly, RACK also suppressed ventricular tachyarrhythmias caused by ischemia-reperfusion by 70%. About 50% of the deaths associated with acute myocardial infarction in humans occur within 1 hour of the event and are attributable to arrhythmias, most often VF.24 Recent clinical studies showed that ischemic preconditioning suppresses postoperative VF or VT in patients with CABG,25 and stable angina preceding myocardial infarction is associated with a lower incidence of ventricular arrhythmia.26 Because PKC plays a main role in ischemic preconditioning,7 we determined the effect of RACK on VF or VT. We previously showed that RACK inhibits human cardiac Na+ channels, activates human delayed rectifying K+ channels expressed in Xenopus oocytes, and inhibits the L-type Ca2+ channel in rat ventricular myocytes.27–29 Cardiac Na+ channels determine cell excitability and are responsible for the conduction velocity of the action potential.27,30 Delayed rectifying K+ channels are important in determining the shape and repolarization of cardiac action potentials.28,31 The deleterious increase in intracellular Ca2+ concentration during ischemia-reperfusion is caused in part by Ca2+ influx through the L-type Ca2+ channels. These ion channels are the target of several antiarrhythmic drugs.30,31 Thus, PKC may suppress ischemia-reperfusion-caused ventricular tachyarrhythmias by regulation of these ion channels and by reducing infarct size in the myocardium.
Our study suggests that treatment with a selective agonist of PKC such as RACK may be useful for patients with ischemic heart disease. Our findings also suggest that sustained delivery of this and other intracellularly acting peptide regulators of PKC can be applied to animal models of chronic diseases in which continuous regulation of PKC activity is desired. However, to evaluate the therapeutic efficacy of RACK as a preconditioning agent in humans, studies of the effects of this peptide in in vivo acute myocardial infarction animal models that have other comorbidity factors such as hypercholesterolemia, diabetes, hypertension, and old age should be carried out.
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Acknowledgments |
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This work was supported by NIH grant HL-52141 (to Dr Mochly-Rosen). Dr Begley was partly supported by a predoctoral award from the American Heart Association.
Disclosure
Dr Mochly-Rosen is a founder of KAI Pharmaceuticals, a pharmaceutical company that aims to bring peptide regulators of PKC to the clinic. However, the research described in this study was carried out in her laboratory at the university, independent of the company and with sole support from the NIH for her university activities.
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Footnotes |
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*Drs Inagaki and Begley contributed equally to this study.
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References |
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 A. D. T. Costa, R. Jakob, C. L. Costa, K. Andrukhiv, I. C. West, and K. D. Garlid The Mechanism by Which the Mitochondrial ATP-sensitive K+ Channel Opening and H2O2 Inhibit the Mitochondrial Permeability Transition J. Biol. Chem., July 28, 2006; 281(30): 20801 - 20808. [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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K. Yamamura, C. Steenbergen, and E. Murphy Protein kinase C and preconditioning: role of the sarcoplasmic reticulum Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2484 - H2490. [Abstract] [Full Text] [PDF]
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 R. Bright and D. Mochly-Rosen The Role of Protein Kinase C in Cerebral Ischemic and Reperfusion Injury Stroke, December 1, 2005; 36(12): 2781 - 2790. [Abstract] [Full Text] [PDF]
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