Cardioprotection by ε-Protein Kinase C Activation From Ischemia
Continuous Delivery and Antiarrhythmic Effect of an ε-Protein Kinase C-Activating Peptide
Background— We previously showed that a selective activator peptide of ε-protein kinase C (PKC), ψεRACK, conferred cardioprotection against ischemia-reperfusion when delivered ex vivo before the ischemic event. Here, we tested whether in vivo continuous systemic delivery of ψεRACK confers sustained cardioprotection against ischemia-reperfusion in isolated mouse hearts and whether ψεRACK treatment reduces infarct size or lethal arrhythmias in porcine hearts in vivo.
Methods and Results— After ψεRACK was systemically administered in mice either acutely or continuously, hearts were subjected to ischemia-reperfusion in an isolated perfused model. Whereas ψεRACK-induced cardioprotection lasted 1 hour after a single intraperitoneal injection, continuous treatment with ψεRACK induced a sustained preconditioned state during the 10 days of delivery. There was no desensitization to the therapeutic effect, no downregulation of εPKC, and no adverse effects after sustained ψεRACK delivery. Porcine hearts were subjected to ischemia-reperfusion in vivo, and ψεRACK was administered by intracoronary injection during the first 10 minutes of ischemia. ψεRACK treatment reduced infarct size (34±2% versus 14±1%, control versus ψεRACK) and resulted in fewer cases of ventricular fibrillation during ischemia-reperfusion (87.5% versus 50%, control versus ψεRACK).
Conclusions— The εPKC activator ψεRACK 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 εPKC after sustained delivery. Thus, ψεRACK may be useful for patients with ischemic heart disease. In addition, the ψεRACK peptide should be a useful pharmacological agent for animal studies in which systemic and sustained modulation of εPKC in vivo is needed.
Received July 24, 2004; revision received September 10, 2004; accepted September 30, 2004.
The hypothesis that the translocation of ε-protein kinase C (PKC) plays a key role in ischemic preconditioning is supported by many studies. Translocation of εPKC is induced by ischemic preconditioning in neonatal rat cultured cardiomyocytes and in the rat and rabbit myocardium.1–3 A selective εPKC antagonist inhibits cardiac protective effects induced by ischemic preconditioning in mice, rats, and rabbits.1,4,5 Conversely, a selective εPKC activator peptide, ψεRACK, confers cardioprotection from ischemia-reperfusion injury in various cell culture and isolated perfused heart models.4,6–9 Importantly, ψεRACK 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 εPKC 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, ψεRACK, confers a sustained therapeutic effect without desensitization and whether ψεRACK treatment during ischemia induces cardioprotection and reduces ventricular tachyarrhythmias in a porcine model in vivo.
The εPKC selective agonist ψεRACK (ψε-receptor for activated C-kinase) derived from εPKC (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 ψεRACK 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 ψεRACK on ischemia-reperfusion-induced damage and on translocation of εPKC.
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-ψεRACK (ψεRACK) 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 ψεRACK 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-ψεRACK (ψεRACK; 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 ψεRACK). 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 ψεRACK in Blood Plasma and Saline
After ψεRACK (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 ψεRACK (Figure 2D). We measured the concentration of ψεRACK in saline over time when 10 mmol/L ψεRACK 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 ψεRACK 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 εPKC and δPKC levels and translocation were determined by SDS-PAGE, followed by Western blot analysis with anti-εPKC and anti-δPKC antibodies (Santa Cruz). δPKC served as control for both protein levels and translocation.
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 χ2 test or Fisher’s exact test for categorical data were used to assess significance (P<0.05).
Intraperitoneal administration of the ψεRACK peptide (conjugated to TAT47–57 as an intracellular carrier) renders the heart resistant to ischemic damage.
To determine whether ψεRACK confers cardioprotection when administered systemically, we injected ψεRACK intraperitoneally into mice. Intraperitoneal delivery of 0.5 or 20 but not 0.05 nmol ψεRACK 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 ψεRACK was specific; injection of the control carrier peptide (TAT47–57) resulted in no cardioprotective effects against ischemia-reperfusion injury.
Duration of Cardioprotection From a Single Administration of ψεRACK
We examined the duration of cardioprotection conferred by a single 20-nmol intraperitoneal injection of ψεRACK. Intraperitoneal administration of ψεRACK 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 ψεRACK injection.
To determine whether the cardioprotection was dependent on enhanced εPKC activation, the duration of increased εPKC translocation in the heart after a single administration of ψεRACK was also determined. Intraperitoneal delivery of ψεRACK resulted in translocation of εPKC in the myocardium 10 and 60 minutes after administration. However, at 90 minutes, when cardioprotection was lost, translocation of εPKC returned to baseline (Figure 2C). These data indicate that cardioprotection induced by a single intraperitoneal administration of ψεRACK was transient because peptide-induced εPKC translocation also was transient.
ψεRACK (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 ψεRACK was ≈1.1 nmol/L (Figure 2D), indicating a 99.99% reduction in peptide concentration. The concentration of ψεRACK 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 ψεRACK by cells.
Continuous Delivery of ψεRACK Confers Sustained Cardioprotection
To obtain sustained treatment, we first examined the stability of the ψεRACK disulfide conjugate in vitro by incubating it in saline at 37°C, and using HPLC-ESI MS, we found that the half-life of ψεRACK 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 ψεRACK could confer sustained cardioprotection. We found that sustained pump delivery of ψεRACK 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 ψεRACK 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).
Next, mice were implanted with pumps containing a range of concentrations of ψεRACK to investigate the dose dependence of peptide-induced cardioprotection. There was a significant correlation between the concentration of ψεRACK 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 ψεRACK for 10 days compared with mice treated with control carrier peptide (Table 1). Thus, sustained systemic delivery of ψεRACK induced a dose-dependent cardioprotection.
Chronic εPKC Activation Does Not Induce Desensitization
To determine whether sustained activation of εPKC by ψεRACK causes downregulation of εPKC or desensitization in the translocation of εPKC, we measured protein level and translocation of εPKC in hearts after continuous delivery of ψεRACK. Neither the overall protein levels of εPKC nor εPKC translocation (Figure 4A and 4B) was decreased after continuous administration of ψεRACK for 10 days compared with treatment for 1 hour. Moreover, protein levels and translocation of δPKC were unaffected by the εPKC-selective agonist (Figure 4C and 4D). Therefore, continuous activation of εPKC in the heart by sustained delivery of the εPKC-selective activator ψεRACK resulted in sustained and selective activation of εPKC without desensitization or downregulation of the enzyme.
In vivo ψεRACK delivery during ischemia conferred cardioprotection and reduced ventricular tachyarrhythmias in a porcine model.
We studied the cardioprotective effect of ψεRACK delivery during ischemia in an in vivo model. ψεRACK delivery resulted in no acute changes in blood pressure or heart rate in pigs (Table 2). ψεRACK delivery reduced infarct size (14±1% versus 34±2% in ψεRACK 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 ψεRACK versus control; P<0.05). ψεRACK delivery resulted in fewer cases of VF and VT (50% versus 87.5% in ψεRACK 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, εPKC activation during ischemia reduced infarct size and suppressed incidence of VF and VT during ischemia.
As in the mouse study, there was no evidence for adverse effects after ψεRACK delivery in pigs on physiological and pathological examination.
In this study, we found that continuous administration of ψεRACK 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 εPKC activation induced by continuous delivery of ψεRACK did not lead to downregulation of the enzyme or desensitization to the cardioprotective effect demonstrate a unique feature of ψεRACK 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 εPKC even after 48 hour of treatment.23 The lack of desensitization or downregulation of εPKC after sustained treatment with ψεRACK (Figure 4) may be due to mild activation of this isozyme by ψεRACK treatment (20% to 30% increase over basal), which is not sufficient to trigger downregulation of εPKC.
We previously expressed the ψεRACK peptide in cardiac myocytes of transgenic mice during postnatal development using an αMHC expression vector. Expression of ψεRACK also resulted in sustained activation of εPKC in cardiac myocytes and sustained protection of the myocardium from ischemic insult without desensitization or downregulation of εPKC.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 ψεRACK 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 ψεRACK; 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 ψεRACK 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, ψεRACK 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 εPKC plays a main role in ischemic preconditioning,7 we determined the effect of ψεRACK on VF or VT. We previously showed that ψεRACK 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, εPKC 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 εPKC such as ψεRACK 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 ψεRACK 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.
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.
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.
↵*Drs Inagaki and Begley contributed equally to this study.
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