Koichi Inagaki

Inhibition of δ-Protein Kinase C Protects Against Reperfusion Injury of the Ischemic Heart In Vivo

Background—Current treatment for acute myocardial infarction (AMI) focuses on reestablishing blood flow (reperfusion). Paradoxically, reperfusion itself may cause additional injury to the heart. We previously found that &dgr;-protein kinase C (&dgr;PKC) inhibition during simulated ischemia/reperfusion in isolated rat hearts is cardioprotective. We focus here on the role for &dgr;PKC during reperfusion only, using an in vivo porcine model of AMI. Methods and Results—An intracoronary application of a selective &dgr;PKC inhibitor to the heart at the time of reperfusion reduced infarct size, improved cardiac function, inhibited troponin T release, and reduced apoptosis. Using 31P NMR in isolated perfused mouse hearts, we found a faster recovery of ATP levels in hearts treated with the &dgr;PKC inhibitor during reperfusion only. Conclusions—Reperfusion injury after cardiac ischemia is mediated, at least in part, by &dgr;PKC activation. This study suggests that including a &dgr;PKC inhibitor at reperfusion may improve the outcome for patients with AMI.

Additive Protection of the Ischemic Heart Ex Vivo by Combined Treatment With δ-Protein Kinase C Inhibitor and ε-Protein Kinase C Activator

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 &dgr;-PKC inhibitor (&dgr;V1-1) and &egr;-PKC activator (&psgr;&egr;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 &dgr;V1-1 but not &psgr;&egr;RACK during reperfusion improved cardiac function and decreased creatine phosphokinase release. In contrast, pretreatment with &psgr;&egr;RACK but not &dgr;V1-1, followed by a 10-minute washout before ischemia/reperfusion, also improved cardiac function and decreased creatine phosphokinase release. Furthermore, administration of &psgr;&egr;RACK before ischemia followed by &dgr;V1-1 during reperfusion only conferred greater cardioprotective effects than that obtained by each peptide treatment alone. Both the &dgr;-PKC inhibitor and &egr;-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—&dgr;-PKC inhibitor prevents reperfusion injury, and &egr;-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.

Cardiac Endothelin-1 Plays a Critical Role in the Functional Deterioration of Left Ventricles During the Transition From Compensatory Hypertrophy to Congestive Heart Failure in Salt-Sensitive Hypertensive Rats

BACKGROUND To investigate whether endogenous ET-1 participates in an adaptive process of left ventricular hypertrophy (LVH) or a maladaptive process from LVH to congestive heart failure (CHF), we used a Dahl salt-sensitive (DS) rat model, in which systemic hypertension caused compensated concentric LVH at the age of 11 weeks followed by marked LV dilatation and global hypokinesis at the age of 17 weeks. METHODS AND RESULTS By specific sandwich enzyme immunoassay, serum and myocardial ET-1 levels at the LVH stage were not elevated compared with age-matched Dahl salt-resistant (DR) rats, despite the marked increase of LV/body weight ratio (LV/BW). However, at the CHF stage, serum and LV ET-1 levels increased by 3. 8-fold and 5.4-fold, respectively. LV ET-1 contents had close relationships with the fractional shortening (r=0.763) and the systolic wall stress (r=0.858) measured by in vivo transthoracic echocardiography. Immunohistochemistry demonstrated that the remarkably increased ET-1 in LV is located mainly in cardiomyocytes. By competitive reverse transcriptase-polymerase chain reaction, LV prepro-ET-1 mRNA levels increased by 4.1-fold in CHF rats. We randomized 11-week-old LVH rats to chronic treatment with the endothelin receptor antagonist bosentan (Bos, 100 mg. kg-1. d-1, n=14), the alpha1-receptor antagonist doxazosin (Dox, 1 mg. kg-1. d-1, n=12), or vehicle (Cont, n=14). Bos treatment did not alter the LV geometry and function at 15 weeks; however, it attenuated the decrease of LV fractional shortening by 51% (P<0.01) without reducing the LV/BW at 17 weeks. Conversely, Dox, which decreased the blood pressure to the same extent as Bos, did not affect the progression of LV dysfunction. Bos (93%; P<0.0001 versus Cont) but not Dox (42%; P=0.8465 versus Cont) ameliorated the survival rate at 17 weeks (Cont; 36%). CONCLUSIONS The accelerated myocardial synthesis of ET-1 contributes directly to LV contractile dysfunction during the transition from LVH to CHF. Unelevated levels of LV ET-1 at the established LVH stage and lack of effects on LV mass by chronic bosentan treatment suggest that myocardial growth is mediated through alternative pathways. These studies indicate that chronic ET antagonism may provide an additional strategy for heart failure therapy in humans.

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 &egr;-protein kinase C (PKC), &psgr;&egr;RACK, conferred cardioprotection against ischemia-reperfusion when delivered ex vivo before the ischemic event. Here, we tested whether in vivo continuous systemic delivery of &psgr;&egr;RACK confers sustained cardioprotection against ischemia-reperfusion in isolated mouse hearts and whether &psgr;&egr;RACK treatment reduces infarct size or lethal arrhythmias in porcine hearts in vivo. Methods and Results—After &psgr;&egr;RACK was systemically administered in mice either acutely or continuously, hearts were subjected to ischemia-reperfusion in an isolated perfused model. Whereas &psgr;&egr;RACK-induced cardioprotection lasted 1 hour after a single intraperitoneal injection, continuous treatment with &psgr;&egr;RACK induced a sustained preconditioned state during the 10 days of delivery. There was no desensitization to the therapeutic effect, no downregulation of &egr;PKC, and no adverse effects after sustained &psgr;&egr;RACK delivery. Porcine hearts were subjected to ischemia-reperfusion in vivo, and &psgr;&egr;RACK was administered by intracoronary injection during the first 10 minutes of ischemia. &psgr;&egr;RACK treatment reduced infarct size (34±2% versus 14±1%, control versus &psgr;&egr;RACK) and resulted in fewer cases of ventricular fibrillation during ischemia-reperfusion (87.5% versus 50%, control versus &psgr;&egr;RACK). Conclusions—The &egr;PKC activator &psgr;&egr;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 &egr;PKC after sustained delivery. Thus, &psgr;&egr;RACK may be useful for patients with ischemic heart disease. In addition, the &psgr;&egr;RACK peptide should be a useful pharmacological agent for animal studies in which systemic and sustained modulation of &egr;PKC in vivo is needed.

Anti-Ischemic Effect of a Novel Cardioprotective Agent, JTV519, Is Mediated Through Specific Activation of δ-Isoform of Protein Kinase C in Rat Ventricular Myocardium

BACKGROUND A new 1,4-benzothiazepine derivative, JTV519, has a strong protective effect against Ca(2+) overload-induced myocardial injury. We investigated the effect of JTV519 on ischemia/reperfusion injury in isolated rat hearts. METHODS AND RESULTS At 30 minutes of reperfusion after 30-minute global ischemia, the percent recovery of left ventricular developed pressure was improved, and the creatine phosphokinase and lactate dehydrogenase leakage was reduced in a concentration-dependent manner when JTV519 was administered in the coronary perfusate both at 5 minutes before the induction of ischemia and at the time of reperfusion. The myocardial protective effect of JTV519 was completely blocked by pretreatment of the heart with GF109203X, a specific protein kinase C (PKC) inhibitor. In contrast, the effect of JTV519 was not affected by alpha(1)-, A(1)-, and B(2)-receptor blockers that couple with PKC in the cardiomyocyte. Both immunofluorescence images and immunoblots of JTV519-treated left ventricular myocardium and isolated ventricular myocytes demonstrated that this agent induced concentration-dependent translocation of the delta-isoform but not the other isoforms of PKC to the plasma membrane. CONCLUSIONS The mechanism of cardioprotection by JTV519 against ischemia/reperfusion injury involves isozyme-specific PKC activation through a receptor-independent mechanism. This agent may provide a novel pharmacological approach for the treatment of patients with acute coronary diseases via a subcellular mechanism mimicking ischemic preconditioning.

Reinduction of T-Type Calcium Channels by Endothelin-1 in Failing Hearts In Vivo and in Adult Rat Ventricular Myocytes In Vitro

Background—In ventricular myocardium, the T-type Ca2+ current (ICa,T), which is temporarily observed during fetal and neonatal periods, has been shown to reappear in failing/remodeling hearts. However, its pathophysiological regulation has not been elucidated. Methods and Results—We utilized Dahl salt-sensitive (DS) rats with hypertension at the stage of concentric left ventricular (LV) hypertrophy (11 weeks old, LVH) and at the heart failure stage (16 to 18 weeks old, CHF). Some were treated with bosentan (100 mg/kg per day) during the period from LVH to CHF. In LVH, neither the presence of ICa,T (measured in the freshly isolated LV myocytes) nor an increase in &agr;-1G mRNA expression were detected. This condition was associated with increases in tissue angiotensin II (AII) but not with endothelin (ET)-1 peptides. In contrast, in CHF, when the tissue AII remained elevated and ET-1 de novo increased, ICa,T was recorded in most of the cells (−0.87±0.18 pA/pF at −30 mV, P <0.01 versus LVH). This was associated with a significant increase in the &agr;-1G mRNA level. The chronic bosentan treatment eliminated both the elevation of &agr;-1G mRNA level and ICa,T from the cells, whereas it did not affect the cell size and membrane capacitance. In addition, 48-hour exposure to ET-1 but not AII induced ICa,T in normal adult myocytes in culture from Sprague-Dawley rats. Conclusions—ICa,T channels reappear in failing but not in hypertrophied LV cardiomyocytes in a manner depending on the tissue ET-1 activation.

Protein Kinase C δ (δPKC)-Annexin V Interaction A REQUIRED STEP IN δPKC TRANSLOCATION AND FUNCTION

Protein kinase C (PKC) plays a critical role in diseases such as cancer, stroke, and cardiac ischemia, and participates in a variety of signal transduction pathways such as apoptosis, cell proliferation, and tumor suppression. Though much is known about PKC downstream signaling events, the mechanisms of regulation of PKC activation and subsequent translocation have not been elucidated. Protein-protein interactions regulate and determine the specificity of many cellular signaling events. Such a specific protein-protein interaction is described here between δPKC and annexin V. We demonstrate, at physiologically relevant conditions, that a transient interaction between annexin V and δPKC occurs in cells after δPKC stimulation, but before δPKC translocates to the particulate fraction. Evidence of δPKC-annexin V binding is provided also by FRET and by in vitro binding studies. Dissociation of the δPKC-annexin V complex requires ATP and microtubule integrity. Furthermore, depletion of endogenous annexin V, but not annexin IV, with siRNA inhibits δPKC translocation following PKC stimulation. A rationally designed eight amino acid peptide, corresponding to the interaction site for δPKC on annexin V, inhibits δPKC translocation and δPKC-mediated function as evidenced by its protective effect in a model of myocardial infarction. Our data indicate that translocation of δPKC is not simply a diffusion-driven process, but is instead a multi-step event regulated by protein-protein interactions. We show that following cell activation, δPKC-annexin V binding is a transient and an essential step in the function of δPKC, thus identifying a new role for annexin V in PKC signaling and a new step in PKC activation.

Sustained pharmacological inhibition of δPKC protects against hypertensive encephalopathy through prevention of blood-brain barrier breakdown in rats

Hypertensive encephalopathy is a potentially fatal condition associated with cerebral edema and the breakdown of the blood-brain barrier (BBB). The molecular pathways leading to this condition, however, are unknown. We determined the role of deltaPKC, which is thought to regulate microvascular permeability, in the development of hypertensive encephalopathy using deltaV1-1 - a selective peptide inhibitor of deltaPKC. As a model of hypertensive encephalopathy, Dahl salt-sensitive rats were fed an 8% high-salt diet from 6 weeks of age and then were infused s.c. with saline, control TAT peptide, or deltaV1-1 using osmotic minipumps. The mortality rate and the behavioral symptoms of hypertensive encephalopathy decreased significantly in the deltaV1-1-treated group relative to the control-treated group, and BBB permeability was reduced by more than 60%. Treatment with deltaV1-1 was also associated with decreased deltaPKC accumulation in capillary endothelial cells and in the endfeet of capillary astrocytes, which suggests decreased microvasculature disruption. Treatment with deltaV1-1 prevented hypertension-induced tight junction disruption associated with BBB breakdown, which suggests that deltaPKC may specifically act to dysregulate tight junction components. Together, these results suggest that deltaPKC plays a role in the development of hypertension-induced encephalopathy and may be a therapeutic target for the prevention of BBB disruption.

Pharmacological Inhibition of ε-Protein Kinase C Attenuates Cardiac Fibrosis and Dysfunction in Hypertension-Induced Heart Failure

Studies on genetically manipulated mice suggest a role for ϵ-protein kinase C (ϵPKC) in cardiac hypertrophy and in heart failure. The potential clinical relevance of these findings was tested here using a pharmacological inhibitor of ϵPKC activity during the progression to heart failure in hypertensive Dahl rats. Dahl rats, fed an 8% high-salt diet from the age of 6 weeks, exhibited compensatory cardiac hypertrophy by 11 weeks, followed by heart failure at ≈17 weeks and death by the age of ≈20 weeks (123±3 days). Sustained treatment between weeks 11 and 17 with the selective ϵPKC inhibitor ϵV1-2 or with an angiotensin II receptor blocker olmesartan prolonged animal survival by ≈5 weeks (ϵV1-2: 154±7 days; olmesartan: 149±5 days). These treatments resulted in improved fractional shortening (ϵV1-2: 58±2%; olmesartan: 53±2%; saline: 41±6%) and decreased cardiac parenchymal fibrosis when measured at 17 weeks without lowering blood pressure at any time during the treatment. Combined treatment with ϵV1-2, together with olmesartan, prolonged animal survival by 5 weeks (37 days) relative to olmesartan alone (from 160±5 to 197±14 days, respectively) and by ≈11 weeks (74 days) on average relative to saline-treated animals, suggesting that the pathway inhibited by ϵPKC inhibition is not identical to the olmesartan-induced effect. These data suggest that an ϵPKC-selective inhibitor such as ϵV1-2 may have a potential in augmenting current therapeutic strategies for the treatment of heart failure in humans.

Suppression of Graft Coronary Artery Disease by a Brief Treatment With a Selective εPKC Activator and a δPKC Inhibitor in Murine Cardiac Allografts

Background—Inhibiting delta protein kinase C (&dgr;PKC) during reperfusion and activating epsilon PKC (&egr;PKC) before ischemia each limits cardiac ischemic injury. Here, we examined whether limiting ischemia–reperfusion injury inhibits graft coronary artery disease (GCAD) and improves murine cardiac allografting. Methods and Results—Hearts of FVB mice (H-2q) were transplanted into C57BL/6 mice (H-2b). &egr;PKC activator (&psgr;&egr;RACK) was injected intraperitoneally (20 nmol) into donor mice 20 minutes before procurement. Hearts were then perfused with &psgr;&egr;RACK (1.5 nmol) through the inferior vena cava (IVC) and subsequently submerged in &psgr;&egr;RACK (0.5 &mgr;mol/L) for 20 minutes at 4°C. Before reperfusion, the peritoneal cavity of recipients was irrigated with &dgr;PKC inhibitor (&dgr;V1–1, 300 nmol); control animals were treated with normal saline. The total ischemic time to the organ was 50 minutes. Two hours after transplantation, production of inflammatory cytokines and adhesion molecules, cardiomyocyte apoptosis, and caspase-3 and caspase-9 (but not caspase-8) activities were significantly reduced in the PKC regulator-treated group. Fas ligand levels (but not Fas) were also significantly reduced in this group. Importantly, GCAD indices, production of inflammatory cytokines, and adhesion molecules were significantly decreased and cardiac allograft function was significantly better as measured up to 30 days after transplantation. ConclusionsAn &egr;PKC activator and a &dgr;PKC inhibitor together reduced GCAD. Clinically, these PKC isozyme regulators may be useful for organ preservation and prevention of ischemia-reperfusion injury and graft coronary artery disease in cardiac transplantation.