A38 Ahstrufs, ISHR North American Section 24th Annual Meeting

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ENHANCED PROTECTlON OF THE ISCHEMIC HEART BY COMBINED TREATMENT WITH 8PKC INHIBITOR AND EPKC ACTlVATOR Koichi lnagaki 81 Daria Mochly-Rosen Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA

Protein kinase C (PKC) plays a major role in cardioprotection from ischemia/reperfusion injury. Using HIV-1 Tat-protein-derived peptide to mediate rapid and efficient transmembrane delivery of peptides regulators of PKC isozymes, we examined the effect of intracoronary delivery of GPKC inhibitor and EPKC activator on infarct size and functional recovery from ischemia/reperfusion damage in isolated perfused rat hearts, ex vivo. When the selective GPKC inhibitor was administered at the beginning of reperfusion, there was an improved cardiac function and a decrease in CPK release during 30 minutes of repetfusion after 20 minutes of ischemia. Pretreatment with the EPKC activator before ischemia/reperfusion also improved cardiac function and decreased CPK release during reperfusion. Furthermore, administration of the EPKC activator before ischemia followed with GPKC inhibitor during reperfusion conferred greater cardioprotective effects than that obtained by each peptide treatment alone. In conclusion, GPKC inhibitor prevents reperfusion injury and EPKC activator mimics ischemic preconditioning. Furthermore, treatment with both peptides confers additional cardioprotective effects. Therefore, these peptide regulators of PKC may provide a novel pharmacological approach for the treatment of patients with acute myocardial infarction.

23 DELTA PKC IN CEREBRAL ISCHEMIC AND REPERFUSION INJURY IN VIVO Rachel Bright, Jeffrey M. Dembner, Midori A. Yenari, Gary K. Steinberg, and Daria Mochly- Rosen Dept. of Molecular Pharmacology and Dept. of Neurosurgery, Stanford University, Stanford, CA 94305

Protein kinase C (PKC) has been implicated in mediating ischemic injury and protection in various organs. In particular, increases in protein and RNA levels of GPKC are observed in response to ischemic insult in the brain. We report here that mediation of GPKC activity with a rationally designed, isozyme specific peptide inhibitor of GPKC reduces the extent of ischemic/reperfusion injury to the brain in viva. HYPOTHESIS: We hypothesized that GPKC plays a role in cerebral ischemic/ reperfusion injury, and inhibition of GPKC activity reduces the extent of injury. METHODS: Transient focal ischemia was induced by two hour it&alumina1 suture occlusion of the middle cerebral artery in rats. The GPKC inhibitor was injected into the internal carotid artery at a final blood concentration of 1 pM. Brains were removed at 24 hours and stained with 2, 3, 5-triphenyl-tetrazolium chloride (TTC). RESULTS: Treatment with the GPKC antagonist decreased infarct size when administered 2 hours post ischemia (7.6*3.7%; P=O.O25) compared to controls (26.3+5.1%). These data correlate to a decrease in GPKC translocation as assayed by Western blot analysis from brain homogenates. CONCLUSIONS: These results suggest that GPKC mediates cerebral ischemic/reperfusion injury in viva, and that the GPKC inhibitor peptide could be useful clinically for the treatment of

22A ROLE FOR DELTA PKC IN CARDIAC REPERFUSION INJURY IN VIVO

Leon Chen, Koichi Ingaki, Fumiaki Ikeno, Felix Lee, Paul Yock, and Daria Mochly-Rosen

Department of Molecular Pharmacology and Department of Cardiovascular Medicine Stanford University, Stanford, CA 94305

Protein Kinase C has been implicated in cardio- protection from ischemic damage. Through the use of peptide modulators of PKC isozymes, we have shown that either epsilon PKC activation or delta PKC inhibition mediates protection from ischemic damage. Epsilon PKC activation mediates preconditioning, but the role of de1 ta PKC is still unclear. HYPOTHESIS: We hypothesized that delta PKC functions in reperfusion injury, or damage that occurs when re-oxygenated blood is restored after an ischemic event. METHODS: In an in vivo porcine model of acute myocardial infarction, pigs were treated with a delta PKC inhibitor for the last 1 min. of a 30-min. occlusion. Cardiac function and infarct size was determined immediately and after 5 days of reperfusion. RESULTS: Application of a delta PKC inhibitor innnedi ately before reperfusion reduces infarct size by 70%, improves ejection fraction and reduces hypokinetic area, in vivo. CONCLUSIONS: These results suggest that delta PKC plays a role in reperfusion injury and that the delta PKC inhi bitor peptide could be useful clinically for the treatment of acute myocardial infarction.

24 PRESERVING MYOCARDIAL cGMP CONTENT DURING EARLY REPERFUSION IS CRITICAL TO RECOVERY FOLLOWING HYPOTHERMIC STORAGE Rod MacArthur, Alexander S. Clanachan, Arvind Koshal, Manoj Gandhi & Barry A. Finegan. Depts. of Surgery 8 Anesthesiology, University of Alberta, Edmonton, Alberta

Elevation of cGMP following ischemia is cardioprotective. This study assessed the effect of preserving cGMP content during reperfusion of hearts subjected to prolonged hypothermic ischemia (HI). LV mechanical function was assessed during working mode perfusion with crystalloid (Krebs) solution in six groups of rat hearts that were 1) fresh (no HI), 2) transplanted heterotopically (HT) and blood reperfused (60 min) without HI or storage, 3-4) subjected to 8 hr HI in absence or presence of sodium nitroprusside (SNP, 200 pmol/L), and 5-6) 5 hr HI as for groups 3-4 but HT and blood reperfused (60 min). The content of cGMP was reduced by HI and was increased 7- fold in SNP-treated groups. SNP improved recovery of LV function in hearts reperfused with Krebs but not in HT hearts where cGMP content decreased during blood repeffusion. Myeloperoxidase activity was elevated in both blood-reperfused groups, and was unaltered by SNP pretreatment. Exogenous glucose metabolism was not affected by SNP. The differential effects observed following in vivo blood vs. crystalloid reperfusion suggest that maintaining cGMP content throughout early reperfusion is essential for myocyte protection. Augmenting cGMP during HI alone is not sufficient to achieve this goal or to inhibit leukocyte infiltration during _. stroke. blood repertusron