Combined treatment with epsilon protein kinase C activator and delta protein kinase C inhibitor ameliorates ischemia reperfusion injury and graft coronary artery disease in murine cardiac allografts

Abstract

Inhibiting delta protein kinase C (δPKC) or activating epsilon PKC (ϵPKC) limits damage from experimental ischemia. We asked whether these isozymes limit ischemia reperfusion (I/R) injury and graft coronary artery disease (GCAD) in transplanted hearts and so examined whether PKC regulator affects their appearance in murine cardiac allografts. Methods: Hearts of FVB mice (H-2q) were transplanted into C57BL/6 mice (H-2b). ϵPKC activator (ΨϵRACK, 20 nmol) was injected i.p. into donor mice 20 min before cardiac procurement. During procurement, hearts were perfused retrogradely with ΨϵRACK (1.5 nmol) via the IVC. Procured hearts were submerged in ΨϵRACK (0.5 μM) for 20 min at 4°C. Prior to reperfusion, the peritoneal cavity was irrigated with δPKC inhibitor (δV1-1, 300 nM). Control animals were treated with normal saline. Grafts were procured after 2 h of reperfusion (n = 5 each group) and analyzed for TNF-α, IL-1β, MCP-1, ICAM-1, and VCAM-1 production by ELISA; for apoptosis by TUNEL and by caspase-2, -3, -8, and -9 activities; and for Fas and Fas ligand expression by ELISA. In Additional animals (n = 7 each groups given i.p. cyclosporine A 20 mg/kg/d), GCAD indices were analyzed at 30 d. Results: At 2 h, TNF-α, IL-1β, MCP-1, ICAM-1, and VCAM-1 production, cardiomyocyte apoptosis, and caspase-3 and -9 activities were reduced in the treatment group (p < 0.001, p < 0.002, and p < 0.03, respectively) without a difference in caspase-2 or -8 activity. Fas ligand but not Fas expression was reduced in the treatment group (p = 0.02). Luminal narrowing (18 ± 6 vs 47 ± 9%, p < 0.001), intima/media ratio (0.19 ± 0.05 vs 0.65 ± 0.16, p < 0.001), and % of diseased vessels (46 ± 18 vs 98 ± 4%, p < 0.001) were decreased at 30 d. Conclusions: ϵPKC activator and δPKC inhibitor together reduce I/R injury and subsequent GCAD. This treatment inhibits the mitochondrial pathway on the induction of apoptosis Clinically, these peptides may be useful for organ preservation and prevention of I/R injury and GCAD.