Abstract P51: Grape Seed Proanthocyanidins Protect Cardiomyocytes from Ischemia and Reperfusion Injury via Akt -NOS Signaling
Ischemia/reperfusion (I/R) injury in cardiomyocytes is related to excess reactive oxygen species (ROS) and can be modulated by nitric oxide (NO). In previous work with grape seed proanthocyanidin extract (GSPE), we have demonstrated attenuation of oxidant stress in chick cardiomyocytes exposed to H202, but the mechanism for such protection remains unknown. In the present study, we investigated whether acute administration of GSPE could protect chick cardiomyocytes against ischemia/reperfusion (I/R) injury and whether such protection was related to NO generation. GSPE treatment significantly attenuated cell death resulting from I/R in a concentration-dependent manner. This reduced cell death [18.0 ± 1.8% GSPE (50 ìg/ml) vs. 42.3 ± 3.0% I/R control, p<0.001] was associated with restored contractility (6/6 vs. 0/6, respectively) in cardiomyocytes. GSPE given during reperfusion also stimulated a significant increase in NO release compared to I/R control cells. Co-treatment of GSPE with the NOS inhibitor Nù-nitro-L-arginine methyl ester (L-NAME, 200 ìÌ) significantly reduced the GSPE-induced NO generated at reperfusion and reversed the GSPE protective phenotype [32.7 ± 2.7% (GSPE + L-NAME) vs. 18.0 ± 1.8% GSPE alone, p<0.01]. Since NO production may be regulated by phosphorylation of eNOS via Akt, we examined the role of Akt in GSPE protection against I/R injury. The Akt inhibitor API-2 (10 ìÌ) reversed GSPE-induced protection [44.3% ± 2.2% (GSPE + API-2) vs. 27.0% ± 4.3% (GSPE alone), p<0.01] and attenuated the elevated phosphorylation level of Akt at Ser473 seen in GSPE-treated cells as compared to control I/R. Further, API-2 attenuated the NO response triggered by GSPE. Blocking both NOS activation (L-NAME) and Akt (API-2) resulted in decreased NO levels similar to using each inhibitor independently. These data suggest that, in the context of GSPE, Akt lies upstream of eNOS, leading to activated eNOS, and increased NO production. This increased NO production during reperfusion then plays a role in protecting cardiomyocytes from I/R injury. Thus, GSPE may offer a unique therapeutic approach to protect cardiomyocytes from oxidant-induced cell injury.