N-n-Butyl haloperidol iodide protects against hypoxia/reoxygenation-induced cardiomyocyte injury by modulating protein kinase C activity

N-n-Butyl halopendol iodide (F-2), a novel compound derived from halopendol, protects against the damaging effects of ischemia/reperfusion (I/R) injury in vitro and in vivo We tested whether the myocardial protection of F-2 on cardiomyocyte hypoxia/reoxygenation (H/R) injury is mediated by modulating protein kinase C (PKC) activity in primary cultured cardiomyocytes Primary cultures of ventricular cardiomyocytes underwent 2-h hypoxia and 30-min reoxygenation Total PKC activity was measured, and the translocation pattern of PKC alpha, beta II, delta and epsilon isoforms was assessed by fractionated western blot analysis We investigated the association of PKC isoform translocation and H/R-induced injury in the presence and absence of the specific inhibitors and activator Measurements included cell damage evaluated by creatine kinase (CK) release, and apoptosis measured by annexin V-FITC assay In primary cultured cardiomyocytes exposed to H/R, PKC alpha, delta and epsilon were translocated, with no change in PKC beta II activity. Total PKC activity, CK release and apoptosis were increased after H/R. Treatment with the conventional PKC inhibitor Go6976 reduced early growth response-1 (Egr-1) protein expression and attenuated apoptosis. The PKC epsilon inhibitor peptide epsilon V1-2 increased H/R injury without influencing Egr-1 expression. Pretreatment with F-2 inhibited translocation of PKC alpha, Increased translocation of PKC epsilon, and relieved the CK release and apoptosis. The protection of F-2 was blocked in part by the conventional PKC activator thymeleatoxin (TXA) and epsilon V1-2 peptide F-2 significantly alleviated H/R-induced injury, which might be attributed to the combined benefits of inhibiting PKC alpha and activating PKC epsilon. (C) 2010 Elsevier Inc All rights reserved