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

Abstract

N- n-Butyl haloperidol iodide (F 2), a novel compound derived from haloperidol, 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 PKCα, βII, δ and ɛ 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, PKCα, δ and ɛ were translocated, with no change in PKCβII activity. Total PKC activity, CK release and apoptosis were increased after H/R. Treatment with the conventional PKC inhibitor Gő6976 reduced early growth response-1 (Egr-1) protein expression and attenuated apoptosis. The PKCɛ inhibitor peptide ɛV1–2 increased H/R injury without influencing Egr-1 expression. Pretreatment with F 2 inhibited translocation of PKCα, increased translocation of PKCɛ, and relieved the CK release and apoptosis. The protection of F 2 was blocked in part by the conventional PKC activator thymeleatoxin (TXA) and ɛV1–2 peptide. F 2 significantly alleviated H/R-induced injury, which might be attributed to the combined benefits of inhibiting PKCα and activating PKCɛ.