KR-31378, a novel benzopyran analog, attenuates hypoxia-induced cell death via mitochondrial K ATP channel and protein kinase C-ɛ in heart-derived H9c2 cells

Abstract

A novel compound KR-31378 [(2 S,3 S,4 R)- N″-cyano- N-(6-amino-3,4-dihydro-3-hydroxy-2-methly-2-dimethoxy-methly-2 H-benzo-pyran-4-yl)- N-benzylguanidine] has been demonstrated as an anti-ischemic agent in rat heart and brain. Here, we report the effects of this compound on hypoxia-induced cell death and possible signaling pathways in heart-derived H9c2 cells. Treatment with KR-31378 (3–30 μM) 1 h before and during hypoxia significantly reduced hypoxia-induced cell death in a concentration-dependent manner. In addition, increase in hypoxia-induced transferase UTP nick end labeling (TUNEL)-positive cells was reduced by KR-31378, suggesting its antiapoptotic potential in H9c2 cells. The protective effect conferred by KR-31378 (10 μM) was abolished by cotreatment with 5-hydroxydecanoate (5HD), a specific blocker of the mitochondrial K ATP (mtK ATP) channel, but not by HMR-1883 (1-[[5-[2-(5-chloro- o-anisamido)ethyl]-methoxyphenyl]sulfonyl]-3-methylthiourea), a specific blocker of the sarcolemmal K ATP channel. We observed that the treatment with KR-31378 could increase the expression of protein kinase C (PKC)-ɛ protein, but not other PKC isotypes (-α, -β, -δ, -ζ), in the particulate fraction. This increased level of PKC-ɛ was sustained during the hypoxic period up to 8 h. In addition, our results showed that treatment with KR-31378 induced the expression of PKC-ɛ mRNA as early as 15 min after the treatment. A specific inhibitor for PKC-ɛ isoform, ɛV1-2, completely blocked the protective effect of KR-31378 against hypoxia-induced cell death. In conclusion, our results suggest that KR-31378 can protect cultured H9c2 cells from hypoxia-induced death via the mtK ATP channel and PKC-ɛ.