Sublethal Simulated Ischemia Promotes Delayed Resistance Against Ischemia via ATP-Sensitive (K + ) Channels in Murine Myocytes: Role of PKC and iNOS

Abstract

In this study, we examined whether sublethal simulated ischemia (SSI) induces delayed cellular protection in mouse cardiac myocytes, and whether the delayed cellular protection depends on the activation of protein kinase C-ϵ (PKC-ϵ), inducible nitric oxide synthase (iNOS), and ATP-sensitive K⁺ (K_ATP) channels against subsequent sustained simulated ischemia (SI). The following groups of mouse cardiac myocytes were studied: (a) SI: incubation with SI buffer for 1 h; (b) SSI: incubation with SSI buffer for 30 min; (c) SSI + PKC inhibitor, chelerythrine chloride (CCl): SSI and 1µM CCl; (d) SSI + iNOS inhibitor, S-methylthiourea (SMT): SSI and 100 nM SMT; (e) SSI + K_ATP channel blocker, glibenclamide (Glb): SSI and 50 µM Glb; (f) SSI+ mitochondrial K_ATP channel blocker, 5-hydroxydecanoate (5-HD): SSI and 50 µM 5-HD. The release of lactate dehydrogenase into the medium and the amount remaining in the cells was measured, and A₁ adenosine receptor, PKC-ϵ, and iNOS were detected through western blot analysis. The delayed cellular protection acquired due to SSI showed a decreased release of lactate dehydrogenase (%) from 46.51 ± 1.60 to 37.00 ± 1.34 (p < 0.001) and was blocked by CCl (47.08 ± 0.95), SMT (48.08 ± 1.18), Glb (45.88 ± 1.31), and 5-HD (47.20 ± 1.56). Simultaneously, SSI-induced up-regulation of A₁ adenosine receptor, PKC-ϵ, iNOS, and opening of both membrane and mitochondrial K_ATP channels also was observed compared with controls.