Cyclic GMP-dependent protein kinase Iα attenuates necrosis and apoptosis following ischemia/reoxygenation in adult cardiomyocyte

Cyclic GMP-dependent protein kinases protein kinase G (PKG) I alpha and PKGI beta are major mediators of cGMP signaling in the cardiovascular system. PKGI alpha is present in the heart, although its role in protection against ischemia/reperfusion injury is not known. We investigated the direct effect of PKGI alpha against necrosis and apoptosis following simulated ischemia (SI) and reoxygenation (RO) in cardiomyocytes. Adult rat cardiomyocytes were infected with adenoviral vectors containing hPKGI alpha or catalytically inactive mutant hPKGI alpha K390A. After 24 h, the cells were subjected to 90 min of SI and 2h RO for necrosis (trypan blue exclusion and lactate dehydrogenase release) or 18 h RO for apoptosis studies. To evaluate the role of K-ATP channels, subgroups of cells were treated with 5-hydroxydecanoate (100 mu M), HMR1098 (30 mu M), or glibenclamide (50 mu M), the respective blockers of mitochondrial, sarcolemmal, or both types of K-ATP channels prior to SI. The necrosis observed in 33.7 +/- 1.6% of total myocytes in the SI-RO control group was reduced to 18.6 +/- 0.8% by PKGI alpha (mean +/- S.E., n = 7, p < 0.001). Theapoptosisobservedin 17.9 +/- 1.3% of total myocytes in the SI-RO control group was reduced to 6.0 +/- 0.6% by PKGI alpha (mean +/- S.E., n = 7, p < 0.001). In addition, PKGI alpha inhibited the activation of caspase-3 after SI-RO in myocytes. Myocytes infected with the inactive PKGI alpha K390A mutant showed no protection. PKGI alpha enhanced phosphorylation of Akt, ERK1/2, and JNK, increasedBcl-2, induciblenitric-oxidesynthase, endothelial nitric-oxide synthase, and decreased Bax expression. 5-Hydroxydecanoate and glibenclamide abolished PKGI alpha-mediated protection against necrosis and apoptosis. However, HMR1098, had no effect. A scavenger of reactive oxygen species, as well as inhibitors of phosphatidylinositol 3-kinase, ERK, JNK1, and NOS, also blocked PKGI alpha-mediated protection against necrosis and apoptosis. These results show that opening of mitochondrial KATP channels and generation of reactive oxygen species, in association with phosphorylation of Akt, ERK, and JNK, and increased expression of NOS and Bcl-2, play an essential role in the protective effect of PKGI alpha.