Cyclic GMP-dependent protein kinases protein kinase G (PKG) Ialpha and
PKGIbeta are major mediators of cGMP signaling in the cardiovascular system.
PKGIalpha is present in the heart, although its role in protection against
ischemia/reperfusion injury is not known. We investigated the direct effect of
PKGIalpha against
necrosis and apoptosis following simulated
ischemia (SI) and reoxygenation (RO) in cardiomyocytes. Adult rat cardiomyocytes were infected with adenoviral vectors containing hPKGIalpha or catalytically inactive mutant hPKGIalphaK390A. After 24 h, the cells were subjected to 90 min of SI and 2 h 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 microm), HMR1098 (30 microm), or
glibenclamide (50 microm), 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
PKGIalpha (mean +/- S.E., n = 7, p < 0.001). The apoptosis observed in 17.9 +/- 1.3% of total myocytes in the SI-RO control group was reduced to 6.0 +/- 0.6% by
PKGIalpha (mean +/- S.E., n = 7, p < 0.001). In addition,
PKGIalpha inhibited the activation of
caspase-3 after SI-RO in myocytes. Myocytes infected with the inactive PKGIalphaK390A mutant showed no protection.
PKGIalpha enhanced phosphorylation of Akt, ERK1/2, and JNK, increased Bcl-2,
inducible nitric-oxide synthase,
endothelial nitric-oxide synthase, and decreased Bax expression.
5-Hydroxydecanoate and
glibenclamide abolished
PKGIalpha-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
PKGIalpha-mediated protection against
necrosis and apoptosis. These results show that opening of mitochondrial K(
ATP) 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
PKGIalpha.