title: Cyclic GMP-dependent protein kinase Ialpha attenuates necrosis and apoptosis following ischemia/reoxygenation in adult cardiomyocyte.
abstract: 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.
authors.affiliation: Department of Internal Medicine, Division of Cardiology, Virginia Commonwealth University Medical Center, Richmond, VA 23298, USA.
authors.forename: Anindita
authors.initials: A
authors.lastname: Das
authors.forename: Albert
authors.initials: A
authors.lastname: Smolenski
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Lohmann
authors.forename: Rakesh C
authors.initials: RC
authors.lastname: Kukreja
mesh_terms: D000818:Animals; D017209:Apoptosis; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000494:pharmacology; D008297:Male; D017202:Myocardial Ischemia / Q000201:enzymology* / Q000473:pathology; D009336:Necrosis; D019001:Nitric Oxide Synthase / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D015221:Potassium Channels / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D016166:Free Radical Scavengers; D015221:Potassium Channels; D047428:Protein Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; C117780:mitochondrial K(ATP) channel; D019001:Nitric Oxide Synthase; D017869:Cyclic GMP-Dependent Protein Kinases; D010100:Oxygen