Protein kinase G-dependent cardioprotective mechanism of phosphodiesterase-5 inhibition involves phosphorylation of ERK and GSK3beta.
Sildenafil, a potent inhibitor of phosphodiesterase-5 (PDE-5) induces powerful protection against myocardial ischemia-reperfusion injury. PDE-5 inhibition increases cGMP levels that activate cGMP-dependent protein kinase (PKG). However, the cause and effect relationship of PKG in sildenafil-induced cardioprotection and the downstream targets of PKG remain unclear. Adult ventricular myocytes were treated with sildenafil and subjected to simulated ischemia and reoxygenation. Sildenafil treatment significantly decreased cardiomyocyte necrosis and apoptosis. The PKG inhibitors, KT5823, guanosine 3',5'-cyclic monophosphorothioate, 8-(4-chloro-phenylthio) (R(p)-8-pCPT-cGMPs), or DT-2 blocked the anti-necrotic and anti-apoptotic effect of sildenafil. Selective knockdown of PKG in cardiomyocytes with adenoviral vector containing short hairpin RNA of PKG also abolished sildenafil-induced protection. Furthermore, intra-coronary infusion of sildenafil in Langendorff-isolated mouse hearts prior to ischemia-reperfusion significantly reduced myocardial infarct size after 20 min ischemia and 30 min reperfusion, which was abrogated by KT5823. Sildenafil significantly increased PKG activity in intact hearts and cardiomyocytes. Sildenafil also enhanced the Bcl-2/Bax ratio, phosphorylation of Akt, ERK1/2, and glycogen synthase kinase 3beta. All these changes (except Akt phosphorylation) were significantly blocked by KT5823 and short hairpin RNA of PKG. These studies provide the first evidence for an essential role of PKG in sildenafil-induced cardioprotection. Moreover, our results demonstrate that sildenafil activates a PKG-dependent novel signaling cascade that involves activation of ERK and inhibition of glycogen synthase kinase 3beta leading to cytoprotection.
Das
Anindita
A
Division of Cardiology, Department of Internal Medicine, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Xi
Lei
L
Kukreja
Rakesh C
RC
D000818:Animals; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism* / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008954:Models, Biological; D032383:Myocytes, Cardiac / Q000378:metabolism; D058986:Phosphodiesterase 5 Inhibitors; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction
D058986:Phosphodiesterase 5 Inhibitors; D010726:Phosphodiesterase Inhibitors; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; C000605508:Gsk3b protein, rat; D017869:Cyclic GMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D038362:Glycogen Synthase Kinase 3; D006152:Cyclic GMP