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.