Abstract 217: Sildenafil (Viagra)-Induces Protection Against Ischemia-Reperfusion Injury in Cardiomyocytes and Isolated Perfused Heart Through Activation of cGMP-Dependent Protein Kinase and Phosphorylation of Akt, ERK1/2 and GSK-3β
Background: Sildenafil (SIL), a selective inhibitor of phosphodiesterese type-5 (PDE-5) induces remarkable protection against myocardial ischemia-reperfusion (I-R) injury. PDE-5 inhibition increases cGMP levels that in turn activate cGMP-dependent protein kinase (PKG). However, the cause and effect relationship of PKG in SIL-induced cardioprotection and novel downstream targets activated through PKG remain unknown.
Methods & Results: Ventricular myocytes were isolated from adult male ICR mice and exposed to 40 min of simulated ischemia (SI) with or without 1 hr pre-incubation of SIL (1 μM). Myocyte necrosis and apoptosis were determined after 1 hr or 18 hrs of reoxygenation (RO), using trypan blue or TUNEL assay, respectively. Pretreatment with SIL protected cardiomyocytes after SI-RO (necrosis 15.1 ± 2.2% and apoptosis 4.7 ± 0.4%, n = 4, p < 0.01) as compared with controls (necrosis 42.4 ± 2.9% and apoptosis 22.5 ± 1.9%). Co-incubation of KT5823 (2 μM), a selective PKG inhibitor blocked both anti-necrotic and anti-apoptotic protection. Selective knockdown of PKG in myocytes with adenoviral vector containing short hairpin RNA (shRNA) of PKG also abolished SIL-induced protection against necrosis/apoptosis. Further evidence was obtained in intact mouse hearts subjected to 20 min of zero-flow global I and 30 min of R in Langendorff mode. SIL (1 μM) was infused intra-coronarily for 10 min prior to I. KT5823 (1 mg/kg) was administered (i.p.) 1 hr before heart isolation. SIL reduced infarct size in post-ischemic heart (16.0 ± 3.0%, n = 6, p < 0.05) as compared to control (29.4 ± 2.4%). The protection by SIL was abolished by KT5823. PKG activity was determined and Western blots were performed to evaluate phosphorylation levels of Akt, GSK3β, ERK, p38 proteins after SIL treatment with or without KT5823. SIL pretreatment increased PKG activity by 196% in heart and 201% in myocytes (n = 3, p < 0.05). Interestingly, SIL enhanced the phosphorylation of Akt, ERK1/2, and GSK3β in both cardiomyocytes and intact hearts, which was blocked by KT5823.
Conclusion: Activation of PKG by sildenafil treatment plays an essential role in protection against I-R injury. Moreover, phosphorylation of Akt, ERK1/2 and GSK3β appears to be intimately linked with PKG-dependent survival pathway.