Abstract P30: Sildenafil Ameliorates Hypoxia/Reoxygenation-induced Autophagy in Cardiac Myocytes
Background: Sildenafil (SNL) was recently shown to protect myocytes from ischemia minimizing necrosis and apoptosis. Autophagy, which was originally described as a survival mechanism against starvation, seems to contribute, in form of autophagic cell death, morphologically characterized by an accumulation of autophagic vacuoles (AV), to the pathogenesis of various heart conditions, including IRI.
Aim: To investigate the anti-autophagic role and mechanism of action of SNL in cardiac cells exposed to hypoxia(H)/reoxygenation(R).
Methods and Results: HL-1 cardiomyocytes were either transfected with the AV marker GFP-LC3; or co-transfected with GPF-LC3 and the mitochondrion-targeted DsRed plasmid; or transfected with GFP-LC3, before incubation with LysoTracker Red, to label lysosomes. Subsequently, cells were pretreated with SNL (10 uM) for 1 hr, before exposure to 2 hrs H (0.2% O2), and 2 hrs R. Autophagy was quantified as GFP-LC3 accumulation into AV; and colocalization of AV with mitochondria and lysosomes (autophagolysosome formation). HR increased AV formation (17.2±2.8% positive cells vs. 6.5±1.2% of control; p<0.05), as well as their colocalization with mitochondria (8.5±2.1% vs 1.3±0.4%; p<0.05) and lysosomes (23±4.2% vs 4.6±0.8%; p<0.05). SNL ameliorated H/R-induced AV formation (8.9±1.3%; p<0.05), as well as colocalization of AV with mitochondria (2.6±0.8%; p<0.05) and lysosomes (7.8±1.5%; p<0.05). In line with this finding, Western blot (WB) analysis documented in SNL-treated cells a 3-fold decrease in HR-induced processing of endogenous LC3 (p<0.05). With respect to its anti-autophagic activity, we evaluated the effects of SNL on the expression and phosphorylation levels of mTOR and Akt, whose activation was shown to prevent the occurrence of autophagy. WB analysis documented that incubation of HL1 cells with SNL significantly increased expression and phosphorylation levels of mTOR, both reduced by HR, while induced phosphorylation, though not upregulation, of Akt, as compared to untreated cells exposed to HR.
Conclusions: We showed for the first time that SNL can reduce H/R-induced autophagy in cardiac myocytes. Thus, the protective effects of SNL on the myocardium may be further expanded to include its impact on autophagy.