A NOVEL ROLE OF SIRT1 IN SILDENAFIL INDUCED CARDIOPROTECTION IN MICE

Phosphodiesterase-5 inhibitor, sildenafil (SIL) protects against myocardial ischemia/reperfusion (I-R) injury. We hypothesized that SIL-induced protection may be mediated through activation of SIRT1, an enzyme which deacetylates proteins involved in cellular stress response. Adult male ICR mice were treated with SIL (0.7mg/kg ip), Resveratrol (RSV) (5mg/kg ip) (positive control), or saline (0.2 ml ip). The hearts were harvested 24 h later and homogenized for SIRT1 activity analysis. Both SIL and RSV increased cardiac SIRT1 activity (P\u3c0.001) as compared to Saline. Adult mouse ventricular cardiomyocytes pre-treated with either SIL or RSV (1µM) in vitro also upregulated SIRT1 activity (P\u3c0.05). SIL also reduced infarct size following 30 min. ischemia and 24 h reperfusion in vivo. Sirtinol (5mg/kg in 10% DMSO, ip), a SIRT1 inhibitor abolished the infarct-limiting effect of SIL and RSV (P\u3c0.001). In conclusion, activation of SIRT1 by SIL plays an essential role in cardioprotection against I-R injury

Sirtuin 1 (SIRT1) activation mediates sildenafil induced delayed cardioprotection against ischemia-reperfusion injury in mice.

BackgroundIt has been well documented that phosphodiesterase-5 inhibitor, sildenafil (SIL) protects against myocardial ischemia/reperfusion (I-R) injury. SIRT1 is part of the class III Sirtuin family of histone deacetylases that deacetylates proteins involved in cellular stress response including those related to I-R injury.Objective/hypothesisWe tested the hypothesis that SIL-induced cardioprotection may be mediated through activation of SIRT1.MethodsAdult male ICR mice were treated with SIL (0.7 mg/kg, i.p.), Resveratrol (RSV, 5 mg/kg, a putative activator of SIRT1 used as the positive control), or saline (0.2 mL). The hearts were harvested 24 hours later and homogenized for SIRT1 activity analysis.ResultsBoth SIL- and RSV-treated mice had increased cardiac SIRT1 activity (PConclusionOur study shows that activation of SIRT1 following SIL treatment plays an essential role in mediating the SIL-induced cardioprotection against I-R injury. This newly identified SIRT1-activating property of SIL may have enormous therapeutic implications

SIRT1 deacetylase activity in the heart tissues from the mice pretreated (<i>i.p.</i>) with saline (0.2 mL), RSV (5 mg/kg), and SIL (0.7 mg/kg) 24 hours prior to 30 min of regional ischemia and 24 hours of reperfusion.

<p>The SIRT1 inhibitor - sirtinol (5 mg/kg) was administered (<i>i.p.</i>) 30 min prior to the onset of ischemia. The Sham group serves as surgical controls, whereas Saline and DMSO groups serve as the drug solvent/vehicle controls. Data are Mean ± SE (n = 3/group). *<i>P</i><0.0001 vs. Control group. #<i>P</i><0.05 vs. all other groups.</p

Enhanced SIRT1 deacetylase activity in heart tissues from the mice treated <i>i.p.</i> with single dose of resveratrol (RSV, 5 mg/kg) or sildenafil (SIL, 0.7 mg/kg) for 24 hours (Graph A); *P<0.001 vs. Saline (n = 3/group).

<p>Graph B shows SIRT1 deacetylase activity in isolated cardiomyocytes following 1(1 µM, n = 3/group) <i>in vitro</i>. *P<0.05 vs. the untreated control group.</p

Cardiac expression of SIRT1 in mice pretreated (<i>i.p.</i>) with saline (0.2 mL), RSV (5 mg/kg), and SIL (0.7 mg/kg) 24 hours prior to heart tissue collection.

<p><i>Top:</i> Western blots showing protein expression of SIRT1 and Actin (as the loading control). <i>Bottom:</i> Densitometric quantification of the Western blotting bands. The values of SIRT1 are normalized against Actin for each sample (n = 3/group, *indicates <i>P</i><0.05 vs. Saline).</p

Figure 2

<p>A. Myocardial Infarct Size. Infarct size was significantly (P<0.001 vs. saline controls) reduced in the Sildenafil-treated, Resveratrol-treated, Sildenafil+DMSO-treated, and Resveratrol+DMSO-treated groups. Sirtinol abolished the cardioprotective effects of both Sildenafil and Resveratrol. n = 6/Group, * = P<0.001 vs. Saline Controls, # = P<0.05 vs. other groups. B. Area at Risk<b>.</b> The area at risk was presented as percentage of the total left ventricular area that underwent ischemia. n = 6/group, no significant difference was found among all groups.</p

Illustrated description of the experimental protocols using an <i>in vivo</i> model of myocardial infarction induced by 30 min of regional ischemia and 24 hours of reperfusion.

<p>Note that the drug pretreatments via <i>i.p.</i> injection were carried out 24 hours prior to the onset of ischemia at the following dosage: Saline (0.2 ml, served as the Control group); RES (Resveratrol, 5 mg/kg); SIL (Sildenafil, 0.7 mg/kg).</p