The Mitochondrial Mechanism Underlying Resveratrol And NO-induced Cardioprotection Against Ischemia/reperfusion Injury

The role of PDE5in resveratrol-induced cardioprotection against ischemia/reperfusionAcute myocardial infarction (AM) is a serious disease endangering human health. Ischemia/reperfusion injury (IRI) that occurred during the AMI is still has no ideal treatment method. The mechanisms underlying it are urgently needed to guide clinical intervention. Most of the researches about the cardioprotective effect of resveratrol have focused on the pharmacological preconditioning mechanisms. Nitric oxide (NO), adenosine receptors), PI3kinase and mitogen-activated protein kinase (MAPK) have been proposed to mediate the cardioprotective effect of resveratrol-induced pharmacological preconditioning. The glycogen synthase kinase3β (GSK-3β) and mitochondrial permeability transition pore (mPTP) have been proposed to play important roles in myocardial ischemia/reperfusion injury. Our previous study demonstrated that resveratrol prevents cardiac reperfusion injury by targeting the mPTP through inactivation of GSK-3β via the cGMP/PKG signaling pathway. But the exact cellular and molecular mechanism by which resveratrol induces cardioprotection remains unclear. cGMP is a major intracellular second messenger and transduce signaling information, while PKG is considered to be the most important downstream signal. The intracellular cGMP level is regulated by both guanylyl cyclase that promotes the synthesis of cGMP using NO and phosphodiesterase (PDE) that induces the hydrolysis of cGMP. Increasing the amount of NO or inhibition the activity of PDE5, theoretically can increase the intracellular level of cGMP and then activate cGMP/PKG signal pathway. The purpose of this study was to explore the molecular mechanism by which resveratrol increases intracellular cGMP leading cardioprotection against reperfusion injury, focusing on the roles of NO and PDE5.Rat cardiomyocytes were isolated enzymatically. Fluorescence dye4-amino-5-methylamino-2’,7’-difluorofluorescein (DAF-FM) was used to image NO. Fluorescence images were obtained with confocal microscopy. Isolated rat hearts were subjected to30min regional ischemia followed by2h of reperfusion. Myocardial samples were collected from the risk zone for PDE5activity, cGMP levels, and western blot analysis. Infarct size was measured by TTC staining. Mitochondrial swelling was measured spectrophotometrically as a decrease in absorbance at520nm (A520).Cardiomyocytes treated with resveratrol for10min did not show a significant increase in DAF-FM fluorescence intensity, indicating that resveratrol does not produce NO. In contrast, resveratrol significantly reduced PDE5activity and increased cGMP levels at reperfusion in the heart, indicating that the cardioprotective effect of resveratrol is not mediated by guanylyl cyclase but is dependent on PDE5. The non-selective PDE inhibitor3-isobutyl-l-methylxanthirie (IBMX) could mimic the cardioprotective effect of resveratrol by reducing infarct size through modulation of the mPTP opening. In addition, resveratrol enhanced the phosphorylation of Vasodilator-Stimulated Phosphoporotein (VASP) and GSK-3β, an effect that was partially blocked by PKG inhibitor KT5823.These data suggest that Inhibition of PDE5leading to the increase in intracellular cGMP accounts for the cardioprotective effect of resveratrol on reperfusion injury through prevention of the mPTP opening via the cGMP/PKG/GSK-3p signal pathway.Conclusion:1. Resveratrol reduced PDE5activity but increased cGMP and phosphorylation of VASP and GSK-3β levels at reperfusion in the heart.2. The non-selective PDE inhibitor IBMX could mimic the cardioprotective effect of resveratrol by reducing infarct size and preventing the mPTP opening.3. Resveratrol protects the heart against ischemia reperfusion injury by activating of cGMP/PKG signal pathway through inhibition the activity of PDE5. Roles of mitochondrial Src tyrosine kinase and zinc in nitric oxide-induced cardioprotection against ischemia/reperfusion injuryWhile nitric oxide (NO) induces cardioprotection by targeting the mitochondrial permeability transition pore (mPTP), the precise mito-chondrial signaling events that mediate the action of NO remain unclear. The purpose of this study was to test whether NO induces cardio-protection against ischemia/reperfusion by inhibiting oxidative stress through mitochondrial zinc and Src tyrosine kinase. The NO donor S-nitroso-N-acetyl penicillamine (SNAP) given before the onset of ischemia reduced cell death in rat cardiomyocytes subjected to simulated ischemia/reperfusion, and this was abolished by the zinc chelator N,N,N’,N’-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN) and the Src tyrosine kinase inhibitor PP2. SNAP also prevented loss of mitochondrial membrane potential (△ψm) at reperfusion, an effect that was blocked by TPEN and PP2. SNAP increased mitochondrion-free zinc upon reperfusion and enhanced mitochondrial Src phosphorylation in a zinc-dependent manner. SNAP inhibited both mitochondrial complex I activity and mitochondrial reactive oxygen species (ROS) generation at reperfusion through zinc and Src tyrosine kinase. Finally, the anti-infarct effect of SNAP was abrogated by TPEN and PP2applied at reperfusion in isolated rat hearts.In conclusion, these data suggest that NO induces cardioprotection at reperfusion by targeting mitochondria through attenuation of oxidative stress resulted from the inhibition of complex I at reperfusion. Activation of mitochondrial Src tyrosine kinase by zinc may account for the inhibition of complex I.