On the basis of our previous reports that cardioprotection induced by ischaemic preconditioning induces autophagy and that resveratrol, a polyphenolic antioxidant present in grapes and red wine induces preconditioning-like effects, we sought to determine if resveratrol could induce autophagy. Resveratrol at lower doses (0.1 and 1 µM in H9c2 cardiac myoblast cells and 2.5 mg/kg/day in rats) induced cardiac autophagy shown by enhanced formation of autophagosomes and its component LC3-II after hypoxia–reoxygenation or ischaemia–reperfusion. The autophagy was attenuated with the higher dose of resveratrol. The induction of autophagy was correlated with enhanced cell survival and decreased apoptosis. Treatment with rapamycin (100 nM), a known inducer of autophagy, did not further increase autophagy compared with resveratrol alone. Autophagic inhibitors, wortmannin (2 µM) and 3-methyladenine (10 mM), significantly attenuated the resveratrol-induced autophagy and induced cell death. The activation of mammalian target of rapamycin (mTOR) was differentially regulated by low-dose resveratrol, i.e. the phosphorylation of mTOR at serine 2448 was inhibited, whereas the phosphorylation of mTOR at serine 2481 was increased, which was attenuated with a higher dose of resveratrol. Although resveratrol attenuated the activation of mTOR complex 1, low-dose resveratrol significantly induced the expression of Rictor, a component of mTOR complex 2, and activated its downstream survival kinase Akt (Ser 473). Resveratrol-induced Rictor was found to bind with mTOR. Furthermore, treatment with Rictor siRNA attenuated the resveratrol-induced autophagy. Our results indicate that at lower dose, resveratrol-mediated cell survival is, in part, mediated through the induction of autophagy involving the mTOR-Rictor survival pathway.

We investigated whether enhanced expression of αB crystallin, a stress‐inducible molecular chaperone of the small heat shock family, can protect myocardial contractile apparatus against ischemia reperfusion (I/R) injury. Transgenic mice overexpressing αB crystallin were generated using the 0.76 kb rat αB crystallin cDNA cloned into a pCAGGS plasmid driven by a human cytomegalovirus expression system. Southern analysis confirmed transgene integration and Northern and Western blotting characterized expression (3.1‐fold and 6.9‐fold elevations in myocardial mRNA and protein levels, respectively). Extent of functional recovery overa3hreperfusion period following a 20 min ischemic period in transgenic and wild‐type mouse hearts was assessed using an ex vivo work‐performing heart preparation. The transgenic group displayed significantly higher values of DP at R45 min (29.14±1.9mmHgvs. 17.6±0.7 mm Hg), R60 min (31.56±1.7mmHgvs. 17.8±0.8 mm Hg), and R75 min (32.5±2.2 mm Hg vs. 16.9±0.9 mm Hg), and of dLVP/dt at R45 min (1740.2±111.5 mm Hg.s ⁻¹ vs. 548.7±82.2 mm Hg.s ⁻¹ ) and R60 min (1199.8±104.6 mm Hg.s ⁻¹ vs. 466.9±61.1 mm Hg.s ⁻¹ ). The transgenic group also displayed development of less oxidative stress, decreased extent of infarction, and attenuated cardiomyocyte apoptotic cell death. Transgene overexpression of αB crystallin was therefore successful in diminishing the independent contributory effects of both necrosis and apoptosis on I/R‐induced cell death.—Ray, P. S., Martin, J. L., Swanson, E. A., Otani, H., Dillmann, W. H., Das, D. K. Transgene overexpression of aB crystallin confers simultaneous protection against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion. FASEB J. 15, 393‐402 (2001)

Background— The signaling pathways that control ischemia/reperfusion-induced cardiomyocyte apoptosis in heart have not been fully defined. In this study, we investigated whether Akt signaling has a role in the antiapoptotic pathways of preconditioning against hypoxia/reoxygenation (H/R). Methods and Results— Primary cultures of adult rat ventricular myocytes (ARVMs) were subjected to preconditioning (PC) by exposing the cells to 10 minutes of hypoxia followed by 30 minutes of reoxygenation. Non-PC and PC myocytes were subjected to 90 minutes of hypoxia followed by 120 minutes of reoxygenation. Hypoxic-PC protected the myocytes from subsequent H/R injury, as evidenced by decreased apoptosis and LDH release and increased cell viability. H/R-induced cytochrome c release and activation of caspase-3 and -9 were blocked by PC. This protective effect was inhibited by treating the cells with LY294002 (50 μmol/L), a PI3 kinase inhibitor, for 10 minutes before and during PC. PC also induced phosphorylation of Akt and BAD. Protein levels of Bcl-2 in mitochondria were maintained in PC. ARVMs were infected with either a control adenovirus (Adeno lac-Z), an adenovirus expressing dominant-negative Akt, or an adenovirus expressing constitutively active Akt. Ectopic overexpression of constitutively active Akt protected ARVMs from apoptosis induced by hypoxia/reoxygenation compared with Adeno lac-Z. In contrast, dominant negative Akt overexpression abolished the antiapoptotic effect of PC. Conclusions— Our data demonstrated that in adult cardiomyocytes, the antiapoptotic effect of PC against H/R requires Akt signaling leading to phosphorylation of BAD, inhibition of cytochrome c release, and prevention of caspase activation.

Resveratrol ( trans-3,4′,5-trihydroxystilbene), a recently described grape-derived polyphenolic antioxidant, has been found to protect the heart from ischemic-reperfusion injury. The present study sought to determine the mechanism of cardioprotection by investigating the ability of resveratrol to precondition the heart. Isolated perfused rat hearts were randomly divided into six groups: group Iwas perfused for 15 min with Kreb-Henseleit buffer (KHB) only; group II was perfused with 10 μM resveratrol; group III was perfused with 10 μM resveratrol plus 100 μM N^G-nitro-l-arginine methyl ester (l-NAME), a nonselective nitric oxide (NO) synthase (NOS) inhibitor; group IV was perfused with 10 μM resveratrol plus 100 μM aminoguanidine (AG), an inducible NOS (iNOS) blocker; and groups V and VI consisted of hearts perfused withl-NAME and AG, respectively. The perfusion was then switched to working mode, and all hearts were made globally ischemic for 30 min followed by 2 h of reperfusion. Preconditioning of the hearts with resveratrol provided cardioprotection as evidenced by improved postischemic ventricular functional recovery (developed pressure and aortic flow) and reduced myocardial infarct size and cardiomyocyte apoptosis. Resveratrol-mediated cardioprotection was completely abolished by both l-NAME and AG. In a separate study, hearts were examined for iNOS mRNA induction. Resveratrol caused an induction of the expression of iNOS mRNA beginning at 30 min after reperfusion, increasing steadily up to 60 min of reperfusion, and then decreasing progressively up to 2 h after reperfusion. Preperfusion of the hearts with AG almost completely blocked the induction of iNOS. The results of our study demonstrate that resveratrol can pharmacologically precondition the heart in a NO-dependent manner.