Impact of 6-mo caloric restriction on myocardial ischemic tolerance: possible involvement of nitric oxide-dependent increase in nuclear Sirt1

Abstract

Ischemic tolerance decreases with aging, and the cardioprotective effect of ischemic preconditioning (IPC) is impaired in middle-aged animals. We have demonstrated that short-term caloric restriction (CR) improves myocardial ischemic tolerance in young and old animals via the activation of adiponectin-AMP-activated protein kinase (AMPK)-mediated signaling. However, it is unknown whether prolonged CR confers cardioprotection in a similar manner. Furthermore, little is known regarding the myocardial expression of silent information regulator 1 (Sirt1; which reportedly mediates various aspects of the CR response) with prolonged CR. Thus, 6-mo-old male Fischer-344 rats were randomly divided into ad libitum (AL) and CR groups. Six months later, isolated perfused hearts were subjected to 25 min of global ischemia followed by 120 min of reperfusion with or without IPC. CR improved the recovery of left ventricular function and reduced infarct size after ischemia-reperfusion and restored the IPC effect. Serum adiponectin levels increased, but myocardial levels of total and phosphorylated AMPK did not change with prolonged CR. Total levels of Sirt1 did not change with CR; however, in the nuclear fraction, CR significantly increased Sirt1 and decreased acetyl-histone H3. Eleven rats from each group were given N-nitro-l-arginine methyl ester in their drinking water for 4 wk before death. In these hearts, chronic inhibition of nitric oxide synthase prevented the increase in nuclear Sirt1 content by CR and abrogated CR-induced cardioprotection. These results demonstrate that 1) prolonged CR improves myocardial ischemic tolerance and restores the IPC effect in middle-aged rats and 2) CR-induced cardioprotection is associated with a nitric oxide-dependent increase in nuclear Sirt1 content.

Figures

Fig. 1.

Effect of prolonged caloric restriction (CR) on lactate dehydrogenase (LDH) and creatine kinase (CK) activity released into the perfusate during reperfusion as well as infarct size. A: CK activity. B: LDH activity. C: representative triphenyltetrazolium chloride staining. D: infarct size. AL group, ad libitum group; IPC, ischemic preconditioning. IPC(−) and IPC(+) groups indicate animals not subjected or subjected to IPC, respectively. Data are expressed as means ± SE. *P < 0.05 vs. the corresponding AL group; +P < 0.05 vs. the corresponding IPC(−) group.

Fig. 2.

Western immunoblot analyses for AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). A: representative Western immunoblots showing the expression of phosphorylated (P-)AMPK-α (Thr172) and total AMPK. B: densitometric analysis of P-AMPK-α (Thr172) and total AMPK signals. C: representative Western immunoblots showing the expression of P-ACC (Ser79) and total ACC. D: densitometric analysis of P-ACC (Ser79) and total ACC signals. Densitometric measurements of protein immunoreactivity are expressed as percentages of the average value measured in the AL group. Data are expressed as means ± SE.

Fig. 3.

Western immunoblot analyses for total silent information regulator 1 (Sirt1) protein and its subcellular distribution. A: representative Western immunoblots showing the expression of total Sirt1 protein. B: densitometric analysis of Sirt1 signals. C: representative Western immunoblots showing the expression of Sirt1, GAPDH, and p300 in each fraction. D: densitometric analysis of Sirt1 in each fraction. Densitometric measurements of protein immunoreactivity are expressed as percentages of the average value measured in the AL group. CF, cytosolic fraction; NF, nuclear fraction; m/o, months old. Data are expressed as means ± SE. *P < 0.05 vs. the corresponding AL group.

Fig. 4.

Western immunoblot analyses for histone H3 and cytochrome c as well as caspase-3 activity. A: representative Western immunoblots showing the expression of total and acetyl-histone H3 (Lys9 and Lys14) in the NF. B: densitometric analysis of acetyl-histone H3 (Lys9 and Lys14). C: representative Western immunoblots showing the expression of cytochrome c and GAPDH in each fraction. D: densitometric analysis of cytochrome c/GAPDH in the CF. E: caspase-3 activity in the CF. MF, mitochondrial fraction. Data are expressed as means ± SE. *P < 0.05 vs. the corresponding AL group.

Fig. 5.

Effect of chronic administration of N-nitro-l-arginine methyl ester (l-NAME) on systolic blood pressure, nuclear Sirt1, and CR-induced cardioprotection. A: systolic blood pressure measured by the tail-cuff method. B: representative Western immunoblots showing the expression of Sirt1 in each fraction. C: densitometric analysis of Sirt1 in the NF. D: infarct size. l-NAME(−) and l-NAME(+) groups indicate animals not treated or treated with l-NAME, respectively. Data are means ± SE. *P < 0.05 vs. the corresponding AL group; #P < 0.05 vs. the corresponding l-NAME(−) group.