N-Acetylcysteine Attenuates Diabetic Myocardial Ischemia Reperfusion Injury through Inhibiting Excessive Autophagy

Abstract

Background. Excessive autophagy is a major mechanism of myocardial ischemia reperfusion injury (I/RI) in diabetes with enhanced oxidative stress. Antioxidant N-acetylcysteine (NAC) reduces myocardial I/RI. It is unknown if inhibition of autophagy may represent a mechanism whereby NAC confers cardioprotection in diabetes. Methods and Results. Diabetes was induced in Sprague-Dawley rats with streptozotocin and they were treated without or with NAC (1.5 g/kg/day) for four weeks before being subjected to 30-minute coronary occlusion and 2-hour reperfusion. The results showed that cardiac levels of 15-F2t-Isoprostane were increased and that autophagy was evidenced as increases in ratio of LC3 II/I and protein P62 and AMPK and mTOR expressions were significantly increased in diabetic compared to nondiabetic rats, concomitant with increased postischemic myocardial infarct size and CK-MB release but decreased Akt and eNOS activation. Diabetes was also associated with increased postischemic apoptotic cell death manifested as increases in TUNEL positive cells, cleaved-caspase-3, and ratio of Bax/Bcl-2 protein expression. NAC significantly attenuated I/RI-induced increases in oxidative stress and cardiac apoptosis, prevented postischemic autophagy formation in diabetes, and reduced postischemic myocardial infarction (all p < 0.05). Conclusions. NAC confers cardioprotection against diabetic heart I/RI primarily through inhibiting excessive autophagy which might be a major mechanism why diabetic hearts are less tolerant to I/RI.

Figure 1

The effects of NAC on heart function and infract size (IS) in diabetic rats. (a) Infarct size (IS) is expressed as a percentage of the area at risk (AAR). (b) CK-MB release. Ischemia reperfusion (I/R) was achieved by 30-minute ischemia followed by 2-hour reperfusion in diabetic rats with or without NAC. Ctrl: nondiabetic control; D4w: 4-week diabetes; D4w + I/R: 4-week diabetic rats with ischemia/reperfusion; D4w + I/R + NAC: 4-week diabetic rats treated with N-acetylcysteine (NAC) and were subjected to ischemia/reperfusion. Dates are expressed as mean ± SEM (n = 6 per group). p < 0.05 versus D group before ischemia; ^∗p < 0.05, ^∗∗p < 0.01, and ns: p > 0.05 (no statistical significance).

Figure 2

Effects of NAC on plasma 15-F2t-IsoP, IL-6, and TNF-α releasing. (a) Plasma level of IL-6, (b) plasma level of TNF-α, and (c) plasma level of 15-F2t-IsoP. Ischemia reperfusion (I/R) was achieved by 30-minute ischemia followed by 2-hour reperfusion in diabetic rats with or without NAC. Ctrl: nondiabetic control; D4w: 4-week diabetes; D4w + I/R: 4-week diabetic rats with ischemia/reperfusion; D4w + I/R + NAC: 4-week diabetic rats treated with N-acetylcysteine (NAC) and were subjected to ischemia/reperfusion. Dates are expressed as mean ± SEM (n = 6 per group), ^∗p < 0.05  ^∗∗p < 0.01.

Figure 3

Effects of NAC on apoptosis. (a) Myocardial cell apoptosis assessed by TUNEL, (b) ratio of Bax/Bcl-2, and (c) expression of cleaved-caspase-3. Ischemia reperfusion (I/R) was achieved by 30-minute ischemia followed by 2-hour reperfusion in diabetic rats with or without NAC. Ctrl: nondiabetic control; D4w: 4-week diabetes; D4w + I/R: 4 weeks' diabetic rats with ischemia/reperfusion; D4w + I/R + NAC: 4 weeks' diabetic rats treated with N-acetylcysteine (NAC) and were subjected to ischemia/reperfusion. Dates are expressed as mean ± SEM (n = 6 per group), ^∗p < 0.05.

Figure 4

Effects of NAC on autophagy pathway. (a) Protein AMPK expression; (b) p-AMPK; (c) ratio of LC3 II/I; (d) protein P62 expression; (e) protein mTOR expression. Ischemia reperfusion (I/R) was achieved by 30-minute ischemia followed by 2-hour reperfusion in diabetic rats with or without NAC. Ctrl: nondiabetic control; D4w: 4-week diabetes; D4w + I/R: 4 weeks' diabetic rats with ischemia/reperfusion; D4w + I/R + NAC: 4 weeks' diabetic rats treated with N-acetylcysteine (NAC) and were subjected to ischemia/reperfusion. Dates are expressed as mean ± SEM (n = 6 per group), ^∗p < 0.05, and ^∗∗p < 0.01.

Figure 5

Changes of PTEN, Akt, and eNOS after NAC treatment. (a) Protein p-PTEN expression; (b) protein p-Akt expression; (c) protein p-eNOS expression. Ischemia reperfusion (I/R) was achieved by 30-minute ischemia followed by 2-hour reperfusion in diabetic rats with or without NAC. Ctrl: nondiabetic control; D4w: 4-week diabetes; D4w + I/R: 4 weeks' diabetic rats with ischemia/reperfusion; D4w + I/R + NAC: 4 weeks' diabetic rats treated with N-acetylcysteine (NAC) and were subjected to ischemia/reperfusion. Dates are expressed as mean ± SEM (n = 6 per group), ^∗p < 0.05.