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. 2016 Oct 12;15(1):146.
doi: 10.1186/s12933-016-0460-z.

N-acetylcysteine attenuates myocardial dysfunction and postischemic injury by restoring caveolin-3/eNOS signaling in diabetic rats

Wating Su  1 , Yuan Zhang  1 , Qiongxia Zhang  1 , Jinjin Xu  1 , Liying Zhan  1 , Qiqi Zhu  2 , Qingquan Lian  2 , Huimin Liu  1 , Zhong-Yuan Xia  1 , Zhengyuan Xia  3   4 , Shaoqing Lei  5
Affiliations

N-acetylcysteine attenuates myocardial dysfunction and postischemic injury by restoring caveolin-3/eNOS signaling in diabetic rats

Wating Su et al. Cardiovasc Diabetol. .

Abstract

Background: Patients with diabetes are prone to develop cardiac hypertrophy and more susceptible to myocardial ischemia-reperfusion (I/R) injury, which are concomitant with hyperglycemia-induced oxidative stress and impaired endothelial nitric oxide (NO) synthase (eNOS)/NO signaling. Caveolae are critical in the transduction of eNOS/NO signaling in cardiovascular system. Caveolin (Cav)-3, the cardiomyocytes-specific caveolae structural protein, is decreased in the diabetic heart in which production of reactive oxygen species are increased. We hypothesized that treatment with antioxidant N-acetylcysteine (NAC) could enhance cardiac Cav-3 expression and attenuate caveolae dysfunction and the accompanying eNOS/NO signaling abnormalities in diabetes.

Methods: Control or streptozotocin-induced diabetic rats were either untreated or treated with NAC (1.5 g/kg/day, NAC) by oral gavage for 4 weeks. Rats in subgroup were randomly assigned to receive 30 min of left anterior descending artery ligation followed by 2 h of reperfusion. Isolated rat cardiomyocytes or H9C2 cells were exposed to low glucose (LG, 5.5 mmol/L) or high glucose (HG, 25 mmol/L) for 36 h before being subjected to 4 h of hypoxia followed by 4 h of reoxygenation (H/R).

Results: NAC treatment ameliorated myocardial dysfunction and cardiac hypertrophy, and attenuated myocardial I/R injury and post-ischemic cardiac dysfunction in diabetic rats. NAC attenuated the reductions of NO, Cav-3 and phosphorylated eNOS and mitigated the augmentation of O2-, nitrotyrosine and 15-F2t-isoprostane in diabetic myocardium. Immunofluorescence analysis demonstrated the colocalization of Cav-3 and eNOS in isolated cardiomyocytes. Immunoprecipitation analysis revealed that diabetic conditions decreased the association of Cav-3 and eNOS in isolated cardiomyocytes, which was enhanced by treatment with NAC. Disruption of caveolae by methyl-β-cyclodextrin or Cav-3 siRNA transfection reduced eNOS phosphorylation. NAC treatment attenuated the reductions of Cav-3 expression and eNOS phosphorylation in HG-treated cardiomyocytes or H9C2 cells. NAC treatment attenuated HG and H/R induced cell injury, which was abolished during concomitant treatment with Cav-3 siRNA or eNOS siRNA.

Conclusions: Hyperglycemia-induced inhibition of eNOS activity might be consequences of caveolae dysfunction and reduced Cav-3 expression. Antioxidant NAC attenuated myocardial dysfunction and myocardial I/R injury by improving Cav-3/eNOS signaling.

Keywords: Caveolin-3; Diabetes; Diabetic cardiomyopathy; Myocardial ischemia–reperfusion injury; N-acetylcysteine.

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Figures

Fig. 1
Fig. 1
N-acetylcysteine treatment attenuated myocardial dysfunction in diabetic rats. Control (C) or STZ-induced diabetic rats were either untreated (D) or treated with antioxidant N-acetylcysteine (1.5 g/kg/day, NAC) by oral gavage for 4 weeks. All the rats were lightly anesthetized and performed transthoracic echocardiography. a–f Heart rate (a), fractional shortening (FS) (b), fractional left ventricular posterior wall thickening (FLVPW) (c), ejection fraction (EF) (d), left ventricular isovolumic relaxation time (IVRT) (e), and the ratio of peak velocity of early and late diastolic filling (E/A) (f). All the results are expressed as mean ± SD, n = 7 per group. Differences were determined by using one-way ANOVA followed by Tukey’s test. *P < 0.05 vs. all the other groups
Fig. 2
Fig. 2
Effects of N-acetylcysteine treatment on the levels of 15-F2t-IsoP, NO, nitrotyrosine and O2 − in diabetic myocardium, as well as NO and O2 − levels in isolated cardiomyocytes. Control (C) or STZ-induced diabetic rats were either untreated (D) or treated with antioxidant N-acetylcysteine (1.5 g/kg/day, NAC) by oral gavage for 4 weeks,and adult rats cardiomyocytes were isolated and prepared. a–e In situ O2 − production in LV sections stained by DHE (a), myocardial levels of 15-F2t-IsoP (b), NO (c), nitrotyrosine (d) and O2 − in the absence and presence of L-NAME (e); f, g cardiomyocytes levels of NO (f) and O2 − (g) production in the absence and presence of L-NAME. All the results are expressed as mean ± SD, n = 7. Differences of O2 − in the absence and presence of L-NAME were determined by using two-way repeated-measures ANOVA followed by Bonferroni’s post hoc test, the others were determined by using one-way ANOVA followed by Tukey’s test. *P < 0.05 vs. all the other groups. MLU, mean light unit
Fig. 3
Fig. 3
Expression of Cav-3 and p-eNOS in diabetic hearts, and their association in isolated cardiomyocytes. a, b Representative western blot of Cav-3 expression with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control (a) and p-eNOS expression compared with total eNOS expression (b) in myocardium from the control (c), diabetic (d) and N-acetylcysteine-treated (1.5 g/kg/day, NAC) rats. c Confocal laser microscopic image of isolated cardiomyocytes from non-diabetic rats underwent standard immunofluorescence staining with Cav-3 and eNOS. d The lysates of isolated rat cardiomyocytes from various groups containing equal amount of total protein were subjected to immunoprecipitation (IP) with Cav-3 antibody (Ab) and analyzed by immunoblot (IB) with eNOS and Cav-3 antibody. All the results are expressed as mean ± SD, n = 7. Differences were determined by using one-way ANOVA followed by Tukey’s test. *P < 0.05 vs. all the other groups
Fig. 4
Fig. 4
Expression of p-eNOS and Cav-3 in cultured cardiomyocytes and H9C2 cells after various treatment in LG (5.5 mmol/L) and HG (25 mmol/L) conditions. a Representative western blot of p-eNOS (Ser1177) in comparison with total eNOS expression in cardiomyoytes exposed to HG in the presence of N-acetylcysteine (NAC, 1 mmol/L) or methyl-b-cyclodextrin (CD, 10 μmol/L); b–e Representative western blot of Cav-3 (b) and p-eNOS (Ser1177) (c), and levels of O2 − (d) and NO (e) in H9C2 cells transfected with Cav-3 siRNA or treated with NAC (1 mmol/L). All the results are expressed as mean ± SD, n = 7. Differences were determined by using one-way ANOVA followed by Tukey’s test. #P < 0.05 vs. corresponding group without siRNA treatment, *P < 0.05 vs. all the other groups
Fig. 5
Fig. 5
N-acetylcysteine attenuated myocardial ischemia–reperfusion (I/R) injury in diabetic rats. Control (C) or STZ-induced diabetic rats were either untreated (D) or treated with antioxidant N-acetylcysteine (1.5 g/kg/day, NAC) by oral gavage for 4 weeks. Then the rats were anaesthetized and subjected to myocardial I/R achieved by occluding the left anterior descending coronary artery for 30 min followed by reperfusion for 2 h. Sham-operated rats underwent the same surgical procedures without ligation. a–d infarct size (a), CK-MB (b), plasma 15-F2t-Isoprostane (15-F2t-IsoP, c), and cardiac 15-F2t-IsoP (d). All the results are expressed as mean ± SD, n = 7. Differences of infarct size among the groups were determined by using one-way ANOVA followed by Tukey’s test, the others were determined by using two-way repeated-measures ANOVA followed by Bonferroni’s post hoc test. *P < 0.05 vs. the other groups in sham or I/R conditions
Fig. 6
Fig. 6
LDH release and 15-F2t-IsoP levels in cultured cardiomyocytes and H9C2 cells subjected to HG stimulation and hypoxia/reoxygenation (H/R) insult. Isolated cardiomyocytes from non-diabetic rats were treated with or without N-acetylcysteine (NAC, 1 mmol/L) during LG (5.5 mmol/L) or HG (25 mmol/L) conditions for 36 h, or H9C2 cells were transfected with Cav-3 siRNA or eNOS siRNA and treated with or without NAC during LG and HG conditions, then exposed to 4 h of hypoxia followed by 4 h of reoxygenation. a–d LDH release (a) and 15-F2t-IsoP (b) in cultured medium of cardiomyocytes, and LDH release (c) and 15-F2t-IsoP (d) in cultured medium of H9C2 cells. All the results are expressed as mean ± SD, n = 7. Differences were determined by using two-way repeated-measures ANOVA followed by Bonferroni’s post hoc test. *P < 0.05 vs. the other groups in normoxia or H/R conditions
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