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. 2020 Jun;24(12):6670-6679.
doi: 10.1111/jcmm.15318. Epub 2020 Apr 29.

Diabetes aggravates myocardial ischaemia reperfusion injury via activating Nox2-related programmed cell death in an AMPK-dependent manner

Chunyan Wang  1   2 , Lijie Zhu  1 , Wenlin Yuan  1 , Lingbin Sun  1 , Zhengyuan Xia  3 , Zhongjun Zhang  1 , Weifeng Yao  4
Affiliations

Diabetes aggravates myocardial ischaemia reperfusion injury via activating Nox2-related programmed cell death in an AMPK-dependent manner

Chunyan Wang et al. J Cell Mol Med. 2020 Jun.

Abstract

Cardiovascular diseases such as myocardial ischaemia have a high fatality rate in patients with diabetes. This study was designed to expose the crosstalk between oxidative stress and AMPK, a vital molecule that controls biological energy metabolism, in myocardial ischaemia reperfusion injury (I/RI) in diabetic rats. Diabetes was stimulated in rats using streptozotocin injection. Rats were separated on random into control, control + I/R, Diabetes, Diabetes + I/R, Diabetes + I/R + N-acetylcysteine and Diabetes + I/R + Vas2870 groups. Myocardial infarct size was determined, and the predominant Nox family isoforms were analysed. In vitro, the H9C2 cells were administered excess glucose and exposed to hypoxia/reoxygenation to mimic diabetes and I/R. The AMPK siRNA or AICAR was used to inhibit or activate AMPK expression in H9C2 cells, respectively. Then, myocardial oxidative stress and programmed cell death were measured. Diabetes or high glucose levels were found to aggravate myocardial I/RI or hypoxia/reoxygenation in H9C2 cells, as demonstrated by an increase in myocardial infarct size or lactate dehydrogenase levels, oxidative stress generation and induction of programmed cell death. In diabetic rat hearts, cardiac Nox1, Nox2 and Nox4 were all heightened. The suppression of Nox2 expression using Vas2870 or Nox2-siRNA treatment in vivo or in vitro, respectively, protected diabetic rats from myocardial I/RI. AMPK gene knockout increased Nox2 protein expression while AMPK agonist decreased Nox2 expression. Therefore, diabetes aggravates myocardial I/RI by generating of Nox2-associated oxidative stress in an AMPK-dependent manner, which led to the induction of programmed cell death such as apoptosis, pyroptosis and ferroptosis.

Keywords: AMPK; Nox2; diabetes; myocardial ischaemia reperfusion injury; programmed cell death.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Increase in myocardial oxidative stress and programmed cell death after myocardial I/R injury in diabetic hearts. A, The infarct size (IS) in the Con and diabetic rats determined by TTC and Evans blue staining; B, Post‐ischaemia IS expressed as percentage of IS to the area at risk (AAR); C, SOD activity during myocardial I/RI in Con and diabetic rats; D, MDA release during myocardial I/RI in Con and diabetic rats; E, 4‐HNE staining; F, Changes in relative 4‐HNE levels in Con and diabetic rats; G, The WB bands showing the protein expression of NLRP3, Cleaved caspase‐3, GPX4 and GAPDH; H, The change in protein expression levels of NLRP3 during myocardial I/RI in Con and diabetic rats; I, TUNEL staining; J, TUNEL positive/total myocytes; K, the change in protein expression levels of cleaved caspase‐3 in Con and diabetic rats; L, The change in protein expression levels of GPX4 during myocardial I/RI in Con and diabetic rats. Myocardial ischaemia reperfusion (I/R) was achieved by 30 min ischaemia and 120 min reperfusion. Data are expressed as mean ± SEM, n = 6 per group. *P < 0.05, **P < 0.01
FIGURE 2
FIGURE 2
Nox inhibition attenuates post‐ischaemic programmed cell death and cardiac injury in diabetic rats. A, WB bands depicting the protein expression of Nox1, Nox2, Nox4 and GAPDH; B, The change in the protein expression of Nox1 in Con and diabetic rats; C, The change in protein expression of Nox2 in Con and diabetic rats; D, The change in protein expression of Nox4 in Con and diabetic rats; E, 4‐HNE and TTC staining; F, The change of IS in response to treatment with or without Vas2870 in diabetic rats; G, Tn‐T release during myocardial I/RI in response to treatment with or without Vas2870 in diabetic rats; H, The relative 4‐HNE levels during myocardial I/RI in diabetic rats, with or without Vas2870 treatment; I, MDA release during myocardial I/RI in diabetic rats, with or without Vas2870 treatment; J, WB bands depicting protein expression of cleaved caspase‐3, NLRP3 and GPX4; K, SOD release during myocardial I/RI in diabetic rats, with or without Vas2870 treatment; L, The change in the protein expression of cleaved caspase‐3 during myocardial I/RI in diabetic rats, with or without Vas2870 treatment; M, The change in the protein expression of NLRP3 during myocardial I/RI in diabetic rats, with or without Vas2870 treatment; N, The change in protein expression of GPX4 during myocardial I/RI in diabetic rats, with or without VAS2870 treatment. Myocardial ischaemia reperfusion(I/R) was achieved by 30 min ischaemia and 120 min reperfusion. Data are expressed as mean ± SEM, n = 6 per group. *P < 0.05, **P < 0.01
FIGURE 3
FIGURE 3
Nox2 gene knockdown attenuated post‐hypoxic oxidative stress and programmed cell death in cardiomyocytes exposed to high glucose. A, LDH release during hypoxia reoxygenation (H/R) in normal (NG) and (HG), with or without siRNA‐mediated knockdown of Nox2; B, WB band showing protein expression of Nox2, NLRP3, Cleaved caspase‐3, GPX4 and GAPDH; C, the change in protein expression of Nox2 during H/R in NG and HG, with or without siRNA Nox2; D, The change in protein expression of NLRP3 during H/R in NG and HG, with or without siRNA Nox2; E, The change in protein expression of cleaved caspase‐3 during H/R in NG and HG, with or without siRNA Nox2; F, The change in protein expression of GPX4 during H/R in NG and HG, with or without siRNA Nox2. In the HG group, H9C2 cells were subjected 30 mmol/L glucose treatment for 48 h, H/R was achieved by 6 h hypoxia exposure and 12 h reperfusion. Data are expressed as mean ± SEM of two independent experiments, each performed in triplicates. n = 6 per group. *P < 0.05, **P < 0.01
FIGURE 4
FIGURE 4
AMPK works upstream of Nox2 in diabetic hearts. A, The change in protein expression of AMPK during myocardial I/RI in Con and diabetic rats, with or without Vas2870 treatment; B, The change in protein expression of p‐AMPK during myocardial I/RI in Con and diabetic rats, with or without Vas2870 treatment; C, The change in protein expression of AMPK during H/R in NG and HG, with or without siRNA Nox2; D, The change in protein expression of p‐AMPK during H/R in NG and HG, with or without siRNA Nox2; E, The change in protein expression of p‐AMPK during H/R in NG and HG, with or without siRNA AMPK, or agonist AICAR. Myocardial ischaemia reperfusion(I/R) was achieved by 30 min ischaemia and 120 min reperfusion; in the HG group, H9C2 cells were subjected 30 mmol/L glucose treatment for 48 h, H/R was achieved by 6 h hypoxia exposure and 12 h reperfusion. Data are expressed as mean ± SEM of two independent experiments, each performed in triplicates. n = 6 per group. * P < 0.05, **P < 0.01
FIGURE 5
FIGURE 5
AMPK attenuated post‐ischaemic myocardial injury and programmed cell death in diabetic rats. A, WB bands depicting the protein expression of AMPK, p‐AMPKA and GAPDH; B, The change in protein expression of AMPK; C, The change in protein expression of p‐AMPK; D, LDH levels; E, SOD activity levels; F, MDA levels; G, The change in protein expression of cleaved caspase‐3; H, The change in protein expression of NLRP3; I, The change in protein expression of GPX4 during H/R in NG and HG, with or without siRNA AMPK, or agonist AICAR. In the HG group, H9C2 cells were subjected to 30 mmol/L glucose treatment for 48 h, H/R was achieved by 6 h hypoxia exposure and 12 h reperfusion. Data are expressed as mean ± SEM of two independent experiments, each performed in triplicates. n = 6 per group. *P < 0.05, **P < 0.01
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