Sirtuin 3 deficiency exacerbates diabetic cardiomyopathy via necroptosis enhancement and NLRP3 activation

Abstract

Sirtuin 3 (SIRT3) is a potential therapeutic target for cardiovascular, metabolic, and other aging-related diseases. In this study, we investigated the role of SIRT3 in diabetic cardiomyopathy (DCM). Mice were injected with streptozotocin (STZ, 60 mg/kg, ip) to induce diabetes mellitus. Our proteomics analysis revealed that SIRT3 expression in the myocardium of diabetic mice was lower than that of control mice, as subsequently confirmed by real-time PCR and Western blotting. To explore the role of SIRT3 in DCM, SIRT3-knockout mice and 129S1/SvImJ wild-type mice were injected with STZ. We found that diabetic mice with SIRT3 deficiency exhibited aggravated cardiac dysfunction, increased lactate dehydrogenase (LDH) level in the serum, decreased adenosine triphosphate (ATP) level in the myocardium, exacerbated myocardial injury, and promoted myocardial reactive oxygen species (ROS) accumulation. Neonatal rat cardiomyocytes were transfected with SIRT3 siRNA, then exposed to high glucose (HG, 25.5 mM). We found that downregulation of SIRT3 further increased LDH release, decreased ATP level, suppressed the mitochondrial membrane potential, and elevated oxidative stress in HG-treated cardiomyocytes. SIRT3 deficiency further raised expression of necroptosis-related proteins including receptor-interacting protein kinase 1 (RIPK1), RIPK3, and cleaved caspase 3, and upregulated the expression of inflammation-related proteins including NLR family pyrin domain-containing protein 3 (NLRP3), caspase 1 p20, and interleukin-1β both in vitro and in vivo. Collectively, SIRT3 deficiency aggravated hyperglycemia-induced mitochondrial damage, increased ROS accumulation, promoted necroptosis, possibly activated the NLRP3 inflammasome, and ultimately exacerbated DCM in the mice. These results suggest that SIRT3 can be a molecular intervention target for the prevention and treatment of DCM.

Keywords: NLRP3; RIPK3; ROS; Sirtuin 3; diabetic cardiomyopathy; mitochondria; necroptosis.

Fig. 1. SIRT3 expression is decreased in the myocardium of the STZ-induced diabetic mice.

Eight-week-old male C57BL/6 mice were injected intraperitoneally daily with STZ (60 mg/kg, DM group) or sodium citrate buffer (control group) for 5 days. a After 12 weeks, quantification of the relative protein in the myocardium in the mice was measured using tandem mass tag (TMT) mass spectrometry, and a heat map was generated. b SIRT3 mRNA expression in the myocardium was measured by real-time PCR. c SIRT3 protein expression in the myocardium was detected by western blotting. Significance was determined by one-way ANOVA. Data are presented as the means ± SEM. **P < 0.01 vs control, n = 5.

Fig. 2. SIRT3 deficiency aggravates cardiac dysfunction in the diabetic mice.

Eight-week-old male wild-type (WT) 129S1/SvImJ mice and SIRT3-knockout (SIRT3-KO) mice were injected intraperitoneally daily with STZ (60 mg/kg, DM group) or sodium citrate buffer (control group) for 5 days. a, b The level of fasting blood glucose (FBG) was detected at different time points. c After 12 weeks, typical two-dimensional M-mode echocardiography and pulse Doppler ultrasound measurements were recorded. d Ejection fraction (EF) and fractional shortening (FS) were calculated. e The ratio of the early diastolic peak (E) to the late diastolic peak (A) of mitral valve blood flow was measured. Significance was determined by one-way ANOVA. Data are presented as the means ± SEM. ^*P < 0.05, ^**P < 0.01 vs the control group of the same genotype; ^##P < 0.01 vs the DM group of WT mice, n = 8.

Fig. 3. SIRT3 deficiency exacerbates myocardial injury in the diabetic mice.

After 12 weeks, the left ventricle of the myocardium was collected. a The sample was stained with HE and photographed. Bar = 50 μm. b The ultrastructure of the myocardium was examined with transmission electron microscopy. Bar = 2 μm (upper) and 1 μm (lower). c Lactate dehydrogenase (LDH) level in the serum was detected. d Adenosine triphosphate (ATP) level in the myocardium was measured. Significance was determined by one-way ANOVA. Data are presented as the means ± SEM. ^**P < 0.01 vs the control group of the same genotype; ^#P < 0.05, ^##P < 0.01 vs the DM group of WT mice, n = 8.

Fig. 4. SIRT3 deficiency promotes myocardial necroptosis in the diabetic mice.

After 12 weeks, the left ventricle of the myocardium was collected. a The level of superoxide anion in the myocardium was measured by DHE fluorescence probe. Bar = 75 μm. b The rate of apoptosis was assessed by TUNEL staining. Bar = 50 μm. c–f The protein expression levels of RIPK1, RIPK3, caspase 3, and MLKL in the myocardium were detected by Western blotting. Significance was determined by one-way ANOVA. Data are presented as the means ± SEM. ^**P < 0.01 vs the control group of the same genotype; ^##P < 0.01 vs the DM group of WT mice, n = 6.

Fig. 5. SIRT3 silencing exacerbates cell injury and enhances oxidative stress in the high-glucose-stimulated cardiomyocytes.

SIRT3 siRNA and nonspecific control (NC) siRNA were transfected into cardiomyocytes. a, b After 48 h, SIRT3 mRNA and protein expression levels were measured by real-time PCR and Western blotting, respectively. ^**P < 0.01 vs the NC group, n = 6. c After 4 h, the cardiomyocytes were stimulated with normal glucose (5.5 mmol/L, NG) or high glucose (25.5 mmol/L, HG) for 48 h. LDH level in the medium was measured. d ATP levels in cardiomyocytes were measured. e The mitochondrial membrane potential (Δψm) of the cardiomyocytes was measured with JC-1 staining. Bar = 50 μm. f Mitochondrial superoxide was detected with MitoSOX. Mitochondrial localization of the MitoSOX signal was confirmed by MitoTracker green. Bar = 75 μm. Significance was determined by one-way ANOVA. Data are presented as the means ± SEM. ^**P < 0.01 vs the NG-treated cardiomyocytes transfected with the same siRNA; ^##P < 0.01 vs the NC + HG-treated cardiomyocytes, n = 8.

Fig. 6. SIRT3 silencing promotes necroptosis in the high-glucose-stimulated cardiomyocytes.

SIRT3 siRNA and nonspecific control (NC) siRNA were transfected into cardiomyocytes. After 4 h, the cardiomyocytes were stimulated with normal glucose (5.5 mmol/L, NG) or high glucose (25.5 mmol/L, HG) for 48 h. a The rate of apoptosis was assessed by TUNEL staining. Bar = 75 μm. b, c RIPK1 and RIPK3 were stained with immunofluorescent Alexa Fluor 488 (green)-conjugated IgG. The nuclei were stained using DAPI (blue). Bar = 25 μm. d–g The protein expression levels of RIPK1, RIPK3, caspase 3, and MLKL in the cardiomyocytes were measured by Western blotting. Significance was determined by one-way ANOVA. Data are presented as the means ± SEM. ^*P < 0.05, ^**P < 0.01 vs NG-treated cardiomyocytes transfected with the same siRNA; ^##P < 0.01 vs NC + HG-treated cardiomyocytes, n = 6.

Fig. 7. SIRT3 deficiency promotes NLRP3 activation in the myocardium of the diabetic mice and high-glucose-stimulated cardiomyocytes.

a–c The protein expression levels of NLRP3, caspase 1, and IL-1β in the myocardium were measured by Western blotting. Significance was determined by one-way ANOVA. Data are presented as the means ± SEM. ^*P < 0.05, ^**P < 0.01 vs the control group of the same genotype; ^#P < 0.05 vs the DM group of WT mice, n = 6. d Mitochondrial NLRP3 was stained using immunofluorescent Alexa Fluor 488 (green)-conjugated IgG and colocalized with MitoTracker red . The nuclei were stained using DAPI (blue). Bar = 25 μm. e NLRP3 and caspase 1 were stained with immunofluorescent Alexa Fluor 488 (green)- and Cy3 (red)-conjugated IgG. The nuclei were stained using DAPI (blue). Bar = 25 μm.

Fig. 8. Illustration of the mechanism by which SIRT3 deficiency exacerbates diabetic cardiomyopathy.

SIRT3 deficiency aggravated hyperglycemia-induced mitochondrial damage, increased ROS accumulation, promoted necroptosis and cell rupture, possibly activated the NLRP3 inflammasome, and finally exacerbated diabetic cardiomyopathy in the mice.