Palmitic acid, but not high-glucose, induced myocardial apoptosis is alleviated by N‑acetylcysteine due to attenuated mitochondrial-derived ROS accumulation-induced endoplasmic reticulum stress

Abstract

Pharmacological inhibition of reactive oxygen species (ROS) is a potential strategy to prevent diabetes-induced cardiac dysfunction. This study was designed to investigate precise effects of antioxidant N‑acetylcysteine (NAC) in alleviating diabetic cardiomyopathy (DCM). Echocardiography and histologic studies were performed 12 weeks after streptozocin injection. Protein levels involved in endoplasmic reticulum stress (ERS) and apoptosis were analyzed by western blotting in diabetic hearts or high-glucose (HG, 30 mM)- and palmitic acid (PA, 300 μM)-cultured neonatal rat cardiomyocytes (NRCMs). ROS generation and structural alterations of mitochondria were also assessed. We report that NAC alleviated diabetes-induced cardiac abnormality, including restored ejection fraction (EF %), fraction shortening (FS %), peak E to peak A ratio (E/A) and reduced cardiac hypertrophy and fibrosis. These effects were concomitant with blocked ERS and apoptosis, as evidenced by inactivation of phosphorylated inositol-requiring enzyme-1α (IRE1α)/spliced X-box binding protein 1 (XBP1), phosphorylated protein kinase-like kinase (PERK)/phosphorylated eukaryotic initiation factor 2α (eIF2α) and glucose-regulated protein 78 (GRP78)/activating transcription factor 6 (ATF6α)/C/EBP homologous protein (CHOP) pathways, as well as suppressed Bcl-2-associated X protein (BAX)/B-cell lymphoma-2 (Bcl-2) and cleaved caspase 3 expressions. Mechanistically, PA mediated excessive mitochondrial ROS generation and oxidative stress, which were antagonized by NAC and Mito-TEMPO, a mitochondrial ROS inhibitor. No effects were noted by addition of apocynin, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, and NADPH oxidase 4 (NOX 4) and NOX 2 expressions were not altered, indicating that PA-induced ROS generation is independent of NADPH oxidases. Most intriguingly, HG failed to promote ROS production despite its ability to promote ERS and apoptosis in NRCMs. Collectively, these findings indicate that NAC primarily abrogates PA-mediated mitochondrial ROS through ERS and therefore alleviates myocardial apoptosis but has little effect on HG-induced cardiac injury. This uncovers a potential role for NAC in formulating novel cardioprotective strategies in DCM patients.

Fig. 1. T2DM model displayed hyperglycemia and hyperlipemia.

Type 2 diabetic rats model were established by using high-fat diet combined with a single-dose injection of STZ (35 mg/kg) and blood sample were processed for a fasting blood glucose levels (FBG), b serum insulin levels, c cholesterol levels, d triglyceride levels, e serum adiponectin levels, and f low-density lipoprotein levels. g Blood glucose concentrations during an oral glucose tolerance test (OGTT) were recorded. Data are presented as the mean±SEM; n = 5–8, *P < 0.05, **P < 0.01 vs Control group; ^#P < 0.05 vs T2DM group

Fig. 2. NAC ameliorates the cardiac dysfunction and morphological abnormity induced by type 2 diabetes.

a, b Myocardial hypertrophy and fibrosis were assessed by H&E staining and MASSON trichrome staining in control and T2DM animals with or without NAC administration; scale bar: 100 μm. c Transthoracic echocardiography was performed to observe changes in cardiac function and morphology in control and T2DM animals with or without NAC treatment. d Evaluation of the ejection fraction (EF %), e fraction shortening (FS %), f peak E to peak A (E/A) ratio, g LV internal dimension at systole (LVIDs), h LV internal dimension at diastole (LVIDd) and i ratio of heart weight to body weight (HW/BW). j Cardiac mRNA expression of atrial natriuretic peptide (ANP) and beta-myosin heavy chain (β-MHC). The data are expressed as the mean±SEM; n = 5–8, *P < 0.05, **P < 0.01 vs control group; ^#P < 0.05, ^##P < 0.01 vs T2DM group

Fig. 3. NAC mitigates the apoptosis induced by ERS in diabetic hearts.

a Representative western blot analysis of Bcl-2, Bax, caspase 3 and cleaved caspase 3. b–d Representative and quantitative images of the protein expression of ERS markers, including p-IRE1α/ sXBP1, p-PERK/p-eIF2α and the GRP78/ATF6/CHOP pathway. GAPDH was used as a loading control. All values are normalized to the GAPDH band. The data are expressed as the mean±SEM; n = 5–8, *P < 0.05, **P < 0.01 vs control group; ^#P < 0.05, ^##P < 0.01 vs T2DM group

Fig. 4. High-glucose- or PA-induced hypertrophic responses were suppressed by treatment with NAC.

a Cultured NRCMs were incubated with 30 mM glucose or 300 μM PA for 24 h. The cell surfaces were measured by staining with rhodamine-phalloidin; scale bar, 50 μm. b The mRNA levels of atrial natriuretic peptide (ANP) were determined by qRT-PCR. c The mRNA levels of beta-myosin heavy chain (β-MHC) were determined by qRT-PCR. The results were normalized to GAPDH and presented as the mean±SEM of three independent experiments. *P < 0.05, **P <0.01 vs control group; ^#P < 0.05, ^##P < 0.01 vs group-matched HG or PA-treated NRCMs

Fig. 5. NAC attenuated ERS and apoptosis in PA-treated cardiomyocytes but not in cells treated with high glucose.

a Representative western blot analysis of Bcl-2, Bax and cleaved caspase 3 in NRCMs after treatment with HG (30 mM) or PA (300 μM) with or without NAC (1 mM). b–d ERS-associated signaling pathways, such as p-IRE1α/sXBP1 (b), p-PERK/p-eIF2α (c) and GRP78/ATF6/CHOP (d) were also examined by western blotting in NRCMs. GAPDH was used as a loading control. ImageJ software was used to measure the band intensity, and each band intensity was normalized to GAPDH. e Annexin V/PI double staining was performed by flow cytometry to detect apoptosis in NRCMs. All data are presented as the mean±SEM of three independent experiments. *P < 0.05, **P < 0.01 vs control group; ^#P < 0.05, ^##P < 0.01 vs PA-treated NRCMs

Fig. 6. NAC attenuates mitochondrial ROS generation and oxidative stress in cells treated with PA.

a DCFH-DA staining was performed to determine total ROS levels in NRCMs after being treated with high glucose (30 mM) and PA (300 μM) in the presence or absence of NAC (1 mM) for 24 h; scale bar, 100 μm. b 4-HNE, NOX 4 and NOX 2 protein expressions were examined by western blotting in NRCMs. c The ultrastructure of NRCMs was observed using a transmission electron microscope (TEM). The black box indicates mitochondria and it was amplified in the rectangle; scale bars: 5 μM. d DCFH-DA staining was performed to determine total ROS levels in NRCMs after being treated with PA with or without apocynin (100 μM), Mito-TEMPO (10 μM) and NAC (1 mM) for 24 h; scale bar, 100 μm. e Mitochondrial ROS levels were determined by staining with MitoSOX after treatment with PA with or without apocynin (100 μM), Mito-TEMPO (10 μM) and NAC (1 mM) for 24 h; scale bar, 50 μm. f NOX 4 and NOX 2 protein expressions were examined by performing western blot after NRCMs were treated with PA with or without apocynin (100 μM), Mito-TEMPO (10 μM) and NAC (1 mM) for 24 h. All western blotting used GAPDH as loading control and ImageJ software was used to measure band intensity and each band intensity was normalized to GAPDH. The fluorescence was imaged and analyzed by using the Operetta® High Content Imaging System. The data were presented as mean±SEM of three independent experiments. ^*P < 0.05, ^**P < 0.01 vs control group; ^#P < 0.05, ^##P < 0.01 vs PA-treated NRCMs; ^$$P < 0.01 vs PA+apocynin-treated NRCMs

Fig. 7. Mitochondrial ROS contributes to PA-induced ERS and hypertrophy.

a GRP78, p-IRE1α, p-eIF2α, p-PERK and CHOP protein levels were detected by performing western blotting in NRCMs after being incubated with PA (300 μM) with or without apocynin (100 μM), Mito-TEMPO (10 μM) and NAC (1 mM). GAPDH was used as loading control. b Western blotting was performed to examine Bcl-2 and Bax expressions to reflect apoptosis. ImageJ software was used to measure band intensity and each band intensity was normalized to GAPDH. c, d NRCMs were incubated with 300 μM PA for 24 h and then cell surfaces were measured by staining with rhodamine-phalloidin; scale bar, 50 μm. e The mRNA levels of atrial natriuretic peptide (ANP) were determined by qRT-PCR. f The mRNA levels of beta-myosin heavy chain (β-MHC) were determined by qRT-PCR. All data were presented as mean±SEM of three independent experiments. *P < 0.05, **P < 0.01 vs control group; ^#P < 0.05, ^##P < 0.01 vs PA-treated NRCMs; ^$P < 0.05, ^$$P < 0.01 vs PA+apocynin-treated NRCMs