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Aims: To investigate the role of FOXO1 in STAT3 activation and mitochondrial quality control in the diabetic heart. Methods: Type 1 diabetes mellitus (T1DM) was induced in rats by a single intraperitoneal injection of 60 mg · kg−1 streptozotocin (STZ), while type 2 diabetes mellitus (T2DM) was induced in rats with a high‐fat diet through intraperitoneal injection of 35 mg · kg−1 STZ. Primary neonatal mouse cardiomyocytes and H9c2 cells were exposed to low glucose (5.5 mM) or high glucose (HG; 30 mM) with or without treatment with the FOXO1 inhibitor AS1842856 (1 μM) for 24 hours. In addition, the diabetic db/db mice (aged 8 weeks) and sex‐ and age‐matched non‐diabetic db/+ mice were treated with vehicle or AS1842856 by oral gavage for 15 days at a dose of 5 mg · kg−1 · d−1. Results: Rats with T1DM or T2DM had excessive cardiac FOXO1 activation, accompanied by decreased STAT3 activation. Immunofluorescence and immunoprecipitation analysis showed colocalization and association of FOXO1 and STAT3 under basal conditions in isolated cardiomyocytes. Selective inhibition of FOXO1 activation by AS1842856 or FOXO1 siRNA transfection improved STAT3 activation, mitophagy and mitochondrial fusion, and decreased mitochondrial fission in isolated cardiomyocytes exposed to HG. Transfection with STAT3 siRNA further reduced mitophagy, mitochondrial fusion and increased mitochondrial fission in HG‐treated cardiomyocytes. AS1842856 alleviated cardiac dysfunction, pathological damage and improved STAT3 activation, mitophagy and mitochondrial dynamics in diabetic db/db mice. Additionally, AS1842856 improved mitochondrial function indicated by increased mitochondrial membrane potential and adenosine triphosphate production and decreased mitochondrial reactive oxygen species production in isolated cardiomyocytes exposed to HG. Conclusions: Excessive FOXO1 activation during diabetes reduces STAT3 activation, with subsequent impairment of mitochondrial quality, ultimately promoting the development of diabetic cardiomyopathy. [ABSTRACT FROM AUTHOR]

Diabetic hearts are more susceptible to myocardial ischemia/reperfusion (I/R) injury and less sensitive to ischemic postconditioning (IPostC), but the underlying mechanisms remain unclear. PKC β 2 is preferentially overactivated in diabetic myocardium, in which autophagy status is abnormal. This study determined whether hyperglycemia-induced PKC β 2 activation resulted in autophagy abnormality and compromised IPostC cardioprotection in diabetes. We found that diabetic rats showed higher cardiac PKC β 2 activation and lower autophagy than control at baseline. However, myocardial I/R further increased PKC β 2 activation and promoted autophagy status in diabetic rats. IPostC significantly attenuated postischemic infarct size and CK-MB, accompanied with decreased PKC β 2 activation and autophagy in control but not in diabetic rats. Pretreatment with CGP53353, a selective inhibitor of PKC β 2, attenuated myocardial I/R-induced infarction and autophagy and restored IPostC-mediated cardioprotection in diabetes. Similarly, CGP53353 could restore hypoxic postconditioning (HPostC) protection against hypoxia reoxygenation- (HR-) induced injury evidenced by decreased LDH release and JC-1 monomeric cells and increased cell viability. These beneficial effects of CGP53353 were reversed by autophagy inducer rapamycin, but could be mimicked by autophagy inhibitor 3-MA. It is concluded that selective inhibition of PKC β 2 could attenuate myocardial I/R injury and restore IPostC-mediated cardioprotection possibly through modulating autophagy in diabetes., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2020 Yafeng Wang et al.)

Background: Patients with diabetes are more vulnerable to myocardial ischemia reperfusion injury (IRI), which is involved in PKC β 2 activation and mitochondrial dysfunction. Glycine has been documented as a cytoprotective agent to attenuate diabetes-related abnormalities and reduce myocardial IRI, but the underlying mechanisms are still unclear. We determined whether glycine could attenuate high glucose- (HG-) and hypoxia/reoxygenation- (H/R-) induced injury by inhibiting PKC β 2 activation and improving mitochondrial quality in cultured H9C2 cells., Methods: H9C2 cells were either exposed to low glucose (LG) or HG conditions with or without treatment of glycine or CGP53353 (a selective inhibitor of PKC β 2) for 48 h, then subjected to 4 h of hypoxia followed by 2 h of reoxygenation (H/R). Cell viability, lactate dehydrogenase (LDH) release, mitochondrial membrane potential (MMP), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration were detected using corresponding commercial kits. Mitochondrial quality control-related proteins (LC-3II, Mfn-2, and Cyt-C) and PKC β 2 activation were detected by Western blot., Results: HG stimulation significantly decreased cell viability and SOD activity and increased LDH release, MDA production, and PKC β 2 activation as compared to LG group, all of which changes were further increased by H/R insult. Glycine or CGP53353 treatment significantly reduced the increase of LDH release, MDA production, PKC β 2 activation, and Cyt-C expression and the decrease of cell viability, SOD activity, MMP, Mfn-2 expression, and LC-3II/LC-3I ratio induced by HG and H/R stimulation., Conclusions: Supplementary glycine protects H9C2 cells from HG- and H/R-induced cellular injury by suppressing PKC β 2 activation and improving mitochondria quality.

Continuous treatment with organic nitrates causes nitrate tolerance and endothelial dysfunction, which is involved with protein kinase C (PKC) signal pathway and NADPH oxidase activation. We determined whether chronic administration with nitroglycerine compromises the protective effects of propofol against tumor necrosis factor (TNF-) induced toxicity in endothelial cells by PKC- β2 dependent NADPH oxidase activation. Primary cultured human umbilical vein endothelial cells were either treated or untreated with TNF- α (40 ng/mL) alone or in the presence of the specific PKC- β2 inhibitor CGP53353 (1 μM)), nitroglycerine (10 μM), propofol (100 μM), propofol plus nitroglycerin, or CGP53353 plus nitroglycerine, respectively, for 24 hours. TNF-α increased the levels of superoxide, Nox (nitrate and nitrite), malondialdehyde, and nitrotyrosine production, accompanied by increased protein expression of p-PKC-β2, gP91phox, and endothelial cell apoptosis, whereas all these changes were further enhanced by nitroglycerine. CGP53353 and propofol, respectively, reduced TNF-α induced oxidative stress and cell toxicity. CGP53353 completely prevented TNF- α induced oxidative stress and cell toxicity in the presence or absence of nitroglycerine, while the protective effects of propofol were neutralized by nitroglycerine. It is concluded that nitroglycerine comprises the protective effects of propofol against TNF-α stimulation in endothelial cells, primarily through PKC-β2 dependent NADPH oxidase activation.