High-glucose-induced apoptosis, ROS production and pro-inflammatory response in cardiomyocytes is attenuated by metformi
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Ó Indian Academy of Sciences (0123456789().,-volV) (0123456789().,-volV)
High-glucose-induced apoptosis, ROS production and pro-inflammatory response in cardiomyocytes is attenuated by metformin treatment via PP2A activation GANG CHENG and LIHUAN LI* Department of Anesthesiology, Fuwai Cardiovascular Hospital (State Key Laboratory of Cardiovascular Disease, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China *Corresponding author (Email, [email protected]) MS received 25 March 2020; accepted 14 September 2020 Metformin has been shown to ameliorate diabetic cardiomyopathy. In the present research we investigated whether metformin would reduce cardiomyocyte apoptosis that was induced by high-glucose stimulation in vitro via activation of PP2A. Primary human and rat cardiomyocytes were subject to high-glucose stimulation. Okadaic acid was used to inhibit PP2A activity. Cell viability and apoptosis was assessed using CCK-8 and by flow cytometry, respectively. Release of HMGB1, TNFa or IL-6 was analyzed by ELISA. Oxidative stress was evaluated by measuring cellular ROS and mitochondrial superoxide level. PP2A activity was evaluated by Serine/ Threonine phosphatase assay system or analyzing Y307 phosphorylation level of PP2A catalytic domain (PP2Ac) by Western blot and the association between PP2Ac and a4 by co-immunoprecipitation. Activation of the NF-jB signaling pathway was assessed by detecting Ser32 phosphorylation level of IjBa as well as nuclear entry of p65 protein by Western blot. Activation of the GSK3b/MCL1 signaling pathway was assessed by detecting Ser9 phosphorylation level of GSK3b and protein level of MCL1. We found Metformin pre-treatment attenuated human and rat cardiomyocytes apoptosis, HMGB1, TNFa and IL-6 release and ROS production that were induced by high-glucose stimulation, and these effects of metformin could be blocked by okadaic acid treatment. Metformin reduced the upregulation of PP2Ac pY307 and the PP2Ac-a4 association, which was not affected by okadaic acid treatment. Metformin pre-treatment reduced NF-jB activation in human and rat cardiomyocytes apoptosis that was elicited by high-glucose stimulation, and this effect of metformin could be blocked by okadaic acid treatment. GSK3b/MCL1 is not part of metformin activating PP2A induced myocardial cell death inhibition. In conclusion, metformin reduced apoptosis, ROS production and inflammatory response in primary human and rat cardiomyocytes in vitro in a PP2A dependent manner. Keywords. Diabetic cardiomyopathy; metformin; protein phosphatase 2A; reactive oxygen species; NF-jB signaling pathway
1. Introduction Diabetic cardiomyopathy is a severe complication and a major cause of death in diabetic patients. Diabetic cardiomyopathy starts with ROS production, apoptosis, necroptosis and inflammation in diabetic cardiomyocytes in response to exceeded blood glucose, the main pathological feature of diabetes (Liang et al. 2017; Yu
et al. 2008). ROS production by the malfunct
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