Journal
JOURNAL OF BIOSCIENCES
Volume 45, Issue 1, Pages -Publisher
INDIAN ACAD SCIENCES
DOI: 10.1007/s12038-020-00096-5
Keywords
Diabetic cardiomyopathy; metformin; protein phosphatase 2A; reactive oxygen species; NF-kappa B signaling pathway
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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 stimulationin vitrovia 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, TNF alpha 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 alpha 4 by co-immunoprecipitation. Activation of the NF-kappa B signaling pathway was assessed by detecting Ser32 phosphorylation level of I kappa B alpha as well as nuclear entry of p65 protein by Western blot. Activation of the GSK3 beta/MCL1 signaling pathway was assessed by detecting Ser9 phosphorylation level of GSK3 beta and protein level of MCL1. We found Metformin pre-treatment attenuated human and rat cardiomyocytes apoptosis, HMGB1, TNF alpha 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-alpha 4 association, which was not affected by okadaic acid treatment. Metformin pre-treatment reduced NF-kappa B 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. GSK3 beta/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 cardiomyocytesin vitroin a PP2A dependent manner.
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