High-glucose-induced apoptosis, ROS production and pro-inflammatory response in cardiomyocytes is attenuated by metformin treatment via PP2A activation

ABSTRACT

Metformin has been shown to ameliorate diabetic cardiomyopathy. In the present research we investigatedwhether metformin would reduce cardiomyocyte apoptosis that was induced by high-glucose stimulation in vitrovia activation of PP2A. Primary human and rat cardiomyocytes were subject to high-glucose stimulation. Okadaicacid was used to inhibit PP2A activity. Cell viability and apoptosis was assessed using CCK-8 and by flowcytometry, respectively. Release of HMGB1, TNFa or IL-6 was analyzed by ELISA. Oxidative stress wasevaluated 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-jBsignaling pathway was assessed by detecting Ser32 phosphorylation level of IjBa as well as nuclear entry of p65protein by Western blot. Activation of the GSK3b/MCL1 signaling pathway was assessed by detecting Ser9phosphorylation level of GSK3b and protein level of MCL1. We found Metformin pre-treatment attenuatedhuman and rat cardiomyocytes apoptosis, HMGB1, TNFa and IL-6 release and ROS production that were inducedby 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 byokadaic acid treatment. Metformin pre-treatment reduced NF-jB activation in human and rat cardiomyocytesapoptosis that was elicited by high-glucose stimulation, and this effect of metformin could be blocked by okadaicacid 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 andrat cardiomyocytes in vitro in a PP2A dependent manner.