GDF-15 Inhibits High Glucose-induced Cardiac Fibroblast Proliferation And Collagen Synthesis Through YAP

Background and purpose:Diabetic cardiomyopathy（DCM）is a cardiomyopathy that is independent of hypertension or coronary heart disease.Cardiomyocyte metabolism disorder and myocardial fibrosis participate in the occurrence of DCM,promote the occurrence of congestive heart failure,early detection of cardiac dysfunction and effective prevention of its pathological progress can significantly improve the quality of life and prognosis of diabetic patients.Cardiac cells are mainly composed of cardiomyocytes and cardiac fibroblasts.During the occurrence and development of cardiac fibrosis,the proliferation of cardiac fibroblasts and the increase of collagen secretion play an important role.GDF-15 is an endocrine hormone that is a member of the TGF-βsuperfamily.GDF-15 expression increased significantly under pathological conditions（such as cardiovascular disease,metabolic disease,cancer,etc.）and under physiological conditions（such as fasting,cold,Hypoxia,etc.）.Studies have shown that exogenous administration of GDF-15 has anti-obesity and anti-diabetic effects,but the role and mechanism of GDF-15 in diabetic cardiomyopathy are still unknown.This study aims to explore the role and mechanism of GDF-15 and YAP in the proliferation and collagen synthesis of cardiac fibroblasts induced by high glucose.Method:After the primary cardiac fibroblasts are separated and to be adhered,the cells are exposed to serum-free Low Glucose（LG）medium(5.5 mmol·L^-1 D-glucose),and the cells undergo the following treatments（1）High Glucose（HG）medium(30 mmol·L^-1 D-glucose)treatment for 24 h;（2）Exogenous GDF-15 treatment for 24 h under high glucose conditions;（3）The overexpression of YAP virus was treated for 24 hours and the YAP virus overexpression or YAP inhibitor Verteporfin（VP）was treated for 24 hours under high glucose conditions.After the treatment was completed,the following experiments were performed:CCK8 kit was performed to the proliferation activity of cardiac fibroblasts;Western Blot was used to detect the protein expression levels of cardiac fibroblasts Proliferating cell nuclear antigen PCNA,p-YAP S127,T-YAP andβ-actin;q PCR was used to detect the expression of CollagenⅠand CollagenⅢmRNA levels of cardiac fibrosis markers.Results:1.High glucose promoted cardiac fibroblast proliferation and collagen deposition.CCK8 experiment showed that cell proliferation increased significantly after high glucose treatment（P<0.001）.Western Blot showed that PCNA protein levels were significantly increased under high glucose conditions（P<0.01）,q PCR showed that high glucose treatment significantly promoted CollagenⅠand CollagenⅢmRNA levels in cardiac fibroblasts（P<0.001）.2.High glucose promotes cardiac fibroblast proliferation and collagen production through YAP.CCK8 experiment showed that the cell proliferation induced by high glucose was significantly reduced after VP treatment（P<0.001）.Western Blot results showed that PCNA protein levels were significantly reduced under VP treatment conditions（P<0.001）,while overexpression of YAP significantly increased PCNA protein levels（P<0.01）,and further increased under high glucose conditions,q PCR showed that VP treatment significantly inhibited CollagenⅠand CollagenⅢmRNA levels（P<0.01）.3.GDF-15 inhibits the proliferation and collagen deposition of cardiac fibroblasts induced by high glucose through YAP.CCK8 experiment showed that the cell proliferation induced by high glucose was significantly reduced after GDF-15 treatment（P<0.05）.Western Blot results showed that PCNA decreased after GDF-15 treatment（P<0.05）,and the phosphorylation level of YAP S127 increased after GDF-15 treatment（P<0.05）,q PCR results showed that CollagenⅠ（P<0.001）and CollagenⅢ（P<0.05）mRNA levels increased by high glucose were significantly suppressed under GDF-15 treatmentConclusion:1.High glucose can induce cardiac fibroblast proliferation and collagen production through YAP;2.GDF-15 can activate YAP S127 site phosphorylation,thereby inhibiting YAP activity,thereby exerting the function of inhibiting cardiac fibroblast proliferation and collagen synthesis.