Abstract 1068 MicroRNA-101-3p regulates gene signatures of murine heart to induce senescence and inflammation: a plausible biomarker of cardiomyopathy

Cardiovascular diseases (CVDs) are the leading cause of death globally. Therefore early detection of the disease is pivotal in saving millions of lives. In mammalian heart, the cardiomyocytes (CM) exit cell cycle soon after birth. T-box transcription factor 20 (Tbx20) stimulates adult CM proliferation post-injury to restore homeostasis. Recently we have shown that during ER stress induced cardiomyopathy and diabetes, Tbx20 and Bmp2 signaling is upregulated with concomitant increase in CM proliferation. However, prolonged ER stress resulted in downregulation of Tbx20 whereas the expression of Bmp2 is augmented in murine heart (Das et al., J Biol Chem 2023). This led us to unravel the mechanism behind the differential regulation of Tbx20 and Bmp2 during ER stress in murine heart (approved by Institutional Animal Ethics Committee). CM and fibroblasts are the most abundant cell types present in the heart. Using real-time PCR analysis we observed higher levels of Tbx20 in CM compared to fibroblasts. On the contrary, Bmp2 inhibitor Noggin is expressed more in fibroblasts compared to CM. Since miRNAs often control gene signatures during cardiomyopathy, therefore, we hypothesized that miRNAs regulates this differential expression profile of Tbx20 and Bmp2 in murine heart during ER stress mediated cardiomyopathy and diabetes. In silico analysis revealed miR-101-3p targets both Tbx20 and Bmp2 inhibitor Noggin. Upregulation of miR-101-3p in murine heart during prolonged ER stress and diabetes is associated with concomitant decrease in expression of Tbx20. In vitro, treatment of CM and fibroblast with increasing concentration of ER stress inducer tunicamycin and prolonged hyperglycemic stress resulted in upregulation of miR-101-3p. Treatment with chemical chaperone 4-Phenylbutyric acid, a selective inhibitor of ER stress resulted in downregulation of miR-101-3p in CM as compared to cells treated with increasing concentration of tunicamycin thus potentiating that increase in the level of miR-101-3p is mediated by ER stress. Immunofluorescence analysis and immunoblotting showed decrease in the levels of miR-101-3p in vitro resulted in augmented expression of Tbx20. Since downregulation of Tbx20 resulted in decreased proliferation, therefore, we looked into the expression of senescent markers. Tbx20 downregulation is accompanied with heightened expression of senescent markers p21, p16 in murine heart as evidenced by immunoblotting and co-labelling experiments. In CM, siRNA mediated knockdown of Tbx20 resulted in increased senescence. Thus, ER stress induced augmentation of Tbx20 suppresses senescence phenotype which is reversed upon prolonging the stress. Furthermore, in fibroblasts, miRNA mediated decrease in Noggin resulted in increased expression of Bmp2 with concomitant augmentation of inflammatory markers Tnfα and Il6. Taken together, our data shows the novel function of miR-101-3p in regulating the expression of Tbx20 and Bmp2 to induce senescence and inflammation of murine heart during ER stress mediated cardiomyopathy and diabetes. Cure for CVDs are one of the important aspects of modern translational medicine, therefore development of therapeutic strategies for the treatment of adult cardiovascular diseases is invaluable. Our study showed a gradual increase in the expression of Bmp2 throughout the progression of cardiomyopathy, therefore the study establishes miR-101-3p and Bmp2 as plausible biomarkers for early detection of cardiomyopathy. The work was supported by project grant sponsored by the Department of Science and Technology-Science and Engineering Research Board to AS (DST-SERB, grant no.: CRG/2020/000348). Shreya Das is a Senior Research Fellow funded by University Grant Commission, India.