MiR-101-3p Promotes Cardiac Senescence and Inflammation via Targeting Tbx20 and Bmp2 to Perturb Cardiac Homeostasis

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, with limited cardiac regeneration hindering recovery in damaged hearts. We previously demonstrated that T-box transcription factor 20 (Tbx20) and bone morphogenetic protein 2 (Bmp2) are crucial for cardiac homeostasis by promoting cardiomyocyte proliferation following endoplasmic reticulum (ER) stress. Here we showed that various stressors (ER stress, diabetes, type2 myocardial infarction, high-fat diet) over shorter and longer durations in vivo lead to distinct expression patterns of Tbx20 and Bmp2 in cardiomyocytes and fibroblasts. In vitro, stress induction resulted in similar expression patterns of Tbx20 and Bmp2, initially increasing in H9c2 cardiomyocytes before showing a sharp decline. In contrast, Bmp2 significantly increased in primary rat adult cardiac fibroblasts during increasing stress. MicroRNAs (miRNAs) are pleiotropic regulators of cardiac development and disease, and are promising therapeutic interventions for regulating cardiac regeneration. Upon delineating the cause of the differential regulation, in silico analysis revealed the presence of putative miR-101-3p binding site in the 3'UTR of tbx20 and Bmp2 inhibitor noggin (nog) gene, which was corroborated by dual-luciferase reporter assay. The expression of miR-101-3p was elevated upon prolonged stress across all the cardiac injury models. In vitro, increasing stress resulted in increased expression of miR-101-3p. MiR-101-3p agonist suppressed and antagonist elevated the expression of Tbx20 in H9c2 cardiomyocytes. Ectopic overexpression of miR-101-3p or siRNA-mediated knockdown of Tbx20 in H9c2 cardiomyocytes heightened the expression of senescence markers (p21, p16, γH₂AX). Furthermore, miR-101-3p targeted Nog under stress, indirectly raising Bmp2 and inflammatory response (TNF-α and IL6) in primary cardiac fibroblasts, thereby exacerbating cardiomyopathy. Inhibition of miR-101-3p reversed its inhibitory effect on Tbx20 and Nog. This study uncovers a novel regulatory mechanism where miR-101-3p acts as a repressor of cardiac genes to induce cardiac senescence and inflammation, positioning miR-101-3p as a therapeutic target for cardiomyopathy.