MicroRNA 29b and Cardiotonic Steroid‐Induced Cardiac Fibrosis in Adult Cardiac Fibroblasts

Abstract

Cardiotonic steroid (CTS)-induced changes in Na/K-ATPase (NKA) signaling regulates cardiac fibrosis in vitro and in vivo in chronic kidney disease. While the signaling pathways connecting CTS induced NKA signaling and fibrosis have been studied, epigenetic regulation of these processes remain poorly understood. Previously, we found decreased expression of microRNA (miR) 29b-3p and increased collagen 1A1 (COL1A1) in cardiac tissues of Sprague Dawley (SD) rats with CKD. In silico analyses revealed that this miR is a negative regulator of fibrosis. The current study used primary cultures of adult cardiac fibroblasts (CF) from SD rats to determine the effects of CTS-induced NKA signaling in mediating miR-29b and fibrosis using qPCR and western blotting. Increases in (COL1A1) mRNA greater than 1.8-fold (p<0.01) were observed in animals treated with ouabain vs control cells. Col1a1 protein expression was significantly increased by more than 1.8-fold (p<0.001) versus controls. Additionally, miR-29b was significantly decreased by more than 1.6-fold (p<0.01) versus controls. To evaluate miR-29b's role in CTS-induced changes in expression of COL1A1 in CF we transfected cells with three concentrations of miR-29b mimic. Subsequently, these cells were treated with ouabain for 24-hrs. Transfection with miR-29b mimcs successfully reversed ouabain-induced decreases in miR-29b expression and blocked ouabain-induced increases in COL1A1 expression (p>0.05). These studies demonstrate that CTS-induced NKA signaling regulates miR-29b expression, and that exogenous treatment with a miR-29b mimic can block ouabain-induced fibrosis in vitro. Supported by NIH HL-105649 and F32-DK-104615.