miR-208A Targeted Suppression of PDE4D Directly Enhances Myocyte Contractile Function via PKA-Mediated Phosphorylation of cTnI and PLN

Molecular inotropy refers to cardiac myocyte contractile status that can be titrated, positively or negatively, to affect overall heart pump performance. Although inotropic drugs have been in clinical practice for many decades, there is an urgent need to discover new inotropes with unique mechanisms of action for the treatment of heart failure. Here, we investigate the prospect of micro-RNAs to directly modify downstream inotropic signaling pathways for improving contractile function. We focused on miR-208a owing in part to its known restricted expression profile in the myocardium. Results show that acute miR-208a expression, at 4-5 fold over endogenous miR-208a and independent of altered host myosin gene expression, confers significant positive inotropy and faster relaxation compared to miR-208a-mutant and untreated control adult cardiac myocytes (P<0.05). Using in vitro cardiac stress testing, miR-208a amplified the inotropic and relaxation responses to increased stimulation frequency. MiR-208a also promoted fast calcium transient decay with no change in peak calcium accounting in part for enhanced relaxation. To gain insight into mechanism, we analyzed in silico putative miR-208a targets, focusing on potential inotropic signaling targets. Interestingly, we provide evidence that miR-208a has a direct effect to negatively regulate expression of PDE4D, but does not affect PDE5A in myocytes. Consistent with these findings, phosphorylation of cTnI and PLN at PKA sites was increased in myocytes after acute miR-208a expression. Taken together, we show for the first time that miR-208a confers positive inotropy and enhances relaxation in myocytes by PKA mediated phosphorylation of cTnI and PLN through a mechanism of direct suppression of PDE4D. Because heart failure is associated with decreased phosphorylation of cTnI and PLN, miR-208a may represent a new therapeutic modality for enhancing ventricular myocyte performance via the PDE4D-cAMP-PKA signaling pathway.