P324Regulation of scn5a by microRNAs: miR-219 modulates scn5a transcript expression and cardiac rhythm in mice

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The human action potential is initiated by a fast-activating fast-inactivating Na+ current modulated by Nav1.5 channel encoded by SCN5A, in association with its β1 subunit encoded by SCN1B. The role of Nav1.5 in the etiology of many cardiac diseases strongly suggests that proper regulation of cell biology and function of the channel is critical for normal cardiac function. Hence, it is not surprising that numerous recent studies have focused on the regulatory mechanisms of its biosynthetic and degradation processes as well as its subcellular localization. However, the post transcriptional regulation by microRNAs remains unexplored. In this study we investigate the role of microRNAs on the post-trancriptional regulation of SCN5A/Nav1.5.

Functional studies in HL1 cardiomyocytes and luciferase assays in fibroblasts demonstrate that SCN5A is directly (miR-98, miR-106, miR-200, miR-219) and indirectly (miR-125 and miR-153) regulated by multiple microRNAs displaying distinct time-dependent profile and differentially expressed during cardiogenesis. Among all, only miR-219 increased Scn5a expression level, impaired Nav1.5 subcellular localization and altered contraction rhythm of HL1 cardiomyocytes. Transcription inhibition experiments deduced that miR-219 stabilized Scn5a transcript increasing thus Nav1.5 protein quantity and the current amplitude and shifting the voltage-dependent activation of INa in HL-1 cells as recorded by Patch-Clamp. Co-transfection experiments, demonstrated that miR-219 and miR-200 have an opposite, independent and additive effect modulating Scn5a gene expression. In vivo miR-219 injection did not affect normal mouse cardiac rhythm but it rescued it from the bradycardiac effects of Flecaine intoxication. Chimeric miR-219 RNA molecules fine-tune the modulation of Scn5a as demonstrated in HL1 cells and confirmed by luciferase assays. Loss of function miR-219 molecules injection increase the RR interval and correct the bradycardiac effect of flecaine once administrated together as demonstrated by ECG recording in mice.

This study demonstrates the involvement of multiple microRNAs on the regulation of SCN5A. Particularly, miR-219 increased Nav1.5 protein quantity by stabilizing its transcript increasing thus the cellular sodium gating. In vivo, miR-219 over-dose did not affect the normal heart rhythm but it modulated the effect of flecaine. Fine-tune modulation can be achieved by discrete altering a reduced number of nucleotides within the miRNA molecules. Thus, our data suggest that microRNAs, such as miR-219 constitute promising therapeutical tools to treat sodium cardiac arrhythmias.

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