MIR-182 is a Tbx5 effector during heart development in zebrafish

Objective: MicroRNAs, small molecules of 22-25 nt, inhibit translation of target mRNAs and with transcription factors comprise two major layers of gene regulatory networks with strictly interconnected activities. Tbx5, a dosage sensitive gene, is a pivotal player involved in heart/limbs development and its mutations are responsible of the Holt-Oram syndrome (HOS) in human, characterized by upper limb malformations and congenital heart defects (CHD)s both in morphology and electrophysiology. Methods: With the hypothesis that Tbx5 and miRNAs can work cooperatively trough mutual cross-regulation, we performed miRNA-profiling on RNA extracted from E11.5-E12.0 hearts isolated from WT and HOS mice. By a bioinformatic approach we selected the miR-182 resulted differentially expressed in HOS and able to putatively target evolutionary conserved genes related to heart development. The miR-182 was functionally tested in vivo in zebrafish with experiments of transient and stable mis-expression and by in situ hybridization analysis. Results: miR-182 was found to be up-regulated in HOS mouse phenotype. In line with this data, miR-182 overexpression in zebrafish embryos resulted in a dose-dependent cardiac defects. miR-182 overexpression decreases the pool of cardiac progenitor cells by reducing their proliferation rate during early stages of development, affects myocardial cell morphology and ventricular muscle fiber at 48 hpf. By digital droplet PCR analysis we observed that miR-182 overexpression determines the downregulation of some calcium channel genes which were putative miR-182 targets. In Tg(myl7:gCaMP) zebrafish line the miR-182 overexpression caused an alteration of calcium wave across the heart suggesting an impact of miRNA activity on calcium handling. Both transient and stable overexpression of miR-182 caused events of strong arrhythmias and a reduction of heart rate on the whole. Finally, the downregulation of miR-182 was able to partially rescue HOS phenotype in zebrafish Tbx5 knockdown embryos and in Tbx5 mutants. Conclusion: Our approach further support the importance of microRNA regulation in HOS pathology and demonstrate that miR-182 is a conserved Tbx5 effector with implications both in heart development and functions.