Role of TBX5 controlled miRNAs in Holt-Oram Syndrome: deciphering regulatory circuitries responsible for cardiac malformations

The formation of the mature vertebrate heart with separated chambers and valves involves a complex orchestration of gene expression. Numerous genes are critical for cardiac morphogenesis, although their exact functions and their integration with other cardiac regulators is still not completely understood (Harvey, 2002). The T-box gene tbx5 encodes a key transcription factor for vertebrate heart development (Goetz et al., 2006; Horb and Thomsen, 1999). Tbx5 function in the heart is gene dosage sensitive, as both haploinsufficiency and gene duplication give rise to Holt−Oram syndrome (HOS). HOS is a highly penetrant autosomal dominant disease characterized by congenital malformations of the heart and upper limbs, which are two sites of Tbx5 expression (Basson et al., 1997; Hatcher and Basson, 2001; Pierpont et al., 2007). Nonetheless, the molecular mechanisms accounting for gene dosage sensitivity are not completely clarified. Mice heterozygous for mutations in Tbx5 display many of the phenotypic abnormalities of individuals with HOS (Bruneau et al., 2001; Mori et al., 2006). Comparable defects are seen in the zebrafish Tbx5 mutant heartstrings, suggesting that Tbx5 expression and function have been conserved throughout vertebrate evolution (Ahn et al., 2002; Garrity et al., 2002). Besides TFs, microRNAs (miRNAs) play key roles in heart development and cardiac diseases (Cordes and Srivastava, 2009; Liu and Stainier, 2010; van Rooij et al., 2006) TFs and miRNAs comprise two major layers of gene regulatory networks with strictly interconnected activities: TFs control miRNA expression and many miRNA targets are TFs. There is increasing evidence that TFs and miRNAs can work cooperatively through mutual cross-regulation (Yu et al., 2008). In line with this hypothesis, in this thesis work we performed by NGS the miRNA profiling on RNA extracted from E11.5-E12.0 hearts isolated from WT, CRE, Tbx5lox/+ and Tbx5del/+ mice. These analysis allowed the identification of several miRNAs up or downregulated as a consequence of Tbx5 modulation. We focused our attention on miR-182, found to be upregulated in HOS mouse hearts. In order to functionally investigate the effect of miR-182 on heart development we decided to use zebrafish and performed both gain- and loss-of-function experiments (GOF and LOF), in transient as well as in stable deregulation. We demonstrated that miR-182 overexpression in zebrafish embryos resulted in a dose-dependent cardiac defects. In-situ hybridization experiments revealed that the miR-182 overexpression was able to affect patterning of the pools of cardiac progenitor cells during early stages of development. Furthermore, downregulation of miR-182 by morpholino microinjection was able to partially rescue the HOS phenotype in zebrafish Tbx5 knockdown embryos and in Tbx5 mutants. Investigating the effects of miR-182 overexpression on heart beating, we observed strong arrhythmias in a high percentage of miR-182 overexpressing embryos. Interestingly, the miR-182 overexpression caused an alteration of calcium flux suggesting an impact of miRNA activity on calcium handling. In line with this observation, by digital droplet PCR, we demonstrated that some calcium channel genes, identified as putative targets of miR-182, are effectively downregulated in dissected hearts of embryos microinjected with the miR-182. We also revealed that transgenic lines for the stable cardiac deregulation of miR-182 develop cardiac defects comparable with defects generated by transient miR-182 modulation.