Understanding the molecular mechanisms regulating cardiac cell proliferation during the embryonic, fetal and adult life holds a paramount importance in view of developing innovative strategies aimed at inducing myocardial regeneration after cardiac damage. Previous high throughput screening studies in our laboratory identified a series of microRNAs able to trigger cardiomyocyte proliferation and stimulate cardiac regeneration after myocardial infarction.
In the first part of this project, we investigated the mechanism of action of the top ten most effective of these miRNAs, revealing an involvement of the Hippo-YAP pathway in their action. We found that all the investigated miRNAs activated YAP-mediated transcription, nuclear localization of active YAP and increased expression of YAP responsive genes. Of notice, miR-199a-3p, one of the most effective miRNAs exerted its direct effect on two mRNA targets impinging on the Hippo pathway, the inhibitory kinase TAOK1 and the E3 ubiquitin ligase, β−TrCP. Most of the miRNAs inducing proliferation (including miR-199a-3p) also modulated the dynamics of the actin cytoskeleton in the treated cardiomyocytes, which displayed a rounded shape and gross bundles of actin fibers at the cytoplasm periphery. Consistent with these observations, we found that the Cofilin2 mRNA was a direct target of four of the investigated miRNAs and that downregulation of Cofilin2 itself was sufficient to promote cardiomyocyte proliferation, activate nuclear translocation of YAP and stimulate transcription of TEAD-responsive genes.
The second part of the project was aimed at identifying small molecules exerting a mitogenic effect on neonatal cardiomyocytes through an unbiased high-throughput screening (HTS) of a library of 780 FDA-registered drugs. The neuroactive alkaloid harmine was identified as the most powerful molecule at inducing cardiomyocyte proliferation in vitro and heart regeneration after myocardial infarction in vivo. Harmine exerted its activity through the inhibition of the dual specificity phosphorylation-regulated tyrosine kinase, Dyrk1a and, again, the activation of YAP nuclear translocation.
Collectively, these results identify both YAP activation and actin cytoskeleton remodelling as major determinants of cardiomyocyte proliferation and establish the molecular basis for the development of pharmacological therapies to promote heart regeneration through the stimulation of the endogenous capacity of cardiomyocytes to proliferate
