Development of a genetic method to visualize cardiomyocyte renewal in vivo

Most human heart diseases (ischemia, pressure and volume overload, infection, inflammation, congenital disorders) result in a loss of cardiomyocytes. Any cardiac injury is healed by scar and fibrosis, which expose the heart to a further risk of failure. This response can be attributed to the poor regeneration capacity of the adult heart: in contrast to other tissues, the contractile apparatus of the heart is not replaced by new cells either differentiating from a pluripotent cell niche or deriving from symmetrical division of existing cardiomyocytes. Consistently, all currently available treatments aim at either preserving the function of the surviving myocardium or replacing its contractile function by mechanical devices. Although recent work has provided convincing and exciting evidence of a robust regenerative potential of the mammalian heart, this appears to be strictly restricted to the prenatal and perinatal window. To what extent cardiac regeneration also occurs in adulthood and/or could be stimulated by therapeutic interventions is still a matter of investigation. A major limit in this research field is the lack of tools and strategies to properly investigate the process of cardiomyocyte renewal. The work presented in this thesis describes a new genetic method, which we named CycleTrack, able to provide a cumulative and accurate estimate of cardiomyocyte division in vivo. This novel method is based on the expression, in cardiomyocytes, of the Cre recombinase under the control of a 312-bp fragment of the Cyclin B2 promoter, which is exquisitely sensitive to cell proliferation. When an AAV9 vector expressing this construct is delivered to Z/EG mice, carrying a floxed EGFP allele that is expressed upon Cre-mediated recombination, only cardiomyocytes traversing G2/M become irreversibly labelled. CycleTrack has allowed us to visualize cardiomyocyte turnover in neonatal mice with or without apical resection and in adult mice in normal conditions and after myocardial infarction, as well as has provided visual and quantitative evidence of increased cardiomyocyte turnover upon cardiac injection of microRNAs (miR-199a-3p and miR-590-3p) that our previous work has shown to induce cardiac regeneration. This method stands as a promising tool for the identification of proregenerative molecules in vivo.