Acquired control over proliferation and differentiation of ventricular cardiomyocytes to break down the mechanisms of cardiac repair

Abstract Funding Acknowledgements None. Loss of myocardial tissue remains a leading cause of morbidity and mortality by causing heart rhythm disturbances and heart failure. The (pre-)clinical effects of inducing cardiac regeneration by cardiac cell therapy have been disappointing. In order to find new curative options for these disabling cardiac diseases a shift of focus towards steering the heart to regenerate itself is needed. The aim of this study was to generate a robust ventricular cardiomyocyte model in which proliferation and differentiation could be tightly controlled to investigate cardiac regeneration. To accomplish this we have generated a line of immortalized neonatal rat ventricular cardiomyocytes (nrVMs) through expression of the cell cycle regulators cyclin-dependent kinase 4 and cyclin D1 (CDK4-D1). The CDK4-D1 expressing nrVMs were positive for the proliferation marker Ki67 in contrast to control nrVMs. Cardiomyogenic differentiation could still be induced after multiple passages ( passage 10) as evidenced by immunocytochemistry and optical voltage mapping. These contractile and excitable cells were indeed positive for the cardiac-specific sarcomeric proteins alpha actinin and troponin T in a sarcomeric pattern similar to native nrVMs. Passaging of the control nrVM cultures, however, ultimately led to a senescent cell population lacking sarcomeric markers after passage 3. Electrophysiological analysis of immortalized monolayers, after inducing cardiomyogenic differentiation, showed not only uniform propagation of electrical signal throughout the cultures, but also re-entrant activity although at low frequency. In conclusion, these data show that were able to immortalize ventricular cardiomyocytes by preserving their contractile and excitable phenotype upon significant expansion. Further optimisation of this model will lead to a research model of ventricular cardiomyocytes which will give us the opportunity to unravel the process of cardiomyogenesis and uncover mechanisms which could allow us to enhance the ability of the heart to regenerate itself.