Large-scale expansion of ventricular cardiomyocytes for disease modelling and therapeutic testing

The adult mammalian heart has a limited capacity of self-renewal. Therefore, 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. Research in this field is hampered by an absence of a ventricular cardiomyocyte cell line. 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 establish proliferation of ventricular cardiomyocytes expression of the cell cycle regulators cyclin-dependent kinase 4 and cyclin D1 (CDK4-D1) was induced in mammalian ventricular cardiomyocytes by transducing these cells with a lentiviral vector encoding CDK4-D1. A cardiac-specific promotor was used for selective transduction of the cardiomyocytes. After proliferation was induced, single cell plating was used to establish monoclones. Ultimately, multiple clones were selected and characterized (n=32). In order to induce cardiomyogenic differentiation CDK-4 inhibitors were added to the differentiation medium. In five monoclones cardiomyogenic differentiation could still be induced after multiple passages (> passage 25) as evidenced by immunocytochemistry and optical voltage mapping. These cells were indeed positive for the cardiac-specific sarcomeric proteins alpha actinin and troponin T in a sarcomeric pattern similar to native cardiomyocytes (percentage ranging from 65 to 76% ± 5% of the cells). Furthermore, the percentage of cells expressing the cardiac sarcomeric proteins was higher in the monoclones than in the polyclone (65-75 ± 5% compared to 33±6%, respectively). Electrophysiological analysis of the 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. The conduction velocity of these monolayers was also increased compared to the monolayers of the polyclone (7.2 cm/s compared to 2.4 cm/s, respectively). In conclusion, these data show that we were able to immortalize ventricular cardiomyocytes by preserving their contractile and excitable phenotype upon significant expansion. Hence, this 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.