Formation of human cardiomyocytes is impaired in a fibrotic environment: unravelling human cardiac regeneration

Abstract

Abstract Loss of myocardial tissue remains a leading cause of disease and death, as the adult heart has insufficient regenerative potential. The (pre-)clinical effects of inducing cardiac regeneration by cardiac cell therapy have been disappointing. This lack of success may result from the fact that it remains largely unclear how the receiving pathological microenvironment affects this process of cardiomyogenic differentiation of implanted cells, and thereby may (negatively) influence the therapeutic outcome. However, the tools to address this lacuna in a proper manner are lacking, as this requires tightly controllable and quantitative models of cardiomyogenic differentiation. Therefore, we have recently generated lines of conditionally immortalized human CMCs (ciCMCs) through doxycycline-dependent expression of SV40 large T antigen after genetic modification and subsequent clonal expansion. In these cells, proliferation and differentiation can be tightly controlled, allowing cardiomyogenic differentiation to be i) induced on cue, ii) precisely monitored and quantified, and iii) manipulated. The aim of this study is to improve our understanding of cardiomyogenic differentiation of guest (transplanted) cells in the context of the host (receiving) microenvironment. To create pathological microenvironments and study the effects on cardiac differentiation, co-culture experiments with human ciCMCs and cardiac fibroblasts were conducted in different ratios (10%, 30%, 60% and 90% ciCMCs). Cardiomyogenic differentiation was determined by immunostaining for cardiac specific markers and electrophysiological analysis by optical voltage mapping. After 12 days of co-culture with cardiac fibroblasts, the amount of ciCMCs that expressed the sarcomeric protein cardiac troponin T was significantly and increasingly reduced (P<0.01) in the groups with higher amounts of cardiac fibroblasts (39.4±3.9, 33±3.4, 25±1.9, 20.3±2.6, 5±1.7 for 100%, 90%, 60%, 30% and 10% ciCMCs respectively (%, mean±SD). Electrophysiological analysis showed a significantly reduced (P<0.01) conduction velocity in the co-cultures compared to the pure cultures of ciCMCs (19.1±2.1 vs 16.0±0.5, 15.8±0.9, 8.6±0.6, 5±1.67 for 100% vs 90%, 60%, 30% and 10% ciCMCs respectively (cm/s, mean±SD). However, no significant difference in conduction velocity was present between the groups with 10% and 30% ciCMCs and 30% and 60% ciCMCs present. In conclusion, a fibrotic environment has a negative effect on the formation of human cardiomyocytes as revealed by the use of ciCMCs. This not only emphasizes the need to consider the interaction between the guest (transplanted) cells and the host (receiving) microenvironment in cardiac regenerative medicine, but also offers new leads to increase the therapeutic potential of this strategy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Leiden University Fund