Formation of human cardiomyocytes is impaired by fibroblastic paracrine signalling: breaking down the mechanisms involved in rebuilding the heart

Abstract To date (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 cardiac differentiation of implanted cells, and thereby may (negatively) influence the therapeutic outcome. We have recently generated lines of conditionally immortalized human CMCs (ciCMCs) through doxycycline-dependent expression of SV40 large T antigen. In these cells, proliferation and differentiation can be tightly controlled. The aim of this study is to improve our understanding of cardiac differentiation of transplanted cells in the context of the receiving microenvironment from a paracrine perspective. To investigate the role of fibroblastic paracrine signalling on cardiomyogenic differentiation, transwell experiments were conducted. Cardiac differentiation was induced in human ciCMCs while cardiac fibroblasts were cultured in the top inserts in different ratios (human ciCMCs:cardiac fibroblasts 1:1, 2:1 and 3:1). Cardiomyogenic differentiation was determined by immunostaining for cardiac specific markers, quantitative RT-PCR, and electrophysiological analysis. After 12 days of differentiation, under the influence of the fibroblastic paracrine signaling, the amount of ciCMCs that expressed the sarcomeric protein α-actinin and myosin light chain 2a was significantly and increasingly reduced (P<0.01) in the groups cultured under the influence of the cardiac fibroblasts (4.8±0.6, 8.1±1.9, 22.4±4.2 for 1:1, 2:1 and 3:1 human ciCMCs:cardiac fibrolasts respectively [%, mean±SD]) while in absence of the cardiac fibroblasts (control group) 41% (±9.8) human ciCMCs were formed. In corroboration with these results, quantitative RT-PCR showed significant reduction (P<0.05) of all cardiac specific markers on a mRNA level in 1:1 human ciCMC: cardiac fibroblasts cultures compared to the control group. Electrophysiological analysis showed a significantly reduced (P<0.01) conduction velocity in the group cultured under the influence of the highest amount of cardiac fibroblasts compared to the control group (13.1±1.0 vs 10.6±0.9 control group vs 1:1 human ciCMCs:cardiac fibroblasts [cm/s, mean±SD]). Additionally, action potential duration was significantly prolonged (P<0.05) in the 1:1 human ciCMC:cardiac fibroblast cultures compared to the control group (93.6±4.8 vs 129.9±3.5 control group vs 1:1 human ciCMCs:cardiac fibroblasts [ms, mean±SD]). In conclusion, fibroblastic paracrine signalling has a negative effect on the structural and electrical differentiation of human cardiomyocytes. This not only emphasizes the need to consider the interaction between the transplanted cells and the 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