Induced cardiomyocyte maturation: Cardiac transcription factors are necessary but not sufficient.

The process by which fibroblasts are directly reprogrammed into cardiomyocytes involves two stages; initiation and maturation. Initiation represents the initial expression of factors that induce fibroblasts to transdifferentiate into cardiomyocytes. Following initiation, the cell undergoes a period of maturation before becoming a mature cardiomyocyte. We wanted to understand the role of cardiac development transcription factors in the maturation process. We directly reprogram fibroblasts into cardiomyocytes by a combination of miRNAs (miR combo). The ability of miR combo to induce cardiomyocyte-specific genes in fibroblasts was lost following the knockdown of the cardiac transcription factors Gata4, Mef2C, Tbx5 and Hand2 (GMTH). To further clarify the role of GMTH in miR combo reprogramming we utilized a modified CRISPR-Cas9 approach to activate endogenous GMTH genes. Importantly, both miR combo and the modified CRISPR-Cas9 approach induced comparable levels of GMTH expression. While miR combo was able to reprogram fibroblasts into cardiomyocyte-like cells, the modified CRISPR-Cas9 approach could not. Indeed, we found that cardiomyocyte maturation only occurred with very high levels of GMT expression. Taken together, our data indicates that while endogenous cardiac transcription factors are insufficient to reprogram fibroblasts into mature cardiomyocytes, endogenous cardiac transcription factors are necessary for expression of maturation genes.