Abstract
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.
Highlights
Several decades of research have given considerable insight into the early stages of heart development; the processes that drive maturation remain poorly understood
While both GMT/GMTH over-expression and our miR combo reprogram fibroblasts into cardiomyocytes they do so despite very different levels of cardiac transcription factor expression
GMT/GMTH over-expression increases the expression of the GMTH factors by more than 10,000 fold; miR combo induces GMTH expression by 1.5 to 5 fold depending upon the cardiac transcription factor [14, 17]
Summary
Several decades of research have given considerable insight into the early stages of heart development; the processes that drive maturation remain poorly understood. Mesp functions as the master regulator of cardiac development and is believed to control the expression, directly or indirectly, of the transcription factors Gata, Hand, Mef2C, Nkx, and Tbx5 [4, 5] This was demonstrated initially with MyoD; a transcription factor that was identified as an important regulator of muscle development In these early cellular reprogramming studies, over-expression of MyoD was found to convert fibroblasts into muscle cells[8, 9]. Taking cues from cardiac development, several researchers demonstrated that the exogenous over-expression of components of the cardiac transcription factor cascade such as GMT (Gata, Mef2C and Tbx5) and GMTH (GMT plus Hand2) directly reprogrammed fibroblasts into cardiomyocytes [10,11,12]. The ability of miR combo to induce maturation was blocked by cardiac transcription factor knockdown suggesting that while cardiac transcription factors are necessary for maturation, by themselves they are not sufficient to induce maturation
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