Matrix rigidity and cardiac reprogramming

Direct cardiac reprogramming from fibroblasts holds great potential for disease modeling, drug screening, and regeneration. However, cardiac reprogramming remains inefficient in vitro, and induced cardiomyocytes (iCMs) generated in vitro are less mature than those in vivo, suggesting undefined biophysical factors may inhibit cardiac reprogramming. Previous studies mainly used conventional polystyrene dishes, and thus the effect of matrix rigidity on cardiac reprogramming remains unclear. Here, we developed a Matrigel-based hydrogel culture system to determine the effect of matrix rigidity and mechanotransduction on cardiac reprogramming. We found that soft matrix rigidity comparable to myocardium greatly enhanced cardiac reprogramming in combination with Gata4, Hand2, Mef2c, and Tbx5. Mechanistically, soft matrix enhanced cardiac reprogramming via inhibition of Rho/ROCK, actomyosin, and YAP/TAZ pathway, and suppression of fibroblast program, which were activated on rigid substrate. Intriguingly, inhibition of YAP/TAZ further suppressed integrin-mediated signaling to create a positive feedback loop for robust reprogramming. Thus, mechanotransduction may represent a new target for cardiac reprogramming. To identify the effect of ECM stiffness on cardiac reprogramming, we compared the global gene expression patterns of retroviral GHMT-transduced fibroblasts on 8 kPa (similar to native myocardium) and 126 kPa (similar to collagenous bone) hydrogels. RNA was extracted 2 weeks after transduction. Arrays were performed using Clariom S Mouse Arrays. Microarray analyses were performed in duplicate, using independent biological samples.