The mammalian heart is composed of ~30% cardiomyocytes which have limited capacity to regenerate and ~70% non-cardiomyocytes including endothelial cells and cardiac fibroblasts. Direct reprogramming of fibroblasts into cardiomyocytes by forced expression of cardiomyogenic transcription factors, GMT (GATA4, Mef2C, Tbx5) or GHMT (GATA4, Hand2, Mef2C, Tbx5), has recently been demonstrated, suggesting a novel therapeutic strategy for cardiac repair. Despite extensive efforts, the efficiency of direct reprogramming of embryonic or adult fibroblasts into cardiomyocytes has yet to exceed 20%, or 0.1% respectively, leading many in the field to question the clinical translatability of this method.
Here, we demonstrate that pro-fibrotic signaling events governed by transforming growth factor-β (TGF-β) and Rho kinase (ROCK) are concomitantly activated in GHMT-expressing fibroblasts, leading to potent suppression of cardiac reprogramming (
Figure 1
). Remarkably, pharmacological inhibition of TGF-β, or ROCK leads to conversion of ≥ 60% of fibroblasts into highly functional cardiomyocytes, displaying global cardiac gene expression, spontaneous contractility, action potentials and calcium transients. Furthermore, inhibition of TGF-β, or ROCK dramatically enhances the kinetics of cardiac reprogramming, with spontaneously contracting cardiomyocytes emerging in less than two weeks, as opposed to 4 weeks with GHMT alone. These findings provide new insights into the molecular mechanisms underlying cardiac conversion of fibroblasts, and should enhance efforts to generate cardiomyocytes for clinical applications.
Kinetics of Recombinant Adeno-Associated Virus-Mediated Gene Transfer