Abstract
SummaryDirect cardiac reprogramming holds great potential for regenerative medicine. However, it remains inefficient, and induced cardiomyocytes (iCMs) generated in vitro are less mature than those in vivo, suggesting that undefined extrinsic factors may regulate cardiac reprogramming. Previous in vitro studies mainly used hard polystyrene dishes, yet the effect of substrate rigidity on cardiac reprogramming remains unclear. Thus, we developed a Matrigel-based hydrogel culture system to determine the roles of matrix stiffness and mechanotransduction in cardiac reprogramming. We found that soft matrix comparable with native myocardium promoted the efficiency and quality of cardiac reprogramming. Mechanistically, soft matrix enhanced cardiac reprogramming via inhibition of integrin, Rho/ROCK, actomyosin, and YAP/TAZ signaling and suppression of fibroblast programs, which were activated on rigid substrates. Soft substrate further enhanced cardiac reprogramming with Sendai virus vectors via YAP/TAZ suppression, increasing the reprogramming efficiency up to ∼15%. Thus, mechanotransduction could provide new targets for improving cardiac reprogramming.
Highlights
Direct reprogramming generates the desired cell types from fibroblasts without passing through a stem cell state by overexpressing tissue-specific transcription factors (Sadahiro et al, 2015)
Soft extracellular matrix (ECM) Comparable with the Myocardium Promotes Cardiac Reprogramming Given that tissue elasticity of the myocardium (10–20 kPa) is much softer than that of polystyrene dishes (PS) dishes (GPa), we studied the effect of matrix stiffness on cardiac reprogramming (Berry et al, 2006; Engler et al, 2006)
We found that soft substrates (4–14 kPa) resulted in a significantly higher number of beating induced cardiomyocytes (iCMs) with a peak at 8 kPa
Summary
Direct reprogramming generates the desired cell types from fibroblasts without passing through a stem cell state by overexpressing tissue-specific transcription factors (Sadahiro et al, 2015). This new technology has wide applications in disease modeling, drug discovery, and regenerative medicine (Isomi et al, 2019; Sadahiro et al, 2018; Srivastava and DeWitt, 2016). The cardiac reprogramming process remains inefficient and iCMs generated in vitro are immature, suggesting that undefined extrinsic factors may regulate the efficiency and quality of cardiac reprogramming (Qian et al, 2012; Song et al, 2012; Srivastava and DeWitt, 2016; Yamakawa et al, 2015; Zhao et al, 2015). Previous cardiac reprogramming studies have mainly used conventional rigid polystyrene dishes (PS; GPa) that exhibit distinct physical properties from native myocardium (10– 20 kPa) (Berry et al, 2006; Engler et al, 2006); the effects of substrate stiffness and mechanotransduction on cardiac reprogramming remain elusive
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