ECM proteins regulate microRNA-mediated direct reprogramming of fibroblasts into cardiomyocytes via YAP signaling

Direct cardiac reprogramming represents a promising strategy to regenerate damaged myocardium by converting cardiac fibroblasts into induced cardiomyocytes (iCMs). Transient delivery of a four-microRNA cocktail (miRcombo: miR-1, miR-133, miR-208, and miR-499) has been shown to activate cardiac transcriptional programs in adult human cardiac fibroblasts (AHCFs). However, in vitro reprogramming efficiency remains limited compared to significantly higher outcomes observed in vivo, suggesting that microenvironmental cues present in the native myocardium play a crucial role in facilitating lineage conversion. This study investigated how extracellular matrix (ECM) proteins modulate miRcombo-mediated reprogramming.

An in vitro cardiac ECM termed "biomatrix" was developed and characterized from long-term cultured AHCFs. An optimized decellularization protocol was applied to preserve major ECM components, including laminin, fibronectin, and collagen type I, while minimizing residual DNA content. Lipoplexes composed of [2-(2,3-didodecyloxypropyl)-hydroxyethyl] ammonium bromide (DE) and dioleoyl phosphatidylethanolamine (DOPE) were used to transiently transfect AHCFs with miRcombo. Cells were cultured on coatings of individual ECM proteins (laminin, fibronectin, collagen I) or biomatrix to assess the influence of cell-substrate interactions on reprogramming efficiency. Analyses were conducted at 7 and 15 days post-transfection.

Biomatrix significantly enhanced reprogramming efficiency, yielding approximately 20% cardiac Troponin T (cTnT)⁺ cells compared to other substrates. Gene expression analyses demonstrated marked upregulation of cardiac markers TNNT2, ACTC1, and CACNA1C in biomatrix-cultured cells. Structural assessment revealed improved cytoskeletal alignment and sarcomeric organization on laminin and biomatrix, whereas fibronectin and collagen I supported poorer structural maturation. At 3 days post-seeding, fibronectin and collagen I promoted higher proliferation rates and increased nuclear localization of YAP, while laminin and biomatrix reduced YAP activation, favoring cardiac transdifferentiation over proliferation.

These findings demonstrate that ECM biochemical cues are key regulators of direct cardiac reprogramming. Laminin- and biomatrix-enriched microenvironments enhance miRcombo-mediated iCM induction efficiency in vitro, potentially by modulating YAP signaling and balancing proliferation versus transdifferentiation. This study highlights the importance of recapitulating native cardiac microenvironmental signals to improve the efficacy of direct cardiac reprogramming strategies.