ECM proteins regulate microRNA-mediated Direct Reprogramming of Fibroblasts into Cardiomyocytes via YAP signaling

Gerardina Ruocco^1,2Letizia Nicoletti^1,2,3Martina Coletto^1,2Alessia Toccaceli^4,5Valeria Chiono^1,2,3Camilla Paoletti^1,2*

• ¹Department of Mechanical and Aerospace Engineering (DIMEAS), Politecnico di Torino, Corso Duca degli Abruzzi 24, Torino, Italy
• ²The Interuniversity Center for the Promotion of the 3Rs Principles in Teaching and Research, Centro 3R, Torino, Italy
• ³PoliRNA srl, Via Vincenzo Vela, 42, 10128, Torino, Italy
• ⁴University of Barcelona, Department of Physiological Science, Campus Bellvitge, Hospitalet de Llobregat, Barcelona, Spain
• ⁵Instituto de Investigaciòn Biomédica de Bellvitge (IDIBELL), Barcelona, Spain

Direct cardiac reprogramming offers 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 successfully activate cardiac transcriptional programs in adult human cardiac fibroblasts (AHCFs). However, reprogramming efficiency achieved in vitro remains limited, while significantly higher outcomes are reported in vivo, suggesting that microenvironmental cues present in native myocardium This is a provisional file, not the final typeset article play a key role in facilitating lineage conversion. In this study, we investigated how extracellular matrix (ECM) proteins modulate miRcombo-mediated reprogramming. We developed and characterized an in vitro cardiac ECM named "biomatrix" derived from long-term cultured AHCFs. Following an optimized decellularization protocol, biomatrix retained the main ECM proteins, including laminin, fibronectin, and collagen type I, with minimal DNA content. Lipoplexes based on [2-(2,3-didodecyloxypropyl)-hydroxyethyl] ammonium bromide (DE) and dioleoyl phosphatidylethanolamine (DOPE) were used to transiently transfect miRcombo to AHCFs in vitro. Single ECM proteins (laminin, fibronectin and collagen I) and biomatrix coatings were used as culture substrates to underscore how cell-substrate interactions influence miRcombo-mediated reprogramming. Analyses at 7 and 15 days showed biomatrix enhanced reprogramming efficiency, with a higher percentage cardiac Troponin T (cTnT)⁺ cells (~20%) compared with other coatings. Gene expression analysis confirmed the significant upregulation of cardiac markers TNNT2, ACTC1, and CACNA1C in biomatrix-cultured cells. Structural maturation showed well-organized cytoskeletal alignment on laminin and biomatrix, whereas fibronectin and collagen I supported poor sarcomeric organization. Interestingly, at 3 days post cell-seeding, fibronectin and collagen I coatings promoted higher proliferation and nuclear YAP localization, while laminin and biomatrix reduced YAP activation, favoring cardiac transdifferentiation over proliferation. These findings suggest that ECM biochemical cues are key regulators of direct cardiac reprogramming and highlight that biomatrix-and laminin-enriched microenvironments can boost iCM induction efficiency in vitro.