Cardiac fibrosis occurs following insults to the myocardium and is characterized by the abnormal accumulation of non-compliant extracellular matrix (ECM), which compromises cardiomyocyte contractile activity and eventually leads to heart failure. This phenomenon is driven by the activation of cardiac fibroblasts (cFbs) to myofibroblasts and results in changes in ECM biochemical, structural and mechanical properties. The lack of predictive in vitro models of heart fibrosis has so far hampered the search for innovative treatments, as most of the cellular-based in vitro reductionist models do not take into account the leading role of ECM cues in driving the progression of the pathology. Here, we devised a single-step decellularization protocol to obtain and thoroughly characterize the biochemical and micro-mechanical properties of the ECM secreted by activated cFbs differentiated from human induced pluripotent stem cells (iPSCs). We activated iPSC-derived cFbs to the myofibroblast phenotype by tuning basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-β1) signalling and confirmed that activated cells acquired key features of myofibroblast phenotype, like SMAD2/3 nuclear shuttling, the formation of aligned alpha-smooth muscle actin (α-SMA)-rich stress fibres and increased focal adhesions (FAs) assembly. Next, we used Mass Spectrometry, nanoindentation, scanning electron and confocal microscopy to unveil the characteristic composition and the visco-elastic properties of the abundant, collagen-rich ECM deposited by cardiac myofibroblasts in vitro. Finally, we demonstrated that the fibrotic ECM activates mechanosensitive pathways in iPSC-derived cardiomyocytes, impacting on their shape, sarcomere assembly, phenotype, and calcium handling properties. We thus propose human bio-inspired decellularized matrices as animal-free, isogenic cardiomyocyte culture substrates recapitulating key pathophysiological changes occurring at the cellular level during cardiac fibrosis.

Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Electronics Informatics and Bioengineering Politecnico di Milano Milan Italy

International Clinical Research Center Barcelona Spain

International Clinical Research Center St Anne's University Hospital Brno

International Clinical Research Center St Anne's University Hospital Brno; Masaryk University Faculty of Medicine Department of Biomedical Sciences Brno 62500 Czech Republic

International Clinical Research Center St Anne's University Hospital Brno; Masaryk University Faculty of Medicine Department of Biomedical Sciences Brno 62500 Czech Republic; School of Cardiovascular and Metabolic Medicine and Sciences King's College London UK

International Clinical Research Center St Anne's University Hospital Brno; School of Cardiovascular and Metabolic Medicine and Sciences King's College London UK

Optics11 Life Amsterdam The Netherlands

Unit of Vascular Biology and Regenerative Medicine Centro Cardiologico Monzino IRCCS Milan Italy

Unit of Vascular Biology and Regenerative Medicine Centro Cardiologico Monzino IRCCS Milan Italy; Dipartimento di Scienze Biomediche Chirurgiche ed Odontoiatriche Università degli Studi di Milano Milan Italy

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