Cardiac fibroblast mechanoresponse guides anisotropic organization of hiPSC-derived cardiomyocytes in vitro

The human myocardium is a mechanically active tissue typified by the anisotropic organization of cells and extracellular matrix (ECM). Upon injury, the composition of the myocardium changes, resulting in disruption of tissue organization and loss of coordinated contraction. Understanding how anisotropic organization in the adult myocardium is shaped and disrupted by environmental cues is thus critical, not only for unravelling the processes taking place during disease progression, but also for developing regenerative strategies to recover tissue function. Here, we decoupled in vitro the two major physical cues that are inherent in the myocardium: structural ECM and mechanical strain. We show that patterned ECM proteins control the orientation of the two main cell types in the myocardium: human cardiac fibroblasts (cFBs) and cardiomyocytes (hiPSC-CMs), despite their different mechanosensing machinery. Uniaxial cyclic strain, mimicking the local anisotropic deformation of the myocardium, did not affect hiPSC-CMs orientation. It did however induce a reorientation of cFBs, perpendicular to the strain direction, albeit this strain-avoidance response was overruled in the presence of anisotropic structural cues. These findings reveal that the mechanoresponsiveness of cFBs may be a critical handle in controlling myocardial tissue structure and function. To test this, we co-cultured hiPSC-CMs and cFBs in varying cell ratios to reconstruct normal and pathological myocardium. Contrary to the hiPSC-CM monoculture, the co-cultures adopted an anisotropic organization under uniaxial cyclic strain, regardless of the cell ratio. Together, these results identify the cFBs as a therapeutic target to mechanically restore structural organization of the tissue in cardiac regenerative therapies. SIGNIFICANCE STATEMENT Upon cardiac injury, adverse remodeling commonly leads to loss of the anisotropy that is typically found in human adult myocardium. Understanding the role of biophysical cues in shaping and disrupting the anisotropic tissue organization is essential to aid in the progress of cardiac regenerative strategies. Here, we report that the mechanoresponsiveness of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (cFBs) differs significantly, resulting in a strain-induced reorganization response for cFBs but not for hiPSC-CMs. In co-culture with varying cell ratios of cFBs and hiPSC-CMs, the co-cultures adopted an anisotropic organization upon cyclic strain administration. Thus, our study proposes the mechanoresponse of cFBs, a cell type often overlooked in cardiac regenerative strategies, as a handle to restore myocardial architecture and function. ### Competing Interest Statement The authors have declared no competing interest.