Mechanical constraint model to understand remodeling in beating cardiac microtissues

In the mechanically active myocardium, beating cardiomyocytes (CMs) and cardiac fibroblasts (cFBs) are linearly arranged, surrounded by an anisotropic collagen matrix to enable eletromechanical coupling between cells and aid in their coordinated contraction. Upon injury, ischemia results in a massive loss of CMs and remodeling into disorganized fibrotic tissue. Disruption of this highly organized structure does not only result in impaired coordinated contraction but also in compromised differentiation, matrix remodeling and mechanotransduction. Understanding the remodeling processes in beating tissues is therefore essential to (re)engineer structural organization in cardiac tissues in vivo and in vitro. Here, we generated microscale 3D mechanical constraint models of cFBs and hPSC-CMs within collagenous matrices. Micropillars of polydimethylsiloxane were used to constrain remodeling while simultaneously reporting tissue forces during this process. After two days of biaxial tissue formation, constraints were removed in one direction in microtissues consisting of 1) cFBs alone, 2) cFBs in co-culture with beating hPSC-CMs, and 3) cFBs in co-culture with non-beating hPSC-CMs to assess the effect of CM contraction on remodeling towards anisotropic tissue structure. By concurrent use of a viable collagen probe and fluorescently labeled hPSC-CMs, both cell and collagen organization can be followed over time in the same sample. Moreover, tissue contraction forces can be quantified using finite element modeling of the micropillar deflections and cardiac marker expression can be analyzed using confocal microscopy. This study presents an approach to study the dynamic relationship between structural organization, tissue forces, and cellular phenotype. We demonstrated that removal of mechanical constraints in one direction enabled remodeling from disorganized towards aligned cardiac microtissues. Next steps are to systematically compare the remodeling regimes of cardiac microtissues with and without beating CMs and assess whether regaining anisotropy increases the coordinated contraction in these microtissues.