hiPSC-derived cardiac fibroblasts dynamically enhance the mechanical function of hiPSC-derived cardiomyocytes on an engineered substrate

Mitchell Josvai^1,2Jodi Lawson^1,2Harshal Kanade^1,2Meghana Kalluri^1,2Jianhua Zhang³Alana Stempien^1,2Lee L Eckhardt³Timothy J. Kamp^3,4Wendy C Crone^1,2,5,6*

• ¹Department of Biomedical Engineering, College of Engineering, University of Wisconsin-Madison, Madison, United States
• ²Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin, United States
• ³Division of Cardiovascular Medicine, Department of Medicine, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States
• ⁴Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States
• ⁵Department of Mechanical Engineering, College of Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States
• ⁶Department of Nuclear Engineering and Engineering Physics, University of Wisconsin-Madison, Madison, United States

Cardiac fibroblasts (CFs) deposit and turnover the extracellular matrix (ECM) in the heart, as well as secrete soluble factors that play critical roles in development, homeostasis, and disease. Coculture of CFs and human induced pluripotent stem cell (iPSChiPSC)-derived cardiomyocytes (CMs) enhances CM mechanical output, yet the mechanism remains unclear. Here, we use an in vitro engineered platform to compare the effects on CM mechanical function of direct CM-CF Coculture and soluble signaling alone through CF Conditioned Medium to a CM Only monoculture. Mechanical analysis is performed using digital image correlation (DIC) and custom software to quantify the coordination and organization of CM contractile behavior. CM-CF Coculture induces larger CM contractile strains, and an increased rate of spontaneous contraction compared to CM Only. Additionally, CM-CF Cocultures have increased contractile anisotropy and myofibril alignment and faster kinetics. The paracrine effects of fibroblast conditioned medium (FCM) are sufficient to induce larger contractile strains and faster contraction kinetics with these effects remaining after the removal of FCM. However, FCM does not influence CM spontaneous rate, contractile alignment, anisotropy, or relaxation kinetics compared to CM Only control. These data suggest that iPSChiPSC-CFs exert dynamic and multifactorial effects on the mechanical function of iPSChiPSC-CMs and highlight the importance of CFs in both the native heart and in vitro cardiac models. Further, this work demonstrates the applicability of the cocultureconditioned mediummonoculture paradigm to decouple the effects of paracrine factor and cell-cell signaling on iPSChiPSC-CM mechanical function and maturation.