(H-289) Mechanical Resistance to Micro-Tissue Contractility unveils early Structural and Functional Pathology in iPSC Models of Hypertrophic Cardiomyopathy

Introduction:: Hypertrophic cardiomyopathy (HCM) is an inherited cardiac disease that occurs in up to 1 in 200
individuals and is the most common cause of sudden death in the young¹. HCM shares many similarities with
non-genetic, hypertension induced overload cardiomyopathy, but treatments that reverse pathology of overload
cardiomyopathy are ineffective in patients with late-stage HCM². To understand this discrepancy and create an
in vitro platform for developing future therapies, our group developed high-throughput micro-heart muscle
arrays (μHM) using genetically defined cardiomyocytes derived from human induced pluripotent stem cells
(hiPSC). We formed μHM on substrates with varying stiffness^3,4, to understand the impact of mechanical
resistance to contractility on structural abnormalities and pathophysiology on hiPSC-cardiomyocytes (hiPSCCM)
harboring HCM-associated loss-of-function mutations in Myosin Binding Protein C (MYBPC3).

Materials and Methods:: Sylgard 184 stencils were cast off high-resolution 3D-prints (FormLabs) via agar intermediates⁵.
Stencils were physically attached to soft elastomeric substrates grafted covalently with fibronectin³. Substrates
were created by blending Sylgard 527 with Sylgard 184 to yield elastic moduli from 0.4 to 114 kPa^3,4. WTC
(Coriell) hiPSC and isogenic hiPSC harboring heterozygous MYBPC3 knockout mutations (MYBPC3+/-)⁵.
hiPSC-CM were generated through small Wnt signaling manipulation^3-7 and seeded into stencils to form μHM
(Fig. 1a). In some cases, isogenic iPSC-derived epicardial fibroblast were derived by passaging cells with accutase at the mesoderm stage of differentiation, and expanding in the presence of fibroblast growth factor and TGF-β inhibitors⁸. Cardiac fibroblasts were seeded at either 5 or 20 percent volume fraction into μHM, with the remainder of cells being hiPSC-CM.
On μHM day 10, electrophysiology and contractility were assessed optically using BeRST-1 voltage
sensitive dye5, GCaMP6f (Ca²⁺) and fluorescent beads embedded into the substrates via custom Matlab
algorithms^5,6,9. Tissue and cellular structure were assessed via confocal micrographs of 12μm cryosections.

Results, Conclusions, and Discussions:: μHM of both genotypes exhibited organized, aligned sarcomeres. While hiPSC-CM in 2D culture failed to
exhibit robust sarcomere localization of the Ca2+ binding troponin isoform, troponin C (TNNC1), culture in μHM rescued TNNC1 localization. While gross sarcomere alignment did not differ between genotypes, MYBPC3^+/- μHM exhibited micro-scale troponin disorganization and cellular hypertrophy (Fig. 1B).

These structural changes in MYBPC3^+/- μHM were associated with energetically inefficient, prolonged contraction kinetics, along with prolonged action and Ca²⁺ transients (Fig. 1C,D). Ca2+ handling pathology in MYBPC3^+/- μHM was further exacerbated as substrate stiffness increased. Mechanistic studies revealed that pathological calcium handling in the HCM-micro-tissues was neither related to dysfunctional SERCA nor excessive calcium buffering at myofilaments. Instead, studies with the channel blocking drug verapamil suggested that excessive L-type calcium channel activity underlies the pathologic calcium handling, and potentially the hypertrophy we observed in MYBPC3^+/- μHM.

These studies highlight a critical role for mechanical loading as a potential trigger for hypertrophic cardiomyopathy in genotype-positive individuals. Ongoing studies are focused on the role played by cardiac fibroblasts in this hypertrophic response, and in probing the response of iPSC-cardiomyocytes and fibroblasts within the micro-tissues to magnetically-induced dynamic changes in substrate rigidity.

Acknowledgements (Optional): : We thank Dr. Evan Miller (UC Berkeley) for providing BeRST-1 dye. This work was supported by the Departments of Biomedical Engineering and Mechanical Engineering and Material Science at Washington University in St. Louis, the American Heart Association (19CDA34730016 to NH, predoctoral fellowship 828938 to JG) and the National Institutes of Health (R01 HL159094 to NH).

References (Optional): : 1. Watkins et al. New Eng. J. Med. 2011. 2. Teekakirikul et al. J Clin Invest. 2010. 3. Guo et al. ACS Biomater Sci Eng. 2020.
4. Guo et al. Cell Mol Bioeng. 2021. 5. Simmons et al. BioRxiv 2022. 6. Ribeiro et al. Circ Res. 2017.
7. Lian et al. PNAS 2011. 8. Zhang et al. Circ. Res. 2019. 9. Oguntuyo, Schuftan et al. Tissue Eng. C. 2022.