Abstract P1121: Substrate Rigidity And Mybpc3 Genotype Regulate Development Of Hypertrophic Cardiomyopathy Structural And Functional Phenotypes In Human Ipsc-derived Micro-heart Muscle Arrays

Abstract

Introduction: We hypothesized that mechanical loading is a key factor in the early stages of hypertrophic remodeling associated with pathogenesis of hypertrophic cardiomyopathy (HCM). We tested this hypothesis in vitro with human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) bearing heterozygous loss-of-function mutations in Myosin Binding Protein C (MYBPC3). Cardiomyocytes were cultured in engineered micro-heart muscle arrays that were loaded by changing substrate rigidity. Methods: Isogenic control and MYBPC3 +/- hiPSC-CM were seeded into dogbone-shaped molds to form micro-heart muscle atop silicone rubber substrates with rigidity levels ranging from levels of embryonic (0.4 kPa) to fibrotic adult heart (114 kPa). Spontaneously contracting micro-tissues formed within 48 hours. 10 days after formation, tissues were monitored for action potentials (voltage sensitive dye, BeRST-1), Ca 2+ transients (GCaMP6f reporter), and contractility (substrate deflection). Cell and tissue architecture were assessed in 8 μm cryosections, and RNA transcription via qRT-PCR. Results: MYBPC3 +/- and control cardiomyocytes had similar physiology and morphology in 2D monolayers. However, MYBPC3 +/- tissues exhibited marked changes in Ca 2+ handling, which were exacerbated when substrate stiffness increased to the level of healthy heart muscle (15 kPa). This was concurrent with hypertrophy of MYBPC3 +/- cardiomyocytes at 15 kPa. MYBPC3 +/- tissue Ca 2+ handling abnormalities were linked to enhanced Ca 2+ intake rather than SERCA deficiency or excessive myofilament buffering. MYBPC3 +/- tissues exhibited mis-regulation of cardiac troponins on the RNA and protein localization levels, which may underlie the observed structural and physiological changes. Conclusions: Changes in the rigidity of the substrate, mimicking changes in heart muscle caused by blood pressure, pushed hiPSC-cardiac micro-tissues with MYBPC3 mutations to recapitulate key structural and electrophysiological characteristics of HCM. In contrast, control tissues exhibited hypertrophy and abnormal calcium handling, but only at extreme values of substrate stiffness. This suggests that HCM mutations may exaggerate load-dependent hypertrophic remodeling.