Hypertrophic Cardiomyopathy Mutations With Opposite Effects on [latin sharp s]-myosin Biomechanics Show Similar Structural and Biomechanical Phenotypes in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (hipsc-cms)

Introduction: Hypertrophic cardiomyopathy (HCM) is the most prevalent heritable cardiovascular disease, commonly caused by mutations in beta cardiac myosin heavy chain (βMYH). We have measured the kinetics of isolated myosin proteins with different HCM mutations in βMYH and found an intriguing heterogeneity in kinetics and force production: some mutations increase whereas others decrease force and velocity; others result in no change. How these divergent molecular alterations converge to the final HCM phenotype: hypercontractility and cellular hypertrophy is still unknown. hiPSC-CMs provide a powerful tool for studying human cardiomyocyte biology including contractility, hypertrophic growth, and intracellular organization, but are limited by immature phenotypes and population heterogeneity in traditional culture environments. Methods and Results: We developed a micropatterned hydrogel platform that enhances force generation and promotes both structural and molecular hiPSC-CM maturation. Using CRISPR/Cas-9 gene editing in an isogenic hiPSC line, we created hiPSCs with two different βMYH mutations (P710R and D239N) that result in opposite effects on force at the molecular level, using in vitro motility and laser trap methods. Cell morphology was quantified by both fluorescence and transmission electron microscopy (EM) and force generation measured by traction force microscopy. Despite having opposite effects at the single molecule level, both HCM lines showed increased contractile force and cellular hypertrophy compared to isogenic controls. Immunostaining for βMYH and EM revealed organizational and microstructural changes including sarcomere and myofibrillar disarray, and thickened z-discs in cells containing these mutations. Both mutations had alterations in AMPK, MAPK, and calcineurin signaling shown to regulate hypertrophy. Discussion: Divergent biomechanical alterations due to HCM mutations at the single molecule level lead to common cellular-level structural and biomechanical phenotypes through activation of common signaling pathways.