Interplay of Genotype and Substrate Stiffness in Driving the Hypertrophic Cardiomyopathy Phenotype in IPSC-Micro-Heart Muscle Arrays

Introduction: In clinical and animal studies, Hypertrophic Cardiomyopathy (HCM) shares many similarities with non-inherited cardiac hypertrophy induced by pressure overload (hypertension). This suggests a potential role for mechanical stress in priming tissues with mutation-induced changes in the sarcomere to develop phenotypes associated with HCM, including hypercontractility and aberrant calcium handling. Here, we tested the hypothesis that heterozygous loss of function of Myosin Binding Protein C (MYBCP3 , mutations in which account for almost 50% of inherited HCM) combines with environmental stiffness to drive HCM phenotypes.

Methods: We differentiated isogenic control (WTC) and MYBPC3 iPSC into cardiomyocytes using small molecule manipulation of Wnt signaling, and then purified them using lactate media. The purified cardiomyocytes were seeded into "dog bone" shaped stencil molds to form micro-heart muscle arrays (μHM). To mimic changes in myocardial stiffness stemming from pressure overload, we varied the rigidity of the substrates μHM contract against. Stiffness levels ranged from those corresponding to fetal (5 kPa), healthy (15 kPa), pre-fibrotic (30 kPa) to fibrotic (65 kPa) myocardium. Substrates were embedded with a thin layer of fluorescent beads to track contractile force, and parent iPSC were engineered to express the genetic calcium indicator, GCaMP6f. High speed video microscopy and image analysis were used to quantify calcium handling and contractility of μHM.

Results: Substrate rigidity triggered physiological adaptation for both genotypes. However, MYBPC3 μHM showed a lower tolerance to substrate stiffness with the peak traction on 15 kPa, while WTC μHM had peak traction on 30 kPa. MYBPC3 μHM exhibited hypercontractility, which was exaggerated by substrate rigidity. MYBPC3 μHM hypercontractility was associated with longer rise times for calcium uptake and force development, along with higher overall Ca intake.

Conclusion: We found MYBPC3 mutations cause iPSC-μHM to exhibit hypercontractility, and also a lower tolerance for mechanical stiffness. Understanding how genetics work in combination with mechanical stiffness to trigger and/or exacerbate pathophysiology may lead to more effective therapies for HCM.

Supplementary Information: The online version contains supplementary material available at (10.1007/s12195-021-00684-x).