Modeling hypertrophic cardiomyopathy with human induced pluripotent stem cells

Abstract

Hypertrophic cardiomyopathy (HCM), a hereditary cardiac disease, is predominantly caused by mutations in sarcomeric genes. However, the mechanism by which these mutations alter contractile function leading to HCM is not fully understood. Although various animal models have uncovered disease hallmarks, they do not fully recapitulate disease phenotype due to species differences in biological responses, thus limiting the investigation of underlying mechanisms of HCM. Recently, the development of human induced pluripotent stem cell (hiPSC)-based models provides unprecedented opportunities to investigate and characterize HCM pathogenesis and pathophysiology. In this study, a model of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) was established to evaluate the role of two identified mutations, cardiac troponin I (cTnI) R186Q and tropomyosin (Tm) E26Q, in the development of HCM. Both cTnI and Tm belong to thin filaments, which coordinated with Ca2+ to regulate actin-myosin interactions and thus cardiomyocytes contraction. The C-terminal end segment of cTnI has been demonstrated to retain the binding affinity for Tm and function as a myofilament Ca2+ desensitizer. Here, for the first time, the iPSC-CM models bearing cTnI R186Q mutation and Tm E26Q mutation was established respectively, which recapitulated a number of characteristics of HCM phenotypes, including enlarged cellular size, sarcomere disarray, multi-nucleation, abnormal electrophysiological properties and excitation-contraction (E-C) uncoupling. Notably, the cTnI R186Q mutation and Tm E26Q exhibited the similar HCM phenotypes, especially the dysfunctional Ca2+ cycling. The imbalance of Ca2+ homeostasis is likely due to the increased myofilament Ca2+ sensitivity and can be restored by neutralizing the hyper-Ca2+-sensitivity with blebbistatin, the Ca2+ desensitizer. In addition, the long-term Ca2+ overload mediated by cTnI R186Q and Tm E26Q mutation induced the activation of calcineurin-NFAT pathway which also contributes to the development of cardiac hypertrophy. Finally, investigations based on cTnI R186 mutation revealed the relationship between transient receptor potential vanilloid 2 (TRPV2) channel and impaired Ca2+ homeostasis, suggesting TRPV2 may serve as a potential therapeutic target for HCM. While iPSC-CMs models recapitulated HCM phenotypes, and serve as a promising platform for disease modeling and drug screening, the interpretation of disease phenotype might be confound by the variation in the genetic background. Here, I combined CRISPR/Cas9 with single-stranded oligodeoxynucleotides to correct cTnI R186Q mutation seamlessly in HCM iPSCs to generate isogenic controls. At least 3 isogenic corrected hiPSC lines were generated, which retained pluripotency and a normal karyotype without detectable off-target events. The corrected iPSC could efficiently differentiate into functional cardiomyocytes that did not display hypertrophy and myofibril disarray, as observed in iPSC-CMs with cTnI R186Q mutation. In addition, correction of R186Q mutation restored Ca2+ homeostasis and β-adrenergic regulation, as well as E-C coupling. Our results provided a proof of concept that the disease phenotypes were caused by cTnI R186Q mutation, suggesting that this method could be utilized for investigating underlying disease pathways and developing cell therapeutic strategies Taken together, my results suggest that an iPSC-CMs model serve as a reliable and consistent in vitro model to explore the underlying mechanisms and potential therapeutic targets of HCM.