1. Isogenic models of hypertrophic cardiomyopathy unveil differential phenotypes and mechanism-driven therapeutics.
- Author
-
Bhagwan JR, Mosqueira D, Chairez-Cantu K, Mannhardt I, Bodbin SE, Bakar M, Smith JGW, and Denning C
- Subjects
- Actins genetics, Actins metabolism, Arrhythmias, Cardiac complications, Arrhythmias, Cardiac genetics, Arrhythmias, Cardiac physiopathology, Base Sequence, CRISPR-Associated Protein 9 metabolism, CRISPR-Cas Systems, Calcium metabolism, Cardiac Myosins genetics, Cardiac Myosins metabolism, Cardiomyopathy, Hypertrophic genetics, Cardiomyopathy, Hypertrophic physiopathology, Cell Line, Cell Respiration, Gene Expression Regulation, Genes, Reporter, Humans, Induced Pluripotent Stem Cells metabolism, Mutation genetics, Myocardial Contraction, Myocytes, Cardiac metabolism, Myosin Heavy Chains genetics, Myosin Heavy Chains metabolism, Optogenetics, Phenotype, Tissue Engineering, Cardiomyopathy, Hypertrophic pathology, Cardiomyopathy, Hypertrophic therapy, Models, Cardiovascular
- Abstract
Background: Hypertrophic cardiomyopathy (HCM) is a prevalent and complex cardiovascular condition. Despite being strongly associated with genetic alterations, wide variation of disease penetrance, expressivity and hallmarks of progression complicate treatment. We aimed to characterize different human isogenic cellular models of HCM bearing patient-relevant mutations to clarify genetic causation and disease mechanisms, hence facilitating the development of effective therapeutics., Methods: We directly compared the p.β-MHC-R453C and p.ACTC1-E99K HCM-associated mutations in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and their healthy isogenic counterparts, generated using CRISPR/Cas9 genome editing technology. By harnessing several state-of-the-art HCM phenotyping techniques, these mutations were investigated to identify similarities and differences in disease progression and hypertrophic signaling pathways, towards establishing potential targets for pharmacological treatment. CRISPR/Cas9 knock-in of the genetically-encoded calcium indicator R-GECO1.0 to the AAVS1 locus into these disease models resulted in calcium reporter lines., Results: Confocal line scan analysis identified calcium transient arrhythmias and intracellular calcium overload in both models. The use of optogenetics and 2D/3D contractility assays revealed opposing phenotypes in the two mutations. Gene expression analysis highlighted upregulation of CALM1, CASQ2 and CAMK2D, and downregulation of IRF8 in p.β-MHC-R453C mutants, whereas the opposite changes were detected in p.ACTC1-E99K mutants. Contrasting profiles of nuclear translocation of NFATc1 and MEF2 between the two HCM models suggest differential hypertrophic signaling pathway activation. Calcium transient abnormalities were rescued with combination of dantrolene and ranolazine, whilst mavacamten reduced the hyper-contractile phenotype of p.ACTC1-E99K hiPSC-CMs., Conclusions: Our data show that hypercontractility and molecular signaling within HCM are not uniform between different gene mutations, suggesting that a 'one-size fits all' treatment underestimates the complexity of the disease. Understanding where the similarities (arrhythmogenesis, bioenergetics) and differences (contractility, molecular profile) lie will allow development of therapeutics that are directed towards common mechanisms or tailored to each disease variant, hence providing effective patient-specific therapy., Competing Interests: Disclosures I.M. is co-founder of EHT Technologies GmbH., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF