Abstract
BackgroundHypertrophic 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. MethodsWe 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. ResultsConfocal 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. ConclusionsOur 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.
Highlights
Hypertrophic cardiomyopathy (HCM) is a prevalent cardiovascular disease which is the leading cause of sudden cardiac death in young adults, including athletes [1,2]
Revisiting isogenic sets of ACTC1-mutant and MYH7-mutant hiPSCs generated by CRISPR/Cas9 editing
In order to compare the effects of different sarcomeric mutations in HCM, we have utilized previously generated isogenic hiPSC-CM models, whereby CRISPR/Cas9 was used to either introduce the g.MYH7C9123Tmutation in healthy hiPSCs or correct the c.ACTC1G301Avariant in patient lines
Summary
Hypertrophic cardiomyopathy (HCM) is a prevalent cardiovascular disease which is the leading cause of sudden cardiac death in young adults, including athletes [1,2]. Approximately half of HCM patients do not exhibit mutations in known sarcomeric genes [7], and while genetic lesions occur at 1:200 incidence, only 1:500 individuals develop HCM phenotypes [8]. With such an array of potentially causative mutations, HCM has variable penetrance, expressivity and severity [9], which greatly complicates treatment. 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, providing effective patient-specific therapy
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