Isogenic models of hypertrophic cardiomyopathy unveil differential phenotypes and mechanism-driven therapeutics

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.

Mitochondrial DNA: Hotspot for Potential Gene Modifiers Regulating Hypertrophic Cardiomyopathy

“…While sequencing mtDNA of a large cohort of patients would have been advantageous to provide a higher degree of patient-wide validation, the alternative use of isogenic sets of hPSC-CM lines enables a more refined investigation of the contribution of mtDNA mutations to HCM, a disease characterized by highly complex mutation-specific effects [ 21 ]. Additionally, the customary investigation of patient peripheral blood as an alternative to scarcely available cardiac tissue biopsies is not optimal due to tissue-specific mtDNA sequence heteroplasmy [ 44 ] (which is recapitulated in hPSC-CMs).…”

“…Conversely, four variants showed opposing potential effects in the two models, reinforcing the notion that HCM exhibits complex genetic causation. We speculate that these variants reflect the contrasting changes in phenotype induced by the two primary sarcomeric mutations, e.g., while the p.R453C-βMHC change caused hypo-contractility, the p.E99K-ACTC1 mutation led to hyper-contractility [ 21 ].…”

“…These two sets have revealed overall similarities and differences in HCM hallmarks and highlighted variations in functional phenotypes and gene expression profiles between the different cell lines carrying the same mutation. Remarkably, both sarcomeric-mutant models exhibited increased mitochondrial respiration rates relative to isogenic healthy controls, characteristic of the compensatory stage of HCM that precedes energy depletion [ 21 ]. Thus, we utilized these sets of hPSC-CMs to investigate the role of mtDNA to harbor potential gene modifiers that could underlie the different HCM phenotypes observed.…”

Mitochondrial Medicine: Genetic Underpinnings and Disease Modeling Using Induced Pluripotent Stem Cell Technology

“…While these cardiomyopathy hiPSC-CM models focused mostly on mutations in sarcomeric genes that regulate cardiomyocyte contraction and calcium handling, a few have also showed energy depletion phenotypes due to mitochondrial dysfunction (74,75). Importantly, hiPSC-CMs have also been harnessed to specifically model mitochondrial cardiomyopathies as these constitute phenocopies of HCM (40).…”

Transfection of hPSC-Cardiomyocytes Using Viafect™ Transfection Reagent

“…For instance, the overexpression of long non-coding RNA (lncRNA) Ahit in mice was performed to modulate the hypertrophic response and to identify interacting partners and novel disease mechanisms [ 15 ]. Additionally, sustained overexpression of genetically-encoded calcium or voltage sensors may be used to establish reporter cell lines of hPSC-CMs, which currently require suboptimal genome integration or short-lived dyes [ 16 , 17 ]. Finally, overexpression may also be used to promote more efficient cardiomyocyte differentiation and maturation [ 18 ].…”