Abstract 12532: Cellular and Cardiac Microtissue Assays of iPSC-derived Myocytes With the Hypertrophic Cardiomyopathy Mutation in MYH7-Val606Met
Introduction: Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease affecting 1 in 500 people and is the leading cause of sudden cardiac death in young adults. Mutations in β-myosin heavy chain (MYH7) account for one-third of cases, but the underlying pathogenesis remains unclear. In this study, we investigated myocytes derived from CRISPR-Cas9-engineered isogenic induced pluripotent stem cell cardiomyocytes (iPSC-CMs) with a missense mutation Val606Met by single-cell and microtissue functional assays.
Hypothesis: We hypothesize that iPSC-CMs with Val606Met will recapitulate features of HCM in single cell and microtissue assays, such as changes in contractile function and hypertrophic signaling.
Methods & Results: Val606Met mutations in MYH7 (VM/+, VM/VM) were introduced by targeting an iPSC line using CRISPR-Cas9. Transfected iPSCs were isolated by FACS to detect green fluorescent protein, clones were expanded, and mutational events were confirmed by Sanger sequencing. Mutant iPSCs, like isogenic wildtype control iPSCs, were differentiated into cardiomyocytes with >90% efficiency. Microtissue studies of mutant iPSC-CMs exhibited increases in contractility (WT=2.96±0.23μN, VM/+ = 3.51±0.34μN, VM/VM = 4.63±0.51; R2= 0.963). Mutant iPSC-CMs also showed evidence of altered signaling by increases in spontaneous beating rate (WT < VM/+ < VM/VM; R2=0.946), cell size (compared to WT, ~20% increases in both VM/+, p=0.04 and VM/VM, p=0.038), and sarcomere content (compared to WT, ~20% increases in both VM/+, p=4e-06 and VM/VM, p=0.11). These changes are supported by activated expression in myocytes of hypertrophic genes such as NPPA, NPPB, and ACTA2 as well as transcriptional up-regulation of TGF-β.
Conclusions: Isogenic iPSC-CMs with MYH7 variant Val606Met created by CRISPR-Cas9 recapitulate features of HCM in vitro. Our data confirms Val606Met as a pathogenic mutation that causes increased contractility in cardiac microtissues and myocyte hypertrophy. We speculate that increased myocyte-derived TGF-β signaling may contribute to the HCM phenotype. This approach could also be used for better understanding of genotype-phenotype correlations in HCM.
Author Disclosures: C.C. Sheng: None. J.T. Hinson: None. A. Chopra: None. C.S. Chen: Ownership Interest; Modest; Innolign Biomedical. J. Seidman: None. C. Seidman: None.
- © 2015 by American Heart Association, Inc.