Myosin Heavy Chain Converter Domain Mutations Drive Early-Stage Changes in Extracellular Matrix Dynamics in Hypertrophic Cardiomyopathy

Abstract

More than 60% of hypertrophic cardiomyopathy (HCM)-causing mutations are found in the gene loci encoding cardiac myosin-associated proteins including myosin heavy chain (MHC) and myosin binding protein C (MyBP-C). Moreover, patients with more than one independent HCM mutation may be at increased risk for more severe disease expression and adverse outcomes. However detailed mechanistic understanding, especially at early stages of disease progression, is limited. To identify early-stage HCM triggers, we generated single (MYH7 c.2167C>T [R723C] with a known pathogenic significance in the MHC converter domain) and double (MYH7 c.2167C>T [R723C]; MYH6 c.2173C>T [R725C] with unknown significance) myosin gene mutations in human induced pluripotent stem cells (hiPSCs) using a base-editing strategy. Cardiomyocytes (CMs) derived from hiPSCs with either single or double mutation exhibited phenotypic characteristics consistent with later-stage HCM including hypertrophy, multinucleation, altered calcium handling, metabolism and arrhythmia. We then probed mutant CMs at time points prior to the detection of known HCM characteristics. We found MYH7/MYH6 dual mutation dysregulated extracellular matrix (ECM) remodeling, altered integrin expression, and interrupted cell-ECM adhesion by limiting the formation of focal adhesions. These results point to a new phenotypic feature of early-stage HCM and reveal novel therapeutic avenues aimed to delay or prohibit disease onset.

Multiplexed functional assessments ofMYH7variants in human cardiomyocytes at scale

“…This functional assay compares variant frequencies in cardiomyocytes relative to their frequency in hiPSCs, when MYH7 is not expressed [30] and should not exert any functional effect. In line with previous examinations of single, pathogenic MYH7 missense variants [11,27], we demonstrate that multiplexed assessment of survival correctly segregates all tested P/LP variants and 3/4 VUS from Syn variants. HCM patients with pathogenic MYH7 variants can develop enhanced cardiac fibrosis by an unknown mechanism [29]; our data suggests that cardiomyocyte loss could play a role in the development of cardiac fibrosis by skewing cellular proportions toward fibroblasts (which do not express MYH7).…”

“…Pathogenic variants in many genes can disrupt protein stability and reduce abundance [24][25][26], thus, we optimized a FACS-based assay to simultaneously measure MHC-β abundance from the pooled library of MYH7 missense variant hiPSC-CMs. In addition, our lab [27] and others [11] have shown that pathogenic MYH7 variant hiPSC-CMs have reduced survival relative to synonymous controls. We find that both the multiplexed assessment of MHC-β abundance and survival in the MYH7 variant library hiPSC-CMs correctly segregated all tested pathogenic/likely pathogenic (3/3) and synonymous (3/3) variants, providing functional variant effect data for 68 amino acid substitutions.…”

“…Previous reports by our group [27] and others [11] demonstrated hiPSC-CMs with pathogenic MYH7 missense variants (MYH7 E848G/+ or MYH7 R723C/R723C , respectively) have progressively reduced numbers of hiPSC-CMs relative to isogenic controls, thus, we hypothesized that a multiplexed survival assay would segregate hiPSC-CMs with P/LP MYH7 variants from hiPSC-CMs with Syn MYH7 variants. To this end, we examined the final frequencies of five P/LP variants and three Syn variants in hiPSC-CMs at T25 and T47 relative to their initial frequencies at the hiPSC stage, when MYH7 is not expressed/functional [30] (hiPSC and T25 hiPSC-CM NGS data from Friedman et al [19]) (Fig.…”

“…Human induced pluripotent stem cells (hiPSCs) have the capacity to differentiate to nearly any cell type in the body and numerous protocols enable directed differentiation of hiPSCs to specific, disease-relevant cell types [8,9]. For example, hiPSC-CMs are a powerful tool to assess variant effect in clinically-actionable genes and have provided functional evidence for VUS classification [10][11][12][13][14][15][16], however, low-throughput variant generation and assessment methods are outpaced by the discovery of novel VUS [17]. To date, DMS has not been performed in diploid hiPSC derivatives due to low genome-editing rates that have prevented the generation of large variant libraries [18].…”