Myosin Heavy Chain-7 E848G Mutation-Induced Sarcopenia and Hypocontractility in Induced Pluripotent Stem Cell Model of Hypertrophic Cardiomyopathy Are Rescued By Muscle RING Finger 1 Ablation

Introduction:: Heart failure, the inability of the heart to pump adequate blood for the body, affects over 6 million adults and has an economic impact of at least $30 billion in the US, illustrating the pressing need for improved understanding of the disease. Familial hypertrophic cardiomyopathy (HCM) is a genetic cardiomyopathy affecting 1 in 500 US adults, most commonly diagnosed at middle age. Often called a disease of the sarcomere due to frequent pathogenic mutations in sarcomeric proteins, HCM manifests in many patients with left ventricular hypertrophy, heart failure, and systolic dysfunction. Mutation in β-myosin heavy chain (βMHC, encoded by MYH7), a sarcomeric thick filament protein, accounts for 20-40% of HCM cases. Of 3983 MYH7 variants reported in ClinVar as of May 2023, over half are classified as variants of unknown significance (VUS), leaving patients in a “genetic purgatory” with unclear clinical consequences. Furthermore, current understanding lacks a generalizable mechanism through which specific MYH7 variants result in HCM, and no disease-modifying therapy exists. Thus, advancements in understanding of MYH7 variant-associated HCM will improve diagnostic and clinical outcomes. Based on previous work, we hypothesized pathogenic MYH7 variants disrupt sarcomere abundance and contractile function. Here, we used a human induced pluripotent stem cell (hiPSC) model of the HCM-causing MYH7 E848G variant to study the connection between pathogenic MYH7 variants, sarcopenia, and hypocontractility. We targeted the βMHC-associated E3 ubiquitin ligase Muscle RING Finger 1 (MuRF1, encoded by TRIM63) to rescue sarcomere abundance and contractility.

Materials and Methods:: Cardiac tissue samples were obtained from a patient expressing MYH7^wt/E848G variant and lysed for protein content. A stable hiPSC line was generated from peripheral blood mononuclear cells from the patient. Using this line as an isogenic background, we knocked in a plasmid expressing exon 22-40 MYH7 cDNA in-frame with enhanced green fluorescent protein (EGFP) into the endogenous MYH7 locus via CRISPR/Cas9, generating isogenic lines with βMHC-EGFP fusion proteins with or without MYH7 E848G variant. TRIM63 was ablated by CRISPR/Cas9 in the parent line as well. These hiPSCs were differentiated into cardiomyocytes (hiPSC-CMs) for use in experiments. hiPSC-CMs were plated in monolayer at 200k cells/cm² at differentiation day 30 for 10 days in DMEM-based cardiomyocyte growth medium. Cells were fixed for flow cytometry and lysed for western blot at differentiation day 40. 500k differentiation day 25 hiPSC-CMs were cocultured with 100k Hs27a stromal cells in a three dimensional engineered heart tissue (EHT) across flexible PDMS posts for three weeks and stimulated with 10V current at 1Hz for tissue-level contractility measurement. Polyacrylamide gels with 10 kPa Young's Modulus were fabricated onto 25 mm coverslips with 1.0x10⁶ 500 nm fluorescent microspheres embedded and repeating 15x100 μm Matrigel coated rectangles on top of the gel. 110k hiPSC-CMs were seeded on the gel at differentiation day 30 for 7 days. Cells were stimulated with 40V current at 1Hz and imaged at 15 fps via traction force microscopy to detect single-cell contractility. Unstimulated gels were fixed at differentiation day 40 for confocal imaging.

Results, Conclusions, and Discussions:: Comparing MYH7^wt/E848G patient tissue to MYH7^wt/wt patient tissue, we found reduced expression of the following sarcomeric proteins: β-myosin heavy chain (βMHC) (97.8 ± 1.1% reduction), α-sarcomeric actinin (αSA) (84.8 ± 7.3%), and ventricular myosin light chain (MLC-1v) (45.9 ± 13.9%). Isogenic MYH7^wt/E848G hiPSC-CMs also had reduced protein expression of βMHC (22.8%) and αSA (24.5%) relative to MYH7^wt/wt hiPSC-CMs and unchanged mRNA expression. Confocal imaging of single isogenic hiPSC-CMs on 10 kPa hydrogels revealed significantly fewer z-discs per cell for MYH7^wt/E848G hiPSC-CMs (132 ± 16 z-discs) relative to MYH7^wt/wt hiPSC-CMs (327 ± 33 z-discs). These results suggest MYH7 E848G HCM induces broad sarcomere component deficiency. We tested hiPSC-CM contractility using single cell traction force microscopy (TFM) and engineered heart tissues (EHT). Single isogenic MYH7^wt/E848G hiPSC-CMs had lower maximum twitch force (89 ± 18 nN) than MYH7^wt/wt hiPSC-CMs (318 ± 42 nN) by TFM. Isogenic MYH7^wt/E848G EHTs had lower maximum twitch force (118.9 ± 23.7 μN) than MYH7^wt/wt EHTs (287.4 ± 27.7 μN). Genetic ablation of TRIM63 (encoding MuRF1) significantly reduced MYH7^wt/E848G hiPSC-CM cell size by flow cytometry (6.8 ± 1.7%). MYH7^wt/E848GTRIM63^-/- hiPSC-CMs increased protein expression of βMHC (154.7 ± 51.9%), αSA (44.9 ± 25.6%), and MLC-1v (246.9 ± 57.3%) relative to MYH7^wt/E848GTRIM63^+/+ hiPSC-CMs. MYH7^wt/E848GTRIM63^-/- had significantly higher maximum twitch force by TFM (125 ± 15 nN) and EHTs (178.2 ± 9.8 µN) than MYH7^wt/E848GTRIM63^+/+ by TFM (80 ± 10 nN) and EHTs (118.9 ± 23.7 μN). Our results implicate sarcopenia in the pathogenesis of MYH7 E848G HCM and identify MuRF1 as a potential novel therapeutic target for hypocontractile HCM. Further study will determine if these findings are generalizable to other pathogenic HCM variants in MYH7 and other sarcomeric genes.


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