Abstract 17079: MYBPC3 Truncation Mutations Cause Contractile Dysregulation in iPSC-Derived Cardiomyocytes

The mechanism of MYBPC3 (encoding cardiac myosin binding protein C, MyBP-C) truncation mutations, the most common genetic cause of hypertrophic cardiomyopathy, has been incompletely resolved. We hypothesized that truncating MYBPC3 mutations cause myofibrillar protein assembly defects and/or contractile dysfunction. Methods and Results: Control iPSCs were CRISPR/Cas9-edited to create cell lines with homozygous (homCT) and heterozygous (hetCT) C-terminal MYBPC3 truncation mutations and heterozygous MYBPC3 promoter knock-out (hetPROM). HetCT was further edited to add an N-terminal flag tag (FhetCT). Purified cardiomyocytes were assayed at day ~25. RNAseq showed a 50% reduction in MYBPC3 mRNA in hetCT (p<0.01). Full ablation of MyBP-C was demonstrated in homCT but MyBP-C content was not reduced in hetCT or hetPROM by quantitative mass spectrometry. Immunofluorescence and co-IP of FhetCT showed no truncated MyBP-C. Sarcomere assembly, quantified by aligned myofilament number after replating onto 7:1 rectangular micropatterns, did not differ between lines; homCT (23±3), hetCT (23±3), hetPROM (22±3 myofilaments), control (22±3), p=NS. Quantitative mass spectrometry demonstrated that the stoichiometry of other major thick and thin filament proteins was not altered (all p=NS). Maximum force was reduced in homCT (866±366 uN, p<0.001) and hetCT (1213±545 uN, p=0.03) vs controls (1509±441 uN), and not significantly different for hetPROM (1238±360 uN, p=0.07), when measured in single micropatterned iPSC-CMs on 8.7 kPa hydrogels by traction force microscopy. Time to peak contraction was shorter in homCT (0.27 s, p=0.02) vs control (0.42 sec), but not in hetCT (0.46 s) and hetPROM (0.49 s). Contractile deceleration time was reduced only in homCT (0.12 s vs 0.25 s, p=0.002). Conclusions: Heterozygous MYBPC3 truncation mutations result in haploinsufficent mRNA with steady state compensation of MYBP3 protein and no MYBPC3 truncated peptide. Even complete ablation of MyBP-C loss does not alter the overall stoichiometry of other sarcomeric thick and thin filament proteins. MyBP-C ablation and heterozygous truncating mutations impair maximal contractile force, strongly implicating contractile dysregulation as the primary mechanism of MYBPC3-HCM.