Effects of MYBPC3 loss-of-function mutations preceding hypertrophic cardiomyopathy
Adam S. Helms, Vi T. Tang, Thomas S. O’Leary, Sabrina Friedline, Mick Wauchope, Akul Arora, Aaron H. Wasserman, Eric D. Smith, Lap Man Lee, Xiaoquan W. Wen, Jordan A. Shavit, Allen P. Liu, Michael J. Previs, Sharlene M. Day
Adam S. Helms, Vi T. Tang, Thomas S. O’Leary, Sabrina Friedline, Mick Wauchope, Akul Arora, Aaron H. Wasserman, Eric D. Smith, Lap Man Lee, Xiaoquan W. Wen, Jordan A. Shavit, Allen P. Liu, Michael J. Previs, Sharlene M. Day
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Research Article Cardiology

Effects of MYBPC3 loss-of-function mutations preceding hypertrophic cardiomyopathy

Abstract

Mutations in cardiac myosin binding protein C (MyBP-C, encoded by MYBPC3) are the most common cause of hypertrophic cardiomyopathy (HCM). Most MYBPC3 mutations result in premature termination codons (PTCs) that cause RNA degradation and a reduction of MyBP-C in HCM patient hearts. However, a reduction in MyBP-C has not been consistently observed in MYBPC3-mutant induced pluripotent stem cell cardiomyocytes (iPSCMs). To determine early MYBPC3 mutation effects, we used patient and genome-engineered iPSCMs. iPSCMs with frameshift mutations were compared with iPSCMs with MYBPC3 promoter and translational start site deletions, revealing that allelic loss of function is the primary inciting consequence of mutations causing PTCs. Despite a reduction in wild-type mRNA in all heterozygous iPSCMs, no reduction in MyBP-C protein was observed, indicating protein-level compensation through what we believe is a previously uncharacterized mechanism. Although homozygous mutant iPSCMs exhibited contractile dysregulation, heterozygous mutant iPSCMs had normal contractile function in the context of compensated MyBP-C levels. Agnostic RNA-Seq analysis revealed differential expression in genes involved in protein folding as the only dysregulated gene set. To determine how MYBPC3-mutant iPSCMs achieve compensated MyBP-C levels, sarcomeric protein synthesis and degradation were measured with stable isotope labeling. Heterozygous mutant iPSCMs showed reduced MyBP-C synthesis rates but a slower rate of MyBP-C degradation. These findings indicate that cardiomyocytes have an innate capacity to attain normal MyBP-C stoichiometry despite MYBPC3 allelic loss of function due to truncating mutations. Modulating MyBP-C degradation to maintain MyBP-C protein levels may be a novel treatment approach upstream of contractile dysfunction for HCM.

Authors

Adam S. Helms, Vi T. Tang, Thomas S. O’Leary, Sabrina Friedline, Mick Wauchope, Akul Arora, Aaron H. Wasserman, Eric D. Smith, Lap Man Lee, Xiaoquan W. Wen, Jordan A. Shavit, Allen P. Liu, Michael J. Previs, Sharlene M. Day

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Figure 5

Global transcriptomic analysis demonstrates dysregulation of protein quality control across all MYBPC3 loss-of-function mutation types but no alterations in calcium-handling–, NMD-, or hypertrophy-associated gene sets.

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Global transcriptomic analysis demonstrates dysregulation of protein qua...
(A) Transcriptome-wide RNA-Seq analysis was performed and differentially expressed genes were tested for pathway enrichment with a Gene Ontology (GO) approach using GO-Seq, revealing 5 categories of genes significantly dysregulated in MYBPC3-mutant iPSCMs. Statistical significance was calculated using GO-Seq with multiple-testing correction for all biologic pathways tested. Isogenic MYPBC3-mutant iPSCM lines were used to avoid confounding with transcriptome-wide analysis, and 2 different control lines (original control iPSC line and nontargeted control iPSC line) were used (N = 20 total mutant samples; N = 10 total control samples). (B) The top enriched GO categories identified were driven primarily by the same set of protein quality control genes. These genes, shown here, show a consistent pattern of dysregulation when compared individually against control samples and a stronger magnitude of effect in the homozygous frameshift line. The relative abundance was calculated as in Figure 1D, using DESeq2 estimates from negative binomial fits for each gene and with significance calculated by the Wald test. (C–E) Gene set enrichment analysis was also performed for prespecified, manually curated pathways, including NMD, calcium handling, and hypertrophy, none of which demonstrated evidence of dysregulation in the MYBPC3 gene-edited iPSCMs. Heatmaps of representative and highly expressed genes from these pathways are shown in C–E. Each gene’s expression was calculated relative to the average of the controls and shown on a heatmap scale for fold change range of 0.2–1.8.