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Effects of MYBPC3 loss-of-function mutations preceding hypertrophic cardiomyopathy
Adam S. Helms, … , Michael J. Previs, Sharlene M. Day
Adam S. Helms, … , Michael J. Previs, Sharlene M. Day
Published December 26, 2019
Citation Information: JCI Insight. 2020;5(2):e133782. https://doi.org/10.1172/jci.insight.133782.
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Research Article Cardiology

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

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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 6

MyBP-C protein level is dynamically regulated to maintain steady-state equilibrium during myofibrillogenesis despite reduced synthesis rate.

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MyBP-C protein level is dynamically regulated to maintain steady-state e...
MyBP-C synthesis and degradation during cell growth following replating of purified iPSCMs was determined using mass spectroscopy analysis of isotopically labeled proteins, introduced through addition of deuterated leucine to the cell medium 24 hours after replating. Control iPSCMs were compared with iPSCM lines from patients with heterozygous frameshift MYBPC3 mutations (HetFS-P1 and HetFS-P2). (A) Newly synthesized MyBP-C was quantified by appearance of labeled MyBP-C peptides and appeared at a slower rate in frameshift MYBPC3-mutant iPSCMs compared with controls (analysis by comparison of the slopes of the linear regression fits, *P < 0.0001). (B) MyBP-C degradation was quantified by measuring the relative reduction in unlabeled MyBP-C at each time point and was reduced in both HCM samples compared with the control samples at time points more than 48 hours (*P = 0.001, Mann-Whitney). MyBP-C degradation was quantified beginning at 12 hours following labeling because a genotype-independent reduction in MyBP-C occurred in all samples in the first 12 hours following withdrawal of the 2% serum-containing replating medium. (C) The MyBP-C/myosin ratio was quantified at each time point as an indication of MyBP-C stoichiometry. In both control and HCM samples, MyBP-C/myosin reduced markedly in the first 12 hours following 2% serum withdrawal from the replating medium, prior to a return to normal levels, with no significant difference between control and HCM samples. Two samples were collected at each time point for controls and 2 samples each for the frameshift mutation lines. Data for each collection point is shown as the mean ± range for all samples (range not shown when less than the size of the dot).

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