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

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 2

Myofilament stoichiometry, myofibrillogenesis, and early stages of iPSCM growth are not affected by heterozygous or homozygous MYBPC3 mutations.

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Myofilament stoichiometry, myofibrillogenesis, and early stages of iPSCM...
(A) Relative mRNA abundance of the major sarcomere genes (except MYBPC3) is not different among the isogenic MYBC3-mutant lines compared to controls, even in the case of the homozygous mutant line. Relative expression levels and statistical significance were determined from RNA-Seq analysis as in Figure 1D. Read counts for gene paralogs (e.g., MYH6 and MYH7) were coalesced for comparison because the objective was to determine overall stoichiometry of sarcomere gene expression and not effects on developmental state, which may be confounded by some variability in the iPSCM model system. (B) Mass spectroscopy quantification of sarcomere proteins (other than MyBP-C), normalized to total myosin, demonstrates no significant alteration in sarcomere protein levels. (C) Representative images from single 7:1 rectangular patterned iPSCMs demonstrate robust myofibrillar development and alignment among both mutant lines and control lines, including the homozygous mutant line. MyBP-C localization is normal in all heterozygous lines but is not observed when imaged with the same imaging parameters in the homozygous line (see also Supplemental Figure 5; MyBP-C detected with anti-C0-M domain antibody). Insets: ×2.5 magnification. (D) Example of variability in myofibrillar density among control iPSCMs. This variability was observed among all control and mutant iPSCM lines and was used as an approach to quantify myofibrillar growth that would represent a cellular phenotype of cardiac hypertrophy. (E) To quantify myofibrillar hypertrophy, which is characterized by parallel addition of myofibrils, blinded myofibrillar counts on individual micropatterned iPSCMs were performed for each line (N > 100 cells per line) and showed no difference among isogenic lines, including the homozygous line (ANOVA, with P < 0.05 as significant). The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range.