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.
View: Text | PDF
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

×

Figure 1

MYBPC3-mutant iPSCMs exhibit compensated MyBP-C protein levels despite reduced mRNA.

Options: View larger image (or click on image) Download as PowerPoint
MYBPC3-mutant iPSCMs exhibit compensated MyBP-C protein levels despite ...
(A) Diagram of MyBP-C depicts the overall strategy for determining MYBPC3 mutation mechanisms through generation of an allelic series of mutations in the same genetic background, in addition to patient iPSC lines. (B) MyBP-C protein levels were determined from spectral counts by mass spectroscopy analysis and normalized to myosin abundance. MyBP-C levels were equivalent among the gene-edited heterozygous, HCM patient, and control lines, but MyBP-C was undetectable in the homozygous frameshift line (1-way ANOVA used for statistical comparison against controls; *P < 0.05). (C) The mutant MYBPC3 mRNA relative abundance was determined by using the proportion of RNA-Seq read counts from the mutant allele, normalized to total control expression level. For HetPR, no mutant mRNA is expressed from the mutant allele, while HetSS has approximately 50:50 mutant to wild-type allele because the mutant mRNA for this line is not susceptible to NMD. In contrast, the HetFS and HomFS lines have a reduction in mutant mRNA because of NMD. The relative mutant proportion in HetCT and HomCT was compared with HetSS using 1-way ANOVA with P < 0.05 as significant (see Methods). The relative mutant proportion in HetFS and HomFS was compared with HetSS using 1-way ANOVA with P < 0.05 as significant (see Methods). (D) Total MYBC3 mRNA abundance was determined using RNA-Seq for robust normalization with a DESeq2 (55) and remove unwanted variation–sequencing RUV-Seq (56) pipeline (see Methods). Relative abundance was determined using dispersion estimates for each gene fit to a binomial generalized linear model with DESeq2, which yields a log2 fold change estimate and SEM for each gene, converted here to relative abundance on a nonlogarithmic scale. The Wald test was used for statistical significance testing with multiple-testing correction for 16,819 genes tested (adjusted P < 0.05 was considered significant). iPSC, induced pluripotent stem cell; iPSCM, induced pluripotent stem cell cardiomyocyte; NMD, nonsense-mediated RNA decay.