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.
Highlights
Mutations in cardiac myosin binding protein C (MyBP-C, encoded by MYBPC3) are the most common cause of familial hypertrophic cardiomyopathy (HCM), an autosomal dominant inherited cardiomyopathy with incomplete penetrance [1]
In the genome-edited lines, frameshift mutations were introduced at a similar location as the patient lines (Figure 1A), premature termination codons (PTCs)-causing mutations in HCM occur throughout the gene, indicating that the precise location of the frameshift is likely unimportant [1]
We take advantage of gene editing and the early developmental state of iPSC-derived cardiomyocytes to establish the regulatory levels at which MYBPC3 gene expression and MyBP-C protein homeostasis are maintained in the presence of PTC-causing MYBPC3 mutations
Summary
Mutations in cardiac myosin binding protein C (MyBP-C, encoded by MYBPC3) are the most common cause of familial hypertrophic cardiomyopathy (HCM), an autosomal dominant inherited cardiomyopathy with incomplete penetrance [1]. Among patients with HCM, 90% of MYBPC3 mutations are heterozygous frameshift, nonsense, or splice site mutations that result in premature termination codons (PTCs) on 1 allele [1]. As such, these mutations are thought to result in HCM from an allelic loss of function via NMD of PTC-containing transcripts [6], leading to a reduction in MyBP-C content in the sarcomere. These mutations are thought to result in HCM from an allelic loss of function via NMD of PTC-containing transcripts [6], leading to a reduction in MyBP-C content in the sarcomere
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