Evidence From Human Myectomy Samples That MYBPC3 Mutations Cause Hypertrophic Cardiomyopathy Through Haploinsufficiency
Rationale: Most sarcomere gene mutations that cause hypertrophic cardiomyopathy are missense alleles that encode dominant negative proteins. The potential exceptions are mutations in the MYBPC3 gene (encoding cardiac myosin-binding protein-C [MyBP-C]), which frequently encode truncated proteins.
Objective: We sought to determine whether there was evidence of haploinsufficiency in hypertrophic cardiomyopathy caused by MYBPC3 mutations by comparing left ventricular muscle from patients undergoing surgical myectomy with samples from donor hearts.
Methods and Results: MyBP-C protein and mRNA levels were quantitated using immunoblotting and RT-PCR. Nine of 37 myectomy samples had mutations in MYBPC3: 2 missense alleles (Glu258Lys, Arg502Trp) and 7 premature terminations. No specific truncated MyBP-C peptides were detected in whole muscle homogenates of hypertrophic cardiomyopathy tissue. However, the overall level of MyBP-C in myofibrils was significantly reduced (P<0.0005) in tissue containing either a truncation or missense MYBPC3 mutation: 0.76±0.03 compared with 1.00±0.05 in donor and 1.01±0.06 in non-MYBPC3 mutant myectomies.
Conclusions: The absence of any detectable truncated MyBP-C argues against its incorporation in the myofiber and any dominant negative effect. In contrast, the lowered relative level of full length protein in both truncation and missense MYBPC3 mutations argues strongly that haploinsufficiency is sufficient to cause the disease.
Myosin binding protein-C (MyBP-C) is a component of the thick filaments of striated muscles. The human cardiac muscle isoform, encoded by MYBPC3, is composed of 11 globular domains, 8 with homology to IgI and 3 with fibronectin III (Figure 1).1 MyBP-C is likely to have both structural and regulatory roles within the sarcomere, and recent data have suggested that MyBP-C has a role in relaxation and stretch activation.2,3 The physiological importance of MyBP-C has been further highlighted with the discovery of mutations in MYBPC3 as the most commonly identified cause of hypertrophic cardiomyopathy (HCM), typically being found in ≈20% to 25% of patients screened; more than 150 different mutations have been reported.4,5 In striking contrast to all other HCM disease genes, approximately two-thirds of MYBPC3 mutations are predicted to generate a truncated protein product. At present, it is not known whether the autosomal dominant nature of the MYBPC3 mutations results from haploinsufficiency (indicating that functional loss of one copy of the gene cannot be compensated) or a poison peptide effect (by which the mutant proteins interfere with normal sarcomere function). Functional studies on HCM mutants of other proteins have given clear evidence of a poison peptide effect.6 Published studies on the heart muscle of individual patients with different MYBPC3 truncation mutations did not find truncated protein, but one study suggested reduced MyBP-C content.7–9 Data from transgenic mouse models that overexpress truncated cMyBP-C have been conflicting, with support for both mutant protein incorporation and haploinsufficiency.10,11 Mice with both alleles of MyBP-C knocked out are viable12,13; in one model, heterozygous null mice show a slight decrease in MyBPC expression and a late-onset hypertrophy phenotype, consistent with a haploinsufficiency mechanism.12 In this report, we have searched for truncated peptides and reduced MyBP-C quantity in myofibrils from control and affected human heart tissue and find a consistently lower MyBP-C expression in the patients with either truncation or missense MYBPC3 mutations.
We obtained human heart muscle from donor hearts and interventricular septum from HCM patients at surgical myectomy. Genotyping and mRNA analysis was by standard methods. MyBP-C protein was detected in muscle homogenates and myofibrillar fractions using an antibody specific to the N-terminal region of MyBP-C, and the MyBP-C content was quantified relative to the actin content using an anti-actin antibody.
An expanded Materials and Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
We screened for MYBPC3 mutations in a series of left ventricular septum samples from HCM patients undergoing septal myectomy to relieve left ventricular outflow tract obstruction. In 9 of the 39 patients, mutations in MYBPC3, with convincing evidence that they were responsible for HCM, were identified (Figure 1). Two carried previously described missense alleles Glu258Lys (sample code M10) and Arg502Trp (MA); 7 had premature terminations, truncating in domains C3 (same mutation present in M8, MI, MT; predicted molecular mass, 52 kDa), C5 (M9, 90 kDa), C7 (M15, 97kDa; M25, 114 kDa), and C10 (M6, 140 kDa).
Immunoblots were carried out on whole tissue homogenates from the myectomy samples using an antibody specific to the N terminus (C0-C2) of MyBP-C (see Online Figure I). At moderate loading (2 μg of tissue), MyBP-C was detected as a single band (Figure 1B). We did not observe any bands corresponding to the expected truncated protein in M6, M8, M9, M15, M25, MI, or MT at moderate (Figure 1B) or high (Online Figure II) loading. Loading tests indicated that the antibody could detect a concentration of <3% of the main bands.
The quantity of MyBP-C in myofibrils was determined in myectomy samples and compared with nonfailing donor heart muscle (Figure 2). The quantity of MyBP-C relative to actin was consistent between donor heart samples and the mean ratio was used to normalize all of the data. An MyBP-C/actin content significantly lower than donor was found in every myectomy sample containing a MYBPC3 mutation, including the 2 missense mutations (Figure 2B and Online Figure III and Online Table II). The mean MyBP-C/actin ratio in myofibrils of all samples with MYBPC3 mutations was 24±3% lower than donor tissue, but the ratio was unaltered in myectomy samples that did not have a MYBPC3 mutation (myectomy/donor=1.01±0.06).
To examine whether reduced amounts of mutant message contributed to the lower total MyBP-C protein content, wild-type/mutant MYBPC3 mRNA ratios were measured in 4 of the samples using a real-time PCR assay with allele-specific probes (Figure 2C). A moderate decrease in the relative abundance of the mutant transcript in comparison with the wild type was found in 3 samples, including the missense mutation sample M10.
We have been able to systematically assess the effect of both truncation and missense HCM-causing MYBPC3 mutations in human heart muscle by studying a series of 9 samples obtained from patients undergoing surgical myectomy in comparison with donor heart and myectomy samples without a MYBPC3 mutation. In samples with MYBPC3 truncation mutations, we show that no truncated MyBP-C proteins are detectable, either incorporated (as determined by analysis of myofibril fractions) or indeed unincorporated into the sarcomere (from analysis of homogenates). In the analysis of all heart samples of patients bearing MYBPC3 mutations, we find a 24% lower MyBP-C content, thus arguing strongly for haploinsufficiency as the disease mechanism for both truncation and missense mutations. This agrees with, and extends, certain earlier observations.7–9 For the truncation mutants, the measured modest reductions in mRNA (Figure 2C), possibly caused by nonsense-mediated mRNA decay, cannot account for the undetectable levels of mutant MyBP-C protein, and thus degradation of the truncated protein is likely, possibly via the ubiquitin–proteasome system as earlier proposed.14 The presence of normal MyBP-C mRNA from the remaining wild-type allele is apparently not sufficient to yield a full complement of MyBP-C protein; this is in contrast to some other contractile proteins, for example, a heterozygous α-tropomyosin knockout mouse has the normal level of protein in the heart.15 Our surprising finding that missense mutations can cause MyBP-C haploinsufficiency may also be explained by proteolysis of the mutant protein (as reported for 1 heterologously expressed MYBPC3 missense mutant16), although a modest reduction in mutant mRNA (as suggested by the M10 data in Figure 2C) could also account for the reduced full-length protein; the mechanism by which a reduced mRNA level of a missense allele is achieved is unclear. It remains possible that the missense mutant protein does incorporate and has an additional deleterious effect.
Although haploinsufficiency has not been observed with mutations in other HCM genes, we propose that in the case of MYBPC3 mutations, it is sufficient to cause the disease. Functional studies, in which MyBP-C has been partially extracted from fiber preparations to a similar extent to the reduction observed in myectomy tissue, suggest that the measured reduction in protein content is sufficient to have a significant effect on contractility.17,18 The depletion of MyBP-C protein in different samples is not equivalent, and we suggest this may contribute to the observed spectrum of disease severity.
We thank Samantha Harris (UCSD) for the MyBP-C antibody and Cris Dos Remedios (Sydney) for the donor heart muscle.
Sources of Funding
We thank the British Heart Foundation, the Oxford NIHR Comprehensive Biomedical Research Centre, and FP6 EUGeneHeart Programme LSHM-CT-2005-018833 for funding. The contributions of W.J.M. and V.T. were funded, in part, by the Department of Health’s National Institute of Health Research Biomedical Research Centres scheme.
↵*This article was reviewed and accepted during Dr. Marban’s editorship.
Original received April 2, 2009; resubmission received June 8, 2009; revised resubmission received June 23, 2009; accepted June 24, 2009.
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