Evidence From Human Myectomy Samples That MYBPC3 Mutations Cause Hypertrophic Cardiomyopathy Through Haploinsufficiency

Abstract

(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 Ϸ20% 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 viable 12,13 ; 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. MethodsWe obtained human heart muscle from donor hearts andinterventricular 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. ResultsWe 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;

ADP-stimulated contraction: A predictor of thin-filament activation in cardiac disease

“…Finally, haploinsufficiency was also reported in HCM with MYBPC3 missense mutations (23), including the mutation investigated in our study (E258K). Indeed, samples with MYBPC3 mut missense mutations in our study showed a 22 ± 9% reduction in cMyBP-C protein level compared with donors.…”

“…Recent evidence supports that the N-terminal extension of cMyBP-C binds the low-Ca 2+ -state (B-state) position of tropomyosin on actin and interferes with tropomyosin-actin interactions, dislocating tropomyosin into the C-state position (i.e., the presence of cMyBP-C sensitizes the thin filament to Ca 2+ ) (20,21). Because it was previously shown that in Ca 2+ -free conditions (B state) ∼5% of the thin filaments (lacking cMyBP-C) have tropomyosin localized in the C-state position (10), more myofilaments may be in the C state in the presence of cMyBP-C. We (22) and others (23) have shown that cMyBP-C mutations, which are a major cause of HCM, have a reduced level of healthy cMyBP-C protein compared with nonfailing hearts (i.e., haploinsufficiency), which may alter tropomyosin position on the thin filament.…”

“…We (22) and others (23) have shown that cMyBP-C truncating mutations lack the presence of the smaller truncated protein, but have a lower total level of healthy full-length cMyBP-C (i.e., haploinsufficiency). To assess whether the expression level of cMyBP-C in MYBPC3 trunc correlates with ADP sensitivity [as cMyBP-C modulates thin-filament transitions (20,21)], cMyBP-C expression was determined.…”

Myocardial energy depletion and dynamic systolic dysfunction in hypertrophic cardiomyopathy

“…In myectomy specimens from patients with an MYBPC3 truncation mutation, protein content was reduced by 24-33% compared with transplant donor, or non-MYBPC3 myectomy specimens 6,7 .…”

Impact of the Myosin Modulator Mavacamten on Force Generation and Cross‐Bridge Behavior in a Murine Model of Hypercontractility

“…This is mainly because missense mutations in β‐MHC that cause HCM result in incorporation of mutant proteins into the sarcomere, whereas the majority of mutations in cMyBPC that cause HCM often result in the production of truncated cMyBPC peptides, which are not incorporated into the sarcomere as they are likely removed by cellular quality control mechanisms such as non‐sense‐mediated mRNA decay or ubiquitin‐proteasome systems 24. The net result is decreased incorporation of full‐length cMyBPC into the sarcomere leading to haploinsufficiency 7, 8, 25, 26. Functionally, decreased incorporation of cMyBPC in human HCM samples expressing cMyBPC truncation mutations led to accelerated XB kinetics at submaximal Ca 2+ activations, in part, contributing to cardiac hypercontractility and hypertrophy 27.…”