(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;