(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;

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BACKGROUND:Myocardial infarction is diagnosed when biomarkers of cardiac necrosis exceed the 99th centile, although guidelines advocate even lower concentrations for early rule-out. We examined how many myocytes and how much myocardium these concentrations represent. We also examined if dietary troponin can confound the rule-out algorithm.

It is well established that MYBPC3 mutations are the most common cause of hypertrophic cardiomyopathy, accounting for about half of identified mutations. However, when compared with mutations in other myofibrillar proteins that cause hypertrophic cardiomyopathy, MYBPC3 mutations seem to be the odd one out. The most striking characteristic of HCM mutations in MYBPC3 is that many are within introns and are predicted to cause aberrant splicing leading to a frameshift and a premature chain termination, yet the truncated peptides have never been identified in human heart tissue carrying these mutations. Instead of expression of a poison peptide we consistently observe haploinsufficiency of MyBP-C in MYBPC3 mutant human heart muscle. In this review we investigate the mechanism for MyBP-C haploinsufficiency and consider how this haploinsufficiency could cause hypertrophic cardiomyopathy.

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BackgroundStudies of the functional consequences of DCM-causing mutations have been limited to a few cases where patients with known mutations had heart transplants. To increase the number of potential tissue samples for direct investigation we performed whole exon sequencing of explanted heart muscle samples from 30 patients that had a diagnosis of familial dilated cardiomyopathy and screened for potentially disease-causing mutations in 58 HCM or DCM-related genes.ResultsWe identified 5 potentially disease-causing OBSCN mutations in 4 samples; one sample had two OBSCN mutations and one mutation was judged to be not disease-related. Also identified were 6 truncating mutations in TTN, 3 mutations in MYH7, 2 in DSP and one each in TNNC1, TNNI3, MYOM1, VCL, GLA, PLB, TCAP, PKP2 and LAMA4. The mean level of obscurin mRNA was significantly greater and more variable in healthy donor samples than the DCM samples but did not correlate with OBSCN mutations. A single obscurin protein band was observed in human heart myofibrils with apparent mass 960 ± 60 kDa. The three samples with OBSCN mutations had significantly lower levels of obscurin immunoreactive material than DCM samples without OBSCN mutations (45±7, 48±3, and 72±6% of control level).Obscurin levels in DCM controls, donor heart and myectomy samples were the same.Conclusions OBSCN mutations may result in the development of a DCM phenotype via haploinsufficiency. Mutations in the obscurin gene should be considered as a significant causal factor of DCM, alone or in concert with other mutations.

We generated a transgenic mouse model expressing the apical hypertrophic cardiomyopathy-causing mutation ACTC E99K at 50% of total heart actin and compared it with actin from patients carrying the same mutation. The actin mutation caused a higher Ca 2؉ sensitivity in reconstituted thin filaments measured by in vitro motility assay (2.3-fold for mice and 1.3-fold for humans) and in skinned papillary muscle. The mutation also abolished the change in Ca 2؉ sensitivity normally linked to troponin I phosphorylation. MyBP-C and troponin I phosphorylation levels were the same as controls in transgenic mice and human carrier heart samples. ACTC E99K mice exhibited a high death rate between 28 and 45 days (48% females and 22% males). At 21 weeks, the hearts of the male survivors had enlarged atria, increased interstitial fibrosis, and sarcomere disarray. MRI showed hypertrophy, predominantly at the apex of the heart. End-diastolic volume and end-diastolic pressure were increased, and relaxation rates were reduced compared with nontransgenic littermates. End-systolic pressures and volumes were unaltered. ECG abnormalities were present, and the contractile response to ␤-adrenergic stimulation was much reduced. Older mice (29-week-old females and 38-week-old males) developed dilated cardiomyopathy with increased end-systolic volume and continuing increased end-diastolic pressure and slower contraction and relaxation rates. ECG showed atrial flutter and frequent atrial ectopic beats at rest in some ACTC E99K mice. We propose that the ACTC E99K mutation causes higher myofibrillar Ca 2؉ sensitivity that is responsible for the sudden cardiac death, apical hypertrophy, and subsequent development of heart failure in humans and mice.

E40K and E54K mutations in ␣-tropomyosin cause inherited dilated cardiomyopathy.Previously we showed, using Ala-Ser ␣-tropomyosin (AS-␣-Tm) expressed in Escherichia coli, that both mutations decrease Ca 2؉ sensitivity. E40K also reduces V max of actin-Tm-activated S-1 ATPase by 18%. We investigated cooperative allosteric regulation by native Tm, AS-␣-Tm, and the two dilated cardiomyopathy-causing mutants. AS-␣-Tm has a lower cooperative unit size (6.5) than native ␣-tropomyosin (10.0). The E40K mutation reduced the size of the cooperative unit to 3.7, whereas E54K increased it to 8.0. For the equilibrium between On and Off states, the K T value was the same for all actin-Tm species; however, the K T value of actin-Tm-troponin at pCa 5 was 50% less for AS-␣-Tm E40K than for AS-␣-Tm and AS-␣-Tm E54K. K b , the "closed" to "blocked" equilibrium constant, was the same for all tropomyosin species. The E40K mutation reduced the affinity of tropomyosin for actin by 1.74-fold, but only when in the On state (in the presence of S-1). In contrast the E54K mutation reduced affinity by 3.5-fold only in the Off state. Differential scanning calorimetry measurements of AS-␣-Tm showed that domain 3, assigned to the N terminus of tropomyosin, was strongly destabilized by both mutations. Additionally with AS-␣-Tm E54K, we observed a unique new domain at 55°C accounting for 25% of enthalpy indicating stabilization of part of the tropomyosin. The disease-causing mechanism of the E40K mutation may be accounted for by destabilization of the On state of the thin filaments; however, the E54K mutation has a more complex effect on tropomyosin structure and function.