Background-Mutations in the MYBPC3 gene, encoding cardiac myosin-binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy. In the present study, we investigated whether protein composition and function of the sarcomere are altered in a homogeneous familial hypertrophic cardiomyopathy patient group with frameshift mutations in MYBPC3 (MYBPC3 mut ). Methods and Results-Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c.2373dupG, nϭ7; c.2864_2865delCT, nϭ4) and nonfailing donors (nϭ13). Western blots with the use of antibodies directed against cMyBP-C did not reveal truncated cMyBP-C in MYBPC3 mut . Protein expression of cMyBP-C was significantly reduced in MYBPC3 mut by 33Ϯ5%. Cardiac MyBP-C phosphorylation in MYBPC3 mut samples was similar to the values in donor samples, whereas the phosphorylation status of cardiac troponin I was reduced by 84Ϯ5%, indicating divergent phosphorylation of the 2 main contractile target proteins of the ␤-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per crosssectional area of the myocytes in MYBPC3 mut (20.2Ϯ2.7 kN/m 2 ) compared with donor (34.5Ϯ1.1 kN/m 2 ). Moreover, Ca 2ϩ sensitivity was higher in MYBPC3 mut (pCa 50 ϭ5.62Ϯ0.04) than in donor (pCa 50 ϭ5.54Ϯ0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic ␤-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca 2ϩ sensitivity between MYBPC3 mut (pCa 50 ϭ5.46Ϯ0.03) and donor (pCa 50 ϭ5.48Ϯ0.02). Conclusions-Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca 2ϩ sensitivity in MYBPC3 mut is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction.

Rationale: Mutations in the MYBPC3 gene encoding cardiac myosin-binding protein (cMyBP)-C are frequent causes of hypertrophic cardiomyopathy, but the mechanisms leading from mutations to disease remain elusive. Objective: The goal of the present study was therefore to gain insights into the mechanisms controlling the expression of MYBPC3 mutations. Methods and Results: We developed a cMyBP-C knock-in mouse carrying a point mutation. The level of total cMyBP-C mRNAs was 50% and 80% lower in heterozygotes and homozygotes, respectively. Surprisingly, the single G>A transition on the last nucleotide of exon 6 resulted in 3 different mutant mRNAs: missense (exchange of G for A), nonsense (exon skipping, frameshift, and premature stop codon) and deletion/insertion (as nonsense but with additional partial retention of downstream intron, restoring of the reading frame, and almost full-length protein). Inhibition of nonsense-mediated mRNA decay in cultured cardiac myocytes or in vivo with emetine or cycloheximide increased the level of nonsense mRNAs severalfold but not of the other mRNAs. By using sequential protein fractionation and a new antibody directed against novel amino acids produced by the frameshift, we showed that inhibition of the proteasome with epoxomicin via osmotic minipumps increased the level of (near) full-length mutants but not of truncated proteins. Homozygotes exhibited myocyte and left ventricular hypertrophy, reduced fractional shortening, and interstitial fibrosis; heterozygotes had no major phenotype. Conclusions: These data reveal (1) an unanticipated complexity of the expression of a single point mutation in the whole animal and (2) the involvement of both nonsense-mediated mRNA decay and the ubiquitin-proteasome system in lowering the level of mutant proteins. (Circ Res. 2009;105:239-248.)Key Words: cardiomyopathy Ⅲ hypertrophic cardiomyopathy Ⅲ mRNA stability Ⅲ transgenic mice Ⅲ ubiquitin C ardiac myosin-binding protein (cMyBP)-C is a major component of the A-band of the sarcomere, where it interacts with myosin, actin and titin (see elsewhere 1,2 and reviewed previously 3 ). It is exclusively expressed in the heart in humans and mice. 4,5 Its role has been enigmatic for long, but accumulating recent evidence suggests that cMyBP-C is essential for normal diastolic relaxation by inhibiting actin-myosin interactions at low intracellular Ca 2ϩ concentrations. 6 -10 Mutations in MYBPC3 encoding cMyBP-C cause hypertrophic cardiomyopathy (HCM) (reviewed previously 3,11 ).HCM is an autosomal-dominant disease characterized by left ventricular (LV) hypertrophy, which predominantly involves the interventricular septum and is associated with myocardial disarray and interstitial fibrosis. 12 HCM involves more than 450 mutations in at least 13 genes encoding sarcomeric proteins. 11,13 Out of them, mutations in MYBPC3 are frequent. 14 In contrast to other disease genes, in which the majority of the mutations are missense, Ϸ70% of MYBPC3 mutations result in a frameshift creating a premature termination...

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Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease accompanied by structural and contractile alterations. We identified a rare c.740C>T (p.T247M) mutation in ACTN2, encoding α‐actinin 2 in a HCM patient, who presented with left ventricular hypertrophy, outflow tract obstruction, and atrial fibrillation. We generated patient‐derived human‐induced pluripotent stem cells (hiPSCs) and show that hiPSC‐derived cardiomyocytes and engineered heart tissues recapitulated several hallmarks of HCM, such as hypertrophy, myofibrillar disarray, hypercontractility, impaired relaxation, and higher myofilament Ca2+ sensitivity, and also prolonged action potential duration and enhanced L‐type Ca2+ current. The L‐type Ca2+ channel blocker diltiazem reduced force amplitude, relaxation, and action potential duration to a greater extent in HCM than in isogenic control. We translated our findings to patient care and showed that diltiazem application ameliorated the prolonged QTc interval in HCM‐affected son and sister of the index patient. These data provide evidence for this ACTN2 mutation to be disease‐causing in cardiomyocytes, guiding clinical therapy in this HCM family. This study may serve as a proof‐of‐principle for the use of hiPSC for personalized treatment of cardiomyopathies.

Abstract-The role of cardiac myosin-binding protein C (cMyBP-C) in cardiac contraction is still not fully resolved.Experimental ablation of cMyBP-C by various means resulted in inconsistent changes in Ca 2ϩ sensitivity and increased velocity of force of skinned preparations. To evaluate how these effects are integrated in an intact, living myocyte context, we investigated consequences of cMyBP-C ablation in ventricular myocytes and left atria from cMyBP-C knock-out (KO) mice compared with wild-type (WT). At 6 weeks, KO myocytes exhibited mild hypertrophy that became more pronounced by 30 weeks. Isolated cells from KO exhibited markedly lower diastolic sarcomere length (SL) without change in diastolic Ca 2ϩ . The lower SL in KO was partly abolished by the actin-myosin ATPase inhibitors 2,3-butanedione monoxime or blebbistatin, indicating residual actin-myosin interaction in diastole. The relationship between cytosolic Ca 2ϩ and SL showed that KO cells started to contract at lower Ca 2ϩ without reaching a higher maximum, yielding a smaller area of the phase-plane diagram. Both sarcomere shortening and Ca 2ϩ transient were prolonged in KO. Isolated KO left atria exhibited a marked increase in sensitivity to external Ca 2ϩ and, in contrast to WT, continued to develop twitch force at low micromolar Ca 2ϩ . Taken together, the main consequence of cMyBP-C ablation was a defect in diastolic relaxation and a smaller dynamic range of cell shortening, both of which likely result from the increased myofilament Ca 2ϩ sensitivity. Our findings indicate that cMyBP-C functions as a restraint on myosin-actin interaction at low Ca 2ϩ and short SL to allow complete relaxation during diastole. (Circ Res. 2007;101:928-938.)

Background: Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. While ≈50% of patients with HCM carry a sarcomere gene mutation (sarcomere mutation-positive, HCM SMP ), the genetic background is unknown in the other half of the patients (sarcomere mutation-negative, HCM SMN ). Genotype-specific differences have been reported in cardiac function. Moreover, HCM SMN patients have later disease onset and a better prognosis than HCM SMP patients. To define if genotype-specific derailments at the protein level may explain the heterogeneity in disease development, we performed a proteomic analysis in cardiac tissue from a clinically well-phenotyped HCM patient group. Methods: A proteomics screen was performed in cardiac tissue from 39 HCM SMP patients, 11HCM SMN patients, and 8 nonfailing controls. Patients with HCM had obstructive cardiomyopathy with left ventricular outflow tract obstruction and diastolic dysfunction. A novel MYBPC3 2373insG mouse model was used to confirm functional relevance of our proteomic findings. Results: In all HCM patient samples, we found lower levels of metabolic pathway proteins and higher levels of extracellular matrix proteins. Levels of total and detyrosinated α-tubulin were markedly higher in HCM SMP than in HCM SMN and controls. Higher tubulin detyrosination was also found in 2 unrelated MYBPC3 mouse models and its inhibition with parthenolide normalized contraction and relaxation time of isolated cardiomyocytes. Conclusions: Our findings indicate that microtubules and especially its detyrosination contribute to the pathomechanism of patients with HCM SMP . This is of clinical importance since it represents a potential treatment target to improve cardiac function in patients with HCM SMP , whereas a beneficial effect may be limited in patients with HCM SMN .

Homozygous or compound heterozygous frameshift mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C) cause neonatal hypertrophic cardiomyopathy (HCM), which rapidly evolves into systolic heart failure and death within the first year of life. Here we show successful long-term Mybpc3 gene therapy in homozygous Mybpc3-targeted knock-in (KI) mice, which genetically mimic these human neonatal cardiomyopathies. A single systemic administration of adeno-associated virus (AAV9)-Mybpc3 in 1-day-old KI mice prevents the development of cardiac hypertrophy and dysfunction for the observation period of 34 weeks and increases Mybpc3 messenger RNA (mRNA) and cMyBP-C protein levels in a dose-dependent manner. Importantly, Mybpc3 gene therapy unexpectedly also suppresses accumulation of mutant mRNAs. This study reports the first successful long-term gene therapy of HCM with correction of both haploinsufficiency and production of poison peptides. In the absence of alternative treatment options except heart transplantation, gene therapy could become a realistic treatment option for severe neonatal HCM.

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.