👤 Thomas Eschenhagen

Hypertrophic cardiomyopathy (HCM) is a prevalent inherited cardiac disorder marked by left ventricular hypertrophy and hypercontractility. This excessive mechanical workload creates an energetic mismatch in which consumption exceeds production, leading to myocardial energy depletion. Although CK (creatine kinase) plays a key role in cardiac energy homeostasis, its involvement in HCM remains unclear. This study investigates how hypercontractility-driven mitochondrial stress and the resulting increase in mitochondrial H CK function was analyzed using myocardial left ventricular tissue from 92 patients with HCM (with and without pathogenic sarcomere variants) and 30 non-failing human controls. Myofilament and mitochondrial CK isoforms were measured using mRNA analysis, protein immunoblotting, enzyme activity assays, mass spectrometry, and redox-sensitive proteomics. To explore links between hypercontractility, mitochondrial reactive oxygen species, and CK dysfunction, we used isolated cardiomyocytes from wild-type, mitochondrial-targeted catalase-overexpressing, CK knockout (myofilament and mitochondrial CK deletion), HCM-associated Our analysis revealed significant reductions in myofilament and mitochondrial CK protein levels, as well as CK activity, in myocardium of patients with HCM, primarily because of oxidative modifications of CK. In isolated mouse cardiomyocytes from wild-type and CK knockouts, hypercontractility induced by EMD-57033 elevated mitochondrial H This study reveals a mechanistic link between hypercontractility, mitochondrial reactive oxygen species, and CK dysfunction in HCM, perpetuating a cycle of energetic dysfunction. Targeting hypercontractility and oxidative stress through myosin inhibition offers a strategy to restore energy balance and reduce arrhythmic risk in HCM. Show less

Hypertrophic cardiomyopathy (HCM) is an inherited cardiomyopathy often caused by pathogenic variants in MYBPC3 and MYH7, encoding myosin-binding protein C3 and myosin heavy chain 7, respectively. These variants can cause increased actin-myosin crossbridge cycling, resulting in ventricular hypercontractility, but mice lacking Mybpc3 exhibited reduced left ventricular ejection time (LVET) as a sign of systolic dysfunction. In this study, we tested whether LVET is specifically altered in patients carrying MYBPC3 variants by retrospective echocardiographic analysis in two genotype-defined HCM cohorts. LVET was measured by echocardiography and adjusted for heart rate [LVET index (LVETI)] in 166 patients. Variant carriers were stratified for the presence (LVH+) or absence of left ventricular hypertrophy with septal thickness of ≥13 mm (LVH-). Multivariate analysis of variance (MANOVA) was used to identify differences in LVETI between variant carriers and controls with LVETI as the dependent variable, adjusted for sex, age, left ventricular ejection fraction (LVEF), interventricular septal diameter in diastole (IVSd), diastolic dysfunction, left ventricular outflow tract (LVOT) gradient at rest and medication history as confounders. In a total of 166 patients carrying MYBPC3 or MYH7 pathogenic variants (38 ± 3 years, 45% female), we compared the discovery cohort (40 MYBPC3 and 31 MYH7) and the validation cohort ('Valsartan in Attenuating Disease Evolution in Early Sarcomeric HCM'; 54 MYBPC3 and 41 MYH7) with 44 healthy controls. LVETI was lower in MYBPC3 and higher in MYH7 LVH+ patients than in controls in the discovery, validation and pooled cohorts (pooled: MYBPC3 381 ± 19 ms vs. MYH7 437 ± 38 ms, P < 0.001; MYBPC3 vs. controls 411 ± 15 ms, P < 0.001; and MYH7 vs. controls, P < 0.001). Similar findings were seen in LVH- (pooled: MYBPC3 380 ± 16 ms vs. MYH7 437 ± 39 ms, P < 0.001; MYBPC3 vs. controls, P < 0.001). While MYH7 variants were all missense as expected, 87% of the MYBPC3 variants were truncating (including nonsense variants, out-of-frame deletion and splice site variants) and 13% were non-truncating (missense and in-frame deletion). LVETI did not differ between the groups and was significantly lower than the control in both. The data suggest that variants in MYBPC3 and MYH7 result in distinct biophysical consequences, which can be detected by measuring LVETI in patients. The findings may have implications for potential genotype-specific differences in response to therapies targeting sarcomere function. Show less

MYBPC3 is the most frequently affected gene in hypertrophic cardiomyopathy (HCM), which is an autosomal-dominant cardiac disease caused by mutations in sarcomeric proteins. Bi-allelic truncating MYBPC3 mutations are associated with severe forms of neonatal cardiomyopathy. We reprogrammed skin fibroblasts from a HCM patient carrying a heterozygous MYBPC3 truncating mutation into human induced pluripotent stem cells (iPSC) and used CRISPR/Cas9 to generate bi-allelic MYBPC3 truncating mutation and isogenic control hiPSC lines. All lines expressed pluripotency markers, had normal karyotype and differentiated into endoderm, ectoderm and cardiomyocytes in vitro. This set of three lines provides a useful tool to study HCM pathomechanisms. Show less

Hypertrophic cardiomyopathy (HCM) patients are at increased risk of ventricular arrhythmias and sudden cardiac death, which can occur even in the absence of structural changes of the heart. HCM mouse models suggest mutations in myofilament components to affect Ca Show less

Phosphorylation of cardiac myosin-binding protein C (cMyBP-C), encoded by MYBPC3, increases the availability of myosin heads for interaction with actin thus enhancing contraction. cMyBP-C phosphorylation level is lower in septal myectomies of patients with hypertrophic cardiomyopathy (HCM) than in non-failing hearts. Here we compared the effect of phosphomimetic (D282) and wild-type (S282) cMyBP-C gene transfer on the HCM phenotype of engineered heart tissues (EHTs) generated from a mouse model carrying a Mybpc3 mutation (KI). KI EHTs showed lower levels of mutant Mybpc3 mRNA and protein, and altered gene expression compared with wild-type (WT) EHTs. Furthermore, KI EHTs exhibited faster spontaneous contractions and higher maximal force and sensitivity to external [Ca Show less

Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of hypertrophic cardiomyopathy (HCM) carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited ∼50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 5' or 3' pre-trans-splicing molecules represented ∼1% of total MYBPC3 transcripts in healthy hiPSC-CMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in ∼2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1) the feasibility of trans-splicing, although with low efficiency, and (2) efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation. Show less

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac illness and can lead to diastolic dysfunction, sudden cardiac death and heart failure. Treatment of HCM patients is empirical and current pharmacological treatments are unable to stop disease progression or reverse hypertrophy. In this study, we tested if the non-dihydropyridine Ca Left ventricular hypertrophy, diastolic dysfunction and fibrosis are the main features of hypertrophic cardiomyopathy (HCM). Guidelines recommend β-adrenoceptor or Ca Show less

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. The most frequently mutated gene is MYBPC3, encoding cardiac myosin-binding protein-C (cMyBP-C). We compared the pathomechanisms of a truncating mutation (c.2373₂₃₇₄insG) and a missense mutation (c.1591G>C) in MYBPC3 in engineered heart tissue (EHT). EHTs enable to study the direct effects of mutants without interference of secondary disease-related changes. EHTs were generated from Mybpc3-targeted knock-out (KO) and wild-type (WT) mouse cardiac cells. MYBPC3 WT and mutants were expressed in KO EHTs via adeno-associated virus. KO EHTs displayed higher maximal force and sensitivity to external [Ca(2+)] than WT EHTs. Expression of WT-Mybpc3 at MOI-100 resulted in ~73% cMyBP-C level but did not prevent the KO phenotype, whereas MOI-300 resulted in ≥95% cMyBP-C level and prevented the KO phenotype. Expression of the truncating or missense mutation (MOI-300) or their combination with WT (MOI-150 each), mimicking the homozygous or heterozygous disease state, respectively, failed to restore force to WT level. Immunofluorescence analysis revealed correct incorporation of WT and missense, but not of truncated cMyBP-C in the sarcomere. In conclusion, this study provides evidence in KO EHTs that i) haploinsufficiency affects EHT contractile function if WT cMyBP-C protein levels are ≤73%, ii) missense or truncating mutations, but not WT do not fully restore the disease phenotype and have different pathogenic mechanisms, e.g. sarcomere poisoning for the missense mutation, iii) the direct impact of (newly identified) MYBPC3 gene variants can be evaluated. Show less

Cardiac myosin-binding protein C (cMyBP-C) regulates actin-myosin interaction and thereby cardiac myocyte contraction and relaxation. This physiologic function is regulated by cMyBP-C phosphorylation. In our study, reduced site-specific cMyBP-C phosphorylation coincided with increased S-glutathiolation in ventricular tissue from patients with dilated or ischemic cardiomyopathy compared to nonfailing donors. We used redox proteomics, to identify constitutive and disease-specific S-glutathiolation sites in cMyBP-C in donor and patient samples, respectively. Among those, a cysteine cluster in the vicinity of the regulatory phosphorylation sites within the myosin S2 interaction domain C1-M-C2 was identified and showed enhanced S-glutathiolation in patients. In vitro S-glutathiolation of recombinant cMyBP-C C1-M-C2 occurred predominantly at Cys(249), which attenuated phosphorylation by protein kinases. Exposure to glutathione disulfide induced cMyBP-C S-glutathiolation, which functionally decelerated the kinetics of Ca(2+)-activated force development in ventricular myocytes from wild-type, but not those from Mybpc3-targeted knockout mice. These oxidation events abrogate protein kinase-mediated phosphorylation of cMyBP-C and therefore potentially contribute to the reduction of its phosphorylation and the contractile dysfunction observed in human heart failure.-Stathopoulou, K., Wittig, I., Heidler, J., Piasecki, A., Richter, F., Diering, S., van der Velden, J., Buck, F., Donzelli, S., Schröder, E., Wijnker, P. J. M., Voigt, N., Dobrev, D., Sadayappan, S., Eschenhagen, T., Carrier, L., Eaton, P., Cuello, F. S-glutathiolation impairs phosphoregulation and function of cardiac myosin-binding protein C in human heart failure. Show less

Hypertrophic cardiomyopathy (HCM) is often accompanied by increased myofilament Ca(2+) sensitivity and diastolic dysfunction. Recent findings indicate increased late Na(+) current density in human HCM cardiomyocytes. Since ranolazine has the potential to decrease myofilament Ca(2+) sensitivity and late Na(+) current, we investigated its effects in an Mybpc3-targeted knock-in (KI) mouse model of HCM. Unloaded sarcomere shortening and Ca(2+) transients were measured in KI and wild-type (WT) cardiomyocytes. Measurements were performed at baseline (1 Hz) and under increased workload (30 nM isoprenaline (ISO), 5 Hz) in the absence or presence of 10 µM ranolazine. KI myocytes showed shorter diastolic sarcomere length at baseline, stronger inotropic response to ISO, and drastic drop of diastolic sarcomere length under increased workload. Ranolazine attenuated ISO responses in WT and KI cells and prevented workload-induced diastolic failure in KI. Late Na(+) current density was diminished and insensitive to ranolazine in KI cardiomyocytes. Ca(2+) sensitivity of skinned KI trabeculae was slightly decreased by ranolazine. Phosphorylation analysis of cAMP-dependent protein kinase A-target proteins and ISO concentration-response measurements on muscle strips indicated antagonism at β-adrenoceptors with 10 µM ranolazine shifting the ISO response by 0.6 log units. Six-month treatment with ranolazine (plasma level >20 µM) demonstrated a β-blocking effect, but did not reverse cardiac hypertrophy or dysfunction in KI mice. Ranolazine improved tolerance to high workload in mouse HCM cardiomyocytes, not by blocking late Na(+) current, but by antagonizing β-adrenergic stimulation and slightly desensitizing myofilaments to Ca(2+). This effect did not translate in therapeutic efficacy in vivo. Show less

Hypertrophic cardiomyopathy (HCM) is an autosomal-dominant disease with mutations in genes encoding sarcomeric proteins. Previous findings suggest deregulation of the ubiquitin proteasome system (UPS) in HCM in humans and in a mouse model of HCM (Mybpc3-targeted knock-in (KI) mice). In this study we investigated transcript levels of several muscle-specific E3 ubiquitin ligases in KI mice and aimed at identifying novel protein targets. Out of 9 muscle-specific E3 ligases, Asb2β was found with the lowest mRNA level in KI compared to wild-type (WT) mice. After adenoviral-mediated Asb2β transduction of WT neonatal mouse cardiomyocytes with either a WT or inactive Asb2β mutant, desmin was identified as a new target of Asb2β by mass spectrometry, co-immunoprecipitation and immunoblotting. Immunofluorescence analysis revealed a co-localization of desmin with Asb2β at the Z-disk of the sarcomere. Knock-down of Asb2β in cardiomyocytes resulted in higher desmin protein levels. Furthermore, desmin levels were higher in ventricular samples of HCM mice and patients than controls. This study identifies desmin as a new Asb2β target for proteasomal degradation in cardiomyocytes and suggests that accumulation of desmin could contribute to UPS impairment in HCM mice and patients. Show less

Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis. Current treatment is based on beta-adrenoceptor (AR) and calcium channel blockers. Since mice deficient of protein phosphatase-1 inhibitor-1 (I-1), an amplifier in beta-AR signalling, were protected from pathological adrenergic stimulation in vivo, we hypothesized that I-1 ablation could result in an improved outcome in a HCM mouse model. We crossed mice deficient of I-1 with homozygous myosin-binding protein C knock-out ( The data indicate that interference with beta-AR signalling has no long-term benefit in this severe Show less

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. Show less

Myosin-binding protein C (Mybpc3)-targeted knock-in mice (KI) recapitulate typical aspects of human hypertrophic cardiomyopathy. We evaluated whether these functional alterations can be reproduced in engineered heart tissue (EHT) and yield novel mechanistic information on the function of cMyBP-C. EHTs were generated from cardiac cells of neonatal KI, heterozygous (HET) or wild-type controls (WT) and developed without apparent morphological differences. KI had 70% and HET 20% lower total cMyBP-C levels than WT, accompanied by elevated fetal gene expression. Under standard culture conditions and spontaneous beating, KI EHTs showed more frequent burst beating than WT and occasional tetanic contractions (14/96). Under electrical stimulation (6Hz, 37°C) KI EHTs exhibited shorter contraction and relaxation times and a twofold higher sensitivity to external [Ca(2+)]. Accordingly, the sensitivity to verapamil was 4-fold lower and the response to isoprenaline or the Ca(2+) sensitizer EMD 57033 2- to 4-fold smaller. The loss of EMD effect was verified in 6-week-old KI mice in vivo. HET EHTs were apparently normal under basal conditions, but showed similarly altered contractile responses to [Ca(2+)], verapamil, isoprenaline and EMD. In contrast, drug-induced changes in intracellular Ca(2+) transients (Fura-2) were essentially normal. In conclusion, the present findings in auxotonically contracting EHTs support the idea that cMyBP-C's normal role is to suppress force generation at low intracellular Ca(2+) and stabilize the power-stroke step of the cross bridge cycle. Pharmacological testing in EHT unmasked a disease phenotype in HET. The altered drug response may be clinically relevant. Show less

RNA trans-splicing has been explored as a therapeutic option for a variety of genetic diseases, but not for cardiac genetic disease. Hypertrophic cardiomyopathy (HCM) is an autosomal-dominant disease, characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction. MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C) is frequently mutated. We evaluated the 5'-trans-splicing strategy in a mouse model of HCM carrying a Mybpc3 mutation. 5'-trans-splicing was induced between two independently transcribed molecules, the mutant endogenous Mypbc3 pre-mRNA and an engineered pre-trans-splicing molecule (PTM) carrying a FLAG-tagged wild-type (WT) Mybpc3 cDNA sequence. PTMs were packaged into adeno-associated virus (AAV) for transduction of cultured cardiac myocytes and the heart in vivo. Full-length repaired Mybpc3 mRNA represented up to 66% of total Mybpc3 transcripts in cardiac myocytes and 0.14% in the heart. Repaired cMyBP-C protein was detected by immunoprecipitation in cells and in vivo and exhibited correct incorporation into the sarcomere in cardiac myocytes. This study provides (i) the first evidence of successful 5'-trans-splicing in vivo and (ii) proof-of-concept of mRNA repair in the most prevalent cardiac genetic disease. Since current therapeutic options for HCM only alleviate symptoms, these findings open new horizons for causal therapy of the severe forms of the disease.Molecular Therapy-Nucleic Acids (2013) 2, e102; doi:10.1038/mtna.2013.31; published online 2 July 2013. Show less

Exon skipping mediated by antisense oligoribonucleotides (AON) is a promising therapeutic approach for genetic disorders, but has not yet been evaluated for cardiac diseases. We investigated the feasibility and efficacy of viral-mediated AON transfer in a Mybpc3-targeted knock-in (KI) mouse model of hypertrophic cardiomyopathy (HCM). KI mice carry a homozygous G>A transition in exon 6, which results in three different aberrant mRNAs. We identified an alternative variant (Var-4) deleted of exons 5-6 in wild-type and KI mice. To enhance its expression and suppress aberrant mRNAs we designed AON-5 and AON-6 that mask splicing enhancer motifs in exons 5 and 6. AONs were inserted into modified U7 small nuclear RNA and packaged in adeno-associated virus (AAV-U7-AON-5+6). Transduction of cardiac myocytes or systemic administration of AAV-U7-AON-5+6 increased Var-4 mRNA/protein levels and reduced aberrant mRNAs. Injection of newborn KI mice abolished cardiac dysfunction and prevented left ventricular hypertrophy. Although the therapeutic effect was transient and therefore requires optimization to be maintained over an extended period, this proof-of-concept study paves the way towards a causal therapy of HCM. Show less

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from gene mutations to the HCM phenotype remain incompletely understood, partially because current mouse models of HCM do not faithfully reflect the human situation and early hypertrophy confounds the interpretation of functional alterations. The goal of this study was to evaluate whether myofilament Ca(2+) sensitization and diastolic dysfunction are associated or precede the development of left ventricular hypertrophy (LVH) in HCM. We evaluated the function of skinned and intact cardiac myocytes, as well as the intact heart in a recently developed Mybpc3-targeted knock-in mouse model carrying a point mutation frequently associated with HCM. Compared to wild-type, 10-week old homozygous knock-in mice exhibited i) higher myofilament Ca(2+) sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca(2+) transient decay in intact myocytes, and iii) LVH, reduced fractional shortening, lower E/A and E'/A', and higher E/E' ratios by echocardiography and Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast, heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit LVH or systolic dysfunction, but exhibited higher myofilament Ca(2+) sensitivity, faster Ca(2+) transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca(2+) sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca(2+) transients point to compensatory mechanisms directed towards normalization of relaxation. We propose that HCM is a model for diastolic heart failure and this mouse model could be valuable in studying mechanisms and treatment modalities. Show less

Several lines of evidence suggest that alterations of the ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP) may be involved in cardiac diseases. Little is known, however, in hypertrophic cardiomyopathy (HCM). This study studied these pathways in two mouse models of HCM that mainly differ by the presence or absence of truncated mutant proteins. Analyses were performed in homozygous Mybpc3-targeted knock-in (KI) mice, carrying a HCM mutation and exhibiting low levels of mutant cardiac myosin-binding protein C (cMyBP-C), and in Mybpc3-targeted knock-out (KO) mice expressing no cMyBP-C, thus serving as a model of pure cMyBP-C insufficiency. In the early postnatal development of cardiac hypertrophy, both models showed higher levels of ubiquitinated proteins and greater proteasomal activities. To specifically monitor the degradation capacity of the UPS with age, mice were crossed with transgenic mice that overexpress Ub(G76V)-GFP. Ub(G76V)-GFP protein levels were fourfold higher in 1-year-old KI, but not KO mice, suggesting a specific UPS impairment in mice expressing truncated cMyBP-C. Whereas protein levels of key ALP markers were higher, suggesting ALP activation in both mutant mice, their mRNA levels did not differ between the groups, underlying rather defective ALP-mediated degradation. Analysis of key proteins regulated in heart failure did not reveal specific alterations in KI and KO mice. Our data suggest (1) UPS activation in early postnatal development of cardiac hypertrophy, (2) specific UPS impairment in old KI mice carrying a HCM mutation, and (3) defective ALP as a common mechanism in genetically engineered mice with cardiac hypertrophy. Show less

Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric septal hypertrophy and is often caused by mutations in MYBPC3 gene encoding cardiac myosin-binding protein C. In contrast to humans, who are already affected at the heterozygous state, mouse models develop the phenotype mainly at the homozygous state. Evidence from cell culture work suggested that altered proteasome function contributes to the pathogenesis of HCM. Here we tested in two heterozygous Mybpc3-targeted mouse models whether adrenergic stress unmasks a specific cardiac phenotype and proteasome dysfunction. The first model carries a human Mybpc3 mutation (Het-KI), the second is a heterozygous Mybpc3 knock-out (Het-KO). Both models were compared to wild-type (WT) mice. Mice were treated with a combination of isoprenaline and phenylephrine (ISO/PE) or NaCl for 1 week. Whereas ISO/PE induced left ventricular hypertrophy (LVH) with increased posterior wall thickness to a similar extent in all groups, it increased septum thickness only in Het-KI and Het-KO. ISO/PE did not affect the proteasomal chymotrypsin-like activity or β5-subunit protein level in Het-KO or wild-type mice (WT). In contrast, both parameters were markedly lower in Het-KI and negatively correlated with the degree of LVH in Het-KI only. In conclusion, adrenergic stress revealed septal hypertrophy in both heterozygous mouse models of HCM, but proteasome dysfunction only in Het-KI mice, which carry a mutant allele and closely mimic human HCM. This supports the hypothesis that proteasome impairment contributes to the pathophysiology of HCM. Show less

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. The goal of the present study was therefore to gain insights into the mechanisms controlling the expression of MYBPC3 mutations. 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. 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. Show less

Familial hypertrophic cardiomyopathy (FHC) is associated with mutations in 11 genes encoding sarcomeric proteins. Most families present mutations in MYBPC3 and MYH7 encoding cardiac myosin-binding protein C and beta-myosin heavy chain. The consequences of MYH7 mutations have been extensively studied at the molecular level, but controversial results have been obtained with either reduced or augmented myosin motor function depending on the type or homogeneity of myosin studied. In the present study, we took advantage of the accessibility to an explanted heart to analyze for the first time the properties of human homozygous mutant myosin. The patient exhibited eccentric hypertrophy with severely impaired ejection fraction leading to heart transplantation, and carries a homozygous mutation in MYH7 (R403W) and a heterozygous variant in MYBPC3 (V896M). In situ analysis of the left ventricular tissue showed myocyte disarray and hypertrophy plus interstitial fibrosis. In vitro motility assays showed a small, but significant increase in sliding velocity of fluorescent-labeled actin filaments over human mutant cardiac myosin-coated surface compared to control (+18%; P<0.001). Mutant myosin exhibited a large increase in maximal actin-activated ATPase activity (+114%; P<0.05) and Km for actin (+87%; P<0.05) when compared to control. These data show disproportionate enhancement of mechanical and enzymatic properties of human mutant myosin. This suggests inefficient ATP utilization and reduced mechanical efficiency in the myocardial tissue of the patient, which could play an important role in the development of FHC phenotype. Show less