Obstructive hypertrophic cardiomyopathy is associated with reduced expression of vinculin in the intercalated disc

Mutations in the cardiac-specific insert of vinculin, metavinculin, rarely cause hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Subsequently, a missense mutation in the ubiquitously expressed vinculin was discovered in a patient with obstructive HCM. Microscopic examination of both myectomy specimens from patients bearing genetic defects in metavinculin and vinculin showed a marked reduction of vinculin/metavinculin expression in the intercalated disc, but normal expression in the Z-disc. Given that distinct functional domains were altered by the metavinculin and vinculin mutations, we hypothesized that the intercalated disc-specific reduction of vinculin may stem from left ventricular tract obstruction in general rather than rarely observed perturbations in VCL-encoded vinculin. To test this hypothesis, we examined the localization of vinculin/metavinculin in hypertrophied human heart tissue from patients with cardiovascular conditions associated with obstruction and hemodynamic overload using an immunohistochemistry approach. Tissue specimens derived from patients with obstructive HCM and aortic stenosis (AS) showed a universal defect of vinculin/metavinculin expression in the intercalated disc but preserved expression in the cardiac Z-disc, whereas tissue specimens derived from patients with either DCM, hypertensive heart disease (HTN), or pulmonary hypertension (PHTN) exhibited normal expression of vinculin/metavinculin in both the Z- and the intercalated disc despite being associated with hypertrophy. Results of this study suggest that cardiac hypertrophy may be associated with different expression of the marker vinculin/metavinculin depending on the underlying pathophysiology; hemodynamic overload may not affect the localization whereas obstructive disease substantially reduces the expression of vinculin preferentially in the intercalated disc.     

Exercise related ventricular arrhythmias are related to cardiac fibrosis in hypertrophic cardiomyopathy mutation carriers

Aims

Hypertrophic cardiomyopathy (HCM) is a frequent cause of sudden cardiac death (SCD) due to exercise-related ventricular arrhythmias (ERVA); however the pathological substrate is uncertain. The aim was to determine the prevalence of ERVA and their relation with fibrosis as determined by cardiac magnetic resonance imaging (CMR) in carriers of an HCM causing mutation.

Methods

We studied the prevalence and origin of ERVA and related these with fibrosis on CMR in a population of 31 HCM mutation carriers.

Results

ERVA occurred in seven patients (23%) who all showed evidence of fibrosis (100% ERVA(+) vs. 58% ERVA(-), p = 0.04). No ventricular tachycardia or ventricular fibrillation occurred. In patients with ERVA, the extent of fibrosis was significantly larger (8 ± 4% vs. 3 ± 4%, p = 0.02). ERVA originated from areas with a high extent of fibrosis or regions directly adjacent to these areas.

Conclusions

ERVA in HCM mutation carriers arose from the area of fibrosis detected by CMR; ERVA seems closely related to cardiac fibrosis. Fibrosis as detected by CMR should be evaluated as an additional risk factor to further delineate risk of SCD in carriers of an HCM causing mutation.     

Pregnancy in women with hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is increasingly being diagnosed in pregnant women. Women with HCM generally tolerate pregnancy well. The risk is however higher in women who are symptomatic before pregnancy or in those with severe left ventricular outflow tract obstruction. The incidence of arrhythmias does not appear to be increased during pregnancy and maternal mortality is low. Prior to conception, women with HCM should have a risk assessment as well as genetic counselling. During pregnancy beta-blockers should be continued and the judicious use of diuretics may be required to treat symptoms of dyspnoea. A vaginal delivery with regional anaesthesia is usually appropriate. Women should be managed by a specialist multidisciplinary team.     

A myomesin mutation associated with hypertrophic cardiomyopathy deteriorates dimerisation properties

Myomesin plays an important structural and functional role in the M-band of striated muscles. The C-terminal domain 13 of myomesin dimerises and forms antiparallel strands which cross-link neighboring Myosin filaments and titin in the M-line of the sarcomeres. These interactions stabilise the contractile apparatus during striated muscle contraction. Since myomesin is an important component of the M-band we screened the myomesin gene for genetic variants in patients with hypertrophic cardiomyopathy (HCM).We identified the missense mutation V1490I in domain 12 of myomesin in a family with inherited HCM. Analytical ultracentrifugation experiments, circular dichroism spectra, and surface plasmon resonance spectroscopy of myomesin fragments were carried out to investigate the effects of the mutation V1490I on structure and function of myomesin domains 11–13 and 12–13. Both the wild type and mutated myomesin domains My11–13 revealed similar secondary structures and formed stable dimers. Mutated myomesin domains My11–13 and My12–13 dimers revealed a reduced thermal stability and a significantly decreased dimerisation affinity, showing disturbed functional properties of V1490I mutated myomesin. However, monomeric myomesin domains My11–12, i.e. without dimerisation domain 13 showed no difference in thermal stability between wild type and V1490I mutated myomesin.In conclusion, the V1490I mutation associated with HCM lead to myomesin proteins with abnormal functional properties which affect dimerisation properties of myomesin domain 13. These effects may contribute to the pathogenesis of HCM.     

Identification and functional analysis of a caveolin-3 mutation associated with familial hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are caused by mutations in 14 and 15 different disease genes, respectively, in a part of the patients and the disease genes for cardiomyopathy overlap in part with that for limb-girdle muscular dystrophy (LGMD). In this study, we examined an LGMD gene encoding caveolin-3 (CAV3) for mutation in the patients with HCM or DCM. A Thr63Ser mutation was identified in a sibling case of HCM. Because the mutation was found at the residue that is involved in the LGMD-causing mutations, we investigate the functional change due to the Thr63Ser mutation as compared with the LGMD mutations by examining the distribution of GFP-tagged CAV3 proteins. It was observed that the Thr63Ser mutation reduced the cell surface expression of caveolin-3, albeit the change was mild as compared with the LGMD mutations. These observations suggest that HCM is a clinical spectrum of CAV3 mutations.     

ACE I/D polymorphism in Indian patients with hypertrophic cardiomyopathy and dilated cardiomyopathy

Aim The study was carried to determine the association of angiotensin converting enzyme (ACE) insertion/deletion (I/D) polymorphism with the risk of hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM). Methods and results A total of 174 patients diagnosed with cardiomyopathy (118 with HCM, 51 with DCM, and 5 with RCM) and 164 ethnically, age- and gender-matched controls were included in the study. ACE I/D genotyping was performed by PCR. In total, 25.86% of the patients were in New York Heart Association (NYHA) class III and IV at presentation. A total of 67.24% patients had dyspnea, 56.89% had angina pectoris, and 25.28% of the patients had at least one event of syncope. Frequency of occurrence of the disease was more in male patients compared to female patients (P < 0.05). After adjustment for age, sex, body mass index (BMI), and smoking habit, the prevalence of ACE DD genotype, and ACE ‘D’ allele was significantly higher in patients as compared to controls and was associated with increased risk (DD: OR 2.11, 95% CI 1.27–3.52, P < 0.05; ‘D’: OR 1.91, 95% CI 1.08–3.35, P < 0.05). The mean septal thickness was higher for DD and ID genotypes (20.40 ± 3.73 mm and 21.82 ± 5.35 mm, respectively) when compared with II genotype (18.63 ± 6.69 mm) in HCM patients, however, the differences were not significant statistically (P > 0.05). The DCM patients with ID genotype showed significantly decreased left ventricular ejection fraction (LVEF) at enrolment (26.50 ± 8.04%) (P = 0.04). Conclusion Our results suggest that D allele of ACE I/D polymorphism significantly influences the HCM and DCM phenotypes.     

Risk stratification for sudden cardiac death in hypertrophic cardiomyopathy: Dutch cardiologists and the care of mutation carriers

Background. Patients with hypertrophic cardiomyopathy (HCM) and HCM mutation carriers are at risk of sudden cardiac death (SCD). Both groups should therefore be subject to regular cardiological testing – including risk stratification for SCD – according to international guidelines. We evaluated Dutch cardiologists' knowledge of and adherence to international guidelines on risk stratification and prevention of SCD in mutation carriers with and without manifest HCM. Methods. A questionnaire was sent to 1109 Dutch cardiologists (in training) containing case-based questions. Results. The response rate was 21%. Own general knowledge on HCM care was rated as insufficient by 63% of cardiologists. The percentage of correct answers (i.e. in agreement with international guidelines), on the case-based questions ranged from 37 to 96%, being lowest in cases with an unknown number of risk factors for SCD. A substantial portion of correct answers was based on the correct answer ‘ask an expert opinion’. Significantly more correct answers were provided in cases with manifest HCM. There was little difference between the answers of cardiologists with different self-reported levels of knowledge, with different numbers of HCM patients in their practice or with different numbers of carriers without manifest HCM. Conclusion. Knowledge on risk stratification and preventive therapy was mediocre, and knowledge gaps exist, especially on HCM mutation carriers without manifest disease. Fortunately, experts are frequently asked for their opinion which might bring patient care to an adequate level. Hopefully, our results will stimulate cardiologists to follow developments in this field, thereby increasing quality of care for HCM patients and mutation carriers. (Neth Heart J 2009:17:464–9.).     

Structural analysis of obscurin gene in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a cardiac disease characterized by left ventricular hypertrophy with diastolic dysfunction. Molecular genetic studies have revealed that HCM is caused by mutations in genes for sarcomere/Z-band components including titin/connectin and its associate proteins. However, disease-causing mutations can be found in about half of the patients, suggesting that other disease-causing genes remain to be identified. To explore a novel disease gene, we searched for obscurin gene (OBSCN) mutations in HCM patients, because obscurin interacts with titin/connectin. Two linked variants, Arg4344Gln and Ala4484Thr, were identified in a patient and functional analyses demonstrated that Arg4344Gln affected binding of obscurin to Z9-Z10 domains of titin/connectin, whereas Ala4484Thr did not. Myc-tagged obscurin showed that Arg4344Gln impaired obscurin localization to Z-band. These observations suggest that the obscurin abnormality may be involved in the pathogenesis of HCM.     

Ubiquitin-proteasome system impairment caused by a missense cardiac myosin-binding protein C mutation and associated with cardiac dysfunction in hypertrophic cardiomyopathy

The ubiquitin-proteasome system is responsible for the disappearance of truncated cardiac myosin-binding protein C, and the suppression of its activity contributes to cardiac dysfunction. This study investigated whether missense cardiac myosin-binding protein C gene (MYBPC3) mutation in hypertrophic cardiomyopathy (HCM) leads to destabilization of its protein, causes UPS impairment, and is associated with cardiac dysfunction. Mutations were identified in Japanese HCM patients using denaturing HPLC and sequencing. Heterologous expression was investigated in COS-7 cells as well as neonatal rat cardiac myocytes to examine protein stability and proteasome activity. The cardiac function was measured using echocardiography. Five novel MYBPC3 mutations—E344K, ΔK814, Δ2864-2865GC, Q998E, and T1046M—were identified in this study. Compared with the wild type and other mutations, the E334K protein level was significantly lower, it was degraded faster, it had a higher level of polyubiquination, and increased in cells pretreated with the proteasome inhibitor MG132 (50 μM, 6 h). The electrical charge of its amino acid at position 334 influenced its stability, but E334K did not affect its phosphorylation. The E334K protein reduced cellular 20 S proteasome activity, increased the proapoptotic/antiapoptotic protein ratio, and enhanced apoptosis in transfected Cos-7 cells and neonatal rat cardiac myocytes. Patients carrying the E334K mutation presented significant left ventricular dysfunction and dilation. The conclusion is the missense MYBPC3 mutation E334K destabilizes its protein through UPS and may contribute to cardiac dysfunction in HCM through impairment of the ubiquitin-proteasome system.     

ENerGetIcs in hypertrophic cardiomyopathy: traNslation between MRI, PET and cardiac myofilament function (ENGINE study)

Introduction

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant heart disease mostly due to mutations in genes encoding sarcomeric proteins. HCM is characterised by asymmetric hypertrophy of the left ventricle (LV) in the absence of another cardiac or systemic disease. At present it lacks specific treatment to prevent or reverse cardiac dysfunction and hypertrophy in mutation carriers and HCM patients. Previous studies have indicated that sarcomere mutations increase energetic costs of cardiac contraction and cause myocardial dysfunction and hypertrophy. By using a translational approach, we aim to determine to what extent disturbances of myocardial energy metabolism underlie disease progression in HCM.

Methods

Hypertrophic obstructive cardiomyopathy (HOCM) patients and aortic valve stenosis (AVS) patients will undergo a positron emission tomography (PET) with acetate and cardiovascular magnetic resonance imaging (CMR) with tissue tagging before and 4 months after myectomy surgery or aortic valve replacement + septal biopsy. Myectomy tissue or septal biopsy will be used to determine efficiency of sarcomere contraction in-vitro, and results will be compared with in-vivo cardiac performance. Healthy subjects and non-hypertrophic HCM mutation carriers will serve as a control group.

Endpoints

Our study will reveal whether perturbations in cardiac energetics deteriorate during disease progression in HCM and whether these changes are attributed to cardiac remodelling or the presence of a sarcomere mutation per se. In-vitro studies in hypertrophied cardiac muscle from HOCM and AVS patients will establish whether sarcomere mutations increase ATP consumption of sarcomeres in human myocardium. Our follow-up imaging study in HOCM and AVS patients will reveal whether impaired cardiac energetics are restored by cardiac surgery.     

Mutations in myosin S2 alter cardiac myosin-binding protein-C interaction in hypertrophic cardiomyopathy in a phosphorylation-dependent manner

Hypertrophic cardiomyopathy (HCM) is an inherited cardiovascular disorder primarily caused by mutations in the β-myosin heavy-chain gene. The proximal subfragment 2 region (S2), 126 amino acids of myosin, binds with the C0-C2 region of cardiac myosin-binding protein-C to regulate cardiac muscle contractility in a manner dependent on PKA-mediated phosphorylation. However, it is unknown if HCM-associated mutations within S2 dysregulate actomyosin dynamics by disrupting its interaction with C0-C2, ultimately leading to HCM. Herein, we study three S2 mutations known to cause HCM: R870H, E924K, and E930Δ. First, experiments using recombinant proteins, solid-phase binding, and isothermal titrating calorimetry assays independently revealed that mutant S2 proteins displayed significantly reduced binding with C0-C2. In addition, CD revealed greater instability of the coiled-coil structure in mutant S2 proteins compared with S2^Wt proteins. Second, mutant S2 exhibited 5-fold greater affinity for PKA-treated C0-C2 proteins. Third, skinned papillary muscle fibers treated with mutant S2 proteins showed no change in the rate of force redevelopment as a measure of actin–myosin cross-bridge kinetics, whereas S2^Wt showed increased the rate of force redevelopment. In summary, S2 and C0-C2 interaction mediated by phosphorylation is altered by mutations in S2, which augment the speed and force of contraction observed in HCM. Modulating this interaction could be a potential strategy to treat HCM in the future.     

Hypertrophic cardiomyopathy: two homozygous cases with "typical" hypertrophic cardiomyopathy and three new mutations in cases with progression to dilated cardiomyopathy

About 10% of cases of hypertrophic cardiomyopathy (HCM) evolve into dilated cardiomyopathy (DCM) with unknown causes. We studied 11 unrelated patients (pts) with HCM who progressed to DCM (group A) and 11 who showed "typical" HCM (group B). Mutational analysis of the beta-myosin heavy chain (MYH7), myosin-binding protein C (MYBPC3), and cardiac troponin T (TNNT2) genes demonstrated eight mutations affecting MYH7 or MYBPC3 gene, five of which were new mutations. In group A-pts, the first new mutation occurred in the myosin head-rod junction and the second occurred in the light chain-binding site. The third new mutation leads to a MYBPC3 lacking titin and myosin binding sites. In group B, two pts with severe HCM carried two homozygous MYBPC3 mutations and one with moderate hypertrophy was a compound heterozygous for MYBPC3 gene. We identified five unreported mutations, potentially "malignant" defects as for the associated phenotypes, but no specific mutations of HCM/DCM.     

CMR-derived TAPSE measurement: a semi-quantitative method of right ventricular function assessment in patients with hypertrophic cardiomyopathy

Aim

To compare cardiovascular magnetic resonance (CMR)-derived right ventricular fractional shortening (RVFS), tricuspid annular plane systolic excursion with a reference point within the right ventricular apex (TAPSE_in) and with one outside the ventricle (TAPSE_out) with the standard volumetric approach in patients with hypertrophic cardiomyopathy (HCM).

Methods and results

105 patients with HCM and 20 healthy subjects underwent CMR. In patients with HCM, TAPSE_in (r = 0.31, p = 0.001) and RVFS (r = 0.35, p = 0.0002) revealed a significant but weak correlation with right ventricular ejection fraction (RVEF), whereas TAPSE_out (r = 0.57, p < 0.0001) showed a moderate correlation with RVEF. The ability to predict RVEF < 45 % in HCM patients was best for TAPSE_out. In patients with hypertrophic obstructive cardiomyopathy (HOCM), RVEF showed a significant but weak correlation with TAPSE_out (r = 0.36, p = 0.02) and no correlation with TAPSE_in (r = 0.05, p = 0.07) and RVFS (r = 0.02, p = 0.2). In patients with hypertrophic non-obstructive cardiomyopathy (HNCM), there was a moderate correlation between RVEF and TAPSE_out (r = 0.57, p < 0.0001) and a weak correlation with TAPSE_in (r = 0.39, p = 0.001) and RVFS (r = 0.38, p = 0.002). In the 20 healthy controls, there was a strong correlation between RVEF and all semi-quantitative measurements.

Conclusion

CMR-derived TAPSE_in is not suitable to determine right ventricular function in HCM patients. TAPSE_out showed a good correlation with RVEF in HNCM patients but only a weak correlation in HOCM patients. TAPSE_out might be used for screening but the detection of subtle changes in RV function requires the 3D volumetric approach.     

A molecular basis for familial hypertrophic cardiomyopathy: a beta cardiac myosin heavy chain gene missense mutation

A point mutation in exon 13 of the beta cardiac myosin heavy chain (MHC) gene is present in all individuals affected with familial hypertrophic cardiomyopathy (FHC) from a large kindred. This missense mutation converts a highly conserved arginine residue (Arg-403) to a glutamine. Affected individuals from an unrelated family lack this missense mutation, but instead have an alpha/beta cardiac MHC hybrid gene. Identification of two unique mutations within cardiac MHC genes in all individuals with FHC from two unrelated families demonstrates that defects in the cardiac MHC genes can cause this disease. The pathology resulting from a missense mutation at residue 403 further suggests that a critical function of myosin is disrupted by this mutation.     

Circulating biomarkers of hypertrophy and fibrosis in patients with hypertrophic cardiomyopathy assessed by cardiac magnetic resonance

Abstract

Background: Myocardial fibrosis in hypertrophic cardiomyopathy (HCM) is associated with worse clinical outcomes. The availability of circulating biomarkers of myocardial fibrosis and hypertrophy would be helpful in clinical practice.

Objective: The aim of this study was to evaluate usefulness of various biomarkers of myocardial fibrosis and hypertrophy in HCM.

Methods: Levels of biomarkers: soluble ST2 (sST2), galectin-3 (Gal-3), growth differentiation factor-15 (GDF-15), NT-proBNP and high-sensitivity cardiac troponin T (hs-cTnT) were measured in 60 patients with HCM. All patients underwent cardiac magnetic resonance imaging to calculate parameters of hypertrophy and fibrosis.

Results: We observed positive correlations among sST2 levels and left ventricular mass (LVM) (r?=?0.32, p?=?0.012), LV mass indexed for the body surface area (LVMI) (r?=?0.27, p?=?0.036) and maximal wall thickness (MWT) (r?=?0.31, p?=?0.015). No correlation was found between Gal-3 and GDF-15 levels and hypertrophy and fibrosis parameters. We observed positive correlations among hs-cTnT levels and LVM (r?=?0.58, p?<?0.0001), LVMI (r?=?0.48, p?=?0.0001), MWT (r?=?0.31, p?=?0.015) and late gadolinium enhancement (LGE) mass (r?=?0.37, p?=?0.003). There were positive correlations between NT-proBNP levels and LVM (r?=?0.33, p?=?0.01), LVMI (r?=?0.41, p?=?0.001), MWT (r?=?0.42, p?<?0.001) and LGE mass (r?=?0.44, p?<?0.001).

Conclusions: Although no correlation between sST2 levels and myocardial fibrosis was found, sST2 may provide some additional information about hypertrophy extension. NT-proBNP and hs-cTnT are useful biomarkers in assessment of hypertrophy and fibrosis in HCM.     

BIO FOr CARE: biomarkers of hypertrophic cardiomyopathy development and progression in carriers of Dutch founder truncating MYBPC3 variants—design and status

BackgroundHypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disease, commonly caused by truncating variants in the MYBPC3 gene. HCM is an important cause of sudden cardiac death; however, overall prognosis is good and penetrance in genotype-positive individuals is incomplete. The underlying mechanisms are poorly understood and risk stratification remains limited.AimTo create a nationwide cohort of carriers of truncating MYBPC3 variants for identification of predictive biomarkers for HCM development and progression.MethodsIn the multicentre, observational BIO FOr CARe (Identification of BIOmarkers of hypertrophic cardiomyopathy development and progression in Dutch MYBPC3 FOunder variant CARriers) cohort, carriers of the c.2373dupG, c.2827C > T, c.2864_2865delCT and c.3776delA MYBPC3 variants are included and prospectively undergo longitudinal blood collection. Clinical data are collected from first presentation onwards. The primary outcome constitutes a composite endpoint of HCM progression (maximum wall thickness ≥ 20 mm, septal reduction therapy, heart failure occurrence, sustained ventricular arrhythmia and sudden cardiac death).ResultsSo far, 250 subjects (median age 54.9 years (interquartile range 43.3, 66.6), 54.8% male) have been included. HCM was diagnosed in 169 subjects and dilated cardiomyopathy in 4. The primary outcome was met in 115 subjects. Blood samples were collected from 131 subjects.ConclusionBIO FOr CARe is a genetically homogeneous, phenotypically heterogeneous cohort incorporating a clinical data registry and longitudinal blood collection. This provides a unique opportunity to study biomarkers for HCM development and prognosis. The established infrastructure can be extended to study other genetic variants. Other centres are invited to join our consortium.Supplementary InformationThe online version of this article (10.1007/s12471-021-01539-w) contains supplementary material, which is available to authorized users.