Familial Hypertrophic Cardiomyopathy Patients Research Articles

MicroRNA expression profiles in familial hypertrophic cardiomyopathy with myosin-binding protein C3 (MYBPC3) gene mutations

Familial hypertrophic cardiomyopathy (FHCM) is an autosomal dominant inherited disease caused by mutations in genes encoding cardiac sarcomere proteins. MicroRNAs (miRNAs) play an important role in the pathogenesis of FHCM. In the present study, we aimed to determine the miRNA profile in FHCM patients with myosin-binding protein C3 (MYBPC3) gene mutations. We recruited three FHCM patients and age- and sex-matched controls. The three probands all had hypertrophic obstructive cardiomyopathy with severe myocardial hypertrophy, and two of the three had a history of sudden cardiac death, representing a “malignant” phenotype. We then compared the miRNA expression profiles of three FHCM patients carrying MYBPC3 gene mutations with those of the normal control group using miRNA sequencing technology. Differentially expressed miRNAs were verified using real-time polymerase chain reaction (qPCR). Target genes and signaling pathways of the identified differentially expressed miRNAs were predicted using bioinformatics analysis. A total of 33 significantly differentially expressed miRNAs were detected in the peripheral blood of the three probands, of which 28 were upregulated, including miR-208b-3p, and 5 were downregulated. Real-time PCR confirmed the upregulated expression of miR-208b-3p in FHCM patients (P < 0.05). Bioinformatics analysis showed that miR-208b-3p was mainly enriched in 79 target genes including UBE2V2, MED13, YBX1, CNKSR2, GATA4, andSOX5/6, et al. Gene ontology (GO) analysis of target genes showed that miR-208b was mainly involved in the processes of negative regulation of transcription from RNA polymerase II promoter, and regulation of transcription, DNA templated. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the target genes regulated by miR-208b-3p were mainly involved in the Wnt signaling pathway. These findings suggest that FHCM patients with MYBPC3 gene mutations have a specific miRNA expression profile, and that miR-208b-3p is significantly upregulated in cardiac hypertrophy. Our results also indicate that miRNA-208b-3p activates the Wnt signaling pathway through its target gene to promote cardiac hypertrophy.

The Arrhythmogenic Impact of the Familial Hypertrophic Cardiomyopathy-related Cardiac Troponin T mutation I79N

Cardiac troponin (cTn) is a heterotrimeric complex that plays an essential role in cardiac contractility. Each complex is composed of a highly conserved Ca2+ binding subunit (cTnC), an inhibitory subunit (cTnI), and a tropomyosin binding subunit (cTnT). Familial hypertrophic cardiomyopathy (FHC) is the most common inherited cardiomyopathy, and 7% of FHC is associated with mutations found in the cTnT gene. Unlike the general anatomical abnormalities found in FHC patients, hearts from patients harbouring cTnT mutations show less ventricular hypertrophy but significant arrhythmogenesis. This study focuses on the I79N cTnT mutation that is morphologically asymptomatic but associated with a high incidence of sudden cardiac death.

Identification of a novel hypertrophic cardiomyopathy-associated mutation using targeted next-generation sequencing.

Hypertrophic cardiomyopathy (HCM), one of the most common forms of myocardial diseases, is the major cause of sudden cardiac death in young adults and competitive athletes. Analyses of gene mutations associated with HCM are valuable for its molecular diagnosis, genetic counseling, and management of familial HCM. To dissect the relationship between the clinical presentation and gene mutations of HCM, the genetic characterizations of 19 HCM-related genes in 18 patients (8 cases from 6 pedigrees with familial HCM and 10 cases without familial HCM) were detected using next-generation sequencing (NGS). As a result, 12 disease-related mutations were identified in the 18 subjects, including 6 single mutations and 3 double mutations [MYBPC3 (p.Gln998Glu) plus TNNI3 (p.Arg145Gly), PRKAG2 (p.Gly100Ser) plus MYBPC3 (p.Lys1209Serfs*28) and TNNI3 (p.Glu124Gln) plus GLA (p.Trp47*)]. The 3 heterozygous double mutations were discovered for the first time in the malignant familial HCM patients. Of the 6 single mutations, a novel mutation was found in tafazzin (TAZ, p.Ile208Val), and a mutation in β-myosin heavy chain gene (MYH7, p.Arg54Gln), which was reported as rare in the general population, was firstly found in one HCM patient. Identification of novel and rare mutations in HCM patients have added new data to the spectrum of gene mutations associated with this disease. These findings provide an essential basis for the molecular diagnosis and better management of family members at risk of familial HCM.

Impaired contractile function due to decreased cardiac myosin binding protein C content in the sarcomere

Mutations in cardiac myosin binding protein C (MyBP-C) are a common cause of familial hypertrophic cardiomyopathy (FHC). The majority of MyBP-C mutations are expected to reduce MyBP-C expression; however, the consequences of MyBP-C deficiency on the regulation of myofilament function, Ca²⁺ homeostasis, and in vivo cardiac function are unknown. To elucidate the effects of decreased MyBP-C expression on cardiac function, we employed MyBP-C heterozygous null (MyBP-C+/-) mice presenting decreases in MyBP-C expression (32%) similar to those of FHC patients carrying MyBP-C mutations. The levels of MyBP-C phosphorylation were reduced 53% in MyBP-C+/- hearts compared with wild-type hearts. Skinned myocardium isolated from MyBP-C+/- hearts displayed decreased cross-bridge stiffness at half-maximal Ca²⁺ activations, increased steady-state force generation, and accelerated rates of cross-bridge recruitment at low Ca²⁺ activations (<15% and <25% of maximum, respectively). Protein kinase A treatment abolished basal differences in rates of cross-bridge recruitment between MyBP-C+/- and wild-type myocardium. Intact ventricular myocytes from MyBP-C+/- hearts displayed abnormal sarcomere shortening but unchanged Ca²⁺ transient kinetics. Despite a lack of left ventricular hypertrophy, MyBP-C+/- hearts exhibited elevated end-diastolic pressure and decreased peak rate of LV pressure rise, which was normalized following dobutamine infusion. Furthermore, electrocardiogram recordings in conscious MyBP-C+/- mice revealed prolonged QRS and QT intervals, which are known risk factors for cardiac arrhythmia. Collectively, our data show that reduced MyBP-C expression and phosphorylation in the sarcomere result in myofilament dysfunction, contributing to contractile dysfunction that precedes compensatory adaptations in Ca²⁺ handling, and chamber remodeling. Perturbations in mechanical and electrical activity in MyBP-C+/- mice could increase their susceptibility to cardiac dysfunction and arrhythmia.

Cardiac Myosin Binding Protein C Phosphorylation in Human Cardiac Disease

During recent years it has become increasingly evident that cardiac myosin binding protein C (cMyBP-C) exerts an important role in regulation of sarcomere function with consequences for in vivo cardiac performance. The functional role of cMyBP-C is tightly regulated by kinase-mediated phosphorylation. The most important kinase which phosphorylates cMyBP-C in vivo is protein kinase A (PKA), which is activated upon stimulation of the β-adrenergic receptors during exercise. In end-stage failing human myocardium, reduced phosphorylation of cMyBP-C has been reported using 1-dimensional and 2-dimensional gel electrophoresis. This reduced phosphorylation has been attributed to down-regulation and desensitization of the β-adrenergic receptor pathway in end-stage human heart failure. Low levels of cMyBP-C phosphorylation were also found in patients with familial hypertrophic cardiomyopathy (FHC), which is frequently caused by mutations in genes encoding sarcomeric proteins, with one exception: in FHC patients with mutations in the gene which encodes cMyBP-C (MYBPC3), phosphorylation of cMyBP-C was unaltered compared to non-failing donor hearts. cMyBP-C can be phosphorylated in vivo on at least three serine sites (Ser273, Ser282 and Ser 302), all of which are located in the cardiac isoform specific M region. At least one other site should exist in humans. With tandem mass spectrometry we recently identified a fourth phosphorylation site on Ser133, which is present in the Pro-Ala rich region that links the C0 and C1 domains. Preliminary data indicate that Ser133 is not phosphorylated by PKA. Analysis of phosphorylation on Ser133 in failing and donor samples revealed lower levels of Ser133 phosphorylation in several forms of cardiac disease compared to non-failing myocardium. Overall, our studies indicate that diverse cMyBP-C phosphorylation patterns exist in human cardiomyopathies, which may in part underlie sarcomere dysfunction observed in human heart failure.

Reduced Length-Dependent Activation in Human Cardiomyocytes Harboring the Troponin I Mutation R145W

Background: One of the mutated sarcomeric proteins in familial hypertrophic cardiomyopathy (FHC) is Troponin I (cTnI), a down-stream target of the β-adrenergic receptor pathway. Activation of this pathway leads to protein kinase A (PKA)-mediated phosphorylation of cTnI and sarcomere desensitization to Ca2+ to improve muscle relaxation. Moreover, cTnI phosphorylation was shown to enhance sarcomere length-dependent activation (i.e. Frank-Starling mechanism). We investigated if the responsiveness to PKA and to an increase in sarcomere length is impaired by mutant cTnI in human FHC.Methods: A comparison was made between cardiac samples from two FHC patients harboring the R145W mutation in the TNNI3 gene (TNNI3mut) and non-failing donor hearts. Isometric force was measured in Triton-permeabilized cardiomyocytes at different [Ca2+] and sarcomere lengths of 1.8 and 2.2 μm before and after treatment with exogenous PKA.Results: At a sarcomere length of 2.2 μm, TNNI3mut cells (n=30) showed lower maximal force (26.3±4.9 kN/m2) and similar passive force (2.1±0.2 kN/m2) compared to donor cells (n=48; 36.4±2.3 and 2.4±0.3 kN/m2, respectively). In addition, a significantly higher Ca2+-sensitivity (EC50)(2.69±0.16 μM) and a decreased steepness (2.17±0.10) of the force-Ca2+ relation were found in TNNI3mut compared to donor (3.09±0.09 μM and 3.75±0.12, respectively). The increase in Ca2+-sensitivity (i.e. length-dependent activation; ΔEC50) upon an increase in sarcomere length was significantly smaller in TNNI3mut (0.29±0.15) compared to donor (0.76±0.06) cells. Treatment with PKA decreased myofilament Ca2+-sensitivity in TNNI3mut to values observed in donor, but did not correct the reduced length-dependent activation.Conclusions: Our data show preserved responsiveness to PKA in FHC harboring the R145W mutation, but a blunted length-dependent activation, which was not corrected by PKA. Reduced maximal force and perturbed Frank-Starling mechanism, both at baseline and after PKA, may impair contractile performance in human FHC with mutant cTnI.

Profile of MicroRNAs Differentially Produced in Hearts from Patients with Hypertrophic Cardiomyopathy and Sarcomeric Mutations

To the Editor: MicroRNAs (miRNAs)1 regulate cardiac growth and conduction and play an important role in cardiac diseases (1). Several miRNAs are differentially produced in cardiac hypertrophic tissue, compared with normal tissue, and may contribute to the development of cardiomyocyte hypertrophy (2, 3). Hypertrophic cardiomyopathy (HCM) is frequently familial and caused by mutations in sarcomeric genes. To our knowledge, no study has reported the miRNA production profile in HCM tissues with sarcomeric gene mutations. To better define the molecular changes in HCM, we defined the production of well-characterized miRNAs in left ventricular (LV) heart tissue from 5 patients who underwent a cardiac transplantation and a control heart tissue (human LV tissue, Ambion/Applied Biosystems), and we compared their production profiles. Two of the patients were familial HCM patients who were carriers of missense mutations in the MYH7 2 (myosin, heavy chain 7, cardiac muscle, beta) gene (Val822Met and Arg453Cys). Three patients were cases of sporadic LV hypertrophy secondary to heart valve disease. The study was approved by the Ethical Committee of Hospital Universitario Central Asturias (HUCA), and all of the patients provided written informed consent. We isolated total RNA with TRIzol (Invitrogen) and …

A low prevalence of MYH7/MYBPC3 mutations among Familial Hypertrophic Cardiomyopathy patients in India

Familial Hypertrophic Cardiomyopathy (FHC) is an autosomal dominant disorder affecting the cardiac muscle and exhibits varied clinical symptoms because of genetic heterogeneity. Several disease causing genes have been identified and most code for sarcomere proteins. In the current study, we have carried out clinical and molecular analysis of FHC patients from India. FHC was detected using echocardiography and by analysis of clinical symptoms and family history. Disease causing mutations in the β-cardiac myosin heavy chain (MYH7) and Myosin binding protein C3 (MYBPC3) genes were identified using Polymerase Chain Reaction-Deoxyribose Nucleic Acid (PCR-DNA) sequencing. Of the 55 patient samples screened, mutations were detected in only nineteen in the two genes; MYBPC3 mutations were identified in 12 patients while MYH7 mutations were identified in five, two patients exhibited double heterozygosity. All four MYH7 mutations were missense mutations, whereas only 3/9 MYPBC3 mutations were missense mutations. Four novel mutations in MYBPC3 viz. c.456delC, c.2128G>A (p.E710K), c.3641G>A (p.W1214X), and c.3656T>C (p.L1219P) and one in MYH7 viz. c.965C>T (p.S322F) were identified. A majority of missense mutations affected conserved amino acid residues and were predicted to alter the structure of the corresponding mutant proteins. The study has revealed a greater frequency of occurrence of MYBPC3 mutations when compared to MYH7 mutations.

Unequal allelic expression of wild-type and mutated β-myosin in familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease, which in about 30% of the patients is caused by missense mutations in one allele of the β-myosin heavy chain (β-MHC) gene (MYH7). To address potential molecular mechanisms underlying the family-specific prognosis, we determined the relative expression of mutant versus wild-type MYH7-mRNA. We found a hitherto unknown mutation-dependent unequal expression of mutant to wild-type MYH7-mRNA, which is paralleled by similar unequal expression of β-MHC at the protein level. Relative abundance of mutated versus wild-type MYH7-mRNA was determined by a specific restriction digest approach and by real-time PCR (RT-qPCR). Fourteen samples from M. soleus and myocardium of 12 genotyped and clinically well-characterized FHC patients were analyzed. The fraction of mutated MYH7-mRNA in five patients with mutation R723G averaged to 66 and 68% of total MYH7-mRNA in soleus and myocardium, respectively. For mutations I736T, R719W and V606M, fractions of mutated MYH7-mRNA in M. soleus were 39, 57 and 29%, respectively. For all mutations, unequal abundance was similar at the protein level. Importantly, fractions of mutated transcripts were comparable among siblings, in younger relatives and unrelated carriers of the same mutation. Hence, the extent of unequal expression of mutated versus wild-type transcript and protein is characteristic for each mutation, implying cis-acting regulatory mechanisms. Bioinformatics suggest mRNA stability or splicing effectors to be affected by certain mutations. Intriguingly, we observed a correlation between disease expression and fraction of mutated mRNA and protein. This strongly suggests that mutation-specific allelic imbalance represents a new pathogenic factor for FHC.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-011-0205-9) contains supplementary material, which is available to authorized users.

Tropomyosin Phosphorylation in Healthy and Diseased Hearts

Tropomyosin (Tm) is an alpha‐helical coiled coil dimer involved in regulating striated muscle contraction. Although sarcomeric Tm is known to be phosphorylated at the penultimate amino acid Ser‐283, little is known regarding its phosphorylation levels in healthy and cardiac diseased states. The objective of this study was to study Tm phosphorylation levels with respect to age in wild type mice and in mice with familial hypertrophic cardiomyopathy (FHC). Phosphorylation levels were determined by western blotting of 2‐D gel electrophoresis of myofibrillar protein preparations. Results show that cardiac Tm phosphorylation is high during the perinatal period and decreases throughout adulthood in wild type mice. We also found that walls of the different cardiac chambers exhibit differential Tm phosphorylation.Two transgenic mouse models harboring FHC mutations in alpha‐Tm (Glu180Gly, and Asp175Asn) were studied to determine the effect of this disease on cardiac Tm phosphorylation. Results show that Tm phosphorylation is altered in both FHC models. Unlike in wild type mice, Tm phosphorylation levels do not decrease after birth, but rather remain constant. Depending on the role of Tm phosphorylation, modulation of phosphorylation levels in FHC patients may be potentially of therapeutic value.This project was funded by NHLBI HL81680 grant to DFW. HNS was supported by a fellowship from Dubai Medical College.

Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms

Familial hypertrophic cardiomyopathy (FHC) is frequently caused by cardiac myosin-binding protein C (cMyBP-C) gene mutations, which should result in C-terminal truncated mutants. However, truncated mutants were not detected in myocardial tissue of FHC patients and were rapidly degraded by the ubiquitin-proteasome system (UPS) after gene transfer in cardiac myocytes. Since the diversity and specificity of UPS regulation lie in E3 ubiquitin ligases, we investigated whether the muscle-specific E3 ligases atrogin-1 or muscle ring finger protein-1 (MuRF1) mediate degradation of truncated cMyBP-C. Human wild-type (WT) and truncated (M7t, resulting from a human mutation) cMyBP-C species were co-immunoprecipitated with atrogin-1 after adenoviral overexpression in cardiac myocytes, and WT-cMyBP-C was identified as an interaction partner of MuRF1 by yeast two-hybrid screens. Overexpression of atrogin-1 in cardiac myocytes decreased the protein level of M7t-cMyBP-C by 80% and left WT-cMyBP-C level unaffected. This was rescued by proteasome inhibition. In contrast, overexpression of MuRF1 in cardiac myocytes not only reduced the protein level of WT- and M7t-cMyBP-C by >60%, but also the level of myosin heavy chains (MHCs) by >40%, which were not rescued by proteasome inhibition. Both exogenous cMyBP-C and endogenous MHC mRNA levels were markedly reduced by MuRF1 overexpression. Similar to cardiac myocytes, MuRF1-overexpressing (TG) mice exhibited 40% lower levels of MHC mRNAs and proteins. Protein levels of cMyBP-C were 29% higher in MuRF1 knockout and 34% lower in TG than in WT, without a corresponding change in mRNA levels. These data suggest that atrogin-1 specifically targets truncated M7t-cMyBP-C, but not WT-cMyBP-C, for proteasomal degradation and that MuRF1 indirectly reduces cMyBP-C levels by regulating the transcription of MHC.

A new polymorphism in human calmodulin III gene promoter is a potential modifier gene for familial hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in genes encoding sarcomeric proteins. Incomplete penetrance suggests the existence of modifier genes. Calmodulin (CaM) could be of importance given the key role of Ca(2+) for cardiac contractile function and growth. Any variant that affects CaM expression and/or function may impact on FHC clinical expression. We screened the promoter region of human calmodulin III gene (CALM3) and identified a new -34T>A polymorphism with a T-allele frequency of 0.70. The distribution of CALM3 genotypes differed in 180 unrelated FHC patients carrying a known FHC mutation compared with 134 controls, with higher TT-genotype frequency (0.73 vs. 0.51) and lower frequencies of AT- (0.24 vs. 0.37) and AA genotypes (0.03 vs. 0.11; P = 0.0005). To study whether the -34T>A polymorphism could play a modifier role, patients' relatives including both affected and healthy carriers were added. Affected carriers had a 0.56 times higher odds of carrying a T allele than healthy carriers (P = 0.053). We then investigated whether the -34T>A polymorphism affects the promoter activity using luciferase reporter vectors containing either CALM3-T or CALM3-A promoters. The activity of CALM3-T was lower than CALM3-A in HEK293 cells (1.00 +/- 0.19 vs. 2.31 +/- 0.13, P = 0.00001) and in cardiomyocytes (0.96 +/- 0.10 vs. 1.33 +/- 0.08, P = 0.00727). These data suggest that the -34T>A CALM3 polymorphism is a modifier gene for FHC, potentially by affecting expression level of CALM3 and therefore Ca(2+)-handling and development of hypertrophy.

Different Molecular Mechanisms of Force Enhancement by Myosin Head Domain Mutations R723G and R453C

The myosin head transforms chemical energy from ATP hydrolysis into mechanical work to generate isometric force or to drive muscle shortening. During the power stroke, small changes in the catalytic ATPase site of the actin-bound myosin head are coupled to tilting of the light-chain binding domain relative to the catalytic domain. In this mechanism distortion of an elastic element within the actomyosin complex is essential for strain to develop prior to movement.We have studied missense mutations naturally occurring in Familial Hypertrophic Cardiomyopathy (FHC) to test molecular mechanisms and functional roles of specific domains of the myosin head. Slow soleus muscle fibers (expressing the ventricular myosin isoform) of FHC-patients were used for these studies. Previously we found that mutation R723G which is located in the converter domain of the myosin head increases active force and resistance to elastic distortion (fiber stiffness) during contraction, relaxation, and rigor, while cross-bridge cycling kinetics were unchanged. This indicated that the converter is the part of the actomyosin complex where most of the elastic distortion occurs.We now included mutation R453C located near the nucleotide binding pocket of myosin in our functional studies. This mutation was found to also increase active force, however, without affecting fiber stiffness. Instead, mutation R453C increased fiber ATPase activity and the rate constant of force redevelopment (kredev) significantly, which were unchanged by mutation R723G. Thus, both FHC mutations cause force enhancement in muscle fibers from FHC patients, however, by distinctly different mechanisms. R723G affects resistance to elastic distortion of the myosin head while R453C alters cross-bridge cycling kinetics. The data underline the different functional roles of the domains within the myosin head and point to an involvement of residue R453 in human ventricular myosin ATPase activity.

Estimate of the abundance of cardiomyopathic mutations in the β-myosin gene

The steady discovery of new β-myosin mutations causing familial hypertrophic cardiomyopathy (FHCM) suggests that only a fraction of the total number of mutations has been identified so far by clinical screenings. To test this hypothesis, data derived from seven independent genetic studies were re-analyzed using statistical methods developed by ecologists for estimating the number of species in a community.This novel approach reveals that more mutations will continue to be identified as more patients are genotyped, and highlights important differences in the distribution of β-myosin mutations across different populations. Hence, additional molecular sampling will be necessary to complete the census of the β-myosin mutations and facilitate the diagnosis and evaluation of FHCM patients.

Disease-Modifying Mutations in Familial Hypertrophic Cardiomyopathy

Cardiomyopathies are primary disorders of cardiac muscle associated with abnormalities of cardiac wall thickness, chamber size, contraction, relaxation, conduction, and rhythm. They are a major cause of morbidity and mortality at all ages and, like acquired forms of cardiovascular disease, frequently progress into heart failure.1 In contrast to dilated cardiomyopathy (DCM), which is characterized by left or biventricular dilation in association with depressed myocardial contractility,1 familial hypertrophic cardiomyopathy (FHC) is characterized by increased cardiac mass with myocyte and myofibrillar disarray. The disease is associated with electrical instability (ie, atrial and ventricular arrhythmias), which makes it the leading cause of death in young athletes. There is marked diversity in the morphological features and clinical manifestations of both DCM and FHC.2 Article p 1820 Family studies have demonstrated that hypertrophic cardiomyopathy is a heritable disorder that is transmitted as an autosomal-dominant trait or as a sporadic disease. There is neither a racial nor an ethnic predisposition to this condition. Hypertrophic cardiomyopathy is not a rare condition; several noninvasive studies have demonstrated echocardiographic criteria for this disease in 0.2% of young adults (reviewed by Maron3). Genetic linkage studies in FHC have found disease loci on at least 14 different genes, most of them encoding sarcomeric genes; hence, FHC has been called a disease of the sarcomere. In addition, several mutations within the Ras pathway have been shown to cause at least syndromic forms of hypertrophic cardiomyopathy,4,5 pointing to the presence of additional but poorly understood disease-causing and/or modifier genes. Thus far, however, most of the clinical heterogeneity of FHC has been accounted for by the substantial diversity of gene defects that cause the condition (ie, up to 45% of disease-causing mutations have been observed in the β-myosin heavy chain [MHC] gene myh7 and up to 35% in …

Abstract 2729: Atrogin-1 and MuRF1 Mediate Degradation of Cardiac Myosin-binding Protein C by the Ubiquitin-Proteasome System

Background. Familial hypertrophic cardiomyopathy (FHC) is a sarcomeropathy frequently associated with cardiac myosin-binding protein C (cMyBP-C) gene mutations. Most of them result in a frameshift and C-terminal truncated proteins. However, truncated mutants were not detected in myocardial tissue of FHC patients. A recent study showed that a truncated cMyBP-C (M7t) is rapidly and quantitatively degraded by the ubiquitin-proteasome system (UPS) after adenoviral gene transfer in neonatal rat cardiac myocytes (NRCM). Since the diversity and specificity of UPS regulation lies in E3 ubiquitin ligases, which specifically target proteins and direct the ubiquitination process, the present study investigated whether the sarcomere-specific E3 ligases, atrogin-1 and MuRF1, mediate degradation of wild-type (WT) and truncated M7t cMyBP-C. Methods and Results. NRCM were co-infected with atrogin-1 and WT or M7t cMyBP-C adenoviruses. Both overexpressed WT and M7t were co-immunoprecipitated with atrogin-1 suggesting that both forms of cMyBP-C interact with atrogin-1. However, whereas the amount of overexpressed WT cMyBP-C did not change, the M7t protein level was 80% lower in NRCM infected with atrogin-1 than without. This suggests that atrogin-1 is the E3 ligase mediating proteasome degradation of truncated M7t cMyBP-C only. We then investigated whether MuRF1 could regulate the level of endogenous cMyBP-C in MuRF1-KO mice and compared to WT. The heart to body weight ratio and the chymotrypsine-like activity of the proteasome were similar in KO and WT mice. The mRNA levels of cMyBP-C, and other E3 ligases, atrogin-1 and MuRF3, were similar between KO and WT ventricles. Strikingly, the protein level of cMyBP-C was 150% and 30% higher in neonatal and 24 wks-old KO compared to age-matched WT mice. Conclusion . These data suggest that atrogin-1, located at the Z-band, mediates the proteasome degradation of truncated M7t cMyBP-C resulting from a FHC mutation, the lack of MuRF1 is not compensated by other E3 ligases and is not associated with hypertrophy in KO mice, and most importantly, MuRF1, mainly located at the M-band, specifically regulates the protein level of cMyBP-C.

Family hypertrophic cardiomyopathy caused by a 14035c > t mutation in cardiac troponin T gene

To study the disease-causing gene mutation in Chinese patients with familial hypertrophic cardiomyopathy (FHC) and to analyze the correlation between the genotype and the phenotype. Peripheral blood samples were collected from 40 members from a family affected with FHC, and 120 healthy volunteers. PCR was performed to analyze the exons and flanking introns of the cardiac troponin T gene (TNNT2), beta-myosin heavy chain gene (MYH7), and myosin-binding protein C gene (MYBPC3) and the products were sequenced. The clinical data including symptom, physical examination, echocardiography and electrocardiography were collected. A 14035c > t mutation, which causes a missense mutation (R130C) in exon 10 of TNNT2 gene were identified in 4 family members, including the proband, female, aged 53, with the onset at the age of 30. The 4 persons with the 14035c > t mutation, all FHC patients, presented left ventricular dysfunction with a penetrance of 100%. Two of the patients died of sudden cardiac death during follow-up. No mutation was identified in the MYH7 and MYBPC3 genes. The 14035c > t mutation of TNNT2 gene is the causal mutation of FHC which is associated with malignant phenotype with a penetrance of 100%. It is a reasonable procedure in HCM patients with malignant phenotype to screen mutation in the TNNT2 gene.

βmyosin mutations and phenotypic heterogeneity in hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (FHC) is a genetically and clinically heterogeneous disease: the morphologic hallmark is an unexplained thickening of the interventricular septum and the free wall of the left ventricle. Clinical features include dizziness, lightheadedness, life threatening arrhythmias, heart failure and high risk of sudden death. Mutations in most of the sarcomeric proteins have been identified in FHC patients [1]. Several animal models have been generated so far by different groups, providing a molecular explanation for the hypertrophic phenotype detected in the heart of affected individuals.

Impairment of the ubiquitin?proteasome system by truncated cardiac myosin binding protein C mutants

Most cardiac myosin binding protein C (cMyBP-C) gene mutations causing familial hypertrophic cardiomyopathy (FHC) result in C-terminal truncated proteins. However, truncated cMyBP-Cs were undetectable in myocardial tissue of FHC patients. In the present study, we investigated whether truncated cMyBP-Cs are subject to accelerated degradation by the lysosome or ubiquitin-proteasome system (UPS). By using an adenovirus-based approach, we analyzed expression and localization of myc-tagged truncated proteins (M6t 3%, M7t 80% truncation, both mutations have been identified in FHC patients) compared to wild type (WT) in neonatal rat cardiomyocytes. Despite similar mRNA levels, protein expression of M6t and M7t was markedly lower than WT (70+/-4% and 11+/-5% of WT, respectively, p<0.05). M6t exhibited weak incorporation in the sarcomere, whereas M7t was mis-incorporated at the Z-disk and formed ubiquitin-positive aggregates. The lysosome inhibitor bafilomycin only slightly raised the protein level of M7t, whereas the UPS inhibitors lactacystin or MG132 markedly raised M6t and M7t to WT level. Using an adenovirus encoding a fluorescent reporter of UPS activity, we demonstrate that mutant cMyBP-Cs impair the proteolytic capacity of the UPS. Truncated cMyBP-Cs are preferentially degraded by the UPS, which, in turn, may competitively inhibit breakdown of other UPS substrates. Since the UPS plays an important role in a variety of fundamental cellular processes, we propose impairment of this system by mutant cMyBP-Cs as a contributing factor to the pathogenesis of FHC.

Assessment of regional systolic and diastolic dysfunction in familial hypertrophic cardiomyopathy using MR tagging.

Diastolic and systolic left ventricular (LV) dysfunction often significantly contribute to disabling symptoms in familial hypertrophic cardiomyopathy (FHC). This study compares regional LV function (midwall circumferential strain) during systole and diastole in eight FHC patients and six normal volunteers (NVs) using MR tagging. A prospectively-gated fast gradient-echo sequence with an echo-train readout was modified to support complementary spatial modulation of magnetization (CSPAMM) tagging and full cardiac cycle data acquisition using the cardiac phase to order reconstruction (CAPTOR), thus providing tag persistence and data acquisition during the entire cardiac cycle. Total systolic strains in FHC patients were significantly reduced in septal and inferior regions (both P < 0.01). Early-diastolic strain rates were reduced in all regions of the FHC group (all P < 0.03). The combination of CSPAMM and CAPTOR allows regional indices of myocardial function to be quantified throughout the cardiac cycle. This technique reveals regional differences in systolic and diastolic impairment in FHC patients.