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PubMed2002

Cardiomyopathy in zebrafish due to mutation in an alternatively spliced exon of titin.

The zebrafish embryo is transparent and can tolerate absence of blood flow because its oxygen is delivered by diffusion rather than by the cardiovascular system. It is therefore possible to attribute cardiac failure directly to particular genes by ruling out the possibility that it is due to a secondary effect of hypoxia. We focus here on pickwickm171 (pikm171), a recessive lethal mutation discovered in a large-scale genetic screen. There are three other alleles in the pik complementation group with this phenotype (pikm242, pikm740, pikm186; ref. 3) and one allele (pikmVO62H) with additional skeletal paralysis. The pik heart develops normally but is poorly contractile from the first beat. Aside from the edema that inevitably accompanies cardiac dysfunction, development is normal during the first three days. We show by positional cloning that the 'causative' mutation is in an alternatively-spliced exon of the gene (ttn) encoding Titin. Titin is the biggest known protein and spans the half-sarcomere from Z-disc to M-line in heart and skeletal muscle. It has been proposed to provide a scaffold for the assembly of thick and thin filaments and to provide elastic recoil engendered by stretch during diastole. We found that nascent myofibrils form in pik mutants, but normal sarcomeres are absent. Mutant cells transplanted to wildtype hearts remain thin and bulge outwards as individual cell aneurysms without affecting nearby wildtype cardiomyocytes, indicating that the contractile deficiency is cell-autonomous. Absence of Titin function thus results in blockage of sarcomere assembly and causes a functional disorder resembling human dilated cardiomyopathies, one form of which is described in another paper in this issue.

Alternative Splicing↗
PubMed1999

Familial hypertrophic cardiomyopathy associated with a novel missense mutation affecting the ATP-binding region of the cardiac beta-myosin heavy chain.

Mutations in the cardiac beta -myosin heavy chain gene (MYH7), and other genes encoding cardiac sarcomere proteins may cause familial hypertrophic cardiomyopathy (F-HCM), an autosomal dominant disease, characterized by myocardial hypertrophy. We analysed the MYH7 gene in three generations of a family with one borderline and four clinically verified cases of hypertrophic cardiomyopathy, and identified a mutation in exon 7 changing the 190 arginine residue into a threonine residue. The mutation is located in the ATP-binding region of the myosin head and alters the charge in the F-helix close to the phosphate-binding P-loop. The mutation may thus interfere with the coupling between ATP-hydrolysis and the transition into mechanical energy. In conclusion, the novel Arg190Thr mutation in exon 7 of the MYH7 gene is associated with the development of symptomatic myocardial hypertrophy in adults.

Adenosine Triphosphate↗
PubMed2006

Familial dilated cardiomyopathy with troponin T K210del mutation.

BACKGROUND: It has been estimated that more than 30% of patients with idiopathic dilated cardiomyopathy have a familial form of the disease. The most frequent pattern of inheritance is autosomal dominant and several genes or loci have been implicated, coding for sarcomeric or cytoskeleton proteins. Most of the genotype-phenotype correlations are still under study, but a particular mutation, K210del in the troponin T gene, has been identified in four different families with severe forms of DCM. The pathogenesis of this mutation has been inferred by functional studies but its transmission has not been demonstrated, perhaps due to the high mortality of the affected family members. The aim of this work was to investigate the prevalence of the K210del mutation in Portuguese and Mozambican families with dilated cardiomyopathy. METHODS: We evaluated 27 probands with familial DCM. Forty idiopathic (sporadic) DCM patients and 100 non-related healthy individuals were used as controls. Mutational analysis was performed by amplification of exon 13 of the troponin T gene by the polymerase chain reaction (PCR), determination of molecular weight of PCR products and further sequencing. RESULTS: The K210del mutation in the cardiac troponin T gene was identified in one of the DCM families which presented an aggressive form of the disease, with a high incidence of sudden death, and need for heart transplant at young age. One affected member had sustained left ventricular function recovery after diagnosis. CONCLUSIONS: These results reinforce previous work by others, indicating that this mutation is a bad prognostic factor in familial forms of DCM. The K210del mutation in the troponin T gene, like other mutations in the troponin complex, seems to be especially prevalent in families with rapidly progressive DCM or sudden cardiac death at young age.

Adult↗
PubMed1998

[Clinical and molecular genetics of hypertrophic cardiomyopathy].

Recent developments in molecular genetics have allowed to identify mutations in seven genes coding the beta myosin heavy chain, troponin T, alpha tropomyosin, myosin binding protein C, essential and regulatory myosin light chains and troponin I causing hypertrophic cardiomyopathy. These mutations affect critical, evolutionary conserved nucleotides of these genes and influence vital functions of the encoded proteins. As all seven genes encodes sarcomeric proteins in the heart muscle, hypertrophic cardiomyopathy is regarded these days as a disease of the sarcomer. Recent data indicate that some mutations are associated with "malignant" clinical picture, with rapidly developing, severe symptoms of the disease and increased risk of sudden cardiac death while other mutations bear a more favourable prognosis. Apart of the disease causing mutation other factors, including disease modifier genes, are likely to make an impact on the clinical appearance of hypertrophic cardiomyopathy. The knowledge provided by molecular genetics influences the clinical management of the disease even today and based on the investigation of mutation carrying patients new diagnostic criteria was proposed for hypertrophic cardiomyopathy. The challenge for the future is the establishment of routine genetic diagnostics and the development of possible gene therapy.

Cardiomyopathy, Hypertrophic↗
PubMed2006

Leucine 135 of tropomodulin-1 regulates its association with tropomyosin, its cellular localization, and the integrity of sarcomeres.

Tropomodulin-1 (Tmod-1) is a well defined actin-capping protein that interacts with tropomyosin (TM) at the pointed end of actin filaments. Previous studies by others have mapped its TM-binding domain to the amino terminus from amino acid 39 to 138. In this study, we have identified several amino acid residues on Tmod-1 that are important for its interaction with TM5 (a nonmuscle TM isoform). Glutathione S-transferase affinity chromatography and immunoprecipitation assays reveal that Tmod sense mutations of either amino acid 134, 135, or 136 causes various degrees of loss of function of Tmod TM-binding ability. The reduction of TM-binding ability was relatively mild (reduced approximately 20-40%) from the G136A Tmod mutant but more substantially (reduced approximately 50-100%) from the I134D, L135E, and L135V Tmod mutants. In addition, mutation at any of these three sites dramatically alters the subcellular location of Tmod-1 when introduced into mammalian cells. Further analysis of these three mutants uncovered a previously unknown nuclear trafficking function of Tmod-1, and residues 134, 135, and 136 are located within a nuclear export signal motif. As a result, mutation on either residue 134 or residue 135 not only will cause a significant reduction of the Tmod-1 ability to bind to TM5 but also lead to predominant nuclear localization of Tmod-1 by crippling its nuclear export mechanism. The failure of the Tmod mutations to fully associate with TM5 when introduced into neonatal rat cardiomyocytes was also associated with an accelerated and severe fragmentation of sarcomeric structures compared with overexpression of wild type Tmod-1. The multiple losses of function of Tmod engendered by these missense mutations are most severe with the single substitution of residue 135.

Chromatography, Affinity↗
PubMed2003

Idiopathic restrictive cardiomyopathy is part of the clinical expression of cardiac troponin I mutations.

Restrictive cardiomyopathy (RCM) is an uncommon heart muscle disorder characterized by impaired filling of the ventricles with reduced volume in the presence of normal or near normal wall thickness and systolic function. The disease may be associated with systemic disease but is most often idiopathic. We recognized a large family in which individuals were affected by either idiopathic RCM or hypertrophic cardiomyopathy (HCM). Linkage analysis to selected sarcomeric contractile protein genes identified cardiac troponin I (TNNI3) as the likely disease gene. Subsequent mutation analysis revealed a novel missense mutation, which cosegregated with the disease in the family (lod score: 4.8). To determine if idiopathic RCM is part of the clinical expression of TNNI3 mutations, genetic investigations of the gene were performed in an additional nine unrelated RCM patients with restrictive filling patterns, bi-atrial dilatation, normal systolic function, and normal wall thickness. TNNI3 mutations were identified in six of these nine RCM patients. Two of the mutations identified in young individuals were de novo mutations. All mutations appeared in conserved and functionally important domains of the gene. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Adolescent↗
PubMed1997

Molecular mechanisms regulating the myofilament response to Ca2+: implications of mutations causal for familial hypertrophic cardiomyopathy.

In this chapter we consider a current perception of the molecular mechanisms controlling myofilament activation with emphasis on alterations that may occur in familial hypertrophic cardiomyopathy (FHC). FHC is a sarcomeric disease (100) with an autosomal dominant pattern of heritability (27, 51). There is a substantial body of evidence implicating missense mutations in the beta-MHC gene as causal for the development of this disease. Recently, mutations in genes of two thin filament regulatory proteins, cardiac troponin T(cTnT) and alpha-tropomyosin (alpha-Tm), have also been linked to FHC. The commonality among the functional consequences of these mutations remains an important question. This review discusses how these pathological mutations may impact the activation process by disrupting critical structure function relations in both the thick and thin filaments.

Actin Cytoskeleton↗
PubMed1995

Activin A and transforming growth factor-beta stimulate heart formation in axolotls but not rescue cardiac lethal mutants.

In the Mexican axolotl (salamander), Ambystoma mexicanum, a recessive cardiac lethal mutation causes an incomplete differentiation of the myocardium. Mutant hearts lack organized sarcomeric myofibrils and do not contract throughout their lengths. We have previously shown that RNA purified from normal anterior endoderm or from juvenile heart tissue is able to rescue mutant embryonic hearts in an in vitro organ culture system. Under these conditions as many as 55% of formerly quiescent mutant hearts initiate regular contractions within 48 hours. After earlier reports that transforming growth factor-beta 1 and, to a lesser extent, platelet-derived growth factor-BB could substitute for anterior endoderm as a promoter of cardiac mesodermal differentiation in normal axolotl embryos, we decided to examine the effect of growth factors in the cardiac mutant axolotl system. In one type of experiment, stage 35 mutant hearts were incubated in activin A, transforming growth factors-beta 1 or beta 2, platelet-derived growth factor, or epidermal growth factor, but no rescue of mutant hearts was achieved. Considering the possibility that growth factors would only be effective at earlier stages of development, we tested transforming growth factors-beta 1 and beta 5, and activin A on normal and mutant precardiac mesoderm explanted in the absence of endoderm at neurula stage 14. We found that, although these growth factors stimulated heart tube formation in both normal and mutant mesoderm explants, only normal explants contained contractile myocardial tissue. We hypothesize that transforming growth factor-beta superfamily peptides initiate a cascade of responses in mesoderm that result in both changes in cell shape (the basis for heart morphogenesis) and terminal myocardial cytodifferentiation. The cardiac lethal mutation appears to be deficient only in the latter process.

Activins↗
PubMed1999

[Evaluation of the risk of sudden death in hypertrophic cardiomyopathy].

Hypertrophic cardiomyopathy (HCM) is defined as primary hypertrophy of the heart muscle, usually the left ventricle which is not dilated. HCM is a relatively common disease with a prevalence estimated at about 1 in 500. It is a complex disease with relatively stereotypical anatomical features but a very variable clinical presentation with a major risk of complication. All forms may be observed from almost asymptomatic hypertrophy to severe familial forms with multiple cases of sudden death. Over the last few years, molecular studies of the genetic abnormalities responsible for HCM have improved our understanding of the clinical variability of this disease. Schematically, HCM is caused by mutation of one of 4 genes which code the proteins of the sarcomere: the gene of the heavy chain of beta-myosin, the gene of cardiac T-troponin, the gene of alpha-tropomyosin and the gene of protein C linked to cardiac myosin. The main problem for clinicians is not making the diagnosis, which is relatively simple by echocardiography, but to assess the risk of complications, especially in adolescents and young adults. Patients over 40 to 45 years of age pose fewer problems as their disease is generally associated with a better prognosis since they have already survived to that age. There are many prognostic factors of sudden death, a reflection of the multifactorial character of sudden death in this disease. Four major risk factors have been identified: a family history of sudden death, abnormal blood pressure changes on exercise, a history of syncope and non-sustained ventricular tachycardia on 24 or 48-hour Holter monitoring. In children and adolescents, only the first three factors may be used, knowing that syncope, though rare, carries a very poor prognosis. On the other hand, in adults up to 40, all 4 factors are valid. Unfortunately, their positive predictive value is relatively poor, all the patients with one of these risk factors not automatically experiencing sudden death. On the other hand, their negative predictive value is excellent. Therefore, a patient with none of these factors has an excellent prognosis and should be allowed to lead a normal life. The risk is considered to be high when 2 or 3 of the factors are associated, theoretically justifying aggressive management (amiodarone? defibrillator?). Finally, there is no established management protocol in cases with a single risk factor. The discovery of mutations causing HCM will probably open up new methods of assessing the risk of sudden death in this disease. It would seem to be possible to assess the impact of the genotype on prognosis. However, this "genetic stratification" remains the realm of top research teams and is not yet accessible routinely in clinical practice.

Adolescent↗
PubMed2003

Relationship between cardiac protein tyrosine phosphorylation and myofibrillogenesis during axolotl heart development.

The axolotl, Ambystoma mexicanum, is a useful system for studying embryogenesis and cardiogenesis. To understand the role of protein tyrosine phosphorylation during heart development in normal and cardiac mutant axolotl embryonic hearts, we have investigated the state of protein tyrosine residues (phosphotyrosine, P-Tyr) and the relationship between P-Tyr and the development of organized sarcomeric myofibrils by using confocal microscopy, two-dimensional isoelectric focusing (IEF)/SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting analyses. Western blot analyses of normal embryonic hearts indicate that several proteins were significantly tyrosine phosphorylated after the initial heartbeat stage (stage 35). Mutant hearts at stages 40-41 showed less tyrosine phosphorylated staining as compared to the normal group. Two-dimensional gel electrophoresis revealed that most of the proteins from mutant hearts had a lower content of phosphorylated amino acids. Confocal microscopy of stage 35 normal hearts using phosphotyrosine monoclonal antibodies demonstrated that P-Tyr staining gradually increased being localized primarily at cell-cell boundaries and cell-extracellular matrix boundaries. In contrast, mutant embryonic hearts showed a marked decrease in the level of P-Tyr staining, especially at sites of cell-cell and cell-matrix junctions. We also delivered an anti-phosphotyrosine antibody (PY 20) into normal hearts by using a liposome-mediated delivery method, which resulted in a disruption of the existing cardiac myofibrils and reduced heartbeat rates. Our results suggest that protein tyrosine phosphorylation is critical during myofibrillogenesis and embryonic heart development in axolotls.

Ambystoma mexicanum↗
PubMed2000

Myotilin is mutated in limb girdle muscular dystrophy 1A.

We have identified a mutation in the myotilin gene in a large North American family of German descent expressing an autosomal dominant form of limb girdle muscular dystrophy (LGMD1A). We have previously mapped this gene to 5q31. Symptoms of this adult onset disease are progressive weakness of the hip and shoulder girdles, as well as a distinctive dysarthric pattern of speech. Muscle of affected individuals shows degeneration of myofibers, variations in fiber size, fiber splitting, centrally located myonuclei and a large number of autophagic vesicles. Affected muscle also exhibits disorganization and streaming of the Z-line similar to that seen in nemaline myopathy. We have identified a C450T missense mutation in the myotilin gene that is predicted to result in the conversion of residue 57 from threonine to isoleucine. This mutation has not been found in 396 control chromosomes. The mutant allele is transcribed and normal levels of correctly localized myotilin protein are seen in LGMD1A muscle. Myotilin is a sarcomeric protein that binds to alpha-actinin and is localized in the Z-line. The observed missense mutation does not disrupt binding to alpha-actinin.

Actinin↗
PubMed1996

Remodeling of cytoskeleton and triads following activation of v-Src tyrosine kinase in quail myotubes.

To study the cellular signals underlying the regulatory mechanisms involved in maintenance of sarcomeric integrity, we have used quail skeletal muscle cells that reach a high degree of structural maturation in vitro, and also express a temperature-sensitive mutant of the v-Src tyrosine kinase that allows the control of differentiation in a reversible manner. By immunofluorescence and electron microscopy we show that v-Src activity in myotubes leads to an extensive cellular remodeling which affects components of the sarcomeres, the cytoskeleton network and the triad junctions. We have previously shown that activation of v-Src causes a selective dismantling of the I-Z-I segments coupled to the formation of aggregates of sarcomeric actin, alpha-actinin and vinculin, called actin bodies. We now show that intermediate filaments do not participate in the formation of actin bodies, while talin, a component of costameres, does. The I-Z-I segments are completely dismantled within 24 hours of v-Src activity, but the A-bands persist for a longer time, implying distinct pathways for the turnover of sarcomeric subdomains. Immunofluorescence labeling of markers of the triad junctions demonstrates that the localization of the alpha 1 subunit of the dihydropyridine receptor is disrupted earlier than that of the ryanodine receptor after tyrosine kinase activation. Furthermore, the location of junctional sarcoplasmic reticulum and transverse tubule membranes is maintained in myotubes in which the I-Z-I have been removed and the regular disposition of the intermediate filaments is disrupted, supporting a role for sarcoplasmic reticulum in the proper positioning of triad junctions. Altogether these results point to a tyrosine kinase signaling cascade as a mechanism for selectively destabilizing sarcomere subdomains and their tethering to the cytoskeleton and the sarcolemma.

Actins↗
PubMed2003

[Therapy and risk-stratification in hypertrophic cardiomyopathy--a current survey].

Hypertrophic cardiomyopathy (HCM) is a relatively common disease of the cardiac sarcomere with broad heterogeneity in terms of the disease-causing gene mutation, phenotypic expression, therapy and prognosis. Besides the standard drug treatment, there are several therapeutic options available for severe refractory symptomatic HCM with obstruction. Dual-chamber pacing and transcoronary ablation of septal hypertrophy (TASH) have recently emerged as alternatives to myectomy. However, myectomy remains the current gold standard of therapy for HCM until the promising initial follow-up data for TASH can be transferred into a long-term follow-up period, or prospective randomized comparative trials between these therapies are available. However, even now, TASH represents an important therapeutic alternative in patients with relevant co-morbidities and a high operative risk. Despite significant gradient reduction and amelioration of clinical symptoms, none of these treatment strategies has a proven influence on the natural history of HCM. Hence, regarding the long-term prognosis of the disease, risk stratification of sudden cardiac death using non-invasive risk assessment has become of paramount importance, while genotyping might become the determinant and stratifying marker in the near future. At present, according to secondary prevention, treatment with an implanted cardioverter-defibrillator +/- amiodarone therapy is mandatory, while according to primary prevention treatment should particularly depend on the individual risk profile.

Cardiomyopathy, Hypertrophic↗
PubMed2000

Calsequestrin, a calcium sequestering protein localized at the sarcoplasmic reticulum, is not essential for body-wall muscle function in Caenorhabditis elegans.

Calsequestrin is the major calcium-binding protein of cardiac and skeletal muscles whose function is to sequester Ca(2+ )in the lumen of the sarcoplasmic reticulum (SR). Here we describe the identification and functional characterization of a C. elegans calsequestrin gene (csq-1). CSQ-1 shows moderate similarity (50% similarity, 30% identity) to rabbit skeletal calsequestrin. Unlike mammals, which have two different genes encoding cardiac and fast-twitch skeletal muscle isoforms, csq-1 is the only calsequestrin gene in the C. elegans genome. We show that csq-1 is highly expressed in the body-wall muscles, beginning in mid-embryogenesis and maintained through the adult stage. In body-wall muscle cells, CSQ-1 is localized to sarcoplasmic membranes surrounding sarcomeric structures, in the regions where ryanodine receptors (UNC-68) are located. Mutation in UNC-68 affects CSQ-1 localization, suggesting that the two possibly interact in vivo. Genetic analyses of chromosomal deficiency mutants deleting csq-1 show that CSQ-1 is not essential for initiation of embryonic muscle formation and contraction. Furthermore, double-stranded RNA injection resulted in animals completely lacking CSQ-1 in body-wall muscles with no observable defects in locomotion. These findings suggest that although CSQ-1 is one of the major calcium-binding proteins in the body-wall muscles of C. elegans, it is not essential for body-wall muscle formation and contraction.

Amino Acid Sequence↗
PubMed1995

The molecular and cellular biology of heart failure.

We review recent publications that use molecular and cellular biology to explore the diagnosis and treatment of cardiovascular diseases that have relevance to heart failure. Familial hypertrophic cardiomyopathy has now been shown to be due to mutations not only in the previously described beta myosin heavy chain gene, but also in the troponin T and alpha-tropomyosin genes, thus providing some symmetry to the idea that this is a molecular disease of the sarcomere. The basis for a type of familial dilated cardiomyopathy without substantial skeletal muscle involvement, caused by a mutation in the dystrophin gene, has been explored. However, by-and-large, the disease basis for most patients with dilated cardiomyopathy remains a molecular mystery. The role of a polymorphism in the angiotensin-converting enzyme gene was examined as a risk factor for a number of cardiovascular diseases. In animal models, the hypothesis that the devolution from hypertrophy to heart failure includes alterations in the molecular direction of extracellular matrix production gained some support. The experimental foundation was laid this year for the concept of and approach to cardiomyocytoplasty--the molecular and cellular treatment of heart failure by augmentation, repair, or replacement of cardiac myocytes--by experiments in cardiac gene transfer and transgenic animals. Gene causes and cures for restenosis after angioplasty garnered considerable attention. As we gain greater understanding of the molecular basis for disease, we will also have to increase our wisdom in the application of genetic testing.

Animals↗
PubMed2000

Hypercontractile properties of cardiac muscle fibers in a knock-in mouse model of cardiac myosin-binding protein-C.

Myosin-binding protein-C (MyBP-C) is a component of all striated-muscle sarcomeres, with a well established structural role and a possible function for force regulation. Multiple mutations within the gene for cardiac MyBP-C, one of three known isoforms, have been linked to familial hypertrophic cardiomyopathy. Here we generated a knock-in mouse model that carries N-terminal-shortened cardiac MyBP-C. The mutant protein was designed to have a similar size as the skeletal MyBP-C isoforms, whereas known myosin and titin binding sites as well as the phosphorylatable MyBP-C motif were not altered. We have shown that mutant cardiac MyBP-C is readily incorporated into the sarcomeres of both heterozygous and homozygous animals and can still be phosphorylated by cAMP-dependent protein kinase. Although histological characterization of wild-type and mutant hearts did not reveal obvious differences in phenotype, left ventricular fibers from homozygous mutant mice exhibited an increased Ca(2+) sensitivity of force development, particularly at lower Ca(2+) concentrations, whereas maximal active force levels remained unchanged. The results allow us to propose a model of how cMyBP-C may affect myosin-head mobility and to rationalize why N-terminal mutations of the protein in some cases of familial hypertrophic cardiomyopathy could lead to a hypercontractile state.

Animals↗