One third of Danish hypertrophic cardiomyopathy patients with MYH7 mutations have mutations [corrected] in MYH7 rod region.
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Familial hypertrophic cardiomyopathy (FHC) is, in most cases, a disease of the sarcomere, caused by a mutation in one of 10 known sarcomere disease genes. More than 266 mutations have been identified since 1989. The FHC disease gene first characterized MYH7, encodes the cardiac beta-myosin heavy chain, and contains more than 115 of these mutations. However, in most studies, only the region encoding the globular head and the hinge region of the mature cardiac beta-myosin heavy chain have been investigated. Furthermore, most studies carries out screening for mutations in the most prevalent disease genes, and discontinues screening when an apparent disease-associated mutation has been identified. The aim of the present study was to screen for mutations in the rod region of the MYH7 gene in all probands of the cohort, regardless of the known genetic status of the proband. Three disease-causing mutations were identified in the rod region in four probands using capillary electrophoresis single-strand conformation polymorphism as a screening method. All mutations were novel: N1327K, R1712W, and E1753K. Two of the probands had already been shown to carry other FHC-associated mutations. In conclusion, we show that in the Danish cohort we find one third of all MYH7 mutations in the rod-encoding region and we find that two of the patients carrying these mutations also carry mutations in other FHC disease genes stressing the need for a complete screening of all known disease genes in FHC-patients. (+info)
Transmural stretch-dependent regulation of contractile properties in rat heart and its alteration after myocardial infarction.
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The "stretch-sensitization" response is essential to the regulation of heart contractility. An increase in diastolic volume improves systolic contraction. The cellular mechanisms of this modulation, the Frank-Starling law, are still uncertain. Moreover, their alterations in heart failure remains controversial. Here, using left ventricular skinned rat myocytes, we show a nonuniform stretch-sensitization of myofilament activation across the ventricular wall. Stretch-dependent Ca2+ sensitization of myofilaments increases from sub-epicardium to sub-endocardium and is correlated with an increase in passive tension. This passive tension-dependent component of myofibrillar activation is not associated with expression of titin isoforms, changes in troponin I level, and phosphorylation status. Instead, we observe that stretch induces phosphorylation of ventricular myosin light chain 2 isoform (VLC2b) in sub-endocardium specifically. Thus, VLC2b phosphorylation could act as a stretch-dependent modulator of activation tuned within normal heart. Moreover, in postmyocardial infarcted rat, the gradient of stretch-dependent Ca2+ sensitization disappears associated with a lack of VLC2b phosphorylation in sub-endocardium. In conclusion, nonuniformity is a major characteristic of the normal adult left ventricle (LV). The heterogeneous myocardial deformation pattern might be caused not only by the morphological heterogeneity of the tissue in the LV wall, but also by the nonuniform contractile properties of the myocytes across the wall. The loss of a contractile transmural gradient after myocardial infarction should contribute to the impaired LV function. (+info)
A mouse model of cardiac rhabdomyoma generated by loss of Tsc1 in ventricular myocytes.
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Tuberous sclerosis is a hamartoma syndrome due to mutations in TSC1 or TSC2 in which cardiac rhabdomyomas are seen in approximately 60% of patients. These lesions have an unusual natural history as they are usually most prominent immediately after birth and spontaneously resolve in most cases. To develop a mouse model of this lesion, we used a conditional, floxed allele of Tsc1 and a modified myosin light chain 2v allele in which cre recombinase expression occurs in ventricular myocytes. Mice with ventricular loss of Tsc1 had a median survival of 6 months and developed a dilated cardiomyopathy with the occurrence of scattered foci of enlarged ventricular myocytes. The enlarged cells were periodic acid-Schiff positive indicating the presence of excess glycogen and expressed elevated levels of phospho-S6, similar to findings in patient rhabdomyoma cells. The observations confirm that rhabdomyomas occur through a two hit mechanism of pathogenesis. However, the mice showed no evidence of fetal/neonatal demise, and there was no evidence of proliferation in the lesions. We propose that these differences are due to the timing of loss of Tsc1 in the ventricular myocytes and/or the truncated gestational period in the mouse compared with humans, during which progestational hormones may accentuate the growth of patient rhabdomyomas. (+info)
Key role of myosin light chain (MLC) kinase-mediated MLC2a phosphorylation in the alpha 1-adrenergic positive inotropic effect in human atrium.
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OBJECTIVE: Mechanisms of the positive inotropic response to alpha(1)-adrenergic stimulation in the heart remain poorly understood, but recent evidence in rat papillary muscle suggests an important role of regulatory myosin light chain (MLC2) phosphorylation. This study investigated alpha(1)-adrenergic contractile effects and the role of MLC kinase (MLCK)-dependent phosphorylation of MLC2 in human atrial muscle strips. METHODS: Force measurement was performed on electrically stimulated atrial muscle strips (n=140; 20 hearts) in the presence of the beta-blocker nadolol. MLC2a phosphorylation was determined by 2D-polyacrylamide gel electrophoresis and Western blotting of muscle strips that were immediately freeze-clamped following force measurements. RESULTS: The alpha(1)-agonist phenylephrine (PE; 0.3-100 microM) exerted a concentration-dependent, monophasic, sustained positive inotropic effect (86% above basal) that was accompanied by an 80% increase in MLC2a phosphorylation. Desinhibition of MLC phosphatase by the Rho kinase inhibitor Y-27632 (10 microM) reduced the effect of PE by 16%. The MLCK inhibitor wortmannin (10 microM) completely abolished both the PE-induced increase in force and MLC2a phosphorylation. The structurally unrelated MLCK inhibitor ML-7 (10 microM) had similar effects. Neither Y-27632 nor wortmannin or ML-7 affected beta-adrenergic force stimulation. In contrast to our findings in atrial muscle strips, we observed no increase in MLC2v phosphorylation after PE in human ventricular muscle strips and wortmannin failed to inhibit PE-induced force of contraction. CONCLUSION: alpha(1)-Adrenergic receptors mediate a prominent increase in contractile force in human atria that depends on MLCK activity and is accompanied by an increase in MLC2 phosphorylation. (+info)
Versican expression during skeletal/joint morphogenesis and patterning of muscle and nerve in the embryonic mouse limb.
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Versican, an extracellular matrix proteoglycan, has been implicated in limb development and is expressed in precartilage mesenchymal condensations. However, studies have lacked precise spatial and temporal investigation of versican localization during skeletogenesis and its relationship to patterning of muscle and nerve during mammalian limb development. The transgenic mouse line hdf (heart defect), which bears a lacZ reporter construct disrupting Cspg2 encoding versican, allowed ready detection of hdf transgene expression through histochemical analysis. Hdf transgene expression in whole mount heterozygous embryos and localization of versican relative to cartilage, muscle, and nerve tissues in paraffin-embedded limb sections of wild-type embryos from 10.5-14 days postcoitum were evaluated by lacZ histochemistry, immunohistochemistry, and in situ hybridization. Versican was localized within precartilage condensations and nascent cartilages with expression diminishing during maturation of the cartilage model at later time points. Interestingly, versican remained highly expressed in developing synovial joint interzones, suggesting potential function for versican in joint morphogenesis. Isolated myoblasts, incipient skeletal muscle masses, and neurites were not present in areas of strong versican expression within the developing limb. Versican-expressing tissues may reserve space for the future limb skeleton and developing joints and may aid in patterning of muscle and nerve by deterring muscle migration and innervation into these regions. (+info)
Heart malformation is an early response to TCDD in embryonic zebrafish.
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The zebrafish (Danio rerio) has become an attractive vertebrate model for studying developmental processes, and is emerging as a model system for studying the mechanisms by which toxic compounds perturb normal development. When exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) shortly after fertilization, zebrafish embryos exhibit pericardial edema and reduced blood flow by 72 h post fertilization (hpf). To better understand the progression of dioxin toxicity in zebrafish, we have examined the effects of TCDD on heart development. At 72 hpf, TCDD-treated embryos exhibited altered looping, with the atria positioned distinctly posterior to the ventricles, contrary to the looping of control hearts, where the two chambers lied side by side. Moreover, the ventricles in dioxin-exposed hearts became more compact, and the atria elongated in comparison to controls. These defects are not secondary to pericardial edema because they were observed when edema formation was suppressed with osmotic support. In addition to morphological changes, TCDD produced functional deficits in the developing hearts, including blood regurgitation and a striking ventricular standstill that became prevalent by 120 hpf. We also assessed the effect of TCDD on the heart size using stereological measurements, which demonstrated significant reduction in heart tissue volume at 72 hpf. Perhaps our most significant finding was a decrease in the total number of cardiomyocytes in TCDD-exposed embryos by 48 hpf, one day prior to observable effects on peripheral blood flow. We conclude that the developing heart is an important target for TCDD in zebrafish. (+info)
Myocardin is sufficient and necessary for cardiac gene expression in Xenopus.
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Myocardin is a cardiac- and smooth muscle-specific cofactor for the ubiquitous transcription factor serum response factor (SRF). Using gain-of-function approaches in the Xenopus embryo, we show that myocardin is sufficient to activate transcription of a wide range of cardiac and smooth muscle differentiation markers in non-muscle cell types. We also demonstrate that, for the myosin light chain 2 gene (MLC2), myocardin cooperates with the zinc-finger transcription factor Gata4 to activate expression. Inhibition of myocardin activity in Xenopus embryos using morpholino knockdown methods results in inhibition of cardiac development and the absence of expression of cardiac differentiation markers and severe disruption of cardiac morphological processes. We conclude that myocardin is an essential component of the regulatory pathway for myocardial differentiation. (+info)
Concentric zones of active RhoA and Cdc42 around single cell wounds.
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Rho GTPases control many cytoskeleton-dependent processes, but how they regulate spatially distinct features of cytoskeletal function within a single cell is poorly understood. Here, we studied active RhoA and Cdc42 in wounded Xenopus oocytes, which assemble and close a dynamic ring of actin filaments (F-actin) and myosin-2 around wound sites. RhoA and Cdc42 are rapidly activated around wound sites in a calcium-dependent manner and segregate into distinct, concentric zones around the wound, with active Cdc42 in the approximate middle of the F-actin array and active RhoA on the interior of the array. These zones form before F-actin accumulation, and then move in concert with the closing array. Microtubules and F-actin are required for normal zone organization and dynamics, as is crosstalk between RhoA and Cdc42. Each of the zones makes distinct contributions to the organization and function of the actomyosin wound array. We propose that similar rho activity zones control related processes such as cytokinesis. (+info)