RESUMO
The transcription factor ZFH-2 has well-documented roles in Drosophila neurogenesis and other developmental processes. Here we provide the first evidence that ZFH-2 has a role in oogenesis. We demonstrate that ZFH-2 is expressed in the wild-type ovary and that a loss of zfh-2 function produces a mutant ovary phenotype where egg chambers are reduced in number and fused. We also show that a loss of zfh-2 function can suppress a daughterless loss-of-function ovary phenotype suggesting a possible genetic relationship between these two genes in the ovary. We also show that ZFH-2 is located at the boundary between bands and interbands on polytene chromosomes and that at a subset of these sites ZFH-2 colocalizes with the insulator/promoter cofactor CP190.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Feminino , Cromossomos , Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Drosophila/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Folículo Ovariano , Ovário , Cromossomos Politênicos/genéticaRESUMO
Titin's C-zone is an inextensible segment in titin, comprised of 11 super-repeats and located in the cMyBP-C-containing region of the thick filament. Previously we showed that deletion of titin's super-repeats C1 and C2 (TtnΔC1-2 model) results in shorter thick filaments and contractile dysfunction of the left ventricular (LV) chamber but that unexpectedly LV diastolic stiffness is normal. Here we studied the contraction-relaxation kinetics from the time-varying elastance of the LV and intact cardiomyocyte, cellular work loops of intact cardiomyocytes, Ca2+ transients, cross-bridge kinetics, and myofilament Ca2+ sensitivity. Intact cardiomyocytes of TtnΔC1-2 mice exhibit systolic dysfunction and impaired relaxation. The time-varying elastance at both LV and single-cell levels showed that activation kinetics are normal in TtnΔC1-2 mice, but that relaxation is slower. The slowed relaxation is, in part, attributable to an increased myofilament Ca2+ sensitivity and slower early Ca2+ reuptake. Cross-bridge dynamics showed that cross-bridge kinetics are normal but that the number of force-generating cross-bridges is reduced. In vivo sarcomere length (SL) measurements revealed that in TtnΔC1-2 mice the operating SL range of the LV is shifted towards shorter lengths. This normalizes the apparent cell and LV diastolic stiffness but further reduces systolic force as systole occurs further down on the ascending limb of the force-SL relation. We propose that the reduced working SLs reflect titin's role in regulating diastolic stiffness by altering the number of sarcomeres in series. Overall, our study reveals that thick filament length regulation by titin's C-zone is critical for normal cardiac function.
Assuntos
Miofibrilas , Sarcômeros , Animais , Conectina/genética , Camundongos , Contração Muscular , Miócitos Cardíacos , Proteínas Quinases/genética , Sarcômeros/fisiologiaRESUMO
Titin is largely comprised of serially-linked immunoglobulin (Ig) and fibronectin type-III (Fn3) domains. Many of these domains are arranged in an 11 domain super-repeat pattern that is repeated 11 times, forming the so-named titin C-zone in the A-band region of the sarcomere. Each super-repeat is thought to provide binding sites for thick filament proteins, such as cMyBP-C (cardiac myosin-binding protein C). However, it remains to be established which of titin's 11 C-zone super-repeats anchor cMyBP-C as titin contains 11 super-repeats and cMyBP-C is found in 9 stripes only. To study the layout of titin's C-zone in relation to MyBP-C, immunolabeling studies were performed on mouse skinned myocardium with antibodies to titin and cMyBP-C, using both immuno-electron microscopy and super-resolution optical microscopy. Results indicate that cMyBP-C locates near the interface between titin's C-zone super-repeats. Studies on a mouse model in which two of titin's C-zone repeats have been genetically deleted support that the first Ig domain of a super-repeat is important for anchoring cMyBP-C but also Fn3 domains located at the end of the preceding repeat. Furthermore, not all super-repeat interfaces are equal as the interface between super-repeat 1 and 2 (close to titin's D-zone) does not contain cMyBP-C. Finally, titin's C-zone does not extend all the way to the bare zone but instead terminates at the level of the second myosin crown. This study enhances insights in the molecular layout of the C-zone of titin, its relation to cMyBP-C, and its possible roles in cardiomyopathies.
Assuntos
Proteínas de Transporte/metabolismo , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Proteínas Quinases/metabolismo , Sarcômeros/metabolismo , Sarcômeros/ultraestrutura , Animais , Imunofluorescência , Camundongos , Microscopia de FluorescênciaRESUMO
The introduction of next-generation sequencing technology has revealed that mutations in the gene that encodes titin (TTN) are linked to multiple skeletal and cardiac myopathies. The most prominent of these myopathies is dilated cardiomyopathy (DCM). Over 60 genes are linked to the etiology of DCM, but by far, the leading cause of DCM is mutations in TTN with truncating variants in TTN (TTNtvs) associated with familial DCM in â¼ 20% of the cases. Titin is a large (3-4 MDa) and abundant protein that forms the third myofilament type of striated muscle where it spans half the sarcomere, from the Z-disk to the M-line. The underlying mechanisms by which titin mutations induce disease are poorly understood and targeted therapies are not available. Here, we review what is known about TTN mutations in muscle disease, with a major focus on DCM. We highlight that exon skipping might provide a possible therapeutic avenue to address diseases that arise from TTNtvs.
Assuntos
Cardiomiopatias/genética , Conectina/genética , Mutação , Animais , Cardiomiopatias/patologia , Conectina/metabolismo , Humanos , PenetrânciaRESUMO
OBJECTIVE: Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder. METHODS: Using massively parallel sequencing we identified 30 patients from 27 families with 2 pathogenic nonsense, frameshift and/or splice site TTN mutations in trans. We then undertook a detailed analysis of the clinical, histopathological and imaging features of these patients. RESULTS: All patients had prenatal or early onset hypotonia and/or congenital contractures. None had ophthalmoplegia. Scoliosis and respiratory insufficiency typically developed early and progressed rapidly, whereas limb weakness was often slowly progressive, and usually did not prevent independent walking. Cardiac involvement was present in 46% of patients. Relatives of 2 patients had dilated cardiomyopathy. Creatine kinase levels were normal to moderately elevated. Increased fiber size variation, internalized nuclei and cores were common histopathological abnormalities. Cap-like regions, whorled or ring fibers, and mitochondrial accumulations were also observed. Muscle magnetic resonance imaging showed gluteal, hamstring and calf muscle involvement. Western blot analysis showed a near-normal sized titin protein in all samples. The presence of 2 mutations predicted to impact both N2BA and N2B cardiac isoforms appeared to be associated with greatest risk of cardiac involvement. One-third of patients had 1 mutation predicted to impact exons present in fetal skeletal muscle, but not included within the mature skeletal muscle isoform transcript. This strongly suggests developmental isoforms are involved in the pathogenesis of this congenital/early onset disorder. INTERPRETATION: This detailed clinical reference dataset will greatly facilitate diagnostic confirmation and management of patients, and has provided important insights into disease pathogenesis. Ann Neurol 2018;83:1105-1124.
Assuntos
Cardiomiopatia Dilatada/congênito , Conectina/genética , Proteínas Musculares/genética , Músculo Esquelético/patologia , Feminino , Humanos , Masculino , Mutação/genética , Fenótipo , Isoformas de Proteínas/genéticaRESUMO
Nebulin is a giant filamentous protein that is coextensive with the actin filaments of the skeletal muscle sarcomere. Nebulin mutations are the main cause of nemaline myopathy (NEM), with typical adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood. To establish nebulin's functional roles in adult muscle, we studied a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinase (MCK) promotor. Neb cKO mice are born with high nebulin levels in their skeletal muscles, but within weeks after birth nebulin expression rapidly falls to barely detectable levels Surprisingly, a large fraction of the mice survive to adulthood with low nebulin levels (<5% of control), contain nemaline rods and undergo fiber-type switching toward oxidative types. Nebulin deficiency causes a large deficit in specific force, and mechanistic studies provide evidence that a reduced fraction of force-generating cross-bridges and shortened thin filaments contribute to the force deficit. Muscles rich in glycolytic fibers upregulate proteolysis pathways (MuRF-1, Fbxo30/MUSA1, Gadd45a) and undergo hypotrophy with smaller cross-sectional areas (CSAs), worsening their force deficit. Muscles rich in oxidative fibers do not have smaller weights and can even have hypertrophy, offsetting their specific-force deficit. These studies reveal nebulin as critically important for force development and trophicity in adult muscle. The Neb cKO phenocopies important aspects of NEM (muscle weakness, oxidative fiber-type predominance, variable trophicity effects, nemaline rods) and will be highly useful to test therapeutic approaches to ameliorate muscle weakness.
Assuntos
Proteínas Musculares/deficiência , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miopatias da Nemalina/genética , Miopatias da Nemalina/patologia , Sarcômeros/metabolismo , Animais , Modelos Animais de Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Camundongos , Camundongos Knockout , Contração Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/ultraestrutura , Proteínas Musculares/genética , Debilidade Muscular/genética , Debilidade Muscular/patologia , Músculo Esquelético/fisiopatologia , Músculo Esquelético/ultraestrutura , Miopatias da Nemalina/mortalidade , Miosinas/genética , Miosinas/metabolismo , Fenótipo , Sarcômeros/patologiaRESUMO
Titin, the largest protein known, forms a giant filament in muscle where it spans the half sarcomere from Z disk to M band. Here we genetically targeted a stretch of 14 immunoglobulin-like and fibronectin type 3 domains that comprises the I-band/A-band (IA) junction and obtained a viable mouse model. Super-resolution optical microscopy (structured illumination microscopy, SIM) and electron microscopy were used to study the thick filament length and titin's molecular elasticity. SIM showed that the IA junction functionally belongs to the relatively stiff A-band region of titin. The stiffness of A-band titin was found to be high, relative to that of I-band titin (â¼ 40-fold higher) but low, relative to that of the myosin-based thick filament (â¼ 70-fold lower). Sarcomere stretch therefore results in movement of A-band titin with respect to the thick filament backbone, and this might constitute a novel length-sensing mechanism. Findings disproved that titin at the IA junction is crucial for thick filament length control, settling a long-standing hypothesis. SIM also showed that deleting the IA junction moves the attachment point of titin's spring region away from the Z disk, increasing the strain on titin's molecular spring elements. Functional studies from the cellular to ex vivo and in vivo left ventricular chamber levels showed that this causes diastolic dysfunction and other symptoms of heart failure with preserved ejection fraction (HFpEF). Thus, our work supports titin's important roles in diastolic function and disease of the heart.
Assuntos
Conectina/metabolismo , Coração/fisiologia , Miocárdio/metabolismo , Sarcômeros/metabolismo , Sequência de Aminoácidos , Animais , Fenômenos Biomecânicos , Pressão Sanguínea/fisiologia , Western Blotting , Células Cultivadas , Conectina/genética , Ecocardiografia , Perfilação da Expressão Gênica , Modelos Lineares , Mecanotransdução Celular , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Miocárdio/citologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcômeros/ultraestrutura , Homologia de Sequência de AminoácidosRESUMO
Titin is a large filamentous protein that is responsible for the passive force of the cardiac sarcomere. Titin's force is generated by its I-band region, which includes the cardiac-specific N2B element. The N2B element consists of three immunoglobulin domains, two small unique sequence insertions, and a large 575-residue unique sequence, the N2B-Us. Posttranslational modifications of the N2B element are thought to regulate passive force, but the underlying mechanisms are unknown. Increased passive-force levels characterize diastolic stiffening in heart-failure patients, and it is critical to understand the underlying molecular mechanisms and identify therapeutic targets. Here, we used single-molecule force spectroscopy to study the mechanical effects of the kinases calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) and extracellular signal-regulated kinase 2 (ERK2) on the single-molecule mechanics of the N2B element. Both CaMKIIδ and ERK2 were found to phosphorylate the N2B element, and single-molecule force spectroscopy revealed an increase in the persistence length (Lp) of the molecule, indicating that the bending rigidity of the molecule was increased. Experiments performed under oxidizing conditions and with a recombinant N2B element that had a simplified domain composition provided evidence that the Lp increase requires the N2B-Us of the N2B element. Mechanical experiments were also performed on skinned myocardium before and after phosphorylation. The results revealed a large (â¼30%) passive force reduction caused by CaMKIIδ and a much smaller (â¼6%) reduction caused by ERK2. These findings support the notion that the important kinases ERK2 and CaMKIIδ can alter the passive force of myocytes in the heart (although CaMKIIδ appears to be more potent) during physiological and pathophysiological states.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Conectina/química , Conectina/metabolismo , Fenômenos Mecânicos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Miocárdio/metabolismo , Sequência de Aminoácidos , Animais , Fenômenos Biomecânicos , Feminino , Humanos , Camundongos , Dados de Sequência Molecular , Fosforilação , Estresse MecânicoRESUMO
BACKGROUND: Experimentally upregulating compliant titins has been suggested as a therapeutic for lowering pathological diastolic stiffness levels. However, how increasing titin compliance impacts global cardiac function requires in-depth study. We investigate the effect of upregulating compliant titins in a novel mouse model with a genetically altered titin splicing factor; integrative approaches were used from intact cardiomyocyte mechanics to pressure-volume analysis and Doppler echocardiography. METHODS AND RESULTS: Compliant titins were upregulated through deletion of the RNA Recognition Motif of the splicing factor RBM20 (Rbm20(ΔRRM)mice). A genome-wide exon expression analysis and a candidate approach revealed that the phenotype is likely to be dominated by greatly increased lengths of titin's spring elements. At both cardiomyocyte and left ventricular chamber levels, diastolic stiffness was reduced in heterozygous (+/-) Rbm20(ΔRRM)mice with a further reduction in homozygous (-/-) mice at only the intact myocyte level. Fibrosis was present in only -/- Rbm20(ΔRRM) hearts. The Frank-Starling Mechanism was reduced in a graded fashion in Rbm20(ΔRRM) mice, at both the cardiomyocyte and left ventricular chamber levels. Exercise tests revealed an increase in exercise capacity in +/- mice. CONCLUSIONS: Titin is not only important in diastolic but also in systolic cardiac function. Upregulating compliant titins reduces diastolic chamber stiffness owing to the increased compliance of myocytes, but it depresses end-systolic elastance; under conditions of exercise, the beneficial effects on diastolic function dominate. Therapeutic manipulation of the RBM20-based splicing system might be able to minimize effects on fibrosis and systolic function while improving the diastolic function in patients with heart failure.
Assuntos
Fenômenos Biomecânicos/fisiologia , Conectina/fisiologia , Diástole/fisiologia , Elasticidade/fisiologia , Coração/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Conectina/deficiência , Conectina/genética , Ecocardiografia Doppler , Heterozigoto , Homozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Rigidez Vascular/fisiologia , Função Ventricular Esquerda/fisiologiaRESUMO
BACKGROUND: Diastolic dysfunction is a poorly understood but clinically pervasive syndrome that is characterized by increased diastolic stiffness. Titin is the main determinant of cellular passive stiffness. However, the physiological role that the tandem immunoglobulin (Ig) segment of titin plays in stiffness generation and whether shortening this segment is sufficient to cause diastolic dysfunction need to be established. METHODS AND RESULTS: We generated a mouse model in which 9 Ig-like domains (Ig3-Ig11) were deleted from the proximal tandem Ig segment of the spring region of titin (IG KO). Exon microarray analysis revealed no adaptations in titin splicing, whereas novel phospho-specific antibodies did not detect changes in titin phosphorylation. Passive myocyte stiffness was increased in the IG KO, and immunoelectron microscopy revealed increased extension of the remaining titin spring segments as the sole likely underlying mechanism. Diastolic stiffness was increased at the tissue and organ levels, with no consistent changes in extracellular matrix composition or extracellular matrix-based passive stiffness, supporting a titin-based mechanism for in vivo diastolic dysfunction. Additionally, IG KO mice have a reduced exercise tolerance, a phenotype often associated with diastolic dysfunction. CONCLUSIONS: Increased titin-based passive stiffness is sufficient to cause diastolic dysfunction with exercise intolerance.
Assuntos
Diástole/fisiologia , Insuficiência Cardíaca Diastólica/genética , Insuficiência Cardíaca Diastólica/fisiopatologia , Imunoglobulinas/fisiologia , Proteínas Quinases/fisiologia , Fatores Etários , Animais , Cardiomegalia/genética , Cardiomegalia/fisiopatologia , Modelos Animais de Doenças , Elasticidade , Tolerância ao Exercício/fisiologia , Imunoglobulinas/química , Imunoglobulinas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Imunoeletrônica , Fenótipo , Fosforilação/fisiologia , Proteínas Quinases/química , Proteínas Quinases/genética , Estrutura Terciária de Proteína , Sarcômeros/fisiologiaRESUMO
The ability to change an organism's DNA through gene editing is of great importance for the prevention and treatment of genetic and acquired diseases. Rapid progress has been made during the last decade due to the discovery and refinement of CRISPR/Cas9 as an accurate, fast, and reliable genome editing technique. In this issue of the JCI, Lebek et al. present the culmination from a line of work in the Olson laboratory focused on in vivo gene editing of CAMK2D. The paper presents a combined state-of-the-art gene therapy approach that demonstrates how gene therapy can yield cardioprotection in a mouse model and takes notable steps toward potential applicability in patients.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Camundongos , Animais , Humanos , Edição de Genes/métodos , Terapia Genética/métodos , DNARESUMO
Nebulin is a skeletal muscle protein that associates with the sarcomeric thin filaments and has functions in regulating the length of the thin filament and the structure of the Z-disk. Here we investigated the nebulin gene in 53 species of birds, fish, amphibians, reptiles, and mammals. In all species, nebulin has a similar domain composition that mostly consists of â¼30-residue modules (or simple repeats), each containing an actin-binding site. All species have a large region where simple repeats are organized into seven-module super-repeats, each containing a tropomyosin binding site. The number of super-repeats shows high interspecies variation, ranging from 21 (zebrafish, hummingbird) to 31 (camel, chimpanzee), and, importantly, scales with body size. The higher number of super-repeats in large animals was shown to increase thin filament length, which is expected to increase the sarcomere length for optimal force production, increase the energy efficiency of isometric force production, and lower the shortening velocity of muscle. It has been known since the work of A.V. Hill in 1950 that as species increase in size, the shortening velocity of their muscle is reduced, and the present work shows that nebulin contributes to the mechanistic basis. Finally, we analyzed the differentially spliced simple repeats in nebulin's C terminus, whose inclusion correlates with the width of the Z-disk. The number of Z-repeats greatly varies (from 5 to 18) and correlates with the number of super-repeats. We propose that the resulting increase in the width of the Z-disk in large animals increases the number of contacts between nebulin and structural Z-disk proteins when the Z-disk is stressed for long durations.
Assuntos
Proteínas Musculares , Peixe-Zebra , Animais , Proteínas Musculares/genética , Músculo Esquelético , SarcômerosRESUMO
BACKGROUND: Low myocardial cGMP-PKG (cyclic guanosine monophosphate-protein kinase G) activity has been associated with increased cardiomyocyte diastolic stiffness in heart failure with preserved ejection fraction. Cyclic guanosine monophosphate is mainly hydrolyzed by PDE (phosphodiesterases) 5a and 9a. Importantly, PDE9a expression has been reported to be upregulated in human heart failure with preserved ejection fraction myocardium and chronic administration of a PDE9a inhibitor reverses preestablished cardiac hypertrophy and systolic dysfunction in mice subjected to transverse aortic constriction (TAC). We hypothesized that inhibiting PDE9a activity ameliorates diastolic dysfunction. METHODS: To examine the effect of chronic PDE9a inhibition, 2 diastolic dysfunction mouse models were studied: (1) TAC-deoxycorticosterone acetate and (2) Leprdb/db. PDE9a inhibitor (5 and 8 mg/kg per day) was administered to the mice via subcutaneously implanted osmotic minipumps for 28 days. The effect of acute PDE9a inhibition was investigated in intact cardiomyocytes isolated from TAC-deoxycorticosterone acetate mice. Atrial natriuretic peptide together with PDE9a inhibitor were administered to the isolated intact cardiomyocytes through the cell perfusate. RESULTS: For acute inhibition, no cellular stiffness reduction was found, whereas chronic PDE9a inhibition resulted in reduced left ventricular chamber stiffness in TAC-deoxycorticosterone acetate, but not in Leprdb/db mice. Passive cardiomyocyte stiffness was reduced by chronic PDE9a inhibition, with no differences in myocardial fibrosis or cardiac morphometry. PDE9a inhibition increased the ventricular-arterial coupling ratio, reflecting impaired systolic function. CONCLUSIONS: Chronic PDE9a inhibition lowers left ventricular chamber stiffness in TAC-deoxycorticosterone acetate mice. However, the usefulness of PDE9a inhibition to treat high-diastolic stiffness may be limited as the required PDE9a inhibitor dose also impairs systolic function, observed as a decline in ventricular-arterial coordination, in this model.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Disfunção Ventricular Esquerda/tratamento farmacológico , Função Ventricular Esquerda/efeitos dos fármacos , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Animais , Diástole , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/enzimologia , Inibidores de Fosfodiesterase/toxicidade , Disfunção Ventricular Esquerda/enzimologia , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
BACKGROUND: Nebulin is a critical thin filament-binding protein that spans from the Z-disk of the skeletal muscle sarcomere to near the pointed end of the thin filament. Its massive size and actin-binding property allows it to provide the thin filaments with structural and regulatory support. When this protein is lost, nemaline myopathy occurs. Nemaline myopathy causes severe muscle weakness as well as structural defects on a sarcomeric level. There is no known cure for this disease. METHODS: We studied whether sarcomeric structure and function can be improved by introducing nebulin's Z-disk region into a nebulin-deficient mouse model (Neb cKO) through adeno-associated viral (AAV) vector therapy. Following this treatment, the structural and functional characteristics of both vehicle-treated and AAV-treated Neb cKO and control muscles were studied. RESULTS: Intramuscular injection of this AAV construct resulted in a successful expression of the Z-disk fragment within the target muscles. This expression was significantly higher in Neb cKO mice than control mice. Analysis of protein expression revealed that the nebulin fragment was localized exclusively to the Z-disks and that Neb cKO expressed the nebulin fragment at levels comparable to the level of full-length nebulin in control mice. Additionally, the Z-disk fragment displaced full-length nebulin in control mice, resulting in nemaline rod body formation and a worsening of muscle function. Neb cKO mice experienced a slight functional benefit from the AAV treatment, with a small increase in force and fatigue resistance. Disease progression was also slowed as indicated by improved muscle structure and myosin isoform expression. CONCLUSIONS: This study reveals that nebulin fragments are well-received by nebulin-deficient mouse muscles and that limited functional benefits are achievable.
Assuntos
Proteínas Musculares/genética , Miopatias da Nemalina/terapia , Sarcômeros/metabolismo , Animais , Dependovirus/genética , Terapia Genética/métodos , Células HEK293 , Humanos , Camundongos , Contração Muscular , Fadiga Muscular , Proteínas Musculares/metabolismo , Miosinas/genética , Miosinas/metabolismo , Sarcômeros/fisiologia , Sarcômeros/ultraestruturaRESUMO
Following the publication of this paper [1], it was brought to the authors' attention that one of the contributing authors was left off of the paper. The authors apologize for the unfortunate oversight. In this correction paper, they have included Dr. Paola Tonino in the author list section.
RESUMO
Regulating the thin-filament length in muscle is crucial for controlling the number of myosin motors that generate power. The giant protein nebulin forms a long slender filament that associates along the length of the thin filament in skeletal muscle with functions that remain largely obscure. Here nebulin's role in thin-filament length regulation was investigated by targeting entire super-repeats in the Neb gene; nebulin was either shortened or lengthened by 115 nm. Its effect on thin-filament length was studied using high-resolution structural and functional techniques. Results revealed that thin-filament length is strictly regulated by the length of nebulin in fast muscles. Nebulin's control is less tight in slow muscle types where a distal nebulin-free thin-filament segment exists, the length of which was found to be regulated by leiomodin-2 (Lmod2). We propose that strict length control by nebulin promotes high-speed shortening and that dual-regulation by nebulin/Lmod2 enhances contraction efficiency.
RESUMO
Nebulin is a giant protein that winds around the actin filaments in the skeletal muscle sarcomere. Compound-heterozygous mutations in the nebulin gene (NEB) cause typical nemaline myopathy (NM), a muscle disorder characterized by muscle weakness with limited treatment options. We created a mouse model with a missense mutation p.Ser6366Ile and a deletion of NEB exon 55, the Compound-Het model that resembles typical NM. We show that Compound-Het mice are growth-retarded and have muscle weakness. Muscles have a reduced myofibrillar fractional-area and sarcomeres are disorganized, contain rod bodies, and have longer thin filaments. In contrast to nebulin-based severe NM where haplo-insufficiency is the disease driver, Compound-Het mice express normal amounts of nebulin. X-ray diffraction revealed that the actin filament is twisted with a larger radius, that tropomyosin and troponin behavior is altered, and that the myofilament spacing is increased. The unique disease mechanism of nebulin-based typical NM reveals novel therapeutic targets.
Assuntos
Proteínas Musculares/genética , Mutação de Sentido Incorreto , Miofibrilas/metabolismo , Miopatias da Nemalina/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animais , Heterozigoto , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Miofibrilas/patologia , Miofibrilas/ultraestrutura , Miopatias da Nemalina/metabolismo , Sarcômeros/metabolismo , Sarcômeros/patologia , Sarcômeros/ultraestrutura , Tropomiosina/química , Tropomiosina/metabolismo , Troponina/química , Troponina/metabolismo , Difração de Raios XRESUMO
We developed a reproducible and reliable method of using a rubber stamp prospectively to document appearances of the sternal wound. 395 consecutive patients undergoing median sternotomy for cardiac procedures were studied over a period of four months. Data were collected by the Senior House Officer during the ward round for all patients at Day 3 and Day 7 post-operatively. Data were entered for 303 patients using the rubber stamp. 78 patients had no stamp entered in their case notes. 21 patients were recorded as having abnormal wounds, 11 of whom confirmed positive microbiological growth. On the other hand, 282 patients were recorded to have normal wounds, 10 of which had positive bacterial growth. Our initial results have been encouraging with almost 80% compliance and 96% specificity. A simple recording system consisting of proven signs of infection known to medical practitioners was our tool.
Assuntos
Esterno/cirurgia , Infecção da Ferida Cirúrgica/diagnóstico , Humanos , Prevalência , Estudos Prospectivos , Fatores de Risco , Sensibilidade e Especificidade , Infecção da Ferida Cirúrgica/epidemiologia , Infecção da Ferida Cirúrgica/etiologia , Fatores de TempoRESUMO
The giant multi-functional striated muscle protein titin is the third most abundant muscle protein after myosin and actin. Titin plays a pivotal role in myocardial passive stiffness, structural integrity and stress-initiated signaling pathways. The complete sequence of the human titin gene contains three isoform-specific mutually exclusive exons [termed novel exons (novex)] coding for the I-band sequence, named novex-1 (exon 45), novex-2 (exon 46) and novex-3 (exon 48). Transcripts containing either the novex-1 or novex-2 exons code for the novex-1 and novex-2 titin isoforms. The novex-3 transcript contains a stop codon and polyA tail signal, resulting in an unusually small (â¼700 kDa) isoform, referred to as novex-3 titin. This 'tiny titin' isoform extends from the Z-disc (N-terminus) to novex-3 (C-terminus) and is expressed in all striated muscles. Biochemical analysis of novex-3 titin in cardiomyocytes shows that obscurin, a vertebrate muscle protein, binds to novex-3 titin. The novex-3/obscurin complex localizes to the Z-disc region and may regulate calcium, and SH3- and GTPase-associated myofibrillar signaling pathways. Therefore, novex-3 titin could be involved in stress-initiated sarcomeric restructuring.
RESUMO
The contractile machinery of heart and skeletal muscles has as an essential component the thick filament, comprised of the molecular motor myosin. The thick filament is of a precisely controlled length, defining thereby the force level that muscles generate and how this force varies with muscle length. It has been speculated that the mechanism by which thick filament length is controlled involves the giant protein titin, but no conclusive support for this hypothesis exists. Here we show that in a mouse model in which we deleted two of titin's C-zone super-repeats, thick filament length is reduced in cardiac and skeletal muscles. In addition, functional studies reveal reduced force generation and a dilated cardiomyopathy (DCM) phenotype. Thus, regulation of thick filament length depends on titin and is critical for maintaining muscle health.