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Robert J. Bloch - One of the best experts on this subject based on the ideXlab platform.
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Biomechanical Properties of the Sarcolemma and Costameres of Skeletal Muscle Lacking Desmin
'Frontiers Media SA', 2021Co-Authors: Karla P. Garcia-pelagio, Robert J. BlochAbstract:Intermediate filaments (IFs), composed primarily by desmin and keratins, link the myofibrils to each other, to intracellular organelles, and to the sarcolemma. There they may play an important role in transfer of contractile force from the Z-disks and M-lines of neighboring myofibrils to Costameres at the membrane, across the membrane to the extracellular matrix, and ultimately to the tendon (“lateral force transmission”). We measured the elasticity of the sarcolemma and the connections it makes at Costameres with the underlying contractile apparatus of individual fast twitch muscle fibers of desmin-null mice. By positioning a suction pipet to the surface of the sarcolemma and applying increasing pressure, we determined the pressure at which the sarcolemma separated from nearby sarcomeres, Pseparation, and the pressure at which the isolated sarcolemma burst, Pbursting. We also examined the time required for the intact sarcolemma-Costamere-sarcomere complex to reach equilibrium at lower pressures. All measurements showed the desmin-null fibers to have slower equilibrium times and lower Pseparation and Pbursting than controls, suggesting that the sarcolemma and its costameric links to nearby contractile structures were weaker in the absence of desmin. Comparisons to earlier values determined for muscles lacking dystrophin or synemin suggest that the desmin-null phenotype is more stable than the former and less stable than the latter. Our results are consistent with the moderate myopathy seen in desmin-null muscles and support the idea that desmin contributes significantly to sarcolemmal stability and lateral force transmission
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biomechanics of the sarcolemma and Costameres in single skeletal muscle fibers from normal and dystrophin null mice
Journal of Muscle Research and Cell Motility, 2011Co-Authors: Karla P Garciapelagio, Alicia Ortega, H L Gonzalezserratos, Robert J. BlochAbstract:We studied the biomechanical properties of the sarcolemma and its links through Costameres to the contractile apparatus in single mammalian myofibers of Extensor digitorum longus muscles isolated from wild (WT) and dystrophin-null (mdx) mice. Suction pressures (P) applied through a pipette to the sarcolemma generated a bleb, the height of which increased with increasing P. Larger increases in P broke the connections between the sarcolemma and myofibrils and eventually caused the sarcolemma to burst. We used the values of P at which these changes occurred to estimate the tensions and stiffness of the system and its individual elements. Tensions of the whole system and the sarcolemma, as well as the maximal tension sustained by the Costameres, were all significantly lower (1.8–3.3 fold) in muscles of mdx mice compared to WT. Values of P at which separation and bursting occurred, as well as the stiffness of the whole system and of the isolated sarcolemma, were ~2-fold lower in mdx than in WT. Our results indicate that the absence of dystrophin reduces muscle stiffness, increases sarcolemmal deformability, and compromises the mechanical stability of Costameres and their connections to nearby myofibrils.
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erratum to differential distribution of dystrophin and β spectrin at the sarcolemma of fast twitch skeletal muscle fibers
Journal of Muscle Research and Cell Motility, 2010Co-Authors: Mcrae W Williams, Robert J. BlochAbstract:We used double label immunofluorescence and confocal microscopy to examine the organization of β-spectrin and dystrophin at the sarcolemma of fast twitch myofibers in the Extensor Digitorum Longus (EDL) of the rat. Both β-spectrin and dystrophin are concentrated in Costameres, a rectilinear sarcolemmal array composed of longitudinal strands and transverse elements overlying Z and M lines. In contrast, intercostameric regions, lying between these linear structures, contain significant levels of dystrophin but little detectable β-spectrin. The dystrophin-associated proteins, syntrophin and β-dystroglycan, are also concentrated at Costameres but, like dystrophin, are present in intercostameric regions as well. Dystrophin is present at Costameres and intercostameric regions in fast twitch muscles of the mouse but is absent from all regions of the sarcolemma in the mdx mouse, which lacks dystrophin. Areas of the sarcolemma near myonuclei also contain dystrophin without β-spectrin, consistent with the idea that the distribution of dystrophin at the sarcolemma is not dependent on β-spectrin. We conclude that dystrophin is present under all areas of the sarcolemma. The increased fragility of the sarcolemma in patients with Duchennes muscular dystrophy may be explained in part by the absence of dystrophin not only from Costameres, but also from intercostameric regions.
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modeling the membrane Costamere myofibril complex from normal and desmin or dystrophin mice as a distributed elastic system
Biophysical Journal, 2009Co-Authors: Karla P Garciapelagio, Alicia Ortega, H L Gonzalezserratos, Robert J. Bloch, Ivan SantamariaAbstract:We studied the stiffness (k) of the membrane-Costamere-myofibril complex and of the sarcolemma alone in myofibers from control and desmin-null or dystrophin-null (mdx) mice. Negative pressure (P) was applied with an elastimeter through a pipette to the sarcolemma of myofibers, isolated from murine extensor digitorum longus muscles, to form blebs. We analyzed the results using a distributed spring model, based on the presumptive organization of the proteins in the extended complex. The model was solved as a lumped system. From the model, we computed k. We estimated k of the complex from 1450 to 2600, from 1100 to 1600 and from 900 to 1300 dyne/cm for control, desmin null, and dystrophin null myofibers, respectively. Values of k for the sarcolemma alone varied from 1000 to 1900, 700 to 1400 and 700 to 1000 dyne/cm for the same groups., The controls are therefore stiffer than either of the null mutants, and the dystrophin-null is more compliant than either controls or desmin-nulls. We compare the experimental values of k for the complex in control and mutant muscles to the theoretical values obtained by the iteration of k for each protein. Normalizing the experimental k values for control myofibers as 1.00, we found values of 0.73 and 0.52 for the desmin- and dystrophin-null muscles, respectively. Computed theoretical values were 1.0, 0.72 and 0.53, in good agreement with our experimental results. We conclude that the complex of proteins that link myofibrils to the sarcolemma at Costameres can be modeled as a distributed, lumped spring system, in which each protein has a different k. As a result, the, absence of desmin or dystrophin affects the mechanical properties of the complex differently.Supported by MDA to RJB and CONACyT
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Absence of keratin 19 in mice causes skeletal myopathy with mitochondrial and sarcolemmal reorganization.
Journal of cell science, 2007Co-Authors: Michele R Stone, Andrea O'neill, Richard M Lovering, John Strong, Wendy G Resneck, Patrick W Reed, Diana M Toivola, Jeanine A Ursitti, M Bishr Omary, Robert J. BlochAbstract:Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. We examined the contractile properties and morphology of fast-twitch skeletal muscle from mice lacking keratin 19. Tibialis anterior muscles of keratin-19-null mice showed a small but significant decrease in mean fiber diameter and in the specific force of tetanic contraction, as well as increased plasma creatine kinase levels. Costameres at the sarcolemma of keratin-19-null muscle, visualized with antibodies against spectrin or dystrophin, were disrupted and the sarcolemma was separated from adjacent myofibrils by a large gap in which mitochondria accumulated. The costameric dystrophin-dystroglycan complex, which co-purified with gamma-actin, keratin 8 and keratin 19 from striated muscles of wild-type mice, co-purified with gamma-actin but not keratin 8 in the mutant. Our results suggest that keratin 19 in fast-twitch skeletal muscle helps organize Costameres and links them to the contractile apparatus, and that the absence of keratin 19 disrupts these structures, resulting in loss of contractile force, altered distribution of mitochondria and mild myopathy. This is the first demonstration of a mammalian phenotype associated with a genetic perturbation of keratin 19.
James M. Ervasti - One of the best experts on this subject based on the ideXlab platform.
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dystrophin is a microtubule associated protein
Journal of Cell Biology, 2009Co-Authors: Kurt W. Prins, James M. Ervasti, Evelyn Ralston, Jill L Humston, Amisha Mehta, Victoria TateAbstract:Cytolinkers are giant proteins that can stabilize cells by linking actin filaments, intermediate filaments, and microtubules (MTs) to transmembrane complexes. Dystrophin is functionally similar to cytolinkers, as it links the multiple components of the cellular cytoskeleton to the transmembrane dystroglycan complex. Although no direct link between dystrophin and MTs has been documented, Costamere-associated MTs are disrupted when dystrophin is absent. Using tissue-based cosedimentation assays on mice expressing endogenous dystrophin or truncated transgene products, we find that constructs harboring spectrinlike repeat 24 through the first third of the WW domain cosediment with MTs. Purified Dp260, a truncated isoform of dystrophin, bound MTs with a Kd of 0.66 µM, a stoichiometry of 1 Dp260/1.4 tubulin heterodimer at saturation, and stabilizes MTs from cold-induced depolymerization. Finally, α- and β-tubulin expression is increased ∼2.5-fold in mdx skeletal muscle without altering the tubulin–MT equilibrium. Collectively, these data suggest dystrophin directly organizes and/or stabilizes costameric MTs and classifies dystrophin as a cytolinker in skeletal muscle.
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Destabilization of the dystrophin-glycoprotein complex without functional deficits in α-dystrobrevin null muscle
PLoS ONE, 2008Co-Authors: Tina M. Bunnell, Michele A. Jaeger, Daniel P. Fitzsimons, Kurt W. Prins, James M. ErvastiAbstract:α-Dystrobrevin is a component of the dystrophin-glycoprotein complex (DGC) and is thought to have both structural and signaling roles in skeletal muscle. Mice deficient for α-dystrobrevin (adbn−/−) exhibit extensive myofiber degeneration and neuromuscular junction abnormalities. However, the biochemical stability of the DGC and the functional performance of adbn−/− muscle have not been characterized. Here we show that the biochemical association between dystrophin and β-dystroglycan is compromised in adbn−/− skeletal muscle, suggesting that α-dystrobrevin plays a structural role in stabilizing the DGC. However, despite muscle cell death and DGC destabilization, Costamere organization and physiological performance is normal in adbn−/− skeletal muscle. Our results demonstrate that myofiber degeneration alone does not cause functional deficits and suggests that more complex pathological factors contribute to the development of muscle weakness in muscular dystrophy.
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Destabilization of the dystrophin-glycoprotein complex without functional deficits in alpha-dystrobrevin null muscle.
Public Library of Science (PLoS), 2008Co-Authors: Tina M. Bunnell, Michele A. Jaeger, Daniel P. Fitzsimons, Kurt W. Prins, James M. ErvastiAbstract:Alpha-dystrobrevin is a component of the dystrophin-glycoprotein complex (DGC) and is thought to have both structural and signaling roles in skeletal muscle. Mice deficient for alpha-dystrobrevin (adbn(-/-)) exhibit extensive myofiber degeneration and neuromuscular junction abnormalities. However, the biochemical stability of the DGC and the functional performance of adbn(-/-) muscle have not been characterized. Here we show that the biochemical association between dystrophin and beta-dystroglycan is compromised in adbn(-/-) skeletal muscle, suggesting that alpha-dystrobrevin plays a structural role in stabilizing the DGC. However, despite muscle cell death and DGC destabilization, Costamere organization and physiological performance is normal in adbn(-/-) skeletal muscle. Our results demonstrate that myofiber degeneration alone does not cause functional deficits and suggests that more complex pathological factors contribute to the development of muscle weakness in muscular dystrophy
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Destabilization of the Dystrophin-Glycoprotein Complex without Functional Deficits in a-Dystrobrevin Null Muscle
2007Co-Authors: Tina M. Bunnell, Michele A. Jaeger, Daniel P. Fitzsimons, Kurt W. Prins, James M. ErvastiAbstract:a-Dystrobrevin is a component of the dystrophin-glycoprotein complex (DGC) and is thought to have both structural and signaling roles in skeletal muscle. Mice deficient for a-dystrobrevin (adbn 2/2) exhibit extensive myofiber degeneration and neuromuscular junction abnormalities. However, the biochemical stability of the DGC and the functional performance of adbn 2/2 muscle have not been characterized. Here we show that the biochemical association between dystrophin and b-dystroglycan is compromised in adbn 2/2 skeletal muscle, suggesting that a-dystrobrevin plays a structural role in stabilizing the DGC. However, despite muscle cell death and DGC destabilization, Costamere organization and physiological performance is normal in adbn 2/2 skeletal muscle. Our results demonstrate that myofiber degeneration alone does not cause functional deficits and suggests that more complex pathological factors contribute to the development of muscle weakness in muscular dystrophy
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the dystrophin complex forms a mechanically strong link between the sarcolemma and costameric actin
Journal of Cell Biology, 2000Co-Authors: Inna N. Rybakova, Jitandrakumar R. Patel, James M. ErvastiAbstract:The absence of dystrophin complex leads to disorganization of the force-transmitting costameric cytoskeleton and disruption of sarcolemmal membrane integrity in skeletal muscle. However, it has not been determined whether the dystrophin complex can form a mechanically strong bond with any costameric protein. We performed confocal immunofluorescence analysis of isolated sarcolemma that were mechanically peeled from skeletal fibers of mouse hindlimb muscle. A population of γ-actin filaments was stably associated with sarcolemma isolated from normal muscle and displayed a costameric pattern that precisely overlapped with dystrophin. However, costameric actin was absent from all sarcolemma isolated from dystrophin-deficient mdx mouse muscle even though it was localized to Costameres in situ. Vinculin, α-actinin, β-dystroglycan and utrophin were all retained on mdx sarcolemma, indicating that the loss of costameric actin was not due to generalized membrane instability. Our data demonstrate that the dystrophin complex forms a mechanically strong link between the sarcolemma and the costameric cytoskeleton through interaction with γ-actin filaments. Destabilization of costameric actin filaments may also be an important precursor to the Costamere disarray observed in dystrophin-deficient muscle. Finally, these methods will be broadly useful in assessing the mechanical integrity of the membrane cytoskeleton in dystrophic animal models lacking other costameric proteins.
Ju Chen - One of the best experts on this subject based on the ideXlab platform.
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obscurin dependent localization of ankyrin b is required for the organization of sub sarcolemma microtubules localization of dystrophin and sarcolemmal integrity in skeletal muscle
Biophysical Journal, 2013Co-Authors: Davide Randazzo, Bert Blaauw, Angela K Peter, Daniela Rossi, Ju Chen, Emiliana Giacomello, Carlo Reggiani, Stephan Lange, Vincenzo SorrentinoAbstract:Obscurin is a large myofibrillar protein that contains several interacting modules, one of which mediates direct binding to muscle-specific ankyrins. Interaction between obscurin and the muscle-specific ankyrin sAnk1.5 has been found to regulate the organization of the sarcoplasmic reticulum in striated muscles. The organization of dystrophin and β-dystroglycan at Costameres is mediated by additional muscle-specific ankyrin isoforms, namely ankB and ankG, localized at the sub-sarcolemma level. In particular, ankB is responsible for the assembly of sub-sarcolemma microtubules required for the correct delivery of dystrophin and β-dystroglycan at Costameres. On this basis, we investigated whether obscurin might be involved in the sub-sarcolemma localization of ankB and ankG, and eventually in the assembly of dystrophin and β-dystroglycan at Costameres in skeletal muscle fibers. We found that in mice deficient for obscurin, ankB was displaced from its localization at the M-band, while localization of ankG at Z-disk was not affected. In obscurin KO mice, the sub-sarcolemma microtubule cytoskeleton was disrupted and localization at Costameres of dystrophin, but not of β-dystroglycan, was markedly reduced. In addition, these mutant mice displayed sarcolemmal fragility and lower forelimbs muscle strength. Altogether, these results support a model where obscurin, by targeting ankB at the M-band, contributes to the organization of sub-sarcolemma microtubules, localization of dystrophin at Costameres and to maintenance of sarcolemmal integrity. Accordingly, obscurin appears to represent a multifunctional anchoring protein that on one hand establishes interactions with sarcomeric proteins and on the other hand enables complex formation with extra-sarcomeric proteins, like the muscle-specific ankyrin isoforms, that help to connect the sarcomeres with the SR and with the sub-sarcolemmal cytoskeleton.
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Obscurin is required for ankyrinB-dependent dystrophin localization and sarcolemma integrity
Journal of Cell Biology, 2013Co-Authors: Davide Randazzo, S Lorenzini, Enrico Pierantozzi, Bert Blaauw, Angela K Peter, Daniela Rossi, Emiliana Giacomello, Carlo Reggiani, Stephan Lange, Ju ChenAbstract:Obscurin is a large myofibrillar protein that contains several interacting modules, one of which mediates binding to muscle-specific ankyrins. Interaction between obscurin and the muscle-specific ankyrin sAnk1.5 regulates the organization of the sarcoplasmic reticulum in striated muscles. Additional muscle-specific ankyrin isoforms, ankB and ankG, are localized at the subsarcolemma level, at which they contribute to the organization of dystrophin and β-dystroglycan at Costameres. In this paper, we report that in mice deficient for obscurin, ankB was displaced from its localization at the M band, whereas localization of ankG at the Z disk was not affected. In obscurin KO mice, localization at Costameres of dystrophin, but not of β-dystroglycan, was altered, and the subsarcolemma microtubule cytoskeleton was disrupted. In addition, these mutant mice displayed marked sarcolemmal fragility and reduced muscle exercise tolerance. Altogether, the results support a model in which obscurin, by targeting ankB at the M band, contributes to the organization of subsarcolemma microtubules, localization of dystrophin at Costameres, and maintenance of sarcolemmal integrity.
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Obscurin is required for ankyrinB-dependent dystrophin localization and sarcolemma integrity
'Rockefeller University Press', 2013Co-Authors: Randazzo Davide, Lange Stephan, Giacomello Emiliana, Lorenzini Stefania, Rossi Daniela, Pierantozzi Enrico, Blaauw Bert, Reggiani Carlo, Peter, Angela K., Ju ChenAbstract:Obscurin is a large myofibrillar protein that contains several interacting modules, one of which mediates binding to muscle-specific ankyrins. Interaction between obscurin and the muscle-specific ankyrin sAnk1.5 regulates the organization of the sarcoplasmic reticulum in striated muscles. Additional muscle specific ankyrin isoforms, ankB and ankG, are localized at the subsarcolemma level, at which they contribute to the organization of dystrophin and \ue062-dystroglycan at Costameres. In this paper, we report that in mice deficient for obscurin, ankB was displaced from its localization at the M band, whereas localization of ankG at the Z disk was not affected. In obscurin KO mice, localization at Costameres of dystrophin, but not of \ue062-dystroglycan, was altered, and the subsarcolemma microtubule cytoskeleton was disrupted. In addition, these mutant mice displayed marked sarcolemmal fragility and reduced muscle exercise tolerance. Altogether, the results support a model in which obscurin, by targeting ankB at the M band, contributes to the organization of subsarcolemma microtubules, localization of dystrophin at Costameres, and maintenance of sarcolemmal integrity
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the Costamere bridges sarcomeres to the sarcolemma in striated muscle
Progress in Pediatric Cardiology, 2011Co-Authors: Angela K Peter, Hongqiang Cheng, Robert S Ross, Kirk U Knowlton, Ju ChenAbstract:Costameres are sub-membranous, Z-line associated structures found in striated muscle. They have been shown to have important roles in transmission of force from the sarcomere to the sarcolemma and extracellular matrix, maintaining mechanical integrity of the sarcolemma, and orchestrating mechanically related signaling. The Costamere is akin to the more well-known focal adhesion complex present in most cells. The Z-line is a critical structural anchor for the sarcomere, but it is also a hot-spot for muscle cell signaling. Therefore functionally, the Costamere represents a two-way signaling highway tethered between the Z-line and the extracellular matrix, relaying mechanical stress signals from outside the cell to intracellular signaling networks. In this role it can modulate myofibril growth and contraction. The major force generated by sarcomeres is transduced in the lateral direction from the sarcomere to the extracellular matrix through the Costamere. Two major protein complexes have been described at the Costamere: the dystrophin–glycoprotein complex and the integrin–vinculin–talin complex. The importance of these two protein complexes in striated muscle function has between demonstrated both in human disease and mouse models. Members of the dystrophin glycoprotein complex and integrins have both been reported to interact directly with filamin-C, thus linking costameric complexes with those present at the Z-line. Moreover, studies from our labs and others have shown that the Z-line proteins belonging to the PDZ-LIM domain protein family, enigma homolog (ENH) and cypher, may directly or indirectly be involved in this linkage. The following review will focus on the protein components of this linkage, their function in force transmission, and how the dysfunction or loss of proteins within these complexes contributes to muscular disease.
Martin Flück - One of the best experts on this subject based on the ideXlab platform.
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time course of Costamere related alterations in focal adhesion signaling and composition of rat soleus muscle after achilles tenotomy
Data in Brief, 2019Co-Authors: Celine Ferrie, Stephanie Kasper, Florian Wanivenhaus, Martin FlückAbstract:Abstract Sarcolemma-based focal adhesions (Costameres) are a central hub for the cytoskeletal anchoring of myofibrils and mechano-regulated signaling. Here we report the time course of alterations in focal adhesion-associated signaling and fiber composition in rat soleus muscle after Achilles tenotomy. The report includes data from tenotomized muscles and contralateral mock controls to expose whether muscle degeneration after tenotomy is due to the transection of the Achilles tendon, or circumjacent surgical manipulations of the tendon. With respect to the interpretation of the data regarding mechanistic implications of Costamere-associated processes for surgical repair of the detached muscle-tendon complex the reader is referred to the accompanying research article ‘Focal adhesion kinase coordinates Costamere-related JNK signaling with muscle fiber transformation after Achilles tenotomy and tendon reconstruction’ Ferrie et al., 2019.
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quantitative changes in focal adhesion kinase and its inhibitor frnk drive load dependent expression of Costamere components
American Journal of Physiology-regulatory Integrative and Comparative Physiology, 2013Co-Authors: Stephan Klossner, Martin Flück, Severin Ruoss, Anne Cecile DurieuxAbstract:Costameres are mechanosensory sites of focal adhesion in the sarcolemma that reinforce the muscle-fiber composite and provide an anchor for myofibrillogenesis. We hypothesized that elevated content...
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Fibre-type specific concentration of focal adhesion kinase at the sarcolemma: influence of fibre innervation and regeneration
J Exp Biol, 2002Co-Authors: Martin Flück, A Ziemiecki, Rudi Billeter, Michael MüntenerAbstract:In skeletal muscles, focal adhesion complexes (FACs) form part of the Costamere, a sarcolemmal protein complex that enables lateral transfer of forces and ensures the stability of the sarcolemma. The present investigation tested whether localisation of a major assembly factor of FACs, focal adhesion kinase (FAK), to the sarcolemma parallels the known modulation of FACs by fibre type (innervation pattern) and fibre regeneration. Immunohistochemical experiments indicated that FAK is preferentially associated with the sarcolemma in a high proportion (>74 %) of the (slow-twitch) type I and (fast-twitch) type IIA fibres in normal rat soleus (N-SOL) muscle and of the type IIA fibres in extensor digitorum longus (N-EDL) muscle. In contrast, a low proportion (
Nicholas J Severs - One of the best experts on this subject based on the ideXlab platform.
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distinct patterns of dystrophin organization in myocyte sarcolemma and transverse tubules of normal and diseased human myocardium
Circulation, 2000Co-Authors: Raffi Kaprielian, Shirley Stevenson, Stephen Rothery, Michael J Cullen, Nicholas J SeversAbstract:Background—Genetic mutations of dystrophin and associated glycoproteins underlie cell degeneration in several inherited cardiomyopathies, although the precise physiological role of these proteins remains under discussion. We studied the distribution of dystrophin in relation to the force-transducing vinculin-rich Costameres in left ventricular cardiomyocytes from normal and failing human hearts to further elucidate the function of this protein complex. Methods and Results—Single- and double-label immunoconfocal microscopy and parallel high-resolution immunogold fracture-label electron microscopy were used to localize dystrophin and vinculin in human left ventricular myocytes from normal (n=6) and failing hearts (idiopathic dilated cardiomyopathy, n=7, or ischemic heart disease, n=5). In control cardiomyocytes, dystrophin had a continuous distribution at the peripheral sarcolemma, with concentrated bands corresponding to the vinculin-rich Costameres. Intracellular labeling extended along transverse (T) tub...
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dystrophin is not a specific component of the cardiac Costamere
Circulation Research, 1997Co-Authors: Shirley Stevenson, Stephen Rothery, Michael J Cullen, Nicholas J SeversAbstract:Dystrophin is a key component of the subsarcolemmal skeleton of muscle cells, and lack of dystrophin is the direct cause of Duchenne muscular dystrophy. In skeletal muscle, dystrophin is reported to be localized specifically at Costameres, transversely oriented riblike subsarcolemmal plaques that mechanically couple the contractile apparatus to the extracellular matrix. Costameres are characteristically rich in vinculin and are prominent in cardiac as well as skeletal muscle. To define the precise spatial relationship between dystrophin in relation to the Costamere in cardiac muscle, we applied high-resolution single- and double-immunolabeling techniques, under a range of preparative conditions, with visualization of vinculin (as a Costamere marker) and dystrophin by confocal microscopy and by the freeze-fracture cytochemical technique, fracture label. Immunoconfocal visualization revealed dystrophin as a continuous uniform layer at the cytoplasmic surface of the peripheral plasma membrane of the rat cardiac myocyte at both costameric and noncostameric regions. The pattern of labeling was reproducible with three different antibodies and was independent of time and antibody concentration. Platinum/carbon replicas and thin sections of fracture-label specimens permitted high-resolution visualization of the distribution of dystrophin in plan views of the freeze-fractured plasma membrane and in relation to the sarcomeric banding patterns of the underlying myofibrils. These results confirmed no preferential association of dystrophin with Costameres or with any region of the sarcomeres of underlying myofibrils in rat cardiac tissue. We conclude that in contrast to skeletal muscle, dystrophin in cardiac muscle is not exclusively a component of the Costamere.
