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Olli Carpén - One of the best experts on this subject based on the ideXlab platform.
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Upsala Journal of Medical Sciences 2009; 114: 235–241 ORIGINAL ARTICLE Maintenance of muscle mass, fiber size, and contractile function in mice lacking the Z-disc protein Myotilin
2013Co-Authors: Julien Ochala, Olli Carpén, Lars LarssonAbstract:Background. Myofibrillar myopathies constitute a rare group of congenital neuromuscular disorders, frequently associated with mutations in Z-disc proteins such as Myotilin. Myotilin location and interactions with other Z-disc proteins are clearly defined, but its role in the regulation of muscle structure and function remains unknown. The present study aims at investigating this specific role of Myotilin. Methods. Skeletal and cardiac muscles were collected from adult mice with a targeted deletion of Myotilin (myo-/-) and wildtype animals (myo +/+). Results and conclusion. Similar skeletal and cardiac muscle weights were observed in myo-/- and myo +/+ mice. At the muscle cell level, the size and force production of single membrane permeabilized fibers were identical between myo-/- and myo +/+ rodents. Thus, Myotilin does not have a significant influence on muscle mass, muscle fiber size, or regulation of muscle contraction. Alternatively, compensatory over-expressions of other elements including proteins from the same subfamily, or Z-disc proteins such as telethonin, or intermediate filaments may compensate for the lack of Myotilin. Key words: Muscle, Myotilin, single membrane permeabilized muscle fiber, telethoni
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Analysis of Myotilin turnover provides mechanistic insight into the role of Myotilinopathy-causing mutations.
The Biochemical journal, 2011Co-Authors: Pernilla Von Nandelstadh, Rabah Soliymani, Marc Baumann, Olli CarpénAbstract:MFM (myofibrillar myopathies) are caused by mutations in several sarcomeric components, including the Z-disc protein Myotilin. The morphological changes typical of MFM include Z-disc alterations and aggregation of dense filamentous sarcomeric material. The causes and mechanisms of protein aggregation in Myotilinopathies and other forms of MFM remain unknown, although impaired degradation may explain, in part, the abnormal protein accumulation. In the present paper we have studied the mechanisms regulating Myotilin turnover, analysed the consequences of defective Myotilin degradation and tested whether disease-causing Myotilin mutations result in altered protein turnover. The results indicate that Myotilin is a substrate for the Ca 2+ -dependent protease calpain and identify two calpain cleavage sites in Myotilin by MS. We further show that Myotilin is degraded by the proteasome system in transfected COS7 cells and in myotubes, and that disease-causing Myotilinopathy mutations result in reduced degradation. Finally, we show that proteolysis-inhibitor-induced reduction in Myotilin turnover results in formation of intracellular Myotilin and actin-containing aggregates, which resemble those seen in diseased muscle cells. These findings identify for the first time biological differences between wt (wild-type) and mutant Myotilin. The present study provides novel information on the pathways controlling Myotilin turnover and on the molecular defects associated with MFM.
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Solution structure of the first immunoglobulin domain of human Myotilin.
Journal of Biomolecular NMR, 2009Co-Authors: Outi Heikkinen, Perttu Permi, Harri Koskela, Olli Carpén, Jari Ylänne, Ilkka KilpeläinenAbstract:Myotilin is a 57 kDa actin-binding and -bundling protein that consists of a unique serine-rich amino-terminus, two Ig-domains and a short carboxy-terminus with a PDZ-binding motif. Myotilin localizes in sarcomeric Z-discs, where it interacts with several sarcomeric proteins. Point mutations in Myotilin cause muscle disorders morphologically highlighted by sarcomeric disarray and aggregation. The actin-binding and dimerization propensity of Myotilin has been mapped to the Ig-domains. Here we present high-resolution structure of the first Ig-domain of Myotilin (MyoIg1) determined with solution state NMR spectroscopy. Nearly complete chemical shift assignments of MyoIg1 were achieved despite several missing backbone 1H-15N-HSQC signals. The structure derived from distance and dihedral angle restraints using torsion angle dynamics was further refined using molecular dynamics. The structure of MyoIg1 exhibits I-type Ig-fold. The absence of several backbone 1H-15N-HSQC signals can be explained by conformational exchange taking place at the hydrophobic core of the protein.
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Maintenance of muscle mass, fiber size, and contractile function in mice lacking the Z-disc protein Myotilin.
Upsala journal of medical sciences, 2009Co-Authors: Julien Ochala, Olli Carpén, Lars LarssonAbstract:BACKGROUND: Myofibrillar myopathies constitute a rare group of congenital neuromuscular disorders, frequently associated with mutations in Z-disc proteins such as Myotilin. Myotilin location and interactions with other Z-disc proteins are clearly defined, but its role in the regulation of muscle structure and function remains unknown. The present study aims at investigating this specific role of Myotilin. METHODS: Skeletal and cardiac muscles were collected from adult mice with a targeted deletion of Myotilin (myo(-/-)) and wild-type animals (myo(+/+)). RESULTS AND CONCLUSION: Similar skeletal and cardiac muscle weights were observed in myo(-/-) and myo(+/+) mice. At the muscle cell level, the size and force production of single membrane permeabilized fibers were identical between myo(-/-) and myo(+/+) rodents. Thus, Myotilin does not have a significant influence on muscle mass, muscle fiber size, or regulation of muscle contraction. Alternatively, compensatory over-expressions of other elements including proteins from the same subfamily, or Z-disc proteins such as telethonin, or intermediate filaments may compensate for the lack of Myotilin.
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A class III PDZ binding motif in the Myotilin and FATZ families binds enigma family proteins: a common link for Z-disc myopathies.
Molecular and cellular biology, 2008Co-Authors: Pernilla Von Nandelstadh, Olli Carpén, Heli Suila, Mohamed Ismail, Chiara Gardin, Ivano Zara, Anna Belgrano, Giorgio Valle, Georgine FaulknerAbstract:Interactions between Z-disc proteins regulate muscle functions and disruption of these interactions results in muscle disorders. Mutations in Z-disc components Myotilin, ZASP/Cypher, and FATZ-2 (calsarcin-1/myozenin-2) are associated with myopathies. We report here that the Myotilin and the FATZ (calsarcin/myozenin) families share high homology at their final C-terminal five amino acids. This C-terminal E[ST][DE][DE]L motif is present almost exclusively in these families and is evolutionary conserved. We show by in vitro and in vivo studies that proteins from the Myotilin and FATZ (calsarcin/myozenin) families interact via this novel type of class III PDZ binding motif with the PDZ domains of ZASP/Cypher and other Enigma family members: ALP, CLP-36, and RIL. We show that the interactions can be modulated by phosphorylation. Calmodulin-dependent kinase II phosphorylates the C terminus of FATZ-3 (calsarcin-3/myozenin-3) and Myotilin, whereas PKA phosphorylates that of FATZ-1 (calsarcin-2/myozenin-1) and FATZ-2 (calsarcin-1/myozenin-1). This is the first report of a binding motif common to both the Myotilin and the FATZ (calsarcin/myozenin) families that is specific for interactions with Enigma family members.
Michael A Hauser - One of the best experts on this subject based on the ideXlab platform.
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Myotilin is not the causative gene for vocal cord and pharyngeal weakness with distal myopathy vcpdm
Annals of Human Genetics, 2008Co-Authors: Sean M Garvey, Jan Senderek, Jacques S Beckmann, Eric Seboun, Charles E Jackson, Michael A HauserAbstract:Summary Myotilin (MYOT) is a promising candidate gene for Vocal Cord and Pharyngeal Weakness with Distal Myopathy (VCPDM, also known as MPD2). Located within the minimum VCPDM candidate interval, Myotilin mutations also cause a similarly progressive and adult-onset muscle disease. We examined Myotilin in VCPDM patients by sequence analysis, RT-PCR, Southern blotting, and western blotting. We detected no defects in the Myotilin gene, transcript, or protein in VCPDM. We also report several useful SNPs and STRs for the analysis of Myotilin in muscle diseases of suspected, yet unknown genetic origin. We conclude that MYOT mutations likely are not a cause of VCPDM.
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Myotilin overexpression enhances myopathology in the LGMD1A mouse model.
Muscle & nerve, 2008Co-Authors: Sean M Garvey, Yutao Liu, Sara E. Miller, Michael A HauserAbstract:Missense mutations in the Myotilin gene cause limb-girdle muscular dystrophy type 1A (LGMD1A). We set out to examine the effect of overexpression of wild-type Myotilin in an LGMD1A mouse model by crossing wild-type and mutant transgenic mice. Compared to single-transgenic mutant mice, double-transgenic mice overexpressing Myotilin showed more severe muscle degeneration, enhanced myofibrillar aggregation, and earlier onset of aggregation. These data suggest that strategies aimed at lowering total Myotilin levels in LGMD1A patients may be an effective therapeutic approach.
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Transgenic mice expressing the Myotilin T57I mutation unite the pathology associated with LGMD1A and MFM
Human molecular genetics, 2006Co-Authors: Sean M Garvey, Sara E. Miller, Dennis R. Claflin, John A. Faulkner, Michael A HauserAbstract:Myotilin is a muscle-specific Z-disc protein with putative roles in myofibril assembly and structural upkeep of the sarcomere. Several Myotilin point mutations have been described in patients with limb-girdle muscular dystrophy type 1A (LGMD1A), myofibrillar myopathy (MFM), spheroid body myopathy (SBM), three similar adult-onset, progressive and autosomal dominant muscular dystrophies. To further investigate Myotilin’s role in the pathogenesis of these muscle diseases, we have characterized three independent lines of transgenic mice expressing mutant (T57I) Myotilin under the control of the human skeletal actin promoter. Similar to LGMD1A and MFM patients, these mice develop progressive myofibrillar pathology that includes Z-disc streaming, excess myofibrillar vacuolization and plaque-like myofibrillar aggregation. These aggregates become progressively larger and more numerous with age. We show that the mutant Myotilin protein properly localizes to the Z-disc and also heavily populates the aggregates, along with several other Z-disc associated proteins. Whole muscle physiological analysis reveals that the extensor digitorum longus muscle of transgenic mice exhibits significantly reduced maximum specific isometric force compared with littermate controls. Intriguingly, the soleus and diaphragm muscles are spared of any abnormal myopathology and show no reductions in maximum specific force. These data provide evidence that Myotilin mutations promote aggregate-dependent contractile dysfunction. In sum, we have established a promising patho-physiological mouse model that unifies the phenotypes of LGMD1A, MFM and SBM.
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Transgenic mice expressing the Myotilin T57I mutation unite the pathology associated with LGMD1A
2006Co-Authors: Sean M Garvey, Sara E. Miller, Dennis R. Claflin, John A. Faulkner, Michael A HauserAbstract:Myotilin is a muscle-specific Z-disc protein with putative roles in myofibril assembly and structural upkeep of the sarcomere. Several Myotilin point mutations have been described in patients with limb-girdle muscular dystrophy type 1A (LGMD1A), myofibrillar myopathy (MFM), spheroid body myopathy (SBM), three similar adult-onset, progressive and autosomal dominant muscular dystrophies. To further investigate Myotilin’s role in the pathogenesis of these muscle diseases, we have characterized three independent lines of trans-genic mice expressing mutant (T57I) Myotilin under the control of the human skeletal actin promoter. Similar to LGMD1A and MFM patients, these mice develop progressive myofibrillar pathology that includes Z-disc streaming, excess myofibrillar vacuolization and plaque-like myofibrillar aggregation. These aggregates become progressively larger and more numerous with age. We show that the mutant Myotilin protein properly localizes to the Z-disc and also heavily populates the aggregates, along with several other Z-disc associated proteins. Whole muscle physiological analysis reveals that the extensor digitorum longus muscle of transgenic mice exhibits significantly reduced maximum specific isometric force compared with littermate controls. Intriguingly, the soleus and diaphragm muscles are spared of any abnormal myopathol-ogy and show no reductions in maximum specific force. These data provide evidence that Myotilin mutations promote aggregate-dependent contractile dysfunction. In sum, we have established a promising patho-physiological mouse model that unifies the phenotypes of LGMD1A, MFM and SBM
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Myotilin Mutation found in second pedigree with LGMD1A.
American journal of human genetics, 2002Co-Authors: Michael A Hauser, Cecilia Conde, Valeria Kowaljow, Guillermo Zeppa, Ana L. Taratuto, Udana Torian, Jeffery M. Vance, Margaret A. Pericak-vance, Marcy C. Speer, Alberto L. RosaAbstract:Limb-girdle muscular dystrophy 1A (LGMD1A [MIM 159000]) is an autosomal dominant form of muscular dystrophy characterized by adult onset of proximal weakness progressing to distal muscle weakness. We have reported elsewhere a mutation in the Myotilin gene in a large, North American family of German descent. Here, we report the mutation screening of an additional 86 families with a variety of neuromuscular pathologies. We have identified a new Myotilin mutation in an Argentinian pedigree with LGMD1 that is predicted to result in the conversion of serine 55 to phenylalanine (S55F). This mutation has not been found in 392 control chromosomes and is located in the unique N-terminal domain of Myotilin, only two residues from the T57I mutation reported elsewhere. Both T57I and S55F are located outside the α-actinin and γ-filamin binding sites within Myotilin. The identification of two independent pedigrees with the same disease, each bearing a different mutation in the same gene, has long been the gold standard for establishing a causal relationship between defects in a gene and the resultant disease. As a description of the second known pedigree with LGMD1A, this finding constitutes that gold standard of proof that mutations in the Myotilin gene cause LGMD1A.
Monica Moza - One of the best experts on this subject based on the ideXlab platform.
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Targeted deletion of the muscular dystrophy gene Myotilin does not perturb muscle structure or function in mice.
Molecular and cellular biology, 2006Co-Authors: Monica Moza, Georgine Faulkner, Luca Mologni, Ras Trokovic, Juha Partanen, Olli CarpénAbstract:Myotilin, palladin, and myopalladin form a novel small subfamily of cytoskeletal proteins that contain immunoglobulin-like domains. Myotilin is a thin filament-associated protein localized at the Z-disk of skeletal and cardiac muscle cells. The direct binding to F-actin, efficient cross-linking of actin filaments, and prevention of induced disassembly of filaments are key roles of Myotilin that are thought to be involved in structural maintenance and function of the sarcomere. Missense mutations in the Myotilin-encoding gene cause dominant limb girdle muscular dystrophy type 1A and spheroid body myopathy and are the molecular defect that can cause myofibrillar myopathy. Here we describe the generation and analysis of mice that lack Myotilin, myo / mice. Surprisingly, myo / mice maintain normal muscle sarcomeric and sarcolemmal integrity. Also, loss of Myotilin does not cause alterations in the heart or other organs of newborn or adult myo / mice. The mice develop normally and have a normal life span, and their muscle capacity does not significantly differ from wild-type mice even after prolonged physical stress. The results suggest that either Myotilin does not participate in muscle development and basal function maintenance or other proteins serve as structural and functional compensatory molecules when Myotilin is absent.
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Myotilin: a prominent marker of myofibrillar remodelling.
Neuromuscular disorders : NMD, 2006Co-Authors: Lena Carlsson, Olli Carpén, Monica Moza, Lars-eric ThornellAbstract:Myofibrillar remodelling with insertion of sarcomeres is a typical feature of biopsies taken from persons suffering of exercise-induced delayed onset muscle soreness. Here we studied the presence of the sarcomeric protein Myotilin in eccentric exercise related lesions. Myotilin is a component of sarcomeric Z-discs and it binds several other Z-disc proteins, i.e. alpha-actinin, filamin C, F-actin and FATZ. Myotilin has previously been shown to be present in nemaline rods and central cores and to be mutated in limb girdle muscular dystrophy 1A (LGMD1A) and in a subset of myofibrillar myopathies, indicating an important role in Z-disc maintenance. Our findings on non-diseased muscle affected by eccentric exercise give new information on how Myotilin is associated to myofibrillar components upon remodelling. We show that Myotilin was present in increased amount in lesions related to Z-disc streaming and events leading to insertion of new sarcomeres in pre-existing myofibrils and can therefore be used as a marker for myofibrillar remodelling. Interestingly, Myotilin is preferentially associated with F-actin rather than with the core Z-disc protein alpha-actinin during these events. This suggests that Myotilin has a key role in the dynamic molecular events mediating myofibrillar assembly in normal and diseased skeletal muscle.
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Actin-organising properties of the muscular dystrophy protein Myotilin.
Experimental cell research, 2005Co-Authors: Pernilla Von Nandelstadh, Monica Moza, Mikaela Grönholm, Arja Lamberg, Harri Savilahti, Olli CarpénAbstract:Myotilin is a sarcomeric Z-disc protein that binds F-actin directly and bundles actin filaments, although it does not contain a conventional actin-binding domain. Expression of mutant Myotilin leads to sarcomeric alterations in the dominantly inherited limb-girdle muscular dystrophy 1A and in myofibrillar myopathy/desmin-related myopathy. Together, with previous in vitro studies, this indicates that Myotilin has an important function in the assembly and maintenance of Z-discs. This study characterises further the interaction between Myotilin and actin. Functionally important regions in Myotilin were identified by actin pull-down and yeast two-hybrid assays and with a novel strategy that combines in vitro DNA transposition-based peptide insertion mutagenesis with phenotype analysis in yeast cells. The shortest fragment to bind actin was the second Ig domain together with a short C-terminal sequence. Concerted action of the first and second Ig domain was, however, necessary for the functional activity of Myotilin, as verified by analysis of transposon mutants, actin binding and phenotypic effect in mammalian cells. Furthermore, the Ig domains flanked with N- and C-terminal regions were needed for actin-bundling, indicating that the mere actin-binding sequence was insufficient for the actin-regulating activity. None of the four known disease-associated mutations altered the actin-organising ability. These results, together with previous studies in titin and kettin, identify the Ig domain as an actin-binding unit.
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Characterization of mouse Myotilin and its promoter.
Biochemical and biophysical research communications, 2005Co-Authors: Luca Mologni, Maciej Lalowski, Monica Moza, Olli CarpénAbstract:Abstract Myotilin is a sarcomeric protein mutated in two forms of muscle disease, limb-girdle muscular dystrophy type 1A and myofibrillar myopathy. Myotilin is expressed late during human myofibrillogenesis and localizes to Z-discs in mature sarcomere. It interacts with α-actinin, actin, and filamin C, and has strong F-actin-bundling activity. These features suggest an important role for Myotilin in sarcomere organization. In our effort towards the construction of a genetic model for Myotilin-related muscle disorders, we have cloned mouse Myotilin, including its promoter region, and studied the expression in various tissues. Mouse Myotilin is 90% identical with the human orthologue. Northern blot analysis revealed strong mRNA transcripts in skeletal and cardiac muscle, and weak expression in liver and lung tissue. Western blot and RT-PCR analysis showed the presence of one major product in mouse tissues. Analysis of the 5′-flanking region revealed a number of putative regulatory elements that drive expression in differentiating myoblasts. Finally, endogenous Myotilin is induced at later stages of Z-disc assembly in C 2 C 12 cells indicating conservation between mouse and human promoter region.
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The palladin/Myotilin/myopalladin family of actin-associated scaffolds.
International review of cytology, 2005Co-Authors: Carol A. Otey, Monica Moza, Andrew Rachlin, Daniel K. Arneman, Olli CarpénAbstract:The dynamic remodeling of the actin cytoskeleton plays a critical role in cellular morphogenesis and cell motility. Actin‐associated scaffolds are key to this process, as they recruit cohorts of actin‐binding proteins and associated signaling complexes to subcellular sites where remodeling is required. This review is focused on a recently discovered family of three proteins, Myotilin, palladin, and myopalladin, all of which function as scaffolds that regulate actin organization. While Myotilin and myopalladin are most abundant in skeletal and cardiac muscle, palladin is ubiquitously expressed in the organs of developing vertebrates. Palladin's function has been investigated primarily in the central nervous system and in tissue culture, where it appears to play a key role in cellular morphogenesis. The three family members each interact with specific molecular partners: all three bind to α‐actinin; in addition, palladin also binds to vasodilator‐stimulated phosphoprotein (VASP) and ezrin, Myotilin binds to filamin and actin, and myopalladin also binds to nebulin and cardiac ankyrin repeat protein (CARP). Since mutations in Myotilin result in two forms of muscle disease, an essential role for this family member in organizing the skeletal muscle sarcomere is implied.
Sean M Garvey - One of the best experts on this subject based on the ideXlab platform.
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Myotilin is not the causative gene for vocal cord and pharyngeal weakness with distal myopathy vcpdm
Annals of Human Genetics, 2008Co-Authors: Sean M Garvey, Jan Senderek, Jacques S Beckmann, Eric Seboun, Charles E Jackson, Michael A HauserAbstract:Summary Myotilin (MYOT) is a promising candidate gene for Vocal Cord and Pharyngeal Weakness with Distal Myopathy (VCPDM, also known as MPD2). Located within the minimum VCPDM candidate interval, Myotilin mutations also cause a similarly progressive and adult-onset muscle disease. We examined Myotilin in VCPDM patients by sequence analysis, RT-PCR, Southern blotting, and western blotting. We detected no defects in the Myotilin gene, transcript, or protein in VCPDM. We also report several useful SNPs and STRs for the analysis of Myotilin in muscle diseases of suspected, yet unknown genetic origin. We conclude that MYOT mutations likely are not a cause of VCPDM.
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Myotilin overexpression enhances myopathology in the LGMD1A mouse model.
Muscle & nerve, 2008Co-Authors: Sean M Garvey, Yutao Liu, Sara E. Miller, Michael A HauserAbstract:Missense mutations in the Myotilin gene cause limb-girdle muscular dystrophy type 1A (LGMD1A). We set out to examine the effect of overexpression of wild-type Myotilin in an LGMD1A mouse model by crossing wild-type and mutant transgenic mice. Compared to single-transgenic mutant mice, double-transgenic mice overexpressing Myotilin showed more severe muscle degeneration, enhanced myofibrillar aggregation, and earlier onset of aggregation. These data suggest that strategies aimed at lowering total Myotilin levels in LGMD1A patients may be an effective therapeutic approach.
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Transgenic mice expressing the Myotilin T57I mutation unite the pathology associated with LGMD1A and MFM
Human molecular genetics, 2006Co-Authors: Sean M Garvey, Sara E. Miller, Dennis R. Claflin, John A. Faulkner, Michael A HauserAbstract:Myotilin is a muscle-specific Z-disc protein with putative roles in myofibril assembly and structural upkeep of the sarcomere. Several Myotilin point mutations have been described in patients with limb-girdle muscular dystrophy type 1A (LGMD1A), myofibrillar myopathy (MFM), spheroid body myopathy (SBM), three similar adult-onset, progressive and autosomal dominant muscular dystrophies. To further investigate Myotilin’s role in the pathogenesis of these muscle diseases, we have characterized three independent lines of transgenic mice expressing mutant (T57I) Myotilin under the control of the human skeletal actin promoter. Similar to LGMD1A and MFM patients, these mice develop progressive myofibrillar pathology that includes Z-disc streaming, excess myofibrillar vacuolization and plaque-like myofibrillar aggregation. These aggregates become progressively larger and more numerous with age. We show that the mutant Myotilin protein properly localizes to the Z-disc and also heavily populates the aggregates, along with several other Z-disc associated proteins. Whole muscle physiological analysis reveals that the extensor digitorum longus muscle of transgenic mice exhibits significantly reduced maximum specific isometric force compared with littermate controls. Intriguingly, the soleus and diaphragm muscles are spared of any abnormal myopathology and show no reductions in maximum specific force. These data provide evidence that Myotilin mutations promote aggregate-dependent contractile dysfunction. In sum, we have established a promising patho-physiological mouse model that unifies the phenotypes of LGMD1A, MFM and SBM.
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Transgenic mice expressing the Myotilin T57I mutation unite the pathology associated with LGMD1A
2006Co-Authors: Sean M Garvey, Sara E. Miller, Dennis R. Claflin, John A. Faulkner, Michael A HauserAbstract:Myotilin is a muscle-specific Z-disc protein with putative roles in myofibril assembly and structural upkeep of the sarcomere. Several Myotilin point mutations have been described in patients with limb-girdle muscular dystrophy type 1A (LGMD1A), myofibrillar myopathy (MFM), spheroid body myopathy (SBM), three similar adult-onset, progressive and autosomal dominant muscular dystrophies. To further investigate Myotilin’s role in the pathogenesis of these muscle diseases, we have characterized three independent lines of trans-genic mice expressing mutant (T57I) Myotilin under the control of the human skeletal actin promoter. Similar to LGMD1A and MFM patients, these mice develop progressive myofibrillar pathology that includes Z-disc streaming, excess myofibrillar vacuolization and plaque-like myofibrillar aggregation. These aggregates become progressively larger and more numerous with age. We show that the mutant Myotilin protein properly localizes to the Z-disc and also heavily populates the aggregates, along with several other Z-disc associated proteins. Whole muscle physiological analysis reveals that the extensor digitorum longus muscle of transgenic mice exhibits significantly reduced maximum specific isometric force compared with littermate controls. Intriguingly, the soleus and diaphragm muscles are spared of any abnormal myopathol-ogy and show no reductions in maximum specific force. These data provide evidence that Myotilin mutations promote aggregate-dependent contractile dysfunction. In sum, we have established a promising patho-physiological mouse model that unifies the phenotypes of LGMD1A, MFM and SBM
Ichizo Nishino - One of the best experts on this subject based on the ideXlab platform.
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In vivo characterization of mutant Myotilins.
The American journal of pathology, 2012Co-Authors: Etsuko Keduka, Yukiko K. Hayashi, S. Shalaby, Hiroaki Mitsuhashi, Satoru Noguchi, Ikuya Nonaka, Ichizo NishinoAbstract:Myofibrillar myopathy (MFM) is a group of disorders that are pathologically defined by the disorganization of the myofibrillar alignment associated with the intracellular accumulation of Z-disk–associated proteins. MFM is caused by mutations in genes encoding Z-disk–associated proteins, including Myotilin. Although a number of MFM mutations have been identified, it has been difficult to elucidate the precise roles of the mutant proteins. Here, we present a useful method for the characterization of mutant proteins associated with MFM. Expression of mutant Myotilins in mouse tibialis anterior muscle by in vivo electroporation recapitulated both the pathological changes and the biochemical characteristics observed in patients with Myotilinopathy. In mutant Myotilin-expressing muscle fibers, Myotilin aggregates and is costained with polyubiquitin, and Z-disk–associated proteins and myofibrillar disorganization were commonly seen. In addition, the expressed S60C mutant Myotilin protein displayed marked detergent insolubility in electroporated mouse muscle, similar to that observed in human MFM muscle with the same mutation. Thus, in vivo electroporation can be a useful method for evaluating the pathogenicity of mutations identified in MFM.
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Defective Myotilin homodimerization caused by a novel mutation in MYOT exon 9 in the first Japanese limb girdle muscular dystrophy 1A patient.
Journal of neuropathology and experimental neurology, 2009Co-Authors: S. Shalaby, Hiroaki Mitsuhashi, Satoru Noguchi, Ikuya Nonaka, Ichizo Nishino, Chie Matsuda, Narihiro Minami, Yukiko K. HayashiAbstract:Myotilin is a muscle-specific Z disk protein. Several missense mutations in the Myotilin gene (MYOT) have been identified in limb girdle muscular dystrophy (LGMD), myofibrillar myopathy, and distal myopathy patients. All previously reported pathogenic MYOT mutations have been identified only in Exon 2. We sequenced MYOT in 138 patients diagnosed as having LGMD, myofibrillar myopathy, or distal myopathy, and identified a novel MYOT mutation in Exon 9 encoding the second immunoglobulin-like domain in 1 patient with clinically typical LGMD. By light microscopy, there were scattered fibers with rimmed vacuoles and myofibrillary disorganization in the patient's muscle biopsy; accumulation of Z disk proteins was observed by immunohistochemistry. Immunoblot analysis demonstrated that the amount of Myotilin monomer was increased in the patient muscle, but that the Myotilin homodimeric band was decreased. Functional analysis of the Myotilin mutation using a yeast 2-hybrid system revealed defective homodimerization of the mutant Myotilin and decreased interaction between mutant Myotilin and alpha-actinin. The homodimerization defect was further demonstrated by immunoprecipitation. This is the first MYOT mutation outside of Exon 2 in an LGMD type 1A patient and the first MYOT mutation identified in the Japanese population. This mutation in the second immunoglobulin-like domain impairs Myotilin dimerization and alters the binding between Myotilin and alpha-actinin, which is known to be important for actin bundling.
