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This article explores the distribution and composition of the ferritin complex in the adult brain and its connection to neuroferritinopathy (NF), a type of neurodegeneration with brain iron accumulation (NBIA). NF is characterized by symptoms like movement disturbances and tremors. The study identifies a novel variant in the ferritin heavy chain gene (FTH1) that is associated with NF and provides insights into the role of ferritin in both healthy and diseased brains. The authors, a diverse group of researchers, present their findings in a respectful and culturally sensitive manner, making this report a valuable resource for library patrons researching this topic. [Extracted from the article]

Synaptopodin-2 (SYNPO2) is a protein associated with the Z-disc in striated muscle cells. It interacts with α-actinin and filamin C, playing a role in Z-disc maintenance under stress by chaperone-assisted selective autophagy (CASA). In smooth muscle cells, SYNPO2 is a component of dense bodies. Furthermore, it has been proposed to play a role in tumor cell proliferation and metastasis in many different kinds of cancers. Alternative transcription start sites and alternative splicing predict the expression of six putative SYNPO2 isoforms differing by extended amino- and/or carboxy-termini. Our analyses at mRNA and protein levels revealed differential expression of SYNPO2 isoforms in cardiac, skeletal and smooth muscle cells. We identified synemin, an intermediate filament protein, as a novel binding partner of the PDZ-domain in the amino-terminal extension of the isoforms mainly expressed in cardiac and smooth muscle cells, and demonstrated colocalization of SYNPO2 and synemin in both cell types. A carboxy-terminal extension, mainly expressed in smooth muscle cells, is sufficient for association with dense bodies and interacts with α-actinin. SYNPO2 therefore represents an additional and novel link between intermediate filaments and the Z-discs in cardiomyocytes and dense bodies in smooth muscle cells, respectively. In pathological skeletal muscle samples, we identified SYNPO2 in the central and intermediate zones of target fibers of patients with neurogenic muscular atrophy, and in nemaline bodies. Our findings help to understand distinct functions of individual SYNPO2 isoforms in different muscle tissues, but also in tumor pathology. [ABSTRACT FROM AUTHOR]

Desmin mutations cause familial and sporadic cardiomyopathies. In addition to perturbing the contractile apparatus, both desmin deficiency and mutated desmin negatively impact mitochondria. Impaired myocardial metabolism secondary to mitochondrial defects could conceivably exacerbate cardiac contractile dysfunction. We performed metabolic myocardial phenotyping in left ventricular cardiac muscle tissue in desmin knock-out mice. Our analyses revealed decreased mitochondrial number, ultrastructural mitochondrial defects, and impaired mitochondria-related metabolic pathways including fatty acid transport, activation, and catabolism. Glucose transporter 1 and hexokinase-1 expression and hexokinase activity were increased. While mitochondrial creatine kinase expression was reduced, fetal creatine kinase expression was increased. Proteomic analysis revealed reduced expression of proteins involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta-oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Thus, desmin deficiency elicits a secondary cardiac mitochondriopathy with severely impaired oxidative phosphorylation and fatty and amino acid metabolism. Increased glucose utilization and fetal creatine kinase upregulation likely portray attempts to maintain myocardial energy supply. It may be prudent to avoid medications worsening mitochondrial function and other metabolic stressors. Therapeutic interventions for mitochondriopathies might also improve the metabolic condition in desmin deficient hearts. [ABSTRACT FROM AUTHOR]

Abstract Background The formation of contractile myofibrils requires the stepwise onset of expression of muscle specific proteins. It is likely that elucidation of the expression patterns of muscle-specific sarcomeric proteins is important to understand muscle disorders originating from defects in contractile sarcomeric proteins. Methods We investigated the expression profile of a panel of sarcomeric components with a focus on proteins associated with a group of congenital disorders. The analyses were performed in cultured human skeletal muscle cells during myoblast proliferation and myotube development. Results Our culture technique resulted in the development of striated myotubes and the expression of adult isoforms of the sarcomeric proteins, such as fast TnI, fast TnT, adult fast and slow MyHC isoforms and predominantly skeletal muscle rather than cardiac actin. Many proteins involved in muscle diseases, such as beta tropomyosin, slow TnI, slow MyBPC and cardiac TnI were readily detected in the initial stages of muscle cell differentiation, suggesting the possibility of an early role for these proteins as constituent of the developing contractile apparatus during myofibrillogenesis. This suggests that in disease conditions the mechanisms of pathogenesis for each of the mutated sarcomeric proteins might be reflected by altered expression patterns, and disturbed assembly of cytoskeletal, myofibrillar structures and muscle development. Conclusions In conclusion, we here confirm that cell cultures of human skeletal muscle are an appropriate tool to study developmental stages of myofibrillogenesis. The expression of several disease-associated proteins indicates that they might be a useful model system for studying the pathogenesis of muscle diseases caused by defects in specific sarcomeric constituents.

Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs—the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and α-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin's structural role in Z-discs. Myotilin is a scaffold protein in the Z-disc, the boundary between adjacent sarcomeres, aiding structural integrity via multiple interactions, including F-actin and α-actinin-2. An integrative structural model of the complex between myotilin and F-actin reveals that myotilin displaces tropomyosin from F-actin, implying a novel role of myotilin in sarcomere biogenesis beyond a mere interaction hub. [ABSTRACT FROM AUTHOR]

Filamin C (FLNc) and Xin actin-binding repeat-containing proteins (XIRPs) are multi-adaptor proteins that are mainly expressed in cardiac and skeletal muscles and which play important roles in the assembly and repair of myofibrils and their attachment to the membrane. We identified the dystrophin-binding protein aciculin (also known as phosphoglucomutase-like protein 5, PGM5) as a new interaction partner of FLNc and Xin. All three proteins colocalized at intercalated discs of cardiac muscle and myotendinous junctions of skeletal muscle, whereas FLNc and aciculin also colocalized in mature Z-discs. Bimolecular fluorescence complementation experiments in developing cultured mammalian skeletal muscle cells demonstrated that Xin and aciculin also interact in FLNc-containing immature myofibrils and areas of myofibrillar remodeling and repair induced by electrical pulse stimulation (EPS). Fluorescence recovery after photobleaching (FRAP) experiments showed that aciculin is a highly dynamic and mobile protein. Aciculin knockdown in myotubes led to failure in myofibril assembly, alignment and membrane attachment, and a massive reduction in myofibril number. A highly similar phenotype was found upon depletion of aciculin in zebrafish embryos. Our results point to a thus far unappreciated, but essential, function of aciculin in myofibril formation, maintenance and remodeling. [ABSTRACT FROM AUTHOR]

The ASB2α protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hematopoietic differentiation and is proposed to exert its effects by regulating the turnover of specific proteins. Three ASB2α substrates have been described so far: the actin-binding protein filamins, the Mixed Lineage Leukemia protein, and the Janus kinases 2 and 3. To determine the degradation of which substrate drives ASB2α biological effects is crucial for the understanding of ASB2α functions in hematopoiesis. Here, we show that neither endogenous nor exogenously expressed ASB2α induces degradation of JAK proteins in hematopoietic cells. Furthermore, we performed molecular modeling to generate the first structural model of an E3 ubiquitin ligase complex of an ASB protein bound to one of its substrates. [ABSTRACT FROM AUTHOR]

Xin is a striated muscle-specific actin-binding protein whose mRNA expression has been observed in damaged skeletal muscle. Here we demonstrate increased Xin protein expression early postinjury (≤12 h) and localization primarily to the periphery of damaged myofibers. At 1 day postinjury, Xin is colocalized with MyoD, confirming expression in activated satellite cells (SCs). By 5 days postinjury, Xin is evident in newly regenerated myofibers, with a return to preinjury levels by 14 days of regeneration. To determine whether the increased Xin expression is functionally relevant, tibialis anterior muscles of wild-type mice were infected with Xin-short hairpin RNA (shRNA) adenovirus, whereas the contralateral tibialis anterior received control adenovirus (Control). Four days postinfection, muscles were harvested or injured with cardiotoxin and collected at 3, 5, or 14 days thereafter. When compared with Control, Xin-shRNA infection attenuated muscle regeneration as demonstrated by Myh3 expression and fiber areas. Given the colocalization of Xin and MyoD, we isolated single myofibers from infected muscles to investigate the effect of silencing Xin on SC function. Relative to Control, SC activation, but not proliferation, was significantly impaired in Xin-shRNA-infected muscles. To determine whether Xin affects the G0-G1 transition, cell cycle reentry was assessed on infected C2C12 myoblasts using a methylcellulose assay. No difference in reentry was noted between groups, suggesting that Xin contributes to SC activation by means other than affecting G0-G1 transition. Together these data demonstrate a critical role for Xin in SC activation and reduction in Xin expression results in attenuated skeletal muscle repair. [ABSTRACT FROM AUTHOR]

Aims: Xin is a striated muscle-specific F-actin binding protein that has been implicated in cardiomyopathies. In cardiomyocytes, Xin is localized at intercalated discs (IDs). Mice lacking only two of the three Xin isoforms (XinAB−/− mice) develop severe cardiac hypertrophy. To further investigate the function of Xin variants in the mammalian heart, we generated XinABC−/− mice deficient in all Xin isoforms. [ABSTRACT FROM PUBLISHER]

Xin is a muscle-specific actin binding protein of which its role and regulation within skeletal muscle is not well understood. Here we demonstrate that Xin mRNA is robustly upregulated (>6-fold) within 12 h of skeletal muscle injury and is localized to the muscle satellite cell population. RT-PCR confirmed the expression pattern of Xin during regeneration, as well as within primary muscle myoblast cultures, but not other known stem cell populations. Immunohistochemical staining of single myofibers demonstrate Xin expression colocalized with the satellite cell marker Syndecan-4 further supporting the mRNA expression of Xin in satellite cells. In situ hybridization of regenerating muscle 5-7 days postinjury illustrates Xin expression within newly regenerated myofibers. Promoter-reporter assays demonstrate that known myogenic transcription factors [myocyte enhancer factor-2 (MEF2), myogenic differentiation-1 (MyoD), and myogenic factor-5 (Myf-5)] transactivate Xin promoter constructs supporting the muscle-specific expression of Xin. To determine the role of Xin within muscle precursor cells, proliferation, migration, and differentiation analysis using Xin, short hairpin RNA (shRNA) were undertaken in C2C12 myoblasts. Reducing endogenous Xin expression resulted in a 26% increase (P < 0.05) in cell proliferation and a 20% increase (P < 0.05) in myoblast migratory capacity. Skeletal muscle myosin heavy chain protein levels were increased (P < 0.05) with Xin shRNA administration; however, this was not accompanied by changes in myoglobin protein (another marker of differentiation) nor overt morphological differences relative to differentiating control cells. Taken together, the present findings support the hypothesis that Xin is expressed within muscle satellite cells during skeletal muscle regeneration and is involved in the regulation of myoblast function. [ABSTRACT FROM AUTHOR]

Myofibrillar myopathy (MFM) is a human disease that is characterized by focal myofibrillar destruction and pathological cytoplasmic protein aggregations. In an extended German pedigree with a novel form of MFM characterized by clinical features of a limb-girdle myopathy and morphological features of MFM, we identified a co-segregating, heterozygous nonsense mutation (8130G→A; W2710X) in the filamin c gene (FLNC) on chromosome 7q32.1. The mutation is the first found in FLNC and is localized in the dimerization domain of filamin c. Functional studies showed that, in the truncated mutant protein, this domain has a disturbed secondary structure that leads to the inability to dimerize properly. As a consequence of this malfunction, the muscle fibers of our patients display massive cytoplasmic aggregates containing filamin c and several Z-disk-associated and sarcolemmal proteins. [ABSTRACT FROM AUTHOR]

Xin is a protein that is expressed during early developmental stages of cardiac and skeletal muscles. Immunolocalization studies indicated a peripheral localization in embryonic mouse heart, where Xin localizes with bb-catenin and N-cadherin. In adult tissues, Xin is found primarily in the intercalated discs of cardiomyocytes and the myotendinous junctions of skeletal muscle cells, both specialized attachment sites of the myofibrillar ends to the sarcolemma. A large part of the Xin protein consists of unique 16 amino acid repeats with unknown function. We have investigated the characteristics of the Xin repeats by transfection experiments and actin-binding assays and ascertained that, upon expression in cultured cells, these repeats bind to and stabilize the actin-based cytoskeleton. In vitro co-sedimentation assays with skeletal muscle actin indicated that they not only directly bind actin filaments, but also have the capability of arranging microfilaments into networks that sediment upon low-speed centrifugation. Very similar repeats were also found in ‘Xin-repeat protein 2’ (XIRP2), a novel protein that seems to be expressed mainly in striated muscles. Human XIRP2 contains 28 Xin repeats with properties identical to those of Xin. We conclude that the Xin repeats define a novel, repetitive actin-binding motif present in at least two different muscle proteins. These Xin-repeat proteins therefore constitute the first two members of a novel family of actin-binding proteins. [ABSTRACT FROM AUTHOR]

Liver cirrhosis but also portal vein obstruction cause portal hypertension (PHT) and angiogenesis. This study investigated the differences of angiogenesis in cirrhotic and non-cirrhotic PHT with special emphasis on the canonical (Shh/Gli) and non-canonical (Shh/RhoA) hedgehog pathway. Cirrhotic (bile duct ligation/BDL; CCl4 intoxication) and non-cirrhotic (partial portal vein ligation/PPVL) rats received either atorvastatin (15 mg/kg; 7d) or control chow before sacrifice. Invasive hemodynamic measurement and Matrigel implantation assessed angiogenesis in vivo. Angiogenesis in vitro was analysed using migration and tube formation assay. In liver and vessel samples from animals and humans, transcript expression was analyzed using RT-PCR and protein expression using Western blot. Atorvastatin decreased portal pressure, shunt flow and angiogenesis in cirrhosis, whereas atorvastatin increased these parameters in PPVL rats. Non-canonical Hh was upregulated in experimental and human liver cirrhosis and was blunted by atorvastatin. Moreover, atorvastatin blocked the non-canonical Hh-pathway RhoA dependently in activated hepatic steallate cells (HSCs). Interestingly, hepatic and extrahepatic Hh-pathway was enhanced in PPVL rats, which resulted in increased angiogenesis. In summary, statins caused contrary effects in cirrhotic and non-cirrhotic portal hypertension. Atorvastatin inhibited the non-canonical Hh-pathway and angiogenesis in cirrhosis. In portal vein obstruction, statins enhanced the canonical Hh-pathway and aggravated PHT and angiogenesis. [ABSTRACT FROM AUTHOR]

The z-disc is a structural component at the lateral borders of the sarcomere and is important for mechanical stability and contractility of both cardiac and skeletal muscles. Of note, the sarcomeric z-disc also represents a nodal point in cardiomyocyte function and signaling. Mutations of numerous z-disc proteins are associated with cardiomyopathies and muscle diseases. To identify additional z-disc proteins that might contribute to cardiac disease, we employed an in silico screen for cardiac-enriched cDNAs. This screen yielded a previously uncharacterized protein named cardiac-enriched FHL2-interacting protein (CEFIP), which exhibited a heart- and skeletal muscle-specific expression profile. Importantly, CEFIP was located at the z-disc and was up-regulated in several models of cardiomyopathy. We also found that CEFIP overexpression induced the fetal gene program and cardiomyocyte hypertrophy. Yeast two-hybrid screens revealed that CEFIP interacts with the calcineurin-binding protein four and a half LIM domains 2 (FHL2). Because FHL2 binds calcineurin, a phosphatase controlling hypertrophic signaling, we examined the effects of CEFIP on the calcineurin/nuclear factor of activated T-cell (NFAT) pathway. These experiments revealed that CEFIP overexpression further enhances calcineurin-dependent hypertrophic signal transduction, and its knockdown repressed hypertrophy and calcineurin/NFAT activity. In summary, we report on a previously uncharacterized protein CEFIP that modulates calcineurin/NFAT signaling in cardiomyocytes, a finding with possible implications for the pathogenesis of cardiomyopathy. [ABSTRACT FROM AUTHOR]

Synaptopodin-2 (SYNPO2) is a protein associated with the Z-disc in striated muscle cells. It interacts with α-actinin and filamin C, playing a role in Z-disc maintenance under stress by chaperone-assisted selective autophagy (CASA). In smooth muscle cells, SYNPO2 is a component of dense bodies. Furthermore, it has been proposed to play a role in tumor cell proliferation and metastasis in many different kinds of cancers. Alternative transcription start sites and alternative splicing predict the expression of six putative SYNPO2 isoforms differing by extended amino- and/or carboxy-termini. Our analyses at mRNA and protein levels revealed differential expression of SYNPO2 isoforms in cardiac, skeletal and smooth muscle cells. We identified synemin, an intermediate filament protein, as a novel binding partner of the PDZ-domain in the amino-terminal extension of the isoforms mainly expressed in cardiac and smooth muscle cells, and demonstrated colocalization of SYNPO2 and synemin in both cell types. A carboxy-terminal extension, mainly expressed in smooth muscle cells, is sufficient for association with dense bodies and interacts with α-actinin. SYNPO2 therefore represents an additional and novel link between intermediate filaments and the Z-discs in cardiomyocytes and dense bodies in smooth muscle cells, respectively. In pathological skeletal muscle samples, we identified SYNPO2 in the central and intermediate zones of target fibers of patients with neurogenic muscular atrophy, and in nemaline bodies. Our findings help to understand distinct functions of individual SYNPO2 isoforms in different muscle tissues, but also in tumor pathology.

Objective: To determine whether a new indel mutation in the dimerization domain of filamin C (FLNc) causes a hereditary myopathy with protein aggregation in muscle fibers, we clinically and molecularly studied a German family with autosomal dominant myofibrillar myopathy (MFM)., Methods: We performed mutational analysis in 3 generations, muscle histopathology, and proteomic studies of IM protein aggregates. Functional consequences of the FLNC mutation were investigated with interaction and transfection studies and biophysics molecular analysis., Results: Eight patients revealed clinical features of slowly progressive proximal weakness associated with a heterozygous c.8025&#95;8030delCAAGACinsA (p.K2676Pfs*3) mutation in FLNC . Two patients exhibited a mild cardiomyopathy. MRI of skeletal muscle revealed lipomatous changes typical for MFM with FLNC mutations. Muscle biopsies showed characteristic MFM findings with protein aggregation and lesion formation. The proteomic profile of aggregates was specific for MFM-filaminopathy and indicated activation of the ubiquitin-proteasome system (UPS) and autophagic pathways. Functional studies revealed that mutant FLNc is misfolded, unstable, and incapable of forming homodimers and heterodimers with wild-type FLNc., Conclusions: This new MFM-filaminopathy family confirms that expression of mutant FLNC leads to an adult-onset muscle phenotype with intracellular protein accumulation. Mutant FLNc protein is biochemically compromised and leads to dysregulation of protein quality control mechanisms. Proteomic analysis of MFM protein aggregates is a potent method to identify disease-relevant proteins, differentiate MFM subtypes, evaluate the relevance of gene variants, and identify novel MFM candidate genes., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Introduction: Most of the approximately 60 genes that if mutated cause steroid-resistant nephrotic syndrome (SRNS) are highly expressed in the glomerular podocyte, rendering SRNS a "podocytopathy.", Methods: We performed whole-exome sequencing (WES) in 1200 nephrotic syndrome (NS) patients., Results: We discovered homozygous truncating and homozygous missense mutation in SYNPO2 (synaptopodin-2) (p.Lys1124∗ and p.Ala1134Thr) in 2 patients with childhood-onset NS. We found SYNPO2 expression in both podocytes and mesangial cells; however, notably, immunofluorescence staining of adult human and rat kidney cryosections indicated that SYNPO2 is localized mainly in mesangial cells. Subcellular localization studies reveal that in these cells SYNPO2 partially co-localizes with α-actinin and filamin A-containing F-actin filaments. Upon transfection in mesangial cells or podocytes, EGFP-SYNPO2 co-localized with α-actinin-4, which gene is mutated in autosomal dominant SRNS in humans. SYNPO2 overexpression increases mesangial cell migration rate (MMR), whereas shRNA knockdown reduces MMR. Decreased MMR was rescued by transfection of wild-type mouse Synpo2 cDNA but only partially by cDNA representing mutations from the NS patients. The increased mesangial cell migration rate (MMR) by SYNPO2 overexpression was inhibited by ARP complex inhibitor CK666. SYNPO2 shRNA knockdown in podocytes decreased active Rac1, which was rescued by transfection of wild-type SYNPO2 cDNA but not by cDNA representing any of the 2 mutant variants., Conclusion: We show that SYNPO2 variants may lead to Rac1-ARP3 dysregulation, and may play a role in the pathogenesis of nephrotic syndrome., (© 2020 International Society of Nephrology. Published by Elsevier Inc.)

Mechanical force-induced conformational changes in proteins underpin a variety of physiological functions, typified in muscle contractile machinery. Mutations in the actin-binding protein filamin C (FLNC) are linked to musculoskeletal pathologies characterized by altered biomechanical properties and sometimes aggregates. HspB1, an abundant molecular chaperone, is prevalent in striated muscle where it is phosphorylated in response to cues including mechanical stress. We report the interaction and up-regulation of both proteins in three mouse models of biomechanical stress, with HspB1 being phosphorylated and FLNC being localized to load-bearing sites. We show how phosphorylation leads to increased exposure of the residues surrounding the HspB1 phosphosite, facilitating their binding to a compact multidomain region of FLNC proposed to have mechanosensing functions. Steered unfolding of FLNC reveals that its extension trajectory is modulated by the phosphorylated region of HspB1. This may represent a posttranslationally regulated chaperone-client protection mechanism targeting over-extension during mechanical stress.

The Z-disc is a protein-rich structure critically important for the development and integrity of myofibrils, which are the contractile organelles of cross-striated muscle cells. We here used mouse C2C12 myoblast, which were differentiated into myotubes, followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising mature Z-discs. Using a quantitative proteomics approach, we found significant changes in the relative abundance of 387 proteins in myoblasts versus differentiated myotubes, reflecting the drastic phenotypic conversion of these cells during myogenesis. Interestingly, EPS of differentiated myotubes to induce Z-disc assembly and maturation resulted in increased levels of proteins involved in ATP synthesis, presumably to fulfill the higher energy demand of contracting myotubes. Because an important role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape further warranted in-depth analysis. We therefore established, by global phosphoproteomics of EPS-treated contracting myotubes, a comprehensive site-resolved protein phosphorylation map of the Z-disc and found that it is a phosphorylation hotspot in skeletal myocytes, underscoring its functions in signaling and disease-related processes. In an illustrative fashion, we analyzed the actin-binding multiadaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKCα phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. Fluorescence recovery after photobleaching experiments indicated that this phosphorylation modulates FLNc dynamics. Moreover, FLNc lacking the cleaved Ig-like domain 24 exhibited remarkably fast kinetics and exceedingly high mobility. Our data set provides research community resource for further identification of kinase-mediated changes in myofibrillar protein interactions, kinetics, and mobility that will greatly advance our understanding of Z-disc dynamics and signaling., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

We describe a new early-onset neuromuscular disorder due to a homozygous loss-of-function variant in the kyphoscoliosis peptidase gene (KY). A 7.5-year-old girl with walking difficulties from 2 years of age presented with generalized muscle weakness; mild contractures in the shoulders, hips and feet; cavus feet; and lordosis but no scoliosis. She had previously been operated with Achilles tendon elongation. Whole-body MRI showed atrophy and fatty infiltration in the calf muscles. Biopsy of the vastus lateralis muscle showed variability in fiber size, with some internalized nuclei and numerous very small fibers with variable expression of developmental myosin heavy chain isoforms. Some small fibers showed abnormal sarcomeres with thickened Z-discs and small nemaline rods. Whole-exome sequencing revealed a homozygous one-base deletion (c.1071delG, p.(Thr358Leufs*3)) in KY, predicted to result in a truncated protein. Analysis of an RNA panel showed that KY is predominantly expressed in skeletal muscle in humans. A recessive variant in the murine ortholog Ky was previously described in a spontaneously generated mouse mutant with kyphoscoliosis, which developed postnatally and was caused by dystrophy of postural muscles. The abnormal distribution of Xin and Ky-binding partner filamin C in the muscle fibers of our patient was highly similar to their altered localization in ky/ky mouse muscle fibers. We describe the first human case of disease associated with KY inactivation. As in the mouse model, the affected child showed a neuromuscular disorder - but in contrast, no kyphoscoliosis.

Xin is a striated muscle-specific protein that is localized to the myotendinous junction in skeletal muscle. However, in injured mouse muscle, Xin expression is up-regulated and observed throughout skeletal muscle fibers and within satellite cells. In this study, Xin was analyzed by immunofluorescent staining in skeletal muscle samples from 47 subjects with various forms of myopathy, including muscular dystrophies, inflammatory myopathies, mitochondrial/metabolic myopathy, and endocrine myopathy. Results indicate that Xin immunoreactivity is positively and significantly correlated (rs = 0.6175, P = <0.0001) with the severity of muscle damage, regardless of myopathy type. Other muscle damage measures also showed a correlation with severity [Xin actin-binding repeat-containing 2 (rs = -0.7108, P = 0.0006) and collagen (rs = 0.4683, P = 0.0783)]. However, because only Xin lacked immunoreactivity within the healthy muscle belly, any detectable immunoreactivity for Xin was indicative of muscle damage. We also investigated the expression of Xin within the skeletal muscle of healthy individuals subjected to damaging eccentric exercise. Consistent with our previously mentioned results, Xin immunoreactivity was increased 24 hours after exercise in damaged muscle fibers and within the activated muscle satellite cells. Taken together, these data demonstrate Xin as a useful biomarker of muscle damage in healthy individuals and in patients with myopathy. The strong correlation between the degree of muscle damage and Xin immunoreactivity suggests that Xin may be a suitable outcome measure to evaluate disease progression and treatment effects in clinical trials., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The Xin actin-binding repeat-containing proteins Xin and XIRP2 are exclusively expressed in striated muscle cells, where they are believed to play an important role in development. In adult muscle, both proteins are concentrated at attachment sites of myofibrils to the membrane. In contrast, during development they are localized to immature myofibrils together with their binding partner, filamin C, indicating an involvement of both proteins in myofibril assembly. We identify the SH3 domains of nebulin and nebulette as novel ligands of proline-rich regions of Xin and XIRP2. Precise binding motifs are mapped and shown to bind both SH3 domains with micromolar affinity. Cocrystallization of the nebulette SH3 domain with the interacting XIRP2 peptide PPPTLPKPKLPKH reveals selective interactions that conform to class II SH3 domain-binding peptides. Bimolecular fluorescence complementation experiments in cultured muscle cells indicate a temporally restricted interaction of Xin-repeat proteins with nebulin/nebulette during early stages of myofibril development that is lost upon further maturation. In mature myofibrils, this interaction is limited to longitudinally oriented structures associated with myofibril development and remodeling. These data provide new insights into the role of Xin actin-binding repeat-containing proteins (together with their interaction partners) in myofibril assembly and after muscle damage.

Mechanical tension is an ever-present physiological stimulus essential for the development and homeostasis of locomotory, cardiovascular, respiratory, and urogenital systems. Tension sensing contributes to stem cell differentiation, immune cell recruitment, and tumorigenesis. Yet, how mechanical signals are transduced inside cells remains poorly understood. Here, we identify chaperone-assisted selective autophagy (CASA) as a tension-induced autophagy pathway essential for mechanotransduction in muscle and immune cells. The CASA complex, comprised of the molecular chaperones Hsc70 and HspB8 and the cochaperone BAG3, senses the mechanical unfolding of the actin-crosslinking protein filamin. Together with the chaperone-associated ubiquitin ligase CHIP, the complex initiates the ubiquitin-dependent autophagic sorting of damaged filamin to lysosomes for degradation. Autophagosome formation during CASA depends on an interaction of BAG3 with synaptopodin-2 (SYNPO2). This interaction is mediated by the BAG3 WW domain and facilitates cooperation with an autophagosome membrane fusion complex. BAG3 also utilizes its WW domain to engage in YAP/TAZ signaling. Via this pathway, BAG3 stimulates filamin transcription to maintain actin anchoring and crosslinking under mechanical tension. By integrating tension sensing, autophagosome formation, and transcription regulation during mechanotransduction, the CASA machinery ensures tissue homeostasis and regulates fundamental cellular processes such as adhesion, migration, and proliferation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Filaminopathy is a subtype of myofibrillar myopathy caused by mutations in FLNC, the gene encoding filamin C, and histologically characterized by pathologic accumulation of several proteins within skeletal muscle fibers. With the aim to get new insights in aggregate composition, we collected aggregates and control tissue from skeletal muscle biopsies of six myofibrillar myopathy patients harboring three different FLNC mutations by laser microdissection and analyzed the samples by a label-free mass spectrometry approach. A total of 390 proteins were identified, and 31 of those showed significantly higher spectral indices in aggregates compared with patient controls with a ratio >1.8. These proteins included filamin C, other known myofibrillar myopathy associated proteins, and a striking number of filamin C binding partners. Across the patients the patterns were extremely homogeneous. Xin actin-binding repeat containing protein 2, heat shock protein 27, nebulin-related-anchoring protein, and Rab35 could be verified as new filaminopathy biomarker candidates. In addition, further experiments identified heat shock protein 27 and Xin actin-binding repeat containing protein 2 as novel filamin C interaction partners and we could show that Xin actin-binding repeat containing protein 2 and the known interaction partner Xin actin-binding repeat containing protein 1 simultaneously associate with filamin C. Ten proteins showed significant lower spectral indices in aggregate samples compared with patient controls (ratio <0.56) including M-band proteins myomesin-1 and myomesin-2. Proteomic findings were consistent with previous and novel immunolocalization data. Our findings suggest that aggregates in filaminopathy have a largely organized structure of proteins also interacting under physiological conditions. Different filamin C mutations seem to lead to almost identical aggregate compositions. The finding that filamin C was detected as highly abundant protein in aggregates in filaminopathy indicates that our proteomic approach may be suitable to identify new candidate genes among the many MFM patients with so far unknown mutation.

Linkage analysis of the dominant distal myopathy we previously identified in a large Australian family demonstrated one significant linkage region located on chromosome 7 and encompassing 18.6 Mbp and 151 genes. The strongest candidate gene was FLNC because filamin C, the encoded protein, is muscle-specific and associated with myofibrillar myopathy. Sequencing of FLNC cDNA identified a c.752T>C (p.Met251Thr) mutation in the N-terminal actin-binding domain (ABD); this mutation segregated with the disease and was absent in 200 controls. We identified an Italian family with the same phenotype and found a c.577G>A (p.Ala193Thr) filamin C ABD mutation that segregated with the disease. Filamin C ABD mutations have not been described, although filamin A and filamin B ABD mutations cause multiple musculoskeletal disorders. The distal myopathy phenotype and muscle pathology in the two families differ from myofibrillar myopathies caused by filamin C rod and dimerization domain mutations because of the distinct involvement of hand muscles and lack of pathological protein aggregation. Thus, like the position of FLNA and B mutations, the position of the FLNC mutation determines disease phenotype. The two filamin C ABD mutations increase actin-binding affinity in a manner similar to filamin A and filamin B ABD mutations. Cell-culture expression of the c.752T>C (p.Met251)Thr mutant filamin C ABD demonstrated reduced nuclear localization as did mutant filamin A and filamin B ABDs. Expression of both filamin C ABD mutants as full-length proteins induced increased aggregation of filamin. We conclude filamin C ABD mutations cause a recognizable distal myopathy, most likely through increased actin affinity, similar to the pathological mechanism of filamin A and filamin B ABD mutations., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Myofibrillar myopathy (MFM) is a pathologically defined group of hereditary human muscle diseases, characterized by focal myofibrillar destruction and cytoplasmic aggregates that contain several Z-disc-related proteins. The previously reported MFM-associated mutation (8130G --> A; W2710X) in the filamin C gene (FLNC) leads to a partial disturbance of the secondary structure of the dimerization domain of filamin C, resulting in massive protein aggregation in skeletal muscle fibers of the patients. Here, we provide a thorough characterization of the biochemical, biophysical and cellular properties of the mutated filamin C polypeptide. Our experiments revealed that the mutant dimerization domain is less stable and more susceptible to proteolysis. As a consequence, it does not dimerize properly and forms aggregates in vitro. Furthermore, the expression of mutant filamin in cultured cells results in the formation of protein aggregates. The mutant filamin does not associate with wild type filamin. These findings are of great importance to explain the pathomechanism of this disease.

The KY protein has been implicated in a neuromuscular dystrophy in the mouse, but its role in muscle function remains unclear. Here, we show that KY interacts with several sarcomeric cytoskeletal proteins including, amongst others, filamin C and the slow isoform of the myosin-binding protein C. These interactions were confirmed in vitro and because of its central role in skeletal muscle disease, characterized in more detail for filamin C. A role for KY in regulating filamin C function in vivo is supported by the expression analysis of filamin C in the null ky mouse mutant, where distinct irregular subcellular localization of filamin C was found in subsets of muscle fibres, which appears to be a specific outcome of KY deficiency. Furthermore, KY shows protease activity in in vitro assays, and specific degradation of filamin C by KY is shown in transfected cells. Given the enzymatic nature of the KY protein, it is likely that some of the identified partners are catalytic substrates. These results suggest that KY is an intrinsic part of the protein networks underlying the molecular mechanism of several limb-girdle muscular dystrophies, particularly those where interactions between filamin C and disease causing proteins have been shown.

The M lines are structural landmarks in striated muscles, necessary for sarcomeric stability and as anchoring sites for the M isoform of creatine kinase (CK-M). These structures, especially prominent in fast skeletal muscles, are missing in rodent extraocular muscle, a particularly fast and active muscle group. In this study, we tested the hypotheses that 1). myomesin and M protein (cytoskeletal components of the M lines) and CK-M are downregulated in mouse extraocular muscle compared with the leg muscles, gastrocnemius and soleus; and 2). the expression of other cytosolic and mitochondrial CK isoforms is correspondingly increased. As expected, mouse extraocular muscles expressed lower levels of myomesin, M protein, and CK-M mRNA than the leg muscles. Immunocytochemically, myomesin and M protein were not detected in the banding pattern typically seen in other skeletal muscles. Surprisingly, message abundance for the other known CK isoforms was also lower in the extraocular muscles. Moreover, total CK activity was significantly decreased compared with that in the leg muscles. Based on these data, we reject our second hypothesis and propose that other energy-buffering systems may be more important in the extraocular muscles. The downregulation of major structural and metabolic elements and relative overexpression of two adenylate kinase isoforms suggest that the extraocular muscle group copes with its functional requirements by using strategies not seen in typical skeletal muscles.

Background: Muscle LIM protein (MLP) is an essential nuclear regulator of myogenic differentiation. Additionally, it may act as an integrator of protein assembly of the actin-based cytoskeleton. MLP-knockout mice develop a marked cardiac hypertrophy reaction and dilated cardiomyopathy (DCM). MLP is therefore a candidate gene for heritable forms of hypertrophic cardiomyopathy (HCM) and DCM in humans., Methods and Results: We analyzed 1100 unrelated individuals (400 patients with DCM, 200 patients with HCM, and 500 controls) for mutations in the human CRP3 gene that encodes MLP. We found 3 different missense mutations in 3 unrelated patients with familial HCM but detected no mutation in the DCM group or the controls. All mutations predicted an amino acid exchange at highly conserved residues in the functionally important LIM1 domain, which is responsible for interaction with alpha-actinin and with certain muscle-specific transcription factors. Protein-binding studies indicate that mutations in the CRP3 gene lead to a decreased binding activity of MLP to alpha-actinin. All 3 index patients were characterized by typical asymmetrical septal hypertrophy. Family studies revealed cosegregation of clinically affected individuals with the respective mutations in MLP., Conclusion: Here, we present evidence that mutations in the CRP3/MLP gene can cause HCM.

Assembly of muscle sarcomeres is a complex dynamic process and involves a large number of proteins. A growing number of these have regulatory functions and are transiently present in the myofibril. We show here that the novel tubulin-associated RING/B-box protein MURF2 associates transiently with microtubules, myosin and titin during sarcomere assembly. During sarcomere assembly, MURF2 first associates with microtubules at the exclusion of tyrosinated tubulin. Then, MURF2-labelled microtubules associate transiently with sarcomeric myosin and later with A-band titin when non-striated myofibrils differentiate into mature sarcomeres. Finally, MURF2 labelled microtubules disappear from the sarcomere after the incorporation of myosin filaments and the elongation of titin. This suggests that the incorporation of myosin into nascent sarcomeres and the elongation of titin require an active, microtubule-dependent transport process and that MURF2-associated microtubules play a role in the alignment and extension of nascent sarcomeres. MURF2 is expressed in at least four isoforms, of which a 27 kDa isoform is cardiac specific. A C-terminal isoform is generated by alternative reading frame use, a novelty in muscle proteins. In mature cardiac sarcomeres, endogenous MURF2 can associate with the M-band, and is translocated to the nucleus. MURF2 can therefore act as a transient adaptor between microtubules, titin and nascent myosin filaments, as well as being involved in signalling from the sarcomere to the nucleus.