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A transduced mechanical signal arriving at its destination in muscle alters sarcomeric structure and function. A major question addressed is how muscle mass and tension generation are optimized to match actual performance demands so that little energy is wasted. Three cases for improved energy efficiency are examined: the troponin complex for tuning force production, control of the myosin heads in a resting state, and the Z-disc proteins for sarcomere assembly. On arrival, the regulation of protein complexes is often controlled by post-translational modification (PTM), of which the most common are phosphorylation by kinases, deacetylation by histone deacetylases and ubiquitination by E3 ligases. Another branch of signals acts not through peptide covalent bonding but via ligand interactions (e.g. Ca(2+) and phosphoinositide binding). The myosin head and the regulation of its binding to actin by the troponin complex is the best and earliest example of signal destinations that modify myofibrillar contractility. PTMs in the troponin complex regulate both the efficiency of the contractile function to match physiologic demand for work, and muscle mass via protein degradation. The regulation of sarcomere assembly by integration of incoming signaling pathways causing the same PTMs or ligand binding are discussed in response to mechanical loading and unloading by the Z-disc proteins CapZ, α-actinin, telethonin, titin N-termini, and others. Many human mutations that lead to cardiomyopathy and heart disease occur in the proteins discussed above, which often occur at their PTM or ligand binding sites.

Blood-accessible molecular biomarkers are becoming highly attractive tools to assess disease progression and response to therapies in Duchenne muscular dystrophy (DMD) especially in very young patients for whom other outcome measures remain subjective and challenging. In this study, we have standardized a highly specific and reproducible multiplexing mass spectrometry method using the tandem mass tag (TMT) strategy in combination with depletion of abundant proteins from serum and high-pH reversed-phase peptide fractionation. Differential proteome profiling of 4 year-old DMD boys (n = 9) and age-matched healthy controls (n = 9) identified 38 elevated and 50 decreased serum proteins (adjusted P < 0.05, FDR

Background:Telethonin (TCAP) is a Z-disk protein that maintains cytoskeletal integrity and various signaling pathways in cardiomyocytes. TCAP is shown to modulate α-subunit of the human cardiac sodium channel (hNav 1.5) by direct interactions. Several TCAP variants are found in cardiomyopathies. We sought to investigate whether TCAP variants are associated with arrhythmia syndromes., Methods:Mutational analyses for TCAP were performed in 303 Japanese patients with Brugada syndrome, arrhythmogenic right ventricular cardiomyopathy, and J-wave pattern ECG. Using patch-clamp techniques, electrophysiological characteristics of hNav 1.5 were studied in HEK-293 cells stably expressing hNav 1.5 and transiently transfected with wild-type (WT) or variant TCAP., Results:We identified two TCAP variants, c.145G>A:p.E49K and c.458G>A:p.R153H, in four individuals. p.E49K was found in two patients with ARVC or BrS. p.R153H was found in two patients with BrS or J-wave pattern ECG. No patient had variant hNav 1.5. Patch-clamp experiments demonstrated that peak sodium currents were significantly reduced in cells expressing p.R153H and p.E49K compared with WT-TCAP (66%, p.R153H; 72%, p.E49K). Voltage dependency of peak IV curve was rightward-shifted by 5 mV in cells expressing p.E49K compared with WT-TCAP. Voltage dependency of activation was not leftward-shifted by p.R153H, while voltage dependency of steady-state inactivation was leftward-shifted by p.E49K., Conclusions:We found two TCAP variants in the patients with BrS, J-wave pattern ECG, and ARVC that can cause loss-of-function of the hNav 1.5 in heterologous expression systems. Our observation suggests that these variants might impair INa and be associated with the patients' electrophysiological phenotypes. Further studies linking our experimental data to clinical phenotypes are warranted.

Introduction and objectives: Hypertrophic cardiomyopathy (HCM) is a genetically and phenotypically heterogeneous disease; there is still a large proportion of patients with no identified disease-causing mutation. Although the majority of mutations are found in the MYH7 and MYBPC3 genes, mutations in Z-disk-associated proteins have also been linked to HCM. Methods: We assessed a small family with HCM based on family history, physical examination, 12-lead ECG, echocardiogram and magnetic resonance imaging. After exclusion of mutations in eleven HCM disease genes, we performed direct sequencing of the TCAP gene encoding the Z-disk protein titin-cap (also known as telethonin). Results: We present a novel TCAP mutation in a small family affected by HCM. The identified p.C57W mutation showed a very low population frequency, as well as high conservation across species. All of the bioinformatic prediction tools used considered this mutation to be damaging/deleterious. Family members were screened for this new mutation and a co-segregation pattern was detected. Both affected members of this family presented with late-onset HCM, moderate asymmetric left ventricular hypertrophy, atrial fibrillation and heart failure with preserved ejection fraction and low risk of sudden cardiac death. Conclusions: We present evidence supporting the classification of the TCAP p.C57W mutation, encoding the Z-disk protein titin-cap/telethonin as a new likely pathogenic variant of hypertrophic cardiomyopathy, with a specific phenotype in the family under analysis. Resumo: Introdução e objetivos: A miocardiopatia hipertrófica (HCM) é uma doença genética e fenotipicamente heterogénea, existindo ainda uma grande proporção de doentes sem uma mutação patogénica identificada. Embora a maioria das mutações seja encontrada nos genes MYH7 e MYBPC3, mutações em proteínas associadas ao disco Z também foram associadas à HCM. Métodos: Avaliámos uma pequena família com HCM com base na história familiar e exame objetivo, eletrocardiograma de 12 derivações, ecocardiograma e ressonância magnética. Após exclusão de mutações em 11 genes causadores de HCM, realizámos a sequenciação direta do gene TCAP que codifica a proteína titin-cap do disco-Z (também conhecida como teletonina). Resultados: Relatamos aqui uma nova mutação do gene TCAP numa pequena família com HCM. A mutação identificada, p.C57W, mostrou uma frequência populacional muito baixa, bem como alta conservação entre as espécies. Todas as ferramentas de previsão de bioinformática usadas previram que essa mutação seria funcionalmente prejudicial. A presença desta nova mutação foi avaliada nos outros membros da família, tendo-se detetado um padrão de cossegregação. Ambos os membros da família afetados apresentaram HCM de início tardio, com hipertrofia ventricular esquerda assimétrica moderada, fibrilhação auricular e insuficiência cardíaca com fracão de ejeção preservada, com baixo risco de morte súbita cardíaca. Conclusão: Apresentamos evidência que suporta a classificação da mutação TCAP p.C57W, que codifica a proteína titin-cap do disco-Z (teletonina), como uma nova variante provavelmente patogénica da HCM, com um perfil fenotípico específico na família analisada.

Limb girdle muscular dystrophy LGMD R7 telethonin-related is a rare autosomal recessive muscle disorder characterized by proximal muscle weakness of pelvic and shoulder girdles. Mutation in TCAP is responsible for LGMD R7, and the disease has a wide geographic distribution in diverse populations, but genotype-phenotype relationships remain unclear. We collected 5 LGMD R7 patients from three unrelated Chinese families. The average onset age was 16 ± 1.41; the initial symptoms included progressive proximal muscle weakness in limbs, difficulty in fast running, and asymmetric muscle atrophy in calves. Muscle MR imaging showed varying severity of fatty infiltration in the pelvic girdle, thigh, and calf muscles, and the severity of muscle infiltration was related to the length of the disease course. Muscle histopathology revealed aberrantly sized muscle fibers, internal nuclei, split fibers, rimmed vacuoles, monocyte invasion, and necrotic fibers. Sequencing identified one novel and one previously reported TCAP mutation. Our study extends the known distribution of this rare muscular dystrophy and presents the first detailed clinical and genetic characterizations of LGMD R7 cases from the Chinese population. Our work expands the mutation spectrum known for LGMD R7 and emphasizes the need for clinicians to consider TCAP mutations when evaluating patients with symptoms of limb girdle muscular dystrophy.

Transverse (t)-tubules drive the rapid and synchronous Ca2+ rise in cardiac myocytes. The virtual complete loss of atrial t-tubules in heart failure (HF) decreases Ca2+ release. It is unknown if or how atrial t-tubules can be restored and if restored t-tubules are functional.Sheep were tachypaced to induce HF and recovered when pacing was stopped. Serial block face Scanning Electron Microscopy and confocal imaging were used to understand t-tubule ultrastructure and function. Candidate proteins involved in atrial t-tubule recovery were identified by western blot and causality determined using expression studies.Sheep atrial t-tubules reappeared following recovery from HF. Despite being disordered (branched, longer and longitudinally arranged) recovered t-tubules triggered Ca2+ release and were associated with restoration of systolic Ca2+. Telethonin and myotubularin abundance correlated with t-tubule density and altered the density and structure of BIN1-driven tubules in neonatal myocytes. Myotubularin had a greater effect, increasing tubule length and branching, replicating that seen in the recovery atria.Recovery from HF restores atrial t-tubules and systolic Ca2+ and myotubularin facilitates this process. Atrial t-tubule restoration could present a new and viable therapeutic strategy.Brief SummaryThe loss of atrial transverse (t)-tubules and the associated dysfunction in heart failure is reversible and the protein myotubularin plays an important role.

Aims: In cardiac myocytes, the sarcomeric Z-disc protein telethonin is constitutively bis-phosphorylated at C-terminal residues S157 and S161; however, the functional significance of this phosphorylation is not known. We sought to assess the significance of telethonin phosphorylation in vivo, using a novel knock-in (KI) mouse model generated to express non-phosphorylatable telethonin (TcapS157/161A).Methods and Results:TcapS157/161A and wild-type (WT) littermates were characterized by echocardiography at baseline and after sustained β-adrenergic stimulation via isoprenaline infusion. Heart tissues were collected for gravimetric, biochemical, and histological analyses. At baseline, TcapS157/161A mice did not show any variances in cardiac structure or function compared with WT littermates and mutant telethonin remained localized to the Z-disc. Ablation of telethonin phosphorylation sites resulted in a gene-dosage dependent decrease in the cardiac telethonin protein expression level in mice carrying the S157/161A alleles, without any alteration in telethonin mRNA levels. The proteasome inhibitor MG132 significantly increased the expression level of S157/161A telethonin protein in myocytes from TcapS157/161A mice, but not telethonin protein in myocytes from WT mice, indicating a role for the ubiquitin–proteasome system in the regulation of telethonin protein expression level. TcapS157/161A mice challenged with sustained β-adrenergic stimulation via isoprenaline infusion developed cardiac hypertrophy accompanied by mild systolic dysfunction. Furthermore, the telethonin protein expression level was significantly increased in WT mice following isoprenaline stimulation but this response was blunted in TcapS157/161A mice.Conclusion: Overall, these data reveal that telethonin protein turnover in vivo is regulated in a novel phosphorylation-dependent manner and suggest that C-terminal phosphorylation may protect telethonin against proteasomal degradation and preserve cardiac function during hemodynamic stress. Given that human telethonin C-terminal mutations have been associated with cardiac and skeletal myopathies, further research on their potential impact on phosphorylation-dependent regulation of telethonin protein expression could provide valuable mechanistic insight into those myopathies.

The Z-disc acts as a protein-rich structure to tether thin filament in the contractile units, the sarcomeres, of striated muscle cells. Proteins found in the Z-disc are integral for maintaining the architecture of the sarcomere. They also enable it to function as a (bio-mechanical) signalling hub. Numerous proteins interact in the Z-disc to facilitate force transduction and intracellular signalling in both cardiac and skeletal muscle. This review will focus on six key Z-disc proteins: α-actinin 2, filamin C, myopalladin, myotilin, telethonin and Z-disc alternatively spliced PDZ-motif (ZASP), which have all been linked to myopathies and cardiomyopathies. We will summarise pathogenic variants identified in the six genes coding for these proteins and look at their involvement in myopathy and cardiomyopathy. Listing the Minor Allele Frequency (MAF) of these variants in the Genome Aggregation Database (GnomAD) version 3.1 will help to critically re-evaluate pathogenicity based on variant frequency in normal population cohorts.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins - α-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) - and one M-line protein - Myomesin-2 (Myom2) - were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.

Titin truncating variants are a well-established cause of cardiomyopathy; however, the role of titin missense variants is less well understood. Here we describe the generation of a mouse model to investigate the underlying disease mechanism of a previously reported titin A178D missense variant identified in a family with non-compaction and dilated cardiomyopathy. Heterozygous and homozygous mice carrying the titin A178D missense variant were characterised in vivo by echocardiography. Heterozygous mice had no detectable phenotype at any time point investigated (up to 1 year). By contrast, homozygous mice developed dilated cardiomyopathy from 3 months. Chronic adrenergic stimulation aggravated the phenotype. Targeted transcript profiling revealed induction of the foetal gene programme and hypertrophic signalling pathways in homozygous mice, and these were confirmed at the protein level. Unsupervised proteomics identified downregulation of telethonin and four-and-a-half LIM domain 2, as well as the upregulation of heat shock proteins and myeloid leukaemia factor 1. Loss of telethonin from the cardiac Z-disc was accompanied by proteasomal degradation; however, unfolded telethonin accumulated in the cytoplasm, leading to a proteo-toxic response in the mice.We show that the titin A178D missense variant is pathogenic in homozygous mice, resulting in cardiomyopathy. We also provide evidence of the disease mechanism: because the titin A178D variant abolishes binding of telethonin, this leads to its abnormal cytoplasmic accumulation. Subsequent degradation of telethonin by the proteasome results in proteasomal overload, and activation of a proteo-toxic response. The latter appears to be a driving factor for the cardiomyopathy observed in the mouse model. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-021-00853-z.

The limb girdle muscular dystrophies (LGMDs) are genetic muscle diseases with primary skeletal muscle involvement in persons with the ability to walk independently at some point in the disease course. They usually have increased creatine kinase levels along with patterns of fatty and fibrous deposition on muscle imaging and/or dystrophic features on muscle biopsy. Distinctive clinical features provide valuable diagnostic clues to the diagnosis and sometimes treatment of these disorders. The advent of gene and cell-based therapies; gene replacement, editing, and modulation; along with stem cell and small molecule therapies may significantly ameliorate clinical severity in the LGMDs.

Introduction and objectives: Hypertrophic cardiomyopathy (HCM) is a genetically and phenotypically heterogeneous disease; there is still a large proportion of patients with no identified disease-causing mutation. Although the majority of mutations are found in the MYH7 and MYBPC3 genes, mutations in Z-disk-associated proteins have also been linked to HCM. Methods: We assessed a small family with HCM based on family history, physical examination, 12-lead ECG, echocardiogram and magnetic resonance imaging. After exclusion of mutations in eleven HCM disease genes, we performed direct sequencing of the TCAP gene encoding the Z-disk protein titin-cap (also known as telethonin). Results: We present a novel TCAP mutation in a small family affected by HCM. The identified p.C57W mutation showed a very low population frequency, as well as high conservation across species. All of the bioinformatic prediction tools used considered this mutation to be damaging/deleterious. Family members were screened for this new mutation and a co-segregation pattern was detected. Both affected members of this family presented with late-onset HCM, moderate asymmetric left ventricular hypertrophy, atrial fibrillation and heart failure with preserved ejection fraction and low risk of sudden cardiac death. Conclusions: We present evidence supporting the classification of the TCAP p.C57W mutation, encoding the Z-disk protein titin-cap/telethonin as a new likely pathogenic variant of hypertrophic cardiomyopathy, with a specific phenotype in the family under analysis. publishersversion published

Limb girdle muscular dystrophy (LGMD) type 2G is a rare form of muscle disease, described only in a few patients worldwide, caused by mutations in TCAP gene, encoding the protein telethonin. It is characterised by proximal limb muscle weakness associated with distal involvement of lower limbs, starting in the first or second decade of life. We describe the case of a 37-year-old woman of Greek origin, affected by disto-proximal lower limb weakness. No cardiac or respiratory involvement was detected. Muscle biopsy showed myopathic changes with type I fibre hypotrophy, cytoplasmic vacuoles, lipid overload, multiple central nuclei and fibre splittings; ultrastructural examination showed metabolic abnormalities. Next generation sequencing analysis detected a homozygous frameshift mutation in the TCAP gene (c.90_91del), previously described in one Turkish family. Immunostaining and Western blot analysis showed complete absence of telethonin. Interestingly, Single Nucleotide Polymorphism analysis of the 10 Mb genomic region containing the TCAP gene showed a shared homozygous haplotype of both the Greek and the Turkish patients, thus suggesting a possible founder effect of TCAP gene c.90_91del mutation in this part of the Mediterranean area.

Background: Inadequate intake of micronutrients with antioxidant properties is common among older adults and has been associated with higher risk of frailty, adverse functional outcome, and impaired muscle health. However, a causal relationship is less well known. The aim was to determine in old mice the impact of reduced dietary intake of vitamins A/E/B6/B12/folate, selenium, and zinc on muscle mass, oxidative capacity, strength, and physical activity (PA) over time. Methods: Twenty-one-month-old male mice were fed either AIN-93-M (control) or a diet low in micronutrients with antioxidant properties (=LOWOX-B: 50% of mouse recommended daily intake of vitamins A, E, B6, and B12, folate, selenium, and zinc) for 4 months. Muscle mass, grip strength, physical activity (PA), and general oxidative status were assessed. Moreover, muscle fatigue was measured of m. extensor digitorum longus (EDL) during an ex vivo moderate exercise protocol. Effects on oxidative capacity [succinate dehydrogenase (SDH) activity], muscle fibre type, number, and fibre cross-sectional area (fCSA) were assessed on m. plantaris (PL) using histochemistry. Results: After 2 months on the diet, bodyweight of LOWOX-B mice was lower compared with control (P

Background Inadequate intake of micronutrients with antioxidant properties is common among older adults and has been associated with higher risk of frailty, adverse functional outcome, and impaired muscle health. However, a causal relationship is less well known. The aim was to determine in old mice the impact of reduced dietary intake of vitamins A/E/B6/B12/folate, selenium, and zinc on muscle mass, oxidative capacity, strength, and physical activity (PA) over time.Methods Twenty-one-month-old male mice were fed either AIN-93-M (control) or a diet low in micronutrients with antioxidant properties (= LOWOX-B: 50% of mouse recommended daily intake of vitamins A, E, B6, and B12, folate, selenium, and zinc) for 4 months. Muscle mass, grip strength, physical activity (PA), and general oxidative status were assessed. Moreover, muscle fatigue was measured of m. extensor digitorum longus (EDL) during an ex vivo moderate exercise protocol. Effects on oxidative capacity [succinate dehydrogenase (SDH) activity], muscle fibre type, number, and fibre cross-sectional area (fCSA) were assessed on m. plantaris (PL) using histochemistry.Results After 2 months on the diet, bodyweight of LOWOX-B mice was lower compared with control (P Conclusions Reduced dietary intake of vitamins A, E, B6, and B12, folate, selenium, and zinc resulted in a lower oxidative capacity and has major impact on muscle health as shown by decreased force production and PA, without effects on muscle mass. The reduced fCSA in combination with similar SDH activity per fibre might explain the reduced oxidative capacity resulting in the increased fatigue after exercise in LOWOX-B mice.

Background Inadequate intake of micronutrients with antioxidant properties is common among older adults and has been associated with higher risk of frailty, adverse functional outcome, and impaired muscle health. However, a causal relationship is less well known. The aim was to determine in old mice the impact of reduced dietary intake of vitamins A/E/B6/B12/folate, selenium, and zinc on muscle mass, oxidative capacity, strength, and physical activity (PA) over time. Methods Twenty-one-month-old male mice were fed either AIN-93-M (control) or a diet low in micronutrients with antioxidant properties (= LOWOX-B: 50% of mouse recommended daily intake of vitamins A, E, B6, and B12, folate, selenium, and zinc) for 4 months. Muscle mass, grip strength, physical activity (PA), and general oxidative status were assessed. Moreover, muscle fatigue was measured of m. extensor digitorum longus (EDL) during an ex vivo moderate exercise protocol. Effects on oxidative capacity [succinate dehydrogenase (SDH) activity], muscle fibre type, number, and fibre cross-sectional area (fCSA) were assessed on m. plantaris (PL) using histochemistry. Results After 2 months on the diet, bodyweight of LOWOX-B mice was lower compared with control (P

Muscle wasting is observed in the course of many diseases and also during physiological conditions (disuse, aging). Skeletal muscle mass is largely controlled by the ubiquitin-proteasome system and thus by the ubiquitinating enzymes (E2s and E3s) that target substrates for subsequent degradation. MuRF1 is the only E3 ubiquitin ligase known to target contractile proteins (α-actin, myosins) during catabolic situations. MuRF1 is therefore a putative target for preventing muscle wasting. However, MuRF1 depends on E2 ubiquitin conjugating enzymes for ubiquitin chain formation on the substrates. We focused on 14 E2 enzymes that are either expressed in skeletal muscle or up-regulated during atrophying conditions. In this work, we demonstrated that only highly sensitive and complementary interactomic approaches (Surface Plasmon Resonance, Yeast three-Hybrid and split-GFP) allowed the identification of MuRF1 E2 partners. Five E2 enzymes physically interacted with MuRF1, namely E2E1, E2G1, E2J1, E2J2 and E2L3. Moreover, we demonstrated that MuRF1-E2E1 and MuRF1-E2J1 interactions are facilitated by telethonin, a newly identified MuRF1 substrate. We next showed that the 5 identified E2s functionally interacted with MuRF1 since, in contrast to the non-interacting E2D2, their co-expression in HEK293T cells with MuRF1 led to increased telethonin degradation. Finally, we showed that telethonin governed the affinity between MuRF1 and E2E1 or E2J1.We report here the first MuRF1-E2s network, which may prove valuable for deciphering the precise mechanisms involved in the atrophying muscle program and for proposing new therapeutical approaches.

In this study, acute myocardial injuries or necrosis were experimentally induced in calves and pigs by ligation of either the left anterior descending coronary artery or left circumflex branch for 30[Formula: see text]min without reperfusion. Various antibodies directed to structural and functional proteins of the sarcomere, as well as activated proteinases, were employed in immunohistochemistry to compare for their potentials to detect early myocardial injury. For comparison, the histological criteria (designated as “Method A”) of cardiomyocyte necrosis such as nuclear pyknosis, sarcoplasmic fragmentation, flocculation, and/or the presence of a contraction band, and inflammatory infiltration were also scored. Additional criteria (designated “Method B”) of changes in late reversible stage of cell injury such as irregular nuclear shape or hyperchromasia, sarcomplasmic hypereosinophilia with either swelling or atrophy in diameter, and perinuclear vacuolation likely of swollen organelles, were also scored. In this setting, telethonin (T-cap), cardiac troponin I (cTnI), the current gold standard, and Method B, were superior to others in detecting ligation-induced ischemic injury. Other markers were either less specific, or less sensitive, or inconclusive for the current application. In conclusion, telethonin may serve as an early immunohistochemical marker in ligation-induced ischemic myocardial injury due to a combination of biomechanical stress, hypoxia, and possibly additional factor as matrix metalloproteinase activation.

A 29-year-old man, born from consanguineous parents, started with toe walking and frequent falls during his second year of life. He developed weakness in lower limbs during the first decade that subsequently extended to upper limbs. On examination, the patient had weakness in proximal muscles of all four limbs and in the tibialis anterior muscle. In addition, he had bilateral Achilles and patellar contractures, bilateral scapular winging, asymmetric calves and a positive Beevor sign, an upward movement of the umbilicus on contraction of rectus femoris due to weakness in the lower part. The muscle biopsy showed dystrophic changes and lobulated fibers. Genetic analysis through a next-generation sequencing panel of genes related to neuromuscular disorders revealed a novel homozygous nonsense mutation (p.Tyr85*) in the TCAP gene. Subsequent western blot assay showed a complete telethonin deficiency. Our observation expands the phenotypic spectrum of TCAP mutations and indicates that telethonin deficiency should be considered in the differential diagnosis of patients presenting with asymmetric calves and early joint retractions.

Limb girdle muscular dystrophy (LGMD) type 2G is a rare form of muscle disease, described only in a few patients worldwide, caused by mutations in TCAP gene, encoding the protein telethonin. It is characterised by proximal limb muscle weakness associated with distal involvement of lower limbs, starting in the first or second decade of life. We describe the case of a 37-year-old woman of Greek origin, affected by disto-proximal lower limb weakness. No cardiac or respiratory involvement was detected. Muscle biopsy showed myopathic changes with type I fibre hypotrophy, cytoplasmic vacuoles, lipid overload, multiple central nuclei and fibre splittings; ultrastructural examination showed metabolic abnormalities. Next generation sequencing analysis detected a homozygous frameshift mutation in the TCAP gene (c.90_91del), previously described in one Turkish family. Immunostaining and Western blot analysis showed complete absence of telethonin. Interestingly, Single Nucleotide Polymorphism analysis of the 10 Mb genomic region containing the TCAP gene showed a shared homozygous haplotype of both the Greek and the Turkish patients, thus suggesting a possible founder effect of TCAP gene c.90_91del mutation in this part of the Mediterranean area. (C) 2018 Elsevier B.V. All rights reserved.

The formation of myofibrils was analyzed in neonatal mouse cardiomyocytes grown in culture and stained with fluorescent antibodies directed against myofibrillar proteins. The cardiomyocyte cultures also were exposed to siRNA probes to test the role of nonmuscle myosin IIB expression in the formation of myofibrils. In culture, new myofibrils formed in the spreading cell margins surrounding contractile myofibrils previously assembled in utero. Observations indicated that assembly of mature myofibrils occurred in three-stages, as previously reported in cultured mouse skeletal muscle. Premyofibrils, characterized by minisarcomeres with nonmuscle myosin IIB and muscle-specific alpha-actinin bound to actin filaments, formed in the first stage; followed by nascent myofibrils, the second stage when muscle myosin II and titin were first detected. In the mature myofibril stage muscle myosin II filaments aligned in periodic A-Bands; late assembling proteins, including myomesin and telethonin, were integrated in the sarcomeres, and nonmuscle IIB was absent from the sarcomeres. Treatment of the cultured neonatal cardiomyocytes with gene-specific siRNAs for nonmuscle myosin IIB, led to a marked decrease in the formation of premyofibrils, and subsequently of mature myofibrils. Anat Rec, 301:2067-2079, 2018. © 2018 Wiley Periodicals, Inc.

Background Gastrointestinal functional and motility disorders, like irritable bowel syndrome (IBS), have a high prevalence in the Western population and cause significant morbidity and loss of quality of life leading to considerable costs for health care. A decade ago, it has been demonstrated that interstitial cells of Cajal and intestinal smooth muscle cells, cells important for gastrointestinal motility, express the sodium channel alpha subunit Nav1.5. In the heart, aberrant variants in this sodium channel, encoded by SCN5A, are linked to inherited arrhythmia syndromes, like the long-QT syndrome type 3 and Brugada syndrome. Mounting data show a possible contribution of SCN5A mutants to gastrointestinal functional and motility disorders. Two percent of IBS patients harbor SCN5A mutations with electrophysiological evidence of loss- and gain-of-function. In addition, gastrointestinal symptoms are more prevalent in cardiac SCN5A-mutation positive patients. Purpose This review firstly describes the Nav1.5 channel and its physiological role in ventricular cardiomyocytes and gastrointestinal cells, then we focus on the involvement of mutant Nav1.5 in gastrointestinal functional and motility disorders. Future research might uncover novel mutation-specific treatment strategies for SCN5A-encoded gastrointestinal channelopathies.

Left ventricular assist devices (LVADs) were developed as a means of temporary circulatory support, but the mechanical unloading they offer also results in significant reverse remodelling. In selected patients, these improvements are sufficient to allow ultimate device explantation without requiring transplantation; this represents a fundamental shift in our understanding of heart failure. Like heart failure itself, LVADs influence multiple biological systems. The transverse tubules are a system of membrane invaginations in ventricular cardiomyocytes which allow rapid propagation of the action potential throughout the cell. Through their dense concentration of L-type Ca2+ channels in close proximity to intracellular ryanodine receptors, the t-tubules enable synchronous Ca2+ release throughout the cell. The t-tubules’ structure appears to be specifically regulated by mechanical load, such that either the overload of heart failure (or the spontaneously hypertensive rat model) or the profound unloading in a chronically unloaded heart result in impaired t-tubule structure, with ineffective Ca2+ release. While there are multiple molecular pathways which underpin t-tubule regulation, Telethonin (Tcap) appears to be important in regulating the effect of altered loading on the t-tubule system.

Recent innovations in stem cell technologies and the availability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have opened new possibilities for studies and drug testing on human cardiomyocytes in vitro. Still, there are concerns about the precise nature of such ‘reprogrammed’ cells. We have performed an investigation using immunocytochemistry and confocal microscopy on several cellular features using commercially available hiPSC-CMs. For some selected developmentally regulated or cardiac chamber-specific proteins, we have compared the results from hiPSC-derived cardiomyocytes with freshly isolated, ventricular cardiomyocytes from adult rats. The results show that all typical cardiac proteins are expressed in these hiPSC-CMs. Furthermore, intercalated disc-like structures, calcium cycling proteins, and myofibrils are present. However, some of these proteins are only known from early developmental stages of the ventricular myocardium or the diseased adult heart. A heterogeneous expression pattern in the cell population was noted for some muscle proteins, such as for myosin light chains, or incomplete organization in sarcomeres, such as for telethonin. These observations indicate that hiPSC-CMs can be considered genuine human cardiomyocytes of an early developmental state. The here described marker proteins of maturation may become instrumental in future studies attempting the improvement of cardiomyocyte in vitro models.

Telethonin anchors the N-terminal region of titin in the Z-disk of the sarcomere by binding to two immunoglobulin-like (Ig) domains (Z1 and Z2) of titin (Z1Z2). Thereby telethonin plays an important role in myofibril assembly and in muscle development and functional regulation. The expression and purification of recombinant telethonin is very challenging. In previous studies, recombinant telethonin expressed from E. coli was refolded in the presence of Z1Z2. Here, we report various strategies to establish a reliable and efficient protocol for the preparation of telethonin and titin Z1Z2 protein. First, a co-expression strategy was designed to obtain soluble Z1Z2/telethonin complexes. The concentration of antibiotics and the type of expression vector were found to be important for achieving high yields of purified complex. Second, the five cysteine residues of telethonin were mutated to serine to avoid severe problems with cysteine oxidation. Third, a short version of telethonin (telethonin 1-90 ) was designed to avoid the proteolytic degradation observed for longer constructs of the protein. The short telethonin formed a highly stable complex with Z1Z2 with no degradation being observed for 30 days at 4 °C. Fourth, an improved refolding protocol was developed to achieve high yields of Z1Z2/telethonin complex. Finally, based on the crystal structure in which Z1Z2 and telethonin 1-90 assemble into a 2:1 complex, a single chain fusion protein was designed, comprising two Z1Z2 modules that are connected by flexible linkers N- and C-terminally of the telethonin 1-90 . Expression of this fusion protein, named ZTZ, affords high yields of soluble expressed and purified protein.

Telethoninopathy is one of the rarest forms of Limb-girdle muscular dystrophy (LGMD). So far, only a small number of LGMD type 2 G (LGMD2G) patients have been described, mostly patients from Brazil. Here we present a 35-year-old female patient of Turkish ethnicity with LGMD2G due to a novel homozygous frame-shift mutation c.90_91del (p.Ser31Hisfs*11) in the telethonin gene, probably leading to truncated protein or nonsense mediated decay. Myalgia and walking on tiptoes were the first symptoms starting in early childhood, around age 22 proximal, later distal leg muscles became affected. Muscle biopsy showed a degenerative myopathy with lobulated fibers, creatine kinase levels were elevated to 1200 U/l. No cardiomyopathy has been detected but ventricular extrasystoles were treated with verapamil. Even though telethoninopathy represents a rare condition, testing for LGMD2G should be included into the diagnostic work-up of mild myopathies with early toe walking and distal and proximal involvement.

It is important to understand how muscle forms normally in order to understand muscle diseases that result in abnormal muscle formation. Although the structure of myofibrils is well understood, the process through which the myofibril components form organized contractile units is not clear. Based on the staining of muscle proteins in avian embryonic cardiomyocytes, we previously proposed that myofibrils formation occurred in steps that began with premyofibrils followed by nascent myofibrils and ending with mature myofibrils. The purpose of this study was to determine whether the premyofibril model of myofibrillogenesis developed from studies developed from studies in avian cardiomyocytes was supported by our current studies of myofibril assembly in mouse skeletal muscle. Emphasis was on establishing how the key sarcomeric proteins, F-actin, non-muscle myosin II, muscle myosin II, and α-actinin were organized in the three stages of myofibril assembly. The results also test previous reports that non-muscle myosins II A and B are components of the Z-Bands of mature myofibrils, data that are inconsistent with the premyofibril model. We have also determined that in mouse muscle cells, telethonin is a late assembling protein that is present only in the Z-Bands of mature myofibrils. This result of using specific telethonin antibodies supports the approach of using YFP-tagged proteins to determine where and when these YFP-sarcomeric fusion proteins are localized. The data presented in this study on cultures of primary mouse skeletal myocytes are consistent with the premyofibril model of myofibrillogenesis previously proposed for both avian cardiac and skeletal muscle cells.

TCAP (also known as titin-cap or telethonin) is one of the titin interacting Z-disk proteins involved in the regulation and development of normal sarcomeric structure. In this study, we cloned the cDNA and promoter sequences of porcine TCAP gene, which contained a 504 bp full-length coding region. Quantitative real-time PCR (qRT-PCR) analyses showed that porcine TCAP was highly expressed in the skeletal muscle, heart, and kidney. During postnatal muscle development, TCAP expression was down-regulated from 30 days to 120 days in Large White and Meishan pigs. One single nucleotide polymorphism c.334 G>A in exon 2 of the TCAP gene was identified and detected by allele-specific primer-polymerase chain reaction (ASP-PCR). Association analysis revealed that the polymorphism had significant associations (P

Horse musculature has been shaped through evolution by environmental and human factors, which has resulted in several extraordinary adaptations to physical effort. Skeletal muscle plasticity results from the response to mechanical stimulation causing hypertrophy, where sarcomeres increase the muscle&rsquo, s cross-sectional area under the influence of contractile forces. The aim of the present study was the evaluation of transcript abundance of the telethonin (TCAP) gene, which is a part of the sarcomere macromolecular mechanosensory complex in the gluteus medius muscle, and the whole blood of Arabian horses during flat race training. The analysis, performed by quantitative PCR, showed an increase of TCAP transcripts in skeletal muscle. However, in whole blood, the transcript abundance decreased after the first stage of training and further increased after the second phase. The obtained results indicate a lack of similarity of TCAP gene expression in both tissues.

Background: Telethonin mutations are associated with cardiomyopathy through unknown mechanisms. Results: Telethonin is a substrate for CaMK family kinases and exists in a bis-phosphorylated state in cardiomyocytes, in which non-phosphorylated telethonin disrupts transverse tubule organization and intracellular calcium transients. Conclusion: Telethonin phosphorylation is critical for the maintenance of normal cardiomyocyte morphology and calcium handling. Significance: Disruption of phospho-telethonin functions may contribute to pathogenesis in cardiomyopathy., Telethonin (also known as titin-cap or t-cap) is a muscle-specific protein whose mutation is associated with cardiac and skeletal myopathies through unknown mechanisms. Our previous work identified cardiac telethonin as an interaction partner for the protein kinase D catalytic domain. In this study, kinase assays used in conjunction with MS and site-directed mutagenesis confirmed telethonin as a substrate for protein kinase D and Ca2+/calmodulin-dependent kinase II in vitro and identified Ser-157 and Ser-161 as the phosphorylation sites. Phosphate affinity electrophoresis and MS revealed endogenous telethonin to exist in a constitutively bis-phosphorylated form in isolated adult rat ventricular myocytes and in mouse and rat ventricular myocardium. Following heterologous expression in myocytes by adenoviral gene transfer, wild-type telethonin became bis-phosphorylated, whereas S157A/S161A telethonin remained non-phosphorylated. Nevertheless, both proteins localized predominantly to the sarcomeric Z-disc, where they partially replaced endogenous telethonin. Such partial replacement with S157A/S161A telethonin disrupted transverse tubule organization and prolonged the time to peak of the intracellular Ca2+ transient and increased its variance. These data reveal, for the first time, that cardiac telethonin is constitutively bis-phosphorylated and suggest that such phosphorylation is critical for normal telethonin function, which may include maintenance of transverse tubule organization and intracellular Ca2+ transients.

Introduction: Creatine kinase (CK) and myoglobin (Mb) do not possess all good qualities as biomarkers of skeletal muscle damage. We investigated the utility of troponin I (TnI) and telethonin (Tcap) as markers and examined their temporal profiles after skeletal muscle damage. Methods: Plasma profiles were measured before and after exercise in 3 groups: subjects affected by either Becker muscular dystrophy or McArdle disease, and healthy subjects. Results: Mb and TnI appeared early in the blood, and the increase of TnI was only observed in patients with muscle disease. The CK increase was more delayed in plasma. Tcap was not detectable at any time. Conclusions: Our results suggest that TnI is a marker of more severe damage signifying sarcomeric damage, and it could therefore be an important supplement to CK and Mb in clinical practice. Tcap is not useful as a marker for skeletal muscle damage. Muscle Nerve 49: 261–266, 2014

Limb girdle muscular dystrophy type 2 G (LGMD2G) is caused by mutations in the telethonin gene. Only few families were described presenting this disease, and they are mainly Brazilians. Here, we identified one additional case carrying the same common c.157C > T mutation in the telethonin gene but with an atypical histopathological muscle pattern. In a female patient with a long duration of symptoms (46 years), muscle biopsy showed, in addition to telethonin deficiency, the presence of nemaline rods, type 1 fiber predominance, nuclear internalization, lobulated fibers, and mitochondrial paracrystalline inclusions. Her first clinical signs were identified at 8 years old, which include tiptoe walking, left lower limb deformity, and frequent falls. Ambulation loss occurred at 41 years old, and now, at 54 years old, she presented pelvic girdle atrophy, winging scapula, foot deformity with incapacity to perform ankle dorsiflexion, and absent tendon reflexes. The presence of nemaline bodies could be a secondary phenomenon, possibly associated with focal Z-line abnormalities of a long-standing disease. However, these new histopathological findings, characteristic of congenital myopathies, expand muscle phenotypic variability of telethoninopathy.

Supplemental Digital Content is available in the text., Background— High throughput next-generation sequencing techniques have made whole genome sequencing accessible in clinical practice; however, the abundance of variation in the human genomes makes the identification of a disease-causing mutation on a background of benign rare variants challenging. Methods and Results— Here we combine whole genome sequencing with linkage analysis in a 3-generation family affected by cardiomyopathy with features of autosomal dominant left ventricular noncompaction cardiomyopathy. A missense mutation in the giant protein titin is the only plausible disease-causing variant that segregates with disease among the 7 surviving affected individuals, with interrogation of the entire genome excluding other potential causes. This A178D missense mutation, affecting a conserved residue in the second immunoglobulin-like domain of titin, was introduced in a bacterially expressed recombinant protein fragment and biophysically characterized in comparison to its wild-type counterpart. Multiple experiments, including size exclusion chromatography, small-angle x ray scattering, and circular dichroism spectroscopy suggest partial unfolding and domain destabilization in the presence of the mutation. Moreover, binding experiments in mammalian cells show that the mutation markedly impairs binding to the titin ligand telethonin. Conclusions— Here we present genetic and functional evidence implicating the novel A178D missense mutation in titin as the cause of a highly penetrant familial cardiomyopathy with features of left ventricular noncompaction. This expands the spectrum of titin’s roles in cardiomyopathies. It furthermore highlights that rare titin missense variants, currently often ignored or left uninterpreted, should be considered to be relevant for cardiomyopathies and can be identified by the approach presented here.

Background—High throughput next generation sequencing techniques have made whole genome sequencing accessible in clinical practice, however, the abundance of variation in the human genomes makes the identification of a disease-causing mutation on a background of benign rare variants challenging. Methods and Results—Here we combine whole genome sequencing with linkage analysis in a three-generation family affected by cardiomyopathy with features of autosomal dominant left-ventricular non-compaction cardiomyopathy. A missense mutation in the giant protein titin is the only plausible disease-causing variant that segregates with disease amongst the eight surviving affected individuals, with interrogation of the entire genome excluding other potential causes. This A178D missense mutation, affecting a conserved residue in the second immunoglobulin-like domain of titin, was introduced in a bacterially expressed recombinant protein fragment and biophysically characterised in comparison to its wild-type counterpart. Multiple experiments, including size exclusion chromatography, small angle X-ray scattering and circular dichroism spectroscopy suggest partial unfolding and domain destabilisation in the presence of the mutation. Moreover, binding experiments in mammalian cells show that the mutation markedly impairs binding to the titin ligand telethonin. Conclusions—Here we present genetic and functional evidence implicating the novel A178D missense mutation in titin as the cause of a highly penetrant familial cardiomyopathy with features of left-ventricular non-compaction. This expands the spectrum of titin's roles in cardiomyopathies. It furthermore highlights that rare titin missense variants, currently often ignored or left un-interpreted, should be considered to be relevant for cardiomyopathies and can be identified by the approach presented here.

Recent genetic studies have revealed the impact of mutations in associated genes for cardiac sarcomere components leading to dilated cardiomyopathy (DCM). The cardiac sarcomere is composed of thick and thin filaments and a giant muscle protein known as titin or connectin. Titin interacts with T-cap/telethonin in the Z-line region and plays a vital role in regulating sarcomere assembly. Initially, we screened all the variants associated with giant protein titin and analyzed their impact with the aid of pathogenicity and stability prediction methods. V54M mutation found in the hydrophobic core region of the protein associated with abnormal clinical phenotype leads to DCM was selected for further analysis. To address this issue, we mapped the deleterious mutant V54M, modeled the mutant protein complex and deciphered the impact of mutation on binding with its partner telethonin in the titin crystal structure of PDB ID: 1YA5 with the aid of docking analysis. Furthermore, two run molecular dynamics simulation was initiated to understand the mechanistic action of V54M mutation in altering the protein structure, dynamics, and stability. According to the results obtained from the repeated 50 ns trajectory files, the overall effect of V54M mutation was destabilizing and transition of bend to coil in the secondary structure was observed. Furthermore, MMPBSA elucidated that V54M found in the Z-line region of titin, decreases the binding affinity of titin to Z-line proteins T-cap/telethonin thereby hindering the protein-protein interaction.

Current concepts of mechanosensation are general and applicable to almost every cell type. However, striated muscle cells are distinguished by their ability to generate strong forces via actin/myosin interaction, and this process is fine-tuned for optimum contractility. This aspect, unique for actively contracting cells, may be defined as "sensing of the magnitude and dynamics of contractility," as opposed to the well-known concepts of the "perception of extracellular mechanical stimuli." The acto/myosin interaction, by producing changes in ATP, ADP, Pi, and force on a millisecond timescale, may be regarded as a novel and previously unappreciated mechanosensory mechanism. In addition, sarcomeric mechanosensory structures, such as the Z-disc, are directly linked to autophagy, survival, and cell death-related pathways. One emerging example is telethonin and its ability to interfere with p53 metabolism and hence apoptosis (mechanoptosis). In this article, we introduce contractility per se as an important mechanosensory mechanism, and we differentiate extracellular from intracellular mechanosensory effects.

Objective—To identify a causative mutation for dilated cardiomyopathy (DCM) in Doberman Pinschers by sequencing the coding regions of 10 cardiac genes known to be associated with familial DCM in humans. Animals—5 Doberman Pinschers with DCM and congestive heart failure and 5 control mixed-breed dogs that were euthanized or died. Procedures—RNA was extracted from frozen ventricular myocardial samples from each dog, and first-strand cDNA was synthesized via reverse transcription, followed by PCR amplification with gene-specific primers. Ten cardiac genes were analyzed: cardiac actin, α-actinin, α-tropomyosin, β-myosin heavy chain, metavinculin, muscle LIM protein, myosinbinding protein C, tafazzin, titin-cap (telethonin), and troponin T. Sequences for DCM-affected and control dogs and the published canine genome were compared. Results—None of the coding sequences yielded a common causative mutation among all Doberman Pinscher samples. However, 3 variants were identified in the α-actinin gene in the DCM-affected Doberman Pinschers. One of these variants, identified in 2 of the 5 Doberman Pinschers, resulted in an amino acid change in the rod-forming triple coiled-coil domain. Conclusions and Clinical Relevance—Mutations in the coding regions of several genes associated with DCM in humans did not appear to consistently account for DCM in Doberman Pinschers. However, an α-actinin variant was detected in some Doberman Pinschers that may contribute to the development of DCM given its potential effect on the structure of this protein. Investigation of additional candidate gene coding and noncoding regions and further evaluation of the role of α-actinin in development of DCM in Doberman Pinschers are warranted.

The N-terminus of telethonin forms a unique structure linking two titin N-termini at the Z-disc. While a specific role for the C-terminus has not been established, several studies indicate it may have a regulatory function. Using a morpholino approach in Xenopus, we show that telethonin knockdown leads to embryonic paralysis, myocyte defects, and sarcomeric disruption. These myopathic defects can be rescued by expressing full-length telethonin mRNA in morpholino background, indicating that telethonin is required for myofibrillogenesis. However, a construct missing C-terminal residues is incapable of rescuing motility or sarcomere assembly in cultured myocytes. We, therefore, tested two additional constructs: one where four C-terminal phosphorylatable residues were mutated to alanines and another where terminal residues were randomly replaced. Data from these experiments support that the telethonin C-terminus is required for assembly, but in a context-dependent manner, indicating that factors and forces present in vivo can compensate for C-terminal truncation or mutation. Developmental Dynamics 239:1124–1135, 2010. © 2010 Wiley-Liss, Inc.

A myopathy in the Great Dane dog with characteristic pathological and molecular features is reported. Young adults present with progressive weakness and generalised muscle atrophy. To better define this condition, an investigation using histopathology, confocal microscopy, biochemistry and microarray analysis was undertaken. The skeletal muscles of affected dogs exhibited increased oxidative fibre phenotype and core fibre lesions characterised by the disruption of the sarcomeric architecture and the accumulation of mitochondrial organelles. Affected muscles displayed co-ordinated expression of genes consistent with a slow-oxidative phenotype, which was possibly a compensatory response to chronic muscle damage. There was disruption of Z-lines in affected muscles which, at the molecular level, manifested as transcriptional dysregulation of several Z-line associated genes, including alpha-actinin, myotilin, desmin, vimentin and telethonin. The pathology of this canine myopathy is distinct from that of human central core myopathies that are characterised by cores devoid of mitochondria and by the presence of myofibrillar breakdown products.

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.

Tcap/telethonin encodes a Z-disc protein that plays important roles in sarcomere assembly, sarcomere-membrane interaction and stretch sensing. It remains unclear why mutations in Tcap lead to limb-girdle muscular dystrophy 2G (LGMD2G) in human patients. Here, we cloned tcap in zebrafish and conducted genetic studies. We show that tcap is functionally conserved, as the Tcap protein appears in the sarcomeric Z-disc and reduction of Tcap resulted in muscular dystrophy-like phenotypes including deformed muscle structure and impaired swimming ability. However, the observations that Tcap integrates into the sarcomere at a stage after the Z-disc becomes periodic, and that the sarcomere remains intact in tcap morphants, suggest that defective sarcomere assembly does not contribute to this particular type of muscular dystrophy. Instead, a defective interaction between the sarcomere and plasma membrane was detected, which was further underscored by the disrupted development of the T-tubule system. Pertinent to a potential function in stretch sensor signaling, zebrafish tcap exhibits a variable expression pattern during somitogenesis. The variable expression is inducible by stretch force, and the expression level of Tcap is negatively regulated by integrin-link kinase (ILK), a protein kinase that is involved in stretch sensing signaling. Together, our genetic studies of tcap in zebrafish suggested that pathogenesis in LGMD2G is due to a disruption of sarcomere–T-tubular interaction, but not of sarcomere assembly per se. In addition, our data prompted a novel hypothesis that predicts that the transcription level of Tcap can be regulated by the stretch force to ensure proper sarcomere–membrane interaction in striated muscles.

Mechanical stability of bonds and protein interactions has recently become accessible through single molecule mechanical experiments. So far, mechanical information about molecular bond mechanics has been largely limited to a single direction of force application. However, mechanical force acts as a vector in space and hence mechanical stability should depend on the direction of force application. In skeletal muscle, the giant protein titin is anchored in the Z-disk by telethonin. Much of the structural integrity of the Z-disk hinges upon the titin-telethonin bond. In this paper we show that the complex between the muscle proteins titin and telethonin forms a highly directed molecular bond. It is designed to resist ultra-high forces if they are applied in the direction along which it is loaded under physiological conditions, while it breaks easily along other directions. Highly directed molecular bonds match in an ideal way the requirements of tissues subject to mechanical stress.

The gradually discovered sarcomeric proteins play important roles for structural integrity and signal transduction of sarcomere during myofibril genesis. TCAP (also described as telethonin, T-cap), one of the sarcomeric protein genes, is regulated developmentally. In this study, we reported the molecular characteristics of porcine TCAP gene. A 979 bp TCAP cDNA nucleotide sequence was obtained in pig and the deduced amino acid sequence had 92 and 91% identity to those of human and mouse homologous genes, respectively. One SNP was discovered and the allele frequency analysis showed that G allele frequency was low among 221 unrelated pigs from seven breeds. The tissue distribution patterns revealed that TCAP mRNA was expressed abundantly in skeletal and heart muscle tissue. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) results displayed TCAP mRNA was up-regulated in both Tongcheng and Landrace pigs during prenatal skeletal muscle development stages. This study suggested that TCAP gene might be a prospective candidate gene affecting muscle mass and meat quality traits in the pig, and also implicated the possible significance of TCAP on sarcomere assembly.

Marbling of cattle meat is dependent on the coordinated expression of multiple genes. Cattle dramatically increase their intramuscular fat content in the longissimus dorsi muscle between 12 and 27 months of age. We used the annealing control primer (ACP)-differential display RT-PCR method to identify differentially expressed genes (DEGs) that may participate in the development of intramuscular fat between early (12 months old) and late fattening stages (27 months old). Using 20 arbitrary ACP primers, we identified and sequenced 14 DEGs. BLAST searches revealed that expression of the MDH, PI4-K, ferritin, ICER, NID-2, WDNMI, telethonin, filamin, and desmin (DES) genes increased while that of GAPD, COP VII, ACTA1, CamK II, and nebulin decreased during the late fattening stage. The results of functional categorization using the Gene Ontology database for 14 known genes indicated that MDH, GAPD, and COP VII are involved in metabolic pathways such as glycolysis and the TCA cycle, whereas telethonin, filamin, nebulin, desmin, and ACTA1 contribute to the muscle contractile apparatus, and PI4-K, CamK II, and ICER have roles in signal transduction pathways regulated by growth factor or hormones. The final three genes, NID-2, WDNMI, and ferritin, are involved in iron transport and extracellular protein inhibition. The expression patterns were confirmed for seven genes (MDH, PI4-K, ferritin, ICER, nebulin, WDNMI, and telethonin) using real-time PCR. We found that the novel transcription repressor ICER gene was highly expressed in the late fattening stage and during bovine preadipocyte differentiation. This information may be helpful in selecting candidate genes that participate in intramuscular fat development in cattle.

SummaryTitin is a gigantic elastic filament that determines sarcomere ultrastructure and stretch response in vertebrate muscle. It folds into numerous Ig and FnIII domains connected in tandem. Data on interdomain arrangements and dynamics are essential for understanding the function of this filament. Here, we report a mechanistic analysis of the conformational dynamics of two Ig domains from the N terminus of titin, Z1Z2, by using X-ray crystallography, SAXS, NMR relaxation data, and residual dipolar couplings in combination. Z1Z2 preferentially adopts semiextended conformations in solution, with close-hinge arrangements representing low-probability states. Although interdomain contacts are not observed, the linker appears to acquire moderate rigidity via small, local hydrophobic interactions. Thus, Z1Z2 constitutes an adaptable modular system with restricted dynamics. We speculate that its preexistent conformation contributes to the selective recruitment of the binding partner telethonin onto the repetitive surface of the filament. The structural interconversion of four Z1Z2 conformers is analyzed.

The Z-disk region defines the lateral boundary of the sarcomere and requires a high level of mechanical strength to provide a stable framework for large filamentous muscle proteins. The level of complexity at this area is reflected by a large number of protein–protein interactions. Recently, we unraveled how the N-terminus of the longest filament component, the giant muscle protein titin, is assembled into an antiparallel (2:1) sandwich complex by the N-terminal titin-binding segment of the Z-disk ligand telethonin/T-cap [Zou, P., Pinotsis, N., Lange, S., Song, Y.H., Popov, A., Mavridis, I., Mayans, O.M., Gautel, M., Wilmanns, M., 2006. Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk. Nature 439, 229–233]. In this contribution, we present structural data of a related complex of the titin N-terminus with full-length telethonin. The C-terminus of telethonin remains invisible, suggesting that it does not fold into a defined structure even in the presence of titin. In contrast to the structure with truncated telethonin, a dimer of two titin/telethonin complexes is formed within the crystal environment, potentially indicating the formation of higher oligomers. We further investigated the structure and dynamics of this assembly by small-angle X-ray scattering, circular dichroism, and in vivo complementation data. The data consistently indicate the involvement of the C-terminal part of telethonin into the assembly of two titin/telethonin complexes.

During myofibril formation, Z-bodies, small complexes of alpha-actinin and associated proteins, grow in size, fuse and align to produce Z-bands. To determine if there were changes in protein dynamics during the assembly process, Fluorescence Recovery after Photobleaching was used to measure the exchange of Z-body and Z-band proteins with cytoplasmic pools in cultures of quail myotubes. Myotubes were transfected with plasmids encoding Yellow, Green or Cyan Fluorescent Protein linked to the Z-band proteins: actin, alpha-actinin, cypher, FATZ, myotilin, and telethonin. Each Z-band protein showed a characteristic recovery rate and mobility. All except telethonin were localized in both Z-bodies and Z-bands. Proteins that were present both early in development in Z-bodies and later in Z-bands had faster exchange rates in Z-bodies. These results suggest that during myofibrillogenesis, molecular interactions develop between the Z-band proteins that decrease their mobility and increase the stability of the Z-bands. A truncated construct of alpha-actinin, which localized in Z-bands in myotubes, and exhibited a very low rate of exchange, lead to disruption of myofibrils, suggesting the importance of dynamic, intact alpha-actinin molecules for the formation and maintenance of Z-bands. Our experiments reveal the Z-band to be a much more dynamic structure than its appearance in electron micrographs of cross-striated muscle cells might suggest.

The limb-girdle muscular dystrophies are a diverse group of muscle-wasting disorders characteristically affecting the large muscles of the pelvic and shoulder girdles. Molecular genetic analyses have demonstrated causative mutations in the genes encoding a disparate collection of proteins involved in all aspects of muscle cell biology. Muscular dystrophy includes a spectrum of disorders caused by loss of the linkage between the extracellular matrix and the actin cytoskeleton. Within this are the forms of limb-girdle muscular dystrophy caused by deficiencies of the sarcoglycan complex and by aberrant glycosylation of alpha-dystroglycan caused by mutations in the fukutin-related protein gene. However, other forms of this disease have distinct pathophysiological mechanisms. For example, deficiency of dysferlin disrupts sarcolemmal membrane repair, whilst loss of calpain-3 may exert its pathological influence either by perturbation of the IkappaBalpha/NF-kappaB pathway, or through calpain-dependent cytoskeletal remodelling. Caveolin-3 is implicated in numerous cell-signalling pathways and involved in the biogenesis of the T-tubule system. Alterations in the nuclear lamina caused by mutations in laminA/C, sarcomeric changes in titin, telethonin or myotilin at the Z-disc, and subtle changes in the extracellular matrix proteins laminin-alpha2 or collagen VI can all lead to a limb-girdle muscular dystrophy phenotype, although the specific pathological mechanisms remain obscure. Differential diagnosis of these disorders requires the careful application of a broad range of disciplines: clinical assessment, immunohistochemistry and immunoblotting using a panel of antibodies and extensive molecular genetic analyses.

Titin is the largest protein known, and is essential for organising muscle sarcomeres. It has many domains with a variety of functions, and stretches from the Z-line to the M-line in the muscle sarcomere. Close to the M-line, titin contains a kinase domain, which is known to phosphorylate the Z-line protein telethonin in developing muscle (Mayans, O., van der Ven, P. F., Wilm, M., Mues, A., Young, P., Furst, D. O., Wilmanns, M. and Gautel, M. (1998) Nature 395, 863-869). This phosphorylation is thought to be important for initiating or regulating myofibrillogenesis. We used a gene-targeting approach in cultured myoblasts to truncate the titin gene so that the kinase domain and other domains downstream of the kinase were not expressed. We recovered cells in which one allele was targeted. We found that these cells expressed both the full-length and a truncated titin that was approximately 0.2 MDa smaller than the corresponding band from wild-type cells. Myofibrillogenesis in these cells was impaired, in that the myotubes were shorter, and the organisation of the muscle sarcomeres, M- and Z-lines was poorer than in wild-type cells. There was also an overall reduction in levels of titin and skeletal myosin expression. These results suggest that the activity of the titin kinase domain and downstream sequence are important in organising myofibrils both at the M- and the Z-line early in myofibrillogenesis.