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A better understanding of the strains experienced by the Achilles tendon during commonly prescribed exercises and locomotor tasks is needed to improve efficacy of Achilles tendon training and rehabilitation programs. The aim of this study was to estimate in vivo free Achilles tendon strain during selected rehabilitation, locomotor, jumping, and landing tasks. Sixteen trained runners with no symptoms of Achilles tendinopathy participated in this study. Personalized free Achilles tendon moment arm and force-strain curve were obtained from imaging data and used in conjunction with motion capture and surface electromyography to estimate free Achilles tendon strain using electromyogram-informed neuromusculoskeletal modeling. There was a strong correspondence between Achilles tendon force estimates from the present study and experimental data reported in the literature (

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix ‘brace’ that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO)., Necroptosis is a regulated form of inflammatory cell death driven by activated MLKL. Here, the authors identify a mutation in the brace region that confers constitutive activation, leading to lethal inflammation in homozygous mutant mice and providing insight into human mutations in this region.

The purpose of this study was to assess the similarity of free Achilles tendon shape and 3-D geometry between magnetic resonance imaging (MRI) and freehand 3-D ultrasound (3-DUS) imaging methods. Fourteen elite/sub-elite middle-distance runners participated in the study. MRI and 3-DUS scans of the Achilles tendon were acquired on two separate imaging sessions, and all 3-D reconstructions were performed using identical methods. Shape similarity of free Achilles tendon reconstructed from MRI and 3-DUS data was assessed using Jaccard index, Hausdorff distance and root mean square error (RMSE). The Jaccard index, Hausdorff distance and RMSE values were 0.76 ± 0.05, 2.70 ± 0.70 and 0.61 ± 0.10 mm, respectively. The level of agreement between MRI and 3-DUS for free Achilles tendon volume, length and average cross-sectional area (CSA) was assessed using Bland-Altman analysis. Compared to MRI, freehand 3-DUS overestimated volume, length and average CSA by 30.6 ± 15.8 mm3 (1.1% ± 0.6%), 0.3 ± 0.7 mm (0.6% ± 1.9%) and 0.3 ± 1.42 mm2 (0.4% ± 2.0%), respectively. The upper and lower limits of agreement between MRI and 3-DUS for volume, length and average CSA were -0.4 to 61.7 mm3 (-0.2% to 2.3%), -1.0 to 1.5 mm (-3.2% to 4.5%) and -2.5 to 3.1 mm2 (-3.5% to 4.3%), respectively. There were no significant differences between imaging methods in CSA along the length of the tendon. In conclusion, MRI and freehand 3-DUS may be considered equivalent methods for estimating shape and 3-D geometry of the free Achilles tendon. These findings, together with the practical benefits of being able to assess 3-D Achilles tendon shape and geometry in a laboratory environment and under isometric loading, make 3-DUS an attractive alternative to MRI for assessing 3-D free Achilles tendon macro-structure in future studies.

A hallmark of T cell ageing is a loss of effector plasticity. Exercise delays T cell ageing, yet the mechanisms driving the effects of exercise on T cell biology are not well elucidated. T cell plasticity is closely linked with metabolism, and consequently sensitive to metabolic changes induced by exercise. Mitochondrial function is essential for providing the intermediate metabolites necessary to generate and modify epigenetic marks in the nucleus, thus metabolic activity and epigenetic mechanisms are intertwined. In this perspective we propose a role for exercise in CD4+ T cell plasticity, exploring links between exercise, metabolism and epigenetic reprogramming.

ObjectivesAthlete health, training continuity and performance can be impeded as a result of Relative Energy Deficiency in Sport (RED-S). Here we report the point prevalence of symptoms described by the RED-S model in a mixed-sport cohort of Australian female athletes.MethodsElite and pre-elite female athletes (n=112) from eight sports completed validated questionnaires and underwent clinical assessment to assess the point prevalence of RED-S symptoms. Questionnaires included the Depression, Anxiety and Stress Questionnaire (DASS-21), Generalized Anxiety Disorder (GAD-7), Center for Epidemiological Studies Depression Scale (CES-D), SCOFF questionnaire for disordered eating, Low Energy Availability in Females Questionnaire (LEAF-Q), and a custom questionnaire on injury and illness. Clinical assessment comprised resting metabolic rate (RMR) assessment, dual-energy X-ray absorptiometry-derived body composition and bone mineral density, venous and capillary blood samples, and the Mini International Neuropsychiatric Interview (MINI 7.0.2). Descriptive prevalence statistics are presented.ResultsAlmost all (80%) participants (age 19 (range 15–32) years; mass 69.5±10.3 kg; body fat 23.1%±5.0%) demonstrated at least one symptom consistent with RED-S, with 37% exhibiting between two and three symptoms. One participant demonstrated five symptoms. Impaired function of the immunological (28%, n=27), haematological (31%, n=33) and gastrointestinal (47%, n=51) systems were most prevalent. A moderate to high (11%–55%) prevalence of risk of low energy availability was identified via RMR and LEAF-Q, and identified mental illnesses were prevalent in one-third of the assessed cohort.ConclusionSymptoms described by the RED-S model were prevalent in this cohort, supporting the need for improved awareness, monitoring and management of these symptoms in this population.

Identification, evaluation and management of disordered eating (DE) is complex. DE exists along the spectrum from optimised nutrition through to clinical eating disorders (EDs). Individual athletes can move back and forth along the spectrum of eating behaviour at any point in time over their career and within different stages of a training cycle. Athletes are more likely to present with DE than a clinical ED. Overall, there is a higher prevalence of DE and EDs in athletes compared with non-athletes. Additionally, athletes participating in aesthetic, gravitational and weight-class sports are at higher risk of DE and EDs than those in sports without these characteristics. The evaluation and management of DE requires a cohesive team of professional practitioners consisting of, at minimum, a doctor, a sports dietitian and a psychologist, termed within this statement as the core multidisciplinary team. The Australian Institute of Sport and the National Eating Disorders Collaboration have collaborated to provide this position statement, containing guidelines for athletes, coaches, support staff, clinicians and sporting organisations. The guidelines support the prevention and early identification of DE, and promote timely intervention to optimise nutrition for performance in a safe, supported, purposeful and individualised manner. This position statement is a call to action to all involved in sport to be aware of poor self-image and poor body image among athletes. The practical recommendations should guide the clinical management of DE in high performance sport.

ObjectivesGlucocorticoids are commonly prescribed in medicine. When administered via certain routes, glucocorticoids are prohibited for incompetition use by WADA. The glucocorticoid prescribing habits of sports medicine doctors have not been reported.MethodsAn online survey was distributed internationally to physicians working in high-performance sports. The survey queried the doctors about their use of glucocorticoids with athletes and their understanding of WADA’s regulations regarding glucocorticoid use in competition.Results603 sports medicine doctors from 30 different countries participated. The majority (>85%) routinely injected glucocorticoids and/or prescribed glucocorticoids by other routes. There were substantial differences in the common routes of injection as well as types of glucocorticoid used among the physicians from various countries. A relatively small percentage of sports doctors (ConclusionGlucocorticoids are widely prescribed by sports physicians. There is a need to better educate sports physicians on the current WADA regulations in relation to glucocorticoid administration.

The automatic segmentation of Achilles tendon tissues is one of the preliminary steps towards creating a tool for diagnosing, prognosing, or monitoring changes in tendon organization over time. Manual delineation is the current approach of identifying Achilles region-of-interest (ROI), it is a tedious and time-consuming task. In this respect, the current work describes the first steps taken towards creating an automatic approach for Achilles tendon segmentation that utilize the capabilities of Deep Convolutional Neural Networks (CNNs). Firstly, the dataset has been pre-processed and manually segmented to be used as the ground-truth in the training and testing of the proposed automated model. Secondly, the model was trained and validated using three CNN architectures SegNet, ResNet-18 and ResNet-50. Finally, Tversky loss function, 3D augmentation and network ensembling approaches were used to improve the segmentation performance and to tackle challenges such as the limited size of the training dataset and data imbalance. The proposed fully automated segmentation method reached average Dice score of 0.904. In conclusion, this novel study demonstrates that a CNN approach is useful for performing accurate Achilles tendon segmentation in musculoskeletal imaging.

ISSUE ADDRESSED The aim of this study was to characterise lifestyle and training habits of a large cohort of Australian recreational runners. Understanding the health benefits of recreational running and differentiating between the habits of males and females may allow for the development of gender-specific messaging for promoting recreational running as a form of physical activity. METHODS An online questionnaire was used to collect data from 4720 Australian recreational runners. Data on physical, lifestyle and training characteristics of male and female subgroups were compared using chi-square tests. Multiple logistic regression method was used to assess the effect of running experience on the reported clinically significant weight loss. RESULTS The study cohort was 54.1% female and 45.9% male. Smoking was uncommon among surveyed runners. The most typical weekly running distance in the cohort was 20-40 km, usually distributed by 2-5 running sessions. Significantly more males than females reported running over 40 km per week (29.9% vs 18.9%, P < .001) and running at least six sessions per week (11.5% vs 6.7%, P < .001). The majority (72.9%) of runners had normal BMI, and the cohort reported a lower overweight/obesity rate than the Australian population. The logistic regression model indicated that commencing running may lead to a clinically significant weight loss irrespectively of sex, participation in other sports and injury history. CONCLUSION Recreational running was associated with beneficial health outcomes. Commencement of running is associated with weight loss, and regular running supports healthy weight maintenance. Male and female runners had different running preferences which should be taken into account for physical activity promotion. SO WHAT?: Captured health outcomes associated with running and described sex differences in training patterns may assist in development of physical activity promotion programmes involving recreational running, particularly targeting weight loss and healthy weight maintenance.

As Australia's peak high-performance sport agency, the Australian Institute of Sport (AIS) has developed this position statement to address the implications of recent advances in the field of genetics and the ramifications for the health and well-being of athletes. Genetic testing has proven of value in the practice of clinical medicine. There are, however, currently no scientific grounds for the use of genetic testing for athletic performance improvement, sport selection or talent identification. Athletes and coaches should be discouraged from using direct-to-consumer genetic testing because of its lack of validation and replicability and the lack of involvement of a medical practitioner in the process. The transfer of genetic material or genetic modification of cells for performance enhancement is gene doping and should not be used on athletes. There are, however, valid roles for genetic research and the AIS supports genetic research which aims to enhance understanding of athlete susceptibility to injury or illness. Genetic research is only to be conducted after careful consideration of a range of ethical concerns which include the provision of adequate informed consent. The AIS is committed to providing leadership in delivering an ethical framework that protects the well-being of athletes and the integrity of sport, in the rapidly changing world of genomic science.

Objective The use of intravenous fluids in out-of-hospital settings has evolved from the practices used by military and emergency response teams. When used in the elite sporting environment, IV fluid use must comply with the World Anti-Doping Code. Uncertainty can arise as clinicians seek to balance the appropriate use of IV fluids in delivering athlete care against the need for World Anti-Doping Code compliance. Design and method This position statement reviews the current literature and incorporates clinical experiences to present best-practice recommendations on the clinical use of Intravenous fluids in the elite sport environment, framing recommendations in the context of the World Anti-Doping Code. Results and conclusion The World Anti-Doping Code restricts the use of Intravenous fluids in athletes under certain conditions. This report takes into account the World Anti-Doping Code and the risks of Intravenous fluid administration to provide guidelines around the judicious use of IV fluids for: 1. Treatment of severe dehydration in an athlete, 2. Management of exertional heat illness in an athlete, 3. Hypovolaemia because of trauma in sport, 4. Administering medications.

SUMMARYWe have isolated a mouse strain with a single missense mutation in the gene encoding MLKL, the essential effector of necroptotic cell death. The resulting substitution lies within the two-helix ‘brace’ and confers constitutive, RIPK3 independent, killing activity to MLKL. Mice homozygous forMlklD139Vdevelop lethal inflammation within days of birth, implicating the salivary glands and pericardium as hotspots for necroptosis and inflammatory infiltration. The normal development ofMlklD139Vhomozygotes until birth, and the absence of any overt phenotype in heterozygotes provides importantin vivoprecedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common humanMLKLpolymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, CRMO.

Although the oral microbiota is known to play a crucial role in human health, there are few studies of diet x oral microbiota interactions, and none in elite athletes who may manipulate their intakes of macronutrients to achieve different metabolic adaptations in pursuit of optimal endurance performance. The aim of this study was to investigate the shifts in the oral microbiome of elite male endurance race walkers from Europe, Asia, the Americas and Australia, in response to one of three dietary patterns often used by athletes during a period of intensified training: a High Carbohydrate (HCHO, n = 9, with 60% energy intake from carbohydrates, ~8.5 g kg&minus, 1 day&minus, 1 carbohydrate, ~2.1 g kg&minus, 1 protein, 1.2 g kg&minus, 1 fat) diet, a Periodised Carbohydrate (PCHO, n = 10, same macronutrient composition as HCHO, but the intake of carbohydrates is different across the day and throughout the week to support training sessions with high or low carbohydrate availability) diet or a ketogenic Low Carbohydrate High Fat (LCHF, 0.5 g kg&minus, 1 carbohydrate, 78% energy as fat, 2.1 g kg&minus, 1 protein) diet. Saliva samples were collected both before (Baseline, BL) and after the three-week period (Post treatment, PT) and the oral microbiota profiles for each athlete were produced by 16S rRNA gene amplicon sequencing. Principal coordinates analysis of the oral microbiota profiles based on the weighted UniFrac distance measure did not reveal any specific clustering with respect to diet or athlete ethnic origin, either at baseline (BL) or following the diet-training period. However, discriminant analyses of the oral microbiota profiles by Linear Discriminant Analysis (LDA) Effect Size (LEfSe) and sparse Partial Least Squares Discriminant Analysis (sPLS-DA) did reveal changes in the relative abundance of specific bacterial taxa, and, particularly, when comparing the microbiota profiles following consumption of the carbohydrate-based diets with the LCHF diet. These analyses showed that following consumption of the LCHF diet the relative abundances of Haemophilus, Neisseria and Prevotella spp. were decreased, and the relative abundance of Streptococcus spp. was increased. Such findings suggest that diet, and, in particular, the LCHF diet can induce changes in the oral microbiota of elite endurance walkers.

The current pilot study investigates whether oral supplementation of specific collagen peptides improves symptoms and tendon vascularisation in patients with chronic mid-portion Achilles tendinopathy in combination with structured exercise. Participants were given a placebo or specific collagen peptides (TENDOFORTE&reg, ) in combination with a bi-daily calf-strengthening program for 6 months. Group AB received specific collagen peptides for the first 3 months before crossing over to placebo. Group BA received placebo first before crossing over to specific collagen peptides. At baseline (T1), 3 (T2) and 6 (T3) months, Victorian Institute of Sports Assessment&ndash, Achilles (VISA-A) questionnaires and microvascularity measurements through contrast-enhanced ultrasound were obtained in 20 patients. Linear mixed modeling statistics showed that after 3 months, VISA-A increased significantly for group AB with 12.6 (9.7, 15.5), while in group BA VISA-A increased only by 5.3 (2.3, 8.3) points. After crossing over group AB and BA showed subsequently a significant increase in VISA-A of, respectively, 5.9 (2.8, 9.0) and 17.7 (14.6, 20.7). No adverse advents were reported. Microvascularity decreased in both groups to a similar extent and was moderately associated with VISA-A (Rc2:0.68). We conclude that oral supplementation of specific collagen peptides may accelerate the clinical benefits of a well-structured calf-strengthening and return-to-running program in Achilles tendinopathy patients.

PURPOSE. Acute anterior uveitis (AAU) is a common intraocular inflammatory disease. AAU occurs in 30% to 50% of patients with ankylosing spondylitis (AS), and both conditions are strongly associated with human leukocyte antigen (HLA)-B27, implying a shared etiology. This study aims to apply genomewide association study (GWAS) to characterize the genetic associations of AAU and their relationship to the genetics of AS. METHODS. We undertook the GWAS analyses in 2752 patients with AS with AAU (cases) and 3836 patients with AS without AAU (controls). There were 7,436,415 single-nucleotide polymorphisms (SNPs) available after SNP microarray genotyping, imputation, and quality-control filtering. RESULTS. We identified one locus associated with AAU at genomewide significance: rs9378248 (P = 2.69 × 10−8, odds ratio [OR] = 0.78), lying close to HLA-B. Suggestive association was observed at 11 additional loci, including previously reported AS loci ERAP1 (rs27529, P = 2.19 × 10−7, OR = 1.22) and NOS2 (rs2274894, P = 8.22 × 10−7, OR = 0.83). Multiple novel suggestive associations were also identified, including MERTK (rs10171979, P = 2.56 × 10−6, OR = 1.20), KIFAP3 (rs508063, P = 5.64 × 10−7, OR = 1.20), CLCN7 (rs67412457, P = 1.33 × 10−6, OR = 1.25), ACAA2 (rs9947182, P = 9.70 × 10−7, OR = 1.37), and 5 intergenic loci. The SNP-based heritability is approximately 0.5 for AS alone, and is much higher (approximately 0.7) for AS with AAU. Consistent with the high heritability, a genomewide polygenic risk score shows strong power in identifying individuals at high risk of either AS with AAU or AS alone. CONCLUSIONS. We report here the first GWAS for AAU and identify new susceptibility loci. Our findings confirm the strong overlap in etiopathogenesis of AAU with AS, and also provide new insights into the genetic basis of AAU.

Objectives The role of neovascularisation in tendinopathy is still poorly understood, potentially due to technical limitations of conventional power Doppler ultrasound. This study aimed to investigate the association between contrast-enhanced ultrasound (CEUS) microvascular volume (MV), Victorian Institute of Sports Assessment-Achilles (VISA-A) scores and intrinsic Achilles tendon tenderness, as well as two different Power Doppler modes. Design Cross-sectional study. Methods 20 individuals with uni- or bilateral Achilles tendinopathy completed a VISA-A questionnaire, and underwent microvascular volume measurements of the Achilles tendon mid-portion using both conventional, ultrasensitive (SMI™) power Doppler ultrasound and CEUS. Intrinsic tendon tenderness was assessed with sensation detection threshold to extracorporeal shock waves (ESW). Linear Mixed Model analysis was used to determine the association between microvascular volume (MV), VISA-A, and ESW-detection threshold for both symptomatic and asymptomatic Achilles tendons. Results There was a significant association between VISA-A and MV (B = −5.3, 95%CI = [−8.5; −2.0], P = 0.0004), and between MV and symptom duration (B = −1.7, 95%CI = [−3.2; −5.0], P = 0.023). No significant associations were found between power Doppler ultrasound and CEUS-based MV or between CEUS-based MV and ESW-detection threshold. In comparison with conventional power Doppler ultrasound, SMI™ showed on average similar detection capacity for neovessels in the mid-portion of the Achilles tendon, whilst being superior for detecting neovessels within Kager’s fat pad (t = 3.46, 95%CI = [0.27; 1.03], P Conclusions Our results indicate that CEUS-based MV of the Achilles tendon is moderately associated with Achilles tendon symptoms. In accordance, CEUS-detected MV could be a novel target for treatment as it seems to be more sensitive than PDU and is correlated with symptoms.

ObjectiveEstablish the prevalence of illness symptoms, poor sleep quality, poor mental health symptoms, low energy availability and stress-recovery state in an Olympic cohort late in the 3 months prior to the Summer Olympic Games.MethodsOlympic athletes (n=317) from 11 sports were invited to complete questionnaires administered 3 months before the Rio 2016 Olympic Games. These questionnaires included the Depression, Anxiety and Stress Questionnaire, Perceived Stress Scale, Dispositional Resilience Scale, Recovery-Stress Questionnaire (REST-Q-52 item), Low Energy Availability in Females Questionnaire (LEAF-Q), Epworth Sleepiness Scale, Pittsburgh Sleep Quality Index and custom-made questionnaires on probiotic usage and travel. Multiple illness (case) definitions were applied. ORs and attributable fractions in the population were used. Factor analyses were used to explore the relationships between variables.ResultsThe response rate was of 42% (male, n=47, age 25.8±4.1 years; female, n=85, age 24.3±3.9 years). Low energy availability was associated with sustaining an illness in the previous month (upper respiratory, OR=3.8, 95% CI 1.2 to 12). The main factor relating to illness pertained to a combination of anxiety and stress-recovery states (as measured by the REST-Q-52 item). All participants reported at least one episode of illness in the last month (100% prevalence).ConclusionsAll participants reported at least one illness symptom in the previous month. Low energy availability was a leading variable associated with illness in Olympic-class athletes. The estimates duration of symptoms ranged from 2 to 7 days. Factor analyses show the interdependence of various health domains and support multidisciplinary care.

Background: With the increasing capacity for remote collection of both data and samples for medical research, a thorough assessment is needed to determine the association of population characteristics and recruitment methodologies with response rates. Objective: The aim of this research was to assess population representativeness in a two-stage study of health and injury in recreational runners, which consisted of an epidemiological arm and genetic analysis. Methods: The cost and success of various classical and internet-based methods were analyzed, and demographic representativeness was assessed for recruitment to the epidemiological survey, reported willingness to participate in the genetic arm of the study, actual participation, sample return, and approval for biobank storage. Results: A total of 4965 valid responses were received, of which 1664 were deemed eligible for genetic analysis. Younger age showed a negative association with initial recruitment rate, expressed willingness to participate in genetic analysis, and actual participation. Additionally, female sex was associated with higher initial recruitment rates, and ethnic origin impacted willingness to participate in the genetic analysis (all P

Background Achilles tendinopathy is the most prevalent tendon disorder in people engaged in running and jumping sports. Aetiology of Achilles tendinopathy is complex and requires comprehensive research of contributing risk factors. There is relatively little research focussing on potential biomedical risk factors for Achilles tendinopathy. The purpose of this systematic review is to identify studies and summarise current knowledge of biomedical risk factors of Achilles tendinopathy in physically active people. Methods Research databases were searched for relevant articles followed by assessment in accordance with PRISMA statement and standards of Cochrane collaboration. Levels of evidence and quality assessment designation were implemented in accordance with OCEBM levels of evidence and Newcastle-Ottawa Quality Assessment Scale, respectively. Results A systematic review of the literature identified 22 suitable articles. All included studies had moderate level of evidence (2b) with the Newcastle-Ottawa score varying between 6 and 9. The majority (17) investigated genetic polymorphisms involved in tendon structure and homeostasis and apoptosis and inflammation pathways. Overweight as a risk factor of Achilles tendinopathy was described in five included studies that investigated non-genetic factors. COL5A1 genetic variants were the most extensively studied, particularly in association with genetic variants in the genes involved in regulation of cell-matrix interaction in tendon and matrix homeostasis. It is important to investigate connections and pathways whose interactions might be disrupted and therefore alter collagen structure and lead to the development of pathology. Polymorphisms in genes involved in apoptosis and inflammation, and Achilles tendinopathy did not show strong association and, however, should be considered for further investigation. Conclusions This systematic review suggests that biomedical risk factors are an important consideration in the future study of propensity to the development of Achilles tendinopathy. The presence of certain medical comorbidities and genetic markers should be considered when contemplating the aetiology of Achilles tendinopathy. Further elucidation of biomedical risk factors will aid in the understanding of tendon pathology and patient risk, thereby informing prevention and management strategies for Achilles tendinopathy. Trial Registration PROSPERO CRD42016036558

The existence of a feedback mechanism regulating the precise amounts of muscle structural proteins, such as actin and the actin-associated protein tropomyosin (Tm), in the sarcomeres of striated muscles is well established. However, the regulation of nonmuscle or cytoskeletal actin and Tms in nonmuscle cell structures has not been elucidated. Unlike the thin filaments of striated muscles, the actin cytoskeleton in nonmuscle cells is intrinsically dynamic. Given the differing requirements for the structural integrity of the actin thin filaments of the sarcomere compared with the requirement for dynamicity of the actin cytoskeleton in nonmuscle cells, we postulated that different regulatory mechanisms govern the expression of sarcomeric versus cytoskeletal Tms, as key regulators of the properties of the actin cytoskeleton. Comprehensive analyses of tissues from transgenic and knock-out mouse lines that overexpress the cytoskeletal Tms, Tm3 and Tm5NM1, and a comparison with sarcomeric Tms provide evidence for this. Moreover, we show that overexpression of a cytoskeletal Tm drives the amount of filamentous actin.

Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is a variably severe neuromuscular disorder for which no effective treatment is available. Although a number of genes have been identified in which mutations can cause NM, the pathogenetic mechanisms leading to the phenotypes are poorly understood. To address this question, we examined gene expression patterns in an NM mouse model carrying the human Met9Arg mutation of alpha-tropomyosin slow (Tpm3). We assessed five different skeletal muscles from affected mice, which are representative of muscles with differing fiber-type compositions, different physiological specializations and variable degrees of pathology. Although these same muscles in non-affected mice showed marked variation in patterns of gene expression, with diaphragm being the most dissimilar, the presence of the mutant protein in nemaline muscles resulted in a more similar pattern of gene expression among the muscles. This result suggests a common process or mechanism operating in nemaline muscles independent of the variable degrees of pathology. Transcriptional and protein expression data indicate the presence of a repair process and possibly delayed maturation in nemaline muscles. Markers indicative of satellite cell number, activated satellite cells and immature fibers including M-Cadherin, MyoD, desmin, Pax7 and Myf6 were elevated by western-blot analysis or immunohistochemistry. Evidence suggesting elevated focal repair was observed in nemaline muscle in electron micrographs. This analysis reveals that NM is characterized by a novel repair feature operating in multiple different muscles.

The functional diversity of the actin microfilaments relies in part on the actin binding protein tropomyosin (Tm). The muscle-specific Tms regulate actin-myosin interactions and hence contraction. However, there is less known about the roles of the numerous cytoskeletal isoforms. We have shown previously that a cytoskeletal Tm, Tm5NM1, defines a Z-line adjacent cytoskeleton in skeletal muscle. Recently, we identified a second cytoskeletal Tm in this region, Tm4. Here we show that Tm4 and Tm5NM1 define separate actin filaments; the former associated with the terminal sarcoplasmic reticulum (SR) and other tubulovesicular structures. In skeletal muscles of Tm5NM1 knockout (KO) mice, Tm4 localization was unchanged, demonstrating the specificity of the membrane association. Tm5NM1 KO muscles exhibit potentiation of T-system depolarization and decreased force rundown with repeated T-tubule depolarizations consistent with altered T-tubule function. These results indicate that a Tm5NM1-defined actin cytoskeleton is required for the normal excitation–contraction coupling in skeletal muscle.

The organisation of structural proteins in muscle into highly ordered sarcomeres occurs during development, regeneration and focal repair of skeletal muscle fibers. The involvement of cytoskeletal proteins in this process has been documented, with nonmuscle γ-actin found to play a role in sarcomere assembly during muscle differentiation and also shown to be up-regulated in dystrophic muscles which undergo regeneration and repair [Lloyd et al.,2004; Hanft et al.,2006]. Here, we show that a cytoskeletal tropomyosin (Tm), Tm4, defines actin filaments in two novel compartments in muscle fibers: a Z-line associated cytoskeleton (Z-LAC), similar to a structure we have reported previously [Kee et al.,2004], and longitudinal filaments that are orientated parallel to the sarcomeric apparatus, present during myofiber growth and repair/regeneration. Tm4 is upregulated in paradigms of muscle repair including induced regeneration and focal repair and in muscle diseases with repair/regeneration features, muscular dystrophy and nemaline myopathy. Longitudinal Tm4-defined filaments also are present in diseased muscle. Transition of the Tm4-defined filaments from a longitudinal to a Z-LAC orientation is observed during the course of muscle regeneration. This Tm4-defined cytoskeleton is a marker of growth and repair/regeneration in response to injury, disease state and stress in skeletal muscle. Cell Motil. Cytoskeleton 2008. © 2007 Wiley-Liss, Inc.