Efficacy and muscle safety assessment of fukutin-related protein gene therapy.

Limb-girdle muscular dystrophy type R9 (LGMDR9) is a muscle-wasting disease that begins in the hip and shoulder regions of the body. This disease is caused by mutations in fukutin-related protein (FKRP), a glycosyltransferase critical for maintaining muscle cell integrity. Here we investigated potential gene therapies for LGMDR9 containing an FKRP expression construct with untranslated region (UTR) modifications. Initial studies treated an aged dystrophic mouse model (FKRPP448L) with adeno-associated virus vector serotype 6 (AAV6). Grip strength improved in a dose- and time-dependent manner, injected mice exhibited fewer central nuclei and serum creatine kinase levels were 3- and 5-fold lower compared to those in non-injected FKRPP448L mice. Treatment also partially stabilized the respiratory pattern during exercise and improved treadmill running, partially protecting muscle from exercise-induced damage. Western blotting of C2C12 myotubes using a novel rabbit antibody confirmed heightened translation with the UTR modifications. We further explored the question of FKRP toxicity in wild-type mice using high doses of two additional muscle-tropic capsids: AAV9 and AAVMYO1. No toxic effects were detected with either therapeutic agent. These data further support the feasibility of gene therapy to treat LGMDR9.

An observational human study investigating the effect of anabolic androgenic steroid use on the transcriptome of skeletal muscle and whole blood using RNA-Seq.

BACKGROUND: The effects of Anabolic Androgenic Steroids (AAS) are largely illustrated through Androgen Receptor induced gene transcription, yet RNA-Seq has yet to be conducted on human whole blood and skeletal muscle. Investigating the transcriptional signature of AAS in blood may aid AAS detection and in muscle further understanding of AAS induced hypertrophy. METHODS: Males aged 20-42 were recruited and sampled once: sedentary controls (C), resistance trained lifters (RT) and resistance trained current AAS users (RT-AS) who ceased exposure ≤ 2 or ≥ 10 weeks prior to sampling. RT-AS were sampled twice as Returning Participants (RP) if AAS usage ceased for ≥ 18 weeks. RNA was extracted from whole blood and trapezius muscle samples. RNA libraries were sequenced twice, for validation purposes, on the DNBSEQ-G400RS with either standard or CoolMPS PE100 reagents following MGI protocols. Genes were considered differentially expressed with FDR < 0.05 and a 1.2- fold change. RESULTS: Cross-comparison of both standard reagent whole blood (N = 55: C = 7, RT = 20, RT-AS ≤ 2 = 14, RT-AS ≥ 10 = 10, RP = 4; N = 46: C = 6, RT = 17, RT-AS ≤ 2 = 12, RT-AS ≥ 10 = 8, RP = 3) sequencing datasets, showed that no genes or gene sets/pathways were differentially expressed between time points for RP or between group comparisons of RT-AS ≤ 2 vs. C, RT, or RT-AS ≥ 10. Cross-comparison of both muscle (N = 51, C = 5, RT = 17, RT-AS ≤ 2 = 15, RT-AS ≥ 10 = 11, RP = 3) sequencing (one standard & one CoolMPS reagent) datasets, showed one gene, CHRDL1, which has atrophying potential, was upregulated in RP visit two. In both muscle sequencing datasets, nine differentially expressed genes, overlapped with RT-AS ≤ 2 vs. RT and RT-AS ≤ 2 vs. C, but were not differentially expressed with RT vs. C, possibly suggesting they are from acute doping alone. No genes seemed to be differentially expressed in muscle after the long-term cessation of AAS, whereas a previous study found long term proteomic changes. CONCLUSION: A whole blood transcriptional signature of AAS doping was not identified. However, RNA-Seq of muscle has identified numerous differentially expressed genes with known impacts on hypertrophic processes that may further our understanding on AAS induced hypertrophy. Differences in training regimens in participant groupings may have influenced results. Future studies should focus on longitudinal sampling pre, during and post-AAS exposure to better control for confounding variables.

The MMAAS Project: An Observational Human Study Investigating the Effect of Anabolic Androgenic Steroid Use on Gene Expression and the Molecular Mechanism of Muscle Memory.

OBJECTIVE: It remains unknown whether myonuclei remain elevated post anabolic-androgenic steroid (AAS) usage in humans. Limited data exist on AAS-induced changes in gene expression. DESIGN: Cross-sectional/longitudinal. SETTING: University. PARTICIPANTS: Fifty-six men aged 20 to 42 years. INDEPENDENT VARIABLES: Non-resistance-trained (C) or resistance-trained (RT), RT currently using AAS (RT-AS), of which if AAS usage ceased for ≥18 weeks resampled as Returning Participants (RP) or RT previously using AAS (PREV). MAIN OUTCOME MEASURES: Myonuclei per fiber and cross-sectional area (CSA) of trapezius muscle fibers. RESULTS: There were no significant differences between C (n = 5), RT (n = 15), RT-AS (n = 17), and PREV (n = 6) for myonuclei per fiber. Three of 5 returning participants (RP1-3) were biopsied twice. Before visit 1, RP1 ceased AAS usage 34 weeks before, RP2 and RP3 ceased AAS usage ≤2 weeks before, and all had 28 weeks between visits. Fiber CSA decreased for RP1 and RP2 between visits (7566 vs 6629 µm 2 ; 7854 vs 5677 µm 2 ) while myonuclei per fiber remained similar (3.5 vs 3.4; 2.5 vs 2.6). Respectively, these values increased for RP3 between visits (7167 vs 7889 µm 2 ; 2.6 vs 3.3). CONCLUSIONS: This cohort of past AAS users did not have elevated myonuclei per fiber values, unlike previous research, but reported AAS usage was much lower. Training and AAS usage history also varied widely among participants. Comparable myonuclei per fiber numbers despite decrements in fiber CSA postexposure adheres with the muscle memory mechanism, but there is variation in usage relative to sampling date and low numbers of returning participants.

ACTN3 genotype influences skeletal muscle mass regulation and response to dexamethasone.

Homozygosity for the common ACTN3 null polymorphism (ACTN3 577X) results in α-actinin-3 deficiency in ~20% of humans worldwide and is linked to reduced sprint and power performance in both elite athletes and the general population. α-Actinin-3 deficiency is also associated with reduced muscle mass, increased risk of sarcopenia, and altered muscle wasting response induced by denervation and immobilization. Here, we show that α-actinin-3 plays a key role in the regulation of protein synthesis and breakdown signaling in skeletal muscle and influences muscle mass from early postnatal development. We also show that α-actinin-3 deficiency reduces the atrophic and anti-inflammatory response to the glucocorticoid dexamethasone in muscle and protects against dexamethasone-induced muscle wasting in female but not male mice. The effects of α-actinin-3 deficiency on muscle mass regulation and response to muscle wasting provide an additional mechanistic explanation for the positive selection of the ACTN3 577X allele in recent human history.

Integrating Transwomen and Female Athletes with Differences of Sex Development (DSD) into Elite Competition: The FIMS 2021 Consensus Statement.

Sport is historically designated by the binary categorization of male and female that conflicts with modern society. Sport's governing bodies should consider reviewing rules determining the eligibility of athletes in the female category as there may be lasting advantages of previously high testosterone concentrations for transwomen athletes and currently high testosterone concentrations in differences in sex development (DSD) athletes. The use of serum testosterone concentrations to regulate the inclusion of such athletes into the elite female category is currently the objective biomarker that is supported by most available scientific literature, but it has limitations due to the lack of sports performance data before, during or after testosterone suppression. Innovative research studies are needed to identify other biomarkers of testosterone sensitivity/responsiveness, including molecular tools to determine the functional status of androgen receptors. The scientific community also needs to conduct longitudinal studies with specific control groups to generate the biological and sports performance data for individual sports to inform the fair inclusion or exclusion of these athletes. Eligibility of each athlete to a sport-specific policy needs to be based on peer-reviewed scientific evidence made available to policymakers from all scientific communities. However, even the most evidence-based regulations are unlikely to eliminate all differences in performance between cisgender women with and without DSD and transwomen athletes. Any remaining advantage held by transwomen or DSD women could be considered as part of the athlete's unique makeup.

Sport and exercise genomics: the FIMS 2019 consensus statement update.

Rapid advances in technologies in the field of genomics such as high throughput DNA sequencing, big data processing by machine learning algorithms and gene-editing techniques are expected to make precision medicine and gene-therapy a greater reality. However, this development will raise many important new issues, including ethical, moral, social and privacy issues. The field of exercise genomics has also advanced by incorporating these innovative technologies. There is therefore an urgent need for guiding references for sport and exercise genomics to allow the necessary advancements in this field of sport and exercise medicine, while protecting athletes from any invasion of privacy and misuse of their genomic information. Here, we update a previous consensus and develop a guiding reference for sport and exercise genomics based on a SWOT (Strengths, Weaknesses, Opportunities and Threats) analysis. This SWOT analysis and the developed guiding reference highlight the need for scientists/clinicians to be well-versed in ethics and data protection policy to advance sport and exercise genomics without compromising the privacy of athletes and the efforts of international sports federations. Conducting research based on the present guiding reference will mitigate to a great extent the risks brought about by inappropriate use of genomic information and allow further development of sport and exercise genomics in accordance with best ethical standards and international data protection principles and policies. This guiding reference should regularly be updated on the basis of new information emerging from the area of sport and exercise medicine as well as from the developments and challenges in genomics of health and disease in general in order to best protect the athletes, patients and all other relevant stakeholders.

Association Between Hematological Parameters and Iron Metabolism Response After Marathon Race and ACTN3 Genotype.

α-Actinin-3 (ACTN3 R577X, rs.1815739) polymorphism is a genetic variation that shows the most consistent influence on metabolic pathway and muscle phenotype. XX genotype is associated with higher metabolic efficiency of skeletal muscle; however, the role of ACTN3 polymorphism in oxygen transport and utilization system has not yet been investigated. Therefore, the aim of this study was to determine the influence of ACTN3 polymorphisms on hematological and iron metabolism response induced by marathon race. Eighty-one Brazilian amateur male endurance runners participated in the study. Blood samples and urine were collected before; immediately after; and 1, 3, and 15 days after the marathon race. Urine, hematological parameters, iron metabolism, and ACTN3 genotyping analyses were performed. The marathon race induced a decrease in erythrocytes, Hb, and Ht, and an increase in hematuria, creatinine, myoglobin, red cell distribution width, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, direct and indirect bilirubin and erythropoietin. Moreover, an elevation immediately or 1 day after the marathon race follows a reduction 3 or 15 days after the marathon race were observed on transferrin saturation and iron and transferrin levels. Hematological parameters and iron metabolism changes induced by marathon race were not observed in XX genotypes. Hematuria and decreased erythrocytes, Hb, Ht, and iron and transferrin levels were observed only in RR and/or RX genotypes but not in XX genotypes. The percentage of runners with hematuria, leukocyturia, iron deficiency, creatinine, myoglobin, and bilirubin imbalance was higher in RR compared to XX genotypes. ACTN3 polymorphism is associated with iron metabolism and hematological responses after endurance exercise. Despite these results being based on a small sample, they highlight a protective role of the XX genotype on hematological and renal changes induced by long-distance exercise. Therefore, these findings should be further replicated.

Cullin-3 dependent deregulation of ACTN1 represents a new pathogenic mechanism in nemaline myopathy.

Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness, fiber atrophy and presence of nemaline bodies within myofibers. However, the understanding of underlying pathomechanisms is lacking. Recently, mutations in KBTBD13, KLHL40 and KLHL41, three substrate adaptors for the E3-ubiquitin ligase Cullin-3, have been associated with early-onset nemaline myopathies. We hypothesized that deregulation of Cullin-3 and its muscle protein substrates may be responsible for the disease development. Using Cullin-3 knockout mice, we identified accumulation of non-muscle alpha-Actinins (ACTN1 and ACTN4) in muscles of these mice, which we also observed in KBTBD13 patients. Our data reveal that proper regulation of Cullin-3 activity and ACTN1 levels is essential for normal muscle and neuromuscular junction development. While ACTN1 is naturally downregulated during myogenesis, its overexpression in C2C12 myoblasts triggered defects in fusion, myogenesis and acetylcholine receptor clustering; features that we characterized in Cullin-3 deficient mice. Taken together, our data highlight the importance for Cullin-3 mediated degradation of ACTN1 for muscle development, and indicate a new pathomechanism for the etiology of myopathies seen in Cullin-3 knockout mice and nemaline myopathy patients.

Is evolutionary loss our gain? The role of ACTN3 p.Arg577Ter (R577X) genotype in athletic performance, ageing, and disease.

A common null polymorphism in the ACTN3 gene (rs1815739:C>T) results in replacement of an arginine (R) with a premature stop codon (X) at amino acid 577 in the fast muscle protein α-actinin-3. The ACTN3 p.Arg577Ter allele (aka p.R577* or R577X) has undergone positive selection, with an increase in the X allele frequency as modern humans migrated out of Africa into the colder, less species-rich Eurasian climates suggesting that the absence of α-actinin-3 may be beneficial in these conditions. Approximately 1.5 billion people worldwide are completely deficient in α-actinin-3. While the absence of α-actinin-3 influences skeletal muscle function and metabolism this does not result in overt muscle disease. α-Actinin-3 deficiency (ACTN3 XX genotype) is constantly underrepresented in sprint/power performance athletes. However, recent findings from our group and others suggest that the ACTN3 R577X genotype plays a role beyond athletic performance with effects observed in ageing, bone health, and inherited muscle disorders such as McArdle disease and Duchenne muscle dystrophy. In this review, we provide an update on the current knowledge regarding the influence of ACTN3 R577X on skeletal muscle function and its potential biological and clinical implications. We also outline future research directions to explore the role of α-actinin-3 in healthy and diseased populations.

The Effect of ACTN3 Gene Doping on Skeletal Muscle Performance.

Loss of expression of ACTN3, due to homozygosity of the common null polymorphism (p.Arg577X), is underrepresented in elite sprint/power athletes and has been associated with reduced muscle mass and strength in humans and mice. To investigate ACTN3 gene dosage in performance and whether expression could enhance muscle force, we performed meta-analysis and expression studies. Our general meta-analysis using a Bayesian random effects model in elite sprint/power athlete cohorts demonstrated a consistent homozygous-group effect across studies (per allele OR = 1.4, 95% CI 1.3-1.6) but substantial heterogeneity in heterozygotes. In mouse muscle, rAAV-mediated gene transfer overexpressed and rescued α-actinin-3 expression. Contrary to expectation, in vivo "doping" of ACTN3 at low to moderate doses demonstrated an absence of any change in function. At high doses, ACTN3 is toxic and detrimental to force generation, to demonstrate gene doping with supposedly performance-enhancing isoforms of sarcomeric proteins can be detrimental for muscle function. Restoration of α-actinin-3 did not enhance muscle mass but highlighted the primary role of α-actinin-3 in modulating muscle metabolism with altered fatiguability. This is the first study to express a Z-disk protein in healthy skeletal muscle and measure the in vivo effect. The sensitive balance of the sarcomeric proteins and muscle function has relevant implications in areas of gene doping in performance and therapy for neuromuscular disease.

Androgen Action via the Androgen Receptor in Neurons Within the Brain Positively Regulates Muscle Mass in Male Mice.

Although it is well established that exogenous androgens have anabolic effects on skeletal muscle mass in humans and mice, data from muscle-specific androgen receptor (AR) knockout (ARKO) mice indicate that myocytic expression of the AR is dispensable for hind-limb muscle mass accrual in males. To identify possible indirect actions of androgens via the AR in neurons to regulate muscle, we generated neuron-ARKO mice in which the dominant DNA binding-dependent actions of the AR are deleted in neurons of the cortex, forebrain, hypothalamus, and olfactory bulb. Serum testosterone and luteinizing hormone levels were elevated twofold in neuron-ARKO males compared with wild-type littermates due to disruption of negative feedback to the hypothalamic-pituitary-gonadal axis. Despite this increase in serum testosterone levels, which was expected to increase muscle mass, the mass of the mixed-fiber gastrocnemius (Gast) and the fast-twitch fiber extensor digitorum longus hind-limb muscles was decreased by 10% in neuron-ARKOs at 12 weeks of age, whereas muscle strength and fatigue of the Gast were unaffected. The mass of the soleus muscle, however, which consists of a high proportion of slow-twitch fibers, was unaffected in neuron-ARKOs, demonstrating a stimulatory action of androgens via the AR in neurons to increase the mass of fast-twitch hind-limb muscles. Furthermore, neuron-ARKOs displayed reductions in voluntary and involuntary physical activity by up to 60%. These data provide evidence for a role of androgens via the AR in neurons to positively regulate fast-twitch hind-limb muscle mass and physical activity in male mice.

Progress and prospects of gene therapy clinical trials for the muscular dystrophies.

Clinical trials represent a critical avenue for new treatment development, where early phases (I, I/II) are designed to test safety and effectiveness of new therapeutics or diagnostic indicators. A number of recent advances have spurred renewed optimism toward initiating clinical trials and developing refined therapies for the muscular dystrophies (MD's) and other myogenic disorders. MD's encompass a heterogeneous group of degenerative disorders often characterized by progressive muscle weakness and fragility. Many of these diseases result from mutations in genes encoding proteins of the dystrophin-glycoprotein complex (DGC). The most common and severe form among children is Duchenne muscular dystrophy, caused by mutations in the dystrophin gene, with an average life expectancy around 25 years of age. Another group of MD's referred to as the limb-girdle muscular dystrophies (LGMDs) can affect boys or girls, with different types caused by mutations in different genes. Mutation of the α-sarcoglycan gene, also a DGC component, causes LGMD2D and represents the most common form of LGMD. Early preclinical and clinical trial findings support the feasibility of gene therapy via recombinant adeno-associated viral vectors as a viable treatment approach for many MDs. In this mini-review, we present an overview of recent progress in clinical gene therapy trials of the MD's and touch upon promising preclinical advances.

Analysis of the ACTN3 heterozygous genotype suggests that α-actinin-3 controls sarcomeric composition and muscle function in a dose-dependent fashion.

A common null polymorphism (R577X) in ACTN3 causes α-actinin-3 deficiency in ∼ 18% of the global population. There is no associated disease phenotype, but α-actinin-3 deficiency is detrimental to sprint and power performance in both elite athletes and the general population. However, despite considerable investigation to date, the functional consequences of heterozygosity for ACTN3 are unclear. A subset of studies have shown an intermediate phenotype in 577RX individuals, suggesting dose-dependency of α-actinin-3, while others have shown no difference between 577RR and RX genotypes. Here, we investigate the effects of α-actinin-3 expression level by comparing the muscle phenotypes of Actn3(+/-) (HET) mice to Actn3(+/+) [wild-type (WT)] and Actn3(-/-) [knockout (KO)] littermates. We show reduction in α-actinin-3 mRNA and protein in HET muscle compared with WT, which is associated with dose-dependent up-regulation of α-actinin-2, z-band alternatively spliced PDZ-motif and myotilin at the Z-line, and an incremental shift towards oxidative metabolism. While there is no difference in force generation, HET mice have an intermediate endurance capacity compared with WT and KO. The R577X polymorphism is associated with changes in ACTN3 expression consistent with an additive model in the human genotype-tissue expression cohort, but does not influence any other muscle transcripts, including ACTN2. Overall, ACTN3 influences sarcomeric composition in a dose-dependent fashion in mouse skeletal muscle, which translates directly to function. Variance in fibre type between biopsies likely masks this phenomenon in human skeletal muscle, but we suggest that an additive model is the most appropriate for use in testing ACTN3 genotype associations.

Therapy of Genetic Disorders-Novel Therapies for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is an inherited, progressive muscle wasting disorder caused by mutations in the dystrophin gene. An increasing variety of approaches are moving towards clinical testing that all aim to restore dystrophin production and to enhance or preserve muscle mass. Gene therapy methods are being developed to replace the defective dystrophin gene or induce dystrophin production from mutant genes. Stem cell approaches are being developed to replace lost muscle cells while also bringing in new dystrophin genes. This review summarizes recent progress in the field with an emphasis on clinical applications.

NF1 is a critical regulator of muscle development and metabolism.

There is emerging evidence for reduced muscle function in children with neurofibromatosis type 1 (NF1). We have examined three murine models featuring NF1 deficiency in muscle to study the effect on muscle function as well as any underlying pathophysiology. The Nf1(+/-) mouse exhibited no differences in overall weight, lean tissue mass, fiber size, muscle weakness as measured by grip strength or muscle atrophy-recovery with limb disuse, although this model lacks many other characteristic features of the human disease. Next, muscle-specific knockout mice (Nf1muscle(-/-)) were generated and they exhibited a failure to thrive leading to neonatal lethality. Intramyocellular lipid accumulations were observed by electron microscopy and Oil Red O staining. More mature muscle specimens lacking Nf1 expression taken from the limb-specific Nf1Prx1(-/-) conditional knockout line showed a 10-fold increase in muscle triglyceride content. Enzyme assays revealed a significant increase in the activities of oxidative metabolism enzymes in the Nf1Prx1(-/-) mice. Western analyses showed increases in the expression of fatty acid synthase and the hormone leptin, as well as decreased expression of a number of fatty acid transporters in this mouse line. These data support the hypothesis that NF1 is essential for normal muscle function and survival and are the first to suggest a direct link between NF1 and mitochondrial fatty acid metabolism.

ACTN3 genotype influences muscle performance through the regulation of calcineurin signaling.

α-Actinin-3 deficiency occurs in approximately 16% of the global population due to homozygosity for a common nonsense polymorphism in the ACTN3 gene. Loss of α-actinin-3 is associated with reduced power and enhanced endurance capacity in elite athletes and nonathletes due to "slowing" of the metabolic and physiological properties of fast fibers. Here, we have shown that α-actinin-3 deficiency results in increased calcineurin activity in mouse and human skeletal muscle and enhanced adaptive response to endurance training. α-Actinin-2, which is differentially expressed in α-actinin-3-deficient muscle, has higher binding affinity for calsarcin-2, a key inhibitor of calcineurin activation. We have further demonstrated that α-actinin-2 competes with calcineurin for binding to calsarcin-2, resulting in enhanced calcineurin signaling and reprogramming of the metabolic phenotype of fast muscle fibers. Our data provide a mechanistic explanation for the effects of the ACTN3 genotype on skeletal muscle performance in elite athletes and on adaptation to changing physical demands in the general population. In addition, we have demonstrated that the sarcomeric α-actinins play a role in the regulation of calcineurin signaling.

Gene replacement therapies for duchenne muscular dystrophy using adeno-associated viral vectors.

The muscular dystrophies collectively represent a major health challenge, as few significant treatment options currently exist for any of these disorders. Recent years have witnessed a proliferation of novel approaches to therapy, spanning increased testing of existing and new pharmaceuticals, DNA delivery (both anti-sense oligonucleotides and plasmid DNA), gene therapies and stem cell technologies. While none of these has reached the point of being used in clinical practice, all show promise for being able to impact different types of muscular dystrophies. Our group has focused on developing direct gene replacement strategies to treat recessively inherited forms of muscular dystrophy, particularly Duchenne and Becker muscular dystrophy (DMD/BMD). Both forms of dystrophy are caused by mutations in the dystrophin gene and all cases can in theory be treated by gene replacement using synthetic forms of the dystrophin gene. The major challenges for success of this approach are the development of a suitable gene delivery shuttle, generating a suitable gene expression cassette able to be carried by such a shuttle, and achieving safe and effective delivery without elicitation of a destructive immune response. This review summarizes the current state of the art in terms of using adeno-associated viral vectors to deliver synthetic dystrophin genes for the purpose of developing gene therapy for DMD.

Animal models of muscular dystrophy.

The muscular dystrophies (MDs) represent a diverse collection of inherited human disorders, which affect to varying degrees skeletal, cardiac, and sometimes smooth muscle (Emery, 2002). To date, more than 50 different genes have been implicated as causing one or more types of MD (Bansal et al., 2003). In many cases, invaluable insights into disease mechanisms, structure and function of gene products, and approaches for therapeutic interventions have benefited from the study of animal models of the different MDs (Arnett et al., 2009). The large number of genes that are associated with MD and the tremendous number of animal models that have been developed preclude a complete discussion of each in the context of this review. However, we summarize here a number of the more commonly used models together with a mixture of different types of gene and MD, which serves to give a general overview of the value of animal models of MD for research and therapeutic development.