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The absence of dystrophin hypersensitizes skeletal muscle of lower and higher vertebrates to eccentric contraction (ECC)-induced strength loss. Loss of strength can be accompanied by transient and reversible alterations to sarcolemmal excitability and disruption, triad dysfunction, and aberrations in calcium kinetics and reactive oxygen species production. The degree of ECC-induced strength loss, however, appears dependent on several extrinsic and intrinsic factors such as vertebrate model, skeletal muscle preparation (in vivo, in situ, or ex vivo), skeletal muscle hierarchy (single fiber versus whole muscle and permeabilized versus intact), strength production, fiber branching, age, and genetic background, among others. Consistent findings across research groups show that dystrophin-deficient fast(er)-twitch muscle is hypersensitive to ECCs relative to wildtype muscle, but because preparations are highly variable and sensitivity to ECCs are used repeatedly to determine efficacy of many preclinical treatments, it is critical to evaluate the impact of skeletal muscle preparations on sensitivity to ECC-induced strength loss in dystrophin-deficient skeletal muscle. Here, we review and discuss variations in skeletal muscle preparations to evaluate the factors responsible for variations and discrepancies between research groups. We further highlight that dystrophin-deficiency, or loss of the dystrophin–glycoprotein complex in skeletal muscle, is not a prerequisite for accelerated strength loss-induced by ECCs.

INTRODUCTION/AIMS Clinical trials addressing treatments for Duchenne muscular dystrophy (DMD) require reliable and valid measurement of muscle contractile function across all disease severity levels. In this work we aimed to evaluate a protocol combining voluntary and evoked contractions to measure strength and excitability of wrist extensor muscles for safety, feasibility, reliability, and discriminant validity between males with DMD and controls. METHODS Wrist extensor muscle strength and excitability were assessed in males with DMD (N = 10; mean ± standard deviation: 15.4 ± 5.9 years of age), using the Brooke Upper Extremity Rating Scale (scored 1-6), and age-matched healthy male controls (N = 15; 15.5 ± 5.0 years of age). Torque and electromyographic (EMG) measurements were analyzed under maximum voluntary and stimulated conditions at two visits. RESULTS A protocol of multiple maximal voluntary contractions (MVCs) and evoked twitch contractions was feasible and safe, with 96% of the participants completing the protocol and having a less than 7% strength decrement on either measure for both DMD patients and controls (P ≥ .074). Reliability was excellent for voluntary and evoked measurements of torque and EMG (intraclass correlation coefficient [ICC] over 0.90 and over 0.85 within and between visits, respectively). Torque, EMG, and timing of twitch-onset measurements discriminated between DMD and controls (P

The actions of selective estrogen receptor modulators are tissue dependent. The primary objective of the current study was to determine the tissue selective effects of bazedoxifene (BZA) on the musculoskeletal system of ovariectomized (OVX) female mice, focusing on the strengths of muscle-bone pairs in the lower hindlimb. Treatment with BZA after ovariectomy (OVX+BZA) did not prevent body or fat mass gains (P < 0.05). In vivo plantarflexor muscle isometric torque was not affected by treatment with BZA (P = 0.522). Soleus muscle peak isometric, concentric and eccentric tetanic force production were greater in OVX+BZA mice compared to OVX+E2 mice (P ≤ 0.048) with no effect on maximal isometric specific force (P = 0.228). Tibia from OVX+BZA mice had greater cortical cross-sectional area and moment of inertia than OVX mice treated with placebo (P < 0.001), but there was no impact of BZA treatment on cortical bone mineral density, cortical thickness, tibial bone ultimate load or stiffness (P ≥ 0.086). Overall, these results indicate that BZA may be an estrogen receptor agonist in skeletal muscle, as it has previously been shown in bone, providing minor benefits to the musculoskeletal system.

The greatest muscle strength adaptations to repeated bouts of eccentric contractions (ECC) occur after the initial injury, with little to no change in subsequent bouts. However, because of the disparity in injury models, it is unknown whether three or more bouts provide further adaptation. Therefore, we performed a systematic review of the literature to evaluate whether a third bout of skeletal muscle ECC impacts immediate strength loss and rate of strength recovery compared with a second bout. A search of the literature in Web of Science, SCOPUS, Medline, and the American College of Sports Medicine database was conducted between May and September 2019 using the keywords eccentric contraction or lengthening contraction and muscle and repeated or multiple, and bout. Eleven studies with 12 experimental groups, using 72 human subjects, 48 mice, and 11 rabbits, met the inclusion criteria. A meta-analysis using a random effects model and effect sizes (ESs; Hedges' g) calculated from the standardized mean differences was completed. Calculated ESs for immediate strength loss provided no evidence that a third bout of ECC results in greater loss of strength compared with a second bout (ES = -0.12, 95% confidence interval [CI] = -0.41 to 0.17). Furthermore, the rate of strength recovery was not different between a second and third bout (ES = -0.15, 95% CI = -1.01 to 0.70). These results indicate a third bout of skeletal muscle ECC does not improve indices of strength loss or rate of strength recovery compared with a second bout. Therefore, coaches and athletes should expect some level of persistent weakness after each of their initial training sessions involving ECC, and the faster recovery of strength deficits in the second bout documented by previous research is not different from a third bout.

Skeletal muscle is sensitive to environmental cues that are first present in utero. Maternal overnutrition is a model of impaired muscle development leading to structural and metabolic dysfunction in adult life. In this study, we investigated the effect of an obesogenic maternal environment on growth and postnatal myogenesis in the offspring. Male C57BL/6J mice born to chow- or high-fat-diet-fed mothers were allocated to four different groups at the end of weaning. For the following 10 wk, half of the pups were maintained on the same diet as their mother and half of the pups were switched to the other diet (chow or high-fat). At 12 wk of age, muscle injury was induced using an intramuscular injection of barium chloride. Seven days later, mice were humanely killed and muscle tissue was harvested. A high-fat maternal diet impaired offspring growth patterns and downregulated satellite cell activation and markers of postnatal myogenesis 7 days after injury without altering the number of newly synthetized fibers over the whole 7-day period. Importantly, a healthy postnatal diet could not reverse any of these effects. In addition, we demonstrated that postnatal myogenesis was associated with a diet-independent upregulation of three miRNAs, mmu-miR-31–5p, mmu-miR-136–5p, and mmu-miR-296–5p. Furthermore, in vitro analysis confirmed the role of these miRNAs in myocyte proliferation. Our findings are the first to demonstrate that maternal overnutrition impairs markers of postnatal myogenesis in the offspring and are particularly relevant to today’s society where the incidence of overweight/obesity in women of childbearing age is increasing.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by loss of repression of the DUX4 gene; however, the DUX4 protein is rare and difficult to detect in human muscle biopsies, and pathological mechanisms are obscure. FSHD is also a chronic disease that progresses slowly over decades. We used the sporadic, low-level, muscle-specific expression of DUX4 enabled by the iDUX4pA-HSA mouse to develop a chronic long-term muscle disease model. After 6 months of extremely low sporadic DUX4 expression, dystrophic muscle presented hallmarks of FSHD histopathology, including muscle degeneration, capillary loss, fibrosis, and atrophy. We investigated the transcriptional profile of whole muscle as well as endothelial cells and fibroadiopogenic progenitors (FAPs). Strikingly, differential gene expression profiles of both whole muscle and, to a lesser extent, FAPs, showed significant overlap with transcriptional profiles of MRI-guided human FSHD muscle biopsies. These results demonstrate a pathophysiological similarity between disease in muscles of iDUX4pA-HSA mice and humans with FSHD, solidifying the value of chronic rare DUX4 expression in mice for modeling pathological mechanisms in FSHD and highlighting the importance FAPs in this disease.

Skeletal muscle dysfunction may contribute to the progression and severity of amyotrophic lateral sclerosis (ALS). In the present study, we characterized the skeletal muscle pathophysiology in an inducible transgenic mouse model (rNLS8) that develops a TAR-DNA binding protein (TDP-43) proteinopathy and ALS-like neuropathology and disease progression; representative of >90% of all familial and sporadic ALS cases. As we previously observed elevated levels of miR-23a in skeletal muscle of patients with familial and sporadic ALS, we also investigated the effect of miR-23a suppression on skeletal muscle pathophysiology and disease severity in rNLS8 mice. Five weeks after disease onset TDP-43 protein accumulation was observed in tibialis anterior (TA), quadriceps (QUAD) and diaphragm muscle lysates and associated with skeletal muscle atrophy. In the TA muscle TDP-43 was detected in muscle fibres that appeared atrophied and angular in appearance and that also contained β-amyloid aggregates. These fibres were also positive for neural cell adhesion molecule (NCAM), but not embryonic myosin heavy chain (eMHC), indicating TDP-43/ β-amyloid localization in denervated muscle fibres. There was an upregulation of genes associated with myogenesis and NMJ degeneration and a decrease in the MURF1 atrophy-related protein in skeletal muscle. Suppression of miR-23a impaired rotarod performance and grip strength and accelerated body weight loss during early stages of disease progression. This was associated with increased AchRα mRNA expression and decreased protein levels of PGC-1α. The TDP-43 proteinopathy-induced impairment of whole body and skeletal muscle functional performance is associated with muscle wasting and elevated myogenic and NMJ stress markers. Suppressing miR-23a in the rNLS8 mouse model of ALS contributes to an early acceleration of disease progression as measured by decline in motor function.

Mutation to the gene encoding dystrophin can cause Duchenne muscular dystrophy (DMD) and increase the sensitivity to stress in vertebrate species, including the mdx mouse model of DMD. Behavioral stressors can exacerbate some dystrophinopathy phenotypes of mdx skeletal muscle and cause hypotension-induced death. However, we have discovered that a subpopulation of mdx mice present with a wildtype-like response to mild (forced downhill treadmill exercise) and moderate (scruff restraint) behavioral stressors. These "stress-resistant" mdx mice are more physically active, capable of super-activating the hypothalamic-pituitary-adrenal and renin-angiotensin-aldosterone pathways following behavioral stress and they express greater levels of mineralocorticoid and glucocorticoid receptors in striated muscle relative to "stress-sensitive" mdx mice. Stress-resistant mdx mice also presented with a less severe striated muscle histopathology and greater exercise and skeletal muscle oxidative capacity at rest. Most interestingly, female mdx mice were more physically active following behavioral stressors compared to male mdx mice; a response abolished after ovariectomy and rescued with estradiol. We demonstrate that the response to behavioral stress greatly impacts disease severity in mdx mice suggesting the management of stress in patients with DMD be considered as a therapeutic approach to ameliorate disease progression.

This thesis describes the integration of different techniques - tumor tissue, liquid biopsy and radiomics analyses - to improve genomics-guided personalized cancer treatment

Objectives To evaluate pterins as diagnostic biomarkers of exercise-induced stress. Design Systematic review of the literature. Methods MEDLINE, Scopus and Web of Science were searched in March 2019 for relevant literature. We only considered in vivo studies of healthy humans that reported measurement of a pterin(s) in response to exercise or sport with no underlying prior disease or complication. Relevant articles were independently reviewed and resolved by consensus. Results We included 29 studies with 644 participants. We classified articles by running/hiking, cycling, rugby, mixed martial arts (MMA) or other. Eighty-six percent of studies measured a significant increase in a pterin in response to exercise. Changes in pterin concentrations were within 24 h of the exercise-stimulus in 79% of studies and 17% measured a change from baseline greater than 48 h post-exercise (49% did not measure or report beyond 48 h). Neopterin or total neopterin (neopterin + 7,8-dihydroneopterin) were the primary pterin measured (28 studies) and they were equally sensitive to exercise regardless of whether the stimulus was running, cycling, rugby, MMA or other. Conclusions Neopterin and total neopterin increase in response to exercise-induced stress. Pterins may have limited capacity for monitoring long-term stress beyond 48 h but further research is required.

Objective Phosphatidylethanolamine (PtdEtn) is a major phospholipid in mammals. It is synthesized via two pathways, the CDP-ethanolamine pathway in the endoplasmic reticulum and the phosphatidylserine (PtdSer) decarboxylase (PSD) pathway in the mitochondria. While the CDP-ethanolamine pathway is considered the major route for PtdEtn synthesis in most mammalian tissues, little is known about the importance of the PSD pathway in vivo, especially in tissues enriched with mitochondria such as skeletal muscle. Therefore, we aimed to examine the role of the mitochondrial PSD pathway in regulating PtdEtn homeostasis in skeletal muscle in vivo. Methods To determine the functional significance of this pathway in skeletal muscle in vivo, an adeno-associated viral vector approach was employed to knockdown PSD expression in skeletal muscle of adult mice. Muscle lipid and metabolite profiling was performed using mass spectrometry. Results PSD knockdown disrupted muscle phospholipid homeostasis leading to an ∼25% reduction in PtdEtn and an ∼45% increase in PtdSer content. This was accompanied by the development of a severe myopathy, evident by a 40% loss in muscle mass as well as extensive myofiber damage as shown by increased DNA synthesis and central nucleation. In addition, PSD knockdown caused marked accumulation of abnormally appearing mitochondria that exhibited severely disrupted inner membrane integrity and reduced OXPHOS protein content. Conclusions The PSD pathway has a significant role in maintaining phospholipid homeostasis in adult skeletal muscle. Moreover, PSD is essential for maintenance of mitochondrial integrity and skeletal muscle mass., Highlights • The PSD pathway has an important role in regulating muscle phospholipid homeostasis. • Disrupting the PSD pathway caused marked muscle wasting and myofibre damage. • Knockdown of PSD caused accumulation of mitochondria with ultrastructural defects. • PSD is important in regulating mitochondrial integrity and skeletal muscle mass.

Eccentric contractions (ECCs) induce force loss in several skeletal muscles of dystrophin-deficient mice (mdx), with the exception of the soleus (Sol). The eccentric force : isometric force (ECC : ISO), expression level of utrophin, fiber type distribution, and sarcoendoplasmic reticulum calcium ATPase expression are factors that differ between muscles and may contribute to the sensitivity of mdx skeletal muscle to ECC. Here, we confirm that the Sol of mdx mice loses only 13% force compared to 87% in the extensor digitorum longus (EDL) following 10 ECC of isolated muscles. The Sol has a greater proportion of fibers expressing Type I myosin heavy chain (MHC) and expresses 2.3-fold more utrophin compared to the EDL. To examine the effect of ECC : ISO, we show that the mdx Sol is insensitive to ECC at ECC : ISO up to 230 ± 15%. We show that the peroneus longus (PL) muscle presents with similar ECC : ISO compared to the EDL, intermediate force loss (68%) following 10 ECC, and intermediate fiber type distribution and utrophin expression relative to EDL and Sol. The combined absence of utrophin and dystrophin in mdx/utrophin-/- mice rendered the Sol only partially susceptible to ECC and exacerbated force loss in the EDL and PL. Most interestingly, the expression levels of cytoplasmic β- and γ-actins correlate inversely with a given muscle's sensitivity to ECC; EDL

Patients with Duchenne muscular dystrophy (DMD) lack the protein dystrophin, which is a critical molecular component of the dystrophin-glycoprotein complex (DGC). Dystrophin is hypothesized to function as a molecular shock absorber that mechanically stabilizes the sarcolemma of striated muscle through interaction with the cortical actin cytoskeleton via its N-terminal half and with the transmembrane protein β-dystroglycan via its C-terminal region. Utrophin is a fetal homologue of dystrophin that can subserve many dystrophin functions and is therefore under active investigation as a dystrophin replacement therapy for DMD. Here, we report the first mechanical characterization of utrophin using atomic force microscopy (AFM). Our data indicate that the mechanical properties of spectrin-like repeats in utrophin are more in line with the PEVK and Ig-like repeats of titin rather than those reported for repeats in spectrin or dystrophin. Moreover, we measured markedly different unfolding characteristics for spectrin repeats within the N-terminal actin-binding half of utrophin compared to those in the C-terminal dystroglycan-binding half, even though they exhibit identical thermal denaturation profiles. Our results demonstrate dramatic differences in the mechanical properties of structurally homologous utrophin constructs and suggest that utrophin may function as a stiff elastic element in series with titin at the myotendinous junction.

Mutation to the dystrophin gene causes skeletal muscle weakness in patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). Deliberation continues regarding implications of prescribing exercise for these patients. The purpose of this study was to determine whether isometric resistance exercise (~10 tetanic contractions/session) improves skeletal muscle strength and histopathology in the mdx mouse model of DMD. Three isometric training sessions increased in vivo isometric torque (22%) and contractility rates (54%) of anterior crural muscles of mdx mice. Mice expressing a BMD-causing missense mutated dystrophin on the mdx background showed comparable increases in torque (22%), while wild-type mice showed less change (11%). Increases in muscle function occurred within 1 h and peaked 3 days posttraining; however, the adaptation was lost after 7 days unless retrained. Six isometric training sessions over 4 wk caused increased isometric torque (28%) and contractility rates (22–28%), reduced fibrosis, as well as greater uniformity of fiber cross-sectional areas, fewer embryonic myosin heavy-chain-positive fibers, and more satellite cells in tibialis anterior muscle compared with the contralateral untrained muscle. Ex vivo functional analysis of isolated extensor digitorum longus (EDL) muscle from the trained hindlimb revealed greater absolute isometric force, lower passive stiffness, and a lower susceptibility to eccentric contraction-induced force loss compared with untrained EDL muscle. Overall, these data support the concept that exercise training in the form of isometric tetanic contractions can improve contractile function of dystrophin-deficient muscle, indicating a potential role for enhancing muscle strength in patients with DMD and BMD. NEW & NOTEWORTHY We focused on adaptive responses of dystrophin-deficient mouse skeletal muscle to isometric contraction training and report that in the absence of dystrophin (or in the presence of a mutated dystrophin), strength and muscle histopathology are improved. Results suggest that the strength gains are associated with fiber hypertrophy, reduced fibrosis, increased number of satellite cells, and blunted eccentric contraction-induced force loss in vitro. Importantly, there was no indication that the isometric exercise training was deleterious to dystrophin-deficient muscle.

Aim Loss of dystrophin causes oxidative stress and affects nitric oxide synthase-mediated vascular function in striated muscle. Because tetrahydrobiopterin is an antioxidant and co-factor for nitric oxide synthase, we tested the hypothesis that tetrahydrobiopterin would be low in mdx mice and humans deficient for dystrophin. Methods Tetrahydrobiopterin and its metabolites were measured at rest and in response to exercise in Duchenne and Becker muscular dystrophy patients, age-matched male controls as well as wild-type, mdx and mdx mice transgenically overexpressing skeletal muscle-specific dystrophins. Mdx mice were also supplemented with tetrahydrobiopterin and pathophysiology was assessed. Results Duchenne muscular dystrophy patients had lower urinary dihydrobiopterin + tetrahydrobiopterin/specific gravity1.020 compared to unaffected age-matched males and Becker muscular dystrophy patients. Mdx mice had low urinary and skeletal muscle dihydrobiopterin + tetrahydrobiopterin compared to wild-type mice. Overexpression of dystrophins that localize neuronal nitric oxide synthase restored dihydrobiopterin + tetrahydrobiopterin in mdx mice to wild-type levels while utrophin overexpression did not. Mdx mice and Duchenne muscular dystrophy patients did not increase tetrahydrobiopterin during exercise and in mdx mice tetrahydrobiopterin deficiency was likely because of lower levels of sepiapterin reductase in skeletal muscle. Tetrahydrobiopterin supplementation improved skeletal muscle strength, resistance to fatiguing and injurious contractions in vivo, increased utrophin and capillary density of skeletal muscle and lowered cardiac muscle fibrosis and left ventricular wall thickness in mdx mice. Conclusion These data demonstrate that impaired tetrahydrobiopterin synthesis is associated with dystrophin loss and treatment with tetrahydrobiopterin improves striated muscle histopathology and skeletal muscle function in mdx mice.