Branched-chain amino acids and L-alanine supplementation ameliorate calcium dyshomeostasis in sarcopenia: New insights for nutritional interventions.

Introduction: During aging, sarcopenia and decline in physiological processes lead to partial loss of muscle strength, atrophy, and increased fatigability. Muscle changes may be related to a reduced intake of essential amino acids playing a role in proteostasis. We have recently shown that branched-chain amino acid (BCAA) supplements improve atrophy and weakness in models of muscle disuse and aging. Considering the key roles that the alteration of Ca2+-related homeostasis and store-operated calcium entry (SOCE) play in several muscle dysfunctions, this study has been aimed at gaining insight into the potential ability of BCAA-based dietary formulations in aged mice on various players of Ca2+ dyshomeostasis. Methods: Seventeen-month-old male C57BL/6J mice received a 12-week supplementation with BCAAs alone or boosted with two equivalents of L-alanine (2-Ala) or with dipeptide L-alanyl-L-alanine (Di-Ala) in drinking water. Outcomes were evaluated on ex vivo skeletal muscles indices vs. adult 3-month-old male C57BL/6J mice. Results: Ca2+ imaging confirmed a decrease in SOCE and an increase of resting Ca2+ concentration in aged vs. adult mice without alteration in the canonical components of SOCE. Aged muscles vs. adult muscles were characterized by a decrease in the expression of ryanodine receptor 1 (RyR1), the Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) pump, and sarcalumenin together with an alteration of the expression of mitsugumin 29 and mitsugumin 53, two recently recognized players in the SOCE mechanism. BCAAs, particularly the formulation BCAAs+2-Ala, were able to ameliorate all these alterations. Discussion: These results provide evidence that Ca2+ homeostasis dysfunction plays a role in the functional deficit observed in aged muscle and supports the interest of dietary BCAA supplementation in counteracting sarcopenia-related SOCE dysregulation.

Myasthenia Gravis Treatment: From Old Drugs to Innovative Therapies with a Glimpse into the Future.

Myasthenia gravis (MG) is a rare autoimmune disease that causes debilitating muscle weakness due to impaired neuromuscular transmission. Since most (about 80-90%) MG patients present autoantibodies against the acetylcholine receptor, standard medical therapy consists of symptomatic treatment with acetylcholinesterase inhibitors (e.g., pyridostigmine). In addition, considering the autoimmune basis of MG, standard therapy includes immunomodulating agents, such as corticosteroids, azathioprine, cyclosporine A, and cyclophosphamide. New strategies have been proposed for the treatment of MG and include complement blockade (i.e., eculizumab, ravulizumab, and zilucoplan) and neonatal Fc receptor antagonism (i.e., efgartigimod and rozanolixizumab). The aim of this review is to provide a detailed overview of the pre- and post-marketing evidence on the five pharmacological treatments most recently approved for the treatment of MG, by identifying both preclinical and clinical studies registered in clinicaltrials.gov. A description of the molecules currently under evaluation for the treatment of MG is also provided.

LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

The potential role of liver kinase B1 (LKB1) in the altered activation of the master metabolic and epigenetic regulator adenosine monophosphate-activated protein kinase (AMPK) in Duchenne muscular dystrophy has not been investigated so far. Hence, we analyzed both gene and protein levels of LKB1 and its related targets in gastrocnemius muscles of adult C57BL/10 mdx mice and D2 mdx mice, a model with a more severe dystrophic phenotype, as well as the sensitivity of the LKB1-AMPK pathway to AMPK activators, such as chronic exercise. Our data show, for the first time, a reduction in the levels of LKB1 and accessory proteins, MO25 and STRADα, in both mdx strains versus the respective wild type, which was further impaired by exercise, in parallel with a lack of further phosphorylation of AMPK. The AMPK-like kinase salt-inducible kinase (SIK) and class II histone deacetylases, along with expression of the HDAC target gene Mef2c, were also altered, supporting an impairment of LKB1-SIK-class II histone deacetylase signaling. Our results demonstrate that LKB1 may be involved in dystrophic progression, paving the way for future preclinical studies.

Growth hormone secretagogues modulate inflammation and fibrosis in mdx mouse model of Duchenne muscular dystrophy.

Introduction: Growth hormone secretagogues (GHSs) exert multiple actions, being able to activate GHS-receptor 1a, control inflammation and metabolism, to enhance GH/insulin-like growth factor-1 (IGF-1)-mediated myogenesis, and to inhibit angiotensin-converting enzyme. These mechanisms are of interest for potentially targeting multiple steps of pathogenic cascade in Duchenne muscular dystrophy (DMD). Methods: Here, we aimed to provide preclinical evidence for potential benefits of GHSs in DMD, via a multidisciplinary in vivo and ex vivo comparison in mdx mice, of two ad hoc synthesized compounds (EP80317 and JMV2894), with a wide but different profile. 4-week-old mdx mice were treated for 8 weeks with EP80317 or JMV2894 (320 µg/kg/d, s.c.). Results: In vivo, both GHSs increased mice forelimb force (recovery score, RS towards WT: 20% for EP80317 and 32% for JMV2894 at week 8). In parallel, GHSs also reduced diaphragm (DIA) and gastrocnemius (GC) ultrasound echodensity, a fibrosis-related parameter (RS: ranging between 26% and 75%). Ex vivo, both drugs ameliorated DIA isometric force and calcium-related indices (e.g., RS: 40% for tetanic force). Histological analysis highlighted a relevant reduction of fibrosis in GC and DIA muscles of treated mice, paralleled by a decrease in gene expression of TGF-ß1 and Col1a1. Also, decreased levels of pro-inflammatory genes (IL-6, CD68), accompanied by an increment in Sirt-1, PGC-1α and MEF2c expression, were observed in response to treatments, suggesting an overall improvement of myofiber metabolism. No detectable transcript levels of GHS receptor-1a, nor an increase of circulating IGF-1 were found, suggesting the presence of a novel receptor-independent mechanism in skeletal muscle. Preliminary docking studies revealed a potential binding capability of JMV2894 on metalloproteases involved in extracellular matrix remodeling and cytokine production, such as ADAMTS-5 and MMP-9, overactivated in DMD. Discussion: Our results support the interest of GHSs as modulators of pathology progression in mdx mice, disclosing a direct anti-fibrotic action that may prove beneficial to contrast pathological remodeling.

Preclinical Ultrasonography in Rodent Models of Neuromuscular Disorders: The State of the Art for Diagnostic and Therapeutic Applications.

Ultrasonography is a safe, non-invasive imaging technique used in several fields of medicine, offering the possibility to longitudinally monitor disease progression and treatment efficacy over time. This is particularly useful when a close follow-up is required, or in patients with pacemakers (not suitable for magnetic resonance imaging). By virtue of these advantages, ultrasonography is commonly used to detect multiple skeletal muscle structural and functional parameters in sports medicine, as well as in neuromuscular disorders, e.g., myotonic dystrophy and Duchenne muscular dystrophy (DMD). The recent development of high-resolution ultrasound devices allowed the use of this technique in preclinical settings, particularly for echocardiographic assessments that make use of specific guidelines, currently lacking for skeletal muscle measurements. In this review, we describe the state of the art for ultrasound skeletal muscle applications in preclinical studies conducted in small rodents, aiming to provide the scientific community with necessary information to support an independent validation of these procedures for the achievement of standard protocols and reference values useful in translational research on neuromuscular disorders.

Branched-Chain Amino Acids and Di-Alanine Supplementation in Aged Mice: A Translational Study on Sarcopenia.

In age-related sarcopenia, the gradual loss of skeletal muscle mass, function and strength is underpinned by an imbalanced rate of protein synthesis/breakdown. Hence, an adequate protein intake is considered a valuable strategy to mitigate sarcopenia. Here, we investigated the effects of a 12-week oral supplementation with branched-chain amino acids (BCAAs: leucine, isoleucine, and valine) with recognized anabolic properties, in 17-month-old (AGED) C57BL/6J male mice. BCAAs (2:1:1) were formulated in drinking water, alone or plus two L-Alanine equivalents (2ALA) or dipeptide L-Alanyl-L-Alanine (Di-ALA) to boost BCAAs bioavailability. Outcomes were evaluated on in/ex vivo readouts vs. 6-month-old (ADULT) mice. In vivo hind limb plantar flexor torque was improved in AGED mice treated with BCAAs + Di-ALA or 2ALA (recovery score, R.S., towards ADULT: ≥20%), and all mixtures significantly increased hind limb volume. Ex vivo, myofiber cross-sectional areas were higher in gastrocnemius (GC) and soleus (SOL) muscles from treated mice (R.S. ≥ 69%). Contractile indices of isolated muscles were improved by the mixtures, especially in SOL muscle (R.S. ≥ 20%). The latter displayed higher mTOR protein levels in mice supplemented with 2ALA/Di-ALA-enriched mixtures (R.S. ≥ 65%). Overall, these findings support the usefulness of BCAAs-based supplements in sarcopenia, particularly as innovative formulations potentiating BCAAs bioavailability and effects.

On the use of D2.B10-Dmdmdx/J (D2.mdx) Versus C57BL/10ScSn-Dmdmdx/J (mdx) Mouse Models for Preclinical Studies on Duchenne Muscular Dystrophy: A Cautionary Note from Members of the TREAT-NMD Advisory Committee on Therapeutics.

The C57BL/10ScSn-Dmdmdx/J (mdx) mouse model has been used by researchers for decades as a model to study pathology of and develop therapies for Duchenne muscular dystrophy. However, the model is relatively mildly affected compared to the human situation. Recently, the D2.B10-Dmdmdx/J (D2.mdx) mouse model was suggested as a more severely affected and therefore better alternative. While the pathology of this model is indeed more pronounced early in life, it is not progressive, and increasing evidence suggest that it actually partially resolves with age. As such, caution is needed when using this model. However, as preclinical experts of the TREAT-NMD advisory committee for therapeutics (TACT), we frequently encounter study designs that underestimate this caveat. We here provide context for how to best use the two models for preclinical studies at the current stage of knowledge.

ß-Dystroglycan Restoration and Pathology Progression in the Dystrophic mdx Mouse: Outcome and Implication of a Clinically Oriented Study with a Novel Oral Dasatinib Formulation.

ROS-activated cSrc tyrosine kinase (TK) promotes the degradation of ß-dystroglycan (ß-DG), a dystrophin-glycoprotein complex component, which may reinforce damaging signals in Duchenne muscular dystrophy (DMD). Therefore, cSrc-TK represents a promising therapeutic target. In mdx mice, a 4-week subcutaneous treatment with dasatinib (DAS), a pan-Src-TKs inhibitor approved as anti-leukemic agent, increased muscle ß-DG, with minimal amelioration of morphofunctional indices. To address possible dose/pharmacokinetic (PK) issues, a new oral DAS/hydroxypropyl(HP)-ß-cyclodextrin(CD) complex was developed and chronically administered to mdx mice. The aim was to better assess the role of ß-DG in pathology progression, meanwhile confirming DAS mechanism of action over the long-term, along with its efficacy and tolerability. The 4-week old mdx mice underwent a 12-week treatment with DAS/HP-ß-CD10% dissolved in drinking water, at 10 or 20 mg/kg/day. The outcome was evaluated via in vivo/ex vivo disease-relevant readouts. Oral DAS/HP-ß-CD efficiently distributed in mdx mice plasma and tissues in a dose-related fashion. The new DAS formulation confirmed its main upstream mechanism of action, by reducing ß-DG phosphorylation and restoring its levels dose-dependently in both diaphragm and gastrocnemius muscle. However, it modestly improved in vivo neuromuscular function, ex vivo muscle force, and histopathology, although the partial recovery of muscle elasticity and the decrease of CK and LDH plasma levels suggest an increased sarcolemmal stability of dystrophic muscles. Our clinically oriented study supports the interest in this new, pediatric-suitable DAS formulation for proper exposure and safety and for enhancing ß-DG expression. This latter mechanism is, however, not sufficient by itself to impact on pathology progression. In-depth analyses will be dedicated to elucidating the mechanism limiting DAS effectiveness in dystrophic settings, meanwhile assessing its potential synergy with dystrophin-based molecular therapies.

Alteration of STIM1/Orai1-Mediated SOCE in Skeletal Muscle: Impact in Genetic Muscle Diseases and Beyond.

Intracellular Ca2+ ions represent a signaling mediator that plays a critical role in regulating different muscular cellular processes. Ca2+ homeostasis preservation is essential for maintaining skeletal muscle structure and function. Store-operated Ca2+ entry (SOCE), a Ca2+-entry process activated by depletion of intracellular stores contributing to the regulation of various function in many cell types, is pivotal to ensure a proper Ca2+ homeostasis in muscle fibers. It is coordinated by STIM1, the main Ca2+ sensor located in the sarcoplasmic reticulum, and ORAI1 protein, a Ca2+-permeable channel located on transverse tubules. It is commonly accepted that Ca2+ entry via SOCE has the crucial role in short- and long-term muscle function, regulating and adapting many cellular processes including muscle contractility, postnatal development, myofiber phenotype and plasticity. Lack or mutations of STIM1 and/or Orai1 and the consequent SOCE alteration have been associated with serious consequences for muscle function. Importantly, evidence suggests that SOCE alteration can trigger a change of intracellular Ca2+ signaling in skeletal muscle, participating in the pathogenesis of different progressive muscle diseases such as tubular aggregate myopathy, muscular dystrophy, cachexia, and sarcopenia. This review provides a brief overview of the molecular mechanisms underlying STIM1/Orai1-dependent SOCE in skeletal muscle, focusing on how SOCE alteration could contribute to skeletal muscle wasting disorders and on how SOCE components could represent pharmacological targets with high therapeutic potential.

BCAAs and Di-Alanine supplementation in the prevention of skeletal muscle atrophy: preclinical evaluation in a murine model of hind limb unloading.

Skeletal muscle atrophy occurs in response to various pathophysiological stimuli, including disuse, aging, and neuromuscular disorders, mainly due to an imbalance of anabolic/catabolic signaling. Branched Chain Amino Acids (BCAAs: leucine, isoleucine, valine) supplements can be beneficial for counteracting muscle atrophy, in virtue of their reported anabolic properties. Here, we carried out a proof-of-concept study to assess the in vivo/ex vivo effects of a 4-week treatment with BCAAs on disuse-induced atrophy, in a murine model of hind limb unloading (HU). BCAAs were formulated in drinking water, alone, or plus two equivalents of L-Alanine (2 ALA) or the dipeptide L-Alanyl-L-Alanine (Di-ALA), to boost BCAAs bioavailability. HU mice were characterized by reduction of body mass, decrease of soleus - SOL - muscle mass and total protein, alteration of postural muscles architecture and fiber size, dysregulation of atrophy-related genes (Atrogin-1, MuRF-1, mTOR, Mstn). In parallel, we provided new robust readouts in the HU murine model, such as impaired in vivo isometric torque and ex vivo SOL muscle contractility and elasticity, as well as altered immune response. An acute pharmacokinetic study confirmed that L-ALA, also as dipeptide, enhanced plasma exposure of BCAAs. Globally, the most sensitive parameters to BCAAs action were muscle atrophy and myofiber cross-sectional area, muscle force and compliance to stress, protein synthesis via mTOR and innate immunity, with the new BCAAs + Di-ALA formulation being the most effective treatment. Our results support the working hypothesis and highlight the importance of developing innovative formulations to optimize BCAAs biodistribution.

Circadian Genes as Exploratory Biomarkers in DMD: Results From Both the mdx Mouse Model and Patients.

Duchenne muscular dystrophy (DMD) is a rare genetic disease due to dystrophin gene mutations which cause progressive weakness and muscle wasting. Circadian rhythm coordinates biological processes with the 24-h cycle and it plays a key role in maintaining muscle functions, both in animal models and in humans. We explored expression profiles of circadian circuit master genes both in Duchenne muscular dystrophy skeletal muscle and in its animal model, the mdx mouse. We designed a customized, mouse-specific Fluidic-Card-TaqMan-based assay (Fluid-CIRC) containing thirty-two genes related to circadian rhythm and muscle regeneration and analyzed gastrocnemius and tibialis anterior muscles from both unexercised and exercised mdx mice. Based on this first analysis, we prioritized the 7 most deregulated genes in mdx mice and tested their expression in skeletal muscle biopsies from 10 Duchenne patients. We found that CSNK1E, SIRT1, and MYOG are upregulated in DMD patient biopsies, consistent with the mdx data. We also demonstrated that their proteins are detectable and measurable in the DMD patients' plasma. We suggest that CSNK1E, SIRT1, and MYOG might represent exploratory circadian biomarkers in DMD.

Ultrasonography validation for early alteration of diaphragm echodensity and function in the mdx mouse model of Duchenne muscular dystrophy.

The mdx mouse model of Duchenne muscular dystrophy is characterized by functional and structural alterations of the diaphragm since early stages of pathology, closely resembling patients' condition. In recent years, ultrasonography has been proposed as a useful longitudinal non-invasive technique to assess mdx diaphragm dysfunction and evaluate drug efficacy over time. To date, only a few preclinical studies have been conducted. Therefore, an independent validation of this method by different laboratories is needed to increase results reliability and reduce biases. Here, we performed diaphragm ultrasonography in 3- and 6-month-old mdx mice, the preferred age-window for pharmacology studies. The alteration of diaphragm function over time was measured as diaphragm ultrasound movement amplitude. At the same time points, a first-time assessment of diaphragm echodensity was performed, as an experimental index of progressive loss of contractile tissue. A parallel evaluation of other in vivo and ex vivo dystrophy-relevant readouts was carried out. Both 3- and 6-month-old mdx mice showed a significant decrease in diaphragm amplitude compared to wild type (wt) mice. This index was well-correlated either with in vivo running performance or ex vivo isometric tetanic force of isolated diaphragm. In addition, diaphragms from 6-month-old dystrophic mice were also highly susceptible to eccentric contraction ex vivo. Importantly, we disclosed an age-dependent increase in echodensity in mdx mice not observed in wt animals, which was independent from abdominal wall thickness. This was accompanied by a notable increase of pro-fibrotic TGF-ß1 levels in the mdx diaphragm and of non-muscle tissue amount in diaphragm sections stained by hematoxylin & eosin. Our findings corroborate the usefulness of diaphragm ultrasonography in preclinical drug studies as a powerful tool to monitor mdx pathology progression since early stages.

Ergogenic Effect of BCAAs and L-Alanine Supplementation: Proof-of-Concept Study in a Murine Model of Physiological Exercise.

BACKGROUND: Branched-chain amino acids (BCAAs: leucine, isoleucine, valine) account for 35% of skeletal muscle essential amino acids (AAs). As such, they must be provided in the diet to support peptide synthesis and inhibit protein breakdown. Although substantial evidence has been collected about the potential usefulness of BCAAs in supporting muscle function and structure, dietary supplements containing BCAAs alone may not be effective in controlling muscle protein turnover, due to the rate-limiting bioavailability of other AAs involved in BCAAs metabolism. METHODS: We aimed to evaluate the in vivo/ex vivo effects of a 4-week treatment with an oral formulation containing BCAAs alone (2:1:1) on muscle function, structure, and metabolism in a murine model of physiological exercise, which was compared to three modified formulations combining BCAAs with increasing concentrations of L-Alanine (ALA), an AA controlling BCAAs catabolism. RESULTS: A preliminary pharmacokinetic study confirmed the ability of ALA to boost up BCAAs bioavailability. After 4 weeks, mix 2 (BCAAs + 2ALA) had the best protective effect on mice force and fatigability, as well as on muscle morphology and metabolic indices. CONCLUSION: Our study corroborates the use of BCAAs + ALA to support muscle health during physiological exercise, underlining how the relative BCAAs/ALA ratio is important to control BCAAs distribution.

"The Social Network" and Muscular Dystrophies: The Lesson Learnt about the Niche Environment as a Target for Therapeutic Strategies.

The muscle stem cells niche is essential in neuromuscular disorders. Muscle injury and myofiber death are the main triggers of muscle regeneration via satellite cell activation. However, in degenerative diseases such as muscular dystrophy, regeneration still keep elusive. In these pathologies, stem cell loss occurs over time, and missing signals limiting damaged tissue from activating the regenerative process can be envisaged. It is unclear what comes first: the lack of regeneration due to satellite cell defects, their pool exhaustion for degeneration/regeneration cycles, or the inhibitory mechanisms caused by muscle damage and fibrosis mediators. Herein, Duchenne muscular dystrophy has been taken as a paradigm, as several drugs have been tested at the preclinical and clinical levels, targeting secondary events in the complex pathogenesis derived from lack of dystrophin. We focused on the crucial roles that pro-inflammatory and pro-fibrotic cytokines play in triggering muscle necrosis after damage and stimulating satellite cell activation and self-renewal, along with growth and mechanical factors. These processes contribute to regeneration and niche maintenance. We review the main effects of drugs on regeneration biomarkers to assess whether targeting pathogenic events can help to protect niche homeostasis and enhance regeneration efficiency other than protecting newly formed fibers from further damage.

Effect of exercise on telomere length and telomere proteins expression in mdx mice.

In Duchenne muscular dystrophy (DMD), telomere shortening has been postulated to contribute to the failure of regenerative activity promoting the premature senescence of satellite cells. The aim of the present study was to investigate the telomere length and the expression of telomeric repeat-binding factor-1 (TRF1), poly (ADP-ribose) polymerase-1 (PARP1) and mouse telomerase reverse transcriptase (MTERT) in gastrocnemius, tibialis anterior and diaphragm muscles of the murine model of DMD, the mdx mouse and whether a chronic protocol of forced exercise impacts on them. Our results confirmed a telomere shortening in mdx muscles, more evident in the diaphragm, in which exercise induced a greater shortening than in wild-type mice. Moreover, we showed for the first time in mdx an increased TRF1 and PARP1 expression and an augmented activity of MTERT, further enhanced by exercise. These results reinforce the hypothesis that a deregulation of mechanisms involved in telomere length occurs and may pave the way for the test of compounds targeting proteins modulating telomere maintenance as a novel strategy to treat dystrophinopathies.

Dasatinib/HP-ß-CD Inclusion Complex Based Aqueous Formulation as a Promising Tool for the Treatment of Paediatric Neuromuscular Disorders.

New scientific findings have recently shown that dasatinib (DAS), the first-choice oral drug in the treatment of chronic myeloid leukemia (CML) for adult patients who are resistant or intolerant to imatinib, is also potentially useful in the paediatric age. Moreover, recent preclinical evidences suggest that this drug could be useful for the treatment of Duchenne muscular dystrophy, since it targets cSrc tyrosin kinase. Based on these considerations, the purpose of this work was to use the strategy of complexation with hydroxypropyl-ß-cyclodextrin (HP-ß-CD) in order to obtain an aqueous preparation of DAS, which is characterized by a low water solubility (6.49 × 10-4 mg/mL). Complexation studies demonstrated that HP-ß-CD is able to form a stable host-guest inclusion complex with DAS with a 1:1 apparent formation constant of 922.13 M-1, as also demonstrated by the Job's plot, with an increase in DAS aqueous solubility of about 21 times in the presence of 6% w/v of HP-ß-CD (0.014 mg/mL). The inclusion complex has been prepared in the solid state by lyophilization and characterized by Fourier Transform Infrared (FT-IR), Nuclear Magnetic Resonance (NMR), Differential Scanning Calorimetry (DSC) techniques, and its dissolution profile was studied at different pH values. Moreover, in view of potential use of DAS for Duchenne muscular dystrophy, the cytotoxic effect of the inclusion complex has been assessed on C2C12 cells, a murine muscle satellite cell line. In parallel, a one-week oral treatment was performed in wild type C57Bl/6J mice to test both palatability and the exposure levels of the new oral formulation of the compound. In conclusion, this new inclusion complex could allow the development of a liquid and solvent free formulation to be administered both orally and parenterally, especially in the case of an administration in paediatric age.

A long-term treatment with taurine prevents cardiac dysfunction in mdx mice.

Taurine is an amino acid abundantly present in heart and skeletal muscle. Duchenne muscular dystrophy (DMD) is a genetic disorder in which the absence of dystrophin leads to skeletal muscle wasting and heart failure. An altered taurine metabolism has been described in dystrophic animals and short-term taurine administration exerts promising amelioration of early muscular alterations in the mdx mouse model of DMD. To reinforce the therapeutic and nutraceutical taurine potential in DMD, we evaluated the effects of a long-term treatment on cardiac and skeletal muscle function of mdx mice in a later disease stage. Taurine was administered in drinking water (1 g/kg/day) to wt and mdx mice for 6 months, starting at 6 months of age. Ultrasonography evaluation of heart and hind limb was performed, in parallel with in vivo and ex vivo functional tests and biochemical, histological and gene expression analyses. 12-month-old mdx mice showed a significant worsening of left ventricular function parameters (shortening fraction, ejection fraction, stroke volume), which were significantly counteracted by the taurine treatment. In parallel, histologic signs of damage were reduced by taurine along with the expression of proinflammatory myocardial IL-6. Interestingly, no effects were observed on hind limb volume and percentage of vascularization or on in vivo and ex vivo muscle functional parameters, suggesting a tissue-specific action of taurine in relation to the disease phase. A trend toward increase in taurine was found in heart and quadriceps from treated animals, paralleled by a slight decrease in mdx mice plasma. Our study provides evidences that taurine can prevent late heart dysfunction in mdx mice, further corroborating the interest on this amino acid toward clinical trials.

Data on protein abundance alteration induced by chronic exercise in mdx mice model of Duchenne muscular dystrophy and potential modulation by apocynin and taurine.

Here we present original data related to the research paper entitled "Proteome analysis in dystrophic mdx mouse muscle reveals a drastic alteration of Key Metabolic and Contractile Proteins after chronic exercise and the potential modulation by anti-oxidant compounds" (Gamberi et al., 2018) [1]. The dystrophin-deficient mdx mouse is the most common animal model for Duchenne muscular dystrophy. The mdx mice phenotype of the disorder is milder than in human sufferers and it can be worsened by chronic treadmill exercise. Apocynin and taurine are two antioxidant compounds proved to be beneficial on some pathology related parameters (Schröder and Schoser, 2009) [2]. This article reports the detailed proteomic data on protein abundance alterations, in tibialis anterior muscle of mdx mice, induced by chronic exercise protocol. A selected group of mdx mice was also treated with apocynin and taurine during this protocol. Detailed MS data, comparison between mdx vs wild type, exercised mdx vs wild type, and complete analysis of spot variation are provided. Furthermore, in wild type mice subjected to the same exercise protocol, the abundance of key proteins, resulted modified in exercised mdx, were analyzed by western blot.

Effect of a long-term treatment with metformin in dystrophic mdx mice: A reconsideration of its potential clinical interest in Duchenne muscular dystrophy.

The pharmacological stimulation of AMP-activated protein kinase (AMPK) via metabolic enhancers has been proposed as potential therapeutic strategy for Duchenne muscular dystrophy (DMD). Metformin, a widely-prescribed anti-hyperglycemic drug which activates AMPK via mitochondrial respiratory chain, has been recently tested in DMD patients in synergy with nitric oxide (NO)-precursors, with encouraging results. However, preclinical data supporting the use of metformin in DMD are still poor, and its actions on skeletal muscle appear controversial. Therefore, we investigated the effects of a long-term treatment with metformin (200 mg/kg/day in drinking water, for 20 weeks) in the exercised mdx mouse model, characterized by a severe mechanical-metabolic maladaptation. Metformin significantly ameliorated histopathology in mdx gastrocnemius muscle, in parallel reducing TGF-ß1 with a recovery score (r.s) of 106%; this was accompanied by a decreased plasma matrix-metalloproteinase-9 (r.s. 43%). In addition, metformin significantly increased mdx diaphragm twitch and tetanic tension ex vivo (r.s. 44% and 36%, respectively), in spite of minor effects on in vivo weakness. However, no clear protective actions on dystrophic muscle metabolism were observed, as shown by the poor metformin effect on AMPK activation measured by western blot, on the expression of mechanical-metabolic response genes analyzed by qPCR, and by the lack of fast-to-slow fiber-type-shift assessed by SDH staining in tibialis anterior muscle. Similar results were obtained in the milder phenotype of sedentary mdx mice. The lack of metabolic effects could be, at least partly, due to metformin inability to increase low mdx muscle levels of l-arginine, l-citrulline and taurine, found by HPLC. Our findings encourage to explore alternative, metabolism-independent mechanisms of action to differently repurpose metformin in DMD, supporting its therapeutic combination with NO-sources.