Loss of endogenous estrogen alters mitochondrial metabolism and muscle clock-related protein Rbm20 in female mdx mice.

Female carriers of a Duchenne muscular dystrophy (DMD) gene mutation manifest exercise intolerance and metabolic anomalies that may be exacerbated following menopause due to the loss of estrogen, a known regulator of skeletal muscle function and metabolism. Here, we studied the impact of estrogen depletion (via ovariectomy) on exercise tolerance and muscle mitochondrial metabolism in female mdx mice and the potential of estrogen replacement therapy (using estradiol) to protect against functional and metabolic perturbations. We also investigated the effect of estrogen depletion, and replacement, on the skeletal muscle proteome through an untargeted proteomic approach with TMT-labelling. Our study confirms that loss of estrogen in female mdx mice reduces exercise capacity, tricarboxylic acid cycle intermediates, and citrate synthase activity but that these deficits are offset through estrogen replacement therapy. Furthermore, ovariectomy downregulated protein expression of RNA-binding motif factor 20 (Rbm20), a critical regulator of sarcomeric and muscle homeostasis gene splicing, which impacted pathways involving ribosomal and mitochondrial translation. Estrogen replacement modulated Rbm20 protein expression and promoted metabolic processes and the upregulation of proteins involved in mitochondrial dynamics and metabolism. Our data suggest that estrogen mitigates dystrophinopathic features in female mdx mice and that estrogen replacement may be a potential therapy for post-menopausal DMD carriers.

Adenylosuccinic Acid Is a Non-Toxic Small Molecule In Vitro and In Vivo.

Adenylosuccinic acid (ASA) is a small molecule dicarboxylate that could be a strong clinical development candidate for inherited myopathies involving dysregulated purine nucleotide metabolism. Currently, there are no published pharmacokinetic/dynamic or toxicology data available, although 10-year clinical trial data on Duchenne muscular dystrophy patients suggests it is a chronically safe drug. In this study, we tested the toxicity of ASA to cultured myoblasts in vitro and its acute systemic toxicity in mice. ASA is a non-toxic small molecule with an LD50 > 5000 mg/kg. Some background necrotic foci in the liver, kidney and gastrointestinal tract were shown that are likely incidental but warrant follow-up sub-/chronic oral exposure studies.

Dimethyl fumarate modulates the dystrophic disease program following short-term treatment.

New medicines are urgently required to treat the fatal neuromuscular disease Duchenne muscular dystrophy (DMD). Dimethyl fumarate (DMF) is a potent immunomodulatory small molecule nuclear erythroid 2-related factor 2 activator with current clinical utility in the treatment of multiple sclerosis and psoriasis that could be effective for DMD and rapidly translatable. Here, we tested 2 weeks of daily 100 mg/kg DMF versus 5 mg/kg standard-care prednisone (PRED) treatment in juvenile mdx mice with early symptomatic DMD. Both drugs modulated seed genes driving the DMD disease program and improved force production in fast-twitch muscle. However, only DMF showed pro-mitochondrial effects, protected contracting muscles from fatigue, improved histopathology, and augmented clinically compatible muscle function tests. DMF may be a more selective modulator of the DMD disease program than PRED, warranting follow-up longitudinal studies to evaluate disease-modifying impact.

FKBP25 regulates myoblast viability and migration and is differentially expressed in in vivo models of muscle adaptation.

FKBP25 (FKBP3 gene) is a dual-domain PPIase protein that consists of a C-terminal PPIase domain and an N-terminal basic tilted helix bundle (BTHB). The PPIase domain of FKBP25 has been shown to bind to microtubules, which has impacts upon microtubule polymerisation and cell cycle progression. Using quantitative proteomics, it was recently found that FKBP25 was expressed in the top 10% of the mouse skeletal muscle proteome. However, to date there have been few studies investigating the role of FKBP25 in non-transformed systems. As such, this study aimed to investigate potential roles for FKBP25 in myoblast viability, migration and differentiation and in adaptation of mature skeletal muscle. Doxycycline-inducible FKBP25 knockdown in C2C12 myoblasts revealed an increase in cell accumulation/viability and migration in vitro that was independent of alterations in tubulin dynamics; however, FKBP25 knockdown had no discernible impact on myoblast differentiation into myotubes. Finally, a series of in vivo models of muscle adaptation were assessed, where it was observed that FKBP25 protein expression was increased in hypertrophy and regeneration conditions (chronic mechanical overload and the mdx model of Duchenne muscular dystrophy) but decreased in an atrophy model (denervation). Overall, the findings of this study establish FKBP25 as a regulator of myoblast viability and migration, with possible implications for satellite cell proliferation and migration and muscle regeneration, and as a potential regulator of in vivo skeletal muscle adaptation.

Adenylosuccinic Acid: An Orphan Drug with Untapped Potential.

Adenylosuccinic acid (ASA) is an orphan drug that was once investigated for clinical application in Duchenne muscular dystrophy (DMD). Endogenous ASA participates in purine recycling and energy homeostasis but might also be crucial for averting inflammation and other forms of cellular stress during intense energy demand and maintaining tissue biomass and glucose disposal. This article documents the known biological functions of ASA and explores its potential application for the treatment of neuromuscular and other chronic diseases.

Cachectic muscle wasting in acute myeloid leukaemia: a sleeping giant with dire clinical consequences.

Acute myeloid leukaemia (AML) is a haematological malignancy with poor survival odds, particularly in the older (>65 years) population, in whom it is most prevalent. Treatment consists of induction and consolidation chemotherapy to remit the cancer followed by potentially curative haematopoietic cell transplantation. These intense treatments are debilitating and increase the risk of mortality. Patient stratification is used to mitigate this risk and considers a variety of factors, including body mass, to determine whether a patient is suitable for any or all treatment options. Skeletal muscle mass, the primary constituent of the body lean mass, may be a better predictor of patient suitability for, and outcomes of, AML treatment. Yet skeletal muscle is compromised by a variety of factors associated with AML and its clinical treatment consistent with cachexia, a life-threatening body wasting syndrome. Cachectic muscle wasting is associated with both cancer and anticancer chemotherapy. Although not traditionally associated with haematological cancers, cachexia is observed in AML and can have dire consequences. In this review, we discuss the importance of addressing skeletal muscle mass and cachexia within the AML clinical landscape in view of improving survivability of this disease.

Chemotherapy-Induced Myopathy: The Dark Side of the Cachexia Sphere.

Cancer cachexia is a debilitating multi-factorial wasting syndrome characterised by severe skeletal muscle wasting and dysfunction (i.e., myopathy). In the oncology setting, cachexia arises from synergistic insults from both cancer-host interactions and chemotherapy-related toxicity. The majority of studies have surrounded the cancer-host interaction side of cancer cachexia, often overlooking the capability of chemotherapy to induce cachectic myopathy. Accumulating evidence in experimental models of cachexia suggests that some chemotherapeutic agents rapidly induce cachectic myopathy, although the underlying mechanisms responsible vary between agents. Importantly, we highlight the capacity of specific chemotherapeutic agents to induce cachectic myopathy, as not all chemotherapies have been evaluated for cachexia-inducing properties-alone or in clinically compatible regimens. Furthermore, we discuss the experimental evidence surrounding therapeutic strategies that have been evaluated in chemotherapy-induced cachexia models, with particular focus on exercise interventions and adjuvant therapeutic candidates targeted at the mitochondria.

Metronomic 5-Fluorouracil Delivery Primes Skeletal Muscle for Myopathy but Does Not Cause Cachexia.

Skeletal myopathy encompasses both atrophy and dysfunction and is a prominent event in cancer and chemotherapy-induced cachexia. Here, we investigate the effects of a chemotherapeutic agent, 5-fluorouracil (5FU), on skeletal muscle mass and function, and whether small-molecule therapeutic candidate, BGP-15, could be protective against the chemotoxic challenge exerted by 5FU. Additionally, we explore the molecular signature of 5FU treatment. Male Balb/c mice received metronomic tri-weekly intraperitoneal delivery of 5FU (23 mg/kg), with and without BGP-15 (15 mg/kg), 6 times in total over a 15 day treatment period. We demonstrated that neither 5FU, nor 5FU combined with BGP-15, affected body composition indices, skeletal muscle mass or function. Adjuvant BGP-15 treatment did, however, prevent the 5FU-induced phosphorylation of p38 MAPK and p65 NF-B subunit, signalling pathways involved in cell stress and inflammatory signalling, respectively. This as associated with mitoprotection. 5FU reduced the expression of the key cytoskeletal proteins, desmin and dystrophin, which was not prevented by BGP-15. Combined, these data show that metronomic delivery of 5FU does not elicit physiological consequences to skeletal muscle mass and function but is implicit in priming skeletal muscle with a molecular signature for myopathy. BGP-15 has modest protective efficacy against the molecular changes induced by 5FU.

Standard of care versus new-wave corticosteroids in the treatment of Duchenne muscular dystrophy: Can we do better?

BACKGROUND: Pharmacological corticosteroid therapy is the standard of care in Duchenne Muscular Dystrophy (DMD) that aims to control symptoms and slow disease progression through potent anti-inflammatory action. However, a major concern is the significant adverse effects associated with long term-use. MAIN: This review discusses the pros and cons of standard of care treatment for DMD and compares it to novel data generated with the new-wave dissociative corticosteroid, vamorolone. The current status of experimental anti-inflammatory pharmaceuticals is also reviewed, with insights regarding alternative drugs that could provide therapeutic advantage. CONCLUSIONS: Although novel dissociative steroids may be superior substitutes to corticosteroids, other potential therapeutics should be explored. Repurposing or developing novel pharmacological therapies capable of addressing the many pathogenic features of DMD in addition to anti-inflammation could elicit greater therapeutic advantages.

Adenylosuccinic acid: a novel inducer of the cytoprotectant Nrf2 with efficacy in Duchenne muscular dystrophy.

Adenylosuccinic acid (ASA) modifies Duchenne muscular dystrophy (DMD) progression in dystrophic mdx mice and human DMD patients. Despite an established role for ASA in augmenting metabolism and cellular energy homeostasis, our previous data suggests an undiscovered ulterior mode of action capable of modifying DMD disease course. Here, we identify ASA as a novel inducer of nuclear factor erythroid 2-related factor-2 (Nrf2), master regulator of the antioxidant and cytoprotective response to cell stress.

Idebenone: When an antioxidant is not an antioxidant.

Idebenone is a well described drug that was initially developed against dementia. The current literature widely portrays this molecule as a potent antioxidant and CoQ10 analogue. While numerous papers seem to support this view, a closer look indicates that the pharmacokinetics of idebenone do not support these claims. A major discrepancy between achievable tissue levels, especially in target tissues such as the brain, and doses required to show the proposed effects, significantly questions our current understanding. This review explains how this has happened and highlights the discrepancies in the current literature. More importantly, based on some recent discoveries, a new framework is presented that can explain the mode of action of this molecule and can align formerly contradictory results. Finally, this new appreciation of the molecular activities of idebenone provides a rational approach to test idebenone in novel indications that might have not been considered previously for this drug.

Targeting Nrf2 for the treatment of Duchenne Muscular Dystrophy.

Imbalances in redox homeostasis can result in oxidative stress, which is implicated in various pathological conditions including the fatal neuromuscular disease Duchenne Muscular Dystrophy (DMD). DMD is a complicated disease, with many druggable targets at the cellular and molecular level including calcium-mediated muscle degeneration; mitochondrial dysfunction; oxidative stress; inflammation; insufficient muscle regeneration and dysregulated protein and organelle maintenance. Previous investigative therapeutics tended to isolate and focus on just one of these targets and, consequently, therapeutic activity has been limited. Nuclear erythroid 2-related factor 2 (Nrf2) is a transcription factor that upregulates many cytoprotective gene products in response to oxidants and other toxic stressors. Unlike other strategies, targeted Nrf2 activation has the potential to simultaneously modulate separate pathological features of DMD to amplify therapeutic benefits. Here, we review the literature providing theoretical context for targeting Nrf2 as a disease modifying treatment against DMD.

Calming the (Cytokine) Storm: Dimethyl Fumarate as a Therapeutic Candidate for COVID-19.

COVID-19 has rapidly spread worldwide and incidences of hospitalisation from respiratory distress are significant. While a vaccine is in the pipeline, there is urgency for therapeutic options to address the immune dysregulation, hyperinflammation and oxidative stress that can lead to death. Given the shared pathogenesis of severe cases of COVID-19 with aspects of multiple sclerosis and psoriasis, we propose dimethyl fumarate as a viable treatment option. Currently approved for multiple sclerosis and psoriasis, dimethyl fumarate is an immunomodulatory, anti-inflammatory and anti-oxidative drug that could be rapidly implemented into the clinic to calm the cytokine storm which drives severe COVID-19.

The Failed Clinical Story of Myostatin Inhibitors against Duchenne Muscular Dystrophy: Exploring the Biology behind the Battle.

Myostatin inhibition therapy has held much promise for the treatment of muscle wasting disorders. This is particularly true for the fatal myopathy, Duchenne Muscular Dystrophy (DMD). Following on from promising pre-clinical data in dystrophin-deficient mice and dogs, several clinical trials were initiated in DMD patients using different modality myostatin inhibition therapies. All failed to show modification of disease course as dictated by the primary and secondary outcome measures selected: the myostatin inhibition story, thus far, is a failed clinical story. These trials have recently been extensively reviewed and reasons why pre-clinical data collected in animal models have failed to translate into clinical benefit to patients have been purported. However, the biological mechanisms underlying translational failure need to be examined to ensure future myostatin inhibitor development endeavors do not meet with the same fate. Here, we explore the biology which could explain the failed translation of myostatin inhibitors in the treatment of DMD.

Nitric Oxide (NO) and Duchenne Muscular Dystrophy: NO Way to Go?

The discordance between pre-clinical success and clinical failure of treatment options for Duchenne Muscular Dystrophy (DMD) is significant. The termination of clinical trials investigating the phosphodiesterase inhibitors, sildenafil and tadalafil (which prolong the second messenger molecule of nitric oxide (NO) signaling), are prime examples of this. Both attenuated key dystrophic features in the mdx mouse model of DMD yet failed to modulate primary outcomes in clinical settings. We have previously attempted to modulate NO signaling via chronic nitrate supplementation of the mdx mouse but failed to demonstrate beneficial modulation of key dystrophic features (i.e., metabolism). Instead, we observed increased muscle damage and nitrosative stress which exacerbated MD. Here, we highlight that acute nitrite treatment of human DMD myoblasts is also detrimental and suggest strategies for moving forward with NO replacement therapy in DMD.

The Paradoxical Effect of PARP Inhibitor BGP-15 on Irinotecan-Induced Cachexia and Skeletal Muscle Dysfunction.

Chemotherapy-induced muscle wasting and dysfunction is a contributing factor to cachexia alongside cancer and increases the risk of morbidity and mortality. Here, we investigate the effects of the chemotherapeutic agent irinotecan (IRI) on skeletal muscle mass and function and whether BGP-15 (a poly-(ADP-ribose) polymerase-1 (PARP-1) inhibitor and heat shock protein co-inducer) adjuvant therapy could protect against IRI-induced skeletal myopathy. Healthy 6-week-old male Balb/C mice (n = 24; 8/group) were treated with six intraperitoneal injections of either vehicle, IRI (30 mg/kg) or BGP-15 adjuvant therapy (IRI+BGP; 15 mg/kg) over two weeks. IRI reduced lean and tibialis anterior mass, which were attenuated by IRI+BGP treatment. Remarkably, IRI reduced muscle protein synthesis, while IRI+BGP reduced protein synthesis further. These changes occurred in the absence of a change in crude markers of mammalian/mechanistic target of rapamycin (mTOR) Complex 1 (mTORC1) signaling and protein degradation. Interestingly, the cytoskeletal protein dystrophin was reduced in both IRI- and IRI+BGP-treated mice, while IRI+BGP treatment also decreased ß-dystroglycan, suggesting significant remodeling of the cytoskeleton. IRI reduced absolute force production of the soleus and extensor digitorum longus (EDL) muscles, while IRI+BGP rescued absolute force production of the soleus and strongly trended to rescue force output of the EDL (p = 0.06), which was associated with improvements in mass. During the fatiguing stimulation, IRI+BGP-treated EDL muscles were somewhat susceptible to rupture at the musculotendinous junction, likely due to BGP-15's capacity to maintain the rate of force development within a weakened environment characterized by significant structural remodeling. Our paradoxical data highlight that BGP-15 has some therapeutic advantage by attenuating IRI-induced skeletal myopathy; however, its effects on the remodeling of the cytoskeleton and extracellular matrix, which appear to make fast-twitch muscles more prone to tearing during contraction, could suggest the induction of muscular dystrophy and, thus, require further characterization.

Dimethyl Fumarate and Its Esters: A Drug with Broad Clinical Utility?

Fumaric acid esters (FAEs) are small molecules with anti-oxidative, anti-inflammatory and immune-modulating effects. Dimethyl fumarate (DMF) is the best characterised FAE and is approved and registered for the treatment of psoriasis and Relapsing-Remitting Multiple Sclerosis (RRMS). Psoriasis and RRMS share an immune-mediated aetiology, driven by severe inflammation and oxidative stress. DMF, as well as monomethyl fumarate and diroximel fumarate, are commonly prescribed first-line agents with favourable safety and efficacy profiles. The potential benefits of FAEs against other diseases that appear pathogenically different but share the pathologies of oxidative stress and inflammation are currently investigated.

Sodium nitrate co-supplementation does not exacerbate low dose metronomic doxorubicin-induced cachexia in healthy mice.

The purpose of this study was to determine whether (1) sodium nitrate (SN) treatment progressed or alleviated doxorubicin (DOX)-induced cachexia and muscle wasting; and (2) if a more-clinically relevant low-dose metronomic (LDM) DOX treatment regimen compared to the high dosage bolus commonly used in animal research, was sufficient to induce cachexia in mice. Six-week old male Balb/C mice (n = 16) were treated with three intraperitoneal injections of either vehicle (0.9% NaCl; VEH) or DOX (4 mg/kg) over one week. To test the hypothesis that sodium nitrate treatment could protect against DOX-induced symptomology, a group of mice (n = 8) were treated with 1 mM NaNO3 in drinking water during DOX (4 mg/kg) treatment (DOX + SN). Body composition indices were assessed using echoMRI scanning, whilst physical and metabolic activity were assessed via indirect calorimetry, before and after the treatment regimen. Skeletal and cardiac muscles were excised to investigate histological and molecular parameters. LDM DOX treatment induced cachexia with significant impacts on both body and lean mass, and fatigue/malaise (i.e. it reduced voluntary wheel running and energy expenditure) that was associated with oxidative/nitrostative stress sufficient to induce the molecular cytotoxic stress regulator, nuclear factor erythroid-2-related factor 2 (NRF-2). SN co-treatment afforded no therapeutic potential, nor did it promote the wasting of lean tissue. Our data re-affirm a cardioprotective effect for SN against DOX-induced collagen deposition. In our mouse model, SN protected against LDM DOX-induced cardiac fibrosis but had no effect on cachexia at the conclusion of the regimen.

Exercise May Ameliorate the Detrimental Side Effects of High Vitamin D Supplementation on Muscle Function in Mice.

Vitamin D is commonly prescribed to normalize deficiencies and to treat osteoporosis. However, the effect vitamin D supplements have on skeletal muscle health is equivocal. Although vitamin D is known to play a role in the various processes that maintain muscle integrity and function, recent studies utilizing high bolus dose vitamin D supplementation has demonstrated an increased risk of falls. Thus, the aim of this study was to investigate the effects of high vitamin D supplementation on skeletal muscle function with and without exercise enrichment. Four-week old C57BL/10 mice (n = 48) were separated into either normal vitamin D (1500 IU/kg diet; unsupplemented) or high vitamin D (20,000 IU/kg diet; supplemented) treatment groups. Each dietary group was further separated into interventional subgroups where mice either remained sedentary or received exercise-enrichment for 8 weeks in the form of voluntary running. Following the intervention period, whole body in vivo and ex vivo contractile analysis were performed. High vitamin D supplementation decreased force production in the slow-twitch soleus muscles of sedentary mice (p < .01); however, exercise normalized this effect. Eight weeks of exercise did not improve fatigue resistance of the extensor digitorum longus (EDL) or soleus muscles in unsupplemented mice, likely due to low levels of activation in these muscles. In contrast, fatigability was improved in the EDL (p < .01) and even more so in the soleus (p < .001) in the supplemented exercise-enriched group. Our data highlights that increasing vitamin D levels above normal reduces postural muscle force as seen in the soleus. Thus, unnecessary vitamin D supplementation may contribute to the increased risk of falls observed in some studies. Interestingly, when vitamin D supplementation was combined with exercise, force production was effectively restored, and fatigue resistance improved, even in muscles lowly activated. Regular exercise may modulate the effects of vitamin D on skeletal muscle, and be recommended for individuals receiving vitamin D supplements. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.