Iron overload and impaired iron handling contribute to the dystrophic pathology in models of Duchenne muscular dystrophy.

BACKGROUND: Oxidative stress is implicated in the pathophysiology of Duchenne muscular dystrophy (DMD, caused by mutations in the dystrophin gene), which is the most common and severe of the muscular dystrophies. To our knowledge, the distribution of iron, an important modulator of oxidative stress, has not been assessed in DMD. We tested the hypotheses that iron accumulation occurs in mouse models of DMD and that modulation of iron through the diet or chelation could modify disease severity. METHODS: We assessed iron distribution and total elemental iron using LA-ICP-MS on skeletal muscle cross-sections of 8-week-old Bl10 control mice and dystrophic mdx mice (with moderate dystrophy) and dystrophin/utrophin-null mice (dko, with severe dystrophy). In addition, mdx mice (4 weeks) were treated with either an iron chelator (deferiprone 150 mg/kg/day) or iron-enriched feed (containing 1% added iron as carbonyl iron). Immunoblotting was used to determine the abundance of iron- and mitochondria-related proteins. (Immuno)histochemical and mRNA assessments of fibrosis and inflammation were also performed. RESULTS: We observed a significant increase in total elemental iron in hindlimb muscles of dko mice (+50%, P < 0.05) and in the diaphragm of mdx mice (+80%, P < 0.05), with both tissues exhibiting severe pathology. Iron dyshomeostasis was further evidenced by an increase in the storage protein ferritin (dko: +39%, P < 0.05) and ferroportin compared with Bl10 control mice (mdx: +152% and dko: +175%, P < 0.05). Despite having features of iron overload, dystrophic muscles had lower protein expression of ALAS-1, the rate-limiting enzyme for haem synthesis (dko -44%, P < 0.05), and the haem-containing protein myoglobin (dko -54%, P < 0.05). Deferiprone treatment tended to decrease muscle iron levels in mdx mice (-30%, P < 0.1), which was associated with lower oxidative stress and fibrosis, but suppressed haem-containing proteins and mitochondrial content. Increasing iron via dietary intervention elevated total muscle iron (+25%, P < 0.05) but did not aggravate the pathology. CONCLUSIONS: Muscles from dystrophic mice have increased iron levels and dysregulated iron-related proteins that are associated with dystrophic pathology. Muscle iron levels were manipulated by iron chelation and iron enriched feed. Iron chelation reduced fibrosis and reactive oxygen species (ROS) but also suppressed haem-containing proteins and mitochondrial activity. Conversely, iron supplementation increased ferritin and haem-containing proteins but did not alter ROS, fibrosis, or mitochondrial activity. Further studies are required to investigate the contribution of impaired ferritin breakdown in the dysregulation of iron homeostasis in DMD.

Spatiotemporal Mapping Reveals Regional Gastrointestinal Dysfunction in mdx Dystrophic Mice Ameliorated by Oral L-arginine Supplementation.

BACKGROUND/AIMS: Patients with Duchenne muscular dystrophy exhibit significant, ongoing impairments in gastrointestinal (GI) function likely resulting from dysregulated nitric oxide production. Compounds increasing neuronal nitric oxide synthase expression and/or activity could improve GI dysfunction and enhance quality of life for dystrophic patients. We used video imaging and spatiotemporal mapping to identify GI dysfunction in mdx dystrophic mice and determine whether dietary intervention to enhance nitric oxide could alleviate aberrant colonic activity in muscular dystrophy. METHODS: Four-week-old male C57BL/10 and mdx mice received a specialized diet either with no supplementation (control) or supplemented (1 g/kg/day) with L-alanine, L-arginine, or L-citrulline for 8 weeks. At the conclusion of treatment, mice were sacrificed by cervical dislocation and colon motility examined by spatiotemporal (ST) mapping ex vivo. RESULTS: ST mapping identified increased contraction number in the mid and distal colon of mdx mice on control and L-alanine supplemented diets relative to C57BL/10 mice (P < 0.05). Administration of either L-arginine or L-citrulline attenuated contraction number in distal colons of mdx mice relative to C57BL/10 mice. CONCLUSIONS: GI dysfunction in Duchenne muscular dystrophy has been sadly neglected as an issue affecting quality of life. ST mapping identified regional GI dysfunction in the mdx dystrophic mouse. Dietary interventions to increase nitric oxide signaling in the GI tract reduced the number of colonic contractions and alleviated colonic constriction at rest. These findings in mdx mice reveal that L-arginine can improve colonic motility and has potential therapeutic relevance for alleviating GI discomfort, improving clinical care, and enhancing quality of life in Duchenne muscular dystrophy.

Glycine administration attenuates progression of dystrophic pathology in prednisolone-treated dystrophin/utrophin null mice.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by progressive muscle wasting and weakness and premature death. Glucocorticoids (e.g. prednisolone) remain the only drugs with a favorable impact on DMD patients, but not without side effects. We have demonstrated that glycine preserves muscle in various wasting models. Since glycine effectively suppresses the activity of pro-inflammatory macrophages, we investigated the potential of glycine treatment to ameliorate the dystrophic pathology. Dystrophic mdx and dystrophin-utrophin null (dko) mice were treated with glycine or L-alanine (amino acid control) for up to 15 weeks and voluntary running distance (a quality of life marker and strong correlate of lifespan in dko mice) and muscle morphology were assessed. Glycine increased voluntary running distance in mdx mice by 90% (P < 0.05) after 2 weeks and by 60% (P < 0.01) in dko mice co-treated with prednisolone over an 8 week treatment period. Glycine treatment attenuated fibrotic deposition in the diaphragm by 28% (P < 0.05) after 10 weeks in mdx mice and by 22% (P < 0.02) after 14 weeks in dko mice. Glycine treatment augmented the prednisolone-induced reduction in fibrosis in diaphragm muscles of dko mice (23%, P < 0.05) after 8 weeks. Our findings provide strong evidence that glycine supplementation may be a safe, simple and effective adjuvant for improving the efficacy of prednisolone treatment and improving the quality of life for DMD patients.

Glycine metabolism in skeletal muscle: implications for metabolic homeostasis.

PURPOSE OF REVIEW: The review summarizes the recent literature on the role of glycine in skeletal muscle during times of stress. RECENT FINDINGS: Supplemental glycine protects muscle mass and function under pathological conditions. In addition, mitochondrial dysfunction in skeletal muscle leads to increased cellular serine and glycine production and activation of NADPH-generating pathways and glutathione metabolism. These studies highlight how glycine availability modulates cellular homeostasis and redox status. SUMMARY: Recent studies demonstrate that supplemental glycine effectively protects muscles in a variety of wasting models, including cancer cachexia, sepsis, and reduced caloric intake. The underlying mechanisms responsible for the effects of glycine remain unclear but likely involve receptor-mediated responses and modulation of intracellular metabolism. Future research to understand these mechanisms will provide insight into glycine's therapeutic potential. Our view is that glycine holds considerable promise for improving health by protecting muscles during different wasting conditions.

Glycine supplementation during calorie restriction accelerates fat loss and protects against further muscle loss in obese mice.

BACKGROUND & AIM: Calorie restriction (CR) reduces co-morbidities associated with obesity, but also reduces lean mass thereby predisposing people to weight regain. Since we demonstrated that glycine supplementation can reduce inflammation and muscle wasting, we hypothesized that glycine supplementation during CR would preserve muscle mass in mice. METHODS: High-fat fed male C57BL/6 mice underwent 20 days CR (40% reduced calories) supplemented with glycine (1 g/kg/day; n = 15, GLY) or l-alanine (n = 15, ALA). Body composition and glucose tolerance were assessed and hindlimb skeletal muscles and epididymal fat were collected. RESULTS: Eight weeks of a high-fat diet (HFD) induced obesity and glucose intolerance. CR caused rapid weight loss (ALA: 20%, GLY: 21%, P < 0.01), reduced whole-body fat mass (ALA: 41%, GLY: 49% P < 0.01), and restored glucose tolerance to control values in ALA and GLY groups. GLY treated mice lost more whole-body fat mass (14%, p < 0.05) and epididymal fat mass (26%, P < 0.05), less lean mass (27%, P < 0.05), and had better preserved quadriceps muscle mass (4%, P < 0.01) than ALA treated mice after 20 d CR. Compared to the HFD group, pro-inflammatory genes were lower (P < 0.05), metabolic genes higher (P < 0.05) and S6 protein phosphorylation lower after CR, but not different between ALA and GLY groups. There were significant correlations between %initial fat mass (pre CR) and the mRNA expression of genes involved in inflammation (r = 0.51 to 0.68, P < 0.05), protein breakdown (r = -0.66 to -0.37, P < 0.05) and metabolism (r = -0.59 to -0.47, P < 0.05) after CR. CONCLUSION: Taken together, these findings suggest that glycine supplementation during CR may be beneficial for preserving muscle mass and stimulating loss of adipose tissue.

Citrulline does not prevent skeletal muscle wasting or weakness in limb-casted mice.

BACKGROUND: Increasing arginine (Arg) availability reduces atrophy in cultured skeletal muscle cells. Supplementation with its metabolic precursor citrulline (Cit) is more effective at improving skeletal muscle Arg concentrations. OBJECTIVE: We tested the hypothesis that Cit supplementation would attenuate skeletal muscle atrophy and loss of function during hindlimb immobilization in mice. METHODS: Male C57BL/6JArc mice underwent 14 d of unilateral hindlimb immobilization/plaster casting and were supplemented with ~0.81 g Cit · kg⁻¹ · d⁻¹ (CIT group) or Ala (ALA group) mixed into their food. The uncasted contralateral limb (internal control) and an uncasted group (CON) served as controls. Muscle atrophy was evaluated with mass, fiber area, and in situ muscle function. RESULTS: Tibialis anterior (TA) muscle mass [ALA: 37.6 ± 0.92 mg; CIT: 38.3 ± 1.25 mg] and peak tetanic force (ALA: 1150 ± 38.5 mN; CIT: 1150 ± 52.0 mN) were lower (P < 0.001) in the ALA (53.9 ± 0.42 mg) and CIT (1760 ± 28.5 mN) groups than in the CON group. No difference was found between ALA and CIT groups for TA mass, fiber area, or peak force. The mRNA expression of the nitric oxide synthase 2, inducible (Nos2; ~15-fold) and B-cell chronic lymphoid leukemia/lymphoma 2/adenovirus E1B 19 kDa interacting protein 3 (Bnip3; ~17-fold) genes and the ratio of microtubule-associated protein light chain 3BII to 3BI (LC3BII:LC3BI) (50.5% ± 17.7%) were higher (P < 0.05) in the ALA group than in the CON group, suggesting increased autophagy. In the CIT group, Bnip3 mRNA was lower (-70%; P < 0.05) and Nos2 mRNA tended to be lower (-45%; P = 0.05) than in the ALA group, whereas LC3BII:LC3BI was not different from the CON group. CONCLUSIONS: Cit treatment of male mice did not affect therapeutically relevant outcome measures such as skeletal muscle mass and peak muscle force after 14 d of hindlimb immobilization.

Leucine as a treatment for muscle wasting: a critical review.

Amino acids are potent modulators of protein turnover and skeletal muscle cells are highly sensitive to changes in amino acid availability. During amino acid abundance increased activity of mTORC1 drives protein synthesis and growth. In skeletal muscle, it has been clearly demonstrated that of all the amino acids, leucine is the most potent stimulator of mTORC1 and protein synthesis in vitro and in vivo. As such, leucine has received considerable attention as a potential pharmaconutrient for the treatment of numerous muscle wasting conditions. However, despite a multitude of studies showing enhanced acute protein synthesis with leucine or leucine-rich supplements in healthy individuals, additional leucine intake does not necessarily enhance protein synthesis during muscle wasting conditions. In addition, long-term, placebo controlled, iso-caloric studies in humans consistently show no beneficial effect of leucine supplementation on skeletal muscle mass or function. This review, critically evaluates the therapeutic potential of leucine to attenuate the skeletal muscle wasting associated with ageing, cancer and immobilization/bed rest. It also highlights the impact of inflammation on amino acid sensing, mTORC1 activation and stimulation of protein synthesis and challenges the underlying hypothesis that the acute activation of mTORC1 and stimulation of protein synthesis by leucine increases in muscle mass over time. We conclude that leucine, as a standalone nutritional intervention, is not effective in the prevention of muscle wasting. Future work should focus on identifying and utilizing other nutrients or treatments that sensitize skeletal muscle to leucine, thereby enhancing its therapeutic potential for muscle wasting conditions.

Glycine administration attenuates skeletal muscle wasting in a mouse model of cancer cachexia.

BACKGROUND AND AIMS: The non-essential amino acid, glycine, is often considered biologically neutral, but some studies indicate that it could be an effective anti-inflammatory agent. Since inflammation is central to the development of cancer cachexia, glycine supplementation represents a simple, safe and promising treatment. We tested the hypothesis that glycine supplementation reduces skeletal muscle inflammation and preserves muscle mass in tumor-bearing mice. METHODS: To induce cachexia, CD2F1 mice received a subcutaneous injection of PBS (control, n = 12) or C26 tumor cells (n = 32) in accordance with the protocols developed by Murphy et al. [Murphy KT, Chee A, Trieu J, Naim T, Lynch GS. Importance of functional and metabolic impairments in the characterization of the C-26 murine model of cancer cachexia. Dis Models Mech 2012;5(4):533-545.]. Subcutaneous injections of glycine (n = 16) or PBS (n = 16) were administered daily for 21 days and at the conclusion of treatment, selected muscles, tumor and adipose tissue were collected and prepared for Real-Time RT-PCR or western blot analysis. RESULTS: Glycine attenuated the loss of fat and muscle mass, blunted increases in markers of inflammation (F4/80, P = 0.01 & IL-6 mRNA, P = 0.01) and atrophic signaling (MuRF, P = 0.047; atrogin-1, P = 0.04; LC3B, P = 0.06 and; BNIP3, P = 0.10) and tended to attenuate the loss of body mass (P = 0.07), muscle function (P = 0.06), and oxidative stress (GSSG/GSH, P = 0.06 and DHE, P = 0.07) seen in tumor-bearing mice. Preliminary studies that compared the effect of glycine administration with isonitrogenous doses of alanine or citrulline showed that the observed protective effect was specific to glycine. CONCLUSIONS: Glycine protects skeletal muscle from cancer-induced wasting and loss of function, reduces the oxidative and inflammatory burden, and reduces the expression of genes associated with muscle protein breakdown in cancer cachexia. Importantly, these effects were glycine specific.

Hsp72 preserves muscle function and slows progression of severe muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca(2+), which activates inflammatory and muscle degenerative pathways. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca(2+)) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.

Therapeutic approaches for muscle wasting disorders.

Muscle wasting and weakness are common in many disease states and conditions including aging, cancer cachexia, sepsis, denervation, disuse, inactivity, burns, HIV-acquired immunodeficiency syndrome (AIDS), chronic kidney or heart failure, unloading/microgravity, and muscular dystrophies. Although the maintenance of muscle mass is generally regarded as a simple balance between protein synthesis and protein degradation, these mechanisms are not strictly independent, but in fact they are coordinated by a number of different and sometimes complementary signaling pathways. Clearer details are now emerging about these different molecular pathways and the extent to which these pathways contribute to the etiology of various muscle wasting disorders. Therapeutic strategies for attenuating muscle wasting and improving muscle function vary in efficacy. Exercise and nutritional interventions have merit for slowing the rate of muscle atrophy in some muscle wasting conditions, but in most cases they cannot halt or reverse the wasting process. Hormonal and/or other drug strategies that can target key steps in the molecular pathways that regulate protein synthesis and protein degradation are needed. This review describes the signaling pathways that maintain muscle mass and provides an overview of some of the major conditions where muscle wasting and weakness are indicated. The review provides details on some therapeutic strategies that could potentially attenuate muscle atrophy, promote muscle growth, and ultimately improve muscle function. The emphasis is on therapies that can increase muscle mass and improve functional outcomes that will ultimately lead to improvement in the quality of life for affected patients.

Hyperbaric oxygen increases the contractile function of regenerating rat slow muscles.

UNLABELLED: Human trials of hyperbaric oxygen (HBO) treatment of sports-related muscle injuries are equivocal. Although most human skeletal muscles are composed of mixed muscle fiber types, it is unclear whether HBO affects fiber types differently. PURPOSE: We tested the hypothesis that HBO can enhance the functional properties of regenerating rat soleus muscles that are composed predominantly of slow fibers. METHODS: After intramuscular injection of bupivacaine hydrochloride to induce the degeneration of all fibers within the soleus muscle, treated rats received daily HBO treatment at 3 atmospheres absolute. RESULTS: In untreated rats, injured muscles demonstrated a reduced force-producing capacity (control soleus vs injured soleus, 220.3 +/- 2.5 vs 157.6 +/- 3.3 kN.m(-2) at 25 d postinjury, respectively, P < 0.05) and contained smaller regenerating muscle fibers than uninjured soleus muscles (fiber cross sectional area in control soleus vs injured soleus, 2289 +/- 164 vs 1154 +/- 92 microm 2 at 25 d postinjury, respectively, P < 0.05). The regenerating soleus muscles of HBO-treated rats demonstrated a greater force-producing capacity as a percentage of contralateral control muscles than the regenerating muscles from untreated rats at 14 d postinjury (regenerating HBO-soleus peak tension and untreated soleus peak tension, 42.9 +/- 1.9 and 35.8 +/- 3.9% of contralateral control muscles, respectively, P < 0.05), but no effect of treatment was observed at 25 d postinjury. CONCLUSION: HBO enhanced the contractile properties of regenerating rat soleus muscles after myotoxic injury, but this improvement was not sustained for the duration of the regenerative process. The data indicate that the outcome of HBO treatment of a muscle injury may be influenced by the fiber type composition of the injured muscle.