Trigonelline is an NAD+ precursor that improves muscle function during ageing and is reduced in human sarcopenia.

Mitochondrial dysfunction and low nicotinamide adenine dinucleotide (NAD+) levels are hallmarks of skeletal muscle ageing and sarcopenia1-3, but it is unclear whether these defects result from local changes or can be mediated by systemic or dietary cues. Here we report a functional link between circulating levels of the natural alkaloid trigonelline, which is structurally related to nicotinic acid4, NAD+ levels and muscle health in multiple species. In humans, serum trigonelline levels are reduced with sarcopenia and correlate positively with muscle strength and mitochondrial oxidative phosphorylation in skeletal muscle. Using naturally occurring and isotopically labelled trigonelline, we demonstrate that trigonelline incorporates into the NAD+ pool and increases NAD+ levels in Caenorhabditis elegans, mice and primary myotubes from healthy individuals and individuals with sarcopenia. Mechanistically, trigonelline does not activate GPR109A but is metabolized via the nicotinate phosphoribosyltransferase/Preiss-Handler pathway5,6 across models. In C. elegans, trigonelline improves mitochondrial respiration and biogenesis, reduces age-related muscle wasting and increases lifespan and mobility through an NAD+-dependent mechanism requiring sirtuin. Dietary trigonelline supplementation in male mice enhances muscle strength and prevents fatigue during ageing. Collectively, we identify nutritional supplementation of trigonelline as an NAD+-boosting strategy with therapeutic potential for age-associated muscle decline.

Plasma beta-hydroxy-beta-methylbutyrate availability after enteral administration during critical illness after trauma: An exploratory study.

BACKGROUND: During critical illness skeletal muscle wasting occurs rapidly. Although beta-hydroxy-beta-methylbutyrate (HMB) is a potential treatment to attenuate this process, the plasma appearance and muscle concentration is uncertain. METHODS: This was an exploratory study nested within a blinded, parallel group, randomized clinical trial in which critically ill patients after trauma received enteral HMB (3 g daily) or placebo. Plasma samples were collected at 0, 60, and 180 min after study supplement administration on day 1. Needle biopsies of the vastus lateralis muscle were collected (baseline and day 7 of the HMB treatment intervention period). An external standard curve was used to calculate HMB concentrations in plasma and muscle. RESULTS: Data were available for 16 participants (male n = 12 (75%), median [interquartile range] age 50 [29-58] years) who received placebo and 18 participants (male n = 14 (78%), age 49 [34-55] years) who received HMB. Plasma HMB concentrations were similar at baseline but increased after HMB (T = 60 min: placebo 0.60 [0.44-1.31] µM; intervention 51.65 [22.76-64.72] µM). Paired muscle biopsies were collected from 11 participants (placebo n = 7, HMB n = 4). Muscle HMB concentrations were similar at baseline between groups (2.35 [2.17-2.95]; 2.07 [1.78-2.31] µM). For participants in the intervention group who had the repeat biopsy within 4 h of HMB administration, concentrations were greater (7.2 and 12.3 µM) than those who had the repeat biopsy >4 h after HMB (2.7 and 2.1 µM). CONCLUSION: In this exploratory study, enteral HMB administration increased plasma HMB availability. The small sample size limits interpretation of the muscle HMB findings.

Beta-hydroxy-beta-methylbutyrate supplementation and functional outcomes in multitrauma patients: A pilot randomized controlled trial.

BACKGROUND: Beta-hydroxy-beta-methylbutyrate (HMB) is a nutrition supplement that may attenuate muscle wasting from critical illness. This trial aimed to determine feasibility of administering a blinded nutrition supplement in the intensive care unit (ICU) and continuing it after ICU discharge. METHODS: Single-center, parallel-group, blinded, placebo-controlled, randomized feasibility trial. After traumatic injury necessitating admission to ICU, participants were randomized to receive an enteral study supplement of 3 g of HMB (intervention) or placebo daily for 28 days or until hospital discharge. Primary outcome was feasibility of administering the study supplement, quantified as protocol adherence. Secondary outcomes included change in quadriceps muscle thickness, measured weekly until day 28 or hospital discharge by using ultrasound and analyzed by using a linear mixed model. RESULTS: Fifty randomized participants (intervention, n = 26; placebo, n = 24) showed comparable baseline characteristics. Participants received 862 (84.3%) of the 1022 prescribed supplements during hospitalization with 543 (62.8%) delivered via an enteral feeding tube. The median (IQR) number of study supplements successfully administered per participant was 19.5 (13.0-24.0) in the intervention group and 16.5 (8.5-23.5) in the placebo group. Marked loss of quadriceps muscle thickness occurred in both groups, with the point estimate favoring attenuated muscle loss with the intervention, albeit with wide CIs (mean intervention difference after 28 days, 0.26 cm [95% CI, -0.13 to 0.64]). CONCLUSION: A blinded, placebo-controlled, randomized clinical trial of daily enteral HMB supplementation for up to 28 days in hospital is feasible. Any effect of HMB supplementation to attenuate muscle wasting after traumatic injury remains uncertain.

Age-Related Changes in Skeletal Muscle Iron Homeostasis.

Sarcopenia is an age-related condition of slow, progressive loss of muscle mass and strength, which contributes to frailty, increased risk of hospitalization and mortality, and increased health care costs. The incidence of sarcopenia is predicted to increase to >200 million affected older adults worldwide over the next 40 years, highlighting the urgency for understanding biological mechanisms and developing effective interventions. An understanding of the mechanisms underlying sarcopenia remains incomplete. Iron in the muscle is important for various metabolic functions, including oxygen supply and electron transfer during energy production, yet these same chemical properties of iron may be deleterious to the muscle when either in excess or when biochemically unshackled (eg, in ferroptosis), it can promote oxidative stress and induce inflammation. This review outlines the mechanisms leading to iron overload in muscle with aging and evaluates the evidence for the iron overload hypothesis of sarcopenia. Based on current evidence, studies are needed to (a) determine the mechanisms leading to iron overload in skeletal muscle during aging; and (b) investigate whether skeletal muscles are functionally deficient in iron during aging leading to impairments in oxidative metabolism.

Time-resolved phosphoproteome and proteome analysis reveals kinase signaling on master transcription factors during myogenesis.

Myogenesis is governed by signaling networks that are tightly regulated in a time-dependent manner. Although different protein kinases have been identified, knowledge of the global signaling networks and their downstream substrates during myogenesis remains incomplete. Here, we map the myogenic differentiation of C2C12 cells using phosphoproteomics and proteomics. From these data, we infer global kinase activity and predict the substrates that are involved in myogenesis. We found that multiple mitogen-activated protein kinases (MAPKs) mark the initial wave of signaling cascades. Further phosphoproteomic and proteomic profiling with MAPK1/3 and MAPK8/9 specific inhibitions unveil their shared and distinctive roles in myogenesis. Lastly, we identified and validated the transcription factor nuclear factor 1 X-type (NFIX) as a novel MAPK1/3 substrate and demonstrated the functional impact of NFIX phosphorylation on myogenesis. Altogether, these data characterize the dynamics, interactions, and downstream control of kinase signaling networks during myogenesis on a global scale.

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.

Albumin and C-reactive protein relate to functional and body composition parameters in patients admitted to geriatric rehabilitation after acute hospitalization: findings from the RESORT cohort.

PURPOSE: Albumin and C-reactive protein (CRP) are non-specific markers of inflammation, which could affect muscle tissue during acute hospitalization. We investigated the association between albumin and CRP during acute hospitalization with functional and body composition parameters in patients admitted to geriatric rehabilitation. METHODS: The REStORing Health of Acutely Unwell AdulTs (RESORT) cohort includes geriatric rehabilitation patients assessed for change in activities of daily living (ADL, using the Katz index) during acute hospitalization, and subsequently for Katz ADL, gait speed (GS), handgrip strength (HGS) and skeletal muscle mass index (SMI) at geriatric rehabilitation admission. Albumin and CRP average (median), variation (interquartile range), and maximum or minimum were collected from serum samples, and were examined for their association with functional and body composition parameters using multivariable linear regression analysis adjusted for age, sex and length of acute hospital stay. RESULTS: 1769 Inpatients were included for analyses (mean age 82.6 years ± 8.1, 56% female). Median length of acute hospitalization was 7 [IQR 4, 13] days and median number of albumin and CRP measurements was 5 [IQR 3, 12] times. ADL declined in 89% of patients (median - 3 points, IQR - 4, - 2). Lower average albumin, higher albumin variation and lower minimum albumin were associated with larger declines in ADL and with lower ADL, GS, HGS and SMI at geriatric rehabilitation admission. Higher average and maximum CRP were associated with lower GS. CONCLUSION: Inflammation, especially lower albumin concentrations, during acute hospitalization is associated with lower physical function at geriatric rehabilitation admission.

ß-Hydroxy-ß-methylbutyrate (HMB) supplementation and functional outcomes in multi-trauma patients: a study protocol for a pilot randomised clinical trial (BOOST trial).

BACKGROUND: There are no therapies proven to diminish the muscle wasting that occurs in patients after major trauma who are admitted to the intensive care unit (ICU). ß-Hydroxy-ß-methylbutyrate (HMB) is a nutrition intervention that may attenuate muscle loss and, thereby, improve recovery. The primary aim of this study is to determine the feasibility of a blinded randomised clinical trial of HMB supplementation to patients after major trauma who are admitted to the ICU. Secondary aims are to establish estimates for the impact of HMB when compared to placebo on muscle mass and nutrition-related patient outcomes. METHODS: This prospective, single-centre, blinded, randomised, placebo-controlled, parallel-group, feasibility trial with allocation concealment will recruit 50 participants over 18 months. After informed consent, participants will be randomised [1:1] to receive either the intervention (three grams of HMB dissolved in either 150 ml of orange juice for those allowed oral intake or 150 ml of water for those being enterally fed) or placebo (150 ml of orange juice for those allowed oral intake or 150 ml of water for those being enterally fed). The intervention will be commenced in ICU, continued after ICU discharge and ceased at hospital discharge or day 28 post randomisation, whichever occurs first. The primary outcome is the feasibility of administering the intervention. Secondary outcomes include change in muscle thickness using ultrasound and other nutritional and patient-centred outcomes. DISCUSSION: This study aims to determine the feasibility of administering HMB to critically ill multi-trauma patients throughout ICU admission until hospital discharge. Results will inform design of a larger randomised clinical trial. TRIAL REGISTRATION: The protocol is registered with Australian New Zealand Clinical Trials Registry (ANZCTR) ANZCTR: 12620001305910 . UTN: U1111-1259-5534.

Investigating the Potential for Sulforaphane to Attenuate Gastrointestinal Dysfunction in mdx Dystrophic Mice.

Gastrointestinal (GI) dysfunction is an important, yet understudied condition associated with Duchenne muscular dystrophy (DMD), with patients reporting bloating, diarrhea, and general discomfort, contributing to a reduced quality of life. In the mdx mouse, the most commonly used mouse model of DMD, studies have confirmed GI dysfunction (reported as altered contractility and GI transit through the small and large intestine), associated with increased local and systemic inflammation. Sulforaphane (SFN) is a natural isothiocyanate with anti-inflammatory and anti-oxidative properties via its activation of Nrf2 signalling that has been shown to improve aspects of the skeletal muscle pathology in dystrophic mice. Whether SFN can similarly improve GI function in muscular dystrophy was unknown. Video imaging and spatiotemporal mapping to assess gastrointestinal contractions in isolated colon preparations from mdx and C57BL/10 mice revealed that SFN reduced contraction frequency when administered ex vivo, demonstrating its therapeutic potential to improve GI function in DMD. To confirm this in vivo, four-week-old male C57BL/10 and mdx mice received vehicle (2% DMSO/corn oil) or SFN (2 mg/kg in 2% DMSO/corn oil) via daily oral gavage five days/week for 4 weeks. SFN administration reduced fibrosis in the diaphragm of mdx mice but did not affect other pathological markers. Gene and protein analysis revealed no change in Nrf2 protein expression or activation of Nrf2 signalling after SFN administration and oral SFN supplementation did not improve GI function in mdx mice. Although ex vivo studies demonstrate SFN's therapeutic potential for reducing colon contractions, in vivo studies should investigate higher doses and/or alternate routes of administration to confirm SFN's potential to improve GI function in DMD.

Dystrophin deficiency disrupts muscle clock expression and mitochondrial quality control in mdx mice.

Impaired oxidative capacity and mitochondrial function contribute to the dystrophic pathology in muscles of patients with Duchenne muscular dystrophy (DMD) and in relevant mouse models of the disease. Emerging evidence suggests an association between disrupted core clock expression and mitochondrial quality control, but this has not been established in muscles lacking dystrophin. We examined the diurnal regulation of muscle core clock and mitochondrial quality control expression in dystrophin-deficient C57BL/10ScSn-Dmdmdx (mdx) mice, an established model of DMD. Male C57BL/10 (BL/10; n = 18) and mdx mice (n = 18) were examined every 4 h beginning at the dark cycle. Throughout the entire light-dark cycle, extensor digitorum longus (EDL) muscles from mdx mice had decreased core clock mRNA expression (Arntl, Cry1, Cry2, Nr1d2; P < 0.05) and disrupted mitochondrial quality control mRNA expression related to biogenesis (decreased; Ppargc1a, Esrra; P < 0.05), fission (increased; Dnm1l; P < 0.01), fusion (decreased; Opa1, Mfn1; P < 0.05), and autophagy/mitophagy (decreased: Bnip3; P < 0.05; increased: Becn1; P < 0.05). Cosinor analysis revealed a decrease in the rhythmicity parameters mesor and amplitude for Arntl, Cry1, Cry2, Per2, and Nr1d1 (P < 0.001) in mdx mice. Diurnal oscillations in Esrra, Sirt1, Map1lc3b, and Sqstm1 were absent in mdx mice, along with decreased mesor and amplitude of Ppargc1a mRNA expression (P < 0.01). The expression of proteins involved in mitochondrial biogenesis (decreased: PPARGC1A, P < 0.05) and autophagy/mitophagy (increased: MAP1LC3BII, SQSTM1, BNIP3; P < 0.05) were also dysregulated in tibialis anterior muscles of mdx mice. These findings suggest that dystrophin deficiency in mdx mice impairs the regulation of the core clock and mitochondrial quality control, with relevance to DMD and related disorders.

Iron accumulation in skeletal muscles of old mice is associated with impaired regeneration after ischaemia-reperfusion damage.

BACKGROUND: Oxidative stress is implicated in the insidious loss of muscle mass and strength that occurs with age. However, few studies have investigated the role of iron, which is elevated during ageing, in age-related muscle wasting and blunted repair after injury. We hypothesized that iron accumulation leads to membrane lipid peroxidation, muscle wasting, increased susceptibility to injury, and impaired muscle regeneration. METHODS: To examine the role of iron in age-related muscle atrophy, we compared the skeletal muscles of 3-month-old with 22- to 24-month-old 129SvEv FVBM mice. We assessed iron distribution and total elemental iron using laser ablation inductively coupled plasma mass spectrometry and Perls' stain on skeletal muscle cross-sections. In addition, old mice underwent ischaemia-reperfusion (IR) injury (90 min ischaemia), and muscle regeneration was assessed 14 days after injury. Immunoblotting was used to determine lipid peroxidation (4HNE) and iron-related proteins. To determine whether muscle iron content can be altered, old mice were treated with deferiprone (DFP) in the drinking water, and we assessed its effects on muscle regeneration after injury. RESULTS: We observed a significant increase in total elemental iron (+43%, P < 0.05) and lipid peroxidation (4HNE: +76%, P < 0.05) in tibialis anterior muscles of old mice. Iron was further increased after injury (adult: +81%, old: +135%, P < 0.05) and associated with increased lipid peroxidation (+41%, P < 0.05). Administration of DFP did not impact iron or measures of lipid peroxidation in skeletal muscle or modulate muscle mass. Increased muscle iron concentration and lipid peroxidation were associated with less efficient regeneration, evident from the smaller fibres in cross-sections of tibialis anterior muscles (-24%, P < 0.05) and an increased percentage of fibres with centralized nuclei (+4124%, P < 0.05) in muscles of old compared with adult mice. Administration of DFP lowered iron after IR injury (PRE: -32%, P < 0.05 and POST: -41%, P < 0.05), but did not translate to structural improvements. CONCLUSIONS: Muscles from old mice have increased iron levels, which are associated with increased lipid peroxidation, increased susceptibility to IR injury, and impaired muscle regeneration. Our results suggest that iron is involved in effective muscle regeneration, highlighting the importance of iron homeostasis in muscle atrophy and muscle repair.

Functional electrical stimulation in-bed cycle ergometry in mechanically ventilated patients: a multicentre randomised controlled trial.

PURPOSE: To investigate the effect of functional electrical stimulation-assisted cycle ergometry (FES-cycling) on muscle strength, cognitive impairment and related outcomes. METHODS: Mechanically ventilated patients aged ≥18 years with sepsis or systemic inflammatory response syndrome were randomised to either 60 min of FES-cycling >5 days/week while in the intensive care unit (ICU) plus usual care rehabilitation versus usual care rehabilitation alone, with evaluation of two primary outcomes: (1) muscle strength at hospital discharge and (2) cognitive impairment at 6-month follow-up. RESULTS: We enrolled 162 participants, across four study sites experienced in ICU rehabilitation in Australia and the USA, to FES-cycling (n=80; mean age±SD 59±15) versus control (n=82; 56±14). Intervention participants received a median (IQR) of 5 (3-9) FES-cycling sessions with duration of 56 (34-63) min/day plus 15 (10-23) min/day of usual care rehabilitation. The control group received 15 (8-15) min/day of usual care rehabilitation. In the intervention versus control group, there was no significant differences for muscle strength at hospital discharge (mean difference (95% CI) 3.3 (-5.0 to 12.1) Nm), prevalence of cognitive impairment at 6 months (OR 1.1 (95% CI 0.30 to 3.8)) or secondary outcomes measured in-hospital and at 6 and 12 months follow-up. CONCLUSION: In this randomised controlled trial, undertaken at four centres with established rehabilitation programmes, the addition of FES-cycling to usual care rehabilitation did not substantially increase muscle strength at hospital discharge. At 6 months, the incidence of cognitive impairment was almost identical between groups, but potential benefit or harm of the intervention on cognition cannot be excluded due to imprecision of the estimated effect. TRIAL REGISTRATION NUMBER: ACTRN 12612000528853, NCT02214823.

Metabolic remodeling of dystrophic skeletal muscle reveals biological roles for dystrophin and utrophin in adaptation and plasticity.

OBJECTIVES: Preferential damage to fast, glycolytic myofibers is common in many muscle-wasting diseases, including Duchenne muscular dystrophy (DMD). Promoting an oxidative phenotype could protect muscles from damage and ameliorate the dystrophic pathology with therapeutic relevance, but developing efficacious strategies requires understanding currently unknown biological roles for dystrophin and utrophin in dystrophic muscle adaptation and plasticity. METHODS: Combining whole transcriptome RNA sequencing and mitochondrial proteomics with assessments of metabolic and contractile function, we investigated the roles of dystrophin and utrophin in fast-to-slow muscle remodeling with low-frequency electrical stimulation (LFS, 10 Hz, 12 h/d, 7 d/wk, 28 d) in mdx (dystrophin null) and dko (dystrophin/utrophin null) mice, two established preclinical models of DMD. RESULTS: Novel biological roles in adaptation were demonstrated by impaired transcriptional activation of estrogen-related receptor alpha-responsive genes supporting oxidative phosphorylation in dystrophic muscles. Further, utrophin expression in dystrophic muscles was required for LFS-induced remodeling of mitochondrial respiratory chain complexes, enhanced fiber respiration, and conferred protection from eccentric contraction-mediated damage. CONCLUSIONS: These findings reveal novel roles for dystrophin and utrophin during LFS-induced metabolic remodeling of dystrophic muscle and highlight the therapeutic potential of LFS to ameliorate the dystrophic pathology and protect from contraction-induced injury with important implications for DMD and related muscle disorders.

Protein Type, Protein Dose, and Age Modulate Dietary Protein Digestion and Phenylalanine Absorption Kinetics and Plasma Phenylalanine Availability in Humans.

BACKGROUND: Dietary protein ingestion stimulates muscle protein synthesis by providing amino acids to the muscle. The magnitude and duration of the postprandial increase in muscle protein synthesis rates are largely determined by dietary protein digestion and amino acid absorption kinetics. OBJECTIVE: We assessed the impact of protein type, protein dose, and age on dietary protein digestion and amino acid absorption kinetics in vivo in humans. METHODS: We included data from 18 randomized controlled trials with a total of 602 participants [age: 53 ± 23 y; BMI (kg/m2): 24.8 ± 3.3] who consumed various quantities of intrinsically l-[1-13C]-phenylalanine-labeled whey (n = 137), casein (n = 393), or milk (n = 72) protein and received intravenous infusions of l-[ring-2H5]-phenylalanine, which allowed us to assess protein digestion and phenylalanine absorption kinetics and the postprandial release of dietary protein-derived phenylalanine into the circulation. The effect of aging on these processes was assessed in a subset of 82 young (aged 22 ± 3 y) and 83 older (aged 71 ± 5 y) individuals. RESULTS: A total of 50% ± 14% of dietary protein-derived phenylalanine appeared in the circulation over a 5-h postprandial period. Casein ingestion resulted in a smaller (45% ± 11%), whey protein ingestion in an intermediate (57% ± 10%), and milk protein ingestion in a greater (65% ± 13%) fraction of dietary protein-derived phenylalanine appearing in the circulation (P < 0.001). The postprandial availability of dietary protein-derived phenylalanine in the circulation increased with the ingestion of greater protein doses (P < 0.05). Protein digestion and phenylalanine absorption kinetics were attenuated in older when compared with young individuals, with 45% ± 10% vs. 51% ± 14% of dietary protein-derived phenylalanine appearing in the circulation, respectively (P = 0.001). CONCLUSIONS: Protein type, protein dose, and age modulate dietary protein digestion and amino acid absorption kinetics and subsequent postprandial plasma amino acid availability in vivo in humans. These trials were registered at clinicaltrials.gov as NCT00557388, NCT00936039, NCT00991523, NCT01317511, NCT01473576, NCT01576848, NCT01578590, NCT01615276, NCT01680146, NCT01820975, NCT01986842, and NCT02596542, and at http://www.trialregister.nl as NTR3638, NTR3885, NTR4060, NTR4429, and NTR4492.

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 Protects Muscle Cells From Wasting in vitro via mTORC1 Signaling.

Glycine supplementation can protect skeletal muscles of mice from cancer-induced wasting, but the mechanisms underlying this protection are not well-understood. The aim of this study was to determine whether exogenous glycine directly protects skeletal muscle cells from wasting. C2C12 muscle cells were exposed to non-inflammatory catabolic stimuli via two models: serum withdrawal (SF) for 48 h; or incubation in HEPES buffered saline (HBS) for up to 5 h. Cells were supplemented with glycine or equimolar concentrations of L-alanine. SF- and HBS-treated myotubes (with or without L-alanine) were ~20% and ~30% smaller than control myotubes. Glycine-treated myotubes were up to 20% larger (P < 0.01) compared to cells treated with L-alanine in both models of muscle cell atrophy. The mTORC1 inhibitor rapamycin prevented the glycine-stimulated protection of myotube diameter, and glycine-stimulated S6 phosphorylation, suggesting that mTORC1 signaling may be necessary for glycine's protective effects in vitro. Increasing glycine availability may be beneficial for muscle wasting conditions associated with inadequate nutrient intake.

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.

Deletion of suppressor of cytokine signaling 3 (SOCS3) in muscle stem cells does not alter muscle regeneration in mice after injury.

Muscles of older animals are more susceptible to injury and regenerate poorly, in part due to a persistent inflammatory response. The janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway mediates inflammatory signaling and is tightly regulated by the suppressor of cytokine signaling (SOCS) proteins, especially SOCS3. SOCS3 expression is altered in the muscle of aged animals and may contribute to the persistent inflammation and impaired regeneration. To test this hypothesis, we performed myotoxic injuries on mice with a tamoxifen-inducible deletion of SOCS3 specifically within the muscle stem cell compartment. Muscle stem cell-specific SOCS3 deletion reduced muscle mass at 14 days post-injury (-14%, P < 0.01), altered the myogenic transcriptional program, and reduced myogenic fusion based on the number of centrally-located nuclei per muscle fiber. Despite the delay in myogenesis, muscles with a muscle stem cell-specific deletion of SOCS3 were still able to regenerate after a single bout or multiple bouts of myotoxic injury. A reduction in SOCS3 expression in muscle stem cells is unlikely to be responsible for the incomplete muscle repair in aged animals.