Nicotinamide Phosphoribosyltransferase-elevated NAD+ biosynthesis prevents muscle disuse atrophy by reversing mitochondrial dysfunction.

BACKGROUND: It is well known that muscle disuse atrophy is associated with mitochondrial dysfunction, which is implicated in reduced nicotinamide adenine dinucleotide (NAD+ ) levels. Nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in NAD+ biosynthesis, may serve as a novel strategy to treat muscle disuse atrophy by reversing mitochondrial dysfunction. METHODS: To investigate the effects of NAMPT on the prevention of disuse atrophy of skeletal muscles predominantly composed of slow-twitch (type I) or fast-twitch (type II) fibres, rabbit models of rotator cuff tear-induced supraspinatus muscle atrophy and anterior cruciate ligament (ACL) transection-induced extensor digitorum longus (EDL) atrophy were established and then administered NAMPT therapy. Muscle mass, fibre cross-sectional area (CSA), fibre type, fatty infiltration, western blot, and mitochondrial function were assayed to analyse the effects and molecular mechanisms of NAMPT in preventing muscle disuse atrophy. RESULTS: Acute disuse of the supraspinatus muscle exhibited significant loss of mass (8.86 ± 0.25 to 5.10 ± 0.79 g; P < 0.001) and decreased fibre CSA (3939.6 ± 136.1 to 2773.4 ± 217.6 µm2 , P < 0.001), which were reversed by NAMPT (muscle mass 6.17 ± 0.54 g, P = 0.0033; fibre CSA, 3219.8 ± 289.4 µm2 , P = 0.0018). Disuse-induced impairment of mitochondrial function were significantly improved by NAMPT, including citrate synthase activity (40.8 ± 6.3 to 50.5 ± 5.6 nmol/min/mg, P = 0.0043), and NAD+ biosynthesis (279.9 ± 48.7 to 392.2 ± 43.2 pmol/mg, P = 0.0023). Western blot revealed that NAMPT increases NAD+ levels by activating NAMPT-dependent NAD+ salvage synthesis pathway. In supraspinatus muscle atrophy due to chronic disuse, a combination of NAMPT injection and repair surgery was more effective than repair in reversing muscle atrophy. Although the predominant composition of EDL muscle is fast-twitch (type II) fibre type that differ from supraspinatus muscle, its mitochondrial function and NAD+ levels are also susceptible to disuse. Similar to the supraspinatus muscle, NAMPT-elevated NAD+ biosynthesis was also efficient in preventing EDL disuse atrophy by reversing mitochondrial dysfunction. CONCLUSIONS: NAMPT-elevated NAD+ biosynthesis can prevent disuse atrophy of skeletal muscles that predominantly composed with either slow-twitch (type I) or fast-twitch (type II) fibres by reversing mitochondrial dysfunction.

Disuse muscle atrophy-improving effect of ninjin'yoeito in a mouse model.

Disuse syndrome indicates psychosomatic hypofunction caused by excess rest and motionless and muscle atrophy is termed disuse muscle atrophy. Disuse muscle atrophy-induced muscle weakness and hypoactivity further induces muscle atrophy, leading to a vicious cycle, and this is considered a factor causing secondary sarcopenia and subsequently frailty. Since frailty finally leads to a bedridden state requiring nursing, in facing a super-aging society, intervention for a risk factor of frailty, disuse muscle atrophy, is important. However, the main treatment of disuse muscle atrophy is physical therapy and there are fewer effective preventive and therapeutic drugs. The objective of this study was to search for Kampo medicine with a disuse muscle atrophy-improving effect. Ninjin'yoeito is classified as a qi-blood sohozai (dual supplement) in Chinese herbal medicine, and it has an action supplementing the spleen related to muscle. In addition, improvement of muscle mass and muscle weakness by ninjin'yoeito in a clinical study has been reported. In this study, the effect of ninjin'yoeito on disuse muscle atrophy was investigated. A disuse muscle atrophy model was prepared using male ICR mice. After surgery applying a ring for tail suspension, a 1-week recovery period was set. Ninjin'yoeito was administered by mixing it in the diet for 1 week after the recovery period, followed by tail suspension for 14 days. Ninjin'yoeito administration was continued until autopsy including the hindlimb suspension period. The mice were euthanized and autopsied immediately after completion of tail suspension, and the hindlimb muscles were collected. The food and water intakes during the hindlimb unloaded period, wet weight of the collected muscle, and muscle synthesis and muscle degradation-related factors in blood and muscle were evaluated. Ingestion of ninjin'yoeito inhibited tail suspension-induced reduction of the soleus muscle wet weight. In addition, an increase in the blood level of a muscle synthesis-related factor, IGF-1, and promotion of phosphorylation of mTOR and 4E-BP1 in the soleus muscle were observed. It was suggested that ninjin'yoeito has a disuse muscle atrophy-improving action. Promotion of the muscle synthesis pathway was considered the action mechanism of this.

Nusinersena: eficácia, segurança e custo-efetividade para o tratamento da atrofia muscular espinhal / Nusinersena: efficacy, safety and cost-effectiveness for the treatment of spinal muscular atrophy

As Atrofias Musculares Espinhais (AME) são um grupo de doenças neuromusculares hereditárias raras (incidência de cerca de 1 para cada 11.000 nascidos vivos).Caracterizam-se pela degeneração dos neurônios motores na medula espinhal e tronco encefálico, resultando em fraqueza muscular progressiva. As AME são categorizadas em subtipos clínicos (tipo I, II, III e IV) com base na idade de início dos sintomas e sua gravidade. O medicamento Nusinersena é eficaz, seguro e custo efetivo para o tratamento de pessoas com Atrofia Muscular Espinhal?

A gestão municipal de saúde de Campo Grande-MS recebeu solicitação de acesso ao medicamento de alto custo Nusinersena (Spinraza®) para crianças diagnosticadas com AME. Diante da necessidade de aprofundamento dos conhecimentos acerca da doença e possibilidades de tratamento, a Secretaria Municipal de Saúde encomendou este estudo para elucidar melhor a tomada de decisão da gestão. Estudos evidenciam que o Nusinersena prolonga a sobrevida livre de ventilação para portadores de AME tipo I e melhora a função motora para portadores de AME tipo I e II. Estas revisões indicam que a maior potencialidade da terapia ocorre nos estágios iniciais da doença pois existe uma janela de oportunidade para ação do medicamento com vistas a resgatar ou estabilizar a função do neurônio motor. Ou seja, melhores respostas ocorreram em crianças mais novas. No entanto, não há cura completa da doença, apenas melhora da sintomatologia5. Em abril de 2019, o Ministério da Saúde incorporou o medicamento Nusinersena para portadores de AME tipo I, ficando em aberto a cobertura dos tipos II,III e IV. O perfil de segurança e tolerabilidade do Nusinersena são aceitáveis, mas há escassez de dados sobre sua eficácia e desdobramentos a longo prazo. Devido aos custos extremamente elevados deste medicamento (análise baseada em preços oficiais) ele se tornou não custo-efetivo. Em agosto de 2018, a CONITEC recomendou a não incorporação do medicamento ao SUS, porém a Advocacia Geral da União recomendou uma nova submissão, feita pela empresa produtora onde foi aprovada em março de 2019 (para portadores de AME tipo I). Não houve acréscimo de novas evidências ou redução de preço que justificassem a mudança de decisão.

Chronic Ouabain Prevents Na,K-ATPase Dysfunction and Targets AMPK and IL-6 in Disused Rat Soleus Muscle.

Sustained sarcolemma depolarization due to loss of the Na,K-ATPase function is characteristic for skeletal muscle motor dysfunction. Ouabain, a specific ligand of the Na,K-ATPase, has a circulating endogenous analogue. We hypothesized that the Na,K-ATPase targeted by the elevated level of circulating ouabain modulates skeletal muscle electrogenesis and prevents its disuse-induced disturbances. Isolated soleus muscles from rats intraperitoneally injected with ouabain alone or subsequently exposed to muscle disuse by 6-h hindlimb suspension (HS) were studied. Conventional electrophysiology, Western blotting, and confocal microscopy with cytochemistry were used. Acutely applied 10 nM ouabain hyperpolarized the membrane. However, a single injection of ouabain (1 µg/kg) prior HS was unable to prevent the HS-induced membrane depolarization. Chronic administration of ouabain for four days did not change the α1 and α2 Na,K-ATPase protein content, however it partially prevented the HS-induced loss of the Na,K-ATPase electrogenic activity and sarcolemma depolarization. These changes were associated with increased phosphorylation levels of AMP-activated protein kinase (AMPK), its substrate acetyl-CoA carboxylase and p70 protein, accompanied with increased mRNA expression of interleikin-6 (IL-6) and IL-6 receptor. Considering the role of AMPK in regulation of the Na,K-ATPase, we suggest an IL-6/AMPK contribution to prevent the effects of chronic ouabain under skeletal muscle disuse.

ActRIIB:ALK4-Fc alleviates muscle dysfunction and comorbidities in murine models of neuromuscular disorders.

Patients with neuromuscular disorders suffer from a lack of treatment options for skeletal muscle weakness and disease comorbidities. Here, we introduce as a potential therapeutic agent a heterodimeric ligand-trapping fusion protein, ActRIIB:ALK4-Fc, which comprises extracellular domains of activin-like kinase 4 (ALK4) and activin receptor type IIB (ActRIIB), a naturally occurring pair of type I and II receptors belonging to the TGF-ß superfamily. By surface plasmon resonance (SPR), ActRIIB:ALK4-Fc exhibited a ligand binding profile distinctly different from that of its homodimeric variant ActRIIB-Fc, sequestering ActRIIB ligands known to inhibit muscle growth but not trapping the vascular regulatory ligand bone morphogenetic protein 9 (BMP9). ActRIIB:ALK4-Fc and ActRIIB-Fc administered to mice exerted differential effects - concordant with SPR results - on vessel outgrowth in a retinal explant assay. ActRIIB:ALK4-Fc induced a systemic increase in muscle mass and function in wild-type mice and in murine models of Duchenne muscular dystrophy (DMD), amyotrophic lateral sclerosis (ALS), and disuse atrophy. Importantly, ActRIIB:ALK4-Fc improved neuromuscular junction abnormalities in murine models of DMD and presymptomatic ALS and alleviated acute muscle fibrosis in a DMD model. Furthermore, in combination therapy ActRIIB:ALK4-Fc increased the efficacy of antisense oligonucleotide M12-PMO on dystrophin expression and skeletal muscle endurance in an aged DMD model. ActRIIB:ALK4-Fc shows promise as a therapeutic agent, alone or in combination with dystrophin rescue therapy, to alleviate muscle weakness and comorbidities of neuromuscular disorders.

Countering disuse atrophy in older adults with low-volume leucine supplementation.

Older adults are at increased risk of being bedridden and experiencing negative health outcomes including the loss of muscle tissue and functional capacity. We hypothesized that supplementing daily meals with a small quantity (3-4 g/meal) of leucine would partially preserve lean leg mass and function of older adults during bed rest. During a 7-day bed rest protocol, followed by 5 days of inpatient rehabilitation, healthy older men and women (67.8 ± 1.1 yr, 14 men; 6 women) were randomized to receive isoenergetic meals supplemented with leucine (LEU, 0.06 g/kg/meal; n = 10) or an alanine control (CON, 0.06 g/kg/meal; n = 10). Outcomes were assessed at baseline, following bed rest, and after rehabilitation. Body composition was measured by dual-energy X-ray absorptiometry. Functional capacity was assessed by knee extensor isokinetic and isometric dynamometry, peak aerobic capacity, and the short physical performance battery. Muscle fiber type, cross-sectional area, signaling protein expression levels, and single fiber characteristics were determined from biopsies of the vastus lateralis. Leucine supplementation reduced the loss of leg lean mass during bed rest (LEU vs. CON: -423 vs. -1035 ± 143 g; P = 0.008) but had limited impact on strength or endurance-based functional outcomes. Similarly, leucine had no effect on markers of anabolic signaling and protein degradation during bed rest or rehabilitation. In conclusion, providing older adults with supplemental leucine has minimal impact on total energy or protein consumption and has the potential to partially counter some, but not all, of the negative effects of inactivity on muscle health.NEW & NOTEWORTHY Skeletal muscle morphology and function in older adults was significantly compromised by 7 days of disuse. Leucine supplementation partially countered the loss of lean leg mass but did not preserve muscle function or positively impact changes at the muscle fiber level associated with bed rest or rehabilitation. Of note, our data support a relationship between myonuclear content and adaptations to muscle atrophy at the whole limb and single fiber level.

Allopurinol partially prevents disuse muscle atrophy in mice and humans.

Disuse muscle wasting will likely affect everyone in his or her lifetime in response to pathologies such as joint immobilization, inactivity or bed rest. There are no good therapies to treat it. We previously found that allopurinol, a drug widely used to treat gout, protects muscle damage after exhaustive exercise and results in functional gains in old individuals. Thus, we decided to test its effect in the prevention of soleus muscle atrophy after two weeks of hindlimb unloading in mice, and lower leg immobilization following ankle sprain in humans (EudraCT: 2011-003541-17). Our results show that allopurinol partially protects against muscle atrophy in both mice and humans. The protective effect of allopurinol is similar to that of resistance exercise which is the best-known way to prevent muscle mass loss in disuse human models. We report that allopurinol protects against the loss of muscle mass by inhibiting the expression of ubiquitin ligases. Our results suggest that the ubiquitin-proteasome pathway is an appropriate therapeutic target to inhibit muscle wasting and emphasizes the role of allopurinol as a non-hormonal intervention to treat disuse muscle atrophy.

The complex of PAMAM-OH dendrimer with Angiotensin (1-7) prevented the disuse-induced skeletal muscle atrophy in mice.

Angiotensin (1-7) (Ang-(1-7)) is a bioactive heptapeptide with a short half-life and has beneficial effects in several tissues - among them, skeletal muscle - by preventing muscle atrophy. Dendrimers are promising vehicles for the protection and transport of numerous bioactive molecules. This work explored the use of a neutral, non-cytotoxic hydroxyl-terminated poly(amidoamine) (PAMAM-OH) dendrimer as an Ang-(1-7) carrier. Bioinformatics analysis showed that the Ang-(1-7)-binding capacity of the dendrimer presented a 2:1 molar ratio. Molecular dynamics simulation analysis revealed the capacity of neutral PAMAM-OH to protect Ang-(1-7) and form stable complexes. The peptide coverage ability of the dendrimer was between ~50% and 65%. Furthermore, an electrophoretic mobility shift assay demonstrated that neutral PAMAM-OH effectively bonded peptides. Experimental results showed that the Ang-(1-7)/PAMAM-OH complex, but not Ang-(1-7) alone, had an anti-atrophic effect when administered intraperitoneally, as evaluated by muscle strength, fiber diameter, myofibrillar protein levels, and atrogin-1 and MuRF-1 expressions. The results of the Ang-(1-7)/PAMAM-OH complex being intraperitoneally injected were similar to the results obtained when Ang-(1-7) was systemically administered through mini-osmotic pumps. Together, the results suggest that Ang-(1-7) can be protected for PAMAM-OH when this complex is intraperitoneally injected. Therefore, the Ang-(1-7)/PAMAM-OH complex is an efficient delivery method for Ang-(1-7), since it improves the anti-atrophic activity of this peptide in skeletal muscle.

Beta-agonist stimulation ameliorates the phenotype of spinal and bulbar muscular atrophy mice and patient-derived myotubes.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease characterized by the loss of lower motor neurons. SBMA is caused by expansions of a polyglutamine tract in the gene coding for androgen receptor (AR). Expression of polyglutamine-expanded AR causes damage to motor neurons and skeletal muscle cells. Here we investigated the effect of ß-agonist stimulation in SBMA myotube cells derived from mice and patients, and in knock-in mice. We show that treatment of myotubes expressing polyglutamine-expanded AR with the ß-agonist clenbuterol increases their size. Clenbuterol activated the phosphatidylinositol-3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway and decreased the accumulation of polyglutamine-expanded AR. Treatment of SBMA knock-in mice with clenbuterol, which was started at disease onset, ameliorated motor function and extended survival. Clenbuterol improved muscle pathology, attenuated the glycolytic-to-oxidative metabolic alterations occurring in SBMA muscles and induced hypertrophy of both glycolytic and oxidative fibers. These results indicate that ß-agonist stimulation is a novel therapeutic strategy for SBMA.

Role of endoplasmic reticulum stress in disuse osteoporosis.

Osteoporosis is a major skeletal disease with low bone mineral density, which leads to an increased risk of bone fracture. Salubrinal is a synthetic chemical that inhibits dephosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) in response to endoplasmic reticulum (ER) stress. To understand possible linkage of osteoporosis to ER stress, we employed an unloading mouse model and examined the effects of salubrinal in the pathogenesis of disuse osteoporosis. The results presented several lines of evidence that osteoclastogenesis in the development of osteoporosis was associated with ER stress, and salubrinal suppressed unloading-induced bone loss. Compared to the age-matched control, unloaded mice reduced the trabecular bone area/total area (B.Ar/T.Ar) as well as the number of osteoblasts, and they increased the osteoclasts number on the trabecular bone surface in a time-dependent way. Unloading-induced disuse osteoporosis significantly increased the expression of Bip, p-eIF2α and ATF4 in short-term within 6h of tail suspension, but time-dependent decreased in HU2d to HU14d. Furthermore, a significant correlation of ER stress with the differentiation of osteoblasts and osteoclasts was observed. Administration of salubrinal suppressed the unloading-induced decrease in bone mineral density, B.Ar/T.Ar and mature osteoclast formation. Salubrinal also increased the colony-forming unit-fibroblasts and colony-forming unit-osteoblasts. It reduced the formation of mature osteoclasts, suppressed their migration and adhesion, and increased the expression of Bip, p-eIF2α and ATF4. Electron microscopy showed that rough endoplasmic reticulum expansion and a decreased number of ribosomes on ER membrane were observed in osteoblast of unloading mice, and the abnormal ER expansion was significantly improved by salubrinal treatment. A TUNEL assay together with CCAAT/enhancer binding protein homologous protein (CHOP) expression indicated that ER stress-induced osteoblast apoptosis was rescued by salubrinal. Collectively, the results support the notion that ER stress plays a key role in the pathogenesis of disuse osteoporosis, and salubrinal attenuates unloading-induced bone loss by altering proliferation and differentiation of osteoblasts and osteoclasts via eIF2α signaling.

Androgens have antiresorptive effects on trabecular disuse osteopenia independent from muscle atrophy.

Aging hypogonadal men are at increased risk of osteoporosis and sarcopenia. Testosterone is a potentially appealing strategy to prevent simultaneous bone and muscle loss. The androgen receptor (AR) mediates antiresorptive effects on trabecular bone via osteoblast-lineage cells, as well as muscle-anabolic actions. Sex steroids also modify the skeletal response to mechanical loading. However, it is unclear whether the effects of androgens on bone remain effective independent of mechanical stimulation or rather require indirect androgen effects via muscle. This study aims to characterize the effects and underlying mechanisms of androgens on disuse osteosarcopenia. Adult male mice received a unilateral botulinum toxin (BTx) injection, and underwent sham surgery or orchidectomy (ORX) without or with testosterone (ORX+T) or dihydrotestosterone (ORX+DHT) replacement. Compared to the contralateral internal control hindlimb, acute trabecular number and bone volume loss was increased by ORX and partially prevented DHT. T was more efficient and increased BV/TV in both hindlimbs over sham values, although it did not reduce the detrimental effect of BTx. Both androgens and BTx regulated trabecular osteoclast surface as well as tartrate-resistant acid phosphatase expression. Androgens also prevented BTx-induced body weight loss but did not significantly influence paralysis or muscle atrophy. BTx and ORX both reduced cortical thickness via endosteal expansion, which was prevented by T but not DHT. In long-term follow-up, the residual trabecular bone volume deficit in sham-BTx hindlimbs was prevented by DHT but T restored it more efficiently to pre-treatment levels. Conditional AR deletion in late osteoblasts and osteocytes or in the satellite cell lineage increased age-related trabecular bone loss in both hindlimbs without influencing the effect of BTx on trabecular osteopenia. We conclude that androgens have antiresorptive effects on trabecular disuse osteopenia which do not require AR actions on bone via muscle or via osteocytes.

Signal transducer and activator of transcription 3 signaling as a potential target to treat muscle wasting diseases.

PURPOSE OF REVIEW: The review summarizes our current knowledge of the role of signal transducer and activator of transcription 3 (STAT3) signaling in skeletal muscle regeneration and the maintenance of muscle mass. RECENT FINDINGS: STAT3 signaling plays a pivotal role in regulating the function of multiple cell types in skeletal muscle. This includes muscle stem cells, myofibers, and macrophages. It regulates muscle stem cell function by antagonizing self-renewal. STAT3 also functions in myofibers to regulate skeletal muscle mass. This is highly relevant under pathological conditions where STAT3 activation promotes protein degradation and muscle atrophy. Transient pharmacological inhibition of STAT3 partially prevents muscle wasting. However, the mechanisms responsible for the improvement of muscle condition are not currently well understood. This is because of the complexity of the system, as STAT3 has a critical role in regulating the function of several cell types residing in skeletal muscle. SUMMARY: Muscle wasting is associated with several human diseases such as muscle dystrophies or cancer cachexia. However, currently there are no effective treatments for this condition, and there is a critical need to identify new potential targets for the development of efficient therapeutic approaches.

A small-molecule Nrf1 and Nrf2 activator mitigates polyglutamine toxicity in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA, also known as Kennedy's disease) is one of nine neurodegenerative disorders that are caused by expansion of polyglutamine-encoding CAG repeats. Intracellular accumulation of abnormal proteins in these diseases, a pathological hallmark, is associated with defects in protein homeostasis. Enhancement of the cellular proteostasis capacity with small molecules has therefore emerged as a promising approach to treatment. Here, we characterize a novel curcumin analog, ASC-JM17, as an activator of central pathways controlling protein folding, degradation and oxidative stress resistance. ASC-JM17 acts on Nrf1, Nrf2 and Hsf1 to increase the expression of proteasome subunits, antioxidant enzymes and molecular chaperones. We show that ASC-JM17 ameliorates toxicity of the mutant androgen receptor (AR) responsible for SBMA in cell, fly and mouse models. Knockdown of the Drosophila Nrf1 and Nrf2 ortholog cap 'n' collar isoform-C, but not Hsf1, blocks the protective effect of ASC-JM17 on mutant AR-induced eye degeneration in flies. Our observations indicate that activation of the Nrf1/Nrf2 pathway is a viable option for pharmacological intervention in SBMA and potentially other polyglutamine diseases.

Sclerostin antibody prevented progressive bone loss in combined ovariectomized and concurrent functional disuse.

Osteoporosis is characterized by low bone mass and compromised trabecular architecture, and is commonly occurred in post-menopausal women with estrogen deficiency. In addition, prolonged mechanical unloading, i.e., long term bed rest, can exaggerate the bone loss. Sclerostin is a Wnt signaling antagonist and acts as a negative regulator for bone formation. A sclerostin-neutralizing antibody (Scl-Ab) increased bone mineral density in women with postmenopausal osteoporosis and healthy men. The objective of this study was to characterize the condition of bone loss in ovariectomized (OVX) rats with concurrent mechanical unloading and evaluate the effect of sclerostin antibody treatment in mitigating the prospective severe bone loss conditions in this model. Four-month-old OVX- or sham-operated female SD rats were used in this study. They were subjected to functional disuse induced by hind-limb suspension (HLS) or free ambulance after 2days of arrival. Subcutaneous injections with either vehicle or Scl-Ab at 25mg/kg were made twice per week for 5weeks from the time of HLS. µCT analyses demonstrated a significant decrease in distal metaphyseal trabecular architecture integrity with HLS, OVX and HLS+OVX (bone volume fraction decreased by 29%, 71% and 87% respectively). The significant improvements of various trabecular bone parameters (bone volume fraction increased by 111%, 229% and 297% respectively as compared with placebo group) with the administration of Scl-Ab are associated with stronger mechanical property and increased bone formation by histomorphometry. These results together indicate that Scl-Ab prevented the loss of trabecular bone mass and cortical bone strength in OVX rat model with concurrent mechanical unloading. The data suggested that monoclonal sclerostin-neutralizing antibody represents a promising therapeutic approach for severe osteoporosis induced by estrogen deficiency with concurrent mechanical unloading.

Angiotensin-(1-7) attenuates disuse skeletal muscle atrophy in mice via its receptor, Mas.

Immobilization is a form of disuse characterized by a loss of strength and muscle mass. Among the main features are decreased IGF-1/Akt signalling and increased ubiquitin-proteasome pathway signalling, which induce greater myosin heavy chain degradation. Activation of the classical renin-angiotensin system (RAS) causes deleterious effects in skeletal muscle, including muscle wasting. In contrast, angiotensin-(1-7) [Ang-(1-7)], a peptide of the non-classical RAS, produces beneficial effects in skeletal muscle. However, the role of Ang-(1-7) in skeletal muscle disuse atrophy and independent of classical RAS activation has not been evaluated. Therefore, we assessed the functions of Ang-(1-7) and the Mas receptor in disuse muscle atrophyin vivousing unilateral cast immobilization of the hind limb in male, 12-week-old wild-type (WT) and Mas-knockout (Mas KO) mice for 1 and 14 days. Additionally, we evaluated the participation of IGF-1/IGFR-1/Akt signalling and ubiquitin-proteasome pathway expression on the effects of Ang-(1-7) immobilization-induced muscle atrophy. Our results found that Ang-(1-7) prevented decreased muscle strength and reduced myofiber diameter, myosin heavy chain levels, and the induction of atrogin-1 and MuRF-1 expressions, all of which normally occur during immobilization. Analyses indicated that Ang-(1-7) increases IGF-1/IGFR-1/Akt pathway signalling through IGFR-1 and Akt phosphorylation, and the concomitant activation of two downstream targets of Akt, p70S6K and FoxO3. These anti-atrophic effects of Ang-(1-7) were not observed in Mas KO mice, indicating crucial participation of the Mas receptor. This report is the first to propose anti-atrophic effects of Ang-(1-7) via the Mas receptor and the participation of the IGF-1/IGFR-1/Akt/p70S6K/FoxO3 mechanism in disuse skeletal muscle atrophy.

Pioglitazone suppresses neuronal and muscular degeneration caused by polyglutamine-expanded androgen receptors.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by the expansion of a CAG repeat in the androgen receptor (AR) gene. Mutant AR has been postulated to alter the expression of genes important for mitochondrial function and induce mitochondrial dysfunction. Here, we show that the expression levels of peroxisome proliferator-activated receptor-γ (PPARγ), a key regulator of mitochondrial biogenesis, were decreased in mouse and cellular models of SBMA. Treatment with pioglitazone (PG), an activator of PPARγ, improved the viability of the cellular model of SBMA. The oral administration of PG also improved the behavioral and histopathological phenotypes of the transgenic mice. Furthermore, immunohistochemical and biochemical analyses demonstrated that the administration of PG suppressed oxidative stress, nuclear factor-κB (NFκB) signal activation and inflammation both in the spinal cords and skeletal muscles of the SBMA mice. These findings suggest that PG is a promising candidate for the treatment of SBMA.

Endoplasmic reticulum stress in spinal and bulbar muscular atrophy: a potential target for therapy.

Spinal and bulbar muscular atrophy is an X-linked degenerative motor neuron disease caused by an abnormal expansion in the polyglutamine encoding CAG repeat of the androgen receptor gene. There is evidence implicating endoplasmic reticulum stress in the development and progression of neurodegenerative disease, including polyglutamine disorders such as Huntington's disease and in motor neuron disease, where cellular stress disrupts functioning of the endoplasmic reticulum, leading to induction of the unfolded protein response. We examined whether endoplasmic reticulum stress is also involved in the pathogenesis of spinal and bulbar muscular atrophy. Spinal and bulbar muscular atrophy mice that carry 100 pathogenic polyglutamine repeats in the androgen receptor, and develop a late-onset neuromuscular phenotype with motor neuron degeneration, were studied. We observed a disturbance in endoplasmic reticulum-associated calcium homeostasis in cultured embryonic motor neurons from spinal and bulbar muscular atrophy mice, which was accompanied by increased endoplasmic reticulum stress. Furthermore, pharmacological inhibition of endoplasmic reticulum stress reduced the endoplasmic reticulum-associated cell death pathway. Examination of spinal cord motor neurons of pathogenic mice at different disease stages revealed elevated expression of markers for endoplasmic reticulum stress, confirming an increase in this stress response in vivo. Importantly, the most significant increase was detected presymptomatically, suggesting that endoplasmic reticulum stress may play an early and possibly causal role in disease pathogenesis. Our results therefore indicate that the endoplasmic reticulum stress pathway could potentially be a therapeutic target for spinal and bulbar muscular atrophy and related polyglutamine diseases.

Skeletal muscle disuse atrophy is not attenuated by dietary protein supplementation in healthy older men.

Short successive periods of muscle disuse, due to injury or illness, can contribute significantly to the loss of muscle mass with aging (sarcopenia). It has been suggested that increasing the protein content of the diet may be an effective dietary strategy to attenuate muscle disuse atrophy. We hypothesized that protein supplementation twice daily would preserve muscle mass during a short period of limb immobilization. Twenty-three healthy older (69 ± 1 y) men were subjected to 5 d of one-legged knee immobilization by means of a full-leg cast with (PRO group; n = 11) or without (CON group; n = 12) administration of a dietary protein supplement (20.7 g of protein, 9.3 g of carbohydrate, and 3.0 g of fat) twice daily. Two d prior to and immediately after the immobilization period, single-slice computed tomography scans of the quadriceps and single-leg 1 repetition maximum strength tests were performed to assess muscle cross-sectional area (CSA) and leg muscle strength, respectively. Additionally, muscle biopsies were collected to assess muscle fiber characteristics as well as mRNA and protein expression of selected genes. Immobilization decreased quadriceps' CSAs by 1.5 ± 0.7% (P < 0.05) and 2.0 ± 0.6% (P < 0.05), and muscle strength by 8.3 ± 3.3% (P < 0.05) and 9.3 ± 1.6% (P < 0.05) in the CON and PRO groups, respectively, without differences between groups. Skeletal muscle myostatin, myogenin, and muscle RING-finger protein-1 (MuRF1) mRNA expression increased following immobilization in both groups (P < 0.05), whereas muscle atrophy F-box/atrogen-1 (MAFBx) mRNA expression increased in the PRO group only (P < 0.05). In conclusion, dietary protein supplementation (∼20 g twice daily) does not attenuate muscle loss during short-term muscle disuse in healthy older men. This trial was registered at clinicaltrials.gov as NCT01588808.

Additive effect of PTH (1-34) and zoledronate in the prevention of disuse osteopenia in rats.

Immobilization is known to cause a rapid bone loss due to increased osteoclastic bone resorption and decreased osteoblastic bone formation. Zoledronate (Zln) is a potent anti-resorptive pharmaceutical, while intermittent PTH is a potent bone anabolic agent. The aim of the present study was to investigate whether PTH or Zln alone or in combination could prevent immobilization-induced osteopenia. Immobilization was achieved by injecting 4IU Botox (BTX) into the right hind limb musculature. Seventy-two 16-week-old female Wistar rats were randomized into 6 groups; baseline (Base), control (Ctrl), BTX, BTX+PTH, BTX+Zln, and BTX+PTH+Zln. PTH (1-34) (80µg/kg) was given 5days/week and Zln (100µg/kg) was given once at study start. The animals were killed after 4weeks of treatment. The bone properties were evaluated using DEXA, µCT, dynamic bone histomorphometry, and mechanical testing. BTX resulted in lower femoral trabecular bone volume fraction (BV/TV) (-25%, p<0.05), lower tibial trabecular bone formation rate (BFR/BS) (-29%, p<0.05), and lower bone strength (Fmax) at the distal femur (-19%, p<0.001) compared with Ctrl. BTX+PTH resulted in higher femoral BV/TV (+31%, p<0.05), higher tibial trabecular BFR/BS (+297%, p<0.05), and higher Fmax at the distal femur (+11%, p<0.05) compared with BTX. BTX+Zln resulted in higher femoral BV/TV (+36%, p<0.05), lower tibial trabecular BFR/BS (-93%, p<0.05), and higher Fmax at the distal femur (+10%, p<0.05) compared with BTX. BTX+PTH+Zln resulted in higher femoral BV/TV (+70%, p<0.001), higher tibial trabecular BFR/BS (+59%, p<0.05), and higher Fmax at the distal femur (+32%, p<0.001) compared with BTX. In conclusion, BTX-induced immobilization led to lower BV/TV, BFR/BS, and Fmax. In general, PTH or Zln alone prevented the BTX-induced osteopenia, whereas PTH and Zln given in combination not only prevented, but also increased BV/TV and BFR/BS, and maintained Fmax at the distal femoral metaphysis compared with Ctrl.

ß-Hydroxy-ß-methylbutyrate (HMB) enhances the proliferation of satellite cells in fast muscles of aged rats during recovery from disuse atrophy.

Loss of myonuclei by apoptosis is thought to contribute to sarcopenia. We have previously shown, that the leucine metabolite, ß-hydroxy-ß-methylbutyrate (HMB) suppresses apoptotic signaling and the apoptotic index (the ratio of apoptotic positive to apoptotic negative myonuclei) during muscle disuse and during reloading periods after disuse in aged rats. However, it was not clear if the apoptotic signaling indexes were due only to preservation of myonuclei or if perhaps the total myogenic pool increased as a result of HMB-mediated satellite cell proliferation as this would have also reduced the apoptotic index. In this study, we tested the hypothesis that HMB would augment myogenic cells (satellite cells) proliferation during muscle recovery (growth) after a period of disuse in senescent animals. The hindlimb muscles of 34 month old Fisher 344 × Brown Norway rats were unloaded for 14 days by hindlimb suspension (HLS), and then reloaded for 14 days. The rats received either Ca-HMB (340 mg/kg body weight; n = 16), or the vehicle (n = 10) by gavage throughout the experimental period. HMB prevented the functional decline in maximal plantar flexion isometric force production during the reloading period, but not during HLS. HMB-treatment enhanced the proliferation of muscle stem cells as shown by a greater percentage of satellite cells that had proliferated (more BrdU positive, Pax-7 positive, and more Pax7/Ki67 positive nuclei) and as a result, more differentiated stem cells were present (more MyoD/myogenin positive myonuclei), relative to total myonuclei, in reloaded plantaris muscles as compared to reloaded muscles from vehicle-treated animals. Furthermore HMB increased the nuclear protein abundance of proliferation markers, inhibitor of differentiation-2 and cyclin A, as compared to vehicle treatment in reloaded muscles. Although HMB increased phosphorylated Akt during reloading, other mTOR related proteins were not altered by HMB treatment. These data show that HMB improved the proliferation of muscle stem cells in fast twitch plantaris muscles. Enhanced satellite cell proliferation leading to increased differentiated myonuclei should increase the transcriptional potential to support muscle hypertrophic changes and functional changes in sarcopenic muscles, and this could partly explain the reduced apoptotic index in HMB treated muscles. Indeed, muscle mass and fiber cross-sectional area were significantly greater in plantaris muscles from HMB-treated animal muscles after reloading as compared to vehicle-treated animals.