Influence of resistance exercise intensity and metabolic stress on anabolic signaling and expression of myogenic genes in skeletal muscle

Introduction: We investigated the effect of resistance exercise intensity and exercise‐induced metabolic stress on the activation of anabolic signaling and expression of myogenic genes in skeletal muscle. Methods: Ten strength‐trained athletes performed high‐intensity [HI, 74% of 1‐repetition maximum (RM)], middle‐intensity (MI, 54% 1RM), or middle‐intensity (54% 1RM) no‐relaxation exercise (MIR). Kinase phosphorylation level and myogenic gene expression in muscle samples were evaluated before, 45 min, 5 h, and 20 h after exercise. Results: The lactate concentration in MI was approximately 2‐fold lower than in the 2 other sessions, and was highest in MIR. The phosphorylation level of extracellular kinase 1/2^{Thr202/Tyr204} after exercise was related to metabolic stress. Metabolic stress induced a decrease in myostatin mRNA expression, whereas mechano‐growth factor mRNA level depended on exercise intensity. Conclusions: This study demonstrates that both intensity and exercise‐induced metabolic stress can be manipulated to affect muscle anabolic signaling. Muscle Nerve 51: 434–442, 2015     

Myostatin DNA vaccine increases skeletal muscle mass and endurance in mice

Myostatin is a transforming growth factor-beta family member that acts as a negative regulator of skeletal muscle growth. In mice, genetic disruption of the myostatin gene leads to a marked increase in body weight and muscle mass. Similarly, pharmacological interference with myostatin in vivo in mdx knockout mice results in a functional improvement of the dystrophic phenotype. Consequently, myostatin is an important therapeutic target for treatment of diseases associated with muscle wasting. To construct a therapeutic DNA vaccine against myostatin, we coupled the foreign, immunodominant T-helper epitope of tetanus toxin to the N terminus of myostatin, and BALB/c mice were immunized with the recombinant vector. Sera from vaccinated mice showed the presence of specific antibodies against the recombinant protein. In addition, body weight, muscle mass, and grip endurance of vaccinated mice were significantly increased. Our study provides a novel, pharmacological strategy for treatment of diseases associated with muscle wasting.     

Reduced skeletal muscle satellite cell number alters muscle morphology after chronic stretch but allows limited serial sarcomere addition

Introduction: Muscles add sarcomeres in response to stretch, presumably to maintain optimal sarcomere length. Clinical evidence from patients with cerebral palsy, who have both decreased serial sarcomere number and reduced satellite cells (SCs), suggests a hypothesis that SCs may be involved in sarcomere addition. Methods: A transgenic Pax7‐DTA mouse model underwent conditional SC depletion, and their soleii were then stretch‐immobilized to assess the capacity for sarcomere addition. Muscle architecture, morphology, and extracellular matrix (ECM) changes were also evaluated. Results: Mice in the SC‐reduced group achieved normal serial sarcomere addition in response to stretch. However, muscle fiber cross‐sectional area was significantly smaller and was associated with hypertrophic ECM changes, consistent with fibrosis. Conclusions: While a reduced SC population does not hinder serial sarcomere addition, SCs play a role in muscle adaptation to chronic stretch that involves maintenance of both fiber cross‐sectional area and ECM structure. Muscle Nerve 55 : 384–392, 2017     

Schwann cell dedifferentiation‐associated demyelination leads to exocytotic myelin clearance in inflammatory segmental demyelination

Schwann cells (SCs), which form the peripheral myelin sheath, have the unique ability to dedifferentiate and to destroy the myelin sheath under various demyelination conditions. During SC dedifferentiation‐associated demyelination (SAD) in Wallerian degeneration (WD) after axonal injury, SCs exhibit myelin and junctional instability, down‐regulation of myelin gene expression and autophagic myelin breakdown. However, in inflammatory demyelinating neuropathy (IDN), it is still unclear how SCs react and contribute to segmental demyelination before myelin scavengers, macrophages, are activated for phagocytotic myelin digestion. Here, we compared the initial SC demyelination mechanism of IDN to that of WD using microarray and histochemical analyses and found that SCs in IDN exhibited several typical characteristics of SAD, including actin‐associated E‐cadherin destruction, without obvious axonal degeneration. However, autophagolysosome activation in SAD did not appear to be involved in direct myelin lipid digestion by SCs but was required for the separation of SC body from destabilized myelin sheath in IDN. Thus, lysosome inhibition in SCs suppressed segmental demyelination by preventing the exocytotic myelin clearance of SCs. In addition, we found that myelin rejection, which might also require the separation of SC cytoplasm from destabilized myelin sheath, was delayed in SC‐specific Atg7 knockout mice in WD, suggesting that autophagolysosome‐dependent exocytotic myelin clearance by SCs in IDN and WD is a shared mechanism. Finally, autophagolysosome activation in SAD was mechanistically dissociated with the junctional destruction in both IDN and WD. Thus, our findings indicate that SAD could be a common myelin clearance mechanism of SCs in various demyelinating conditions.     

Continuous endurance-type exercise training does not modulate satellite cell content in obese type 2 diabetes patients

Endurance-type exercise training represents a cornerstone in type 2 diabetes treatment. However, the effects of prolonged continuous, endurance-type exercise on muscle fiber characteristics remain equivocal. Fifteen obese male type 2 diabetes patients (61 ± 6 years) participated in a 6-month continuous, endurance-type exercise program. Muscle biopsies were collected before, and after 2 and 6 months of intervention. Muscle fiber type-specific composition, size, and satellite cell (SC) and myonuclear content were determined by immunohistochemistry. Although continuous endurance-type exercise training lowered total body weight and reduced fat mass, no changes were observed in leg lean mass. At baseline, SC content was significantly lower in type II compared with type I muscle fibers. No change in SC content was observed after exercise training. Continuous endurance-type exercise training lowers fat mass, but it does not increase leg lean mass and/or modulate muscle fiber characteristics in type 2 diabetes patients.     

Schwann cells seeded in acellular nerve grafts improve functional recovery

Introduction: This study evaluated whether Schwann cells (SCs) from different nerve sources transplanted into cold‐preserved acellular nerve grafts (CP‐ANGs) would improve functional regeneration compared with nerve isografts. Methods: SCs isolated and expanded from motor and sensory branches of rat femoral and sciatic nerves were seeded into 14mm CP‐ANGs. Growth factor expression, axonal regeneration, and functional recovery were evaluated in a 14‐mm rat sciatic injury model and compared with isografts. Results: At 14 days, motor or sensory‐derived SCs increased expression of growth factors in CP‐ANGs versus isografts. After 42 days, histomorphometric analysis found CP‐ANGs with SCs and isografts had similar numbers of regenerating nerve fibers. At 84 days, muscle force generation was similar for CP‐ANGs with SCs and isografts. SC source did not affect nerve fiber counts or muscle force generation. Conclusions: SCs transplanted into CP‐ANGs increase functional regeneration to isograft levels; however SC nerve source did not have an effect. Muscle Nerve 49 : 267–276, 2014     

Single muscle fiber contractile properties in adults with muscular dystrophy treated with MYO‐029

Myostatin inhibitors are being investigated as treatments for myopathies. We assessed single muscle fiber contractile properties before and after 6 months of study drug in 6 patients with facioscapulohumeral, Becker, and limb‐girdle muscular dystrophy. Five of the patients received MYO‐029, a myostatin inhibitor, and 1 received placebo. The chemically skinned single muscle fiber preparation was used to measure single fiber force, specific force, maximum unloaded shortening velocity, power, and specific power in type I and IIa fibers from each subject. In 4 of 5 patients who received MYO‐029, improvement was seen in single muscle fiber contractile properties; thus, there may be a beneficial effect of myostatin inhibition on muscle physiology at the cellular level. No improvement was seen in the patient who received placebo. This finding may be clinically relevant in spite of the fact that quantitative muscle strength measurements in our patients did not improve. Further studies of myostatin inhibition as a treatment for muscular dystrophy are warranted, and single muscle fiber contractile studies are a useful assay for muscle function at the cellular level. Muscle Nerve 39: 3–9, 2009     

Fibroblast‐derived tenascin‐C promotes Schwann cell migration through β1‐integrin dependent pathway during peripheral nerve regeneration

Peripheral nerve regeneration requires precise coordination and dynamic interaction among various types of cells in the tissue. It remains unclear, however, whether the cellular crosstalk between fibroblasts and Schwann cells (SCs) is related to phenotype modulation of SCs, a critical cellular process after peripheral nerve injury. In this study, microarray analysis revealed that a total of 6,046 genes were differentially expressed in the proximal nerve segment after sciatic nerve transection in rats, and bioinformatics analysis further identified tenascin‐C (TNC), an extracellular matrix (ECM) protein, as a key gene regulator. TNC was abundantly produced by nerve fibroblasts accumulating at the lesion site, rather than by SCs as usually expected. TNC significantly promoted SC migration without effects on SC proliferation in primary culture. In co‐culture of fibroblasts and SCs, inhibition of TNC expression either by siRNA transfection or antibody blockade could suppress SC migration, while TNC‐stimulated SC migration was mediated by TNC binding to β1‐integrin receptor in SCs through activation of Rac1 effectors. The in vivo evidence showed that exogenous TNC protein enhanced SC migration and axonal regrowth. Our results highlight that TNC‐mediated cellular interaction between fibroblasts and SCs may regulate SC migration through β1‐integrin‐dependent pathway during peripheral nerve regeneration. GLIA 2016;64:374–385     

Disruption of laminin in the peripheral nervous system impedes nonmyelinating Schwann cell development and impairs nociceptive sensory function

The mechanisms controlling the differentiation of immature Schwann cells (SCs) into nonmyelinating SCs is not known. Laminins are extracellular matrix proteins critical for myelinating SC differentiation, but their roles in nonmyelinating SC development have not been established. Here, we show that the peripheral nerves of mutant mice with laminin‐deficient SCs do not form Remak bundles, which consist of a single nonmyelinating SC interacting with multiple unmyelinated axons. These mutant nerves show aberrant L1 and neural cell adhesion molecule (N‐CAM) expression pattern during development. The homophilic and heterophilic interactions of N‐CAM are also impaired in the mutant nerves. Other molecular markers for nonmyelinating SCs, including Egr‐1, glial fibrillary acidic protein, and AN2/NG2, are all absent in adult mutant nerves. Analysis of expression of SC lineage markers demonstrates that nonmyelinating SCs do not develop in mutant nerves. Additionally, mutant mice are insensitive to heat stimuli and show a decreased number of C‐fiber sensory neurons, indicating reduced nociceptive sensory function. These results show that laminin participates in nonmyelinating SC development and Remak bundle formation and suggest a possible role for laminin deficiency in peripheral sensory neuropathies. © 2008 Wiley‐Liss, Inc.     

Satellite cell pool expansion is affected by skeletal muscle characteristics

Introduction: We investigated changes in satellite cell (SC) pool size after an acute bout of strenuous exercise and evaluated the influence of baseline SC count and fiber type. Methods: Participants completed a downhill running (DHR) intervention (5 × 8 min, 2‐min rest; 80% VO _2max; ?10% gradient). Muscle biopsies were taken 7 days before VO _2max and 7–9 days after the DHR intervention. Delayed‐onset muscle soreness (DOMS) and creatine kinase activity (CK) were measured on days 1, 2, 7, and 9 post‐DHR. SCs were identified by Pax7 and laminin staining. Relative distribution of MHC isoforms was determined by electrophoresis. Results: DOMS and CK peaked on day 1 post‐DHR (P < 0.01). The SC pool increased (26%) after DHR (P = 0.005). SCs/total myonuclei after recovery correlated with baseline SCs (r = 0.979, P = 0.003) and VO _2max (r = 0.956, P = 0.011), whereas change in SC pool (Pax7⁺ cells/total myonuclei: recovery minus baseline) tended to correlate with percent MHC II (r = 0.848; P = 0.06). Conclusion: Interindividual physiological characteristics affect SC pool expansion after a single bout of DHR and are influenced by VO _2max. Muscle Nerve, 2013     

Inhibition of myostatin with emphasis on follistatin as a therapy for muscle disease

In most cases, pharmacologic strategies to treat genetic muscle disorders and certain acquired disorders, such as sporadic inclusion body myositis, have produced modest clinical benefits. In these conditions, inhibition of the myostatin pathway represents an alternative strategy to improve functional outcomes. Preclinical data that support this approach clearly demonstrate the potential for blocking the myostatin pathway. Follistatin has emerged as a powerful antagonist of myostatin that can increase muscle mass and strength. Follistatin was first isolated from the ovary and is known to suppress follicle‐stimulating hormone. This raises concerns for potential adverse effects on the hypothalamic–pituitary–gonadal axis and possible reproductive capabilities. In this review we demonstrate a strategy to bypass off‐target effects using an alternatively spliced cDNA of follistatin (FS344) delivered by adeno‐associated virus (AAV) to muscle. The transgene product is a peptide of 315 amino acids that is secreted from the muscle and circulates in the serum, thus avoiding cell‐surface binding sites. Using this approach our translational studies show increased muscle size and strength in species ranging from mice to monkeys. Adverse effects are avoided, and no organ system pathology or change in reproductive capabilities has been seen. These findings provide the impetus to move toward gene therapy clinical trials with delivery of AAV‐FS344 to increase size and function of muscle in patients with neuromuscular disease. Muscle Nerve 39: 283–296, 2009     

Activin IIB receptor blockade attenuates dystrophic pathology in a mouse model of duchenne muscular dystrophy

Modulation of transforming growth factor‐β (TGF‐β) signaling to promote muscle growth holds tremendous promise for the muscular dystrophies and other disorders involving the loss of functional muscle mass. Previous studies have focused on the TGF‐β family member myostatin and demonstrated that inhibition of myostatin leads to muscle growth in normal and dystrophic mice. We describe a unique method of systemic inhibition of activin IIB receptor signaling via adeno‐associated virus (AAV)‐mediated gene transfer of a soluble form of the extracellular domain of the activin IIB receptor to the liver. Treatment of mdx mice with activin IIB receptor blockade led to increased skeletal muscle mass, increased force production in the extensor digitorum longus (EDL), and reduced serum creatine kinase. No effect on heart mass or function was observed. Our results indicate that activin IIB receptor blockade represents a novel and effective therapeutic strategy for the muscular dystrophies. Muscle Nerve, 2010     

Effect of constitutive inactivation of the myostatin gene on the gain in muscle strength during postnatal growth in two murine models

Introduction: The effect of constitutive inactivation of the gene encoding myostatin on the gain in muscle performance during postnatal growth has not been well characterized. Methods: We analyzed 2 murine myostatin knockout (KO) models, (i) the Lee model (KO^Lee) and (ii) the Grobet model (KO^Grobet), and measured the contraction of tibialis anterior muscle in situ. Results: Absolute maximal isometric force was increased in 6‐month‐old KO^Lee and KO^Grobet mice, as compared to wild‐type mice. Similarly, absolute maximal power was increased in 6‐month‐old KO^Lee mice. In contrast, specific maximal force (relative maximal force per unit of muscle mass was decreased in all 6‐month‐old male and female KO mice, except in 6‐month‐old female KO^Grobet mice, whereas specific maximal power was reduced only in male KO^Lee mice. Conclusions: Genetic inactivation of myostatin increases maximal force and power, but in return it reduces muscle quality, particularly in male mice. Muscle Nerve 55 : 254–261, 2017     

Therapeutic strategies for muscular dystrophy by myostatin inhibition]

Myostatin is a member of the TGF-beta superfamily that is expressed predominantly in skeletal muscle and functions as a negative regulator of skeletal muscle mass. Myostatin inhibition, therefore, has tremendous potential for increasing muscle mass clinically to treat patients with muscle wasting diseases. Systemic administration of a myostatin neutralizing antibody in mdx mice (a model of Duchenne muscular dystrophy) resulted in an increase in skeletal muscle mass and strength. A human anti-myostatin monoclonal antibody, MYO-029 is under clinical trials in patients with muscular dystrophy in the USA and Europe. Additional approaches to myostatin inhibition have been shown to have beneficial effects in vivo. Blockade of myostatin activity with the myostatin prodomain resulted in increases in muscle mass, enhanced muscle function, and histological improvement of the dystrophic muscle in mdx mice and mutant caveolin-3 transgenic mice (a model of LGMD1C). Treatment with an extracellular ligand-binding domain of the myostatin receptor, ActRIIB, resulted in prominent muscle mass increases in LGMD1C model mice. These findings indicate that myostatin inhibition could lead to effective therapeutics to treat muscular dystrophy. However, therapeutic indication against various types of muscular dystrophy as well as safety of the treatment should be established for the future clinical application.     

Intermittent stretching induces fibrosis in denervated rat muscle

Introduction: Stretching (St) has been used for treating denervated muscles. However, its effectiveness and safety claims require further study. Methods: Rats were divided into: (1) those with denervated (D) muscles, evaluated 7 or 15 days after sciatic nerve crush injury; (2) those with D muscles submitted to St during 7 or 15 days; and (3) those with normal muscles. Muscle fiber cross‐sectional area, serial sarcomere number, sarcomere length, and connective tissue density were measured. MMP‐2, MMP‐9, TIMP‐1, TGF‐β1, and myostatin mRNAs were determined by real‐time polymerase chain reaction. MMP‐2 and MMP‐9 activity was evaluated by zymography. Collagen I was localized using immunofluorescence. Results: St did not prevent muscle atrophy due to denervation, but it increased fibrosis and collagen I deposition at day 15. St also upregulated MMP‐9 and TGF‐β1 gene expressions at day 7, and myostatin at day 15. Conclusions: Stretching denervated muscle does not prevent atrophy, but it increases fibrosis via temporal modulation of TGF‐β1/myostatin and MMP‐9 cascades. Muscle Nerve 53 : 118–126, 2016     

Myogenic,matrix, and growth factor mRNA expression in human skeletal muscle: Effect of contraction intensity and feeding

Introduction: We examined short‐term (3‐hour) and long‐term (12‐week) training effects after heavy load [HL; 70% 1RM] and light load (LL; 16% 1RM) exercise. Methods: mRNA expression of genes involved in skeletal muscle remodeling were analyzed and muscle activity (EMG measurements) was measured. Results: Relative muscle activity differed between HL and LL resistance exercise, whereas median power frequency was even, suggesting an equal muscle‐fiber‐type recruitment distribution. mRNA expression of Myf6, myogenin, and p21 was mostly increased, and myostatin was mostly depressed by HL resistance exercise. No major differences were seen in atrophy‐related genes between HL and LL resistance exercise. No changes were seen over 12‐week training for any of the targets. Conclusions: Resistance exercise at LL and HL elevated the expression of genes involved in skeletal muscle hypertrophy, although the greatest response was from HL. However, no long‐term effect from either LL or HL resistance exercise was seen on basal levels of the mRNA targets. Muscle Nerve 47: 748–759, 2013     

SB431542 treatment promotes the hypertrophy of skeletal muscle fibers but decreases specific force

The small molecule inhibitor SB431542 inhibits activin type I receptors. The muscle growth‐inhibitor myostatin binds to and signals via these receptors. The aim of this study was to test the hypothesis that SB431542 can inhibit myostatin‐related Smad signaling and induce muscle growth in cultured C2C12 myotubes and increase growth and specific force in cultured Xenopus muscle fibers. The effect of SB431542 was assessed in vitro on C2C12 myotubes and ex vivo using mature Xenopus muscle fibers. SB431542 treatment reduced myostatin‐induced C‐terminal Smad2 phosphorylation and resulted in the formation of enlarged myotubes. However myogenin expression was unchanged, while p70 S6k phosphorylation at Thr389, total myosin heavy chain, and the rate of protein synthesis were all reduced. Mature Xenopus muscle fibers that were treated with SB431542 had a higher fiber cross‐sectional area but decreased specific force production than control. SB431542 can initially antagonize myostatin signaling, but long‐term unexpected signaling effects occur. Muscle fibers hypertrophy, but their specific force decreases compared to control. Muscle Nerve, 2010     

Low‐volume resistance exercise attenuates the decline in strength and muscle mass associated with immobilization

We determined the effectiveness of low‐volume resistance exercise (EX) for the attenuation of loss of muscle mass and strength during leg immobilization. Men (N = 5) and women (N = 12, age 24 ± 5 years, body mass index 25.4 ± 3.6 kg/m²) were divided into two groups: exercise (EX; n = 12) and control (CON; n = 5). Subjects wore a knee brace on one leg that prevented weight bearing for 14 days. Resistance exercise (EX; 80% of maximal) was performed by the immobilized limb every other day. Immobilization induced a significant reduction (P < 0.05) in muscle fiber and thigh cross‐sectional area (CSA), isometric knee extensor, and plantarflexor strength in the CON (P < 0.01) but not in the EX group. There were significant losses in triceps surae CSA in the CON and EX groups (P < 0.05), but the losses were greater in CON subjects (P < 0.01). A minimal volume (140 contractions in 14 days) of resistive exercise is an effective countermeasure against immobilization‐induced atrophy of the quadriceps femoris but is only partially effective for the triceps surae. Muscle Nerve, 2010     

Negative effect of clenbuterol on physical capacities and neuromuscular control of muscle atrophy in adult rats

Introduction: Clenbuterol has been used to alleviate chronic obstructive pulmonary disease and elicit an anabolic response in muscles. The aim of this study was to determine the influence of muscle mass variation on physical capacities in rats. Methods: The left hindlimbs of Wistar rats were immobilized for 20 days in plantarflexion with a splint and then remobilized for 16 days. The effect of a non‐myotoxic dose of clenbuterol during the immobilization period was evaluated. Physical capacities were coordination, free locomotion, grip strength, and bilateral deficit. Results: Immobilization induced a loss of muscle mass, coordination, and strength without any effect on free locomotion. The positive anabolic effect of clenbuterol did not prevent a loss of physical capacities resulting from immobilization. Conclusions: Muscle mass correlated strongly with coordination and isometric strength in untreated rats. Anabolic effect, fiber phenotype modification, and perturbation in neuromuscular communication with clenbuterol improved muscle mass, but it altered physical capacities. Muscle Nerve 52 : 1078–1087, 2015