Different pattern of aquaporin-4 expression in extensor digitorum longus and soleus during early development

Aquaporin-4 (AQP4) is the neuromuscular water channel expressed at the sarcolemma of mammalian fast-twitch fibers that mediates a high water transport rate, which is important during muscle activity. Clinical interest in the neuromuscular expression of AQP4 has increased as it is associated with the protein complex formed by dystrophin, the product of the gene affected in Duchenne muscular dystrophy. The expression of AQP4 during development has not been characterized. In this study, we analyzed the expression of AQP4 in extensor digitorum longus (EDL) and soleus, a fast- and slow-twitch muscle, respectively, during the first weeks after birth. The results show that AQP4 expression in both types of skeletal muscle occurs postnatally. The time course of expression of AQP4 in the two types of muscles was also different. Whereas the expression of AQP4 protein levels in the EDL showed a progressive increase during the first month after birth, reaching levels found in adults by day 24, the levels of the protein in the soleus showed a transient peak between day 12 and day 24 and declined thereafter, an effect that may be related to the transient high number of fast motor units innervating the soleus muscle during this time. The results suggest that AQP4 expression in skeletal muscle is under neuronal influence and contribute to the understanding of the molecular events of fiber differentiation during development.     

Upregulation of MHC class I in transgenic mice results in reduced force‐generating capacity in slow‐twitch muscle

Expression of major histocompatibility complex (MHC) class I in skeletal muscle fibers is an early and consistent finding in inflammatory myopathies. To test if MHC class I has a primary role in muscle impairment, we used transgenic mice with inducible overexpression of MHC class I in their skeletal muscle cells. Contractile function was studied in isolated extensor digitorum longus (EDL, fast‐twitch) and soleus (slow‐twitch) muscles. We found that EDL was smaller, whereas soleus muscle was slightly larger. Both muscles generated less absolute force in myopathic compared with control mice; however, when force was expressed per cross‐sectional area, only soleus muscle generated less force. Inflammation was markedly increased, but no changes were found in the activities of key mitochondrial and glycogenolytic enzymes in myopathic mice. The induction of MHC class I results in muscle atrophy and an intrinsic decrease in force‐generation capacity. These observations may have important implications for our understanding of the pathophysiological processes of muscle weakness seen in inflammatory myopathies. Muscle Nerve, 2008     

IGF-I treatment improves the functional properties of fast- and slow-twitch skeletal muscles from dystrophic mice

Although insulin-like growth factor-I (IGF-I) has been proposed for use by patients suffering from muscle wasting conditions, few studies have investigated the functional properties of dystrophic skeletal muscle following IGF-I treatment. 129P1 ReJ-Lama2(dy) (129 ReJ dy/dy) dystrophic mice suffer from a deficiency in the structural protein, laminin, and exhibit severe muscle wasting and weakness. We tested the hypothesis that 4 weeks of IGF-I treatment ( approximately 2 mg/kg body mass, 50 g/h via mini-osmotic pump, subcutaneously) would increase the mass and force producing capacity of skeletal muscles from dystrophic mice. IGF-I treatment increased the mass of the extensor digitorum longus (EDL) and soleus muscles of dystrophic mice by 20 and 29%, respectively, compared with untreated dystrophic mice (administered saline-vehicle only). Absolute maximum force (P(o)) of the EDL and soleus muscle was increased by 40 and 32%, respectively, following IGF-I treatment. Specific P(o) (sP(o)) was increased by 23% in the EDL muscles of treated compared with untreated mice, but in the soleus muscle sP(o) was unchanged. IGF-I treatment increased the proportion of type IIB and type IIA fibres and decreased the proportion of type I fibres in the EDL muscles of dystrophic mice. In the soleus muscles of dystrophic mice, IGF-I treatment increased the proportion of type IIA fibres and decreased the proportion of type I fibres. Average fibre cross-sectional area was increased in the EDL and soleus muscles of treated compared with untreated mice. We conclude that IGF-I treatment ameliorates muscle wasting and improves the functional properties of skeletal muscles of dystrophic mice. The findings have important implications for the role of IGF-I in ameliorating muscle wasting associated with the muscular dystrophies.     

Role of ubiquitin–proteasome proteolysis in muscle fiber destruction in experimental chloroquine‐induced myopathy

Previous studies have documented the presence of rimmed vacuoles, atrophic fibers, and increased lysosomal cathepsin activity in skeletal muscle from animal models of chloroquine‐induced myopathy, suggesting that muscle fibers in this type of myopathy may be degraded via the lysosomal‐proteolysis pathway. Given recent evidence of abnormal ubiquitin accumulation in rimmed vacuoles, in this study we chose to examine the significance of the ubiquitin–proteasome proteolytic system in the process of muscle fiber destruction in experimental chloroquine myopathy. Expression of ubiquitin, 26S proteasome proteins, and ubiquitin ligases, such as muscle‐specific RING finger‐1 (MuRF‐1) and atrogin‐1/muscle atrophy F‐box protein (MAFbx), was analyzed in innervated and denervated rat soleus muscles after treatment with either saline or chloroquine. Abnormal accumulation of rimmed vacuoles was observed only in chloroquine‐treated denervated muscles. Ubiquitin and proteasome immunostaining, and ubiquitin, MuRF‐1, and atrogin‐1/MAFbx mRNAs were significantly increased in denervated soleus muscles from saline‐ and chloroquine‐treated rats when compared with contralateral innervated muscles. Further, ubiquitin and ubiquitin ligase mRNA levels were higher in denervated muscles from chloroquine‐treated rats when compared with saline‐treated rats. These data demonstrate increased proteasomes and ubiquitin in denervated muscles from chloroquine‐treated rats and suggest that the ubiquitin–proteasome proteolysis pathway as well as the lysosomal‐proteolysis pathway mediate muscle fiber destruction in experimental chloroquine myopathy. Muscle Nerve 39: 521–528, 2009     

Contractile properties of slow and fast skeletal muscles from protease activated receptor‐1 null mice

Introduction: Protease‐activated receptors (PARs) may play a role in skeletal muscle development. We compared the contractile properties of slow‐twitch soleus muscles and fast‐twitch extensor digitorum longus (EDL) muscles from PAR‐1 null and littermate control mice. Methods: Contractile function was measured using a force transducer system. Fiber type proportions were determined using immunohistochemistry. Results: Soleus muscles from PAR‐1 null mice exhibited longer contraction times, a leftward shift in the force–stimulation frequency relationship, and decreased fatiguability compared with controls. PAR‐1 null soleus muscles also had increased type 1 and decreased type IIb/x fiber numbers compared with controls. In PAR‐1 null EDL muscles, no differences were found, except for a slower rate of fatigue compared with controls. Conclusions: The absence of PAR‐1 results in a slower skeletal muscle contractile phenotype, likely due to an increase in type I and a decrease in type IIb/x fiber numbers. Muscle Nerve 50: 991–998, 2014     

Effects of exercise and muscle type on BDNF,NT‐4/5, and TrKB expression in skeletal muscle

Muscle‐derived neurotrophins are thought to contribute to the adaptation of skeletal muscle to exercise, but the effects of brief exercise interventions on BDNF, NT‐4/5, and trkB are not understood. RNA was extracted for RT‐PCR from soleus and medial gastrocnemius of Sprague‐Dawley rats exercised on a treadmill at speeds up to 20 m/min at 5% incline for 5 or 10 days. BDNF expression was elevated in soleus following 5 days (184%, P < 0.001) but not 10 days of exercise. NT‐4/5 and trkB were not affected at either time‐point. BDNF mRNA was significantly higher in soleus at rest when compared with medial gastrocnemius (193%, P < 0.05). No significant effects of muscle type were detected for NT‐4/5 and trkB. Our results indicate differential control of BDNF expression between soleus and medial gastrocnemius following 5 days of exercise. BDNF may be a protein with an uncharacterized contribution to the acute adaptation of skeletal muscle to exercise, whereas NT‐4/5 shows no response. Muscle Nerve, 2009     

Effects of leukemia inhibitory factor on rat skeletal muscles are modulated by clenbuterol

Leukemia inhibitory factor (LIF) is implicated in skeletal muscle regeneration, but the effect of exogenous LIF on uninjured muscles is not known. We tested the hypothesis that LIF administration would stimulate muscle hypertrophy, with an increased effect during clenbuterol-induced fiber remodeling. Rats received daily injections of either saline or LIF, and either regular or clenbuterol-supplemented drinking water for 4 weeks. In the slow-twitch soleus muscles of LIF-treated rats, specific force (sP(o)) and muscle fiber size were increased by approximately 13% and approximately 26%, respectively, compared to saline-treated rats. In the soleus muscles of rats receiving LIF and clenbuterol, compared to rats receiving clenbuterol alone, maximum isometric tension (P(o)) was approximately 19% greater. LIF alone did not affect the properties of fast-twitch extensor digitorum longus (EDL) muscles, but in rats receiving LIF and clenbuterol, compared to clenbuterol alone, EDL fiber size and muscle mass were increased by approximately 20% and approximately 10%, respectively. The hypertrophic effects of exogenous LIF on uninjured skeletal muscles indicate that LIF may have application in the treatment of conditions characterized by muscle wasting.     

Parvalbumin in cross-reinnervated and denervated muscles

The extensor digitorum longus (EDL) muscle was cross-reinnervated by the soleus (SOL) nerve, leading to the well-known transformation toward a slow muscle. Nine weeks after the operation, the quantitative analysis of the Ca2+-binding protein, parvalbumin (PV), using high-performance liquid chromatography, showed a threefold reduction of PV in the cross-reinnervated EDL muscle. Denervation of the EDL muscle, which leads to an increase of the half-relaxation time, resulted in a 20% decrease of the PV concentration within 4 days. This significant lower PV level was detectable prior to any change of the myofibrillar adenosine triphosphatase (ATPase). Normal PV concentrations were reached after 9 weeks following self-reinnervation of the EDL muscle. The experiments support the view that PV is involved in the relaxation of rat fast skeletal muscles and that its expression is dependent on nerve-muscle interaction. Since PV changes preceded histochemical changes after denervation, this protein may be a sensitive marker for early stages of neuromuscular disturbances.     

Inflammatory response during slow‐ and fast‐twitch muscle regeneration

Introduction: Skeletal muscles are characterized by their unique ability to regenerate. Injury of a so‐called fast‐twitch muscle, extensor digitorum longus (EDL), results in efficient regeneration and reconstruction of the functional tissue. In contrast, slow‐twitch muscle (soleus) fails to properly reconstruct and develops fibrosis. This study focuses on soleus and EDL muscle regeneration and associated inflammation. Methods: We determined differences in the activity of neutrophils and M1 and M2 macrophages using flow cytometry and differences in the levels of proinflammatory cytokines using Western blotting and immunolocalization at different times after muscle injury. Results: Soleus muscle repair is accompanied by increased and prolonged inflammation, as compared to EDL. The proinflammatory cytokine profile is different in the soleus and ED muscles. Conclusions: Muscle repair efficiency differs by muscle fiber type. The inflammatory response affects the repair efficiency of slow‐ and fast‐twitch muscles. Muscle Nerve 55 : 400–409, 2017     

Activated central galanin type 1 receptor alleviated insulin resistance in diabetic rat muscle

Evidence indicates that central galanin is involved in regulation of insulin resistance in animals. This study investigates whether type 1 galanin receptor (GAL1) in the brain mediates the ameliorative effect of galanin on insulin resistance in skeletal muscles of type 2 diabetic rats. Rats were intracerebroventricularly (i.c.v.) injected with galanin(1–13)‐bradykinin(2–9) amide (M617), a GAL1 agonist, and/or Akti‐1/2, an Akt inhibitor, via caudal veins once per day for 10 days. Insulin resistance in muscle tissues was evaluated by glucose tolerance and 2‐[N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino]‐2‐deoxyglucose (2‐NBDG) tests, peroxisome proliferator‐activated receptor‐γ (PPARγ), glucose transporter 4 (GLUT4) mRNA expression levels, Akt phosphorylation, and GLUT4 and vesicle‐associated membrane protein 2 (VAMP2) concentration at plasma membranes in muscle cells. The results show that i.c.v. treatment with M617 increased glucose tolerance, 2‐NBDG uptake, PPARγ levels, Akt phosphorylation, GLUT4 protein, and GLUT4 mRNA expression levels as well as GLUT4 and VAMP2 concentration at plasma membranes. All increases may be blocked by pretreatment with Akti‐1/2. These results suggest that activated central GAL1 may trigger the Akt signaling pathway to alleviate insulin resistance in muscle cells. Therefore, the impact of galanin on insulin resistance is mediated mainly by GAL1 in the brain, and the GAL1 agonist may be taken as a potential antidiabetic agent for treatment of type 2 diabetes mellitus. © 2016 Wiley Periodicals, Inc.     

Expression of genes related to myostatin signaling during rat skeletal muscle longitudinal growth

In this study we investigated the gene expression of proteins related to myostatin (MSTN) signaling during skeletal muscle longitudinal growth. To promote muscle growth, Wistar male rats were submitted to a stretching protocol for different durations (12, 24, 48, and 96 hours). Following this protocol, soleus weight and length and sarcomere number were determined. In addition, expression levels of the genes that encode MSTN, follistatin isoforms 288 and 315 (FLST288 and FLST315), follistatin‐like 3 protein (FLST‐L3), growth and differentiation factor–associated protein‐1 (GASP‐1), activin IIB receptor (ActIIB), and SMAD‐7 were determined by real‐time polymerase chain reaction. Prolonged stretching increased soleus weight, length, and sarcomere number. In addition, MSTN gene expression was increased at 12–24 hours, followed by a decrease at 96 hours when compared with baseline values. FLST isoforms, FLST‐L3, and GASP‐1 mRNA levels increased significantly over all time‐points. ActIIB gene expression decreased quickly at 12–24 hours. SMAD‐7 mRNA levels showed a late increase at 48 hours, which peaked at 96 hours. The gene expression pattern of inhibitory proteins related to MSTN signaling suggests a strong downregulation of this pathway in response to prolonged stretching. Muscle Nerve, 2009     

Effects of moderate‐ and high‐intensity chronic exercise on brain‐derived neurotrophic factor expression in fast and slow muscles

Introduction: Brain‐derived neurotrophic factor (BDNF) protein expression is sensitive to cellular activity. In the sedentary state, BDNF expression is affected by the muscle phenotype. Methods: Eighteen Wistar rats were divided into the following 3 groups: sedentary (S); moderate‐intensity training (MIT); and high‐intensity training (HIT). The training protocol lasted 8 weeks. Forty‐eight hours after training, total RNA and protein levels in the soleus and plantaris muscles were obtained. Results: In the plantaris, the BDNF protein level was lower in the HIT than in the S group (P < 0.05). A similar effect was found in the soleus (without significant difference). In the soleus, higher Bdnf mRNA levels were found in the HIT group (P < 0.001 vs. S and MIT groups). In the plantaris muscle, similar Bdnf mRNA levels were found in all groups. Conclusions: These results indicate that high‐intensity chronic exercise reduces BDNF protein level in fast muscles and increases Bdnf mRNA levels in slow muscles. Muscle Nerve 53: 446–451, 2016     

不同浓度氯氮平对小鼠空腹血糖和骨骼肌葡萄糖转运蛋白4基因表达的影响

目的探讨氯氮平对雄性C57BL/6小鼠空腹血糖和骨骼肌葡萄糖转运蛋白4(GLUT4)基因表达的影响。方法将63只雄性C57BL/6小鼠随机分为3组,每组21只,分别灌胃给予蒸馏水、氯氮平4mg/kg及氯氮平20mg/kg,于给药后3h、1周、4周以试纸法测定各组空腹血糖,用逆转录-聚合酶链反应测定GLUT4mRNA表达。结果(1)灌药后3h、1周氯氮平4mg/kg组和氯氮平20mg/kg组空腹血糖和GLUT4mRNA的表达与空白对照组相比,差异无统计学意义(P>0.05);(2)灌药后4周氯氮平4mg/kg组和20mg/kg组的空腹血糖值[(5.6±0.5)mmol/L和(5.8±0.5)mmol/L]高于空白对照组[(4.6±0.6)mmol/L],而GLUT4mRNA的表达(0.50±0.14和0.48±0.12)却低于空白对照组(0.85±0.27),差异均有统计学意义(P<0.01)。结论氯氮平可以慢性升高空腹血糖,降低GLUT4mRNA的表达,可能是抗精神病药长期应用后血糖升高的发生机制之一。     

Effects of tail suspension on serum testosterone and molecular targets regulating muscle mass

Introduction: The contribution of reduced testosterone levels to tail suspension (TS)‐induced muscle atrophy remains equivocal. The molecular mechanism by which testosterone regulates muscle mass during TS has not been investigated. Methods: Effects of TS on serum testosterone levels, muscle mass, and expression of muscle atrophy‐ and hypertrophy‐inducing targets were measured in soleus (SOL) and extensor digitorum longus (EDL) muscles after testosterone administration during 1, 5, and 14 days of TS in male mice. Results: TS produced an increase followed by a transient drop in testosterone levels. Muscle atrophy was associated with downregulation of Igf1 and upregulation of Mstn, Redd1, Atrogin‐1, and MuRF1 mRNA with clear differences in Igf1, Mstn, and MAFbx/Atrogin‐1 gene expression between SOL and EDL. Testosterone supplementation did not affect muscle mass or protein expression levels during TS. Conclusions The known anabolic effects of testosterone are not sufficient to ameliorate loss of muscle mass during TS. Muscle Nerve 52 : 278–288, 2015     

Low dose formoterol administration improves muscle function in dystrophic mdx mice without increasing fatigue

The beta(2)-adrenoceptor agonist (beta(2)-agonist), formoterol, has been shown to cause muscle hypertrophy in rats even when administered at the micromolar dose of 25 micro g/kg/day. We investigated whether a similar low dose of formoterol could improve muscle function in the dystrophic mdx mouse. Ten-week-old male mdx and wild-type (C57BL/10) mice were administered formoterol (25 micro g/kg/day, i.p.) for 4 weeks. Formoterol treatment increased extensor digitorum longus (EDL) and soleus muscle mass, increased median muscle fibre size in diaphragm, EDL, and soleus muscles, and increased maximum force producing capacity in skeletal muscles of both wild-type and mdx mice. In contrast to other studies where beta(2)-agonists have been administered to mice and rats, generally at higher doses, low dose formoterol treatment did not increase the fatiguability of EDL, soleus or diaphragm muscles. Although others have found formoterol can decrease ubiquitin mRNA and proteasome activity when administered to tumour bearing rats at high doses (2mg/kg/day), in the present study low dose formoterol treatment did not alter ubiquitin or the E1 and E3 ubiquitin ligases in diaphragm muscles of wild-type or mdx mice, but it did reduce the level of ubiquitinated proteins in diaphragm of wild-type mice. The findings indicate that formoterol has considerably more powerful anabolic effects on skeletal muscle than older generation beta(2)-agonists (like clenbuterol and albuterol), and has considerable therapeutic potential for muscular dystrophies and other neuromuscular disorders where muscle wasting is indicated.     

Recovery of rat skeletal muscles after partial denervation is enhanced by treatment with nifedipine

Following partial denervation of adult rat skeletal muscle intact axons sprout to reinnervate denervated muscle fibres and increase their territory. The extent of this increase is limited and may depend on the ability of axon terminals to form and maintain synaptic contacts with the denervated muscle fibres. Here we tested the possibility whether reducing Ca²⁺ entry into presynaptic nerve terminals through dihydropyridine sensitive channels may allow more nerve–muscle contacts to be formed and maintained. Hindlimb muscles of adult Wistar rats were partially denervated by removing a small segment of the L4 or L5 spinal nerve on one side. A nifedipine-containing silastic rubber strip was subsequently implanted close to the partially denervated soleus or extensor digitorum longus (EDL) muscles in some animals. In control experiments silastic strips which did not contain nifedipine were used. Several weeks later isometric contractions were recorded, to determine the effect of (a) partial denervation and (b) nifedipine treatment on force output and motor unit numbers. The tension produced by nifedipine treated partially denervated muscles was 82% and 79% of the unoperated contralateral value for soleus and EDL, respectively. This was significantly greater than in untreated muscles, which only produced 61% and 48%, respectively. Mean motor unit force was also significantly larger with nifedipine treatment. Histological analysis revealed that a significantly larger proportion of the total number of muscle fibres remained in nifedipine-treated partially denervated muscles (soleus, 90% and EDL, 101%) compared with untreated muscles (soleus, 51% and EDL, 66%). Thus the number of neuromuscular contacts was increased with nifedipine treatment.     

Putative effects of nerve extract on carbonic anhydrase III expression in rat muscles

Carbonic anhydrase III (CA III), the predominant CA isoform in skeletal muscle is very sensitive to neuronal influences. We aimed to determine whether CA III expression could be influenced by neurotrophic factor(s) present in sciatic nerve extract (SNE). Intact muscles were thus compared with denervated soleus (SOL), extensor digitorum longus (EDL), and tibialis anterior (TA) muscles injected daily for 7 days with saline solution (SS) or with SNE. CA III activity was significantly increased in SS-treated EDL and TA muscles compared to control (CTR), while SNE injections partially prevented this increase. There was no significant difference for CA III activity in the SOL between CTR, SS, and SNE groups. The CA III mRNA increase observed in response to denervation was reduced by 40% in SNE-treated EDL and TA muscles. While SOL CA III mRNA level was not affected by denervation, a 52% decrease was observed with SNE. We concluded that neuronal modulation of CA III expression in type II fibers may involve a neurotrophic component. © 1994 John Wiley & Sons, Inc.