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Skeletal muscles comprise hundreds of individual muscle fibers, with each possessing specialized contractile properties. Skeletal muscles are recognized as being highly plastic, meaning that the physiological properties of single muscle fibers can change with appropriate use. During fiber type transitions, one myosin heavy chain isoform is exchanged for another and over time the fundamental nature of the fiber adapts to become a different fiber type. Within the rat triceps surae complex, the soleus muscle starts out as a muscle comprised of a mixture type IIA and type I fibers. As neonatal rats grow and mature, the soleus undergoes a near complete transition into a muscle with close to 100% type I fibers at maturity. We used immunohistochemistry and single fiber SDS-PAGE to track the transformation of type IIA into type I fibers. We found that transitioning fibers progressively incorporate new myofibrils containing type I myosin into existing type IIA fibers. During this exchange, distinct type I-containing myofibrils are segregated among IIA myofibrils. The individual myofibrils within existing muscle fibers thus appear to represent the functional unit that is exchanged during fiber type transitions that occur as part of normal muscle development.

Mangled extremities were classically managed by amputation. But over the past few decades, with the advancement in surgical techniques, an increased number of limb salvages have been possible. As muscles usually get damaged in such grievous injuries, a thorough understanding of muscle regeneration may give a better insight into muscle healing in these injuries. Muscles are composed of slow and fast fibers which can be represented by slow and fast myosin, respectively. There are some animal studies which reported differential regeneration of slow and fast muscle fibers during muscle healing. We conducted this pilot study to find out whether the same holds true for muscle healing in mangled extremities also. This pilot study is designed in 15 patients with lower limb mangled extremities presenting to trauma center of PGIMER, Chandigarh, who were operated within 24 h of injury to see whether muscle healing in mangled extremities follows the same pattern. Biopsies were taken during initial surgery conducted within 24 h of injury and on the 7th day of injury when patient was posted again for secondary wound closure procedure or revision amputation. The biopsy samples were subjected to histopathological and immunohistochemistry examination using antibodies against fast and slow myosin. We found that the regenerating muscle fibers in the biopsy sample taken on the 7th day of injury showed only slow muscle fibers with the absence of fast muscle fibers when compared with the initial biopsy results showing differential regeneration of slow muscle fibers.

Exercise intolerance is a primary symptom of heart failure (HF); however, the specific contribution of central and peripheral factors to this intolerance is not well described. The hyperbolic relationship between exercise intensity and time to exhaustion (speed-duration relationship) defines exercise tolerance but is underused in HF. We tested the hypotheses that critical speed (CS) would be reduced in HF, resting central functional measurements would correlate with CS, and the greatest HF-induced peripheral dysfunction would occur in more oxidative muscle. Multiple treadmill-constant speed runs to exhaustion were used to quantify CS and D' (distance coverable above CS) in healthy control (Con) and HF rats. Central function was determined via left ventricular (LV) Doppler echocardiography [fractional shortening (FS)] and a micromanometer-tipped catheter [LV end-diastolic pressure (LVEDP)]. Peripheral O 2 delivery-to-utilization matching was determined via phosphorescence quenching (interstitial Po 2 , Po 2 is ) in the soleus and white gastrocnemius during electrically induced twitch contractions (1 Hz, 8V). CS was lower in HF compared with Con (37 ± 1 vs. 44 ± 1 m/min, P < 0.001), but D' was not different (77 ± 8 vs. 69 ± 13 m, P = 0.6). HF reduced FS (23 ± 2 vs. 47 ± 2%, P < 0.001) and increased LVEDP (15 ± 1 vs. 7 ± 1 mmHg, P < 0.001). CS was related to FS ( r  = 0.72, P = 0.045) and LVEDP ( r  = -0.75, P = 0.02) only in HF. HF reduced soleus Po 2 is at rest and during contractions (both P < 0.01) but had no effect on white gastrocnemius Po 2 is ( P > 0.05). We show in HF rats that decrements in central cardiac function relate directly with impaired exercise tolerance (i.e., CS) and that this compromised exercise tolerance is likely due to reduced perfusive and diffusive O 2 delivery to oxidative muscles. NEW & NOTEWORTHY We show that critical speed (CS), which defines the upper boundary of sustainable activity, can be resolved in heart failure (HF) animals and is diminished compared with controls. Central cardiac function is strongly related with CS in the HF animals, but not controls. Skeletal muscle O 2 delivery-to-utilization dysfunction is evident in the more oxidative, but not glycolytic, muscles of HF rats and is explained, in part, by reduced nitric oxide bioavailability.

We examined the lateral gastrocnemius (LG), plantaris (PL), and extensor digitorum longus (EDL) muscles to determine whether differential activation of the calpain system is related to the degree of atrophy in these fast-twitch skeletal muscles during hibernation in Daurian ground squirrels ( Spermophilus dauricus ). Results from morphological indices showed various degrees of atrophy in the order LG > PL > EDL. Furthermore, all three muscles underwent fast-to-slow fiber-type conversion in hibernation. In regard to the calpain system in the LG muscle, cytosolic Ca 2+ increased significantly in hibernation, followed by recovery in posthibernation. Furthermore, calpastatin expression significantly decreased, and calpain 1 and 2 expression significantly increased, which may be responsible for the increased degradation of desmin during hibernation compared with that during summer activity. In the EDL muscle, Ca 2+ overload was observed during interbout arousal, and calpastatin showed an increase during hibernation and interbout arousal, which could explain the increased levels of troponin T during both periods compared with levels during summer activity. These findings suggest that cytosolic Ca 2+ overload and subsequent calpain 1 and 2 activation may be an important mechanism of LG muscle atrophy during hibernation. Cytosolic Ca 2+ homeostasis and high expression of calpain inhibitor calpastatin during hibernation may also be an important mechanism for the EDL muscle to maintain muscle mass. Thus, the differential activation of the calpain system and selective degradation of downstream substrates may be involved in muscle atrophy of different fast-twitch muscles during hibernation. NEW & NOTEWORTHY We found that the extent of both muscle atrophy and calpain system activation differed in fast-twitch lateral gastrocnemius (LG), plantaris (PL), and extensor digitorum longus (EDL) skeletal muscles in hibernating Daurian ground squirrels, but similar hierarchies in the order of LG > PL > EDL. The differential activation of the calpain system and selective degradation of downstream substrates may be involved in muscle atrophy in different fast-twitch muscles during hibernation.

Although exercise affects the function and structure of skeletal muscle, our knowledge regarding the biomedical alterations induced by different intensities of exercise is incomplete. Here we report on the changes in biomarker levels and myofiber constitution in the rat soleus muscle induced by exercise intensity. Male adult rats at 7 weeks of age were divided into 3 groups by exercise intensity, which was set based on the accumulated lactate levels in the blood using a treadmill: stationary control (0 m/min), aerobic exercise (15 m/min), and anaerobic exercise (25 m/min). The rats underwent 30 min/day treadmill training at different exercise intensities for 14 days. Immediately after the last training session, the soleus muscle was dissected out in order to measure the muscle biomarker levels and evaluate the changes in the myofibers. The mRNA expression of citrate synthase, glucose-6-phosphate dehydrogenase, and Myo D increased with aerobic exercise, while the mRNA expression of myosin heavy-chain I and Myo D increased in anaerobic exercise. These results suggest that muscle biomarkers can be used as parameters for the muscle adaptation process in aerobic/anaerobic exercise. Interestingly, by 14 days after the anaerobic exercise, the number of type II (fast-twitch) myofibers had decreased by about 20%. Furthermore, many macrophages and regenerated fibers were observed in addition to the injured fibers 14 days after the anaerobic exercise. Constitutional changes in myofibers due to damage incurred during anaerobic exercise are necessary for at least about 2 weeks. These results indicate that the changes in the biomarker levels and myofiber constitution by exercise intensity are extremely important for understanding the metabolic adaptations of skeletal muscle during physical exercise.

The relationship between the extracellular signal-regulated kinase 1 and 2 (ERK1/2), one of the mitogen-activated protein kinases (MAPKs), and mammalian skeletal muscle fiber phenotype is unclear. We looked at this relationship in three in vivo conditions in male Wistar rats. First, the levels of phosphorylated (active) ERK1/2 protein were closely associated with the fiber type composition of sedentary rat hindlimb muscles: highest in the superficial portion of the gastrocnemius (100% fast fibers), lower in the plantaris (~ 80% fast fibers), and lowest in the soleus (~ 15% fast fibers). Second, during growth, there was a gradual decrease in the percentage of fast fibers from 40% at 3 weeks to 1.5% at 65 weeks and a concomitant gradual decrease in the levels of phosphorylated ERK1/2 in the soleus muscle. Third, sciatic nerve denervation induced a significant decrease in the weight of both the soleus and plantaris, but a slow-to-fast fiber type shift and increase in phosphorylated ERK1/2 protein were observed only in the soleus. Although only a few fast and fast + slow hybrid fibers of the denervated soleus muscle reacted positively to the anti-phosphorylated ERK1/2 antibody by immuno-histochemical analysis, our results suggest that the phosphorylated form of ERK1/2 seems to be closely related to the fast fiber phenotype program. Further evidence for this relationship was provided by the observation that several slow fiber phenotype-specific proteins, i.e., Hsp72, Hsp60, and PGC-1, changed in the opposite direction of the levels of phosphorylated ERK1/2 protein.

A low quadriceps slow-twitch (ST), oxidative (relative to fast-twitch) fiber proportion is prevalent in chronic diseases such Chronic Obstructive Pulmonary Disease (COPD) and is associated with exercise limitation and poor outcomes. Benefits of an increased ST fiber proportion are demonstrated in genetically modified animals. Pathway analysis of published data of differentially expressed genes in mouse ST and FT fibers, mining of our microarray data and a qPCR analysis of quadriceps specimens from COPD patients and controls were performed. ST markers were quantified in C2C12 myotubes with EGF-neutralizing antibody, EGFR inhibitor or an EGFR-silencing RNA added. A zebrafish egfra mutant was generated by genome editing and ST fibers counted. EGF signaling was (negatively) associated with the ST muscle phenotype in mice and humans, and muscle EGF transcript levels were raised in COPD. In C2C12 myotubes, EGFR inhibition/silencing increased ST, including mitochondrial, markers. In zebrafish, egfra depletion increased ST fibers and mitochondrial content. EGF is negatively associated with ST muscle phenotype in mice, healthy humans and COPD patients. EGFR blockade promotes the ST phenotype in myotubes and zebrafish embryos. EGF signaling suppresses the ST phenotype, therefore EGFR inhibitors may be potential treatments for COPD-related muscle ST fiber loss.

Skeletal muscle fiber is largely classified into two types: type 1 (slow-twitch) and type 2 (fast-twitch) fibers. Meat quality and composition of fiber types are thought to be closely related. Previous research showed that overexpression of constitutively active peroxisome proliferator-activated receptor (PPAR)δ, a nuclear receptor present in skeletal muscle, increased type 1 fibers in mice. In this study, we found that hexane extracts of Yamabushitake mushroom (Hericium erinaceus) showed PPARδ agonistic activity in vitro. Eight-week-old C57BL/6J mice were fed a diet supplemented with 5% (w/w) freeze-dried Yamabushitake mushroom for 24 hr. After the treatment period, the extensor digitorum longus (EDL) muscles were excised. The Yamabushitake-supplemented diet up-regulated the PPARδ target genes Pdk4 and Ucp3 in mouse skeletal muscles in vivo. Furthermore, feeding the Yamabushitake-supplemented diet to mice for 8 weeks resulted in a significant increase in muscle endurance. These results indicate that Yamabushitake mushroom contains PPARδ agonistic ligands and that dietary intake of Yamabushitake mushroom could activate PPARδ in skeletal muscle of mice. Unexpectedly, we observed no significant alterations in composition of muscle fiber types between the mice fed control and Yamabushitake-supplemented diets., (© 2019 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.)

Insulin-stimulated glucose uptake (GU) by skeletal muscle is enhanced several hours after acute exercise in rats with normal or reduced insulin sensitivity. Skeletal muscle is composed of multiple fiber types, but exercise's effect on fiber type-specific insulin-stimulated GU in insulin-resistant muscle was previously unknown. Male rats were fed a high-fat diet (HFD; 2 wk) and were either sedentary (SED) or exercised (2-h exercise). Other, low-fat diet-fed (LFD) rats remained SED. Rats were studied immediately postexercise (IPEX) or 3 h postexercise (3hPEX). Epitrochlearis muscles from IPEX rats were incubated in 2-deoxy-[ 3 H]glucose (2-[ 3 H]DG) without insulin. Epitrochlearis muscles from 3hPEX rats were incubated with 2-[ 3 H]DG ± 100 µU/ml insulin. After single fiber isolation, GU and fiber type were determined. Glycogen and lipid droplets (LDs) were assessed histochemically. GLUT4 abundance was determined by immunoblotting. In HFD-SED vs. LFD-SED rats, insulin-stimulated GU was decreased in type IIB, IIX, IIAX, and IIBX fibers. Insulin-independent GU IPEX was increased and glycogen content was decreased in all fiber types (types I, IIA, IIB, IIX, IIAX, and IIBX). Exercise by HFD-fed rats enhanced insulin-stimulated GU in all fiber types except type I. Single fiber analyses enabled discovery of striking fiber type-specific differences in HFD and exercise effects on insulin-stimulated GU. The fiber type-specific differences in insulin-stimulated GU postexercise in insulin-resistant muscle were not attributable to a lack of fiber recruitment, as indirectly evidenced by insulin-independent GU and glycogen IPEX, differences in multiple LD indexes, or altered GLUT4 abundance, implicating fiber type-selective differences in the cellular processes responsible for postexercise enhancement of insulin-mediated GLUT4 translocation.

The type 2 iodothyronine-deiodinase (D2) enzyme converts T4 to T3, and mice deficient in this enzyme [D2 knockout (D2KO) mice] have decreased T3 derived from T4 in skeletal muscle despite normal circulating T3 levels. Because slow skeletal muscle is particularly susceptible to changes in T3 levels, we expected D2 inactivation to result in more pronounced slow-muscle characteristics in the soleus muscle, mirroring hypothyroidism. However, ex vivo studies of D2KO soleus revealed higher rates of twitch contraction and relaxation and reduced resistance to fatigue. Immunostaining of D2KO soleus showed that these properties were associated with changes in muscle fiber type composition, including a marked increase in the number of fast, glycolytic type IIB fibers. D2KO soleus muscle fibers had a larger cross-sectional area, and this correlated with increased myonuclear accretion in myotubes formed from D2KO skeletal muscle precursor cells differentiated in vitro. Consistent with our functional findings, D2KO soleus gene expression was markedly different from that in hypothyroid wild-type (WT) mice. Comparison of gene expression between euthyroid WT and D2KO mice indicated that PGC-1α, a T3-dependent regulator of slow muscle fiber type, was decreased by ∼50% in D2KO soleus. Disruption of Dio2 in the C2C12 myoblast cell line led to a significant decrease in PGC-1α expression and a faster muscle phenotype upon differentiation. These results indicate that D2 loss leads to significant changes in soleus contractile function and fiber type composition that are inconsistent with local hypothyroidism and suggest that reduced levels of PCG-1α may contribute to the observed phenotypical changes., (Copyright © 2019 Endocrine Society.)

Alcoholic myopathy is characterized by the reduction in cross-sectional area (CSA) of muscle fibers and impaired anabolic signaling. The goal of the current study was to investigate the causes and compare the changes in CSA and fiber type composition with the modifications of anabolic and catabolic signaling pathways at the early stages of chronic alcohol consumption in women. Skeletal muscle samples from 5 female patients with alcohol abuse (AL; 43 ± 5 yr old; alcohol abuse duration 5,6 ± 0,6 yr) were compared with the muscle from the control group of 8 healthy women (C; 35 ± 4 yr old). The average daily dose of alcohol consumption was 110 ± 10 ml of pure ethanol. In women patients, a significant decrease in CSA of type I and II muscle fibers, titin and nebulin content, plasma IGF-1 level and total IRS-1, p-Akt and p-4E-BP1 in vastus lateralis was found in comparison with the control group. The p-AMPK level was found to be increased versus the control group. In women patients with chronic alcoholic myopathy 1 ) both fast and slow muscle fibers are subjected to atrophy; 2 ) impairments in IGF-I-dependent signaling and pathways controlling translation initiation (AMPK/mTOR/4E-BP1), but not translation elongation, are observed; 3 ) the level of calpain-1 and ubiquitinated proteins increases, unlike E3 ligases content.

An impaired capacity of muscle to regenerate after critical illness results in long-term functional disability. We previously described in a long-term rat peritonitis model that gastrocnemius displays near-normal histology whereas soleus demonstrates a necrotizing phenotype. We thus investigated the link between the necrotizing phenotype of critical illness myopathy and proteasome activity in these two limb muscles. We studied male Wistar rats that underwent an intraperitoneal injection of the fungal cell wall constituent zymosan or n-saline as a sham-treated control. Rats ( n = 74) were killed at 2, 7, and 14 days postintervention with gastrocnemius and soleus muscle removed and studied ex vivo. Zymosan-treated animals displayed an initial reduction of body weight but a persistent decrease in mass of both lower hindlimb muscles. Zymosan increased chymotrypsin- and trypsin-like proteasome activities in gastrocnemius at days 2 and 7 but in soleus at day 2 only. Activated caspases-3 and -9, polyubiquitin proteins, and 14-kDa fragments of myofibrillar actin (proteasome substrates) remained persistently increased from day 2 to day 14 in soleus but not in gastrocnemius. These results suggest that a relative proteasome deficiency in soleus is associated with a necrotizing phenotype during long-term critical illness. Rescuing proteasome clearance may offer a potential therapeutic option to prevent long-term functional disability in critically ill patients.

Earlier research using muscle tissue demonstrated that postexercise elevation in insulin-stimulated glucose uptake (ISGU) occurs concomitant with greater insulin-stimulated Akt substrate of 160 kDa (AS160) phosphorylation (pAS160) on sites that regulate ISGU. Because skeletal muscle is a heterogeneous tissue, we previously isolated myofibers from rat epitrochlearis to assess fiber type-selective ISGU. Exercise induced greater ISGU in type I, IIA, IIB, and IIBX but not IIX fibers. This study tested if exercise effects on pAS160 correspond with previously published fiber type-selective exercise effects on ISGU. Rats were studied immediately postexercise (IPEX) or 3.5 h postexercise (3.5hPEX) with time-matched sedentary controls. Myofibers dissected from the IPEX experiment were analyzed for fiber type (myosin heavy chain isoform expression) and key phosphoproteins. Isolated muscles from the 3.5hPEX experiment were incubated with or without insulin. Myofibers (3.5hPEX) were analyzed for fiber type, key phosphoproteins, and GLUT4 protein abundance. We hypothesized that insulin-stimulated pAS160 at 3.5hPEX would exceed sedentary controls only in fiber types characterized by greater ISGU postexercise. Values for phosphorylation of AMP-activated kinase substrates (acetyl CoA carboxylase Ser79 and AS160 Ser704 ) from IPEX muscles exceeded sedentary values in each fiber type, suggesting exercise recruitment of all fiber types. Values for pAS160 Thr642 and pAS160 Ser704 from insulin-stimulated muscles 3.5hPEX exceeded sedentary values for type I, IIA, IIB, and IIBX but not IIX fibers. GLUT4 abundance was unaltered 3.5hPEX in any fiber type. These results advanced understanding of exercise-induced insulin sensitization by providing compelling support for the hypothesis that enhanced insulin-stimulated phosphorylation of AS160 is linked to elevated ISGU postexercise at a fiber type-specific level independent of altered GLUT4 expression.

Mitogen-activated protein kinase (MAPK) signaling consists of an array of successively acting kinases. The extracellular signal-regulated kinases 1/2 (ERK1/2) are major components of the greater MAPK cascade that transduce growth factor signaling at the cell membrane. Here we investigated ERK1/2 signaling in skeletal muscle homeostasis and disease. Using mouse genetics, we observed that the muscle-specific expression of a constitutively active MEK1 mutant promotes greater ERK1/2 signaling that mediates fiber-type switching to a slow, oxidative phenotype with type I myosin heavy chain expression. Using a conditional and temporally regulated Cre strategy as well as Mapk1 (ERK2) and Mapk3 (ERK1) genetically targeted mice, MEK1-ERK2 signaling was shown to underlie this fast-to-slow fiber type switching in adult skeletal muscle as well as during development. Physiologic assessment of these activated MEK1-ERK1/2 mice showed enhanced metabolic activity and oxygen consumption with greater muscle fatigue resistance. Moreover, induction of MEK1-ERK1/2 signaling increased dystrophin and utrophin protein expression in a mouse model of limb-girdle muscle dystrophy and protected myofibers from damage. In summary, sustained MEK1-ERK1/2 activity in skeletal muscle produces a fast-to-slow fiber-type switch that protects from muscular dystrophy, suggesting a therapeutic approach to enhance the metabolic effectiveness of muscle and protect from dystrophic disease.

Consequences of running mountain races on muscle damage were investigated by analysing serum muscle enzymes and fibre-type-specific sarcomere proteins. We studied 10 trained amateur and 6 highly trained runners who ran a 35 km and 55 km mountain trail race (MTR), respectively. Levels of creatine kinase (CK), CK-MB isoform (CK-MB), sarcomeric mitochondrial CK (sMtCK), transaminases (AST and ALT), cardiac troponin I (cTnI) and fast (FM) and slow myosin (SM) isoforms, were assessed before, 1 h, 24 h and 48 h after the beginning of MTR. Significant SM increases were found at 24 h in the 55 km group. Levels of CK, CK-MB, AST and cTnI were significantly elevated in both groups following MTR, but in the 55 km group they tended to stabilize in at 48 h. Using pooled data, time-independent serum peaks of SM and CK-MB were significantly correlated. Moreover, concentration of sMtCK was significantly elevated at 1 and 24 h after the race in the 35 km group. Although training volume could confer protection on the mitochondria, the increase in serum CK-MB and SM in the 55 km group might be related to damage to the contractile apparatus type I fibres. Competing in long-distance MTRs might be related to deeper type I muscle fibre damage, even in highly trained individuals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Free amino acids are important components of tastants and flavor precursors in meat. To clarify the correlation between muscle fiber type and free amino acids, we determined the concentrations of various free amino acids and dipeptides in samples of different muscle tissues (n = 21), collected from 26-month-old Japanese Black steers (n = 3) at 2 days postmortem. The proportions of the myosin heavy chain (MyHC), slow (MyHC1) and fast (MyHC2) isoforms were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The contents of free amino acids and dipeptides were measured by high performance liquid chromatography (HPLC). The MyHC isoform composition varied among the tissue samples. The MyHC1 proportion ranged from 6.9% ± 3.9% to 83.3% ± 16.7%. We confirmed that there was a strong positive correlation between MyHC1 composition and total free amino acid concentrations, including those for two dipeptides. Among the 31 measured free amino acids and dipeptides, 11 showed significant positive correlations and five showed significant negative correlations with MyHC1 composition. These results suggest that a high MyHC1 content induces high free amino acid contents in bovine muscles possibly because of greater oxidative metabolism. This high level of free amino acids could contribute to the intense flavor of meat that is rich in slow-twitch fibers., (© 2019 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.)

The aim of the present study was to investigate how myostatin dysfunction affects fast and slow muscle stiffness and viscosity during severe repeated loading. Isolated extensor digitorum longus (EDL) and soleus muscles of young adult female mice of the BEH (dysfunctional myostatin) and BEH+/+ (functional myostatin) strains were subjected to 100 contraction-stretching loading cycles during which contractile and mechanical properties were assessed. BEH mice exhibited greater exercise-induced muscle damage, although the effect was muscle- and age-dependent and limited to the early phases of simulated exercise. The relative reduction of the EDL muscle isometric force recorded during the initial 10-30 loading cycles was greater in BEH mice than in BEH+/+ mice and exceeded that of the soleus muscle of either strain. The induced damage was associated with lower muscle stiffness. The effects of myostatin on the mechanical properties of muscles depend on muscle type and maturity.

Lipin-1 ( Lpin1)-deficient lipodystrophic mice have scant and immature adipocytes and develop transient fatty liver early in life. Unlike normal mice, these mice cannot rely on stored triglycerides to generate adenosine triphosphate (ATP) from the β-oxidation of fatty acids during periods of fasting. To compensate, these mice store much higher amounts of glycogen in skeletal muscle and liver than wild-type mice in order to support energy needs during periods of fasting. Our studies demonstrated that there are phenotypic changes in skeletal muscle fibers that reflect an adaptation to this unique metabolic situation. The phenotype of skeletal muscle (soleus, gastrocnemius, plantaris, and extensor digitorum longus [EDL]) from Lpin1 -/- was evaluated using various methods including immunohistochemistry for myosin heavy chains (Myh) 1, 2, 2a, 2b, and 2x; enzyme histochemistry for myosin ATPase, cytochrome-c oxidase (COX), and succinyl dehydrogenase (SDH); periodic acid-Schiff; and transmission electron microscopy. Fiber-type changes in the soleus muscle of Lpin1 -/- mice were prominent and included decreased Myh1 expression with concomitant increases in Myh2 expression and myosin-ATPase activity; this change was associated with an increase in the presence of Myh1/2a or Myh1/2x hybrid fibers. Alterations in mitochondrial enzyme activity (COX and SDH) were apparent in the myofibers in the soleus, gastrocnemius, plantaris, and EDL muscles. Electron microscopy revealed increases in the subsarcolemmal mitochondrial mass in the muscles of Lpin1 -/- mice. These data demonstrate that lipin-1 deficiency results in phenotypic fiber-specific modulation of skeletal muscle necessary for compensatory fuel utilization adaptations in lipodystrophy.

The skeletal muscle is a crucial tissue for maintaining whole body homeostasis. Aging seems to have a disruptive effect on skeletal muscle homeostasis including proteostasis. However, how aging specifically impacts slow and fast twitch fiber types remains elusive. Muscle proteostasis is largely maintained by the proteasomal system. Here we characterized the proteasomal system in two different fiber types, using a non-sarcopenic aging model. By analyzing the proteasomal activity and amount, as well as the polyubiquitinated proteins and the level of protein oxidation in Musculus soleus (Sol) and Musculus extensor digitorum longus (EDL), we found that the slow twitch Sol muscle shows an overall higher respiratory and proteasomal activity in young and old animals. However, especially during aging the fast twitch EDL muscle reduces protein oxidation by an increase of antioxidant capacity. Thus, under adaptive non-sarcopenic conditions, the two fibers types seem to have different strategies to avoid age-related changes., (Copyright © 2018. Published by Elsevier Inc.)

Guilherme, JPLF, Egorova, ES, Semenova, EA, Kostryukova, ES, Kulemin, NA, Borisov, OV, Khabibova, SA, Larin, AK, Ospanova, EA, Pavlenko, AV, Lyubaeva, EV, Popov, DV, Lysenko, EA, Vepkhvadze, TF, Lednev, EM, Govorun, VM, Generozov, EV, Ahmetov, II, and Lancha Junior, AH. The A-allele of the FTO gene rs9939609 polymorphism is associated with decreased proportion of slow oxidative muscle fibers and over-represented in heavier athletes. J Strength Cond Res 33(3): 691-700, 2019-The purpose of this study was to explore the frequency of the FTO T > A (rs9939609) polymorphism in elite athletes from 2 cohorts (Brazil and Russia), as well as to find a relationship between FTO genotypes and muscle fiber composition. A total of 677 athletes and 652 nonathletes were evaluated in the Brazilian cohort, whereas a total of 920 athletes and 754 nonathletes were evaluated in the Russian cohort. It was found a trend for a lower frequency of A/A genotype in long-distance athletes compared with nonathletes (odds ratio [OR]: 0.65; p = 0.054). By contrast, it was found an increased frequency of the A-allele in Russian power athletes. The presence of the T/A + A/A genotypes rather than T/T increased the OR of being a Russian power athlete compared with matched nonathletes (OR: 1.45; p = 0.002). Different from that observed in combat sports athletes of lighter weight categories, the A-allele was also over-represented in combat sports athletes of heavier weight categories. The presence of the T/A + A/A genotypes rather than T/T increased the OR of being a combat sports athlete of heavier weight categories compared with nonathletes (OR: 1.79; p = 0.018). Regarding the muscle fibers, we found that carriers of the A/A genotype had less slow-twitch muscle fibers than T-allele carriers (p = 0.029). In conclusion, the A/A genotype of the FTO T > A polymorphism is under-represented in athletes more reliant on a lean phenotype and associated with decreased proportion of slow-twitch muscle fibers, while is over-represented in strength and heavier athletes.

Aim: Cachexia is a severe wasting disorder involving loss of body- and muscle mass reducing survival and quality of life in cancer patients. We aim at determining if cachexia is a mere perturbation of the protein balance or if the condition also involves a degenerative loss of myonuclei within the fibre syncytia or loss of whole muscle fibres., Methods: We induced cachexia by xenografting PC3 prostate cancer cells in nu/nu mice. Six weeks later, we counted myonuclei by in vivo microscopic imaging of single live fibres in the extensor digitorum longus muscle (EDL), and the EDL, soleus and tibialis anterior muscles were also harvested for ex vivo histology., Results: The mice lost on average 15% of the whole-body wt. The muscle wet weight of the glycolytic, fast EDL was reduced by 14%, the tibialis anterior by 17%, and the slow, oxidative soleus by 6%. The fibre cross-sectional area in the EDL was reduced by 21% with no loss of myonuclei or any significant reduction in the number of muscle fibres. TUNEL-positive nuclei or fibres with embryonic myosin were rare both in cachectic and control muscles, and haematoxylin-eosin staining revealed no clear signs of muscle pathology., Conclusion: The data suggest that the cachexia induced by xenografted prostate tumours induces a pronounced atrophy not accompanied by a loss of myonuclei or a loss of muscle fibres. Thus, stem cell related treatment might be redundant, and the quest for treatment options should rather focus on intervening with intracellular pathways regulating muscle fibre size., (© 2018 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Background: The pathophysiology of obstruction and swallowing dysfunction in snores and sleep apnea patients remains unclear. Neuropathy and to some extent myopathy have been suggested as contributing causes. Recently we reported an absence and an abnormal isoform of two cytoskeletal proteins, desmin, and dystrophin, in upper airway muscles of healthy humans. These cytoskeletal proteins are considered vital for muscle function. We aimed to investigate for muscle cytoskeletal abnormalities in upper airways and its association with swallowing dysfunction and severity of sleep apnea., Methods: Cytoskeletal proteins desmin and dystrophin were morphologically evaluated in the uvula muscle of 22 patients undergoing soft palate surgery due to snoring and sleep apnea and in 10 healthy controls. The muscles were analysed with immunohistochemical methods, and swallowing function was assessed using videoradiography., Results: Desmin displayed a disorganized pattern in 21 ± 13% of the muscle fibres in patients, while these fibers were not present in controls. Muscle fibres lacking desmin were present in both patients and controls, but the proportion was higher in patients (25 ± 12% vs. 14 ± 7%, p = 0.009). The overall desmin abnormalities were significantly more frequent in patients than in controls (46 ± 18% vs. 14 ± 7%, p < 0.001). In patients, the C-terminus of the dystrophin molecule was absent in 19 ± 18% of the desmin-abnormal muscle fibres. Patients with swallowing dysfunction had 55 ± 10% desmin-abnormal muscle fibres vs. 22 ± 6% in patients without swallowing dysfunction, p = 0.002., Conclusion: Cytoskeletal abnormalities in soft palate muscles most likely contribute to pharyngeal dysfunction in snorers and sleep apnea patients. Plausible causes for the presence of these abnormalities is traumatic snoring vibrations, tissue stretch or muscle overload.

Mitochondria play an important role in providing ATP for muscle contraction. Muscle physiology is compromised in Duchenne muscular dystrophy (DMD) and several studies have shown the involvement of bioenergetics. In this work we investigated the mitochondrial physiology in fibers from fast-twitch muscle (EDL) and slow-twitch muscle (soleus) in the mdx mouse model for DMD and in control C57BL/10J mice. In our study, multiple mitochondrial respiratory parameters were investigated in permeabilized muscle fibers from 12-week-old animals, a critical age where muscle regeneration is observed in the mdx mouse. Using substrates of complex I and complex II from the electron transport chain, ADP and mitochondrial inhibitors, we found in the mdx EDL, but not in the mdx soleus, a reduction in coupled respiration suggesting that ATP synthesis is affected. In addition, the oxygen consumption after addition of complex II substrate is reduced in mdx EDL; the maximal consumption rate (measured in the presence of uncoupler) also seems to be reduced. Mitochondria are involved in calcium regulation and we observed, using alizarin stain, calcium deposits in mdx muscles but not in control muscles. Interestingly, more calcium deposits were found in mdx EDL than in mdx soleus. These data provide evidence that in 12-week-old mdx mice, calcium is accumulated and mitochondrial function is disturbed in the fast-twitch muscle EDL, but not in the slow-twitch muscle soleus.

A previous study demonstrated that leucine upregulates the slow myosin heavy chain mRNA expression in C2C12 cells. However, the role of leucine in slow-twitch muscle fibers expression and mitochondrial function of porcine skeletal muscle satellite cells as well as its mechanism remain unclear. In this study, porcine skeletal muscle satellite cells cultured in differentiation medium were treated with 2 mM leucine for 3 days. Sirt1 inhibitor EX527, AMPK inhibitor compound C, and AMPKα1 siRNA were used to examine its underlying mechanism. Here we showed that leucine increased slow-twitch muscle fibers and mitochondrial function-related gene expression, as well as increased succinic dehydrogenase (SDH) and malate dehydrogenase (MDH) activities. Moreover, leucine increased the protein levels of Sirt1 and phospho-AMPK. We also found that AMPKα1 siRNA, AMPK inhibitor compound C, or Sirt1 inhibitor EX527 attenuated the positive effect of leucine on slow-twitch muscle fibers and mitochondrial function-related gene expression. Finally, we showed that Sirt1 was required for leucine-induced AMPK activation. Our results provide, for the first time, evidence that leucine induces slow-twitch muscle fibers expression and improves mitochondrial function through Sirt1/AMPK signaling pathway in porcine skeletal muscle satellite cells., (© 2018 Japanese Society of Animal Science.)

Skeletal muscle is an important and complex organ with a variety of functions in humans and animals. Skeletal myogenesis is a multistep and complex process, and increasing evidence suggests that microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) play critical roles in skeletal myogenesis. In this study the expression of miR-351-5p is dynamically regulated during skeletal myogenesis in vitro and in vivo. Cell-counting kit-8, qRT-PCR, and EdU immunofluorescence analysis showed that miR-351-5p overexpression promoted the proliferation and inhibited the differentiation of C2C12 myoblast, whereas inhibition of miR-351-5p had the opposite effect. In addition, miR-351-5p mediated the regulation of muscle fiber type transition in vivo. In vitro, loss of miR-351-5p in muscle tissues promoted muscle hypertrophy and increased slow-twitch fibers in the gastrocnemius muscles of mice. Luciferase reporter assay and functional analyses demonstrated that lactamase β ( LACTB) is a direct target of miR-351-5p involved in the regulation of skeletal myogenesis. Expression levels of a myogenesis-associated lncRNA ( lnc-mg) correlated negatively with miR-351-5p and positively with LACTB during C2C12 myoblast proliferation and differentiation. Further analyses showed that lnc-mg acted as a molecular sponge for miR-351-5p, demonstrating its involvement in the negative regulation of LACTB by miR-351-5p during skeletal myogenesis. These findings indicate that miRNA-351-5p functions in skeletal myogenesis by targeting LACTB and is regulated by lnc-mg, supporting the role of the competing endogenous RNA network in skeletal myogenesis.-Du, J., Zhang, P., Zhao, X., He, J., Xu, Y., Zou, Q., Luo, J., Shen, L., Gu, H., Tang, Q., Li, M., Jiang, Y., Tang, G., Bai, L., Li, X., Wang, J., Zhang, S., Zhu, L. MicroRNA-351-5p mediates skeletal myogenesis by directly targeting lactamase β and is regulated by lnc-mg.

The objective of this study was to investigate effects of active immunization against Akirin2 on muscle fiber-type composition in pigs. Here we showed that the titer of Akirin2 antibody in pigs immunized with porcine Akirin2 (pAkirin2) was significantly increased. Active immunization against pAkirin2 decreased succinic dehydrogenase and malate dehydrogenase activities and increased lactate dehydrogenase activity in the longissimus dorsi muscle of pigs. Active immunization against pAkirin2 significantly decreased MyHC I and MyHC IIa mRNA expressions and MyHC I protein expression and increased mRNA expressions of MyHC IIb as well as protein expressions of MyHC IIb and fast-MyHC. mRNA expressions of nuclear factors of activated T cells c1 (NFATc1), transcriptional coactivator PPARγ coactivator-1α, myocyte enhancer factor 2C, and modulatory calcineurin interacting protein 1 exon 4 isform were also notably decreased by active immunization against pAkirin2. Together, our data imply that active immunization against pAkirin2 may result in a slow to fast fiber-type shift in pigs, and which may be mediated by suppression of the calcineurin/NFATc1 signaling pathway.

Muscle fibre types can transform from slow-twitch (slow myosin heavy chain (MyHC)) to fast-twitch (fast MyHC) or vice versa. Leucine plays a vital effect in the development of skeletal muscle. However, the role of leucine in porcine myofibre type transformation and its mechanism are still unclear. In this study, effects of leucine and microRNA-27a (miR-27a) on the transformation of porcine myofibre type were investigated in vitro. We found that leucine increased slow MyHC protein level and decreased fast MyHC protein level, increased the levels of phospho-protein kinase B (Akt)/Akt and phospho-forkhead box 1 (FoxO1)/FoxO1 and decreased the FoxO1 protein level. However, blocking the Akt/FoxO1 signalling pathway by wortmannin attenuated the role of leucine in porcine myofibre type transformation. Over-expression of miR-27a decreased slow MyHC protein level and increased fast MyHC protein level, whereas inhibition of miR-27a had an opposite effect. We also found that expression of miR-27a was down-regulated following leucine treatment. Moreover, over-expression of miR-27a repressed transformation from fast MyHC to slow MyHC caused by leucine, suggesting that miR-27a is interdicted by leucine and then contributes to porcine muscle fibre type transformation. Our finding provided the first evidence that leucine promotes porcine myofibre type transformation from fast MyHC to slow MyHC via the Akt/FoxO1 signalling pathway and miR-27a.

Alaska pollack protein (APP) was previously shown to reduce serum triacylglycerol and the atherogenic index and significantly increase gastrocnemius muscle mass in rats. To determine which myofibers are involved in this observed hypertrophy, we stained the gastrocnemius muscle with fast and slow fiber-specific antibodies and measured the muscle fiber diameter. We observed muscle hypertrophy in both the fast and slow fibers of APP-fed rats. Although muscle hypertrophy leads to drastic lipid changes, the amount of lipids did not differ significantly between casein-fed and APP-fed rats. To determine the lipid changes at the molecular species level and their localization, we performed matrix-assisted laser desorption/ionization mass spectrometry imaging to visualize lipids in the gastrocnemius muscles. We determined that lipid molecules were significantly changed due to APP feeding. Thus, APP feeding changes muscle lipid metabolism, and these metabolic changes might be related to hypertrophy.

Aim This study sought to investigate the effects of two different maternal high-fat diets, during gestation and lactation, on the morphology of the skeletal muscle of the adult offspring rats., Methods: Female Wistar rats were fed Control (C) or High-fat/high-caloric (HH) or High-fat/isocaloric (HI) diet during gestation and lactation. The somatic growth of the offspring was measured throughout lactation. Glucose and insulin tolerance tests were performed at PND61 and PND65, respectively. At PND70, soleus and extensor digitorum longus (EDL) muscles were removed for myofibrillar ATPase staining analysis., Key Findings: HH pups were heavier and longer at weaning but presented same body weight at PND70. No difference among groups in glucose or insulin tolerance tests was observed. In the soleus muscle, HH offspring showed increased proportion and size of type 1 fibres and reduced proportion and number with increased size of type 2A fibres. In EDL muscle, there was no difference in proportion and number of fibres. HH and HI animals presented reduced type 1 and 2A fibres size while HH animals presented increased type 2B fibres size, in EDL muscle., Significance: Maternal HH diet promoted a more oxidative profile in soleus muscle. Though, maternal high-fat/isocaloric diet influenced only fibres size. Glycolytic muscle is more resistant to maternal diet influence. These results emphasize the importance of maternal diet during the critical period of development on muscle morphology of the offspring., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Several studies have compared molecular components between red and white skeletal muscles in mammals. However, mammalian skeletal muscles are composed of mixed types of muscle fibers. In the current study, we analyzed and compared the distributions of titin, lipid, phosphate ions, and fatty acid levels in red and white muscles using a fish model ( Tilapia ), which is rich in red and white muscles, and these are well separated. Oil-red O staining showed that red muscle had more-abundant lipids than did white muscle. A time-of-flight secondary-ion mass spectrometric (TOF-SIMS) analysis revealed that red muscle possessed high levels of palmitic acid and oleic acid, but white muscle contained more phosphate ions. Moreover, elastica-van Gieson (EVG) and Mito-Tracker green FM staining showed that collagen and elastic fibers were highly, respectively, distributed in connective tissues and mitochondria in red muscle. An electron micrographic analysis indicated that red muscle had a relatively higher number of mitochondria and longer sarcomere lengths and Z-line widths, while myofibril diameters were thicker in white muscle. Myofibrillar proteins separated by SDS-PAGE showed that the major giant protein, titin, was highly expressed in white muscle than in red muscle. Furthermore, ratios of titin to myosin heavy chain (MHC) (titin/MHC) were about 1.3 times higher in white muscle than red muscle. We postulated that white muscle is fit for short and strong contractile performance due to high levels of titin and condensed sarcomeres, whereas red muscle is fit for low intensity and long-lasting activity due to high levels of lipids and mitochondria and long sarcomeres.

The pro-inflammatory cytokine interleukin-15 (IL15) and its receptor α (IL15RA) participate in the regulation of musculoskeletal function and metabolism. Deletion of the Il15ra gene in mice increases spontaneous activity, improves fatigue resistance in the glycolytic extensor digitorum longus (EDL) and protects from diet-induced obesity. In humans, IL15RA single-nucleotide polymorphisms (SNPs) have been linked to muscle strength, metabolism and performance in elite endurance athletes. Taken together, these features suggest a possible role for IL15RA in muscle mitochondrial structure and function. Here, we have investigated the consequences of loss of IL15RA on skeletal muscle fiber-type properties and mitochondrial ultrastructure. Immunostaining of the EDL for myosin heavy chain (MyHC) isoforms revealed no significant changes in fiber type. Electron microscopy (EM) analysis of the EDL indicated an overall higher mitochondria content, and increased cristae density in subsarcolemmal and A-band mitochondrial subpopulations. The higher cristae density in Il15ra -/- mitochondria was associated with higher OPA1 and cardiolipin levels. Overall, these data extend our understanding of the role of IL15RA signaling in muscle oxidative metabolism and adaptation to exercise., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Purpose: Cross-sectional studies suggest that training can increase muscle carnosine (MCarn), although longitudinal studies have failed to confirm this. A lack of control for dietary β-alanine intake or muscle fiber type shifting may have hampered their conclusions. The purpose of the present study was to investigate the effects of high-intensity interval training (HIIT) on MCarn., Methods: Twenty vegetarian men were randomly assigned to a control (CON) (n = 10) or HIIT (n = 10) group. High-intensity interval training was performed on a cycle ergometer for 12 wk, with progressive volume (6-12 series) and intensity (140%-170% lactate threshold [LT]). Muscle carnosine was quantified in whole-muscle and individual fibers; expression of selected genes (CARNS, CNDP2, ABAT, TauT, and PAT1) and muscle buffering capacity in vitro (βmin vitro) were also determined. Exercise tests were performed to evaluate total work done, V˙O2max, ventilatory thresholds (VT) and LT., Results: Total work done, VT, LT, V˙O2max, and βmin vitro were improved in the HIIT group (all P < 0.05), but not in CON (P > 0.05). MCarn (in mmol·kg dry muscle) increased in the HIIT (15.8 ± 5.7 to 20.6 ± 5.3; P = 0.012) but not the CON group (14.3 ± 5.3 to 15.0 ± 4.9; P = 0.99). In type I fibers, MCarn increased in the HIIT (from 14.4 ± 5.9 to 16.8 ± 7.6; P = 0.047) but not the CON group (from 14.0 ± 5.5 to 14.9 ± 5.4; P = 0.99). In type IIa fibers, MCarn increased in the HIIT group (from 18.8 ± 6.1 to 20.5 ± 6.4; P = 0.067) but not the CON group (from 19.7 ± 4.5 to 18.8 ± 4.4; P = 0.37). No changes in gene expression were shown., Conclusions: In the absence of any dietary intake of β-alanine, HIIT increased MCarn content. The contribution of increased MCarn to the total increase in βmin vitro appears to be small.

The aim was to determine the effects of enhanced availability of branched-chain amino acids (BCAAs; leucine, isoleucine, and valine) on ammonia detoxification to glutamine (GLN) and protein metabolism in two types of skeletal muscle under hyperammonemic conditions. Isolated soleus (SOL, slow-twitch) and extensor digitorum longus (EDL, fast-twitch) muscles from the left leg of white rats were incubated in a medium with 1 mM ammonia (NH3 group), BCAAs at four times the concentration of the controls (BCAA group) or high levels of both ammonia and BCAA (NH3 + BCAA group). The muscles from the right leg were incubated in basal medium and served as paired controls. L-[1- 14 C]leucine was used to estimate protein synthesis and leucine oxidation, and 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. We observed decreased protein synthesis and glutamate and α-ketoglutarate (α-KG) levels and increased leucine oxidation, GLN levels, and GLN release into medium in muscles in NH3 group. Increased leucine oxidation, release of branched-chain keto acids and GLN into incubation medium, and protein synthesis in EDL were observed in muscles in the BCAA group. The addition of BCAAs to medium eliminated the adverse effects of ammonia on protein synthesis and adjusted the decrease in α-KG found in the NH3 group. We conclude that (i) high levels of ammonia impair protein synthesis, activate BCAA catabolism, enhance GLN synthesis, and decrease glutamate and α-KG levels and (ii) increased BCAA availability enhances GLN release from muscles and attenuates the adverse effects of ammonia on protein synthesis and decrease in α-KG.

This study investigated the effect of arginine on skeletal muscle fiber type transformation in mice and in C2C12 myotubes. Our data showed that dietary supplementation of arginine in mice significantly up-regulated the slow myosin heavy chain (MyHC), troponin I-SS, sirtuin1 (Sirt1) and peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) protein expressions, as well as significantly down-regulated the fast MyHC protein expression. In C2C12 myotubes, arginine significantly increased the protein level of slow MyHC and the number of slow MyHC-positive cells, as well as significantly decreased the protein level of fast MyHC and the number of fast MyHC-positive cells. We also showed that arginine increased the activities of succinic dehydrogenase and malate dehydrogenase and decreased the activity of lactate dehydrogenase in mice and in C2C12 myotubes. Here we found that AMP-activated protein kinase (AMPK) was activated by arginine in mice and in C2C12 myotubes. However, inhibition of AMPK activity by compound C significantly attenuated the effects of arginine on slow MyHC and fast MyHC expressions in C2C12 myotubes. Finally, we showed that inhibition of Sirt1 expression by EX527 attenuated arginine-induced increase in the protein levels of phospho-AMPK and slow MyHC, the mRNA level of nitric oxide synthase (NOS) and the contents of NOS and NO, as well as decrease in fast MyHC protein level. Together, our findings indicated that arginine promotes skeletal muscle fiber type switching from fast-twitch to slow-twitch via Sirt1/AMPK pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Emerging evidence indicates that skeletal muscle possesses endocrine function to secret myokines. Interleukin 6 (IL-6) is a well-characterized myokine that is involved in regulation of metabolism and muscle function. Metabolism type and contractile dynamics vary in different muscle types. It is not clear, however, if IL-6 secretion differs in different muscle types. In this study, we first established an ex vivo approach to test the inducible muscle secretion. Freshly isolated muscles were incubated in Krebs solution at 37 °C with oxygen supply. Secreted IL-6 in the incubation media was measure using Western blot and ELISA assay. We first confirmed that the IL-6 release was inducible by treating the incubated muscle with a cytokine stimulant. We demonstrated that physiological temperature (37 °C) and O 2 supply were essential for the induction of IL-6 release from the incubated muscle, suggesting it is a controlled secretion rather than a spontaneous leak. Using this approach, we found that IL-6 release was only inducible from soleus muscle but not EDL muscle. We further showed that IL-6 protein level was higher in slow oxidative muscle fibers. Moreover, we showed that EDL, although lacks of IL-6 release, surely has inducible secretory function that had different secretory pattern from soleus., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Skeletal muscles are composed of heterogeneous collections of fibers with different metabolic profiles. With varied neuronal innervation and fiber-type compositions, each muscle fulfils specific functions and responds differently to stimuli and perturbations. We assessed individual fibers by mass spectrometry to dissect protein changes after loss of neuronal innervation due to section of the sciatic nerve in mice. This proteomics approach enabled us to quantify ∼600 proteins per individual soleus and EDL (extensor digitorum longus) muscle fiber. Expression of myosin heavy chain (MyHC) in individual fibers enabled clustering of specific fiber types; comparison of fibers from control and denervated muscles with the same MyHC expression revealed restricted regulation of a total of 240 proteins in type-I, -IIa, or -IIb fibers 7 days after denervation. The levels of several mitochondrial and proteasomal proteins were significantly altered, indicating rapid adaption of metabolic processes after denervation. Furthermore, we observed fiber-type-specific regulation of proteins involved in calcium ion binding and transport, such as troponins, parvalbumin, and ATP2A2, indicating marked remodeling of muscle contractility after denervation. This study provides novel insight into how different muscle fiber types remodel their proteomes during muscular atrophy.

Primary human muscle progenitor cells (hMPCs) are commonly used to understand skeletal muscle biology, including the regenerative process. Variability from unknown origin in hMPC expansion capacity occurs independently of disease, age, or sex of the donor. We sought to determine the transcript profile that distinguishes hMPC cultures with greater expansion capacity and to identify biological underpinnings of these transcriptome profile differences. Sorted (CD56+/CD29+) hMPC cultures were clustered by unbiased, K-means cluster analysis into FAST and SLOW based on growth parameters (saturation density and population doubling time). FAST had greater expansion capacity indicated by significantly reduced population doubling time (-60%) and greater saturation density (+200%), nuclei area under the curve (AUC, +250%), and confluence AUC (+120%). Additionally, FAST had fewer % dead cells AUC (-44%, P < 0.05). RNA sequencing was conducted on RNA extracted during the expansion phase. Principal component analysis distinguished FAST and SLOW based on the transcript profiles. There were 2,205 differentially expressed genes (DEgenes) between FAST and SLOW (q value ≤ 0.05); 362 DEgenes met a more stringent cut-off (q value ≤ 0.001 and 2.0 fold-change). DEgene enrichment suggested FAST (vs. SLOW) had promotion of the cell cycle, reduced apoptosis and cellular senescence, and enhanced DNA replication. Novel (RABL6, IRGM1, and AREG) and known (FOXM1, CDKN1A, Rb) genes emerged as regulators of identified functional pathways. Collectively the data suggest that variation in hMPC expansion capacity occurs independently of age and sex and is driven, in part, by intrinsic mechanisms that support the cell cycle.

Cl - channel protein 1 (ClC-1) may be important for excitability and contractility in skeletal muscle, but ClC-1 abundance has not been examined in human muscle. The aim of the present study was to examine ClC-1 abundance in human skeletal muscle, including fiber type specific differences and the effect of exercise training. A commercially available antibody was tested with positive and negative control tissue, and it recognized specifically ClC-1 in the range from 100 to 150 kDa. Abundance of ClC-1 was 38% higher ( P < 0.01) in fast twitch Type IIa muscle fibers than in slow twitch Type I. Muscle ClC-1 abundance did not change with 4 wk of training consisting of 30 min cycling at 85% of maximal heart rate (HR max ) and 3 × 30-s all out sprints or during a 7-wk training period with 10-12 × 30 s uphill cycling and 4-5 × ~4 min cycling at 90%-95% of HR max . ClC-1 abundance correlated negatively ( P < 0.01) with maximal oxygen consumption ( r = -0.552) and incremental exercise performance ( r = -0.546). In addition, trained cyclists had lower ( P < 0.01) ClC-1 abundance than lesser trained individuals. The present observations indicate that a low abundance of muscle ClC-1 may be beneficial for exercise performance, but the role of abundance and regulation of ClC-1 in skeletal muscle of humans with respect to exercise performance and trainability need to be elucidated. NEW & NOTEWORTHY Abundance of the Cl - channel protein 1 (ClC-1) chloride channel may be important for excitability and contractility in human skeletal muscle and may therefore have implications for fatigue development. In this study, we confirmed ClC-1 specificity for a commercially available antibody, and this study is first to our knowledge to determine ClC-1 protein abundance in human muscle by Western blotting. We observed that abundance of ClC-1 was higher in fast compared with slow twitch fibers and lower in trained individuals than in recreationally active.

It is currently unclear as to whether sex hormones are significantly affected by soy or whey protein consumption. Additionally, estrogenic signaling may be potentiated via soy protein supplementation due to the presence of phytoestrogenic isoflavones. Limited also evidence suggests that whey protein supplementation may increase androgenic signaling. Therefore, the purpose of this study was to examine the effects of soy protein concentrate (SPC), whey protein concentrate (WPC), or placebo (PLA) supplementation on serum sex hormones, androgen signaling markers in muscle tissue, and estrogen signaling markers in subcutaneous (SQ) adipose tissue of previously untrained, college-aged men (n = 47, 20 ± 1 yrs) that resistance trained for 12 weeks. Fasting serum total testosterone increased pre- to post-training, but more so in subjects consuming WPC (p < 0.05), whereas serum 17β-estradiol remained unaltered. SQ estrogen receptor alpha (ERα) protein expression and hormone-sensitive lipase mRNA increased with training regardless of supplementation. Muscle androgen receptor (AR) mRNA increased while ornithine decarboxylase mRNA (a gene target indicative of androgen signaling) decreased with training regardless of supplementation (p < 0.05). No significant interactions of supplement and time were observed for adipose tissue ERα/β protein levels, muscle tissue AR protein levels, or mRNAs in either tissue indicative of altered estrogenic or androgenic activity. Interestingly, WPC had the largest effect on increasing type II muscle fiber cross sectional area values (Cohen's d = 1.30), whereas SPC had the largest effect on increasing this metric in type I fibers (Cohen's d = 0.84). These data suggest that, while isoflavones were detected in SPC, chronic WPC or SPC supplementation did not appreciably affect biomarkers related to muscle androgenic signaling or SQ estrogenic signaling. The noted fiber type-specific responses to WPC and SPC supplementation warrant future research.

Neurotransmitter release during trains of activity usually involves two vesicle pools (readily releasable pool, or RRP, and reserve pool, or RP) and two exocytosis mechanisms ("full-collapse" and "kiss-and-run"). However, synaptic terminals are adapted to differing patterns of use and the relationship of these factors to enabling terminals to adapt to differing transmitter release demands is not clear. We have therefore tested their contribution to a terminal's ability to maintain release, or synaptic fatiguability in motor terminals innervating fast-twitch (fatiguable), and postural slow-twitch (fatigue-resistant) muscles. We used electrophysiological recording of neurotransmission and fluorescent dye markers of vesicle recycling to compare the effects of kinase inhibitors of varying myosin light chain kinase (MLCK) selectivity (staurosporine, wortmannin, LY294002 & ML-9) on vesicle pools, exocytosis mechanisms, and sustained neurotransmitter release, using postural-type activity train (20 Hz for 10 min) in these muscles. In both muscles, a small, rapidly depleted vesicle pool (the RRP) was inhibitor insensitive, continuing to release FM1-43, which is a marker of full-collapse exocytosis. MLCK-inhibiting kinases blocked all remaining FM1-43 loss from labelled vesicles. However, FM2-10 release only slowed, indicating continuing kiss-and-run exocytosis. Despite this, kinase inhibitors did not affect transmitter release fatiguability under normal conditions. However, augmenting release in high Ca 2+ entirely blocked the synaptic fatigue-resistance of terminals in slow-twitch muscles. Thus, full-collapse exocytosis from most vesicles (the RP) is not essential for maintaining release during a single prolonged train. However, it becomes critical in fatigue-resistant terminals during high vesicle demand.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that are widely involved in a variety of biological processes. Different skeletal muscle fiber type composition exhibits characteristic differences in functional properties and energy metabolism of skeletal muscle. However, the molecular mechanism by which miRNAs control the different type of muscle fiber formation is still not fully understood. In the present study, we characterized the role of microRNA-139-5p (miR-139-5p) in the regulation of myosin heavy chain (MyHC) isoform expression and its underlying mechanisms. Here we found that the expression of miR-139-5p was significantly higher in mouse slow-twitch muscle than in fast-twitch muscle. Overexpression of miR-139-5p downregulated the expression of MyHC I and MyHC IIa, whereas inhibition of miR-139-5p upregulated them. We also found that the levels of calcineurin (CaN), NFATc1, MEF2C and MCIP1.4, which are the components of CaN/NFAT signaling pathway that has shown to positively regulate slow fiber-selective gene expression, were notably inhibited by miR-139-5p overexpression. Furthermore, treatment of phenylephrine (PE), a α1-adrenoceptor agonist, abolished the inhibitory effect of miR-139-5p on MyHC I and MyHC IIa expression. Together, our findings indicated that the role of miR-139-5p in regulating the MyHC isoforms, especially MyHC I and MyHC IIa, may be achieved through inhibiting CaN/NFAT signaling pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aims: Existing evidence emphasize the role of mitochondrial dysfunction in sarcopenia which is revealed as loss of skeletal muscle mass and neuromuscular junction remodeling. We assessed the effect of low-intensity aerobic training along with blood flow restriction on muscle hypertrophy index, muscle-specific kinase (MuSK), a pivotal protein of the neuromuscular junction and Peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) in aged male rats., Main Methods: Animals groups were control (CTL), sham (Sh), leg blood flow restriction (BFR), exercise (Ex), sham + exercise (Sh + Ex), and BFR plus exercise (BFR + Ex) groups. The exercise groups were trained with low intensity exercise for 10 weeks. 48 h after the last training session, animals were sacrificed under anesthesia. Soleus and EDL muscles were isolated, hypertrophy index was estimated and MuSK and PGC-1α were measured by western blot method., Key Findings: Hypertrophy index enhanced in soleus and Extensor digitorum longus (EDL) muscles of BFR + Ex group (P < 0.01 versus CTL and Sh groups, and P < 0.001 versus other groups). The MuSK protein of soleus and EDL muscles increased in BFR + Ex group (P < 0.01 and P < 0.001, respectively) in comparison with CTL and Sh groups. In BFR + Ex group, the PGC-1α protein increased in both soleus and EDL (P < 0.001 compared to other groups). Also the PGC-1α of soleus muscle was higher in Ex and Sh + Ex groups versus CTL and Sh groups (P < 0.05)., Significance: Findings suggest that low endurance exercise plus BFR improves the MuSK and hypertrophy index of both slow and fast muscles of elderly rats probably through the rise of PGC-1α expression., (Copyright © 2018. Published by Elsevier Inc.)

The molecular mechanism of the failure of contractile function of skeletal muscles caused by oxidative damage to myosin in hyperthyroidism is not fully understood. Using an in vitro motility assay, we studied the effect of myosin damage caused by oxidative stress in experimental hyperthyroidism on the actin-myosin interaction and its regulation by calcium. We found that hyperthyroidism-induced oxidation of myosin is accompanied by a decrease in the sliding velocity of the regulated thin filaments in the in vitro motility assay, and this effect is increased with the duration of the pathological process.

New Findings: What is the central question of this study? Translocation of nNOSμ initiates catabolic signalling via FoxO3a and skeletal muscle atrophy during mechanical unloading. Recent evidence suggests that unloading-induced muscle atrophy and FoxO3a activation are redox sensitive. Will a mimetic of superoxide dismutase and catalase (i.e. Eukarion-134) also mitigate suppression of the Akt-mTOR pathway? What is the main finding and its importance? Eukarion-134 rescued Akt-mTOR signalling and sarcolemmal nNOSμ, which were linked to protection against the unloading phenotype, muscle fibre atrophy and partial fibre-type shift from slow to fast twitch. The loss of nNOSμ from the sarcolemma appears crucial to Akt phosphorylation and is redox sensitive, although the mechanisms remain unresolved., Abstract: Mechanical unloading stimulates rapid changes in skeletal muscle morphology, characterized by atrophy of muscle fibre cross-sectional area and a partial fibre-type shift from slow to fast twitch. Recent studies revealed that oxidative stress contributes to activation of forkhead box O3a (FoxO3a), proteolytic signalling and unloading-induced muscle atrophy via translocation of the μ-splice variant of neuronal nitric oxide synthase (nNOSμ) and activation of FoxO3a. There is limited understanding of the role of reactive oxygen species in the Akt-mammalian target of rapamycin (mTOR) pathway signalling during unloading. We hypothesized that Eukarion-134 (EUK-134), a mimetic of the antioxidant enzymes superoxide dismutase and catalase, would protect Akt-mTOR signalling in the unloaded rat soleus. Male Fischer 344 rats were separated into the following three study groups: ambulatory control (n = 11); 7 days of hindlimb unloading + saline injections (HU, n = 11); or 7 days of HU + EUK-134; (HU + EUK-134, n = 9). EUK-134 mitigated unloading-induced dephosphorylation of Akt, as well as FoxO3a, in the soleus. Phosphorylation of mTOR in the EUK-treated HU rats was not different from that in control animals. However, EUK-134 did not significantly rescue p70S6K phosphorylation. EUK-134 attenuated translocation of nNOSμ from the membrane to the cytosol, reduced nitration of tyrosine residues and suppressed upregulation of caveolin-3 and dysferlin. EUK-134 ameliorated HU-induced remodelling, atrophy of muscle fibres and the 12% increase in type II myosin heavy chain-positive fibres. Attenuation of the unloaded muscle phenotype was associated with decreased reactive oxygen species, as assessed by ethidium-positive nuclei. We conclude that oxidative stress affects Akt-mTOR signalling in unloaded skeletal muscle. Direct linkage of abrogation of nNOSμ translocation with Akt-mTOR signalling during unloading is the subject of future investigation., (© 2018 The Authors. Experimental Physiology © 2018 The Physiological Society.)

Actin is a multi-functional gene family that can be divided into muscle-type actins and non-muscle-type actins. In this study, 37 unigenes encoding actins were identified from RNA-Seq data of Pacific white shrimp, Litopenaeus vannamei. According to phylogenetic analysis, four and three cDNAs belong to cytoplasmic- and heart-type actins and were named LvActinCT and LvActinHT, respectively. 10 cDNAs belong to the slow-type skeletal muscle actins, and 18 belong to the fast-type skeletal muscle actins; they were designated LvActinSSK and LvActinFSK, respectively. Some muscle actin genes formed gene clusters in the genome. Multiple alternative transcription starts sites (ATSSs) were found for LvActinCT1. Based on the early developmental expression profile, almost all LvActins were highly expressed between the early limb bud and post-larval stages. Using LvActinSSK5 as probes, slow-type muscle was localized in pleopod muscle and superficial ventral muscle. We also found three actin genes that were down-regulated in the hemocytes of white spot syndrome virus (WSSV)- and Vibrio parahaemolyticus-infected L. vannamei. This study provides valuable information on the actin gene structure of shrimp, furthers our understanding of the shrimp muscle system and helps us develop strategies for disease control and sustainable shrimp farming.

Adult muscles have a vast adaptation capacity, enabling function switches in response to altered conditions. During intensive physical activity, disease, or aging, adult skeletal muscles change and adjust their functions. The competence to adjust varies among muscles. Muscle-specific molecular mechanisms in healthy and normal conditions could designate changes in physiologic and pathologic conditions. We generated deep mRNA-sequencing data in adult fast and slow mouse muscles, and applying paired analysis, we identified that the muscle-specific signatures are composed of half of the muscle transcriptome. The fast muscles showed a more compact gene network that is concordant with homogenous myofiber typing, compared with the pattern in the slow muscle. The muscle-specific mRNA landscape did not correlate with alternative spicing, alternative polyadenylation, or the expression of muscle transcription factor gene networks. However, we found significant correlation between the differentially expressed noncoding RNAs, microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) and their target genes. More than 25% of the genes expressed in a muscle-specific fashion were found to be targets of muscle-specific miRNAs and lncRNAs. We suggest that muscle-specific miRNAs and lncRNAs contribute to the establishment of muscle-specific transcriptomes in adult muscles.-Raz, V., Riaz, M., Tatum, Z., Kielbasa, S. M., 't Hoen, P. A. C. The distinct transcriptomes of slow and fast adult muscles are delineated by noncoding RNAs.

High-intensity interval training (HIIT) enhances skeletal muscle oxygen delivery and utilization but data are limited regarding fiber-specific adaptations in humans. We examined the effect of 18 sessions of HIIT (10 × 60-sec cycling intervals at ~90% HR max , interspersed by 60-sec of recovery) over 6 weeks on markers of microvascular density and oxidative capacity in type I and II fibers in healthy but sedentary young women (Age: 26 ± 7 years; BMI: 30 ± 4 kg·m -2 ; VO 2peak : 2.16 ± 0.45 L·m -1 ). Immunohistochemical analyses of muscle cross sections revealed a training-induced increase in capillary contacts per fiber in type I fibers (PRE: 4.38 ± 0.37 vs. POST: 5.17 ± 0.80; main effect, P < 0.05) and type II fibers (PRE: 4.24 ± 0.55 vs. POST: 4.92 ± 0.54; main effect, P < 0.05). The capillary-to-fiber ratio also increased after training in type I fibers (PRE: 1.53 ± 1.44 vs. POST: 1.88 ± 0.38; main effect, P < 0.05) and type II fibers (PRE: 1.45 ± 0.19 vs. POST: 1.76 ± 0.27; main effect, P < 0.05). Muscle oxidative capacity as reflected by the protein content of cytochrome oxidase IV also increased after training in type I fibers (PRE: 3500 ± 858 vs. POST: 4442 ± 1377 arbitrary units; main effect, P < 0.01) and type II fibers (PRE: 2632 ± 629 vs. POST: 3863 ± 1307 arbitrary units; main effect, P < 0.01). We conclude that short-term HIIT in previously inactive women similarly increases markers of capillary density and mitochondrial content in type I and type II fibers., (© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Denervation induces skeletal muscle atrophy, which primarily impairs oxidative slow twitch fibers. The underlying mechanism of this phenomenon, however, remains to be addressed. We hypothesize that denervation-induced fiber-specific atrophy may result from the distinct activities of different signaling pathways that are involved in protein synthesis and degradation in fast- and slow-twitch fibers. In this study, 1-month-old male mice were subjected to unilateral sciatic denervation for 4 days. Fast-twitch muscle extensor digitorum longus (EDL) and slow-twitch muscle soleus were collected from the denervated side and the control side of hind limbs. Total and phosphorylated protein levels of key factors of major signaling pathways in these tissues were determined using western blot assay. Our data showed that total AKT and FoxO3 protein levels were upregulated in denervated muscles as compared with control sides. Phosphorylation of AKT and FoxO3 were proportionally enhanced in denervated EDL but not soleus, indicating AKT activation drives phosphorylation of FoxO3 in EDL but not in soleus upon denervation. As a result, FoxO3-targeted atrogenes MurF1 and Atrogin1 protein abundances were reduced in denervated EDL but not altered in soleus. In consistent with this change, polyubiquitination were significantly increased in denervated soleus, but only a slight increase in ubiquitination was found in denervated EDL. Autophagy marker LC3 protein level was significantly increased in both muscle types, but in greater extent in EDL after denervation. IRS1 protein level and active ERK were reduced in both muscles upon denervation, which might contribute to the upregulation of total AKT protein level and FoxO3 abundance in EDL and soleus. Total and phosphorylated AMPK protein levels were increased in denervated soleus but not in EDL. Overall, these data reveal that the key signaling pathways that regulate protein synthesis and degradation are more sensitive in soleus than EDL in response to denervation., (© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Age-related declines in human skeletal muscle performance may be caused, in part, by decreased responsivity of muscle fibers to calcium (Ca 2+ ). This study examined the contractile properties of single vastus lateralis muscle fibers with various myosin heavy chain (MHC) isoforms (I, I/IIA, IIA and IIAX) across a range of Ca 2+ concentrations in 11 young (24.1±1.1years) and 10 older (68.8±0.8years) men and women. The normalized pCa-force curve shifted rightward with age, leading to decreased activation threshold (pCa 10 ) and/or Ca 2+ sensitivity (pCa 50 ) for all MHC isoforms examined. In older adults, the slope of the pCa-force curve was unchanged in MHC I-containing fibers (I, I/IIA), but was steeper in MHC II-containing fibers (IIA, IIAX), indicating greater cooperativity compared to young adults. At sub-maximal [Ca 2+ ], specific force was reduced in MHC I-containing fibers, but was minimally decreased in MHC IIA fibers as older adults produced greater specific forces at high [Ca 2+ ] in these fibers. Lessor pCa 50 in MHC I fibers independently predicted reduced isokinetic knee extensor power across a range of contractile velocities, suggesting that the Ca 2+ response of slow-twitch fibers contributes to whole muscle dysfunction. Our findings show that aging attenuates Ca 2+ responsiveness across fiber types and that these cellular alterations may lead to age-related reductions in whole muscle power output., (Copyright © 2017. Published by Elsevier Inc.)

Skeletal muscle is composed of four types of fibers in mammals; oxidative slow-twitch type I, oxidative fast-twitch IIA, and glycolytic fast-twitch IIB and IIX/D. In this study using C2C12 myotubes, an extract of soybean protein significantly upregulated mRNA level of myosin heavy chain 7 (Myh7), the predominant isoform expressed in oxidative slow-twitch type I and downregulated mRNA levels of Myh4, the predominant isoform expressed in glycolytic fast-twitch IIB. Similarly, its hydrolysate prepared using digestive enzyme also significantly increased Myh7 expression. In contrast, no significant change was observed in Myh4 mRNA level after the hydrolysate treatment. These findings suggest that dietary intake of the soybean protein extract may increase oxidative slow-twitch fiber in skeletal muscle.