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The biophysical environment of membrane phospholipids affects structure, function, and stability of membrane-bound proteins.1,2 Obesity can disrupt membrane lipids, and in particular, alter the activity of sarco/endoplasmic reticulum (ER/SR) Ca2+-ATPase (SERCA) to affect cellular metabolism.3-5 Recent evidence suggests that transport efficiency (Ca2+ uptake / ATP hydrolysis) of skeletal muscle SERCA can be uncoupled to increase energy expenditure and protect mice from diet-induced obesity.6,7 In isolated SR vesicles, membrane phospholipid composition is known to modulate SERCA efficiency.8-11 Here we show that skeletal muscle SR phospholipids can be altered to decrease SERCA efficiency and increase whole-body metabolic rate. The absence of skeletal muscle phosphatidylethanolamine (PE) methyltransferase (PEMT) promotes an increase in skeletal muscle and whole-body metabolic rate to protect mice from diet-induced obesity. The elevation in metabolic rate is caused by a decrease in SERCA Ca2+-transport efficiency, whereas mitochondrial uncoupling is unaffected. Our findings support the hypothesis that skeletal muscle energy efficiency can be reduced to promote protection from obesity.

Estradiol and estrogen receptor α (ERα) have been shown to be important for the maintenance of skeletal muscle strength in females; however, little is known about the roles of estradiol and ERα in male muscle. The purpose of this study was to determine if skeletal muscle ERα is required for optimal contractility in male mice. We hypothesize that reduced ERα in skeletal muscle impairs contractility in male mice. Skeletal muscle-specific knockout (skmERαKO) male mice exhibited reduced strength across multiple muscles and several contractile parameters related to force generation and kinetics compared with wild-type littermates (skmERαWT). Isolated EDL muscle-specific isometric tetanic force, peak twitch force, peak concentric and peak eccentric forces, as well as the maximal rates of force development and relaxation were 11%–21% lower in skmERαKO compared with skmERαWT mice. In contrast, isolated soleus muscles from skmERαKO mice were not affected. In vivo peak torque of the anterior crural muscles was 20% lower in skmERαKO compared with skmERαWT mice. Muscle masses, contractile protein contents, fiber types, phosphorylation of the myosin regulatory light chain, and caffeine-elicited force did not differ between muscles of skmERαKO and skmERαWT mice, suggesting that strength deficits were not due to size, composition, or calcium release components of muscle contraction. These results indicate that in male mice, reduced skeletal muscle ERα blunts contractility to a magnitude similar to that previously reported in females; however, the mechanism may be sexually dimorphic. NEW & NOTEWORTHY: We comprehensively measured in vitro and in vivo contractility of leg muscles with reduced estrogen receptor α (ERα) in male mice and reported that force generation and contraction kinetics are impaired. In contrast to findings in females, phosphorylation of myosin regulatory light chain cannot account for low force production in male skeletal muscle ERα knockout mice. These results indicate that ERα is required for optimal contractility in males and females but via sexually dimorphic means. [ABSTRACT FROM AUTHOR]

The various functions of skeletal muscle (movement, respiration, thermogenesis, etc.) require the presence of oxygen (O2). Inadequate O2 bioavailability (ie, hypoxia) is detrimental to muscle function and, in chronic cases, can result in muscle wasting. Current therapeutic interventions have proven largely ineffective to rescue skeletal muscle from hypoxic damage. However, our lab has identified a mammalian skeletal muscle that maintains proper physiological function in an environment depleted of O2. Using mouse models of in vivo hindlimb ischemia and ex vivo anoxia exposure, we observed the preservation of force production in the flexor digitorum brevis (FDB), while in contrast the extensor digitorum longus (EDL) and soleus muscles suffered loss of force output. Unlike other muscles, we found that the FDB phenotype is not dependent on mitochondria, which partially explains the hypoxia resistance. Muscle proteomes were interrogated using a discovery-based approach, which identified significantly greater expression of the transmembrane glucose transporter GLUT1 in the FDB as compared to the EDL and soleus. Through loss-and-gain-of-function approaches, we determined that GLUT1 is necessary for the FDB to survive hypoxia, but overexpression of GLUT1 was insufficient to rescue other skeletal muscles from hypoxic damage. Collectively, the data demonstrate that the FDB is uniquely resistant to hypoxic insults. Defining the mechanisms that explain the phenotype may provide insight towards developing approaches for preventing hypoxia-induced tissue damage. Graphical Abstract Mouse models of in vivo hindlimb ischemia and ex vivo anoxia exposure were employed to demonstrate the ability of the FDB to function without oxygen. Discovery-based proteomics identified significantly higher expression of the glucose transporter GLUT1 in the FDB as compared to the hypoxia-sensitive EDL and soleus muscles. Loss-and-gain-of-function approaches revealed that GLUT1 is necessary for the FDB phenotype, but insufficient to rescue other skeletal muscles from hypoxic damage. [ABSTRACT FROM AUTHOR]

Assessing contractile function of skeletal muscle in murine models is a commonly employed laboratory technique that investigators utilize to measure the impact of genetic manipulations, drug efficacy, or other therapeutic interventions. Often overlooked is the potential for the strain of the mouse to influence the functional properties of the skeletal muscle. Thus, we sought to characterize commonly assessed isometric force measures in the hindlimb muscles across a variety of mouse strains. Using 6-8-week-old male mice, we measured isometric force, fatigue susceptibility, relaxation kinetics, muscle mass, myofiber cross-sectional area, and fiber type composition of the extensor digitorum longus (EDL) and soleus muscles in C57BL/6NJ, BALB/cJ, FVB/NJ, C57BL/6J, and C57BL/10 mice. The data demonstrate both unique differences and a number of similarities between both muscles in the various genetic backgrounds of mice. Soleus muscle specific force (i.e., force per unit size) exhibited higher variation across strains while specific force of the EDL muscle exhibited minimal variation. In contrast, absolute force differed only in a few mouse strains whereas analysis of muscle morphology revealed many distinctions when compared across all the groups. Collectively, the data suggest that the strain of the mouse can potentially influence the measured biological outcome and may possibly promote a synergistic effect with any genetic manipulation or therapeutic intervention. Thus, it is critical for the investigator to carefully consider the genetic background of the mouse used in the experimental design and precisely document the strain of mouse employed during publication. [ABSTRACT FROM AUTHOR]

We sought to delineate the effects of acute and chronic exercise on the regulation of intracellular nitric oxide (N[O.sub.i]) production in putative endothelial progenitor cells (EPCs). Putative EPC colony-forming units (CFU-EC) were cultured from blood drawn before and after 30 min of treadmill exercise at 75% of maximal oxygen uptake in active (n = 8) and inactive (n = 8) men. CFU-EC were similar between groups at baseline, but increased after exercise in active men only (P = 0.04). CFU-EC expressed Dower NADPH oxidase subunit [gp91.sup.phox] mRNA and elevated endothelial nitric oxide synthase mRNA in active relative to inactive men at baseline (P < 0.05). Acute exercise reduced [gp91.sup.phox] mRNA in CFU-EC of both groups (P < 0.05), whereas [p47.sup.phox] mRNA levels were reduced in the inactive group only (P = 0.02). There were no differences between groups or with acute exercise in xanthine oxidase, superoxide dismutase isoforms, or gluthathione peroxidase-1 mRNA levels. N[O.sub.i] was significantly greater in CFU-EC of active men at baseline (P = 0.004). N[O.sub.i] increased in CFU-EC of inactive men with acute exercise, and in vitro experiments with apocynin indicated the increased N[O.sub.i] production was caused by suppression of NADPH oxidase. However, the increases in N[O.sub.i] with the different treatments in the inactive group did not reach the baseline levels in the active group (P < 0.05). We conclude that acute exercise increases N[O.sub.i] in cells generated by the CFU-EC assay through an NADPH oxidase-inhibition mechanism in sedentary men. However, differences due to chronic exercise must involve additional factors. Our findings support exercise as a means to improve putative EPC function and suggest a novel mechanism that may explain this effect. physical activity; angiogenesis; oxidative stress doi: 10.1152/ajpheart.00347.2009.

Currently, the repertoire of cellular and molecular pathways that control skeletal muscle atrophy and hypertrophy are not well defined. It is possible that intracellular regulatory signaling pathways are active at different times during the muscle hypertrophy process. The hypothesis of the given experiments was that cellular signals related to protein translation would be active at early time points of skeletal muscle regrowth, whereas transcriptional signals would be active at later time points of skeletal muscle regrowth. The phosphorylation status of p38 MAPK and JNK increased at the end of limb immobilization but returned to control values at recovery day 3. Transient increases in phosphorylation and in protein concentration occurred during recovery of soleus muscle mass. Phosphorylation of Akt, [p70.sup.S6k], and signal transducer and activator of transcription 3 (STAT3) peaked on recovery day 3 compared with day 0. Glycogen synthase kinase (GSK)-3[beta] phosphorylation was increased on the sixth and fifteenth recovery day. In addition, transient peaks in seven protein concentrations occurred at different times of recovery: STAT3, calcineurin A (CaNA), CaNB, and [beta]4E-BP1 protein concentrations peaked on the third recovery day; [p70.sup.S6k], STAT3, Akt, and GSK3-[beta] peaked on the sixth recovery day; and GSK3-[beta] peaked on the fifteenth recovery day. The apexes of STAT3 and GSK3-[beta] protein concentrations remained elevated for two recovery time points. Thus the time course of increase in molecules of signaling pathways differed as the young rat soleus muscle regrew from an atrophied state. skeletal muscle; hypertrophy; rehabilitation; atrophy

The purpose of the current study was to examine IGFBP-3, -4, and -5 mRNA and protein expression levels as a function of muscle type, age, and regrowth from an immobilization-induced atrophy in Fischer 344 x Brown Norway rats. IGFBP-3 mRNA expression in the 4-mo-old animals was significantly higher in the red and white portions of the gastrocnemius muscle compared with the soleus muscle. However, there were no significant differences in IGFBP-3 mRNA expression among any of the muscle groups in the 30-mo-old animals. There were no significant differences in IGFBP-5 mRNA expression in any of the tauscie groups, whereas in the 30-mo-old animals there was significantly less IGFBP-5 mRNA expression in the white gastrocnemius compared with the red gastrocnemius muscles. Although IGFBP-3 and -5 proteins were detected in the type I soleus muscle with Western blot analyses, no detection was observed in the type II red and white portions of the gastrocnemius muscle. Aging from adult (18 mo) to old animals (30 mo) was associated with decreases in IGFBP-3 mRNA and protein and IGFBP-5 protein only in the soleus muscle. After 10 days of recovery from 10 days of hindlimb immobilization, IGFBP-3 mRNA and protein increased in soleus muscles from young (4-mo) rats; however, only IGFBP-3 protein increased in the old (30-mo) rats. Whereas there were no changes in IGFBP-5 mRNA expression during recovery, IGFBP-5 protein in the 10-day-recovery soleus muscle did increase in the young, but not in the old, rats. Because one of the functions of IGFBPs is to modulate IGF-I action on muscle size and phenotype, it is hypothesized that IGFBP-3 and -5 proteins may have potential modulatory roles in type I fiber-dominated muscles, aging, and regrowth from atrophy. insulin-like growth factor-binding protein; insulin-like growth factor I; growth; hypertrophy; fiber type

Extracellular vesicles (EVs) are biomarkers and modifiers of human disease. EVs secreted by insulin-responsive tissues like skeletal muscle (SkM) and white adipose tissue (WAT) contribute to metabolic health and disease but the relative abundance of EVs from these tissues has not been directly examined. Human Protein Atlas data and directly measuring EV secretion in mouse SkM and WAT using an ex vivo tissue explant model confirmed that SkM tissue secretes more EVs than WAT. Differences in EV secretion between SkM and WAT were not due to SkM contraction but may be explained by differences in tissue metabolic capacity. We next examined how many EVs secreted from SkM tissue ex vivo and in vivo are myofiber-derived. To do this, a SkM myofiber-specific dual fluorescent reporter mouse was created. Spectral flow cytometry revealed that SkM myofibers are a major source of SkM tissue-derived EVs ex vivo and EV immunocapture indicates that 5% of circulating tetraspanin-positive EVs are derived from SkM myofibers in vivo. Our findings demonstrate that 1) SkM secretes more EVs than WAT, 2) many SkM tissue EVs are derived from SkM myofibers, and 3) SkM myofiber-derived EVs reach the circulation in vivo. These findings advance our understanding of EV secretion between metabolically active tissues and provide direct evidence that SkM myofibers secrete EVs that can reach the circulation in vivo. [ABSTRACT FROM AUTHOR]

The importance of defining sex differences across various biological and physiological mechanisms is more pervasive now than it has been over the past 15-20 years. As the muscle biology field pushes to identify small molecules and interventions to prevent, attenuate, or even reverse muscle wasting, we must consider the effect of sex as a biological variable. It should not be assumed that a therapeutic will affect males and females with equal efficacy or equivalent target affinities under conditions where muscle wasting is observed. With that said, it is not surprising to find that we have an unclear or even a poor understanding of the effects of sex or sex hormones on muscle wasting conditions. Although recent investigations are beginning to establish experimental approaches that will allow investigators to assess the impact of sex-specific hormones on muscle wasting, the field still needs rigorous scientific tools that will allow the community to address critical hypotheses centered around sex hormones. The focus of this review is on female sex hormones, specifically estrogens, and the roles that these hormones and their receptors play in skeletal muscle wasting conditions. With the overall review goal of assembling the current knowledge in the area of sexual dimorphism driven by estrogens with an effort to provide insights to interested physiologists on necessary considerations when trying to assess models for potential sex differences in cellular and molecular mechanisms of muscle wasting. [ABSTRACT FROM AUTHOR]

To determine whether changes in glycogen synthase kinase-3[beta] (GSK-3[beta]) phosphorylation contribute to muscle hypertrophy, we delineated the effects of GSK-3[beta] activity on [C.sub.2][C.sub.12] myotube size. We also examined possible insulin-like growth factor I (IGF-I) signaling of NFAT (nuclear factors of activated T cells)-inducible gene activity and possible modulation of NFAT activation by GSK-3[beta]. Application of IGF-I (250 ng/ml) or LiCl (10 mM) alone (i.e., both inhibit GSK-3[beta] activity) increased the area of [C.sub.2][C.sub.12] myotubes by 80 and 85%, respectively. The application of IGF-I (250 ng/ml) elevated GSK-3[beta] phosphorylation and reduced GSK-3[beta] kinase activity by ~800% and ~25%, respectively. LY-294002 (100 [micro] M) and wortmannin (150 [micro]M), specific inhibitors of phosphatidylinositol 3'-kinase, attenuated IGF-I-induced GSK-3[beta] phosphorylation by 67 and 92%, respectively. IGF-I suppressed the kinase activity of GSK-3[beta]. IGF-I (250 ng/ml), but not LiCl (10 mM), induced an increase in NFAT-activated luciferase reporter activity. Cotransfection of a constitutively active GSK-3[beta] (cGSK-3[beta]) inhibited the induction by IGF-I of NFAT-inducible reporter activity. LiCl, which inhibits GSK3[beta], removed the block by cGSK-3[beta] on IGF-I-inducible NFAT-responsive reporter gene activity. These data suggest that the IGF-I-induced increase in skeletal myotube size is signaled, in part, through the inhibition of GSK-3[beta]. skeletal muscle; signaling; glycogen synthase kinase-3[beta]; insulin-like growth factor I; nuclear factors of activated T cells

Multiple signaling pathways mediate LIF-induced skeletal muscle satellite cell proliferation. Am J Physiol Cell Physiol 283: C204-C211, 2002. First published March 6, 2002; 10.1152/ ajpcell.00574.2001.--There are many known growth factors/ cytokines that induce skeletal muscle satellite cell proliferation. Currently, the signaling mechanisms in which these growth factors/cytokines activate satellite cell proliferation are not completely understood. Here, we sought to determine signaling mechanisms by which leukemia inhibitory factor (LIF) induces satellite cell proliferation in culture. First, we confirmed that LIF induces proliferation of [C.sub.2][C.sub.12] immortalized myoblasts and cultured primary rat satellite cells. In addition, we also found that this increase in proliferation can be inhibited by incubation of the cells in tyrphostin AG 490, a specific inhibitor of Janus-activated kinase (JAK) 2 activity. Furthermore, we also found that incubation of the cells at various time points with LIF (10 ng/ml) induces a significant, transient increase in JAK2 phosphorylation, signal transducers and activators of transcription (STAT3) phosphorylation, and STAT3 transcriptional activity. Increases in the STAT3-sensitive endogenous SOC3 protein followed these transient increases in STAT3 activation. In addition, AG 490 inhibited the increase in STAT3 phosphorylation. Finally, LIF did not change the phosphorylation status of extracellular signal-regulated protein kinase (ERK)1/2 or affect the phosphorylation status of Akt/protein kinase B. However, LY-294002, an inhibitor of phosphoinositide 3-kinase, blocked LIF-induced proliferation of satellite cells. These data suggest that LIF induces satellite cell proliferation by activation of the JAK2-STAT3 signaling pathway, suggesting that this may be an important pathway in muscle growth and/or hypertrophy. cytokine; mitogen; Janus-activated kinase; leukemia inhibitory factor; signal transducers and activators of transcription

This article discusses the relationship between insulin-like growth factor, cell proliferation, muscle growth, and muscular atrophy prevention.

Breast Cancer gene 1 (BRCA1) is a large, multifunctional protein that regulates a variety of mechanisms in multiple different tissues. Our work established that Brca1 is expressed in skeletal muscle and localizes to the mitochondria and nucleus. Here, we propose BRCA1 expression is critical for the maintenance of force production and mitochondrial respiration in skeletal muscle. [ABSTRACT FROM AUTHOR]

The effects of physical activity on the uptake and release of calcium ions from the sarcoplasmic reticulum (SR) are investigated using isolated frog sartorius muscle preparations. Activities are classified as constant fatigue or constant duration. Results from constant fatigue experiments reveal that the depression of SR function is independent of the activity protocol. However, when there is imposition of a constant duration in activity, SR function correlate well with force reduction.

Ozone (O3) is a criteria air pollutant known to increase the morbidity and mortality of cardiopulmonary diseases. This occurs through a pulmonary inflammatory response characterized by increased recruitment of immune cells into the airspace, pro-inflammatory cytokines, and pro-inflammatory lipid mediators. Recent evidence has demonstrated sex-dependent differences in the O3-induced pulmonary inflammatory response. However, it is unknown if this dimorphic response is evident in pulmonary lipid mediator metabolism. We hypothesized that there are sex-dependent differences in lipid mediator production following acute O3 exposure. Male and female C57BL/6J mice were exposed to 1 part per million O3 for 3 h and were necropsied at 6 or 24 h following exposure. Lung lavage was collected for cell differential and total protein analysis, and lung tissue was collected for mRNA analysis, metabololipidomics, and immunohistochemistry. Compared with males, O3-exposed female mice had increases in airspace neutrophilia, neutrophil chemokine mRNA, pro-inflammatory eicosanoids such as prostaglandin E2, and specialized pro-resolving mediators (SPMs), such as resolvin D5 in lung tissue. Likewise, precursor fatty acids (arachidonic and docosahexaenoic acid; DHA) were increased in female lung tissue following O3 exposure compared with males. Experiments with ovariectomized females revealed that loss of ovarian hormones exacerbates pulmonary inflammation and injury. However, eicosanoid and SPM production were not altered by ovariectomy despite depleted pulmonary DHA concentrations. Taken together, these data indicate that O3 drives an increased pulmonary inflammatory and bioactive lipid mediator response in females. Furthermore, ovariectomy increases susceptibility to O3-induced pulmonary inflammation and injury, as well as decreases pulmonary DHA concentrations. [ABSTRACT FROM AUTHOR]