Exercise-Induced Skeletal Muscle Adaptations Alter the Activity of Adipose Progenitor Cells

Exercise decreases adiposity and improves metabolic health; however, the physiological and molecular underpinnings of these phenomena remain unknown. Here, we investigate the effect of endurance training on adipose progenitor lineage commitment. Using mice with genetically labeled adipose progenitors, we show that these cells react to exercise by decreasing their proliferation and differentiation potential. Analyses of mouse models that mimic the skeletal muscle adaptation to exercise indicate that muscle, in a non-autonomous manner, regulates adipose progenitor homeostasis, highlighting a role for muscle-derived secreted factors. These findings support a humoral link between skeletal muscle and adipose progenitors and indicate that manipulation of adipose stem cell function may help address obesity and diabetes.     

Hypoxia Impairs Muscle Function and Reduces Myotube Size in Tissue Engineered Skeletal Muscle

Contemporary tissue engineered skeletal muscle models display a high degree of physiological accuracy compared with native tissue, and therefore may be excellent platforms to understand how various pathologies affect skeletal muscle. Chronic obstructive pulmonary disease (COPD) is a lung disease which causes tissue hypoxia and is characterized by muscle fiber atrophy and impaired muscle function. In the present study we exposed engineered skeletal muscle to varying levels of oxygen (O₂; 21–1%) for 24 h in order to see if a COPD like muscle phenotype could be recreated in vitro, and if so, at what degree of hypoxia this occurred. Maximal contractile force was attenuated in hypoxia compared to 21% O₂; with culture at 5% and 1% O₂ causing the most pronounced effects with 62% and 56% decrements in force, respectively. Furthermore at these levels of O₂, myotubes within the engineered muscles displayed significant atrophy which was not seen at higher O₂ levels. At the molecular level we observed increases in mRNA expression of MuRF‐1 only at 1% O₂ whereas MAFbx expression was elevated at 10%, 5%, and 1% O₂. In addition, p70S6 kinase phosphorylation (a downstream effector of mTORC1) was reduced when engineered muscle was cultured at 1% O₂, with no significant changes seen above this O₂ level. Overall, these data suggest that engineered muscle exposed to O₂ levels of ≤5% adapts in a manner similar to that seen in COPD patients, and thus may provide a novel model for further understanding muscle wasting associated with tissue hypoxia. J. Cell. Biochem. 118: 2599–2605, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

Dihydropyridine Receptor-Ryanodine Receptor Uncoupling in Aged Skeletal Muscle

The mechanisms underlying skeletal muscle functional impairment and structural changes with advanced age are only partially understood. In the present study, we support and expand our theory about alterations in sarcolemmal excitation-sarcoplasmic reticulum Ca²⁺ release-contraction uncoupling as a primary skeletal muscle alteration and major determinant of weakness and fatigue in mammalian species including humans. To test the hypothesis that the number of RYR1 (ryanodine receptor) uncoupled to DHPR (dihydropyridine receptor) increases with age, we performed high-affinity ligand binding studies in soleus, extensor digitorum longus (EDL) and in a pool of several skeletal muscles consisting of a mixture of fast- and slow-twitch muscle fibers in middle-aged (14-month) and old (28-months) Fisher 344 Brown Norway F1 hybrids rats. The number of DHPR, RYR1, the coupling between both receptors expressed as the DHPR/RYR1 maximum binding capacity, and their dissociation constant for high-affinity ligands were measured. The DHPR/RYR1 ratio was significantly reduced in the three groups of muscles (pool: 1.03 ± 0.15 and 0.80 ± 0.11, soleus: 0.44 ± 0.12 and 0.26 ± 0.10, and EDL: 0.95 ± 0.14 and 0.68 ± 0.10, for middle-aged and old muscles, respectively). These data support the concept that DHPR-RYR1 uncoupling results in alterations in the voltage-gated sarcoplasmic reticulum Ca²⁺ release mechanism, decreases in myoplasmic Ca²⁺ elevation in response to sarcolemmal depolarization, reduced Ca²⁺ supply to contractile proteins and reduced contraction force with aging. Received: 26 August 1996/Revised: 30 December 1996     

Elevated Mitochondrial Oxidative Stress Impairs Metabolic Adaptations to Exercise in Skeletal Muscle

Mitochondrial oxidative stress is a complex phenomenon that is inherently tied to energy provision and is implicated in many metabolic disorders. Exercise training increases mitochondrial oxidative capacity in skeletal muscle yet it remains unclear if oxidative stress plays a role in regulating these adaptations. We demonstrate that the chronic elevation in mitochondrial oxidative stress present in Sod2 ^+/- mice impairs the functional and biochemical mitochondrial adaptations to exercise. Following exercise training Sod2 ^+/- mice fail to increase maximal work capacity, mitochondrial enzyme activity and mtDNA copy number, despite a normal augmentation of mitochondrial proteins. Additionally, exercised Sod2 ^+/- mice cannot compensate for their higher amount of basal mitochondrial oxidative damage and exhibit poor electron transport chain complex assembly that accounts for their compromised adaptation. Overall, these results demonstrate that chronic skeletal muscle mitochondrial oxidative stress does not impact exercise induced mitochondrial biogenesis, but impairs the resulting mitochondrial protein function and can limit metabolic plasticity.     

Control of Cell Volume in Skeletal Muscle

Regulation of cell volume is a fundamental property of all animal cells and is of particular importance in skeletal muscle where exercise is associated with a wide range of cellular changes that would be expected to influence cell volume. These complex electrical, metabolic and osmotic changes, however, make rigorous study of the consequences of individual factors on muscle volume difficult despite their likely importance during exercise. Recent charge-difference modelling of cell volume distinguishes three major aspects to processes underlying cell volume control: (i) determination by intracellular impermeant solute; (ii) maintenance by metabolically dependent processes directly balancing passive solute and water fluxes that would otherwise cause cell swelling under the influence of intracellular membrane-impermeant solutes; and (iii) volume regulation often involving reversible short-term transmembrane solute transport processes correcting cell volumes towards their normal baselines in response to imposed discrete perturbations. This review covers, in turn, the main predictions from such quantitative analysis and the experimental consequences of comparable alterations in extracellular pH, lactate concentration, membrane potential and extracellular tonicity. The effects of such alterations in the extracellular environment in resting amphibian muscles are then used to reproduce the intracellular changes that occur in each case in exercising muscle. The relative contributions of these various factors to the control of cell volume in resting and exercising skeletal muscle are thus described.     

Oxidative Stress Induces Caveolin 1 Degradation and Impairs Caveolae Functions in Skeletal Muscle Cells

Increased level of oxidative stress, a major actor of cellular aging, impairs the regenerative capacity of skeletal muscle and leads to the reduction in the number and size of muscle fibers causing sarcopenia. Caveolin 1 is the major component of caveolae, small membrane invaginations involved in signaling and endocytic trafficking. Their role has recently expanded to mechanosensing and to the regulation of oxidative stress-induced pathways. Here, we increased the amount of reactive oxidative species in myoblasts by addition of hydrogen peroxide (H₂O₂) at non-toxic concentrations. The expression level of caveolin 1 was significantly decreased as early as 10 min after 500 μM H₂O₂ treatment. This reduction was not observed in the presence of a proteasome inhibitor, suggesting that caveolin 1 was rapidly degraded by the proteasome. In spite of caveolin 1 decrease, caveolae were still able to assemble at the plasma membrane. Their functions however were significantly perturbed by oxidative stress. Endocytosis of a ceramide analog monitored by flow cytometry was significantly diminished after H₂O₂ treatment, indicating that oxidative stress impaired its selective internalization via caveolae. The contribution of caveolae to the plasma membrane reservoir has been monitored after osmotic cell swelling. H₂O₂ treatment increased membrane fragility revealing that treated cells were more sensitive to an acute mechanical stress. Altogether, our results indicate that H₂O₂ decreased caveolin 1 expression and impaired caveolae functions. These data give new insights on age-related deficiencies in skeletal muscle.     

Glutaric Acid Administration Impairs Energy Metabolism in Midbrain and Skeletal Muscle of Young Rats

A genetic mice model of glutaric acidemia type I (GAI) has recently been developed, however affected animals do not develop the striatal damage characteristic of patients with this disorder. Therefore, the initial aim of the present work was to induce high glutaric acid (GA) concentrations in rat brain similar to those found in GAI patients through subcutaneous injection of GA. High brain GA concentrations (up to 0.60 μmol/g ≅ 0.60mM) were achieved by a single subcutaneous injection of saline-buffered GA (5 μmol/g body weight) to Wistar rats of 7–22 days of life. GA brain levels were about 10-fold lower than in plasma and 5-fold lower than in skeletal and cardiac muscles, indicating that the permeability of the blood brain barrier to GA is low. We also aimed to use this model to investigate neurochemical parameters in the animals. Thus, we evaluated the effect of this model on energy metabolism parameters in midbrain, in which the striatum is localized, as well as in peripheral tissues (skeletal and cardiac muscles) of 22-day-old rats. Control rats were treated with saline in the same volumes. We verified that CO₂ production from glucose was not altered in midbrain of rats treated with GA, indicating a normal functioning of the tricarboxylic acid cycle. Creatine kinase activity was also not changed in midbrain, skeletal and cardiac muscles. In contrast, complex I–III activity of the respiratory chain was inhibited in midbrain (25%), while complexes I–III (25%) and II–III (15%) activities were reduced in skeletal muscle, with no alterations found in cardiac muscle. These data indicate that GA administration moderately impairs cellular energy metabolism in midbrain and skeletal muscle of young rats.     

Lifelong Physical Activity Prevents Aging-Associated Insulin Resistance in Human Skeletal Muscle Myotubes via Increased Glucose Transporter Expression

Both aging and physical inactivity are associated with increased development of insulin resistance whereas physical activity has been shown to promote increased insulin sensitivity. Here we investigated the effects of physical activity level on aging-associated insulin resistance in myotubes derived from human skeletal muscle satellite cells. Satellite cells were obtained from young (22 yrs) normally active or middle-aged (56.6 yrs) individuals who were either lifelong sedentary or lifelong active. Both middle-aged sedentary and middle-aged active myotubes had increased p21 and myosin heavy chain protein expression. Interestingly MHCIIa was increased only in myotubes from middle-aged active individuals. Middle-aged sedentary cells had intact insulin-stimulated Akt phosphorylation however, the same cell showed ablated insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane. On the other hand, middle-aged active cells retained both insulin-stimulated increases in glucose uptake and GLUT4 translocation to the plasma membrane. Middle-aged active cells also had significantly higher mRNA expression of GLUT1 and GLUT4 compared to middle-aged sedentary cells, and significantly higher GLUT4 protein. It is likely that physical activity induces a number of stable adaptations, including increased GLUT4 expression that are retained in cells ex vivo and protect, or delay the onset of middle-aged-associated insulin resistance. Additionally, a sedentary lifestyle has an impact on the metabolism of human myotubes during aging and may contribute to aging-associated insulin resistance through impaired GLUT4 localization.     

Self-Renewal of the Adult Skeletal Muscle Satellite Cell

The concept of the stem cell has evolved in dynamic systems such as those involved inembryonic development and, in the adult, in tissues such as blood and skin which arecontinuously renewed. It has proved difficult to establish whether stem cell mechanismsunderlie the maintenance of the more stable tissues that form the majority of the adultbody. We have investigated skeletal muscle, a low-turnover and largely postmitotictissue which nevertheless maintains a remarkable capacity to regenerate itself followinginjury. The contractile units of muscle are myofibers, elongated syncytial cells eachcontaining many hundreds of postmitotic myonuclei. Satellite cells are resident beneaththe basal lamina of myofibers and function as myogenic precursors during muscleregeneration. We have recently demonstrated that as few as seven Pax7+ satellite cellsassociated with one myofiber can regenerate a hundred or more new myofiberscontaining thousands of myonuclei. Satellite cells also undergo self-renewal, givingthem the ability to participate in multiple rounds of injury-induced regeneration. Thesatellite cell may thus serve as a prototype for stem cell function in stable adult tissues: atissue-specific progenitor which is normally quiescent but which has self-renewalproperties similar to those of better known stem cells.     

Increased Plin2 Expression in Human Skeletal Muscle Is Associated with Sarcopenia and Muscle Weakness

Human aging is associated with a progressive loss of muscle mass and strength and a concomitant fat accumulation in form of inter-muscular adipose tissue, causing skeletal muscle function decline and immobilization. Fat accumulation can also occur as intra-muscular triglycerides (IMTG) deposition in lipid droplets, which are associated with perilipin proteins, such as Perilipin2 (Plin2). It is not known whether Plin2 expression changes with age and if this has consequences on muscle mass and strength. We studied the expression of Plin2 in the vastus lateralis (VL) muscle of both healthy subjects and patients affected by lower limb mobility limitation of different age. We found that Plin2 expression increases with age, this phenomenon being particularly evident in patients. Moreover, Plin2 expression is inversely correlated with quadriceps strength and VL thickness. To investigate the molecular mechanisms underpinning this phenomenon, we focused on IGF-1/p53 network/signalling pathway, involved in muscle physiology. We found that Plin2 expression strongly correlates with increased p53 activation and reduced IGF-1 expression. To confirm these observations made on humans, we studied mice overexpressing muscle-specific IGF-1, which are protected from sarcopenia. These mice resulted almost negative for the expression of Plin2 and p53 at two years of age. We conclude that fat deposition within skeletal muscle in form of Plin2-coated lipid droplets increases with age and is associated with decreased muscle strength and thickness, likely through an IGF-1- and p53-dependent mechanism. The data also suggest that excessive intramuscular fat accumulation could be the initial trigger for p53 activation and consequent loss of muscle mass and strength.     

Neural Control of Myofibrillar ATPase Activity in Rat Skeletal Muscle

THE limb muscles of mammals such as the cat and rat can be divided into the fast-twitch muscles and the slow-twitch muscles. While the absolute contraction speeds vary from species to species the isometric twitch time (the time taken from the start of contraction until the instant of peak tension development) of a slow-twitch muscle is always about three times longer than the isometric twitch time of a fast-twitch muscle. Thus, at 37° C, the isometric twitch time of cat soleus muscle (a slow-twitch muscle) is approximately 70 ms while the isometric twitch time of the flexor hallucis longus muscle (a fast-twitch muscle) is approximately 20 ms. In the rat, the contraction times of the corresponding muscles would be of the order of 36 ms and 12 ms respectively.     

Differentiation-dependent PTPIP51 Expression in Human Skeletal Muscle Cell Culture

Protein tyrosine phosphatase-interacting protein 51 (PTPIP51) expression was analyzed in proliferating and differentiating human myogenic cells cultured in vitro. Satellite cell cultures derived from four different individuals were used in this study. To analyze the expression of PTPIP51, myoblasts were cultured under conditions promoting either proliferation or differentiation. In addition, further differentiation of already-differentiated myobtubes was inhibited by resubmitting the cells to conditions promoting proliferation. PTPIP51 protein and mRNA were investigated in samples taken at defined time intervals by immunostaining, immunoblotting, in situ hybridization, and PCR. Image analyses of fluorescence immunostainings were used to quantify PTPIP51 in cultured myoblasts and myotubes. Myoblasts grown in the presence of epidermal and fibroblast growth factors (EGF and FGF), both promoting proliferation, expressed PTPIP51 on a basic level. Differentiation to multinuclear myotubes displayed a linear increase in PTPIP51 expression. The rise in PTPIP51 protein was paralleled by an augmented expression of muscle-specific proteins, namely, sarcoplasmic reticulum Ca²⁺ ATPase and myosin heavy-chain protein, both linked to a progressive state of myotubal differentiation. This differentiation-induced increase in PTPIP51 was partly reversible by resubmission of differentiated myotubes to conditions boosting proliferation. The results clearly point toward a strong association between PTPIP51 expression and differentiation in human muscle cells. (J Histochem Cytochem 57:425–435, 2009)     

Loss of the Inducible Hsp70 Delays the Inflammatory Response to Skeletal Muscle Injury and Severely Impairs Muscle Regeneration

Skeletal muscle regeneration following injury is a highly coordinated process that involves transient muscle inflammation, removal of necrotic cellular debris and subsequent replacement of damaged myofibers through secondary myogenesis. However, the molecular mechanisms which coordinate these events are only beginning to be defined. In the current study we demonstrate that Heat shock protein 70 (Hsp70) is increased following muscle injury, and is necessary for the normal sequence of events following severe injury induced by cardiotoxin, and physiological injury induced by modified muscle use. Indeed, Hsp70 ablated mice showed a significantly delayed inflammatory response to muscle injury induced by cardiotoxin, with nearly undetected levels of both neutrophil and macrophage markers 24 hours post-injury. At later time points, Hsp70 ablated mice showed sustained muscle inflammation and necrosis, calcium deposition and impaired fiber regeneration that persisted several weeks post-injury. Through rescue experiments reintroducing Hsp70 intracellular expression plasmids into muscles of Hsp70 ablated mice either prior to injury or post-injury, we confirm that Hsp70 optimally promotes muscle regeneration when expressed during both the inflammatory phase that predominates in the first four days following severe injury and the regenerative phase that predominates thereafter. Additional rescue experiments reintroducing Hsp70 protein into the extracellular microenvironment of injured muscles at the onset of injury provides further evidence that Hsp70 released from damaged muscle may drive the early inflammatory response to injury. Importantly, following induction of physiological injury through muscle reloading following a period of muscle disuse, reduced inflammation in 3-day reloaded muscles of Hsp70 ablated mice was associated with preservation of myofibers, and increased muscle force production at later time points compared to WT. Collectively our findings indicate that depending on the nature and severity of muscle injury, therapeutics which differentially target both intracellular and extracellular localized Hsp70 may optimally preserve muscle tissue and promote muscle functional recovery.     

Increased Store-Operated Ca Entry in Skeletal Muscle with Reduced Calsequestrin-1 Expression

Store-operated Ca²⁺ entry (SOCE) contributes to Ca²⁺ handling in normal skeletal muscle function, as well as the progression of muscular dystrophy and sarcopenia, yet the mechanisms underlying the change in SOCE in these states remain unclear. Previously we showed that calsequestrin-1 (CSQ1) participated in retrograde regulation of SOCE in cultured skeletal myotubes. In this study, we used small-hairpin RNA to determine whether knockdown of CSQ1 in adult mouse skeletal muscle can influence SOCE activity and muscle function. Small-hairpin RNA against CSQ1 was introduced into flexor digitorum brevis muscles using electroporation. Transfected fibers were isolated for SOCE measurements using the Mn²⁺ fluorescence-quenching method. At room temperature, the SOCE induced by submaximal depletion of the SR Ca²⁺ store was significantly enhanced in CSQ1-knockdown muscle fibers. When temperature of the bathing solution was increased to 39°C, CSQ1-knockdown muscle fibers displayed a significant increase in Ca²⁺ permeability across the surface membrane likely via the SOCE pathway, and a corresponding elevation in cytosolic Ca²⁺ as compared to control fibers. Preincubation with azumolene, an analog of dantrolene used for the treatment of malignant hyperthermia (MH), suppressed the elevated SOCE in CSQ1-knockdown fibers. Because the CSQ1-knockout mice develop similar MH phenotypes, this inhibitory effect of azumolene on SOCE suggests that elevated extracellular Ca²⁺ entry in skeletal muscle may be a key factor for the pathophysiological changes in intracellular Ca²⁺ signaling in MH.     

A New Chelation Method for Determining ATPase Activity in Skeletal Muscle

Traditional methods for visualizing ATPase in sections use heavy metals that generate visible metal salfide products. These methods use unpleasant and toxic reagents. We report a safer method using a novel ferric ion chelating agent to produce highly specific, low background, and permanent staining of muscle fiber enzymes.