Fast-twitch Extensor Digitorum Longus Muscles Research Articles

Comparative analysis of semaphorin 3A in soleus and EDL muscle satellite cells in vitro toward understanding its role in modulating myogenin expression

Resident myogenic stem cells, satellite cells, up-regulate a secreted multi-functional modulator, semaphorin 3A (Sema3A), exclusively at the early-differentiation phase in response to muscle-crush injury and treatment with hepatocyte growth factor (HGF) or basic fibroblast growth factor (FGF2). Here, we add evidence that the Sema3A expression and secretion induced by the growth factors is significantly higher in primary cultures from adult rat soleus than from the fast-twitch extensor digitorum longus (EDL) muscle. The higher Sema3A response, revealed by quantitative PCR and Western blotting of cell lysates and conditioned media, may account for the higher myogenin expression of soleus muscle satellite cells early in differentiation since addition of recombinant Sema3A stimulates myogenin expression in cultures. These experiments also showed that mRNA expression of plexin A2, which together with neuropilins, constitutes Sema3A composite-receptors, was higher in satellite cells from soleus than EDL with no difference in plexin A1 and A3 and neuropilin-1 and 2 levels. These comparative studies, therefore, highlight a possible Sema3A-plexin A2-myogenin signaling axis that may ensure promoting early differentiation by soleus muscle satellite cells.

Single fiber analyses of glycogen-related proteins reveal their differential association with glycogen in rat skeletal muscle

To understand how glycogen affects skeletal muscle physiology, we examined enzymes essential for muscle glycogen synthesis and degradation using single fibers from quiescent and stimulated rat skeletal muscle. Presenting a shift in paradigm, we show these proteins are differentially associated with glycogen granules. Protein diffusibility and/or abundance of glycogenin, glycogen branching enzyme (GBE), debranching enzyme (GDE), phosphorylase (GP), and synthase (GS) were examined in fibers isolated from rat fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscle. GDE and GP proteins were more abundant (~10- to 100-fold) in fibers from EDL compared with SOL muscle. GS and glycogenin proteins were similar between muscles while GBE had an approximately fourfold greater abundance in SOL muscle. Mechanically skinned fibers exposed to physiological buffer for 10 min showed ~70% total pools of GBE and GP were diffusible (nonbound), whereas GDE and GS were considerably less diffusible. Intense in vitro stimulation, sufficient to elicit a ~50% decrease in intracellular glycogen, increased diffusibility of GDE, GP, and GS (~15-60%) and decreased GBE diffusibility (~20%). Amylase treatment, which breaks α-1,4 linkages of glycogen, indicated differential diffusibilities and hence glycogen associations of GDE and GS. Membrane solubilization (1% Triton-X-100) allowed a small additional amount of GDE and GS to diffuse from fibers, suggesting the majority of nonglycogen-associated GDE/GS is associated with myofibrillar/contractile network of muscle rather than membranes. Given differences in enzymes required for glycogen metabolism, the current findings suggest glycogen particles have fiber-type-dependent structures. The greater catabolic potential of glycogen breakdown in fast-twitch fibers may account for different contraction induced rates of glycogen utilization.

Altered in vitro Proliferation of Mouse SOD1-G93A Skeletal Muscle Satellite Cells

Background: Amyotrophic lateral sclerosis (ALS) is the most common adult-onset neurodegenerative disease characterized by ascending muscle weakness, atrophy and paralysis. Early muscle abnormalities that precede motor neuron loss in ALS may destabilize neuromuscular junctions, and we have previously demonstrated alterations in myogenic regulatory factor (MRF) expression in vivo and in the activation of myofiber-associated skeletal muscle satellite cells (SMSCs) in the mouse model of ALS (SOD1-G93A). Methods: To elucidate niche dependence versus cell-autonomous mutant SOD1 (mSOD1) toxicity in this model, we measured in vitro proliferation potential and MRF and cyclin gene expression in SMSC cultures derived from fast-twitch extensor digitorum longus and slow-twitch soleus muscles of SOD1-G93A mice. Results: SMSCs from early presymptomatic (p40) to terminal, semi-paralytic (p120) SOD1-G93A mice demonstrated generally lower proliferation potential compared with age-matched controls. However, induced proliferation was observed in surgically denervated wild-type animals and SOD1-G93A animals at p90, when critical denervation arises. SMSCs from fast and slow muscles were similarly affected by mSOD1 expression. Lowered proliferation rate was generally corroborated with decreased relative MRF expression levels, although this was most prominent in early age and was modulated by muscle type origin. Cyclins controlling cell proliferation did not show modifications in their mRNA levels; however, the expression of cyclin-dependent kinase inhibitor 1A (Cdkn1a), which is known to promote myoblast differentiation, was decreased in SOD1-G93A cultures. Conclusions: Our data suggest that the function of SMSCs is impaired in SOD1-G93A satellite cells from the earliest stages of the disease when no critical motor neuron loss has been described.

Changes in the activin A‐myostatin‐follistatin system within bone and muscle of aging mice

The activin A‐myostatin‐follistatin system is thought to play an important role in the regulation of muscle and bone mass, but changes in the relative levels of these factors in muscle and bone with age are not well understood. We investigated age‐related changes in the activin A‐myostatin‐follistatin system using femoral bone marrow aspirates from young (12 months) and aged (24 months) mice, and soleus and extensor digitorum longus (EDL) muscles from these same animals. Bone marrow supernatant samples were analyzed using ELISA, and muscle samples were analyzed using real‐time PCR and ELISA. Results indicate that myostatin levels were significantly (+75%) increased in mouse bone marrow with age, whereas levels of activin A and follistatin did not differ significantly between young and aged mice. Myostatin and activin A levels were not significantly altered with age in either soleus or EDL, however follistatin levels decreased by ~30% in mouse EDL with age. These findings suggest that age‐associated loss of muscle mass in the predominantly fast‐twitch EDL muscle may be due in part to declining levels of follistatin. Funding for this research was provided by the Congressionally Directed Medical Research Programs (USAMRAA).

Adaptation of Mouse Skeletal Muscle to Long-Term Microgravity in the MDS Mission

The effect of microgravity on skeletal muscles has so far been examined in rat and mice only after short-term (5–20 day) spaceflights. The mice drawer system (MDS) program, sponsored by Italian Space Agency, for the first time aimed to investigate the consequences of long-term (91 days) exposure to microgravity in mice within the International Space Station. Muscle atrophy was present indistinctly in all fiber types of the slow-twitch soleus muscle, but was only slightly greater than that observed after 20 days of spaceflight. Myosin heavy chain analysis indicated a concomitant slow-to-fast transition of soleus. In addition, spaceflight induced translocation of sarcolemmal nitric oxide synthase-1 (NOS1) into the cytosol in soleus but not in the fast-twitch extensor digitorum longus (EDL) muscle. Most of the sarcolemmal ion channel subunits were up-regulated, more in soleus than EDL, whereas Ca2+-activated K+ channels were down-regulated, consistent with the phenotype transition. Gene expression of the atrophy-related ubiquitin-ligases was up-regulated in both spaceflown soleus and EDL muscles, whereas autophagy genes were in the control range. Muscle-specific IGF-1 and interleukin-6 were down-regulated in soleus but up-regulated in EDL. Also, various stress-related genes were up-regulated in spaceflown EDL, not in soleus. Altogether, these results suggest that EDL muscle may resist to microgravity-induced atrophy by activating compensatory and protective pathways. Our study shows the extended sensitivity of antigravity soleus muscle after prolonged exposition to microgravity, suggests possible mechanisms accounting for the resistance of EDL, and individuates some molecular targets for the development of countermeasures.

Does skeletal muscle fiber branching play a role in the inability of old EDL dystrophic muscle to resist large passive stretches?

to the editor: Hakim et al. ([3][1]) examined the passive mechanical properties of fast-twitch extensor digitorum longus (EDL) muscles from young and old mdx dystrophic mice. They elegantly showed that by increasing passive strain on the dystrophic muscle from 110% of optimal length ( L o) to 160%

Lessons from calsequestrin-1 ablation in vivo: much more than a Ca2+ buffer after all

Calsequestrin type-1 (CASQ1), the main sarcoplasmic reticulum (SR) Ca(2+) binding protein, plays a dual role in skeletal fibers: a) it provides a large pool of rapidly-releasable Ca(2+) during excitation-contraction (EC) coupling; and b) it modulates the activity of ryanodine receptors (RYRs), the SR Ca(2+) release channels. We have generated a mouse lacking CASQ1 in order to further characterize the role of CASQ1 in skeletal muscle. Contrary to initial expectations, CASQ1 ablation is compatible with normal motor activity, in spite of moderate muscle atrophy. However, CASQ1 deficiency results in profound remodeling of the EC coupling apparatus: shrinkage of junctional SR lumen; proliferation of SR/transverse-tubule contacts; and increased density of RYRs. While force development during a twitch is preserved, it is nevertheless characterized by a prolonged time course, likely reflecting impaired Ca(2+) re-uptake by the SR. Finally, lack of CASQ1 also results in increased rate of SR Ca(2+) depletion and inability of muscle to sustain tension during a prolonged tetani. All modifications are more pronounced (or only found) in fast-twitch extensor digitorum longus muscle compared to slow-twitch soleus muscle, likely because the latter expresses higher amounts of calsequestrin type-2 (CASQ2). Surprisingly, male CASQ1-null mice also exhibit a marked increased rate of spontaneous mortality suggestive of a stress-induced phenotype. Consistent with this idea, CASQ1-null mice exhibit an increased susceptibility to undergo a hypermetabolic syndrome characterized by whole body contractures, rhabdomyolysis, hyperthermia and sudden death in response to halothane- and heat-exposure, a phenotype remarkably similar to human malignant hyperthermia and environmental heat-stroke. The latter findings validate the CASQ1 gene as a candidate for linkage analysis in human muscle disorders.

Alterations in intrinsic mitochondrial function with aging are fiber type‐specific and do not explain differential atrophy between muscles

To determine whether mitochondrial dysfunction is causally related to muscle atrophy with aging, we examined respiratory capacity, H(2) O(2) emission, and function of the mitochondrial permeability transition pore (mPTP) in permeabilized myofibers prepared from four rat muscles that span a range of fiber type and degree of age-related atrophy. Muscle atrophy with aging was greatest in fast-twitch gastrocnemius (Gas) muscle (-38%), intermediate in both the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (Sol) muscles (-21%), and non-existent in adductor longus (AL) muscle (+47%). In contrast, indices of mitochondrial dysfunction did not correspond to this differential degree of atrophy. Specifically, despite higher protein expression for oxidative phosphorylation (oxphos) system in fast Gas and EDL, state III respiratory capacity per myofiber wet weight was unchanged with aging, whereas the slow Sol showed proportional decreases in oxphos protein, citrate synthase activity, and state III respiration. Free radical leak (H(2) O(2) emission per O(2) flux) under state III respiration was higher with aging in the fast Gas, whereas state II free radical leak was higher in the slow AL. Only the fast muscles had impaired mPTP function with aging, with lower mitochondrial calcium retention capacity in EDL and shorter time to mPTP opening in Gas and EDL. Collectively, our results underscore that the age-related changes in muscle mitochondrial function depend largely upon fiber type and are unrelated to the severity of muscle atrophy, suggesting that intrinsic changes in mitochondrial function are unlikely to be causally involved in aging muscle atrophy.

Potential benefits of taurine in the prevention of skeletal muscle impairment induced by disuse in the hindlimb-unloaded rat

Hindlimb unloading (HU) in rats induces severe atrophy and a slow-to-fast phenotype transition in postural slow-twitch muscles, as occurs in human disuse conditions, such as spaceflight or bed rest. In rats, a reduction of soleus muscle weight and a decrease of cross-sectional area (CSA) were observed as signs of atrophy. An increased expression of the fast-isoform of myosin heavy chain (MHC) showed the phenotype transition. In parallel the resting cytosolic calcium concentration (restCa) was decreased and the resting chloride conductance (gCl), which regulates muscle excitability, was increased toward the values of the fast-twitch muscles. Here, we investigated the possible role of taurine, which is known to modulate calcium homeostasis and gCl, in the restoration of muscle impairment due to 14-days-HU. We found elevated taurine content and higher expression of the taurine transporter TauT in the soleus muscle as compared to the fast-twitch extensor digitorum longus (EDL) muscle of control rats. Taurine level was reduced in the HU soleus muscle, although, TauT expression was not modified. Taurine oral supplementation (5g/kg) fully prevented this loss, and preserved resting gCl and restCa together with the slow MHC phenotype. Taurine supplementation did not prevent the HU-induced drop of muscle weight or fiber CSA, but it restored the expression of MURF-1, an atrophy-related gene, suggesting a possible early protective effect of taurine. In conclusion, taurine prevented the HU-induced phenotypic transition of soleus muscle and might attenuate the atrophic process. These findings argue for the beneficial use of taurine in the treatment of disuse-induced muscle dysfunction.

In vivo stimulation of oestrogen receptor α increases insulin‐stimulated skeletal muscle glucose uptake

Previous studies suggest oestrogen receptor α (ERα) is involved in oestrogen-mediated regulation of glucose metabolism and is critical for maintenance of whole body insulin action. Despite this, the effect of direct ERα modulation in insulin-responsive tissues is unknown. The purpose of the current study was to determine the impact of ERα activation, using the ER subtype-selective ligand propylpyrazoletriyl (PPT), on skeletal muscle glucose uptake. Two-month-old female Sprague-Dawley rats, ovariectomized for 1 week, were given subcutaneous injections of PPT (10 mg kg⁻¹), oestradiol benzoate (EB; 20 μg kg⁻¹), the ERβ agonist diarylpropionitrile (DPN, 10 mg kg⁻¹) or vehicle every 24 h for 3 days. On the fourth day, insulin-stimulated skeletal muscle glucose uptake was measured in vitro and insulin signalling intermediates were assessed via Western blotting.Activation of ERα with PPT resulted in increased insulin-stimulated glucose uptake into the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL)muscles, activation of insulin signalling intermediates (as measured by phospho-Akt (pAkt) and pAkt substrate (PAS)) and phosphorylation of AMP-activated protein kinase (AMPK). GLUT4 protein was increased only in the EDL muscle. Rats treated with EB or DPN for 3 days did not show an increase in insulin-stimulated skeletal muscle glucose uptake compared to vehicle-treated animals. These new findings reveal that direct activation of ERα positively mediates glucose uptake and insulin action in skeletal muscle. Evidence that oestrogens and ERα stimulate glucose uptake has important implications for understanding mechanisms of glucose homeostasis, particularly in postmenopausal women.

Tumor necrosis factor‐alpha antagonist administration recovers skeletal muscle dysfunction in ovariectomized rats

Skeletal muscles deteriorate after ovariectomy. Molecular pathway of this deterioration has not been defined. Tumor necrosis factor (TNF)-alpha activation is assumed to trigger muscle atrophy and administration of its antagonist is hypothesized to recover this atrophy in rats. Slow-twitch soleus and fast-twitch extensor digitorum longus muscle functions were investigated in intact, ovariectomized (OVX), and OVX plus 10 µg/g/week TNF-alpha antagonist administered female rats. Maximum isometric twitch and tetanic contraction responses were lower in the OVX groups. Maximum isometric twitch amplitudes recovered in the extensor digitorum longus but not in the soleus muscles after TNF-alpha antagonist administration. The decrease in responses to tetanic stimulations recovered in the OVX-TNF group at frequencies higher than 20 Hz in both muscle types. OVX animals body weight was 21% higher than intact animals. Muscle weight to body weight ratios of the OVX groups were higher than the control group which recovered after TNF-alpha antagonist administration. Findings suggest that the functional loss in OVX rat muscles is TNF-alpha pathway dependent. Skeletal muscle atrophy and function after OVX recovered by TNF-alpha antagonist administration.

Is the efficiency of mammalian (mouse) skeletal muscle temperature dependent?

Myosin crossbridges in muscle convert chemical energy into mechanical energy. Reported values for crossbridge efficiency in human muscles are high compared to values measured in vitro using muscles of other mammalian species. Most in vitro muscle experiments have been performed at temperatures lower than mammalian physiological temperature, raising the possibility that human efficiency values are higher than those of isolated preparations because efficiency is temperature dependent. The aim of this study was to determine the effect of temperature on the efficiency of isolated mammalian (mouse) muscle. Measurements were made of the power output and heat production of bundles of muscle fibres from the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles during isovelocity shortening. Mechanical efficiency was defined as the ratio of power output to rate of enthalpy output, where rate of enthalpy output was the sum of the power output and rate of heat output. Experiments were performed at 20, 25 and 30◦C. Maximum efficiency of EDL muscles was independent of temperature; the highest value was 0.31}0.01 (n =5) at 30◦C. Maximum efficiency of soleus preparations was slightly but significantly higher at 25 and 30◦C than at 20◦C; the maximum mean value was 0.48±0.02 (n =7) at 25◦C. It was concluded that maximum mechanical efficiency of isolated mouse muscle was little affected by temperature between 20 and 30◦C and that it is unlikely that differences in temperature account for the relatively high efficiency of human muscle in vivo compared to isolated mammalian muscles.

Sepsis and glucocorticoids downregulate the expression of the nuclear cofactor PGC-1β in skeletal muscle

Muscle wasting during sepsis is at least in part regulated by glucocorticoids and is associated with increased transcription of genes encoding the ubiquitin ligases atrogin-1 and muscle-specific RING-finger protein-1 (MuRF1). Recent studies suggest that muscle atrophy caused by denervation is associated with reduced expression of the nuclear cofactor peroxisome proliferator-activated receptor-γ coactivator (PGC)-1β and that PGC-1β may be a repressor of the atrogin-1 and MuRF1 genes. The influence of other muscle-wasting conditions on the expression of PGC-1β is not known. We tested the influence of sepsis and glucocorticoids on PGC-1β and examined the potential link between downregulated PGC-1β expression and upregulated atrogin-1 and MuRF1 expression in skeletal muscle. Sepsis in rats and mice and treatment with dexamethasone resulted in downregulated expression of PGC-1β and increased expression of atrogin-1 and MuRF1 in the fast-twitch extensor digitorum longus muscle, with less pronounced changes in the slow-twitch soleus muscle. In additional experiments, adenoviral gene transfer of PGC-1β into cultured C2C12 myotubes resulted in a dose-dependent decrease in atrogin-1 and MuRF1 mRNA levels. Treatment of cultured C2C12 myotubes with dexamethasone or PGC-1β small interfering RNA (siRNA) resulted in downregulated PGC-1β expression and increased protein degradation. Taken together, our results suggest that sepsis- and glucocorticoid-induced muscle wasting may, at least in part, be regulated by decreased expression of the nuclear cofactor PGC-1β.

Short-term adenosine monophosphate–activated protein kinase activator 5-aminoimidazole-4-carboxamide-1- β- d-ribofuranoside treatment increases the sirtuin 1 protein expression in skeletal muscle

Adenosine monophosphate–activated protein kinase (AMPK) has been proposed to stimulate mitochondrial biogenesis and fat and glucose metabolism in skeletal muscle. Nicotinamide adenine dinucleotide–dependent histone deacetylase sirtuin 1 (SIRT1) is also thought to play a pivotal role for such metabolic adaptations. The purpose of the present study was to examine the effect of AMPK activation with the administration of AMPK activator 5-aminoimidazole-4-carboxamide-1- β- d-ribofuranoside (AICAR) to rats on skeletal muscle SIRT1 protein expression as well as peroxisome proliferator activated receptor γ coactivator–1 α (PGC-1 α) and glucose transporter 4 (GLUT4) protein expression and hexokinase activity. The AICAR promoted the phosphorylation of AMPK α-subunit (Thr 172) and acetyl–coenzyme A carboxylase (Ser 79) without any change of total AMPK α-subunit or acetyl–coenzyme A carboxylase protein levels in both the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles. The SIRT1 protein expression increased at 24 hours after administration of AICAR in the EDL muscle but not in the soleus muscle. The PGC-1 α protein expression increased in both the soleus and EDL muscles and GLUT4 did in the EDL muscle at 24 hours after an administration of AICAR. The hexokinase activity increased at 18 and 24 hours in the soleus and at 12, 18, and 24 hours in the EDL after an AICAR treatment. These results suggest that short-term AICAR treatment to rats promotes skeletal muscle AMPK phosphorylation and then coincidently increases the SIRT1 protein expression. In addition, such treatment also enhances the PGC-1 α and GLUT4 protein contents and hexokinase activity in skeletal muscle.

Effect of heparin treatment on the expression and activity of different ion-motive P-type ATPase isoforms from mouse extensor digitorum longus muscle during degeneration and regeneration after Bothrops jararacussu venom injection

Ca 2+ ions are essential to myonecrosis, a serious complication of snake envenomation, and heparin seems to counteract this effect. We investigated the effect of local injection of Bothrops jararacussu venom in mouse fast-twitch extensor digitorum longus (EDL) muscle, without or with heparin, on functional/molecular alterations of two central proteins involved in intracellular Ca 2+ homeostasis, sarco(endo)plasmic reticulum Ca 2+-ATPase (SERCA) and Na +/K +-ATPase. EDL-specific SERCA1 isoform expression dropped significantly just after venom administration (up to 60% compared to control EDL values at days 1 and 3; p < 0.05) while SERCA2 and Na +/K +-ATPase α 1 isoform expression increased at the same time (3–6- and 2–3-fold, respectively; p < 0.05). Although not significant, Na +/K +-ATPase α 2 isoform followed the same trend. Except for SERCA2, all proteins reached basal levels at the 7th day. Intravenous heparin treatment did not affect these profiles. Ca 2+-ATPase activity was also decreased during the first days after venom injection, but here heparin was effective to reinstate activity to control levels within 3 days. We also showed that B. jararacussu venom directly inhibited Ca 2+-ATPase activity in a concentration-dependent manner. Our results indicate that EDL SERCA and Na +/K +-ATPase are importantly affected by B. jararacussu venom and heparin has protective effect on activity but not on protein expression.

Iron injections in mice increase skeletal muscle iron content, induce oxidative stress and reduce exercise performance

Iron accelerates the production of reactive oxygen species (ROS). Excessive levels of ROS are thought to accelerate skeletal muscle fatigue and contribute to the loss of skeletal muscle mass and function with age. Patients with an iron overload disease frequently report symptoms of weakness and fatigue, which is attributed to reduced cardiac function. The contribution of skeletal muscle to these symptoms is unknown. Using a mouse model of iron overload, we determined the extent of iron accumulation in skeletal muscle and the concentrations of the iron storage protein ferritin. The level of oxidative stress, changes in antioxidant enzymes and exercise performance were also assessed. Compared with control mice, the iron overloaded mice had elevated levels of iron in the tibialis anterior muscle and a fourfold increase in ferritin light chain. The oxidative stress product malondialdehyde was increased in the iron group compared with the control group, as was the antioxidant enzyme activity of glutathione reductase and glutathione peroxidase. The iron group performed less work on an endurance test and produced less force in a strength test. Body weight and skeletal muscle weight were lower in the iron group following the intervention. Iron loading reduced the weight of the fast-twitch extensor digitorum longus muscle more than the slow-twitch soleus muscle. In summary, iron accumulation in skeletal muscle may play a significant role in the reduced exercise capacity seen in iron overload disorders and in ageing, and may play an underlying role in skeletal muscle atrophy.

AMP‐activated protein kinase response to contractions and treatment with the AMPK activator AICAR in young adult and old skeletal muscle

One characteristic of ageing skeletal muscle is a decline in mitochondrial function. Activation of AMP-activated protein kinase (AMPK) occurs in response to an increased AMP/ATP ratio, which is one potential result of mitochondrial dysfunction. We have previously observed higher AMPK activity in old (O; 30 months) vs young adult (YA; 8 months) fast-twitch muscle in response to chronic overload. Here we tested the hypothesis that AMPK would also be hyperactivated in O vs YA fast-twitch extensor digitorum longus muscles from Fischer(344) x Brown Norway (FBN) rats (n = 8 per group) in response to high-frequency electrical stimulation of the sciatic nerve (HFES) or injection of AICAR, an activator of AMPK. Muscles were harvested immediately after HFES (10 sets of six 3-s contractions, 10 s rest between contractions, 1 min rest between sets) or 1 h after AICAR injection (1 mg (g body weight)(-1) subcutaneously). The phosphorylations of AMPKalpha and acetyl-CoA carboxylase (ACC2; a downstream AMPK target) were both greatly increased (P <or= 0.05) in response to HFES in O muscles, but were either unresponsive (AMPK alpha) or much less responsive (ACC) in YA muscles. AMPK alpha2 activity was also greatly elevated in response to HFES in O muscles (but not YA muscles) despite a lower total AMPK alpha2 protein content in O vs YA muscles. In contrast, AMPK alpha2 activity was equally responsive to AICAR treatment in both age groups. Since mitochondrial content and/or efficiency could potentially underlie AMPK hyperactivation, we measured levels of mitochondrial proteins as well as citrate synthase (CS) activity. While CS activity was increased by 25% in O vs YA muscles, uncoupling protein-3 (UCP-3) protein level was upregulated with age by 353%. Thus, AMPK hyperactivation in response to contractile activity in aged fast-twitch muscle may be the result of compromised cellular energetics and not necessarily due to an inherent defect in responsiveness of the AMPK molecule per se.

Loss of muscle‐specific inositol phosphatase (MIP) and increased phosphatidylinositol 3,5‐bisphosphate (PI(3,5)P2) inhibit muscle contractility and produce a Brody's disease phenotype

MIP knockout mice (KO) in which cellular PI(3,5)P2 is increased display behavioral characteristics that suggest a loss of muscle function and increased fatigability. To test muscle function, fast‐twitch extensor digitorum longus and slow‐twitch soleus muscles were isolated from the control and KO animals used in treadmill experiments 1‐2 weeks after the treadmill tests. Maximum isometric tetanic force was significantly reduced in both muscle types and the force vs. frequency relationship was shifted to the right in KO soleus muscles. Resting force was increased and the relaxation of KO muscles was prolonged. Muscles from KO animals fatigued more quickly, to a greater extent, and recovered to a lesser extent than muscles from the WT controls. In primary myotubes and adult muscle fibers, resting Ca2+ was elevated in the KO and the SR Ca2+ content was decreased. Store‐operated Ca2+ entry (SOCE) was also inhibited in KO myotubes. Perfusion of PI(3,5)P2 in WT myotubes recapitulated the defects in Ca2+ signaling of KO cells. Thus, ablation of MIP increases PI(3,5)P2 and causes muscle weakness and increased fatigability via a reduction of SR Ca2+ content by Ca2+ leak from the SR and inhibition of SOCE. These overall phenotypes recapitulate the symptomatology of Brody disease and certain forms of muscular dystrophies. (Support: TREC NCI Grant U54‐CA116867 to TMN, MB, and CKQ; AHA 0535555N to MB; NIH HL068212 and HL082670 to CKQ)

Dissociation between force and maximal Na+, K+-ATPase activity in rat fast-twitch skeletal muscle with fatiguing in vitro stimulation

This study investigated whether high frequency in vitro stimulation of rat fast-twitch extensor digitorum longus muscle depresses Na(+), K(+)-ATPase (NKA) activity as measured by the maximal in vitro 3-O-MFPase assay. EDL muscles subjected to 10 s continuous 100 Hz stimulation reduced tetanic force by 51.8 +/- 5.1% which recovered to 81.2 +/- 6.1% after 1 min and remained stable over 1 h recovery period. A second bout reduced force by 50.3 +/- 3.8% of initial but had no effect on 3-O-MFPase activity. Three minutes of intermittent stimulation (1 s at 100 Hz and 4 s recovery) resulted in 87.0 +/- 2.8% decline force with slow recovery (62.7 +/- 5.8% of initial after 1 h). The second 3-min bout reduced force by 83.3 +/- 3.6% of initial with no change in maximal 3-O-MFPase activity. These findings contrast previous human studies where fatiguing voluntary exercise depresses maximal NKA activity. This suggests that NKA in rat fast-twitch muscle is resistant to fatigue-induced inactivation under these conditions. Furthermore, the loss of force with fatigue was not related to depressed maximal NKA activity.

Skeletal Muscle-Specific Ablation of raptor, but Not of rictor, Causes Metabolic Changes and Results in Muscle Dystrophy

Mammalian target of rapamycin (mTOR) is a central controller of cell growth. mTOR assembles into two distinct multiprotein complexes called mTOR complex 1 (mTORC1) and mTORC2. Here we show that the mTORC1 component raptor is critical for muscle function and prolonged survival. In contrast, muscles lacking the mTORC2 component rictor are indistinguishable from wild-type controls. Raptor-deficient muscles become progressively dystrophic, are impaired in their oxidative capacity, and contain increased glycogen stores, but they express structural components indicative of oxidative muscle fibers. Biochemical analysis indicates that these changes are probably due to loss of activation of direct downstream targets of mTORC1, downregulation of genes involved in mitochondrial biogenesis, including PGC1alpha, and hyperactivation of PKB/Akt. Finally, we show that activation of PKB/Akt does not require mTORC2. Together, these results demonstrate that muscle mTORC1 has an unexpected role in the regulation of the metabolic properties and that its function is essential for life.