Xin-deficient mice display myopathy, impaired contractility, attenuated muscle repair and altered satellite cell functionality.

AIM: Xin is an F-actin-binding protein expressed during development of cardiac and skeletal muscle. We used Xin-/- mice to determine the impact of Xin deficiency on different aspects of skeletal muscle health, including functionality and regeneration. METHODS: Xin-/- skeletal muscles and their satellite cell (SC) population were investigated for the presence of myopathic changes by a series of histological and immunofluorescent stains on resting uninjured muscles. To further understand the effect of Xin loss on muscle health and its SCs, we studied SCs responses following cardiotoxin-induced muscle injury. Functional data were determined using in situ muscle stimulation protocol. RESULTS: Compared to age-matched wild-type (WT), Xin-/- muscles exhibited generalized myopathy and increased fatigability with a significantly decreased force recovery post-fatiguing contractions. Muscle regeneration was attenuated in Xin-/- mice. This impaired regeneration prompted an investigation into SC content and functionality. Although SC content was not different, significantly more activated SCs were present in Xin-/- vs. WT muscles. Primary Xin-/- myoblasts displayed significant reductions (approx. 50%) in proliferative capacity vs. WT; a finding corroborated by significantly decreased MyoD-positive nuclei in 3 days post-injury Xin-/- muscle vs. WT. As more activated SCs did not translate to more proliferating myoblasts, we investigated whether Xin-/- SCs displayed an exaggerated loss by apoptosis. More apoptotic SCs (TUNEL+/Pax7+) were present in Xin-/- muscle vs. WT. Furthermore, more Xin-/- myoblasts were expressing nuclear caspase-3 compared to WT at 3 days post-injury. CONCLUSION: Xin deficiency leads to a myopathic condition characterized by increased muscle fatigability, impaired regeneration and SC dysfunction.

A novel GFP reporter mouse reveals Mustn1 expression in adult regenerating skeletal muscle, activated satellite cells and differentiating myoblasts.

AIM: Mustn1 has been implicated in myofusion as well as skeletal muscle growth and repair; however, the exact role and spatio-temporal expression of Mustn1 have yet to be fully defined. METHODS: Transgenic mice were generated with a 1512-bp sequence of the Mustn1 promoter directing the expression of GFP (Mustn1(PRO) -GFP). These mice were used to investigate the spatio-temporal expression of Mustn1(PRO) -GFP during skeletal muscle development and adult skeletal muscle repair, as well as various phases of the satellite cell lifespan (i.e. quiescence, activation, proliferation, differentiation). RESULTS: Mustn1(PRO) -GFP expression was observed within somites at embryonic day 12 and developing skeletal muscles at embryonic day 15 and 18. While uninjured adult tibialis anterior muscle displayed no detectable Mustn1(PRO) -GFP expression, cardiotoxin injury robustly elevated Mustn1(PRO) -GFP expression at 3 days post-injury with decreasing levels observed at 5 days and minimal, focal expression seen at 10 days. The expression of Mustn1(PRO) -GFP at 3 days post-injury consistently overlaid with MyoD although the strongest expression of Mustn1(PRO) -GFP was noted in newly formed myotubes that were expressing minimal levels of MyoD. By 5 days post-injury, Mustn1(PRO) -GFP overlaid in all myotubes expressing myogenin although cells were present expressing Mustn1(PRO) -GFP alone. The expression patterns of Mustn1(PRO) -GFP in regenerating muscle preceded the expression of desmin throughout the regenerative time course consistent with Mustn1 being upstream of this myogenic protein. Further, quiescent satellite cells located on freshly isolated, single myofibers rarely expressed Mustn1(PRO) -GFP, but within 24 h of isolation, all activated satellite cells expressed Mustn1(PRO) -GFP. Expression of Mustn1(PRO) -GFP in primary myoblasts diminished with prolonged time in proliferation media. However, in response to serum withdrawal, the expression of Mustn1(PRO) -GFP increased during myofusion (day 2) followed by declining expression thereafter. CONCLUSION: Mustn1(PRO) -GFP is expressed in activated satellite cells and myoblasts but continued time in proliferation media diminished Mustn1(PRO) -GFP expression. However, myoblasts exposed to serum withdrawal increased Mustn1(PRO) -GFP expression consistent with its demonstrated role in myofusion. The in vivo expression pattern of Mustn1 observed in regenerating and developing skeletal muscle is consistent with its presence in satellite cells and its critical role in myofusion.

Voluntary physical activity and leucine correct impairments in muscle protein synthesis in partially pancreatectomised rats.

AIMS/HYPOTHESIS: Poorly controlled type 1 diabetes mellitus can cause reduced skeletal muscle mass and weakness during adolescence, which may affect long-term management of the disease. The aim of this study was to determine whether regular voluntary physical activity and leucine feeding restore rates of protein synthesis and deficits in skeletal muscle mass in a young, hypoinsulinaemic/hyperglycaemic rat model of diabetes. METHODS: Four-week-old male Sprague-Dawley rats were partially pancreatectomised (Px) to induce hypoinsulinaemia/hyperglycaemia and housed with/without access to running wheels for 3 weeks (n = 12-14/group). Sham surgery rats (shams) served as sedentary controls (n = 18). Protein synthesis and markers of protein anabolism were assessed in the fasted state and following leucine gavage. Fibre type and cross-sectional areas of the gastrocnemius muscle were measured using a metachromatic ATPase stain. RESULTS: Compared with sedentary behaviour, regular activity lowered fasting glycaemia and reduced fed hyperglycaemia in Px rats. Active-Px rats, which ran 2.2 ± 0.71 km/night, displayed greater muscle mass and fibre areas similar to shams, while sedentary-Px rats displayed a 20-30% loss in muscle fibre areas. Muscle protein synthesis (basal and in response to leucine gavage) was impaired in sedentary-Px (by ~65%), but not in active-Px rats, when compared with shams. Following leucine gavage, the phosphorylation status of eIF4E binding protein 1 (4E-BP1) and ribosomal S6 kinase 1 (S6K1), markers of mammalian target of rapamycin complex 1 (mTORC1) signalling, increased in shams (by two- and ninefold, respectively) and in active-Px (1.5- and fourfold, respectively) rats, but not in sedentary-Px rats. CONCLUSION/INTERPRETATION: Moderate physical activity in young Px rats normalises impairments in skeletal muscle growth and protein synthesis. These findings illustrate the critical compensatory role that modest physical activity and targeted nutrition can have on skeletal muscle growth during periods of hypoinsulinaemia in adolescent diabetes.

The ontogeny of aerobic and diving capacity in the skeletal muscles of Weddell seals.

Our objective was to determine the ontogenetic changes in the skeletal muscles of Weddell seals that transform a non-diving pup into an elite diving adult. Muscle biopsies were collected from pups, juveniles and adults and analyzed for changes in fiber type, mitochondrial density, myoglobin concentrations and aerobic, lipolytic and anaerobic enzyme activities. The fiber type results demonstrated a decrease in slow-twitch oxidative (Type I) fibers and a significant increase in fast-twitch oxidative (Type IIA) fibers as the animals mature. In addition, the volume density of mitochondria and the activity of lipolytic enzymes significantly decreased as the seals matured. To our knowledge, this is the first quantitative account describing a decrease in aerobic fibers shifting towards an increase in fast-twitch oxidative fibers with a significant decrease in mitochondrial density as animals mature. These differences in the muscle physiology of Weddell seals are potentially due to their three very distinct stages of life history: non-diving pup, novice diving juvenile, and elite deep diving adult. During the first few weeks of life, pups are a non-diving terrestrial mammal that must rely on lanugo (natal fur) for thermoregulation in the harsh conditions of Antarctica. The increased aerobic capacity of pups, associated with increased mitochondrial volumes, acts to provide additional thermogenesis. As these future elite divers mature, their skeletal muscles transform to a more sedentary state in order to maintain the low levels of aerobic metabolism associated with long-duration diving.

p21 is essential for normal myogenic progenitor cell function in regenerating skeletal muscle.

Despite the ability of myogenic progenitor cells (MPCs) to completely regenerate skeletal muscle following injury, little is known regarding the molecular program that regulates their proliferation and differentiation. Although mice lacking the cyclin-dependent kinase inhibitor p21 (p21-/-), develop normally, we report here that p21-/- MPCs display increased cell number and enhanced cell cycle progression compared with wild-type MPCs. Therefore, we hypothesized that p21-/- mice would demonstrate temporally enhanced regeneration following myotrauma. In response to cardiotoxin-induced injury, p21-/- skeletal muscle regeneration was significantly attenuated vs. regenerating wild-type muscle, contrary to the hypothesis. Regenerating p21-/- skeletal muscle displayed increased proliferative (PCNA positive) nuclei coincident with increased apoptotic nuclei (TUNEL positive) compared with wild-type muscle up to 3 wk after injury. Differentiation of p21-/- MPCs was markedly impaired and associated with increased apoptosis compared with wild-type MPCs, confirming that the impaired differentiation of the p21-/- MPCs was a cell autonomous event. No dysregulation of p27, p53, or p57 protein expression in differentiating p21-/- MPCs compared with wild-type MPCs was observed, suggesting that other compensatory mechanisms are responsible for the regeneration that ultimately occurs. On the basis of these findings, we propose that p21 is essential for the coordination of cell cycle exit and differentiation in the adult MPC population and that in the absence of p21, skeletal muscle regeneration is markedly impaired.

Myogenic satellite cells: physiology to molecular biology.

Adult skeletal muscle has a remarkable ability to regenerate following myotrauma. Because adult myofibers are terminally differentiated, the regeneration of skeletal muscle is largely dependent on a small population of resident cells termed satellite cells. Although this population of cells was identified 40 years ago, little is known regarding the molecular phenotype or regulation of the satellite cell. The use of cell culture techniques and transgenic animal models has improved our understanding of this unique cell population; however, the capacity and potential of these cells remain ill-defined. This review will highlight the origin and unique markers of the satellite cell population, the regulation by growth factors, and the response to physiological and pathological stimuli. We conclude by highlighting the potential therapeutic uses of satellite cells and identifying future research goals for the study of satellite cell biology.

Ouabain stimulates unidirectional and net potassium efflux in resting mammalian skeletal muscle.

The present study compared ouabain-sensitive unidirectional K+ flux into (JinK) and out of (JoutK) perfused rat hindlimb skeletal muscle in situ and mouse flexor digitorum brevis (FDB) in vitro. In situ, 5 mM ouabain inhibited 54 +/- 4% of the total JinK in 28 +/- 1 min, and increased the net and unidirectional efflux of K+ within 4 min. In contrast, 1.8 mM ouabain inhibited 40 +/- 8% of the total JinK in 38 +/- 2 min, but did not significantly affect JoutK. In vitro, 1.8 and 0.2 mM ouabain decreased JinK to a greater extent (83 +/- 5%) than in situ, but did not significantly affect 42K loss rate compared with controls. The increase in unidirectional K+ efflux (JoutK) with 5 mM ouabain in situ was attributed to increased K+ efflux through cation channels, since addition of barium (1 mM) to ouabain-perfused muscles returned JoutK to baseline values within 12 min. Perfusion with 5 mM ouabain plus 2 mM tetracaine for 30 min decreased JinK 46 +/- 9% (0.30 +/- 0.03 to 0.16 +/- 0.02 micromol x min(-1) x g(-1)), however tetracaine was unable to abolish the ouabain-induced increase in unidirectional K+ efflux. In both rat hindlimb and mouse FDB, tetracaine had no effect on JoutK. Perfusion of hindlimb muscle with 0.1 mM tetrodotoxin (TTX, a Na+ channel blocker) decreased JinK by 15 +/- 1%, but had no effect on JoutK; subsequent addition of ouabain (5 mM) decreased JinK a further 32 +/- 2%. The ouabain-induced increase in unidirectional K+ efflux did not occur when TTX was perfused prior to and during perfusion with 5 mM ouabain. We conclude that 5 mM ouabain increases the unidirectional efflux of K+ from skeletal muscle through a barium and TTX-sensitive pathway, suggestive of voltage sensitive Na+ channels, in addition to inhibiting Na+/K+-ATPase activity.

An integrative, in situ approach to examining K+ flux in resting skeletal muscle.

The contributions of Na+/K+-ATPase, K+ channels, and the NaK2Cl cotransporter (NKCC) to total and unidirectional K+ flux were determined in mammalian skeletal muscle at rest. Rat hindlimbs were perfused in situ via the femoral artery with a bovine erythrocyte perfusion medium that contained either 86Rb or 42K, or both simultaneously, to determine differences in ability to trace unidirectional K+ flux in the absence and presence of K+-flux inhibitors. In most experiments, the unidirectional flux of K+ into skeletal muscle (J(in)K) measured using 86Rb was 8-10% lower than J(in)K measured using 42K. Ouabain (5 mM) was used to inhibit Na+/K+-ATPase activity, 0.06 mM bumetanide to inhibit NKCC activity, 1 mM tetracaine or 0.5 mM barium to block K+ channels, and 0.05 mM glybenclamide (GLY) to block ATP-sensitive K+ (K(ATP)) channels. In controls, J(in)K remained unchanged at 0.31 +/- 0.03 micromol x g(-1) x min(-1) during 55 min of perfusion. The ouabain-sensitive Na+/K+-ATPase contributed to 50 +/- 2% of basal J(in)K, K+ channels to 47 +/- 2%, and the NKCC to 12 +/- 1%. GLY had minimal effect on J(in)K, and both GLY and barium inhibited unidirectional efflux of K+ (J(out)K) from the cell through K+ channels. Combined ouabain and tetracaine reduced J(in)K by 55 +/- 2%, while the combination of ouabain, tetracaine, and bumetanide reduced J(in)K by 67 +/- 2%, suggesting that other K+-flux pathways may be recruited because the combined drug effects on inhibiting J(in)K were not additive. The main conclusions are that the NKCC accounted for about 12% of J(in)K, and that K(ATP) channels accounted for nearly all of the J(out)K, in resting skeletal muscle in situ.

Paraxanthine, a caffeine metabolite, dose dependently increases [Ca(2+)](i) in skeletal muscle.

It was hypothesized that the caffeine derivative paraxanthine results in subcontracture increases in intracellular calcium concentration ([Ca(2+)](i)) in resting skeletal muscle. Single fibers obtained from mouse flexor digitorum brevis were loaded with a fluorescent Ca(2+) indicator, indo 1-acetoxymethyl ester. After a stable baseline was recorded, the fiber was superfused with physiological salt solution (Tyrode) containing 0.5, 1.0, 2.5, or 5 mM paraxanthine, resulting in [Ca(2+)](i) increases of 6.4 +/- 2.5, 9.7 +/- 3.6, 26.8 +/- 11.7, and 39.6 +/- 9.6 nM, respectively. The increases in [Ca(2+)](i) were transient and were also observed with exposure to 5 mM theophylline and theobromine. Six fibers were exposed to 5 mM paraxanthine followed by 5 mM paraxanthine in the presence of 10 mM procaine (sarcoplasmic reticulum Ca(2+) release channel blocker). There was no increase from baseline [Ca(2+)](i) when fibers were superfused with paraxanthine and procaine, suggesting that the sarcoplasmic reticulum is the primary Ca(2+) source in the paraxanthine-induced response. In separate experiments, intact flexor digitorum brevis (n = 13) loaded with indo 1-acetoxymethyl ester had a significant increase in [Ca(2+)](i) with exposure to 0.01 mM paraxanthine. It is concluded that physiological and low pharmacological concentrations of paraxanthine result in transient, subcontracture increases in [Ca(2+)](i) in resting skeletal muscle, the magnitude of which is related to paraxanthine concentration.

K+ transport in resting rat hind-limb skeletal muscle in response to paraxanthine, a caffeine metabolite.

This study tested the hypothesis that paraxanthine, a caffeine metabolite, stimulates skeletal muscle potassium (K+) transport by an increase in Na+ -K+ ATPase activity. The unidirectional transport of K+ into muscle (J(in)K) was studied using a perfused rat hind limb technique. Using 12 hind limbs, we examined the response to 20 min of paraxanthine perfusion (0.1 mM), followed by 20 min perfusion with 0.1 mM paraxanthine and 5 mM ouabain (n = 5) to irreversibly inhibit Na+ -K+ ATPase activity. Paraxanthine stimulated J(in)K by 23+/-5% within 20 min. Ouabain abolished the paraxanthine-induced stimulation of J(in)K, suggesting the increase in K+ uptake was due to activation of the Na+ -K+ ATPase. To confirm the role of the Na+ -K+ ATPase, 14 hind limbs were perfused for 20 min with 5 mM ouabain prior to 20 min perfusion with 0.1 mM paraxanthine and 5 mM ouabain (n = 6). Ouabain alone resulted in a 41+/-7% decrease in J(in)K within 15 min. Inhibition of ouabain-sensitive J(in)K prevented the paraxanthine-induced increase in J(in)K. Hind limbs (n = 3) were also perfused with 0.1 mM paraxanthine for 60 min to examine the response to longer duration paraxanthine perfusion. The paraxanthine-induced increase in J(in)K continued for the entire 60 min. In another series, hind limbs were perfused with 0.01 (n = 9), 0.1 (n = 9), or 0.5 (n = 6) mM paraxanthine for 15 min. There was no concentration-dependent relationship between J(in)K and paraxanthine concentration, and 0.01, 0.1, and 0.5 mM paraxanthine increased J(in)K similarly (25+/-5, 22+/-4, and 27+/-6%, respectively). The effect of paraxanthine on J(in)K could not be reversed by subsequent perfusion with paraxanthine-free perfusate. Caffeine (0.05-1.0 mM) had no effect on K+ transport. It is concluded that paraxanthine increases J(in)K in resting skeletal muscle by stimulating ouabain-sensitive Na+ -K+ ATPase activity.

Increased flow rate and papaverine increase K+ exchange in perfused rat hind-limb skeletal muscle.

This study tested the hypothesis that increases in perfusate flow rate result in increased rates of unidirectional and net K+ transport in rat hind-limb skeletal muscle at rest. Ten neurally and vascularly isolated hind limbs, with arterial and venous catheters placed proximal to the popliteal region, were perfused for 10-min periods at flow rates (presented in a random order) of 0.27, 0.42, 0.63, 0.84, or 1.05 mL x min(-1) x g(-1). Potassium extraction and unidirectional K+ influx were determined using 42K, and arterial perfusion pressure was measured continuously. Increases in flow rate resulted in decreases in K+ extraction and increases in unidirectional K+ influx, unidirectional K+ efflux, and net K+ efflux. The increases in K+ flux were associated with increases in oxygen uptake, glucose uptake, and lactate release. In separate experiments (n = 5), the vasodilator papaverine (10(-4) M) did not further vasodilate the vasculature of resting hind limbs, suggesting that the hind limbs in this preparation were fully vasodilated. Papaverine, at constant flow, resulted in a nearly 1.5-fold increase in K+ extraction, a doubling of unidirectional K+ influx, and increases in unidirectional K+ efflux and net K+ efflux. It is concluded that physiological increases in flow rate result in increases in K+ transport in isolated, perfused rat hind-limb skeletal muscle. Furthermore, papaverine appeared to induce an increase in skeletal muscle membrane permeability to K+.

Stimulation of Na+, K(+)-pump activity in skeletal muscle by methylxanthines: evidence and proposed mechanisms.

Evidence is presented to support the hypothesis that submillimolar concentrations of methylxanthines stimulate Na+, K(+)-ATPase activity in skeletal muscle. Administration of methylxanthines to skeletal muscle results in plasma membrane hyperpolarization and increased rates of K+ uptake and Na+ efflux. These effects are both dose- and time-dependent and inhibited by blockers of the Na+, K+ ATPase. The mechanisms for stimulation of Na+, K(+)-ATPase activity and the signal transduction pathways are not known. The methylxanthine concentrations required for stimulation of Na+, K(+)-ATPase activity are less than those required to cause a 50% inhibition of phosphodiesterase activity, and therefore increases in cyclic AMP due to inhibition of the enzyme are not involved. Possible mechanisms by which methylxanthines may increase Na+, K(+)-ATPase activity include; (1) a role for increased intracellular [Ca2+]; (2) Ca2+ or adenosine-receptor-mediated increases in intracellular cyclic AMP; and (3) a direct action of methylxanthines on the Na+, K+ ATPase.