Sympathetic innervation in skeletal muscle and its role at the neuromuscular junction.

Neuromuscular junctions are the synapses between motor neurons and skeletal muscle fibers, which mediate voluntary muscle movement. Since neuromuscular junctions are also tightly associated with the capping function of terminal Schwann cells, these synapses have been classically regarded as tripartite chemical synapses. Although evidences from sympathetic innervation of neuromuscular junctions was described approximately a century ago, the essential presence and functional relevance of sympathetic contribution to the maintenance and modulation of neuromuscular junctions was demonstrated only recently. These findings shed light on the pathophysiology of different clinical conditions and can optimize surgical and clinical treatment modalities for skeletal muscle disorders.

Long-term heat acclimation training in mice: Similar metabolic and running performance adaptations despite a lower absolute intensity than training at temperate conditions.

This study investigated the impact of long-term heat acclimation (HA) training on mouse thermoregulation, metabolism, and running performance in temperate (T) and hot (H) environments. Male Swiss mice were divided into 1) Sedentary (SED) mice kept in T (22 °C; SED/T), 2) Endurance Trained mice (ET, 1 h/day, 5 days/week, 8 weeks, 60 % of maximum speed) in T (ET/T), 3) SED kept in H (32 °C; SED/H), and 4) ET in H (ET/H). All groups performed incremental load tests (ILT) in both environments before (pre-ET) and after four and eight weeks of ET. In the pre-ET period, H impaired (∼30 %) performance variables (maximum speed and external work) and increased (1.3 °C) maximum abdominal body temperature compared with T. In T, after four weeks, although ET/H exercised at a lower (∼30 %) absolute intensity than ET/T, performance variables and aerobic power (peak oxygen uptake, VO2peak) were similarly increased in both ET groups compared with SED/T. After eight weeks, the external work was higher in both ET groups compared with SED/T. Only ET/T significantly increased VO2peak (∼11 %) relative to its pre-ET period. In H, only after eight weeks, both ET groups improved (∼19 %) maximum speed and reduced (∼46 %) post-ILT blood lactate concentrations compared with their respective pre-ET values. Liver glycogen content increased (34 %) in both ET groups and SED/H compared with SED/T. Thus, ET/H was performed at a lower absolute intensity but promoted similar effects to ET/T on metabolism, aerobic power, and running performance. Our findings open perspectives for applying HA training as part of a training program or orthopedic and metabolic rehabilitation programs in injured or even obese animals, reducing mechanical load with equivalent or higher physiological demand.

Calcitonin gene-related peptide exerts inhibitory effects on autophagy in the heart of mice.

Calcitonin Gene-Related Peptide (CGRP) is a potent vasodilator peptide widely distributed in the central nervous system and various peripheral tissues, including cardiac muscle. However, its role in heart protein metabolism remains unknown. We examined the acute effects of CGRP on autophagy and the related signaling pathways in the heart mice and cultured neonatal cardiomyocytes. CGRP (100 µg kg-1; s.c.) or 0.9 % saline was injected in awake male C57B16 mice, and the metabolic profile was determined up to 60 min. In fed mice, CGRP drastically increased glycemia and reduced insulinemia, an effect that was accompanied by reduced cardiac phosphorylation levels of Akt at Ser473 without affecting FoxO. Despite these catabolic effects, CGRP acutely inhibited autophagy as estimated by the decrease in LC3II:LC3I and autophagic flux. In addition, the fasting-induced autophagic flux in mice hearts was entirely abrogated by one single injection of CGRP. In parallel, CGRP stimulated PKA/CREB and mTORC1 signaling and increased the phosphorylation of Unc51-like kinase-1 (ULK1), an essential protein in autophagy initiation. Similar effects were observed in cardiomyocytes, in which CGRP also inhibited autophagic flux and stimulated Akt and FoxO phosphorylation. These findings suggest that CGRP in vivo acutely suppresses autophagy in the heart of fed and fasted mice, most likely through the activation of PKA/mTORC1 signaling but independent of Akt.

Insulin/IGF1 signalling mediates the effects of ß2 -adrenergic agonist on muscle proteostasis and growth.

BACKGROUND: Stimulation of ß2 -adrenoceptors can promote muscle hypertrophy and fibre type shift, and it can counteract atrophy and weakness. The underlying mechanisms remain elusive. METHODS: Fed wild type (WT), 2-day fasted WT, muscle-specific insulin (INS) receptor (IR) knockout (M-IR-/- ), and MKR mice were studied with regard to acute effects of the ß2 -agonist formoterol (FOR) on protein metabolism and signalling events. MKR mice express a dominant negative IGF1 receptor, which blocks both INS/IGF1 signalling. All received one injection of FOR (300 µg kg-1 subcutaneously) or saline. Skeletal muscles and serum samples were analysed from 30 to 240 min. For the study of chronic effects of FOR on muscle plasticity and function as well as intracellular signalling pathways, fed WT and MKR mice were treated with formoterol (300 µg kg-1  day-1 ) for 30 days. RESULTS: In fed and fasted mice, one injection of FOR inhibited autophagosome formation (LC3-II content, 65%, P ≤ 0.05) that was paralleled by an increase in serum INS levels (4-fold to 25-fold, P ≤ 0.05) and the phosphorylation of Akt (4.4-fold to 6.5-fold, P ≤ 0.05) and ERK1/2 (50% to two-fold, P ≤ 0.05). This led to the suppression (40-70%, P ≤ 0.05) of the master regulators of atrophy, FoxOs, and the mRNA levels of their target genes. FOR enhanced (41%, P ≤ 0.05) protein synthesis only in fed condition and stimulated (4.4-fold to 35-fold, P ≤ 0.05) the prosynthetic Akt/mTOR/p70S6K pathway in both fed and fasted states. FOR effects on Akt signalling during fasting were blunted in both M-IR-/- and MKR mice. Inhibition of proteolysis markers by FOR was prevented only in MKR mice. Blockade of PI3K/Akt axis and mTORC1, but not ERK1/2, in fasted mice also suppressed the acute FOR effects on proteolysis and autophagy. Chronic stimulation of ß2 -adrenoceptors in fed WT mice increased body (11%, P ≤ 0.05) and muscle (15%, P ≤ 0.05) growth and downregulated atrophy-related genes (30-40%, P ≤ 0.05), but these effects were abolished in MKR mice. Increases in muscle force caused by FOR (WT, 24%, P ≤ 0.05) were only partially impaired in MKR mice (12%, P ≤ 0.05), and FOR-induced slow-to-fast fibre type shift was not blocked at all in these animals. In MKR mice, FOR also restored the lower levels of muscle SDH activity to basal WT values and caused a marked reduction (57%, P ≤ 0.05) in the number of centrally nucleated fibers. CONCLUSIONS: NS/IGF1 signalling is necessary for the anti-proteolytic and hypertrophic effects of in vivo ß2 -adrenergic stimulation and appears to mediate FOR-induced enhancement of protein synthesis. INS/IGF1 signalling only partially contributes to gain in strength and does not mediate fibre type transition induced by FOR.

Involvement of cAMP/Epac/PI3K-dependent pathway in the antiproteolytic effect of epinephrine on rat skeletal muscle.

Very little is known about the signaling pathways by which catecholamines exert anabolic effects on muscle protein metabolism, stimulating protein synthesis and suppressing proteolysis. The present work tested the hypothesis that epinephrine-induced inhibition of muscle proteolysis is mediated through the cAMP/Epac/PI3K-dependent pathway with the involvement of AKT and Foxo. The incubation of extensor digitorum longus (EDL) muscles from rats with epinephrine and/or insulin increased the phosphorylation of AKT and its downstream target Foxo3a, a well-known effect that prevents Foxo translocation to the nucleus and the activation of proteolysis. Similar effects on AKT/Foxo signaling were observed in muscles incubated with DBcAMP (cAMP analog). The stimulatory effect of epinephrine on AKT phosphorylation was completely blocked by wortmannin (selective PI3K inhibitor), suggesting that the epinephrine-induced activation of AKT is mediated through PI3K. As for epinephrine and DBcAMP, the incubation of muscles with 8CPT-2Me-cAMP (selective Epac agonist) reduced rates of proteolysis and increased phosphorylation levels of AKT and Foxo3a. The specific PKA agonist (N6BZ-cAMP) inhibited proteolysis and abolished the epinephrine-induced AKT and Foxo3a phosphorylation. On the other hand, inhibition of PKA by H89 further increased the phosphorylation levels of AKT and Foxo3a induced by epinephrine, DBcAMP or 8CPT-2Me-cAMP. These findings suggest that the antiproteolytic effect of the epinephrine on isolated skeletal muscle may occur through a cAMP/Epac/PI3K-dependent pathway, which leads to the phosphorylation of AKT and Foxo3a. The parallel activation of PKA-dependent pathway also inhibits proteolysis and seems to limit the stimulatory effect of cAMP on AKT/Foxo3a signaling.

Chemical sympathectomy further increases muscle protein degradation of acutely diabetic rats.

The present work investigated the role of the sympathetic nervous system (SNS) in the control of protein degradation in skeletal muscles from rats with streptozotocin (STZ)-induced diabetes. Diabetes (1, 3, and 5 days after STZ) induced a significant increase in the norepinephrine content of soleus and EDL muscles, but it did not affect plasma catecholamine levels. Chemical sympathectomy induced by guanethidine (100 mg/kg body weight, for 1 or 2 days) reduced muscle norepinephrine content to negligible levels (less than 5%), decreased plasma epinephrine concentration, and further increased the high rate of protein degradation in muscles from acutely diabetic rats. The rise in the rate of proteolysis (nmol.mg wet wt(-1).2h(-1)) in soleus from 1-day diabetic sympathectomized rats was associated with increased activities of lysosomal (0.127 +/- 0.008 vs. 0.086 +/- 0.013 in diabetic control) and ubiquitin (Ub)-proteasome-dependent proteolytic pathways (0.154 +/- 0.007 vs. 0.121 +/- 0.006 in diabetic control). Increases in Ca2+-dependent (0.180 +/- 0.007 vs. 0.121 +/- 0.011 in diabetic control) and Ub-proteasome-dependent proteolytic systems (0.092 +/- 0.003 vs. 0.060 +/- 0.002 in diabetic control) were observed in EDL from 1-day diabetic sympathectomized rats. The lower phosphorylation levels of AKT and Foxo3a in EDL muscles from 3-day diabetic rats were further decreased by sympathectomy. The data suggest that the SNS exerts acute inhibitory control of skeletal muscle proteolysis during the early stages of diabetes in rats, probably involving the AKT/Foxo signaling pathway.

Cyclic adenosine monophosphate-phosphodiesterase inhibitors reduce skeletal muscle protein catabolism in septic rats.

We have previously shown that catecholamines exert an inhibitory effect on muscle protein degradation through a pathway involving the cyclic adenosine monophosphate (cAMP) cascade in normal rats. In the present work, we investigated in vivo and in vitro effects of cAMP-phosphodiesterase inhibitors on protein metabolism in skeletal muscle from rats submitted to a model of acute sepsis. The in vivo muscle protein metabolism was evaluated indirectly by measurements of the tyrosine interstitial concentration using microdialysis. Muscle blood flow (MBF) was monitored by ethanol perfusion technique. Sepsis was induced by cecal ligation and puncture and resulted in lactate acidosis, hypotension, and reduction in MBF (-30%; P < 0.05). Three-hour septic rats showed an increase in muscle interstitial tyrosine concentration (approximately 150%), in arterial plasma tyrosine levels (approximately 50%), and in interstitial-arterial tyrosine concentration difference (approximately 200%; P < 0.05). Pentoxifylline (50 mg/kg of body weight, i.v.) infusion during 1 h after cecal ligation and puncture prevented the tumor necrosis factor alpha increase and significantly reduced by 50% (P < 0.05) the interstitial-arterial tyrosine difference concentration. In situ perfusion with isobutylmethylxanthine (IBMX; 10(-3) M) reduced by 40% (P < 0.05) the muscle interstitial tyrosine in both sham-operated and septic rats. Neither pentoxifylline nor IBMX altered MBF. The addition of IBMX (10(-3) M) to the incubation medium increased (P < 0.05) muscle cAMP levels and reduced proteolysis in both groups. The in vitro addition of H89, a protein kinase A inhibitor, completely blocked the antiproteolytic effect of IBMX. The data show that activation of cAMP-dependent pathways and protein kinase A reduces muscle protein catabolism during basal and septic state.

CL 316,243, a selective beta3-adrenergic agonist, inhibits protein breakdown in rat skeletal muscle.

The in vitro effect of CL 316,243 (CL), a selective beta3-adrenoceptor agonist in the rate of overall proteolysis, the activity of proteolytic systems (lysosomal, Ca2+-dependent, ATP-dependent, and ATP-independent) and in the process of protein synthesis was investigated in rat skeletal muscles. The rate of overall proteolysis in soleus muscle from rats incubated with CL (10(-4) and 10(-5) M) or epinephrine (10(-5) M) was significantly decreased. In vitro rates of maximal activity of Ca2+-dependent proteolysis in soleus muscles were decreased by about 41% in the presence of 10(-5) M CL. No change was observed in the activities of the lysosomal, ATP-dependent or ATP-independent proteolytic systems. The anti-proteolytic effect of CL or epinephrine was partially prevented by 10(-5) M SR 59230A, a selective beta3-adrenoceptor antagonist. The increase of proteolysis induced by food deprivation in soleus was abolished by in vitro addition of 10(-5) M CL. No change in proteolysis was observed in extensor digitorum longus (EDL) muscles incubated with any concentration of the beta3-adrenoceptor agonist tested. Rates of protein synthesis were not affected by 10(-4) M CL neither in soleus nor EDL. The data suggest that a beta3-adrenoceptor-mediated inhibition of Ca2+-dependent proteolysis participates of the antiproteolytic effect of catecholamines in oxidative muscles.

Effect of sympathetic denervation on the rate of protein synthesis in rat skeletal muscle.

Rates of protein synthesis were investigated in skeletal muscles from rats submitted to chemical and surgical sympathectomy. Three models of sympathetic denervation were used: 1) treatment with guanethidine (100 mg.kg(-1).day(-1) sc); 2) lumbar sympathetic denervation (surgical excision of the second and third lumbar ganglia of the sympathetic chain, from which arises the postganglionic fibers to the skeletal muscles of rat hindlimb); and 3) adrenodemedullation. Protein synthesis was estimated in isolated soleus muscle by the rate of incorporation of [(14)C]tyrosine (0.1 mM, 0.05 microCi/ml) into total protein. Soleus isolated after 2 and 4 days of chemical sympathectomy or after 3 days of lumbar denervation showed a 17-20% statistically significant decrease in in vitro rates of protein synthesis. These effects were reverted by addition of 10(-5) M isoproterenol or epinephrine in vitro. Neither clenbuterol nor isoproterenol (10(-7), 10(-6), or 10(-5) M) in vitro affected the rate of protein synthesis in soleus from normal rats. On the other hand, clenbuterol or epinephrine (10(-5) M) increased by 20% the rate of protein synthesis in soleus muscles from adrenodemedullated rats and prevented its decrease in muscles from fasted rats. The data suggest that the sympathetic nervous system stimulates protein synthesis in oxidative muscles, probably through the activation of beta(2)-adrenoceptors, especially in situations of hormonal or nutritional deficiency.