Early identification of individuals at increased cardiometabolic risk could allow the implementation of better preventive strategies through earlier interventions. Exaggerated blood pressure response to exercise has been associated with the later development of resting hypertension, cardiovascular mortality, as well as insulin resistance. We aimed to assess how exercise blood pressure at a standardized submaximal workload could help identify individuals with a deteriorated cardiometabolic risk profile.

Introduction: Clinical conduct can influence the healing of injured tissue. Eradication of inflammation seemed a promising strategy to promote musculoskeletal healing until studies showed a delayed/incomplete recovery from partial or complete elimination of inflammation. Endogenous lipid mediators biosynthesized from omega-3 and some from -6 fatty acids are molecules potentially playing important roles in the resolution of inflammation. Using such lipid mediators to treat injuries represents an attractive approach due to their anti-inflammatory and pro-resolving roles. Our goal was to identify the intracellular and/or extracellular targets used by 15-deoxy-delta-12,14Prostaglandin J2(15∆-PGJ2) to stimulate myoblast proliferation. Methods: Expression of D prostanoid (DP) 1 and 2 receptors was evaluated by western blotting. Proliferation of L6 myoblasts incubated with agonists and antagonists of prostaglandin (PG) D2 receptors DP1 and DP2 and of the peroxisome proliferator-activated receptor (PPAR) δ was assessed. Intracellular and extracellular concentrations of 15∆-PGJ2 following L6 cell activation with protease-activated receptor (PAR)-2 agonist were measured by liquid chromatography coupled to tandem mass spectrometry. Results: Both DP1 and DP2 receptors are present in myoblasts. DP1 agonist did not modulate L6 myoblast proliferation, but DP2 and PPARδ agonists induced an increase. DP1 and DP2 antagonists both significantly inhibited 15∆-PGJ2-induced stimulating effect of L6 cell proliferation (60% and 75%, respectively). 15∆-PGJ2was present in the intracellular and extracellular compartments under basal conditions, but was not modulated by PAR-2 receptor activation. Conclusion: L6 muscle cell can produce 15∆-PGJ2 and its effect on cell proliferation likely relies on both DP1 and DP2 receptor activation.

BACKGROUND:Sequential accumulation of M1 and M2 macrophages is critical for skeletal muscle recovery after an acute injury. While M1 accumulation is believed to rely on monocyte infiltration, the mechanisms of M2 accumulation remain controversial, but could involve an infiltrating precursor. Yet, strong depletion of monocytes only partially impairs skeletal muscle healing, supporting the existence of alternative mechanisms to palliate the loss of infiltrating macrophage progenitors. The aims of this study are thus to investigate if proliferation occurs in macrophage subsets within injured skeletal muscles; and to determine if monocyte depletion leads to increased proliferation of macrophages after injury.METHODS:Injury was induced by bupivacaine injection in the tibialis anterior muscle of rats. Blood monocytes were depleted by daily intravenous injections of liposome-encapsulated clodronate, starting 24 h prior to injury. In separate experiments, irradiation of hind limb was also performed to prevent resident cell proliferation. Upon euthanasia, blood and muscles were collected for flow cytometric analyses of macrophage/monocyte subsets.RESULTS:Clodronate induced a 80%-90% depletion of monocyte but only led to 57% and 41% decrease of M1 and M2 macrophage accumulation, respectively, 2 d following injury. Conversely, the number of M1 macrophages in monocyte-depleted rats was 2.4-fold higher than in non-depleted rats 4 d after injury. This was associated with a 16-fold increase in the number of proliferative M1 macrophages, which was reduced by 46% in irradiated animals. Proliferation of M2 macrophages was increased tenfold by clodronate treatment 4 d post injury. The accumulation of M2 macrophages was partially impaired by irradiation, regardless of monocyte depletion.CONCLUSIONS:M1 and M2 subsets proliferate after skeletal muscle injury and their proliferation is enhanced under condition of monocyte depletion. Our study supports the conclusion that both infiltrating and resident precursors could contribute to M1 or M2 macrophage accumulation in muscle injury.

We have shown that feeding cod protein, which is rich in anti-inflammatory arginine, glycine, and taurine, may beneficially modulate the inflammatory response during recovery following skeletal muscle injury; however it is unknown if these amino acids are responsible for this effect. This study was designed to assess whether supplementing casein with an amino acid mixture composed of arginine, glycine, taurine and lysine, matching their respective levels in cod protein, may account for the anti-inflammatory effect of cod protein. Male Wistar rats were fed isoenergetic diets containing either casein, cod protein, or casein supplemented with L-arginine (0.45%), glycine (0.43%), L-taurine (0.17%) and L-lysine (0.44%) (casein+). After 21 days of ad libitum feeding, one tibialis anterior muscle was injured with 200 µl bupivacaine while the saline-injected contra-lateral tibialis anterior was served as sham. Cod protein and casein+ similarly modulated the inflammation as they decreased COX-2 level at day 2 post-injury (cod protein, p=0.014; casein+, p=0.029) and ED1(+) macrophage density at days 2 (cod protein, p=0.012; casein+, p<0.0001), 5 (cod protein, p=0.001; casein+, p<0.0001) and 14 (cod protein, p<0.0001; casein+, p<0.0001) post-injury, and increased ED2(+) macrophage density at days 5 (cod protein, p<0.0001; casein+, p=0.006), 14 (cod protein, p=0.001; casein+, p<0.002) and 28 (cod protein, p<0.009; casein+, p<0.005) post-injury compared with casein. Furthermore, cod protein up-regulated (p=0.037) whereas casein+ tended to up-regulate (p=0.062) myogenin expression at day 5 post-injury compared with casein. In the cod protein-fed group, these changes resulted in greater muscle mass at days 14 (p=0.002), and 28 (p=0.001) post-injury and larger myofiber cross-sectional area at day 28 post-injury compared with casein (p=0.012). No such effects were observed with casein+. These data indicate that anti-inflammatory actions of cod protein, contrary to its effect on muscle mass recovery, are driven by its high levels of arginine, glycine, taurine and lysine.

This study was designed to assess the mechanisms by which dietary cod protein may facilitate skeletal muscle recovery from injury through analyses of the IGF1‐Akt/PKB signaling pathway. Male Wistar rats were fed isoenergetic diets containing either casein (C), cod protein (CP), or casein supplemented with a mixture of arginine, glycine, taurine and lysine (C+), matching their respective levels as in CP. Muscle injury was induced by bupivacaine injection and downstream IGF1‐Akt/PKB effectors were measured post‐injury. At day 2 post‐injury, C+ upregulated (p=0.0008) whereas CP tended to upregulate phospho‐Akt Thr308 compared with C (p=0.075), suggesting that the myogenic effect of CP is due in part to its high levels of arginine, glycine, taurine and lysine. At day 2 post‐injury, CP reduced the expression of MuRF‐1 by 38% compared with C (p=0.03), supporting a decrease in the ubiquitylation of muscle proteins and their degradation. At day 5 post‐injury, CP tended to increase the muscle IGF‐1 level compared with C (p=0.098); phospho‐Akt Ser473, a phosphorylation required for maximum activation of Akt in addition to phosphorylation at Thr308, was also increased (p<0.0001). These findings suggest that at early time‐points of recovery, CP can reduce muscle protein degradation through modulation of the ubiquitination process and upregulate protein synthesis through enhanced Akt activation.Supported by NSERC

ABSTRACT Introduction : Mast cells ( MCs ) can stimulate cell proliferation, but their specific contribution to skeletal muscle regeneration is not well defined. Methods : L6 myoblast proliferation was assessed in coculture with MCs or when grown with MC ‐conditioned media. To address the in vivo implication of MCs in regeneration, rats were treated with cromolyn, and myoblast proliferation, immune cell accumulation, and myogenic factors were assessed in bupivacaine‐injured muscles. Results : In vitro , both procedures increased the L6 cell proliferation rate, and this was tryptase‐dependent. In vivo , MC stabilization increased myoblast proliferation and accumulation of macrophages CD68 and CD163 after injury. This correlated with a sequential increase in MyoD and myogenin protein level expression. Conclusions : MCs can directly stimulate muscle cell proliferation via tryptase. MCs can influence myoblast proliferation in vivo, but this effect seems to be predominantly related to their modulation of macrophage recruitment. The MC is a potential actor in the early stages of muscle healing. Muscle Nerve 48 : 403–414, 2013

Muscle atrophy in chronic obstructive pulmonary disease (COPD) is associated with reduced exercise tolerance, muscle strength, and survival. The molecular mechanisms leading to muscle atrophy in COPD remain elusive. The mitogen-activated protein kinases (MAPKs) such as p38 MAPK and ERK 1/2 can increase levels of MAFbx/Atrogin and MuRF1, which are specifically involved in muscle protein degradation and atrophy. Our aim was to investigate the level of activation of p38 MAPK, ERK 1/2, and JNK in the quadriceps of patients with COPD. A biopsy of the quadriceps was obtained in 18 patients with COPD as well as in 9 healthy controls. We evaluated the phosphorylated as well as total protein levels of p38 MAPK, ERK 1/2, and JNK as well as MAFbx/Atrogin and MuRF1 in these muscle samples. The corresponding mRNA expression was also assessed by RT-PCR. Ratios of phosphorylated to total level of p38 MAPK (P = 0.02) and ERK 1/2 (P = 0.01) were significantly elevated in patients with COPD compared with controls. Moreover, protein levels of MAFbx/Atrogin showed a tendency to be greater in patients with COPD (P = 0.08). mRNA expression of p38 MAPK (P = 0.03), ERK 1/2 (P = 0.02), and MAFbx/Atrogin (P = 0.04) were significantly elevated in patients with COPD. In addition, phosphorylated-to-total p38 MAPK ratio (Pearson's r = -0.45; P < 0.05) and phosphorylated-to-total ERK 1/2 ratio (Pearson's r = -0.47; P < 0.05) were negatively associated with the mid-thigh muscle cross-sectional area. These data support the hypothesis that the MAPKs might play a role in the development of muscle atrophy in COPD.

Feeding cod protein may beneficially influence the time course of inflammation during recovery following skeletal muscle injury. The objective of this study was to identify amino acids in cod protein having a beneficial impact on muscle repair. Male Wistar rats were fed isoenergetic diets containing either casein (C), cod protein (CP), or casein supplemented with arginine (1.9%), glycine (1.9%), taurine (0.7%) and lysine (1.7%) (C+). After 21 d of ad libitum feeding, one tibialis anterior muscle (TA) was injured by injecting 200μl of bupivacaine while the contra‐lateral TA was injected with saline and served as sham. Rats fed CP exhibited higher muscle weight at days 5, 14, and 28 post‐injury and higher myofiber cross‐sectional area at days 5 and 28 post‐injury compared with C. CP and C+ similarly modulated the inflammation as they decreased ED1+ macrophages and COX‐2 level at day 2 post injury compared with casein (p=0.01), and increased ED2+ macrophages compared with C. The C+ diet upregulated (p=0.02) whereas the CP diet tended to upregulate myogenin expression at day 5 post‐injury compared with C (p=0.06). These data support the hypothesis that the beneficial impact of cod protein on skeletal muscle recovery is partly attributed to the anti‐inflammatory and myogenic action of its high arginine, lysine, glycine and taurine levels.Supported by The Natural Sciences and Engineering Research Council of Canada.

Inflammatory cells are traditionally associated with pain, heat, redness and swelling. However, accumulating studies have shown that some of these cells can also contribute to tissue repair. Indeed, neutrophils and macrophages can contribute to the resolution of inflammation and to skeletal muscle regeneration via the release of cytokines and growth factors. We recently showed that tryptase, the most abundant mediator in mast cell granules, could potentially support muscle regeneration by increasing skeletal muscle cell proliferation. PURPOSE: To evaluate if mast cells can stimulate skeletal muscle cell proliferation. METHODS: In vitro: mast cells were isolated from peritoneal cavity of female Wistar rats. L6 muscle cells were cultured with either mast cells activated with compound 48/80 or mast cellderived conditioned media. L6 cell number was determined with CellTiter assay 24h post-seeding. In vivo: muscle injury was induced through a bupivacain injection into the right EDL muscle. Rats received a daily intra-peritoneal injection of 5 bromo-2’deoxyuridine (BrdU) and were treated or not with the mast cell stabilizer cromolyn from 24h before injury. Rats were sacrificed 48 h post injury and immunohistochemistry analyzes were performed. RESULTS: In vitro proliferation of L6 cells cultured with either activated mast cells or mast cell-conditioned media was significantly increased above control (1.30±0.08 fold and 1.24±0.04 fold), respectively. The proliferative effect of conditioned media was lost when APC-366, a tryptase inhibitor, was added. In vivo results shown that, compared to control, mast cell stabilization increased the density of proliferating cells (109,033±8,186 vs 79,678±10,833 cells/mm3), neutrophils (34,116±6,167 vs 15,636±4,201 cells/mm3), macrophages ED1 (35,426±7,517 vs 13,075±4,108 cells/mm3) and macrophages ED2 (21,671±1,676 vs 16,922±715 cells/mm3), respectively. P<0.05. CONCLUSION: Activated mast cells can stimulate skeletal muscle cell proliferation via tryptase release in vitro. However, in vivo this effect was masked by the influence of mast cells on the recruitment of other mitogenic cells such as neutrophils and macrophages. Supported by grants from NSERC.

This study examined the effect of peanut and cod proteins on post-damage skeletal muscle repair, compared with casein. We hypothesized that because of their high arginine content, these proteins would improve the resolution of inflammation and muscle mass recovery following injury. One hundred and twenty-eight male Wistar rats were assigned to isoenergetic diets composed of casein and peanut (experiment 1) or cod protein (experiment 2). After 21 days of feeding, one tibialis anterior muscle (TA) was injured with bupivacaine, while the contralateral TA was injected with saline (sham muscle). Measurements were taken at days 0, 3, 14, and 24 post-injury. Compared with casein, peanut protein reduced muscle mass at days 0 (-12%, p = 0.005) and 14 post-injury in the injured muscle (-13%, p = 0.04), and lowered myofiber cross-sectional area in both the sham (-21%, p = 0.008) and injured muscles (-26%, p = 0.05) at day 24 post-injury, showing that peanut protein has a weak potential to support muscle growth. At day 14 post-injury, muscle mass in the sham (13%, p = 0.02) and injured muscles (12%, p = 0.01) was higher in cod-protein-fed rats, indicating better muscle mass recovery, than in casein-fed rats. Cod protein tended (p = 0.06) to decrease the density of neutrophils (-24%) at day 14 post-injury in the injured muscle, and to decrease the density of ED1(+) macrophages at day 24 post-injury in both sham (-29%, p = 0.03) and injured (-40%, p = 0.01) muscles. No effects were observed for peanut protein. These data indicate that cod protein is better for promoting growth and regeneration of skeletal muscle after trauma, partly because of the improved resolution of inflammation.

It has been recently shown that cod protein can modulate the production of pro‐inflammatory cytokines. We therefore postulated that it may regulate muscle regeneration and promote muscle mass recovery. The aim of this study was to determine the effects of dietary cod protein on skeletal muscle repair after injury compared with casein. Sixty four male Wistar rats were assigned to isoenergetic diets composed of either dietary cod protein or casein. After 21 d of ad libitum feeding, one tibialis anterior muscle (TA) was injured by bupivacaine injection (100μl) at the half proximal region while the contra‐lateral TA was injected with saline and served as sham. TA muscles collected at time 0 served as control. At day 14 post‐injury, values for muscle weight in both contra‐lateral (0.017) and injured groups (0.014) were higher in rats fed the cod protein diet, indicating better muscle mass recovery than in those fed the casein diet. Conversely, the cod protein diet led to lower pro‐inflammatory ED1+ macrophage density in both injured (P=0.008) and contra‐lateral sham (P=0.029) at day 24 post‐injury, suggesting that cod protein modulates the time course of inflammatory cell trafficking. Globally these data suggest that cod protein favors muscle growth and influences the inflammatory response, which potentially impact on recovery following injury. Supported by The Natural Sciences and Engineering Research Council of Canada.

Background Mast cells contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase which activates protease-activated receptor-2 (PAR-2). The possibility that a tryptase/PAR-2 signaling pathway exists in skeletal muscle cell has never been investigated. The aim of this study was to evaluate whether tryptase can stimulate myoblast proliferation and determine the downstream cascade. Methods Proliferation of L6 rat skeletal myoblasts stimulated with PAR-2 agonists (tryptase, trypsin and SLIGKV) was assessed. The specificity of the tryptase effect was evaluated with a specific inhibitor, APC-366. Western blot analyses were used to evaluate the expression and functionality of PAR-2 receptor and to assess the expression of COX-2. COX-2 activity was evaluated with a commercial activity assay kit and by measurement of PGF 2 α production. Proliferation assays were also performed in presence of different prostaglandins (PGs). Results Tryptase increased L6 myoblast proliferation by 35% above control group and this effect was completely inhibited by APC-366. We confirmed the expression of PAR-2 receptor in vivo in skeletal muscle cells and in satellite cells and in vitro in L6 cells, where PAR-2 was found to be functional. Trypsin and SLIGKV increased L6 cells proliferation by 76% and 26% above control, respectively. COX-2 activity was increased following stimulation with PAR-2 agonist but its expression remained unchanged. Inhibition of COX-2 activity by NS-398 abolished the stimulation of cell proliferation induced by tryptase and trypsin. Finally, 15-deoxy-Δ- 12,14 -prostaglandin J 2 (15Δ-PGJ 2 ), a product of COX-2-derived prostaglandin D 2 , stimulated myoblast proliferation, but not PGE 2 and PGF 2 α. Conclusions Taken together, our data show that tryptase can stimulate myoblast proliferation and this effect is part of a signaling cascade dependent on PAR-2 activation and on the downstream activation of COX-2.

Duchenne muscular dystrophy (DMD) is the most frequent muscular dystrophy. Currently, there is no cure for the disease. The transplantation of muscle precursor cells (MPCs) is one of the possible treatments, because it can restore the expression of dystrophin in DMD muscles. In this study, we investigated the effects of myoblasts injected with cardiotoxin on the contractile properties and resistance to eccentric contractions of transplanted and nontransplanted muscles. We used the extensor digitorum longus (EDL) as a model for our study. We conclude that the sole presence of dystrophin in a high percentage of muscle fibers is not sufficient by itself to increase the absolute or the specific force in the EDL of transplanted mdx muscle. This lack of strength increase may be due to the extensive damage that was produced by the cardiotoxin, which was coinjected with the myoblasts. However, the dystrophin presence is sufficient to protect muscle from eccentric damage as indicated by the force drop results.

It is well known that diets high in nuts or peanuts favourably affect plasma lipid concentrations. However, few studies have examined the effects of nut and peanut protein (PP) on body composition and skeletal muscle properties. The present study was aimed at evaluating the effect of dietary PP compared with two animal proteins, casein (C) and cod protein (CP) on body composition, skeletal muscle contractile properties and lipid metabolism in rats. Thirty-two male rats were assigned to one of the following four diets containing either C, CP, PP or C + peanut protein (CPP, 50:50) mixture. After 28d of ad libitum feeding and after 12-h fast, blood, liver and muscle were collected for measurements of plasma and hepatic cholesterol and TAG, plasma glucose and insulin and contractile properties. Rats fed with the low-quality protein, PP, had lower body weight gain, body protein mass, soleus mass and liver weight than those fed with the high-quality dietary proteins, C and CP. PP also caused a deficit in contractile properties in soleus. Likewise, PP increased plasma cholesterol and body fat mass compared with CP. However, these elevations were accompanied with increased hepatic TAG concentrations and lowered intestinal fat excretion. These results show that PP intake alters body composition by reducing skeletal muscle mass and liver weight as well as muscle contractility and lipid metabolism. Adding a complete protein such as C might partially counteract these adverse effects.

Impaired resting metabolism in peripheral muscles potentially contributes to exercise intolerance in chronic obstructive pulmonary disease (COPD). This study investigated the cytosolic energy metabolism of the quadriceps, from glycogen degradation to lactate accumulation, in exercising patients with COPD, in comparison to healthy controls. We measured, in 12 patients with COPD and 10 control subjects, resting and post-cycling exercise quadriceps levels of 1) energy substrates and end products of glycolysis (glycogen, glucose, pyruvate, and lactate) and intermediate markers of glycolysis (glucose-6-phosphate, glucose-1-phosphate, fructose-6-phosphate) and 2) the activity of key enzymes involved in the regulation of glycolysis (phosphofructokinase, lactate dehydrogenase). Exercise intensity (P < 0.01), duration (P = 0.049), and total work (P < 0.01) were reduced in patients with COPD. The variations in energy substrates and end products of glycolysis after cycling exercise were of similar magnitude in patients with COPD and controls. Glucose-6-phosphate (P = 0.036) and fructose-6-phosphate (P = 0.042) were significantly elevated in patients with COPD after exercise. Phosphofructokinase (P < 0.01) and lactate dehydrogenase (P = 0.02) activities were greater in COPD. Muscle glycogen utilization (P = 0.022) and lactate accumulation (P = 0.025) per unit of work were greater in COPD. We conclude that cycling exercise induced changes in quadriceps metabolism in patients with COPD that were of similar magnitude to those of healthy controls. These intramuscular events required a much lower exercise work load and time to occur in COPD. Our data suggest a greater reliance on glycolysis during exercise in COPD, which may contribute to exercise intolerance in COPD.

Peripheral muscle dysfunction associated with chronic diseases is undeniably a growing problem as one of its main causes, chronic obstructive pulmonary disease (COPD), progresses. Among others, muscle atrophy is one component building the concept of muscle dysfunction. Muscle atrophy has a significant impact on patient clinical status, independent of the impairment in lung function. A lot of effort has been devoted lately to increasing our understanding of the relationship between COPD and the initiation and the development of muscle atrophy. A growing body of evidence is showing that the ubiquitin-proteasome system, an ATP-dependent proteolytic pathway, is playing a crucial role in the cascade leading to degradation of contractile proteins, thus promoting the development of muscle atrophy. Interestingly, this system is also involved in essential cellular processes such as response to hypoxemia and muscle tissue regeneration. In this review, existing evidence linking the activity of the ubiquitin-proteasome system and the cellular events taking place in respiratory and peripheral muscles of patients with COPD are reported. Based on this information, the reader should be able to understand the essential role of this pathway in the context of muscle homeostasis and to picture the coming research in this area.