Sébastien S. Dufresne,* Nicolas A. Dumont,* Antoine Boulanger-Piette, Val A. Fajardo, Daniel Gamu, Sandrine-Aurélie Kake-Guena, Rares Ovidiu David, Patrice Bouchard, Éliane Lavergne, Josef M. Penninger, Paul C. Pape, A. Russell Tupling, and Jérôme Frenette Centre Hospitalier Universitaire de Québec-Centre de Recherche du Centre Hospitalier de l’Université Laval, Université Laval, Quebec City, Quebec, Canada; Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada; Faculté de Médecine et des Sciences de la Santé, Département de Physiologie et Biophysique, Université de Sherbrooke, Sherbrooke, Québec, Canada; Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria; and Faculté de Médecine, Département de Réadaptation, Université Laval, Quebec City, Quebec, Canada

Receptor-activator of nuclear factor-κB (RANK), its ligand RANKL, and the soluble decoy receptor osteoprotegerin are the key regulators of osteoclast differentiation and bone remodeling. Here we show that RANK is also expressed in fully differentiated myotubes and skeletal muscle. Muscle RANK deletion has inotropic effects in denervated, but not in sham, extensor digitorum longus (EDL) muscles preventing the loss of maximum specific force while promoting muscle atrophy, fatigability, and increased proportion of fast-twitch fibers. In denervated EDL muscles, RANK deletion markedly increased stromal interaction molecule 1 content, a Ca(2+)sensor, and altered activity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) modulating Ca(2+)storage. Muscle RANK deletion had no significant effects on the sham or denervated slow-twitch soleus muscles. These data identify a novel role for RANK as a key regulator of Ca(2+)storage and SERCA activity, ultimately affecting denervated skeletal muscle function.

Receptor-activator of NF-κB, its ligand RANKL, and the soluble decoy receptor osteoprotegerin are the key regulators of osteoclast differentiation and bone remodeling. Although there is a strong association between osteoporosis and skeletal muscle atrophy/dysfunction, the functional relevance of a particular biological pathway that synchronously regulates bone and skeletal muscle physiopathology still is elusive. Here, we show that muscle cells can produce and secrete osteoprotegerin and pharmacologic treatment of dystrophic mdx mice with recombinant osteoprotegerin muscles. (Recombinant osteoprotegerin-Fc mitigates the loss of muscle force in a dose-dependent manner and preserves muscle integrity, particularly in fast-twitch extensor digitorum longus.) Our data identify osteoprotegerin as a novel protector of muscle integrity, and it potentially represents a new therapeutic avenue for both muscular diseases and osteoporosis.

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.

Receptor‐activator of nuclear factor‐κB (RANK), its ligand RANKL and the soluble decoy receptor osteoprotegerin (OPG) are members of the tumor necrosis factor (TNF) superfamily that control osteoclast differentiation, bone remodelling and osteoporosis. Since bone and skeletal muscle physiopathology occurs synchronously, we tested whether RANK/RANKL pathway is involved in muscle dysfunction following different muscle wasting conditions. Contractile properties of EDL muscles were obtained following 14d of denervation in mice specifically deficient in RANK skeletal muscle or in dystrophic mdx mice treated daily for 10d with OPG [0.3mg/kg], the natural inhibitor of RANKL. The activity and expression of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) were determined in denervated or dystrophic muscles. RANK muscle deletion did not prevent atrophy but greatly reduced the losses of force production and SERCA activity in denervated EDL muscles. Force production of EDL muscles also increased by 140% in OPG‐treated relative to PBS‐treated mdx mice while the treatment with OPG prevented the loss of the fast twitch SERCA1 protein content in dystrophic mice. Together, these data indicate that the deletion or blockage of RANK/RANKL pathway has inotropic effects and increases SERCA activity and expression in fast‐twitch skeletal muscles opening promising new avenues for several muscle diseases.

We describe a new method for determining the concentration of total Ca in whole skeletal muscle samples ([Ca-T](WM) in units of mmoles/kg wet weight) using the Ca-dependent UV absorbance spectra of the Ca chelator BAPTA (1,2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid). Muscle tissue was homogenized in a solution containing 0.15 mM BAPTA and 0.5% sodium dodecyl sulfate (to permeabilize membranes and denature proteins) and then centrifuged. The solution volume was adjusted so that BAPTA captured essentially all of the Ca. [Ca-T](WM) was obtained with Beer's law from the absorbance change produced by adding 1 mM EGTA to capture Ca from BAPTA. Results from mouse, rat, and frog muscles were reasonably consistent with results obtained using other methods for estimating total [Ca] in whole muscles and in single muscle fibers. Results with external Ca removed before determining [Ca-T](WM) indicate that most of the Ca was intracellular, indicative of a lack of bound Ca in the extracellular space. In both fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) muscles from mice, [Ca-T](WM) increased approximately linearly with decreasing muscle weight, increasing approximately twofold with a twofold decrease in muscle weight. This suggests that the Ca concentration of smaller muscles might be increased relative to that in larger muscles, thereby increasing the specific force to compensate for the smaller mass. Knocking out the high capacity Ca-binding protein calsequestrin (CSQ) did not significantly reduce [Ca-T](WM) in mouse EDL or soleus muscle. However, in EDL muscles lacking CSQ, muscle weights were significantly lower than in wild-type (WT) muscles and the values of [Ca-T](WM) were, on average, about half the expected WT values, taking into account the above [Ca-T](WM) versus muscle weight relationship. Because greater reductions in [Ca-T](WM) would be predicted in both muscle types, we hypothesize that there is a substantial increase in Ca bound to other sites in the CSQ knockout muscles.

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.

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

Clinical observations from Buruli ulcer (BU) patients in West Africa suggest that severe Mycobacterium ulcerans infections can cause skeletal muscle contracture and atrophy leading to significant impairment in function. In the present study, male mice C57BL/6 were subcutaneously injected with M. ulcerans in proximity to the right biceps muscle, avoiding direct physical contact between the infectious agent and the skeletal muscle. The histological, morphological, and functional properties of the muscles were assessed at different times after the injection. On day 42 postinjection, the isometric tetanic force and the cross-sectional area of the myofibers were reduced by 31% and 29%, respectively, in the proximate-infected muscles relative to the control muscles. The necrotic areas of the proximate-infected muscles had spread to 7% of the total area by day 42 postinjection. However, the number of central nucleated fibers and myogenic regulatory factors (MyoD and myogenin) remained stable and low. Furthermore, Pax-7 expression did not increase significantly in mycolactone-injected muscles, indicating that the satellite cell proliferation is abrogated by the toxin. In addition, the fibrotic area increased progressively during the infection. Lastly, muscle-specific RING finger protein 1 (MuRF-1) and atrogin-1/muscle atrophy F-box protein (atrogin-1/MAFbx), two muscle-specific E3 ubiquitin ligases, were upregulated in the presence of M. ulcerans . These findings confirmed that skeletal muscle is affected in our model of subcutaneous infection with M. ulcerans and that a better understanding of muscle contractures and weakness is essential to develop a therapy to minimize loss of function and promote the autonomy of BU patients.

Neutrophils are the first leukocytes to invade tendons after an acute injury. They could modulate both the inflammatory response and early repair processes through the release of reactive species, cytokines, growth factors, and proteinases. However, the exact role of these cells in damaged tendons remains unclear. We investigated their role by inducing a transient neutropenia in C57BL/6 male mice using an anti-Ly6C/Ly6G antibody. Placebo mice received only serum. The right Achilles tendon was sectioned and sutured using the 8-strand technique, which allowed immediate weight bearing. A significant increase in macrophage accumulation and cell proliferation was observed in tendons from neutropenic animals compared to the placebo group at days 3 and/or 7 postinjury. However, there was a reduction in cell proliferation in a group of mice depleted in macrophages, indicating that macrophages play a role in cell replication in injured tendons. Lastly, the tendons of neutropenic and placebo mice had similar collagen content and mechanical properties at days 7, 14, and/or 28 postinjury. Our findings demonstrate that neutropenia modulates macrophage accumulation and cell proliferation, but overall, a reduction in neutrophil number has no significant effect on tendon repair.

Besides their hemostatic function, platelets can express key factors involved in tissue healing. However, the role of platelets in tendon healing following acute injury is poorly understood. We investigated this role by injecting male C57BL/6 mice with an antiplatelet antibody to induce thrombocytopenia. Placebo animals received serum only. The right Achilles tendon was sectioned and sutured using the 8-strand technique that allows immediate weight bearing. Platelet depletion did not alter the accumulation of neutrophils and macrophages or cell proliferation. A slight increase in vascularization was observed 7 days postinjury in tendons from thrombocytopenic mice relative to placebo animals, but the effect had disappeared by day 14. Furthermore, collagen content had a tendency to decrease in Achilles tendons under thrombocytopenia when compared with placebo treatment at 7 days posttrauma. This was correlated with a decline in maximal stress sustained by tendons at day 14 but not after 28 days. The impact of thrombocytopenia was otherwise negligible, as force relaxation and stiffness were similar in the two groups. Our findings demonstrate that platelets modulate early tendon repair following rupture, although the effect is limited over time. Nevertheless, platelets are not essential for the recruitment of inflammatory cells, proliferation, angiogenesis, and tendon maturation.

Neutrophils phagocyte necrotic debris and release cytokines, enzymes, and oxidative factors. In the present study, we investigated the contribution of neutrophils to muscle injury, dysfunction, and recovery using an unloading and reloading model. Mice were submitted to 10 days of hindlimb unloading and were transiently depleted in neutrophils with anti-Ly6G/Ly6C antibody prior to reloading. Leukocyte accumulation and muscle function were assessed immunohistologically and functionally in vitro. In addition, soleus muscles submitted to unloading and reloading were incubated in vitro with LPS (100 microg/ml) to determine whether exogenous stimulus would activate neutrophil response and produce extensive muscle damage. Contractile properties were recorded every hour for 6 h, and muscles were subsequently incubated in procion orange to assess muscle damage. Neutrophil depletion affected neither the loss in muscle force nor the time of recovery in atrophied and reloaded soleus muscles. However, atrophied and reloaded soleus muscles that contained high concentration of neutrophils experienced a 20% greater loss in force than atrophied and reloaded soleus muscles depleted in neutrophils following in vitro incubation with LPS. Procion orange dye also confirmed that neutrophils induced a 2.5-fold increase in muscle membrane damage in the presence of LPS. These results show that neutrophil infiltration during modified mechanical loading is highly regulated and efficiently eliminated, with no significant muscle fiber injury unless the activation state of neutrophils is modified by the presence of LPS.