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Skeletal muscle regeneration is severely compromised in the case of extended damage. The current challenge is to find factors capable of limiting muscle degeneration and/or potentiating the inherent regenerative program mediated by a specific type of myoblastic cells, the satellite cells. Recent studies from our groups and others have shown that the bioactive lipid, sphingosine 1-phosphate (S1P), promotes myoblast differentiation and exerts a trophic action on denervated skeletal muscle fibres. In the present study, we examined the effects of S1P on eccentric contraction (EC)-injured extensor digitorum longus muscle fibres and resident satellite cells. After EC, skeletal muscle showed evidence of structural and biochemical damage along with significant electrophysiological changes, i.e. reduced plasma membrane resistance and resting membrane potential and altered Na(+) and Ca(2+) current amplitude and kinetics. Treatment with exogenous S1P attenuated the EC-induced tissue damage, protecting skeletal muscle fibre from apoptosis, preserving satellite cell viability and affecting extracellular matrix remodelling, through the up-regulation of matrix metalloproteinase 9 (MMP-9) expression. S1P also promoted satellite cell renewal and differentiation in the damaged muscle. Notably, EC was associated with the activation of sphingosine kinase 1 (SphK1) and with increased endogenous S1P synthesis, further stressing the relevance of S1P in skeletal muscle protection and repair/regeneration. In line with this, the treatment with a selective SphK1 inhibitor during EC, caused an exacerbation of the muscle damage and attenuated MMP-9 expression. Together, these findings are in favour for a role of S1P in skeletal muscle healing and offer new clues for the identification of novel therapeutic approaches to counteract skeletal muscle damage and disease., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

We recently demonstrated that skeletal muscle differentiation induced by sphingosine 1-phosphate (S1P) requires gap junctions and transient receptor potential canonical 1 (TRPC1) channels. Here, we searched for the signaling pathway linking the channel activity with Cx43 expression/function, investigating the involvement of the Ca(2+)-sensitive protease, m-calpain, and its targets in S1P-induced C2C12 myoblast differentiation. Gene silencing and pharmacological inhibition of TRPC1 significantly reduced Cx43 up-regulation and Cx43/cytoskeletal interaction elicited by S1P. TRPC1-dependent functions were also required for the transient increase of m-calpain activity/expression and the subsequent decrease of PKCα levels. Remarkably, Cx43 expression in S1P-treated myoblasts was reduced by m-calpain-siRNA and enhanced by pharmacological inhibition of classical PKCs, stressing the relevance for calpain/PKCα axis in Cx43 protein remodeling. The contribution of this pathway in myogenesis was also investigated. In conclusion, these findings provide novel mechanisms by which S1P regulates myoblast differentiation and offer interesting therapeutic options to improve skeletal muscle regeneration.

Transient receptor potential canonical (TRPC) channels provide cation and Ca(2+) entry pathways, which have important regulatory roles in many physio-pathological processes, including muscle dystrophy. However, the mechanisms of activation of these channels remain poorly understood. Using siRNA, we provide the first experimental evidence that TRPC channel 1 (TRPC1), besides acting as a store-operated channel, represents an essential component of stretch-activated channels in C2C12 skeletal myoblasts, as assayed by whole-cell patch-clamp and atomic force microscopic pulling. The channel's activity and stretch-induced Ca(2+) influx were modulated by sphingosine 1-phosphate (S1P), a bioactive lipid involved in satellite cell biology and tissue regeneration. We also found that TRPC1 was functionally assembled in lipid rafts, as shown by the fact that cholesterol depletion resulted in the reduction of transmembrane ion current and conductance. Association between TRPC1 and lipid rafts was increased by formation of stress fibres, which was elicited by S1P and abolished by treatment with the actin-disrupting dihydrocytochalasin B, suggesting a role for cytoskeleton in TRPC1 membrane recruitment. Moreover, TRPC1 expression was significantly upregulated during myogenesis, especially in the presence of S1P, implicating a crucial role for TRPC1 in myoblast differentiation. Collectively, these findings may offer new tools for understanding the role of TRPC1 and sphingolipid signalling in skeletal muscle regeneration and provide new therapeutic approaches for skeletal muscle disorders.

Actin cytoskeleton profoundly influence a variety of signaling events, including those related to cell growth, survival and differentiation. Recent evidence have provided insights into the mechanisms underlying the ability of cytoskeleton to regulate signal transduction cascades involved in muscle development. This review will deal with the most recent aspects of this field paying particular attention to the role played by actin dynamics in the induction of skeletal muscle-specific genes.

In many cell systems, sphingosine 1-phosphate (SPP) increases cytosolic Ca2+ concentration ([Ca2+]i) by acting as intracellular mediator and extracellular ligand. We recently demonstrated (Meacci E, Cencetti F, Formigli L, Squecco R, Donati C, Tiribilli B, Quercioli F, Zecchi-Orlandini S, Francini F, and Bruni P. Biochem J 362: 349-357, 2002) involvement of endothelial differentiation gene (Edg) receptors (Rs) specific for SPP in agonist-mediated Ca2+ response of a mouse skeletal myoblastic (C2C12) cell line. Here, we investigated the Ca2+ sources of SPP-mediated Ca2+ transients in C2C12 cells and the possible correlation of ion response to cytoskeletal rearrangement. Confocal fluorescence imaging of C2C12 cells preloaded with Ca2+ dye fluo 3 revealed that SPP elicited a transient Ca2+ increase propagating as a wave throughout the cell. This response required extracellular and intracellular Ca2+ pool mobilization. Indeed, it was significantly reduced by removal of external Ca2+, pretreatment with nifedipine (blocker of L-type plasma membrane Ca2+ channels), and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+ pathway inhibitors. Involvement of EdgRs was tested with suramin (specific inhibitor of Edg-3). Fluorescence associated with Ins(1,4,5)P3Rs and L-type Ca2+ channels was evident in C2C12 cells. SPP also induced C2C12 cell contraction. This event, however, was unrelated to [Ca2+]i increase, because the two phenomena were temporally shifted. We propose that SPP may promote C2C12 cell contraction through Ca2+-independent mechanisms.

Sphingosine 1-phosphate (SPP) is a bioactive lipid that exerts multiple biological effects in a large variety of cell types, acting as either an intracellular messenger or an extracellular ligand coupled to Edg-family receptors (where Edg stands for endothelial differentiation gene). Here we report that in C(2)C(12) myoblasts SPP elicited significant Ca(2+) mobilization. Analysis of the process using a confocal laser-scanning microscope showed that the Ca(2+) response occurred in a high percentage of cells, despite variations in amplitude and kinetics. Quantitative analysis of SPP-induced Ca(2+) transients performed with a spectrophotofluorimeter showed that the rise in Ca(2+) was strictly dependent on availability of extracellular Ca(2+). Cell treatment with pertussis toxin partially prevented the Ca(2+) response induced by SPP, indicating that G(i)-coupled-receptors were involved. Indeed, SPP action was shown to be mediated by agonist-specific Edg receptors. In particular, suramin, an antagonist of the SPP-specific receptor Edg3, as well as down-regulation of Edg3 by cell transfection with antisense oligodeoxyribonucleotides (ODN), significantly reduced agonist-mediated Ca(2+) mobilization. Moreover, an antisense ODN designed to inhibit Edg5 expression also decreased the SPP-induced rise in Ca(2+), although to a lesser extent than that observed by inhibiting Edg3. On the contrary, the SPP response was unaffected in myoblasts loaded with antisense ODN specific for Edg1. Remarkably, the concomitant inhibition of Edg3 and Edg5 receptors abolished the SPP-induced Ca(2+) increase, supporting the notion that Ca(2+) mobilization in C(2)C(12) cells induced by SPP is a receptor-mediated process that involves Edg3 and Edg5, but not Edg1.

Neutrophil accumulation and the consequent production of oxygen-derived free radicals are involved in the pathogenesis of Ischemia-Reperfusion syndrome. In this study we investigated whether a treatment with Vitamin E, which has antioxidant properties, could attenuate the tissue damage by interfering with the influx of neutrophils within the ischemic and reperfused human skeletal muscle. To this purpose, patients undergoing aortic cross-clamping during the surgical repair of aortic abdominal aneurysm were studied as a model of ischemia-reperfusion of the lower limb muscles. Muscle biopsies from the right femoral quadriceps of patients not receiving and receiving Vitamin E pretreatment before surgery were taken: a) after the induction of anaesthesia, as control samples, and b) after a period of ischemia followed by 30 min of reperfusion. The tissue samples were either routinely processed for morphological study and immunohistochemical analysis to detect an altered expression of specific endothelial adhesion proteins, such as E-selectin and ICAM-1. The results obtained showed that Vitamin E administration was able to prevent the accumulation of neutrophils within the ischemic and reperfused muscle. This beneficial effect of Vitamin E was due to its ability to hinder the expression of E-selectin and ICAM-1, molecules known to increase the adhesiveness of endothelium to circulating neutrophils. After treatment with Vitamin E a marked attenuation of the reperfusion injury was also evident. In conclusion, Vitamin E treatment may be considered a valuable tool for protection against the ischemia-reperfusion damage of human skeletal muscle.

Purpose: The biochemical and morphological alterations induced in lower limb skeletal muscle by ischemia-reperfusion (I-R) during aortic surgery and the effect of vitamin E pretreatment were investigated., Methods: Two groups of patients undergoing aortic aneurysm resection, one untreated and one treated with vitamin E, were examined. Quadricep muscle biopsies were taken after induction of anesthesia, at the end of ischemia, and after reperfusion. The malondialdehyde (MDA) content and morphology of biopsies were examined to assess peroxidative processes., Results: Ischemia did not induce an increase in MDA content but did increase neutrophil infiltration in muscle fibers of untreated patients. Reperfusion led to a significant increase in MDA content and to intermyofibrillar edema and mitochondrial swelling. The MDA content was not increased during ischemia and neutrophil infiltration was minimal in vitamin E treated patients. At reperfusion, the MDA content, the ultrastructural injuries and neutrophil infiltration were significantly reduced by the treatment., Conclusions: Vitamin E is effective in reducing the oxidative muscle damage occurring after a period of I-R.

This work was undertaken to assess the role of endothelial E-selectin in the development of neutrophil accumulation into the ischemic and reperfused human skeletal muscle and eventually in the genesis of ischemia-reperfusion syndrome. Twelve patients affected by abdominal aortic aneurysm who were undergoing reconstructive vascular surgery were studied. Muscle biopsies from the right femoral quadriceps were taken (1) immediately after anesthesia, as control samples, (2) before declamping the aorta, as ischemic samples, and (3) 30 minutes after reperfusion and then processed for immunohistochemical and ultrastructural analysis. Immunohistochemistry revealed a strong positive reaction for E-selectin on the venular endothelium during ischemia and reperfusion. Ultrastructural investigation showed that reactivity for E-selectin matched neutrophil accumulation of the skeletal muscle tissue. This phenomenon was dependent upon a complex series of events that included neutrophil adhesion to the inner surface of the postcapillary venules, passage through endothelial intercellular junctions, and migration distally into the interstitial spaces of the skeletal muscle tissue. Neutrophil tissue infiltration was also associated with ultrastructural signs of tissue damage at reperfusion. This is in agreement with accumulating evidence indicating a role for tissue infiltrating neutrophils in the genesis of toxic O2 free radicals. Our data suggest that E-selectin expression on the vascular endothelium of human skeletal muscle may represent a key regulatory point in the process of neutrophil tissue accumulation and indicate an active role for the venular endothelium in the development of human ischemia-reperfusion syndrome.

Our previous study on human skeletal muscle undergoing ischemia and reperfusion has revealed that granulocytes, which infiltrate the muscle tissue in large numbers, play an important role in mediating fibre injuries by producing superoxide anion (O2-) which is responsible for membrane lipid peroxidation. In the current study, five patients undergoing aortic reconstructive surgery were given acetyl-carnitine (2 mg/kg i.v. plus 1 mg/kg/min for 30 min) prior to the induction of ischemia. Muscle biopsies and blood samples were examined: a) after anaesthesia; b) at the end of ischemia; and c) 30 min after reperfusion, with the aim of elucidating whether acetylcarnitine could prevent the infiltration and/or the activation of granulocytes and eventually skeletal muscle injuries. During ischemia and reperfusion complement activation recruited numerous granulocytes into the muscle tissue, but, contrary to the untreated samples, the ability for O2(-)-generation of these cells remained at low levels and was comparable to that of ischemia even when molecular O2 was reintroduced to the tissue. Accordingly, the morphological changes of the postischemic muscle fibers were substantially reduced when compared to the untreated samples; in fact, the mitochondrial swelling was only moderate and the intramitochondrial dense bodies were small and scarce. The current findings support a positive role of acetyl-carnitine in ameliorating the ischemia-reperfusion (I-R)-induced damage of human skeletal muscle.