**11. Sphingolipids in skeletal muscle regeneration**

Sphingolipids (SL) and cholesterol (CHOL) create LR in plasma membrane, but it is the biochemistry of SL that is apparently decisive for skeletal muscle biology including its growth and differentiation. PM sphingomyelin is a target for both acidic and neutral sphingomyeli‐ nases (aSMase and nSMase) bringing ceramide (Cer) as product. Ceramides are LR modulators believed to alter PM fluidity and favor receptor oligomerization [116]. Cer has been suggested to fulfill a second-messenger function but the evidence for this action is scarce and controver‐ sial, while support is emerging for its indirect impact on cellular signaling resulting from changes in membrane structure. Cer could be further converted to sphingosine by ceramidase, whereas sphingosine 1-phosphate is synthesized from sphingosine by a phosphorylation reaction catalyzed by the sphingosine kinases (SKs) SK1 and SK2, which are highly conserved enzymes activated by numerous stimuli including transactivation induced by IGF-1 [117]. From studies carried out on C2C12 myoblast cell line as progeny of mouse satellite cells, it is clear that IGF-1 evokes two mutually exclusive biological responses (hyperplasia vs. hyper‐ trophy). This cytokine plays a key role in skeletal muscle regeneration as it is able to recruit satellite cells and stimulate myoblast proliferation and myogenic differentiation. As mentioned before, myoblasts must not fuse unless they are withdrawn from the cell cycle. How then, IGF-1 regulates two opposite responses? In recent years, the sphingosine 1-phosphate (S1P) attracts special attention with regard to physiology of resident skeletal muscle satellite cells as well as proliferating and differentiating myoblasts. First, several lines of evidence indicate significant role of S1P in skeletal muscle regeneration and repair [85, 117–122]. The extracel‐ lular action of S1P present in micromolar concentrations in peripheral blood is exerted by binding to five specific cell surface heterotrimeric G protein–coupled S1P receptors (S1P1– S1P5). In turn, S1P agonist levels are tightly controlled by the balance between biosynthesis catalyzed by SKs, reversible conversion to sphingosine mediated by specific and nonspecific lipid phosphatases, and S1P lyase (SPL)-dependent degradation. Second, S1P receptors are coupled to one or more G-proteins so they can elicit distinct and even contrasting final cellular effects (Figure 12). In skeletal muscle cells, major role is played by S1P1, S1P2, and S1P3 receptors [117, 119–122]. Actually, in myoblasts, S1P1 and S1P3 receptors via SK activation negatively regulate the mitogenic effect elicited by IGF-1, whereas S1P2 receptor is involved in myogenic effect of the growth factor (Figure 13). Thus, in myoblasts, SK/S1P axis upon IGF-1 action regulates two opposing biological effects – transducing its myogenic response on one side and inhibiting its mitogenic effect on the other. The IGF-1-dependent transactivation of S1P receptors was also observed in other cell types pointing to the conservation of the IGF-1/SK/S1P circuit in tissues other than skeletal muscle [123]. How IGF-1 does affect SKs leading to two divergent biological effects in skeletal muscle? Among the various signaling pathways activated by S1P in skeletal muscle cells, the activation of ERK1/2 and p38 MAPK, both identified as downstream effectors of S1P2 in response to growth factors, was required for cell proliferation and the stimulation of myogenic differentiation, respectively [124]. The inhibition of ERK1/2 activity with specific U0126 metabolic inhibitor prevented SK1 phos‐ phorylation induced by IGF-1, demonstrating that the activation of SK1 induced by the growth factor was mediated by ERK1/2 [116]. Similarly, the S1P-induced differentiation was prevented

in myoblasts where p38 MAPK was inactivated by the overexpression of the dominant negative mutant or by the use of specific p38 MAPK inhibitors SB203580 and SB239063 [118]. **21** 32 [109-110] [110-111] **21** 37 [111, 112] [112-113]

**21** 38 [105] [106]

**24** 4 [124] [126]

**24** 5 [125] [127]

**24** 9 [126] [128]

**24** 13 [127] [129]

**23** 11 Donati et al. 2013 [124] Donati et al. 2013 [125]

**11. Sphingolipids in skeletal muscle regeneration**

128 Muscle Cell and Tissue

Sphingolipids (SL) and cholesterol (CHOL) create LR in plasma membrane, but it is the biochemistry of SL that is apparently decisive for skeletal muscle biology including its growth and differentiation. PM sphingomyelin is a target for both acidic and neutral sphingomyeli‐ nases (aSMase and nSMase) bringing ceramide (Cer) as product. Ceramides are LR modulators believed to alter PM fluidity and favor receptor oligomerization [116]. Cer has been suggested to fulfill a second-messenger function but the evidence for this action is scarce and controver‐ sial, while support is emerging for its indirect impact on cellular signaling resulting from changes in membrane structure. Cer could be further converted to sphingosine by ceramidase, whereas sphingosine 1-phosphate is synthesized from sphingosine by a phosphorylation reaction catalyzed by the sphingosine kinases (SKs) SK1 and SK2, which are highly conserved enzymes activated by numerous stimuli including transactivation induced by IGF-1 [117]. From studies carried out on C2C12 myoblast cell line as progeny of mouse satellite cells, it is clear that IGF-1 evokes two mutually exclusive biological responses (hyperplasia vs. hyper‐ trophy). This cytokine plays a key role in skeletal muscle regeneration as it is able to recruit satellite cells and stimulate myoblast proliferation and myogenic differentiation. As mentioned before, myoblasts must not fuse unless they are withdrawn from the cell cycle. How then, IGF-1 regulates two opposite responses? In recent years, the sphingosine 1-phosphate (S1P) attracts special attention with regard to physiology of resident skeletal muscle satellite cells as well as proliferating and differentiating myoblasts. First, several lines of evidence indicate significant role of S1P in skeletal muscle regeneration and repair [85, 117–122]. The extracel‐ lular action of S1P present in micromolar concentrations in peripheral blood is exerted by binding to five specific cell surface heterotrimeric G protein–coupled S1P receptors (S1P1– S1P5). In turn, S1P agonist levels are tightly controlled by the balance between biosynthesis catalyzed by SKs, reversible conversion to sphingosine mediated by specific and nonspecific lipid phosphatases, and S1P lyase (SPL)-dependent degradation. Second, S1P receptors are coupled to one or more G-proteins so they can elicit distinct and even contrasting final cellular effects (Figure 12). In skeletal muscle cells, major role is played by S1P1, S1P2, and S1P3 receptors [117, 119–122]. Actually, in myoblasts, S1P1 and S1P3 receptors via SK activation negatively regulate the mitogenic effect elicited by IGF-1, whereas S1P2 receptor is involved in myogenic effect of the growth factor (Figure 13). Thus, in myoblasts, SK/S1P axis upon IGF-1 action regulates two opposing biological effects – transducing its myogenic response on one side and inhibiting its mitogenic effect on the other. The IGF-1-dependent transactivation of S1P receptors was also observed in other cell types pointing to the conservation of the IGF-1/SK/S1P circuit in tissues other than skeletal muscle [123]. How IGF-1 does affect SKs leading to two divergent biological effects in skeletal muscle? Among the various signaling pathways activated by S1P in skeletal muscle cells, the activation of ERK1/2 and p38 MAPK, both identified as downstream effectors of S1P2 in response to growth factors, was required for cell proliferation and the stimulation of myogenic differentiation, respectively [124]. The inhibition of ERK1/2 activity with specific U0126 metabolic inhibitor prevented SK1 phos‐ phorylation induced by IGF-1, demonstrating that the activation of SK1 induced by the growth factor was mediated by ERK1/2 [116]. Similarly, the S1P-induced differentiation was prevented

**Figure 12.** Role of sphingosine 1-phosphate on cell proliferation and migration in myoblasts and activated satellite cells, adapted from Donati et al. 2013 [125]. **23** 8 [119] [118]

**23** 14 Donati et al. 2013 [124] Donati et al. 2013 [125] **Figure 13.** Schematic diagram of the biological actions evoked by S1PR transactivation by some growth factors in C2C12 myoblasts, adapted from Donati et al. 2013 [125].

2

One has to keep in mind, however, that S1P receptors are differently expressed in satellite cells, myoblasts, and muscle fibers, moreover their expression may vary in response to the action of particular factor. PDGF stimulates myoblasts proliferation and motility, while these effects are blocked by SK1/S1P1 signaling axis [126]. In contrast, TGF-β1 was demonstrated to convey its detrimental profibrotic effect through S1P3 receptors (Figure 13) [127]. These observations complement widely known activities of PDGF and TGF-β1 in wound healing. In the second intention healing as it is observed in the severe skeletal muscle injury or late stages of myo‐ pathy, the major role is played by myofibroblasts which cause fibrosis, a hallmark of in which myofibers are replaced by progressive deposition of extracellular matrix proteins [128]. The main task is therefore to facilitate skeletal muscle regeneration rather than repair, as the first one restores tissue structure and contractile function while the latter is limited to structural return. There are efforts observed to improve muscle healing by regeneration rather than fibrosis [129]. Collectively, taking into account the knowledge on how sphingosine 1-phos‐ phate influences RSC and how it might prevent muscle fibrosis, it will be interesting to further investigate in this context the crosstalk between IGF-1 and S1P signaling pathway.

#### **12. Concluding remarks**

Primary stem cells in adult skeletal muscle known as satellite cells drive postnatal muscle growth and regeneration-associated hypertrophy. They reside beneath the basal lamina of the myofibers suggesting close contact between the adjacent cytoskeletons and chemical commu‐ nication between the cells. One of the major unexplored areas of satellite cell biology is the identification of signals that are conferred from adjacent myofibers and the surrounding extracellular matrix. Equally important are soluble endocrine, paracrine, and autocrine factors that maintain satellite cells quiescent and control their preference to activate. For example, the maintenance of skeletal muscle requires notch signaling and greatly depends on delta upregulation for RSC activation [130]. In addition to the loss of notch activation, nonregener‐ ating skeletal muscle produces excessive transforming growth factor (TGF)-β (but not myostatin), which induces unusually high levels of TGF-β pSmad3 and interferes with their regenerative capacity [131]. Thus a balance between endogenous pSmad3 and active notch controls the regenerative competence of muscle stem cells, and the deregulation of this balance in the old muscle microniche interferes with regeneration. The molecular mechanisms that regulate satellite cell quiescence, activation, and self renewal (asymmetric divisions) are not well understood, even though a possible clue for the ambiguous behavior of satellite cells could be associated with the membrane segregation of rafts and bioactive lipids such as PS that seems to accelerate myoblasts fusion into myotubes [132]. It seems plausible to affirm sarcolemmal differentiation as the leader constituent of stimulated skeletal muscle progenitors and subse‐ quent populations of daughter cells (myoblasts, myotubes, and juvenile myofibers) involved in myogenic program. Lipid moiety of plasma membrane is not merely a boundary or the component in intercellular communication. Nowadays, it is widely accepted that specific lipids associate to form functional units (LR/C), creating substructures that actively modify its own composition including proteins and triggering a myriad of different signaling pathways. Lipid segregation seems to account for the adaptability of skeletal muscle to a variety of stimuli, where the critical role is played by the myogenic signals represented by growth factors and cytokines. Cholesterol, isoprenoids, dolichols, and sphingolipids all contribute significantly to the physiological responses of skeletal muscle to injury. These bioactive lipids mediate most, if not all, of the signals elicited at the plasma membrane receptors. Recent advances in muscle research suggest key position occupied by sphingosine 1-phosphate, protein prenylation, and "caveolar" and "noncaveolar rafts" in skeletal muscle regeneration process.
