**3. Sarcolemma**

**1. Introduction**

108 Muscle Cell and Tissue

Skeletal muscle growth and regeneration is dependent on the activation of mitotically quiescent resident cells known as skeletal muscle satellite cells (SC) located beneath the basal lamina (integral part of basement membrane) on the plasma membrane (sarcolemma) of adult skeletal muscle fibers. Activated by muscle injury including work overload (i.e., weight lifting), satellite cells proliferate making myogenic precursor cells (myoblasts) that migrate to the site of injury and after withdrawal from the cell cycle fuse collectively or with damaged fibers. The fusion process is mediated by plasma membrane proteins, some of which are the receptors for intermediate of lipid metabolism such as sphingosine 1-phosphate (sphingolipid, S1P). A great deal of plasma membrane surface and integral proteins at the extracellular site is glycosylated and prenylated, the processes indispensable for intracellular protein transport (from endo‐ plasmic reticulum to Golgi apparatus, and from trans-Golgi network to the plasma membrane) as well as for lateral and vertical protein translocation within sarcolemma. In adult skeletal muscle, the self-renewing capacity of satellite cells contributes to muscle growth, and regen‐ eration-associated hypertrophy as skeletal muscle-specific adaptation to workload. Hypertro‐ phy also occurs in satellite cell-depleted skeletal muscle, although in this case neither increase in myonuclei in satellite cell-depleted fibers nor the muscle regenerates after BaCl2-induced severe muscle damage [1]. Accordingly, the biochemistry and structural modifications of plasma membrane are seemingly indispensable for the commitment of satellite cells and their progeny of myoblasts to skeletal muscle renewal. In this review, we hypothesized that changes in the sarcolemmal composition of proteome, glycoproteome, and/or lipidome are the major determinants of satellite cells and muscle fibers to regenerate skeletal muscle. From the experiments and clinical observations related to statin-induced myopathy [2–4] as well as the successful efforts aiming to correct plasma membrane integrity by the modification of skeletal muscle plasma membrane fluidity, we conclude that closer examination of the plasma membrane composition and structural organization might shed more light on the molecular

mechanisms of satellite cell commitment to muscle rejuvenation.

Nowadays, it is obvious that skeletal muscle growth and regeneration is firmly linked to the activity of satellite cells adjacent to extrafusal and intrafusal muscle fibers. Intact skeletal muscle encloses satellite cells in the quiescent state, with a dense nuclear chromatin (hetero‐ chromatin), fine rim of the cytoplasm, and little organelles. Covered by a thin layer of basement membrane, the satellite cell rests closely applied to the sarcolemma of the muscle fiber (Figure 1). The notion that satellite cells donate nuclei to a growing or regenerating fiber, one at a time, is widely known from a half of the century [5]. Although quiescent in normal skeletal muscle, satellite cells (named by Mauro, [6]) become activated and recruited to the cell cycle when there is a requirement to increase myonuclear number [7]. In response to signals accompanying skeletal muscle injury, denervation, exercise, or work overload, the activation reverses the morphology of satellite cells to lower chromatin density (euchromatin), expanded cytoplasm,

**2. Skeletal muscle growth and regeneration**

Plasma membrane in skeletal muscle has several exclusive features related to the structure and composition as well as unique characteristics of membranoskeleton. Nonetheless, some properties are fairly common for any membrane as phospholipids spontaneously form lipid bilayers in aqueous environments due to the amphipathic nature of the molecules with a highly hydrophobic "tail" (acyl chains) and hydrophilic "head" (glycosyl or phosphatidyl) moieties. Lipids in membranes are distributed disproportionately accounting for substantial differences in the extracellular vs. intracellular face of plasma membrane (PM). Anyway, a given mem‐ brane has a stable and specific membrane composition dependent on cell type and organelle, and any changes are observed only in certain physiological situation or pathological anomaly. The asymmetry of the external leaflet of PM (exoplasmic) is featured by highly enriched in choline-containing lipids such as phosphatidylcholine (PC) and sphingomyelin (SM), whereas the cytoplasmic leaflet (protoplasmic) is rich in phosphatidylethanolamine (PE), phosphati‐ dylserine (PS), and other phospholipids [phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol-4-monophosphate (PIP), and phosphatidylinositol-4,5-biphosphate (PIP2)]. The latter participates in cell signaling. Despite cross-sectional diversity, membranes posses lateral asymmetry in basal, lateral, and apical regions [12–13]. Lipids are also capable of displaying different phases under different conditions (mesomorphism). Some lipids, however, as cone-shaped lysophosphatidylcholine (LPC) or PE can form nonlamellar finite structures such as spherical micelles or tubular structures in membranes [14]. Nonlamellar prone lipids are involved in membrane fission and fusion processes with the aim of enzymes such as scramblases, flippases, and floppases. Finally, intact PM is extremely elastic as it reseals after mechanical rupture allowing for the separation of cell fragments (i.e., synaptosomes).

As thin (5–10 nm) lipid bilayer in eukaryotes, PM plays several tasks emerging beyond defining simple cellular boundary. It can organize complex tools for transportation (ion channels, transport proteins, pumps, and invaginations for macromolecules) or molecular sensing (receptors, Figure 2). PM is also essential to control intercellular communication (flow of information) through highly dynamic microdomains acting as platforms for molecule inter‐

**Figure 1.** Electron micrograph of a typical myonucleus (*A*) and a muscle satellite cell (*B*). Muscle satellite cells (S) were identified by their location inside the basal lamina (arrowheads) and outside the sarcolemma (arrows) and an inde‐ pendent cytoplasm. In contrast, a myonucleus (M) is located inside the sarcolemma of the muscle fiber and does not contain an independent cytoplasm. Bar, 1 mm., reprinted from Sinha-Hikim et al. 2003 [133].

actions. Skeletal muscle cell plasma membrane is specifically adapted to resist consequences of muscle fiber shortening during contraction. Additionally, sarcolemma periodically invagi‐ nates, giving rise to the transverse tubule (TT) network liable for sensing depolarization by dihydropyridine receptor (DHP) essential to trigger of calcium flux evoked by activation of ryanodine receptor (RY). Dystrophin–glycoprotein complex (DGC), also known as dystro‐ phin-associated protein complex (DAC), is embedded in sarcolemma (found in other cell types such as astrocytes) and plays paramount role in the aforementioned actions (Figure 3). It is composed of several proteins including, dystrophin (DP), dystrobrevin (DB), syntrophin (SP), dystroglycans (α- and β-DG), and sarcoglycans (α-, β-, δ-, and γ-SG). These proteins are assembled in order to transmit lateral force during isotonic twitch. Subsarcolemmal protein assemblies circumferentially aligned in schedule with the Z-disk or peripheral myofibrils are also known as costameres (assemblies of costameric proteins, Figure 4). They physically couple force-generating sarcomeres with the sarcolemma in striated muscle fibers and are thus considered a pitfall of skeletal muscle, a critical component of striated muscle morphology which, when compromised, is thought to directly contribute to the development of several distinct myopathies also termed sarcolemmopathies (Figure 5). Costameric proteins are found in a cholesterol (CHOL) rich membrane fraction pointing to lipid rafts (LR) as spatial locali‐ zation of DGC [15]. Actually, LR are clustered at the level of DP complex through lamininmediated interaction with dystroglycans [16]. Furthermore, CHOL depletion uncouples β-DG from sarcolemmal domains with related impairment of mechanical activity of skeletal muscle [17] (Figure 6), whereas DP repeats which interact with membrane lipids strengthen the sarcolemma providing flexible support to muscle fiber membranes (Figure 7) [18]. Addition‐ ally, DGC spatial organization is vital for physical interface between SP and sodium channels or neuronal nitric oxide synthase (nNOS), although not at the same time. DGC is regularly distributed along sarcomeres and aligned mainly with Z-disks to connect actin cytoskeleton with extracellular matrix (ECM) protein laminin (Figure 8). Laminin together with collagen IV, fibronectin, perlecan, entactins, agrin, and glycosaminoglycans is a component of basement membrane. Extrinsic protein β-dystroglycan is the laminin receptor and binds membranespanning (integral) α-dystroglycan that mediates interactions with DP and DB [19]. At the focal adhesions facing Z-lines, the integrin receptors (α7β1) connect fibronectin/laminin with actin filaments of sarcomeres (Figure 9). Tallin, vinculin, and paxillin are intermediate filaments forming a lever that hooks up integrins with thin filaments [20, 21]. Caveolin-3 is the muscle-specific form of caveolin found mainly as intrinsic (inner leaflet) membrane protein at the sarcolemma and TT [22]. The functions of caveolin-3, β-DG, DP, and SG are controlled by cholesterol and sphingolipid concentrations in the lipid rafts and caveolae [17–18, 23]. In striated muscle, signal transduction through cellular membranes can be regulated by the interaction of the cytoskeleton with caveolae (C) – caveolin-enriched membrane domains [24]. Mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains (lipid rafts, caveolae, and coated pits) that serve as spatiotemporal platforms for interacting signaling proteins [11]. Lipid rafts (single lipid raft is approximately 50 nm in diameter) are defined as detergent insoluble glycolipid (GL)-enriched planar domains (detergent resistant membranes, DRM) highly enriched in cholesterol (CHOL), SM, glycosphingolipids (GSL), and glycosylphospha‐ tidylinositol (GPI)-anchored proteins. To sum up, membrane lipids are classified into three major groups: glycerol-based lipids (glycosyl-glycerides and phospholipids), sphingolipids (SL) with sphingoid-base backbone (SM and GSL), and cholesterol. Similar LR may differ in

actions. Skeletal muscle cell plasma membrane is specifically adapted to resist consequences of muscle fiber shortening during contraction. Additionally, sarcolemma periodically invagi‐

contain an independent cytoplasm. Bar, 1 mm., reprinted from Sinha-Hikim et al. 2003 [133].

110 Muscle Cell and Tissue

**Figure 1.** Electron micrograph of a typical myonucleus (*A*) and a muscle satellite cell (*B*). Muscle satellite cells (S) were identified by their location inside the basal lamina (arrowheads) and outside the sarcolemma (arrows) and an inde‐ pendent cytoplasm. In contrast, a myonucleus (M) is located inside the sarcolemma of the muscle fiber and does not

their size (fusion and fission), as well as in the proportions of lipids and proteins, somewhat modified by pathophysiological processes or nutritional and/or pharmacological interven‐ tions. Caveolae are dissimilar to LR as they are deficient in (GPI)-anchored proteins and poor in CHOL but rich in caveolins, the structural proteins assembled to stabilize membrane invaginations [25]. In extreme situations, PM may be subjected to disintegration, protein misfolding, and aggregation, and finally profound dysfunction causes cell death by necrosis. How these nanodomains are segregated within plasma membrane is a matter of debate, although cholesterol molecules establish closeness of PL, GL, GSL, SM, and proteins. Choles‐ terol (alcohol) acts as "glue" with hydroxyl group that combines with the phosphate head of phospholipids, whereas the hydrophobic steroid section works together with phospholipids acyl chains. Growing body of evidence points to physicochemical forces (intermolecular forces including electrostatic interactions, hydrogen bonds, Van der Waals forces, and hydration forces) which determine asymmetric geometry of membranes both laterally and in crosssection. Moreover, integral proteins also influence lipid structure in the membrane. One might bear in mind that according to lipidomics, more than 1000 different lipid forms are to be found in plasma membrane. To meet the requirements of fluidity, membrane components are also subject to considerable qualitative and quantitative seasonal changes adjusted by the cell [26]. It is determined by the environmental conditions (i.e., cold vs. heat) but also by needs of adaptation such as hyperplasia/hypertrophy or resistance to different types of stress (shear stress, oxidative stress including irradiation). In either case, physicochemical properties of membranes have to facilitate cell signaling and motility. In turn, cell signaling and motility are influenced by the glycosylation status of PM proteins, both integral represented by receptors and peripheral because they are heavily modified on the external leaflet.

**Figure 2.** Schematic illustration of a biomembrane, depicting membrane lipid asymmetry as well as microdomains en‐ riched in particular lipids and those induced by membrane proteins, reprinted from Escriba et al. [12].

**Figure 3.** The dystrophin–glycoprotein complex network. Shown in red are the constituents of the core dystrophin– glycoprotein complex, which copurify as a highly stable complex from skeletal muscle and which show greatly de‐ creased abundance in dystrophin-deficient muscle. α-Dystroglycan and β-dystroglycan (α-DG, β-DG); the sarcoglycan complex (SGC); sarcospan (SPN); α-dystrobrevin-2 (α-Db 2); syntrophin (SYN). Also shown are structural proteins that interact directly with components of the dystrophin–glycoprotein complex, their direct binding partners, and their lo‐ cation within striated muscle cells. Cytokeratins 8 and 19 (K8/K19). Proteins highlighted in blue are present at in‐ creased levels when dystrophin is absent, reprinted from Ervasti 2007 [23].
