2.5. Skeletal muscle cell
2.5.1. Muscle types
All three muscle types, skeletal, cardiac and smooth, are composed of elongated cells specialized for contraction. Smooth muscles, as the name implies, are non-striated, mainly found in vascular system, digestive tract and in uterus. The non-striated muscle cells are mononucleated and actin and myosin are less ordered than in striated muscle cells. The contraction of smooth muscle cells is slower though of greater extent and can be sustained longer. Cross-striated cardiac muscle is confined to the heart and is rhythmically contractile. Cardiac muscle cells have one nucleus but otherwise the ultrastructure reminds more that of skeletal muscle cells. The rate of the heart beating is under nervous control but many individual cells do not receive direct innervation. The other cross-striated muscle type, skeletal muscle, forms the major muscle component of the body. It is organized into muscles that are responsible for the gross and fine movements of limbs and the maintenance of body position and posture (Tortora & Grabowski 2003).
Skeletal muscles are organs specialized for rapid force production. They consist of a heterogeneous population of multinucleated, striated myofibers held together by connective tissue. The connective tissue that surrounds both individual myofibers and bundles of them, and in which a rich blood supply and a rich supply of nerves travel, is essential for force transduction. At the end of the muscle, the connective tissue continues as a tendon or some other arrangement of collagenous fibers that attaches the muscle to tissues such as those of the skeleton, forming a myotendous junction (MTJ). Skeletal muscle cells are coordinated directly or indirectly by the nervous system and capable of highly complex actions. One or more nerves supply each skeletal muscle, while each myofiber receives a single terminal branch of an alpha efferent axon, which ends at a neuromuscular junction (NMJ) (Williams et al. 1989; Tortora & Grabowski 2003).
2.5.2. Myogenesis
Skeletal muscle fibers are highly differentiated cells, which are formed in various regions of the body. Skeletal muscle development is a multistep pathway, in which mesodermal precursor cells are selected to form myoblasts that are withdrawn from normal cell cycle and subsequently differentiate (Buckingham 1994)(Fig. 2.). When skeletal muscle cells differentiate, thousands of structural and regulatory molecules assemble into the semicrystalline sarcomeric contractile units. As a consequence of this precise assembly, many different classes of proteins function together to convert the molecular interactions of actin and myosin efficiently into the macroscopic movements of contractile activity (Gregorio & Antin 2000).
Myoblasts fuse to form long multinucleated myotubes in which the assembly of myofilaments, as well as the production of muscle-specific proteins, begins. During the differentiation the organization of the cellular organelles and the plasma membrane of the myoblasts changes dramatically, with the consequent formation of a single functional unit. This process involves extensive reorganization of the constituents of the cytoskeleton, the microtubule organizing sites being relocalized at the surface of the nuclei in myotubes, in marked contrast with the classical pericentriolar localization (Lu et al. 2001). Also new muscle specific organelles such as the sarcolemma, the sarcoplasmic reticulum (SR) and the transverse (T-) tubules are generated. (Tassin et al. 1985). Apparently also novel membrane trafficking pathways are needed to communicate with the new organelles and membrane domains. One example of profound reorganization of the existing membrane compartments during myotube formation is reorientation of the Golgi elements from polarized juxtanuclear to perinuclear and interfibrillar distribution (Tassin et al. 1985; Rahkila et al. 1996). Also ER seems to diffuse to a common network between the myonuclei. Simultaneously part of the ER differentiates into SR by the gradual displacement of generic ER proteins by SR-specific proteins. In parallel to this, formation of the T tubules takes place (Flucher et al. 1993) assisted by amphiphysin and caveolin-3 (Lee et al. 2002). In adult myofibers SR forms a prominent membrane compartment that is morphologically strictly organized in a cross-striated fashion. However the localization of the RER, as well as the relationship between SR and ER, have remained obscure until our work (III).
2.5.3. Satellite cells-repair units
About 5 percent of all the myonuclei seen in the light microscope at the periphery of myofibers belong to the satellite cells, a cell population distinct from the myoblasts (Schultz 1989). Satellite cells attach to the surface of the myotubes and are thus situated between the basal lamina and the plasma membrane of the mature myofiber. Satellite cells are responsible for muscle maintenance and the extensive repair and regeneration that skeletal muscles are capable of after injury. They are quiescent myogenic precursor cells, which become activated following disruption of the sarcolemma and muscle necrosis in muscle injury. Satellite cells proliferate and differentiate into myoblasts. As long as the basal lamina remains intact in the injury, the myoblasts fuse with each other to form myotubes, which then mature into a new fiber or fuse with an existing one (Schultz 1989; Russell et al. 1992). The regeneration process is identical to myogenesis during fetal development and is similar irrespective of the injury. In contrast, disruption of the basal lamina results in fibroblast repair of the injured site with scar tissue formation (Bodine-Fowler 1994).
2.5.4. Studying muscle cells in culture
Satellite cells can be induced to proliferate and make up an enriched source of myoblasts that can be extracted from the muscle tissue and propagated in culture. Numerous cell lines have been established from these stem cell populations. Satellite cell derived myoblasts can be cultured to mimic different aspects of myogenesis, from proliferation to withdrawal from the cell cycle, fusion into myotubes, and expression of a gene subset encoding contractile proteins, although the individual events are much slower than during in vivo differentiation (Gregorio & Antin 2000).
Studies on membrane trafficking and other cellular events in muscle cells have mainly been carried out with myoblasts. Established muscle cell lines like the rat L6 or mouse C2C12 cell lines are commercially available. These mononucleated cells are much-used tools, because they are relatively easy to cultivate, and they can be induced to fuse to form multinucleated myotubes on culture dishes. Their differentiation stops, though, at a certain point, and there are aspects of myogenesis, such as the full recapitulation of contractile function and generation of different fiber types or establishment of subcellular domains within the mature myotube, that cannot be achieved in muscle cell cultures (Neville et al 1997). Expression of desmin is the first sign of muscle differentiation. Subsequently, other proteins taking part in myofibrillogenesis appear in precise order and timing of expression. In cultured myotubes the typical array, but not all, of muscle specific proteins are subsequently expressed (Yablonka-Reuveni & Rivera 1994). The main disadvantage of the use of myotubes, though, is the big differences that exist between the cell lines and those that depend on culture conditions.
Since the differentiation is far from complete, the situation in myotubes cannot totally correspond to that in mature myofibers. Mature myofibers have been used in the studies on vesicle trafficking and other cellular events very seldom because their cultivation is rather difficult. In some studies myofibers have been mechanically stripped from muscle tissue but such myofibers cannot survive in culture. In our laboratory we have utilized a method to isolate mature myofibers and culture them on dishes originally developed by Bekoff and Betz (1977). My aim was to use mainly these mature, fully differentiated muscle cells in my studies.
2.5.5. Myofiber
The cellular units of skeletal muscle are the myofibers, each a long cylindrical structure surrounded by a plasma membrane, the sarcolemma. The subcellular architecture of skeletal muscle, shown in Figure 3, is very different from that of mononucleated cells. Muscle fibers are 10 to 100 µm in diameter and from a few millimeters to several centimeters long, and contain up to several thousand nuclei derived from the fusion of myoblasts in fetal and postnatal life. Most of the myofiber nuclei are located peripherally beneath the sarcolemma. The fibers are further composed of myofibrils, membranes, and cytoskeletal network, which anchor the contractile fibrils to the sarcolemma. Myofibrils are composed of repeating contractile units known as sarcomeres, perhaps the most highly ordered macromolecular structures in eucaryotic cells (Gregorio & Antin 2000).
Each sarcomere consists of thick and thin filaments whose arrangement is largely responsible for the cross-striated banding pattern observed under light and electron microscopy. Sarcomeres are delineated at their ends to Z-lines where thin actin filaments of opposite directions are linked together by -actinin dimers (Luther 2000). Z-line is located in the middle of the I-band, which appears lighter in a light microscope and contains mainly actin filaments. Polymers of myosin molecules form the darker A-band. The A band is bisected by a light region called the H band, the major component of which is creatinine kinase. Running through the midline of H band is the M line in which the thick filaments are anchored by several myosin-binding proteins. Thick filaments are connected to giant titin molecules expanding to half of a sarcomere, from Z-line to M-line. Titin is thought to function as a spring and a ruler defining sarcomere length after muscle contraction (Gautel et al. 1999), which happens when actin filaments interact with the myosin filaments so that the thin filaments move past the thick filaments toward the center of the sarcomere thus shortening it.
Although all skeletal myofibers have the same basic sarcomeric organization, a number of distinct types have been described according to structural, physiological and biochemical criteria. Skeletal muscle fibers can be generally classified as fast or slow twitch, based on their contractile and metabolic properties and associated patterns of gene expression (Hughes 1998). Red slow twitch oxidative fibers (type I), are involved in sustained, tonic contractile events and maintain intracellular Ca2+ concentrations at relatively high levels (100-300nM). In contrast, white fast twitch glycolytic fibers (type II) are used for sudden bursts of contraction and are characterized by brief, high-amplitude Ca2+ transients and lower ambient Ca2+ levels (<50nM). These properties of skeletal muscle fibers are dependent on the pattern of motor neuron stimulation, so that tonic motor neuron activity promotes the slow fiber phenotype while infrequent motor neuron firing results in fast fibers (Olson & Williams 2000). Structurally, when compared to type II myofibers, type I myofibers tend to be narrower, have thicker Z and M bands, have more glycogen, and their sarcoplasm is rich in mitochondria. The molecular basis for the functional diversity of myofibers is the expression of specific isoforms of most of the proteins involved in muscle contraction and relaxation. Myofiber classification is based on contraction speed and other physiological properties but predominantly according to specific myosin heavy chain (MyHC) isoforms (Schiaffino & Reggiani 1996; Ralston et al. 2001).
Morphological details vary in different muscle fibers. Skeletal muscles respond to changes in physiological demands by remodeling the architecture of individual fibers. Sarcomeres are added or removed when muscles are held at abnormally long or short lengths, and myofilaments are added or removed when muscle fibers function against abnormally heavy or light loads (Trotter 2002). This leads to changes in overall mass of the tissue. Also the spatial relationship among muscle cells and other components of muscle tissue can change and gene expression can be reprogrammed to alter specialized metabolic and contractile properties of myfibers (Olson & Williams 2000).
2.5.5.1. Protein components and membrane skeleton
The primary protein components of skeletal muscle fibrils, myosin and actin, and the tropomyosin and troponins associated with actin, constitute more than 75% of the total protein of the muscle fiber. The remaining proteins, like titin, nebulin, α-actinin or myomesin, are essential in regulating the spacing, attachment, and precise alignment of the myofilaments.
In skeletal and cardiac muscle, sarcolemmal dystrophin associates with various proteins to form a protein complex, which is thought to play a structural role in linking the actin cytoskeleton to the extracellular matrix. Myofiber function and survival are dependent on this link, which stabilizes the sarcolemma during repeated cycles of contraction and relaxation, and transmits force generated in the muscle sarcomeres to the extracellular matrix (Petrof et al. 1993). Integrins (McDonald et al. 1995) and the dystrophin-associated glycoprotein complex (DGC) take part in maintaining this anchorage. The components of the DGC have been described in detail (Ohlendieck 1996). An extracellular component of it, α-dystroglycan, provides a physical link between the extracellular matrix and the intracellular cytoskeleton. The precise distribution of the DGC on the sarcolemma is not known, though dystrophin is concentrated at the sarcolemma of skeletal muscle fibers in longitudinally oriented strands and in costameres, regions of the sarcolemma that overlie Z and M. The most likely candidate to a protein responsible for coordinating the distribution of proteins at the sarcolemma is actin (Williams & Bloch 1999).
Cytoskeletal structures containing intermediate filaments, like vimentin, desmin and nestin, form a physical link to connect the contracting subunits to the sarcolemma in striated muscles. Intermediate filaments may have an important role in cellular organization during myogenesis and in maintaining structural integrity in mature myofibers by redistributing the stress caused by contractile activity (Vaittinen et al. 1999). In mature myofibers vimentin expression is completely down-regulated, whereas nestin is expressed at low levels adjacent to NMJ and MTJ. Desmin expression increases continuously with advancing maturation and it accumulates finally at the margins of Z-lines, anchoring the Z-lines of adjacent myofibrils together and taking part in connecting myofibrils to the plasma membrane (Lazarides 1980; Tokuyasu et al. 1985).
2.5.6. Specific myofiber membrane compartments and their functional significance
The muscle surface membrane consists of the plasma membrane proper (sarcolemma), and the T-tubule system. The sarcolemma and the T- tubule system are continuous but have distinct lipid and protein compositions. The myofiber plasma membrane contains also two kinds of specialized areas, namely the NMJ and the MTJ.
2.5.6.1. Sarcolemma
Treatment with collagenase or other enzymes is needed to strip connective tissue and basal lamina from the sarcolemma. The exposed surface of the muscle fiber is smooth with cross striation pattern that conforms to the underlying myofibrils. The A band and the Z disc are slightly protruded compared to the I bands, and they all are laterally aligned in register. The degree of fiber stretching before and during fixation causes variations in the surface contour.
A notable feature of the sarcolemma is that it is studded with caveolae, which are considered as a specialized type of lipid rafts. In muscle caveolae contain muscle specific caveolin-3. In differentiating skeletal muscle caveolin-3 has been shown to associate with the developing T-tubules, but in mature skeletal muscle caveolin-3 is restricted to sarcolemmal caveolae and is no longer detectable in T-tubules (Parton et al. 1997). Caveolin-3 co-fractionates with dystrophin and DGC proteins (Song et al. 1996). The exact localization of caveolin-3 with respect to defined surface markers has been studied by Rahkila and co-workers (2001). They found out that the entire muscle sarcolemma seems to be an organized array of caveolin-associated raft and nonraft domains comprising a mosaic of t-tubule domains, sarcolemmal caveolae and β -dystroglycan domains.
2.5.6.2. T-tubules and SR
The A-I junctions in the myofibrils are surrounded by T-tubules. T-tubules are regarded as an extension of the plasma membrane forming junctions with the sarcoplasmic reticulum. The best-known function of T-tubules is to rapidly spread changes in membrane potential throughout the muscle fibers, but they might also serve as irradication channels to provide extracellular fluid direct access to the core of the thick muscle fibers.
T-tubules have been identified as continuous elements running transversely over several myofibrils, which are penetrating to all levels of the myofiber. They are an extensive surface-connected system of membranes, which develop and maintain a protein and lipid composition distinct from the sarcolemma (Flucher 1992; Parton et al. 1997). Some studies have provided evidence for an internal T-tubule compartment, which subsequently fuses with the sarcolemma (Flucher et al. 1993). In other studies it has been suggested that T-tubules form from the repeated budding of caveolae aided by amphiphysin (Lee et al. 2002). Muscle specific caveolin-3 might be required to generate the unique protein and lipid composition of the T-tubule system (Parton et al. 1997), and amphiphysin II has been found essential for organization and normal morphology of the skeletal muscle T-tubules. Amphiphysin II colocalizes with ankyrin and plays a role in T-tubule branching, in anchoring T tubules to their places, and in organizing protein components of T-tubules (Razzaq et al. 2001). Ankyrin-1 isoform has been found to localize to the M line where it binds to obscurin. This interaction may provide a direct link between the SR and myofibrils (Bagnato et al. 2003).
Calcium must be available for the reaction between actin and myosin for contraction to occur. After contraction, the calcium must be removed. This rapid delivery and removal of calcium is accomplished by the combined work of the SR and the T-tubule system. The SR surrounds myofibrils like a net stocking. One network of SR surrounds the A band, and another network surrounds the I band. Where the two networks meet, at the junction of A and I bands, the SR forms terminal cisternae. The SR controls the level of intracellular Ca2+ in cardiac and skeletal muscles by storing and releasing Ca2+. It is known that the SR initially develops as ER and that, as the muscle cell differentiates, it becomes greatly enriched in SR-specific proteins (Volpe et al. 1992; Villa et al. 1993). Three proteins first purified from the SR are the calcium ATPase (SERCA), calsequestrin(CLQ) and ryanodine receptor(RyR). SERCA is responsible for pumping calcium into the lumen of the SR during relaxation, CLQ is the most prominent of intralumenal calcium-binding protein that greatly increases the SR capacity for calcium, and RyR is responsible for calcium release during muscle activation. The most abundant SR protein outside the SR-T-tubule junction is SERCA, which is normally distributed in tubular elements surrounding the Z line and M lines, as well as in elements aligned with the longitudinal axis of the myofiber (Williams & Bloch 1999). In the lumen of the SR, the major protein is CLQ (Jorgensen et al. 1977), an acidic protein that binds to calcium with moderate affinity and high capacity. CLQ is specifically targeted to the junctional SR by its acidic carboxy-terminal end (Nori et al. 1993). CLQ and RyR are functionally coupled. Triadin is an abundant membrane protein in the junctional SR, where it colocalizes with the RyR. It anchors CLQ to the junctional face membrane and mediates the functional coupling between the RyR and CLQ in the lumen of the SR (Franzini-Armstrong et al. 1987; Guo & Campbell 1995). Conversely, all cells contain SR-like specialized domains but in much smaller amounts. Some non-muscle cells, such as the Purkinje cells of the cerebellum, actually have extensive SR-like domains containing muscle-specific isoforms of RyRs and calsequestrin (Franzini-Armstrong 1999). T tubules are located between adjacent terminal cisternae of the SR forming a triad. SR-T-tubule junctions and their association with myofibrils develop in a series of consecutive steps (Flucher 1992; Flucher et al. 1993). The formation of junctions between the two membrane systems occurs concurrently, initiating molecular changes in both membrane systems (Takekura et al. 2001).
When a nerve impulse arrives at the muscle membrane, the plasma membrane depolarizes, and there is a rush of Na+ ions into the muscle cell. The depolarization is transmitted into the depths of the cell along the membranes of the T system. Na+ ions signal the SR to release Ca2+ in to the cytosol, initiating contraction in each myofibril. This series of events is called excitation-contraction (e-c) coupling. Several proteins that are specifically localized to the SR-T-tubule junction play essential roles in e-c coupling. The T-tubular dihydropyridine receptor (DHPR) senses the voltage across the membrane, and activation of this receptor leads to the release of Ca2+ from the SR (Flucher 1992). The RyR/ Ca2+ release channel is localized in the junctional SR and is responsible for the Ca2+ release from the Ca2+ stores. Both RyR and DHPR are needed for appropriate muscle development though neither of them is needed for T-SR docking or for the targeting and/or association of CLQ and triadin in the junctional SR (Felder & Franzini-Armstrong 2002).
2.5.6.3. NMJ
Each myofiber is innervated by an axon terminal and they respond to impulses conducted by motor neurons of the spinal cord or brain stem. A single motor neuron may contact some tens over one thousand myofibers, but each myofiber is innervated by one nerve cell and one axon terminal only. The specialized structure at the contact site between the terminal branches of the axon and muscle is called the motor end plate or the NMJ. At the NMJ the membrane is thrown into deep clefts increasing the receptor area in the myofiber (Ishikawa & Shimada 1982). NMJ is usually located in the middle third of a myofiber. Nerve cells not only serve to instruct the muscle cells to contract but also exert a trophic influence on the muscle cells, which is necessary to maintain their structural integrity. Typically three to five nuclei cluster in the NMJ and protein synthesis as well as many other biochemical processes are very prominent in this junctional area. The hallmark of the NMJ is the high local concentration of the acetylcholine receptor and of several associated proteins, as well as that of mRNAs encoding them (Ralston et al. 1997).
2.5.6.4. MTJ
The MTJ is the morphologically distinct interface between muscle and tendon. Ultimately the contractile forces generated by active myofilaments get transmitted to the tendon through this interface. At the MTJ the end of the muscle fiber abruptly tapers. Characteristic of its membrane domain are longitudinal projections and invaginations, which form many finger-like cytoplasmic projections, mingled with the collagen fibrils of the tendon. The morphology of the interface between a muscle fiber and the tendinous connective tissue looks like an adhesive joint. The folds increase the interfacial area by at least an order of magnitude over the cross-sectional area of the muscle fiber. They also ensure that the stresses applied to the interface are experienced mainly as shear stresses (Williams et al. 1989)
Structurally, the MTJ consists of the actin filaments that extend from the last A-band, actin-binding proteins that bundle the actin filaments together, proteins that link the actin filament bundles to the sarcolemma, transmembrane proteins that link to extracellular components, the external lamina, and proteins that link the external lamina to the collagen-fibril rich matrix outside it. Current evidence supports the view that skeletal muscle fibers have two parallel systems for linking intracellular and extracellular structural proteins, namely the dystrophin-DPG system (Bao et al. 1993) and the α7β 1 integrin system (Trotter 2002). Also desmin is shown to transmit force from myofibrillar force generators to the muscle surface and to the MTJ (Lieber et al. 2002).
2.5.6.5. Membranes of exocytic machinery
In the multinucleated muscle cells the whole exocytic transport machinery appears to be organized differently when compared to the organization of the exocytic machinery in the mononucleated cells (Rahkila et al. 1998). In mononucleated cells ribosomes attached to the surface of RER membranes clearly indicate its location. To locate RER in skeletal myofibers is very difficult, if not impossible, to do by mere morphology.
In ultrathin sections of a myofiber it is difficult to distinguish single ribosomes from single glycogen particles, although they do differ slightly in size and electron density, and glycogen has been shown to locate preferentially at the I bands, along with the glycogenolytic enzymes (Dolken et al. 1975). Horne and Hesketh (1990) found that according to immunostaining of large subunits ribosomes locate mainly to the A bands. But the myosin mRNA localized by in situ hybridization is accumulated primarily at the periphery of muscle cells that are actively involved in myosin synthesis, and even when present in the interior, it shows no preferential association with the A bands. The compartmentalization of myosin isoforms within a muscle cell suggests that myosin might be assembled directly into thick filaments at sites where it is synthesized (Gauthier 1990). In chicken anterior latissimus dorsi (ALD), ribosomes were found to be located between thick filaments, often aligned in rows suggesting that ribosomes are located within the filament lattice to be available for local myosin synthesis (Gauthier & Mason-Savas 1993). It was not shown though which, if any of the ribosomes are involved in the synthesis of new myosin.
During skeletal muscle differentiation, the Golgi complex undergoes a dramatic reorganization (Tassin et al. 1985; Ralston 1993), from the classic, compact juxtanuclear position to dispersed elements that form a belt around each of the myotube nuclei and extend between the nuclei along microtubules like strings of pearls (Rahkila et al. 1997). Each myofiber Golgi complex is made of thousands of small dispersed elements. There is an average of 100 Golgi elements per nucleus, the amount being roughly of the same magnitude as that found in other mammalian cells (Ralston et al. 1999). Golgi distribution at the NMJ seems constant in all fibers and independent of fiber type, but otherwise Golgi distribution is fiber type-specific. In slow-twitch, type I fibers, about 75% of all Golgi elements are located within 1 µm from the sarcolemma, and each nucleus is surrounded by a belt of Golgi elements. In contrast, in the fast twitch type II fibers, most Golgi elements are in the fiber core, and most nuclei only have Golgi elements at their poles. (Ralston et al. 1999; Ralston et al. 2001).
Centrosomal proteins, microtubules, ER exit sites, and Golgi elements are linked and affected by activity. Whether one of them determines the localization of the others is less clear, because very little is known of the link between ER exit sites and microtubules. ER exit sites have been reported to be mostly immobile in HeLa and similar cell types (Hammond & Glick 2000), but their organization changes during muscle differentiation (Lu et al. 2001; Ralston et al. 2001).