Normal Muscle

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Chapter 87 Normal Muscle

Movement is one of the ultimate expressions of the nervous system and depends totally on the contraction of skeletal muscle. As an organ, skeletal muscle is the largest structure of the body and has other functions besides voluntary movement and the generation of force. This chapter reviews the embryology, anatomy, function, and metabolism of muscle and the common diagnostic procedures used for its study.

Embryology and Development

Primitive cells, called premyoblasts, are the precursors of muscle in the embryonic paraxial mesoderm. The paraxial mesoderm coalesces to form segmented epithelial spheres on either side of the notochord, referred to as somites. Craniofacial muscles arise rostral of the first somite from paraxial and prechordal mesoderm [Lu et al., 2002]. Discrete condensations of mesenchymal cells, called dermatomyotomes and located dorsomedial to the notochord, give rise to the axial muscles, and overlying skin and condensations from the lateral somite give rise to the limb muscles (Figure 87-1) [McLennon, 1994; Buckingham et al., 2003]. Connective tissue and tendons arise from the somatopleural mesoderm, somites, and neural crest. Sclerotome, which is ventral in location, forms the skeleton.

Premyoblasts express the paired box transcription factors, Pax-3 and Pax-7. Bone morphogenetic protein 4 (BMP4), released from adjacent neural tube and lateral plate mesoderm, inhibits gene expression of myogenic transcription factors, MyoD and Myf-5. Wnts (Wnt 1, 3, 7a, and 1) and sonic hedgehog signals from adjacent notochord, neural tube, and surface ectoderm activate Myf-5 and MyoD (members of a family of transcription factors called myogenic regulatory factors) in premyoblasts [Pownall et al., 2002]. These committed cells, myoblasts, migrate to the myotome. The myoblasts divide rapidly within the first several weeks of pregnancy, after a quantal cell cycle under the influence of fetal growth factors. The primordial cells stream ventrally and penetrate between the ectoderm and somatopleura. As the cells migrate, they split and recombine, such that individual muscles are formed from several adjacent myotomes. In the limb bud, the cells coalesce into dorsal and ventral masses.

In a coordinated fashion, the MyoD family of growth factors form heterodimeric DNA complexes with other basic helix–loop helix transcription factors to regulate an array of gene expression [Kassar-Duchossoy et al., 2004]. Collectively, the transcription factors govern the assignment to skeletal muscle lineage, migration of progenitor cells from the hypoaxial domain of the dermatomyotome to the limb, condensation of premuscle masses, and formation of primary and secondary myotubes [Cossu and Biressi, 2005].

The maturation of premyoblasts to myoblasts begins with the cessation of DNA synthesis. The postmitotic myoblasts elongate and begin attachment and fusion with other myoblasts, end to end, to form a long and slender primary myotube (Figure 87-2). The process is facilitated by the appearance of several fetal adhesion molecules on the surface of the myoblast [Schnorrer and Dickson, 2004]. Secondary and tertiary myotubes are formed from side-to-side fusion of myoblasts to existing myotubes and require innervation for the process. Satellite cells provide nuclei to the polar ends of the elongating myotube. Individual muscles begin to form after the initial myotubes appear and the ingrowth of innervation. In the absence of innervation, maturation beyond primary myotubes does not occur, and the muscle develops abnormally.

At about 4 weeks’ gestation, the contractile proteins appear and polymerize to form myofilaments, which are produced predominantly in the polar region of the myotube. By the fifth week, the myofilaments aggregate to form the myofibrils, with simultaneous formation of characteristic striations. A microscopic cross-section of the muscle fiber at this stage reveals a tubular structure, with the contractile proteins located around the periphery of the fiber and the center containing the nuclei. The basic shape of an anatomic muscle is apparent by 7 weeks. Movement begins simultaneously with innervation at 8 weeks. In the latter stages of early fetal development, neuron sprouting is intense. Initially, individual mammalian muscle fibers are multiply innervated. Between 16 and 25 weeks’ gestation and in association with secondary myotube formation, all but one synapse is eliminated. There is constant denervation and reinnervation as neuromuscular interaction forms physiologic innervations of the motor units. Multiple motor neurons compete for innervation. Competition weakens some synapses and strengthens others, so that, ultimately, a single input prevails. This process of synaptic elimination is stimulated by local factors produced by muscle [Wyatt and Balice-Gordon, 2003]. Several growth factors and receptors appear on the cell surface to facilitate integration of nerve terminals to the muscle cell. The fetal (g) form of the acetylcholine receptor is present until 31 weeks’ gestation, after which only the adult type (e) is found. Rhythmic movements that are spinally mediated start shortly after innervation. Most of the fetal membrane proteins that promote growth and differentiation, including the major histocompatibility gene products [Kaparti et al., 1988], are not present on normal mature muscle.

At about 16 weeks’ gestation, the nuclei begin migrating to the subsarcolemmal position. Most muscle fibers achieve the histologic features of mature muscle by 25 weeks, though a few continue to have central nuclei. At this stage, the fibers are rounded and loosely arranged within the prominent endomysium. The characteristic polygonal cross-sectional shape becomes apparent after birth.

Distinctive muscle fiber types (see next section) develop after innervation. Primitive fibers, typed as IIc, are undifferentiated and are believed to be precursors of the mature fibers: types I, IIa, and IIb [Landon, 1982]. The myosin in type IIc fibers is immunologically distinct from that of other fiber types [Thornell et al., 1984]. Type I fibers appear at about 18 weeks and are smaller than type II fibers until after birth, when they become somewhat larger. Most muscle fibers in the 20- to 26-week-old fetus are type IIc fibers. Type IIa and IIb fibers appear during the last month of gestation. Only a small percentage of type IIc fibers persist after birth. Differentiation into distinct fiber types is determined by neural influences and causes the biochemical and physiologic diversity of mature fibers. Calcineurin, a Ca2+-calmodulin-regulated phosphatase, plays a critical role in the differentiation of physiologic properties in the muscle fiber [Matlin et al., 2001]. Continuous firing of motoneurons causes an increase in intracellular calcium, which activates calcineurin. Activated calcineurin binds and dephosphorylates two kinds of transcription factors: nuclear factor of activated T cell (NFAT) and myocyte enhancer factor 2 (MEF2), resulting in significant activation of the slow myosin heavy chain 2 gene (slow MyHC2) promoter. In contrast, in fast fibers, high-amplitude calcium sparks induced by infrequent phasic firing of the motor nerves are insufficient to keep activation of calcineurin. When calcineurin is inactivated, phosphorylated NFAT cannot enter the nucleus and the slow fiber-specific program is downregulated, resulting in the predominant transcription of genes encoding fast fiber-specific proteins [Jiang et al., 2004].

Without innervation, muscle fibers atrophy and undergo cell death. Similarly, spinal motor neurons become nonviable without innervating muscle, so that muscle and neuronal development are ultimately interdependent [McLennon,1994].

Satellite cells are mononuclear muscle cells that lie beneath the basement membrane of mature muscle fibers. They were first described by Alexander Mauro in 1960. Satellite cells are abundant in fetal muscle, compose 1–5 percent of the total nuclei of mature muscle, and diminish with aging. The typical satellite cell during embryonic development migrates from dorsal somite to dermatomyotome. Transcription factor Pax-7 is a key protein involved in myogenic determination of satellite cells. Transcription factor Pax-3 is needed for the migration of these cells from the dorsal somite and Pax-7 is required for the transition to forming mature muscle cells. The dependency on Pax-7 is felt to rest on the stage of maturation of the animal. Pax-7 are not essential for maturation of satellite cells in adults [Lepper et al., 2009]. The satellite cells express M-cadherin, c-met, foxk-1, and CD 34 antigens [Wernig et al., 2004; Beauchamp et al., 2000]. Muscle injury activates satellite cell proliferation and maturation into myofibers. This is marked by expression of muscle regulatory factors proteins Myf-5 and MyoD [Martin et al., 2006].

While most of the satellite cells are derived from the primordial somite, they can also be derived from hematopoietic stem cells and vascular progenitor cells (endothelium, pericyte, and mesangioblast) [Shi and Garry, 2006]. They are the primary stem cell for regeneration of injured muscle and are capable of forming not only individual muscle fibers, but also complete muscle fascicles [Alameddine et al., 1989; Anderson, 2000; Schultz, 1985].

The subsequent growth and strength of muscle after birth depend on functional demand, sex, age, training, and other factors. Enlargement of a muscle results from hypertrophy of muscle fibers, rather than from growth of new fibers. Large muscles with a greater workload, such as the quadriceps, have muscle fibers with greater cross-sectional diameters than do other muscles, such as the diaphragm. There is no difference in fiber growth in males and females until puberty, when a noticeable increase appears in males. Training increases the cross-sectional diameter of the muscle fiber, and disuse decreases the diameter. Muscle atrophy associated with aging is caused by loss of muscle fibers, rather than by fiber atrophy, and a decrease in satellite cells which can produce skeletal muscle cells.

Muscle is quite malleable and is under a number of neuronal, hormonal, and usage influences that determine muscle function and contractile properties [Pette, 2001]. This plasticity of muscle results in changes in fiber type distribution, contractile proteins, calcium uptake of the sarcoplasmic reticulum, and altered energy metabolism.

Anatomy and Structure

Morphology

The muscle as an organ comprises all the individual anatomic muscles. Each anatomic muscle has an origin and an insertion on the skeleton, and bridges one or more bony articulations. Contraction of the anatomic muscle thus causes movement across a joint. The anatomic muscle is enclosed within a thick sheet of connective tissue called the epimysium. Separating the muscle into individual fascicles is the perimysium, which is contiguous with the epimysium. Within the perimysium are the nutrient blood vessels, intramuscular nerves, and muscle spindles (Figure 87-3). The confluence of the perimysial and epimysial connective tissue forms the tendons at either end of the muscle belly. The muscle fascicle, which is bounded by perimysial connective tissue, is a wedge-shaped structure comprising several hundred individual muscle fibers. Surrounding each muscle fiber is a network of fine connective tissue, called the endomysium. The terminal axons and a rich capillary network reside within the endomysium. Within the muscle fiber are large groups of myofibrils, which contain myofilaments. Myofilaments are made of contractile proteins.

Although a muscle may have almost any shape, all muscles are composed of almost identical muscle fibers. Muscle architecture can be specialized for force production or excursion, depending on the number of muscle fibers arranged in parallel or in series, respectively. A muscle fiber, or muscle cell, is a multinucleated, long, tubular structure that varies in diameter from 10–20 μm in the infant to about 50–70 μm in the adult. Fiber length varies considerably, depending on the size of the muscle and whether the fibers are arranged in series or in parallel orientation; it can measure several centimeters and can span the entire muscle.

Sarcomere

The most striking feature of skeletal muscle on microscopic examination is the characteristic striations (“striated” muscle), which are especially prominent under polarized light. The striations are caused by the difference in the refractive index of the contractile proteins that are in phase with each other in the myofibrils. A repeating unit is called a sarcomere; it comprises interdigitating myofilaments and is bounded by the Z line (Figure 87-4). The sarcomere has a length of about 2.5–3.0 μm and a diameter of 1.0 μm. The Z disk anchors the thin filaments of actin that extend into each adjacent sarcomere and are located in the I band. The M line bisects the sarcomere and also divides the A band, which is formed by an array of thick filaments composed of myosin. The area within the A band in which the thin and thick filaments do not overlap is called the H band. A refers to anisotropic and I refers to isotropic in connection with the refractile indexes under polarized light.

Contractile and Sarcomeric Proteins

The two major proteins involved in muscle contraction are actin and myosin, which jointly interact with adenosine triphosphate to convert chemical energy to mechanical work. The actin system is complex. The actin molecule has four major domains surrounding a cleft containing adenosine triphosphate or adenosine diphosphate, and is tightly bound to a divalent cation [Pollard, 1993]. There are at least 48 classes of actin-binding proteins. The thin filaments are composed of two chains of about 400 polymerized actin molecules arranged in a double helix (Figure 87-5). The actin molecule has a molecular weight of about 42,000 daltons and is rich in 3-methyl histidine, a unique amino acid found exclusively in muscle. The actin molecule is the same in both fast and slow skeletal muscle fibers. Alpha-actinin is a major component of the Z disk and plays a role in the binding or cross-linking of actin to other cytoskeletal structures [Critchley, 1993]. Capping proteins anchor the barbed end of actin to the Z line [Cooper et al., 2010]. Mutation in the α-actin gene has been associated with congenital myopathy with actin filament aggregate myopathies and nemaline myopathy [Schroder et al., 2004; Nowak et al., 1999], hypertrophic and dilated cardiomyopathies [Marston and Hodgkinson, 2001]. The role of actin structure in its interaction with myosin and in its effect on polymerization of actin molecules is thought to cause the phenotypic variability in clinical presentation. The mechanochemical myosin molecule is a complex protein with a molecular weight of about 500,000 daltons. The myosin molecule consists of at least six polypeptides with two heavy chains and four light chains. The contractile properties of a muscle fiber are related to myosin. Myosin heavy chains are different for type I, IIa, and IIb fibers. The two heavy chains form a double helix that extends along the tail of the molecule. The light chains are of the following three types: two alkali-dissociated and one phosphorylated. The light chains are different for type I and II fibers, but are the same for IIa and IIb fibers. The structure of myosin is hexameric, composed of two heavy, two alkali-dissociated, and two phosphorylated chains. There are nine isoforms of the myosin heavy chain: I, IIA, IIB, IID/X, IIA, α, neonatal, embryonic, and extraocular. The various isoforms of the heavy and light chains create a spectrum of isomyosins that are specific for fast-twitch skeletal muscle, slow-twitch skeletal muscle, cardiac muscle, smooth muscle, brain, and platelets [Whalen, 1985]. Embryonic and neonatal muscle contains distinct sets of isomyosins. Skeletal muscle isomyosins are determined by neural influences (see above). “Pure” muscle fibers contain a single myosin heavy chain isoform. “Hybrid” fibers contain more than one myosin isoform, as in muscle fibers transitioning from one fiber type to another during reinnervation. The maximal force, velocity, and power produced by muscle fibers are determined to a large extent by the properties of myosin isoforms [Lutz and Lieber, 2002]. Mutations in myosin gene MYH7 cause hyaline body myopathy with or without cardiomyopathy and familial cardiomyoneuromyopathy. Respiratory failure occurs with mutations in exons 37 and 38 of the MYH7 gene [Goebel and Laing, 2009; Selcen et al., 2002].

At one end, the myosin molecule evaginates to form globular heads. The myosin head is called the S2 region and is attached to the heavy chains. The S2 region is flexible at either end [Huxley, 1982]. One of each type of light chain is associated with the globular heads. The LC1 (light chain 1), or P light chain, somehow modulates the contractile response by specific phosphatase and kinase, and exists as P1 and P2 isoforms [Westwood et al., 1984]. The cross-links between actin and myosin occur at the globular heads during excitation. Thus, the myosin molecule is a bipolar structure, with the heavy chains of the tail ordered along the backbone of the thick filament and the heads projected from the side. The heads have a repeating helical arrangement that forms about three cross-bridges per repeating unit [Harrington and Rodgers, 1984]. C proteins exist as several isoforms and can be seen as cross-stripes on the A band on electron microscopy [Fishman, 1993]. The function of C proteins is unknown but is probably regulatory of cross-bridge movements.

Actin and myosin have an inherent affinity, and troponin and tropomyosin are regulatory proteins that enable the actin–myosin interaction to be controlled by the calcium ion [Grabarek et al., 1990]. Tropomyosin is another helical structure, comprising two long filaments that reside within the groove of the actin chains [Smillie, 1993]. One tropomysin molecule binds one troponin complex and positions tropinin molecules regularly along the actin filament with a periodicity of 385 A. The troponin complex holds tropomyosin in an “off” or “on” state of contraction position on the actin helix, depending on the level of calcium [Lehman et al., 2001]. There are at least two isoforms of tropomyosin. Fast-twitch skeletal muscle contains both α and β isoforms; slow-twitch muscle contains only β-tropomyosin [Heeley et al., 1985]. Because of the close association with actin, tropomyosin probably also has a structural role.

The troponin molecules are located periodically along tropomyosin and comprise three subunits. Troponin T anchors troponin to tropomyosin. Troponin C binds calcium (Ca2+), which induces a conformational change of the protein and results in activation of actomyosin adenosine triphosphatase, which causes contraction [Gergeley et al., 1993]. Troponin I inhibits actomyosin adenosine triphosphatase. The contraction of striated muscle is regulated by the troponin (Tn) complex, which acts as a Ca2+ sensor. Ca2+ binding to the regulatory sites of troponin C (TnC) strengthens the interaction of TnC with troponin I (TnI), and weakens the inhibitory activity of TnI for actin and the affinity of troponin T (TnT) for tropomyosin (Tm). Tm then moves toward the groove of the helical actin filament, switching on the myosin active site, which leads to muscle contraction. Tn binds to actin-Tm in the absence of Ca2+, and the changes in interactions among these thin filament components in the presence of Ca2+ promote strong binding of myosin to actin [Gomes et al., 2002, Brown and Cohen, 2005]. Mutations of sarcomeric genes for troponin TI and tropomyosin are some of several causes of nemaline myopathy [Wallgren-Pettersson, 2002] and cardiomyopathies [Gomes et al., 2004].

Titin, the largest known protein of about 3–3.7 megadaltons, constitutes about 10 percent of myofibrillary proteins and is important for maintaining tension during stretch and recentering the sarcomere after relaxation [Tskhovrebova and Trinick, 2003]. Opposing molecules of titin span the sarcomere. The NH2 termini of the titin molecules attach to the Z disk, attaching myosin to the Z disk. The COOH ends extend and overlap in the M band, interconnecting myosin and actin, and providing elasticity to the sarcomere. It is encoded by a single gene (TTN0), which is located on the long arm of chromosome 2. The protein spans 3–4 megadaltons and consists of repeating immunoglobulin and fibronectin 111 domains. The first 200 amino acids of the titin molecule reside at the periphery of the Z disk and mark its boundary. Titin interacts with several proteins, including telethonin, ankyrin, filamin, nebulin, and α-actinin. Titin plays a major role in myofibrillogenesis, and over- or under-expression of titin leads to disruption of assembly of the Z disk and sarcomere. It has a major function as a “molecular blueprint” that specifies the precise assembly of structural regulatory and contractile proteins of sarcomere, and it gives the sarcomere its distinct biomechanical properties and integrity during stretch, relaxation, and contraction [Kontrogianni-Konstantopoulos et al., 2009]. Mutations in the titin gene cause familial dilated cardiomyopathy [Geruli et al., 2002].

Nebulin is another large protein of the skeletal muscle sarcomere and measures 500–800 kDa. The COOH terminus is attached to the Z disk, while the NH2 terminus projects into the I band and is closely associated with actin. The NH2 terminal binds to the actin-capping protein, and the COOH terminal binds to titin and myopalladin. Nebulin is encoded by the NEB gene, which is located on the short arm of chromosome 2 [Donner et al., 2004]. Nebulin regulates the length of thin filaments in sarcomeres. Mice with null mutations of the NEB gene have variable length and generate less force. The role of the NEB gene is felt to be in stabilizing thin filaments at a determined length and is critical for generation of optimal force. Like titin, nebulin interacts with several structural and functional proteins [Kontrogianni-Konstantopoulos et al., 2009]. Mutations of nebulin causes nemaline myopathy [Lekhotari et al., 2006] and distal myopathy [Wallgren-Pettersson et al., 2007].

Obscurin is the third and most recently discovered member of a family of giant proteins involved in maintaining the structure of the sarcomere. It measures 720–900 kDa and is encoded by the OBSCN gene, located on the short arm of chromosome 1. Immunohistochemical techniques indicate that obscurin is localized at the M line and Z disk in skeletal and cardiac muscle. Obscurin binds and interacts with numerous muscle proteins, including titin, ankyrin, calmodulin, and myomesin. Obscurin plays an important role in myofibrillogenesis by allowing lateral alignment and fusion of myofibrils into larger bundles. It also has an important part in assembly and stabilization of A bands and M bands in the sarcomere. Mutations of the obscurin gene produce hypertrophic cardiomyopathy by causing loss of binding with titin [Arimura et al., 2007].

Sarcotubular System

A major constituent of the muscle fiber is the sarcotubular system (Figure 87-6). The sarcolemma is the plasma membrane of the muscle cell and is surrounded by the basement membrane and endomysial connective tissue. The sarcolemma is an excitable membrane and shares many properties with the membrane of the neuron. The T tubules are contiguous with the sarcolemma and extend into the interior of the muscle fiber as a tubular system in communication with the sarcolemma. Depolarization of the sarcolemma is propagated throughout the interior of the muscle fiber through this system [Peachey, 1985]. The T tubules project into the interior of the muscle fibers in the area of the junction of the I band and A band, where they come in immediate contact with a second tubular system within the sarcoplasm called the sarcoplasmic reticulum. The T tubule, bounded on either side by the sarcoplasmic reticulum, is a triad. The sarcoplasmic reticulum forms a fine plexus around the myofibrils. Excitation of the sarcolemma and T tubules causes release of calcium from the sarcoplasmic reticulum and initiation of contraction by the myofilaments.

Several important proteins are associated with the junction of the T tubules and the sarcoplasmic reticulum. The voltage sensors of the T-tubule calcium channels are regulated by dihydropyridine receptors. Ryanodine receptors mediate the calcium release of the sarcoplasmic reticulum during muscle activation. Calcium adenosine triphosphatase pumps calcium back into the sarcoplasmic reticulum during relaxation. Calsequestrin is a calcium-binding protein that increases the capacity of calcium in the sarcoplasmic reticulum [Franzini-Armstrong, 1999]. Some patients with myasthenia gravis, especially associated with thymoma, have anti-skeletal muscle antibodies to ryanodine receptor antigens, as well as to titin [Skeie et al., 2003; Suzuki et al., 2009]. Autosomal-dominant and autosomal-recessive mutations of the ryanodine receptor gene (RYR) cause central core disease [Ferreiro et al., 2002; Jungbluth et al., 2005], myopathy with type 1 fiber dominance [Morrison, 2008], and central core disease with rods [Scacheri et al., 2000].

Other features seen on microscopic examination of muscle are nuclei and the sarcoplasm and its organelles. Normally, nuclei are located beneath the sarcolemma, and each muscle fiber has thousands of nuclei. The sarcoplasm contains many of the elements found in the cytoplasm of other tissues. Most important are the mitochondria, which are located primarily in the intramyofibrillary space near the Z line and adjacent to the A bands. Numerous glycogen granules and fat droplets are located in the same areas.

Cytoskeletal Proteins

The structural integrity of the muscle is maintained against the physical forces of contraction exerted on the sarcolemma membrane by an intricate system of cytoskeletal proteins (Figure 87-7). These proteins, through a complex pattern of arrangement, anchor the internal structure of the muscle to the basement membrane. Advances in techniques of molecular biology have led to identification and characterization of several of these proteins (Table 87-1).

Table 87-1 Muscle Proteins and Human Genetic Disease

Protein Location Disease
PLASMA MEMBRANE
Dystrophin Xp21 DMD BMD, DMD
α-Sarcoglycan 17q12–q21 LGMD-2D
β-Sarcoglycan 4q12 LGMD-2E
γ-Sarcoglycan 13q12 LGMD-2C
δ-Sarcoglycan 5q33–34 LGMD-2F
Dysferlin 2p13 LGMD-2D, Miyoshi’s myopathy
Caveolin-3 3p25 LGMD-1C
EXTRACELLULAR MATRIX
α2-Laminin 6q2 Merosin-deficient CMD
Collagen VI 21q22 Ullrich’s CMD, Bethlem’s myopathy
SARCOPLASMIC
Calpain-3 15q15.1 LGMD-2A
TRIM32 9q31–34 LGMD-2H
Myotubularin Xq28 Myotubular myopathy
Desmin 11q21–23 Desmin-related myopathy
Plectin 8q24–qter Epidermolysis bullosa simplex with late-onset muscular dystrophy
GLYCOSYLATION-RELATED
Fukutin 9q31–33 Fukuyama CMD
FKRP 19q1 LGMD-D2, CMD-1C
POMGnT1 1p32–34 Muscle-eye-brain CMD (O-mannose β-1,2-N-acetylglucosaminyl transferase)
POMT1 9q34 Walker–Warburg CMD (O-mannosyltransferase-1)
SARCOMERIC PROTEINS
Titin 2q LGMD-2J
Myotilin 5q22–34 LGMD-1A
Telethonin 17q11–12 LGMD-2G
Actin 1q42 Nemaline myopathy
Tropomyosin-3 1q21–23 Nemaline myopathy
Tropomyosin-2 9p13 Nemaline myopathy
Nebulin 2q21–22 Nemaline myopathy
Slow troponin T 19q13 Nemaline myopathy
Ryanodine receptor (RYR1) 19q13.1 Central core disease, central core disease with rods
Myosin heavy chain (MyH 7) 14q12 Myopathy with hyaline bodies, cardiomyopathy, cardiomyoneuropathy
NUCLEAR PROTEINS
Emerin Xq28 EDMD
Lamin A/C 1q11–21 LGMD-1B

BMD, Becker muscular dystrophy; CMD, congenital muscular dystrophy; DMD, Duchenne muscular dystrophy; EDMD, Emery–Dreifuss muscular dystrophy; LGMD, limb girdle muscular dystrophy.

Recent updates from www.neuromuscular.wustl.edu and www.Genetests.com neuromuscular_wustl.edu.

(Adapted from Vainzof M, Zatz M: Protein defects in neuromuscular diseases, Braz J Med Biol Res 36:543–555, 2003.)

Dystrophin

The identification and characterization of dystrophin led to an understanding of the role of cytoskeletal proteins in providing strength and stability to the muscle membrane. Dystrophin is a large protein molecule of about 427 kilodaltons and is composed of 3685 amino acids. It constitutes 0.01 percent of total muscle protein and 5 percent of the sarcolemmal cytoskeletal proteins [Hoffman et al., 1987]. The protein has the shape of a rod, is about 150 nm long, and is arranged as an antiparallel homodimer. It is abundant at the myotendinous junction [Samitt and Bonilla, 1990] and at the postsynaptic membrane of the neuromuscular junction [Byers et al., 1991]. Immunocytochemical studies have localized dystrophin to the cytoplasmic surface of the sarcolemma [Bies et al., 1992]. Dystrophin and two other structural proteins, spectrin and vinculin, are located at the sites of attachment of sarcomeres to the cytoplasmic membrane overlying both the I bands and M lines [Porter et al., 1992]. These findings demonstrate that dystrophin forms an integral part of a muscle’s cytoskeleton and links the contractile apparatus to the sarcolemma. There is no difference in the expression of dystrophin in fast- and slow-twitch muscle fibers or in intrafusal muscle fibers [Zubrzycka-Gaarn et al., 1988].

Dystrophin has a binding site for the filamentous form of actin at the 5′ end or domain. The central rod domain contains a number of repeats; it demonstrates homology with spectrin and gives the molecule a flexible rod-shaped structure [Pons et al., 1990]. The rod domain is highly conserved in vertebrates, and antibodies against the human dystrophin rod domain cross-react with amphibian and other vertebrate species [Sherratt et al., 1992]. The third domain is rich in cysteine [Suzuki et al., 1992], and the fourth domain, the carboxy terminus, binds with the dystrophin–glycoprotein complex [Ervasti and Campbell, 1991]. The dystrophin–glycoprotein complex consists of dystroglycans (α and β), the sarcoglycans (α,β,γ,δ,ε), sarcospan, the syntrophin, and dystrobrevin [Crawford et al., 2000]. The peripheral members of the complex include neuronal nitric oxide synthase, caveolin, laminin, and merosin [Watkins et al., 2000]. Even though the RNA levels of these proteins are normal in patients with dystrophin deficiency, the protein expression is reduced. The deficiency of these proteins contributes further to the pathologic effects of dystrophin deficiency [Chen et al., 2000].

The gene coding for the protein dystrophin is located on the short arm of the X chromosome near the region Xp21. The dystrophin gene is the largest gene identified so far, covering more than 2.5 megabases (Mb), and contains at least 79 exons [Gutmann and Fischbeck, 1989]. The large size of the gene is responsible for the high rate of mutation in this gene. A 14-kilobase dystrophin messenger RNA is expressed in skeletal, cardiac, and smooth muscle cells. Smaller amounts are expressed in the brain. Isoforms of dystrophin, which are smaller in size, are expressed in nearly all tissues examined. Deficiency of the brain isoform of dystrophin, Dp 140 and Dp 71, is associated with cognitive handicaps. Dystrophin in the brain is important in maintaining synaptic plasticity and participates in cellular signaling pathways involved in modeling synapses [Blake and Kroger, 2000]. Dystrophin isoform Dp 260 is expressed in retina alone [Pillers et al., 1999].

The most important function of dystrophin is to provide mechanical support and structural integrity to the sarcolemma [Lapidos et al., 2004]. Dystrophin is part of the linkage system from the actin cytoskeleton out through the sarcolemmal membrane and basal lamina to the extracellular matrix. Dystrophin plays an important role in maintenance of calcium homeostasis in muscle fibers [Blake et al., 2002]. Muscle fibers deficient in dystrophin show an increase in intracellular calcium and are derived from leaks in the cell membrane and sarcoplasmic reticulum [Carlson, 1998]. Hypercontracted muscle fibers seen on histological examination are the earliest signs of increase in intracellular calcium. The increase in intracellular calcium is believed to have a role in muscle fiber necrosis and apoptosis [Sandri and Carbano, 1999]. Dystrophin within the dystrophin–glycoprotein complex participates in this intracellular signaling. It plays a role in regulation of calcium-dependent kinases in the cell by interacting with calmodulin [Anderson et al., 1996]. Alteration in calcium signaling in dystrophin-deficient muscles plays a major role in altering the normal maturation process of regenerating and developing muscle fibers [Chen et al., 2000].

Through its linkage to neuronal nitric oxide synthase, dystrophin has a role in regulation of blood flow through muscle during exercise. Abnormalities in modulation of neuronal nitric oxide contribute to muscle fiber damage in dystrophinopathies [Sander et al., 2000]. Deletions or abnormalities of the dystrophin gene cause a deficiency or absence of dystrophin, resulting in the X-linked Duchenne’s and Becker’s muscular dystrophies.

Dystrophin–Glycoprotein Complex

Biochemical investigation of dystrophin led to the discovery of a large oligomeric complex of glycoprotein localized in the sarcolemma. This dystrophin–glycoprotein complex binds the muscle cytoskeleton to a component of the extracellular matrix, laminin [Campbell and Kahl, 1989; Ervasti et al., 1990]. The dystrophin–glycoprotein complex consists of cytoskeletal, transmembrane, and extracellular components, and is composed of two subgroups of protein complexes: the dystroglycans and sarcoglycans.

Dystroglycan consists of a transmembrane and an extracellular component encoded by a single messenger RNA. The precursor protein is processed by an unknown protease to α- and β-dystroglycan molecules. The size of dystroglycan molecule is expressed in various tissues and its molecular weight varies, based upon differential glycosylation in those tissues. In muscle α-dystroglycan weighs 43 kDa, and in brain it weighs 120 kDa. The synthesized protein undergoes glycosylation as it passes from endoplasmic reticulum to the sarcolemmal membrane. The α-dystroglycan is a heavily glycosylated protein located on the extracellular side of the sarcolemmal membrane. The glycosylation is mediated by six glycosyl transferases: protein-O-mannosyl transferase 1 (POMT1), protein-O-mannosyl transferase 2 (POMT2), protein-O-mannose 1,2-N-acetylglucosaminyltransferase 1 (POMGnT1), fukutin (FKTN), fukutin-related protein (FKRP), and LARGE. Mutations in these enzymes cause myopathies, and brain and eye malformations [Mercuri et al., 2009].

Alpha-dystroglycan plays an active role in basement membrane assembly. It binds to β-dystroglycan (transmembrane component) with its carboxy-terminal region on the intracellular side. Alpha-dystroglycan binds to several proteins – laminins, agrin, and perlecan – to anchor into the extracellular matrix. Beta-dystroglycan binds to the WW domain and EF hands of the dystrophin molecule. The interactions between beta-dystroglycan, caveolin and dystrophin regulates the insertion of the dystrophin molecule into the sarcolemma. Rapsyn, an essential protein for formation of the neuromuscular junction, binds to β-dystroglycan. The dystroglycan complex plays a pivotal role in linking the cytoskeleton to the extracellular matrix. Dystroglycans with dystrophin are important in the formation of the neuromuscular junction. Binding of agrin to α-dystroglycan is a critical step in the stabilization of acetylcholine receptor clustering at the neuromuscular junction. The agrin-mediated signaling is mediated by muscle-specific tyrosine kinase (MuSK), which is part of the protein complex at the neuromuscular junction. Antibodies directed against MuSK result in myasthenia gravis (the acetylcholine receptor antibody-negative form). The dystroglycan complex also plays an important part in normal migration of neurons in the cerebral cortex [Martin and Freeze, 2003; Michele and Campbell, 2003]. The assembly and integration of these proteins occur in the presence of dystrophin; in its absence, these proteins may not be degraded, properly assembled, or integrated into the sarcolemma [Ibraghimov-Beskrovnaya et al., 1992].

No primary mutations in dystroglycans have been identified in human disease. Defective glycosylation of α-dystroglycans causes congenital muscular dystrophies with brain involvement: Fukuyama’s congenital muscular dystrophy, muscle-eye-brain disease, Walker–Warburg syndrome, and limb girdle muscular dystrophy type 2I [Mercuri et al., 2009].

Sarcoglycans

The sarcoglycan complex consists of α-sarcoglycan (adhalin), β-sarcoglycan, γ-sarcoglycan, and δ-sarcoglycan [Bonnemann et al., 1995]. The sarcoglycan genes α, β, and γ are located on 17q12–21, 4q12, and 13q12 chromosomes, respectively. Expression of α- and γ-sarcoglycan is limited to skeletal and cardiac muscles [Yamamoto et al., 1994]. The sarcoglycans form a tetrameric complex in the Golgi apparatus, and then transition to the sarcolemma as a completely assembled structure. The sarcoglycans consist of a single transmembrane domain, a small intracellular domain, and a large extracellular domain, and their molecular weight ranges from 35 to 50 kilodaltons. The sarcoglycan complex, which is composed of α-, β-, γ-, and δ-sarcoglycan, is part of the dystrophin-associated glycoprotein complex; it acts as a link between the extracellular matrix and the cytoskeleton, confers structural stability to the sarcolemma, and protects muscle fibers from mechanical stress during muscle contraction. Alpha-sarcoglycan is present only in cardiac and skeletal muscles, but the other sarcoglycans are found in smooth muscle cells as well. Gamma-sarcoglycan bind to dystrophin, and δ-sarcoglycan can bind to β-dystroglycan. The sarcoglycans interact with filamin, a protein that is important for actin reorganization and which is involved in signal transduction associated with cell migration.

Mutations in any of the sarcoglycan genes cause destabilization of the complex, produce a decrease in the amount of all sarcoglycan proteins, and cause the different forms of limb girdle muscular dystrophy [Strauband Campbell, 1997]. The diseases associated with sarcoglycan gene mutations (α-sarcoglycan [SGCA], limb girdle muscular dystrophy type 2D; β-sarcoglycan [SGCB], limb girdle muscular dystropy type 2E; γ-sarcoglycan [SGSG], limb girdle muscular dystrophy type 2C; δ-sarcoglycan [SGCD], limb girdle muscular dystrophy type 2F) are rare disorders in the general population but represent a sizable proportion of all muscular dystrophies with normal dystrophin (about 10–20 percent of cases). Limb girdle muscular dystrophy type 2D is the most common sarcoglycanopathy, followed by types 2C and 2E; the rarest form is type 2F [Boito et al., 2003; Manzur and Muntoni, 2009].

The measurement of α-sarcoglycan is a useful screening test to look for sarcoglycan gene mutations in patients with muscular dystrophy [Duggan et al., 1997]. In sarcoglycan knockout mice, α-sarcoglycan has been successfully injected and expressed in tibialis anterior muscle of mice with adeno-associated virus 1 as a vector [Rodino- Klapac et al., 2008]. A phase 1 human trial for limb girdle muscular dystrophy type 2D, with gene transfer of human sarcoglycan with adeno-associated virus, is in progress (http:/www.clinicaltrials.gov).