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.

Fig. 87-3 Morphology of muscle.

en, endomysium; ep, epimysium; f, fascicle; mf, muscle fiber; p, perimysium.

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.

Fig. 87-4 Sarcomere.

A sarcomere extends from Z line to Z line. The thick filaments are made up of myosin and occupy the A band. The thin filaments are made up of actin and make up the I band. The H band is the area in which the thick and thin filaments do not overlap. The M line anchors the thick filaments.

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].

Fig. 87-5 Contractile proteins.

Myosin (M) is located in the thick filaments and is composed of two heavy chains (H) and four light chains located in the head region. The myosin head is divided into two parts: S1 and S2. The thin filaments are composed of actin (A), tropomyosin (Tm), and troponin (Tp).

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.

Fig. 87-6 Sarcotubular system.

The drawing illustrates a myofibril of one sarcomere and the associated membrane systems. m, mitochondrion; pm, plasma membrane; sr, sarcoplasmic reticulum; T, T tubule; tr, triad.

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).

Fig. 87-7 Sarcolemma-associated cytoskeletal structures.

Dystroglycan (Dg) consists of α-Dg, associated with merosin (M), and β-Dl, which binds to dystrophin (D). Syntropin (S) is bound to the C-terminal region of dystrophin. The sarcoglycan complex (Sg) consists of four transmembrane subunits – α, β, γ, and δ – attached to the sarcolemma (Sl).

(Modified from Duggan DJ, et al: Mutations in the sarcoglycan genes in patients with myopathy, N Engl J Med 336:618, 1997).

Table 87-1 Muscle Proteins and Human Genetic Disease

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 http://www.neuromuscular.wustl.edu and http://www.Genetests.com http://neuromuscular_wustl.edu.

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

Nuclear Membrane-Related Proteins: Emerin and Lamin A/C

Emerin is a protein associated with the inner nuclear membrane. It is attached to the inner nuclear membrane by its carboxy terminus and extends into the nucleoplasm. Emerin is composed of 254 amino acids and is coded by a gene on the X chromosome. Emerin is important in the organization of nuclear membrane during cell division [Fairley et al., 1999]. Mutation of this gene causes X-linked Emery–Dreifuss muscular dystrophy [Haraguchi et al., 2004].

Lamins are major components of the nuclear lamina, which is the major structural framework of nuclei in eukaryotic cells. In humans, two forms of type A (A and C) and B (B1 and B2) are present. Lamins have the helical rod portion and nonhelical portions at the amino and carboxy termini. Lamins differ from other intermediate filaments by the presence of a nuclear localization signal close to their carboxy terminus. The lamins interact with themselves, lamin-associated proteins, and chromatin. The lamins play a role in DNA replication, chromatin organization, spatial arrangement of nuclear pores, nuclear growth, and mechanical stability of the nucleus [Loewinger and McKeon, 1988; Maniotis et al., 1997]. Mice with null mutations for lamin A gene show chromatin clumping, nuclear fragmentation, nuclear invaginations, and inclusions in myonuclei at the myotendinous junctions, indicating susceptibility to mechanical stress in nuclear membranes deficient in lamin A [Mittelbronn et al., 2008].

The nuclear lamins belong to type 5 intermediate filaments. Lamin A is expressed during and after differentiation, and lamin B is expressed thoughout development and cell differentiation, but is absent in mature cells. The LMNA gene is located on the 1q21–22 region and has 12 exons. Through alternative splicing, lamins A and C are formed. Lamin A is produced by post-translational modification of precursor prelamin A. Post-translational modification of prelamin A includes farnesylation, proteolytic cleavage of the terminal three amino acids, carboxymethylation, and excision of 15 carboxy-terminal amino acids to produce the mature lamin A. The enzyme zinc metalloproteinase is responsible for the first and second steps in proteolysis. Lamin A has 566 amino acids; lamin C, formed by alternative splicing, has 98 amino acids and shares a similarity with the carboxy-terminus amino acids of lamin A [Jacob and Garg, 2006].

Mutations in the lamin A/C gene cause autosomal-dominant Emery–Dreifuss muscular dystrophy, Charcot–Marie–Tooth disease type 2, dilated cardiomyopathy with conduction defects, Dunnigan-type familial partial lipodystrophy, mandibuloacral dysplasia type B, and restrictive dermopathy [Benedetti and Merlini, 2004; van der Kooi et al., 2002].

Motor Unit

Contraction of a muscle fiber is initiated by depolarization of its anterior horn cell, which is the final common pathway for all cortical and spinal influences that affect movement. Depolarization of the anterior horn cell membrane is propagated along its axon in the peripheral nerve. At the nerve terminal, axon twigs run parallel within the muscle fascicle to the muscle fibers and innervate the fibers at the neuromuscular junction. Each anterior horn cell innervates about 50–200 muscle fibers, depending on the muscle. The anterior horn cell, its peripheral axon, and the innervated muscle fibers compose the motor unit and can be considered a functional unit of the muscle. The muscle fibers within the motor unit are not contiguous but are scattered fairly randomly. In the rat, the motor unit is dispersed over 8–76 percent of the cross-sectional area of the muscle, with some clustering of a few fibers in the motor unit territory [Bodine-Fowler et al., 1990]. When the anterior horn cell discharges, all the muscle fibers innervated by that anterior horn cell contract almost simultaneously. The finer and more coordinated the movement of a muscle, the fewer the fibers in the motor unit. Eye muscles and some hand muscles may have 30–50 fibers in the motor unit, whereas some back muscles have hundreds. In general, the larger the muscle, the larger the size of the motor units [McComas, 1991]. For example, there are about 100 fibers per motor unit in the intrinsic muscles of the hand, 200 in the extensor digitorum brevis, and 900 in the larger muscles.

Neuromuscular Transmission

The neuromuscular junction is formed by specialized portions of the nerve and muscle plasma membrane, which are separated by the synaptic cleft (Figure 87-8). The area on the muscle is the end plate and is located near the center of the muscle at the end-plate zone. The synaptic knob is the terminal dilatation of the nerve axon and contains synaptic vesicles of acetylcholine. The motor neuron action potential is propagated down the motor nerve by saltatory conduction. Na⁺ channels at the nodes of Ranvier produce current. Within the muscle, the motor nerve fiber branches into terminal fibers that individually innervate muscle fibers within the motor unit. The nerve terminals are unmyelinated, and the membrane excitability is maintained by both K⁺ and Na⁺. Antibodies to the K⁺ antigen cause acquired neuromyotonia, such as Isaac’s syndrome [Newsome-Davis et al., 2003; Tan et al., 2008].

Fig. 87-8 Neuromuscular junction.

bm, basement membrane; da, distal axon; jf, junctional folds; pm, plasma membrane; sc, synaptic cleft; tk, terminal knob.

Depolarization of the nerve terminus opens voltage-gated calcium channels at the synaptic knob. As calcium enters the nerve ending, exocytosis of the synaptic vesicles occurs, releasing acetylcholine. Antibodies directed against the voltage-gated calcium channels cause Lambert–Eaton syndrome by reducing the quantal release of acetylcholine [Newsome-Davis, 2004]. Acetylcholine is synthesized from acetyl coenzyme A and choline, and exists in a reserve compartment that is not readily available for release and in a mobilization compartment of vesicles. Each synaptic vesicle contains a quantum of about 10,000 acetylcholine molecules. The vesicles are attached to the active zone of the presynaptic membrane. Fifty to 300 vesicles fuse with a nerve terminus depolarization. The process of vesicle formation, fusion, and release is a complex system involving many proteins [Ruff, 2003].

Acetylcholine combines with specific acetylcholine receptors located on the postsynaptic membrane of the muscle sarcolemma. There are about 15,000/μm² receptors in the junctional fold concentrated at the tops of the secondary folds. The acetylcholine receptor is a transmembrane glycoprotein with a molecular weight of 250,000 daltons and is composed of four subunits: α, β, δ, and γ [Momoi and Lennon, 1982]. The muscle acetylcholine receptor monomer is constructed with two α subunits and one of each of the other subunits in order: α₁, γ, δ, β₁ [Lindstrom, 2003]. The acetylcholine receptor monomer contains two acetylcholine-binding sites and the cation-specific channel, whose opening is regulated. The receptor is barrel-shaped, traverses the sarcolemma, and projects above its surface. The subunits are arranged around the central channel-like staves (Figure 87-9) [Kistler et al., 1982]. The acetylcholine receptors are anchored to dystrophin-related protein complexes and connected to the cytoskeleton by dystrophoglycans and sarcoglycan protein complexes. The structure and function of the acetylcholine receptor are remarkably similar in all species studied. Congenital myasthenic syndromes are caused by genetic mutations of the receptor and other synapse structures, whereas myasthenia gravis is caused by antibodies to receptor proteins.

Fig. 87-9 Acetylcholine receptor.

The receptor projects above the plasma membrane and is composed of five subunits arranged around the central ion channel.

The binding of two acetylcholine molecules with the α subunits of the receptor causes a conformational change in the M2 portion of α-helical structures of each of the five subunits. This change results in a rotational realignment of the helices to open the center of the receptor for a few milliseconds and thereby alter the sodium and potassium permeability by opening ion channels. Increased ion conductance across the sarcolemma causes a localized depolarization of the muscle end plate. A single quantum of acetylcholine interacting with the end plate causes a miniature end-plate potential insufficient to engender a propagated membrane discharge. When depolarization of the terminal axon occurs, 50–300 acetylcholine quanta are released. Sufficient interactions between acetylcholine molecules and receptors cause the summation of potentials to reach the threshold potential for the end plate. Thus, an end-plate potential is achieved, and the depolarization is propagated along the sarcolemma and T tubules. Depolarization of the end plate is a statistical phenomenon; given sufficient numbers of acetylcholine molecules and receptors, an interaction will probably occur, followed by depolarization [Katz and Miledi, 1972]. Interference with the release of acetylcholine or a reduction in the number of receptors lessens the likelihood of successful neuromuscular transmission.

Excitation–Contraction Coupling

The major function of muscle is generating movement or force by contraction. This function is accomplished through a series of events called excitation–contraction coupling, which is the linkage of the electrical and mechanical properties of muscle. Depolarization of the T tubules at the triad is sensed by dihydropyridine (a calcium-channel blocker) receptors, which are associated with charged molecules within the tubular membrane. Movement of these charged molecules, which can be inhibited by dantrolene [Hui, 1983], causes a conformational change in the “feet,” which are structures that bridge the membranes of the T tubules and sarcoplasmic reticulum [Peachey, 1985]. Calsequestrin is a major protein of the sarcoplasmic reticulum that binds calcium and is located close to the sarcoplasmic reticulum calcium efflux channels in register with the “feet” processes [Stadhouders, 1990]. Interaction of these membrane structures at the triad causes the release of calcium from the sarcoplasmic reticulum into the sarcoplasm. During tetany, calcium concentrations decrease in the sarcoplasmic reticulum and increase in the sarcoplasm [Somlyo et al., 1981]. This release of calcium is critical for the mechanics of muscle contractions. Calcium-binding proteins in the sarcoplasm, including calmodulin [Kennelly et al., 1989] and parvalbumin [Heizmann et al., 1990], are essential for regulation of the contractile proteins.

Contraction of muscle is brought about by contractile proteins. These proteins have different forms (isoforms), and the presence of a specific isoform within the fiber determines some of the contraction properties of the muscle. The isoforms are similar but have slightly different properties, such as calcium-binding capacity and adenosine triphosphatase activity. Every fiber is capable of synthesizing all isoforms of the contractile proteins, but the precise composition of isoforms within a muscle fiber depends on the many complex mechanisms that coordinate gene expression and the neural influences that ultimately control fiber typing (see earlier discussion).

When it is released by the sarcoplasmic reticulum, calcium binds with calmodulin; the complex acts as a second messenger that activates the myosin light chain troponin C kinase [Kennelly et al., 1989]. This reaction causes an unmasking of a myosin-binding site by displacing troponin I and tropomyosin. The myosin head swings away from the backbone of the thick filament at the hinge area of the S2 region [Horowits et al., 1989]. The myosin head then attaches to actin at an angle, and the hydrolysis of adenosine triphosphate causes the myosin head to swivel to a more acute angle, moving the chains toward one another (Figure 87-10). Repetition of this event causes shortening of the sarcomere [Vale and Milligan, 2000]. Each attachment, swivel, and reattachment shortens the muscle about 1 percent, or about 14 nm [Ford et al., 1977]. Myosin hydrolyzes adenosine triphosphate slowly, unless combined with actin. Each stroke of the myosin–actin cross-bridge attachment consumes one molecule of adenosine triphosphate: adenosine triphosphate binding to myosin, dissociation of actin and myosin, adenosine triphosphate hydrolysis, reassociation of actin and myosin, and release of hydrolysis products. The rate of stroke cycling and therefore the contraction velocity are dependent on the myosin adenosine triphosphatase isoform [Pollack, 1996]. The exact mechanism of the phase transition that brings about muscle contraction is still not fully known [Brooks, 2003]. The entire process constitutes excitation–contraction coupling.

Fig. 87-10 Myosin head movement.

A and B, During contraction, the myosin head swivels from its resting position (A), toward actin (B). C, With hydrolysis of adenosine triphosphate, the orientation of the head closes to a more acute angle, propelling the thin filament to slide.

The tension generated by a muscle is directly proportional to the number of cross-links between the myosin heads of thick filaments and the actin of the thin filaments, as determined by length–tension relationships [Pollack and Sugi, 1984]. On cross-section, each myosin heavy chain is surrounded by six actin light chains. This geometric array is found in the area of the sarcomere where there are overlapping myofilaments, as in the A band (Figure 87-11). The more foreshortened the sarcomere, the more cross-links between the filaments. However, only the thin filaments occur in the I band. Each muscle has a length at which it generates maximum force and at which the greatest interaction between actin and myosin occurs [Huxley, 1996; Lieber, 1986].

Fig. 87-11 Schematic diagram of myofilament orientation within the sarcomere.

Cross-sections illustrate the relationships of the thick and thin filaments. A, A band. B, H band. C, I band.

Evidence suggests that contraction of a muscle occurs by heavy and light myofilaments sliding toward one another. When a muscle contracts, the distance between the Z lines decreases at the expense of the I and H bands. The A band remains unchanged (Figure 87-12). During stretch, the I band increases in length as the actin filament arrays slide in the direction opposite to the myosin filament arrays; thus, the distance between Z lines increases. This sliding is produced by the formation and breakage of the cross-links between actin and myosin.

Fig. 87-12 Sliding filament mechanism of muscle contraction.

The thick and thin filaments slide toward one another during contraction, causing the distance between the Z lines to shorten.

Neuromuscular transmission is terminated by calcium sequestering within the synaptic knob. Muscle contraction is terminated when the T tubules repolarize; this causes reaccumulation of calcium within the sarcoplasmic reticulum, using a pump that depends on adenosine triphosphate [Peachey, 1985]. Unlike the nerve and muscle plasma membrane, the contraction process has no refractory period. As long as calcium is free in the sarcoplasm and adequate energy stores exist, the muscle will stay contracted. Muscle can stay contracted without excitation of the membrane system, and energy is required for the reuptake of calcium into the sarcoplasmic reticulum. Failure of calcium reuptake from the sarcoplasm when energy stores are depleted inhibits relaxation of the muscle. This mechanism causes rigor mortis and the cramping during exercise found in energy-deficiency disorders, such as McArdle’s disease.

Motor Unit System

The motor unit consists of a single lower motor neuron and the muscle fibers it innervates. The motor unit is the final common pathway of the motor system [Miles, 1994]. The following two control mechanisms are used by the nervous system in generating muscle contraction force:

There is a rank order of recruitment within a motor neuron pool, so that increasingly larger motor units are recruited for increasing demands of force. Smaller motor units are recruited initially [Henneman, 1985]. Similarly, the rate of discharge of motor units is proportional to the degree of force generated. Current information indicates that the firing rates of motor neurons are not controlled individually, but that the nervous system controls the firing rate of motor neuron pools to modulate force generation. This mechanism has been termed common drive [De Luca, 1985]. In general, the recruitment of additional motor units is more important in generating force and power than modulating firing rates [Brooks, 2003].

Motor unit recruitment is the process by which different motor units are activated to produce a specific degree and type of muscle force [Petajan, 1991]. In the graded generation of force, the lower force threshold, slow-twitch motor units are recruited first. Increasing force output raises the firing rate of these first-recruited motor neuron pools in fine gradations. Further force generation requires recruitment of large motor neurons with higher force threshold, fast-twitch motor units. This recruitment causes a 5- to 10-fold increase in the graded force. Normally, a motor unit discharges up to rates of 5–15 Hz before an additional neuron is recruited. The firing rate of the fastest-firing motor unit divided by the number of recruited neurons is normally less than 5 (e.g., three neurons firing at a rate of 15 Hz). When this recruitment ratio is greater than 10 (e.g., two neurons firing at a rate of 20), it suggests a loss of motor units. At maximal exertion, there is fusion of contractions of the slow-twitch motor units. Because of the short twitch duration of the large motor units, they may not achieve contraction fusion with voluntary effort [De Luca et al., 1982]. During normal activity, slow-twitch motor units discharge more frequently and are active more often than fast-twitch motor units [Hennig and Lomo, 1985].

Changes in the firing rate and the number of motor units recruited for a given muscle contraction are under a variety of controls, including the γ afferents, Golgi tendon organs, and Renshaw cells that facilitate or disfacilitate neurons to produce a graded contraction.

Gamma Efferent System

The γ efferent system plays a major role in controlling muscle contraction via its effects on the muscle spindle. The muscle spindle is a specialized sensory organ located within the perimysial connective tissue (Figure 87-13). It is an encapsulated structure containing 2–10 small muscle fibers (intrafusal fibers) and special sensory nerve endings. The intrafusal fibers comprise nuclear bag fibers, nuclear chain fibers, and an intermediate type of fiber. Each has distinct ultrastructural and histochemical characteristics [Barker et al., 1972]. The intrafusal fibers of the spindle are arranged in parallel with the extrafusal fibers of the muscle.

Fig. 87-13 Muscle spindle.

cap, Capsule; get, γ efferent with trail ending; gep, γ efferent with plate ending; II, type II afferent with flower-spray ending; Ia, type Ia efferent with annulospiral ending; nbf, nuclear bag fiber; ncf, nuclear chain fiber.

The sensory endings are of two types [Scalzi and Price, 1972]. The primary endings (annulospiral) are supplied by large, myelinated, rapidly conducting Ia afferent fibers, and end in a spiral or cagelike arrangement around nuclear bag fibers. These endings are sensitive to both stretch of the spindle and the velocity of stretch. Secondary endings of group II sensory nerves terminate in a variety of endings, including cylindrical, flat, and annulospiral or ring-shaped endings, mostly on nuclear chain fibers [Schroder et al., 1989]. Secondary endings are sensitive to the stretch of the spindle.

The fusimotor fibers compose as much as 30 percent of the total number of ventral root fibers. Most are γ efferents, although a few are β efferents [Barker et al., 1972]. Multiterminal plate endings (i.e., flower-spray) are located on nuclear bag fibers from myelinated γ efferents and probably cause dynamic innervation. Trail endings, however, primarily innervate nuclear chain fibers and arise from smaller myelinated and unmyelinated fibers. Trail fibers supply static innervation.

The afferent fibers of primary endings synapse directly on the anterior horn cells, which supply the extrafusal fibers of the same muscle. The secondary ending afferents probably synapse on motor neurons of other muscles [Scalzi and Price, 1972]. Stretch of a muscle causes distortion of the primary endings and generates a signal to the motor neuron to cause contraction. This signal is the mechanism of the deep tendon reflex. If the muscle were shortened, spindle discharge would decrease, causing muscle relaxation; therefore, spindles serve as a feedback mechanism to maintain muscle length and tone.

The γ efferent system operates in concert with the α motor neuron to effect movement [van der Meulen et al., 1972]. Stimulation of the γ motor neuron causes stretching of the nuclear bag fibers, distortion of the primary endings, and reflex stimulation of the α motor neuron, as discussed previously. The γ motor neurons are regulated by several descending tracts, whose main purpose is to maintain the sensitivity to stretch throughout muscle contraction so that different muscle groups can adjust to the needs of postural control.

The other major sensory innervation of muscle is the Golgi tendon organ [Stuart et al., 1972], which is a netlike structure located around the tendon fascicles supplied by fast-conducting myelinated afferent fibers. The output causes stimulation of inhibitory internuncial neurons, which, in turn, synapse on α motor neurons. The sensory organ is arranged in series with the muscle and thus is more sensitive to changes in muscle force than to changes in length. Excessive stretch produces an inhibitory influence on the α motor neurons.

Clinical Biochemistry

Several muscle sarcoplasmic enzymes are used in the clinical evaluation of muscle disease. These enzymes are released from the muscle fiber when it is damaged. Of these enzymes, aldolase and creatine kinase are used most commonly, especially the latter. Creatine kinase exists as several isozymes, including M and B. BB is found almost exclusively in brain tissue, whereas MM is found almost exclusively in muscle and heart tissue. MB is found in several tissues. Determination of creatine kinase isozymes is helpful in differentiating brain from muscle disease, but not muscle from heart disease [Vainzof et al., 1985]. Creatine kinase is not found in the liver or erythrocytes.

The normal range of creatine kinase enzyme activity in the blood varies with age, gender, and daily activities. In an inactive male, however, this range may rise to 2–3 times that of the upper limits. After vigorous extended activity, such as running a marathon or a similar effort, plasma creatine kinase activity may approach 10 times that of normal [Brown et al., 1982]. Elevation of plasma creatine kinase also occurs after trauma, seizures [Wyllie et al., 1985], electroconvulsive therapy [Taylor et al., 1981], and injections. Increased activities of these enzymes are suggestive, but not diagnostic, of myopathic disease. Creatine kinase can also be mildly elevated in neuropathic diseases. Other enzymes released include transaminases, lactate dehydrogenase, carbonic anhydrase III, and pyruvate kinase. None is as useful as creatine kinase. Serum myoglobin also correlates with muscle breakdown [Norregaard-Hansen and Hein-Sorensen, 1982]. Other biochemical methods that have been used in investigating muscle disease include measuring urinary excretion of creatine and 3-methyl histidine. Their clinical application has not been commonly accepted for various reasons.

Electromyography

Electromyography is the recording of electrical activity by a needle electrode of action potentials of muscle fibers firing singly or in groups near the electrode [Daube, 1991].

The electromyography study has three aspects. The first aspect is the recording of activity during insertion of the needle electrode. The insertional activity is a reflection of the mechanical damage caused by the needle. Inserting the needle electrode or adjusting the needle usually causes a brief, spontaneous discharge of the surrounding muscle fibers. Abnormal activity on insertion, such as prolonged discharges or activation of spontaneous trains of discharges, indicates disease.

The second part of clinical electromyography is recording the spontaneous resting properties of the muscle. Normally, muscle is electrically silent during rest. End-plate noise can be recorded if the needle is placed near the end-plate zone. End-plate noise is generated by miniature end-plate potentials and single fiber discharges produced by needle irritation [Shahani and Young, 1985]. Spontaneous activities, such as fibrillations, positive sharp waves, fasciculations, or others, usually indicate underlying disease, most commonly neuropathic disorders. Spontaneous repetitive discharges are found in various neuropathic and myopathic disorders, such as the myotonias.

The third part of electromyography is recording motor unit potentials to evaluate the electrical properties of muscle during contraction. The motor unit potential usually has two or three phases and is 5–15 milliseconds in duration. The voltage usually does not exceed 5 mV. With a primary muscle disease, a frequent finding is brief, small-amplitude, polyphasic potentials. With chronic neuropathic disease, a common finding is prolonged, large-amplitude, polyphasic potentials. With graded contraction, an increase in the firing rate of individual motor units occurs. Further effort causes an increase in the number of recruited motor units and is proportional to the degree of muscle contractions. Thus, motor units are recruited in response to the degree of effort, and there is a recruitment order of motor units that is relatively fixed [Henneman et al., 1974]. Single-fiber electromyography is a technique for studying the characteristics of neuromuscular transmission and the localized organization of muscle fibers within a motor unit [Sanders et al., 1996]. A special needle electrode is introduced into the muscle at multiple sites. The number of single muscle fiber action potentials belonging to one motor unit recorded in a given area gives a quantitative measure of the fiber density. Recording from two fibers of the same motor unit enables one to measure “jitter,” or the mean consecutive difference in time between the firing of the two fibers, which is usually between 10 and 55 milliseconds. An increase in jitter is commonly observed with defects of neurotransmission. Single-fiber electromyography also detects blocking when the second fiber fails to discharge, which is the basis for the decremental response during repetitive stimulation in patients with myasthenia gravis. These subjects are discussed in other chapters.

Muscle Biopsy

Muscle biopsy is the definitive test for diagnosing specific muscle diseases and is indicated in the child with suspected neuromuscular disease; however, muscle biopsy is less likely to yield diagnostic findings if creatine kinase and electromyography readings are normal. Muscle biopsy may not be diagnostic early in the course of an illness when the abnormalities are scant and a sampling error can mask the pathologic condition. The pathologic condition may also escape diagnosis late in the course of a disease if the muscle is so altered that no distinguishing features exist (“end-stage” muscle).

The method of muscle biopsy has been reviewed in detail elsewhere [Dubowitz and Brook, 1973]. It can be performed by either an open biopsy or a needle biopsy technique [Heckmatt et al., 1984]. Either method is relatively simple and can be performed on an outpatient basis. The muscle chosen for biopsy should be one affected clinically, but not severely so.

To obtain optimal results, the material must be handled by experienced technicians and interpreted by those familiar with neuromuscular diseases. Routine histologic stains often used in evaluating a muscle biopsy include the hematoxylin-eosin stain, modified Gomori trichrome stain, periodic acid–Schiff stain for glycogen, and oil red O or Sudan black stain for lipid. The hematoxylin-eosin stain is used most often for evaluating muscle fibers, nuclei, and other cellular constituents. The trichrome stain is used especially for evaluating connective tissue, intramuscular nerves, and certain features of the muscle fiber, such as mitochondria. Other histologic stains are often used for special purposes.

Histochemistry often reveals the most significant information. Adenosine triphosphatase reactions are used to determine fiber typing. The random distribution of the fiber type on the adenosine triphosphatase reaction is a major feature of normal muscle. Normally, there are about equal numbers of type I, IIa, and IIb fibers, although up to 50 percent of a single type can be found in some muscles [Dubowitz and Brook, 1973]. Oxidative stains and the periodic acid–Schiff stain also reflect fiber typing (see Table 87-3). The oxidative reactions that include succinic dehydrogenase and nicotinamine-adenine dinucleotide transreductase are helpful in identifying mitochondria and the sarcotubular system. Many other histochemical reactions facilitate the study of specific diseases, such as the myophosphorylase reaction that aids in diagnosis of McArdle’s disease. Figure 87-14 illustrates the major histologic and histochemical features of muscle.

Fig. 87-14 Muscle histology and histochemistry.

Serial sections are stained as follows: A, Hematoxylin and eosin. B, Modified Gomori trichrome. C, Myosin adenosine triphosphate, pH 9.4. D, Myosin adenosine triphosphate, pH 4.6. E, Myosin adenosine triphosphate, pH 4.3. F, Nicotine-adenine dinucleotide transreductase. G, Succinic dehydrogenase. H, Myophosphorylase. I, Periodic acid–Schiff stain.

Immunohistochemistry is becoming increasingly important for the study of normal and diseased muscle. The absence of dystrophin on immunohistochemical staining of muscle tissue is diagnostic of Duchenne’s and related muscular dystrophies. Certain congenital myopathies can be classified based on immunohistochemical staining of other cytoskeletal proteins [Duggan et al., 1997; Philpot et al., 1995; Wallgren-Pettersson et al., 1995].

Electron microscopy is rarely needed for accurate diagnosis, but it is often necessary in disorders of the mitochondria and to determine the ultrastructure of central cores, nemaline rods, and other inclusions in congenital myopathy [Younger and Gordon, 1996].

Muscle Imaging

Ultrasonography, computed tomography, and magnetic resonance imaging are used in the diagnosis and management of patients with neuromuscular disease [Clague et al., 1995]. Ultrasonography is a cost-effective method for localization of diseased muscle for biopsy purposes. It is useful for imaging dystrophic muscle [Heckmatt and Dubowitz, 1988]. Magnetic resonance imaging is particularly useful for the diagnosis of inflammatory muscle disease. Inflammation is easily identified and can be distinguished from other causes of weakness [Stonecipher et al., 1994].

Molecular Genetics

Techniques for mapping and sequencing the human genome have brought about an entirely new approach to the diagnosis of human neuromuscular disease [Nawrotzki et al., 1996]. High deletion frequencies, unstable tandem repeat sequences, genomic duplications, and triplet repeat expansions are common abnormalities that enable a molecular genetic diagnosis to be made for such diseases as Duchenne’s and Becker’s muscular dystrophy, some forms of limb girdle dystrophy, myotonic muscular dystrophy, congenital muscular dystrophy, and many others (see Table 87-1). Often, the defective gene product, such as dystrophin in the X-linked dystrophies, can be measured or visualized in muscle samples for diagnostic purposes.