Muschi-Anatomie,Fiziologie Si Biochimie

5 Muscle: Anatomy, Physiology, and BiochemistryJody A. dAntzig • EugEniA C. PAChECo-PinEdo • yAlE E. goldmAn

93

KEY POINTS

Muscles have a complex developmental process and structure.

Various types of muscle fibers are specialized in terms of metabolic requirements, fatigue susceptibility, speed, and power.

The structure, function, and protein isoform expression can change rapidly and are sensitive to the type of innervation and level of activity (i.e., plasticity).

The smallest functional unit of muscle, the sarcomere, is com-posed of an almost crystalline array of filamentous proteins that transduce metabolic energy into motion and work.

Muscles are connected to the skeleton through collagenous tendons and the epimysium.

Skeletal muscle contraction is controlled by the central nervous system through motoneurons, the neuromuscular junction, and the intricate internal membrane system of the muscle fiber.

Force is transmitted to the exterior through two sets of protein cell adhesion complexes: integrins and dystroglycans.

Mature muscle fibers can regenerate after insult using components of the myogenesis process.

Approximately 660 skeletal muscles support and move the body under the control of the central nervous system. They constitute up to 40% of the adult human body mass. Most skeletal muscles are fastened by collagenous tendons across joints in the skeleton. The transduction of chemi-cal energy into mechanical work by the muscle cells leads to muscle shortening and consequent movement. A high degree of specialization in this tissue is evident from the intricate architecture and kinetics of the intracellular mem-brane systems, the contractile proteins, and the molecular components that transmit force extracellularly to the base-ment membrane and tendons. Muscle cells normally exhibit wide variations in activity level and are able to adapt in size, isoenzyme composition, membrane organization, and energetics. In pathologic states, they often become decon-ditioned. These examples of plasticity can be surprisingly swift and extensive. This chapter outlines the structure and function of muscle and its relationship to the associ-ated connective tissue. It also introduces the basis for the highly adaptive response to altered functional demands and diseases. Two excellent Web sites can be accessed for further information.1,2

MUSCLE DEVELOPMENT

During embryogenesis, connective tissue, bone, and skeletal muscle are derived from mesodermal cells of the somites (Fig. 5-1). Developmental studies have shown that highly regulated sequential expression of transcription factors such as Sonic Hedgehog, bone morphogenetic proteins, and oth-ers3-5 are secreted from nearby structures, including the neu-ral tube, notochord, dorsal ectoderm, and lateral mesoderm.3 These patterning factors participate in the commitment and delamination of mesodermal muscle precursors, which later migrate to the limb buds under the influence of other mor-phogenetic transcription factors.6-8 Once in the limb bud, the cells proliferate and differentiate under the stimulation of muscle regulatory factors such as myogenesis determining factor (MyoD), myogenic factor 5 (Myf-5), myogenic regula-tory factor 4 (Mrf-4), and myogenin6,8; Myf-5 strongly pro-motes myoblast proliferation, whereas MyoD predominantly induces cell cycle arrest and differentiation (see Fig. 5-1).

The migrating mesodermal precursors withdraw from the cell cycle, differentiate into spindle-shaped myoblasts (see Fig. 5-1), and begin to synthesize embryonic isoforms of muscle-specific proteins9 (Fig. 5-2A). Myoblasts align in columns and fuse to produce multinucleated primary myo-tubes (see Figs. 5-1 and 5-2B and C). The contractile organ-elle, the myofibril, comprises long columns of sarcomeres and self-assembles on cytoskeletal scaffolding (see Fig. 5-2D and E).10-12 Myofibrils first appear near the periphery of the myotubes (see Fig. 5-2D) and fill in toward the center of the newly formed skeletal muscle fibers. Myofibrillogenesis con-tinues until the cytoplasm is packed with parallel, laterally aligned sarcomeres (see Fig. 5-2E). Myoblasts continue to proliferate, and later generations elongate, fuse, and differ-entiate within the basal laminae of the primary myotubes, forming independent secondary myotubes.11,12 The nuclei migrate from the center to the periphery, where they remain in mature, multinucleated muscle fibers (see Fig. 5-2D and E). Central nuclei in adult muscle biopsies are thus diagnos-tic of abnormal muscle cell turnover (see Chapter 78). Both primary and secondary myotubes develop into phenotypi-cally distinct muscle fiber types by sequentially expressing a series of embryonic, neonatal, and mature isoforms of the contractile proteins (Table 5-1).5,12 Some of the develop-mental and mature isoforms of the contractile proteins that appear in fast, slow, and cardiac muscle are encoded by mul-tigene families, whereas others are differentially expressed by alternative splicing of messenger RNA.

An extracellular matrix of type IV collagen, proteogly-cans, fibronectin, and laminin is secreted by the myotubes to form the basal lamina, which fully ensheathes the fiber membrane (sarcolemma) except at the neuromuscular

94 DANTZIG | Muscle: ANAToMy, PhysIoloGy, AND BIocheMIsTry

junction.13 Cell matrix interactions also feed back to the gene expression apparatus, as shown by experiments in which the lineage of cultured mesenchymal stem cells was modulated by the mechanical stiffness of the substrate.14

Under normal conditions, the number of muscle fibers within a skeletal muscle is virtually constant throughout life. Myogenic stem cells (satellite cells) remain situated in mature muscle fibers between the sarcolemma and basal lamina (see Fig. 5-1), providing a reservoir for muscle repair and growth.15 When a muscle fiber is damaged or necrosed, mitogenic factors and peptide regulators released from the damaged cells5,16 trigger satellite cells to proliferate and migrate into the affected area, guided by the basal lamina. Some satellite cells differentiate into myoblasts (see Fig. 5-1, red arrow, and following steps), fuse, and form new muscle fibers. Others replenish the satellite cell niche. The supply of these stem cells is limited, however, which diminishes the potential for repair in severe degenerative conditions. Recent studies suggest that other stem cell populations, such as muscle side populations17 and a small percentage of bone marrow cells,18-21 may also participate in muscle repair and satellite cell replenishment.

Many of the genes involved in muscle myogenesis are also induced in the muscle repair process.5,6 For this reason,

knowledge of the mechanism of myogenesis may assist us in understanding regeneration and lead to new therapeutic strategies for treating muscle injuries and diseases.

STRUCTURE

MUSCLE TISSUE

Parallel, aligned bundles of skeletal muscle fibers make up approximately 85% of muscle tissue. Nerves, blood sup-ply, and connective tissue structures that provide support, elasticity, and force transmission to the skeleton (discussed later) constitute the remaining volume. Muscle fibers range in length from a few millimeters to many centimeters and range in diameter from 10 to 150 μm. This elongated shape is determined by the organization of the contractile proteins that occupy the majority of the sarcoplasm. Each muscle has a limited range of shortening that is amplified into large motions by lever systems of the skeleton, usually operating at a mechanical disadvantage. Variations in the geometric arrangements of the fibers—parallel, fan shaped, fusiform (spindle-like), or pennate (feather-like)—determine some of the mechanical properties. For example, the slant of the fibers in a pennate muscle increases the magnitude of force

Epaxialdermomyotome

Hypaxialdermomyotome

Delamination

Mesodermalprecursor cellsSomite

Sclerotome

Notochord

NogginShhWntsBMPNotch

Muscle damage

IGF-1Pitx-2Pax-7

Satellite cell activation

NotchReplenishment

Satellitecells

Myofiber

Innervationand maturation

Multinucleatedearly myotubeMrf4

MLPPax7

MyogeninMef 2Six-1MyoDMyf-6(Mrf4)Slug(snail)

Myoblasts

Differentiationand fusion

Myf-5MyoDCyclin D1IdRb

Determination

Myoblast precursorsin the limb bud

Proliferation

Mesodermalprogenitor cells

Pax-3C-MetMsx-1Mox-2SixMyf-5Myo D

Pax 3C-Met / HGFLbx1

Migration

Pitx2Pax-3C-Met

Numb

Presomitic andsomitic stages

Neuraltube

Sarcolemma

Basal Lamina

Figure 5-1 Myogenesis and cell regeneration of skeletal muscle. The long red arrow indicates the initiation of muscle regeneration due to disease or injury. The somite is composed of a dermomyotome and a sclerotome. BMP, bone morphogenetic protein; c-Met, hepatocyte growth factor receptor; hGF, hepatocyte growth factor; Id, originally identified as dominant negative antagonists of the basic helix-loop-helix transcription factor family; IGF-1, insulin-like growth factor 1; lbx1, ladybird homeobox homolog 1; Mef-2, myocyte enhancing factor 2; MlP, muscle lIM protein; Mox-2, membrane glycoprotein; Mrf-4, myogenic regulatory factor 4; Msx-1, homeobox msh-like 1; Myf-5, myogenic factor 5; Myf-6, myogenic factor 6; MyoD, myogenesis determining factor; Pax-3, paired box gene 3; Pax-7, paired box gene 7; Pitx2, paired-like homeodomain transcription factor 2; rb, product of the reti-noblastoma tumor suppressor gene; shh, sonic hedgehog; six-1, homolog of Drosophila sine oculis homeobox 1; slug(snail), muscle lIM protein; Wnt, wingless-type mouse mammary tumor virus integration site family.

95PArT 1 | sTrucTure AND FuNcTIoN oF BoNe, JoINTs, AND coNNecTIVe TIssue

generation at the expense of speed and range of movement, compared with a similarly sized muscle with fibers aligned parallel to the tendons.22 Muscles designed for strength (e.g., gastrocnemius) are typically pennate, whereas those designed for speed (e.g., biceps) tend to have parallel fibers. Muscles are commonly arranged around joints as antagonis-tic pairs, facilitating bidirectional motion. When one muscle (the agonist) contracts, another (its antagonist) is relaxed and passively extended. Their roles reverse to actively gen-erate the opposite motion, unless it occurs passively by the force of gravity.

An extensive network of connective tissue, forming the endomysium, surrounds each muscle fiber. Fine nerve branches and the small capillaries, necessary for the exchange of nutrients and metabolic waste products, penetrate this layer. The endomysium is continuous with the perimysium, a connective tissue network that ensheathes small parallel bundles of muscle fibers known as fasciculi, intrafusal fibers, larger nerves, and blood vessels. The epimysium encom-passes the whole muscle. All three layers of connective tis-sue contain collagen, mostly types I, III, IV, and V; types IV and V predominate in the basement membranes sur-rounding each skeletal muscle fiber. The α12α2-chain com-position of the collagen IV isoform is the most prevalent and provides the mechanical stability and flexibility of the basal lamina.23,24 The perimysium and endomysium merge at the junction between the muscle fibers and the tendons, aponeuroses, and fasciae. These layers give the attachment

sites great tensile strength and distribute axial force into shear forces over a larger surface area.

FIBER TYPES

Muscles adapt to their specific functions. In any given muscle, part of this adaptation arises from its composition and the organization of fiber types. Fibers can be classified according to their size, twitch duration, speed of contrac-tion, balance between aerobic and glycolytic metabolism, and resistance to fatigue (Table 5-2). In addition, isoen-zymes of the signaling, regulatory, and contractile proteins and the surface area of the sarcoplasmic reticulum (SR) membranes are key features that distinguish the functional properties of the different fiber types. For instance, the dura-tion of the twitch is influenced by the rates of release and reuptake of calcium ions (Ca2+) by the SR, and the velocity of shortening is determined by myosin isoenzyme composi-tion. Type IIB (fast) fibers are less “red” than the other fiber types because they contain less of the iron-heme–containing proteins, myoglobin, and mitochondrial cytochromes. Clas-sification schemes are not, however, absolute, because some groups of fibers have composite or intermediate functional, ultrastructural, and histochemical characteristics.

During development, fiber-type specificity may be partly determined before innervation.25 Although the biological events and signals responsible for designating functional specialization in muscle fibers are not fully understood,

Actin

Non-muscle α-actinin

Non-muscle myosin IIB

Muscle α-actin

Muscle myosin II filamentsTitin Nuclei

A B C

D

E

Stress fibers

Myoblasts

Premyofibrils

Fusing myoblasts

Nascent myofibrils

Primary myotube

Secondary myotubes

Myotubes

Mature myofibrils

Mature myofibrils

Figure 5-2 Developmental progression of myoblasts during fusion into myotubes. A, unicellular myoblasts. B, Initial fusion of myoblasts. C, Multinucleated myotubes. D, Myoblast fusing to multinucleated myotube; interface between myotube and mature myofibril. E, Mature myofibril.


classic cross-innervation experiments demonstrated that innervation can dynamically specify and modify the type of muscle fiber.26 After cross-innervation, the functional and histological properties listed in Table 5-2 shift toward the target fiber type over a few weeks’ time, indicating the ability of muscles to adapt and remodel in accordance with the pat-tern of neuronal activity.

EVENTS DURING MUSCLE CONTRACTION

NEURAL CONTROL

Normally, a skeletal muscle fiber is activated briefly and then relaxes. This twitch, lasting for 5 to 40 msec, is initi-ated by an action potential propagated from the central nervous system along an alpha motoneuron, synaptic transmission across the neuromuscular junction, and an action potential in the muscle sarcolemma. Muscle fibers are typically innervated at one or two sites along their length by branches of an alpha motoneuron axon arising from the ventral horn of the spinal column. A motoneu-ron and the approximately 5 to 1600 homogeneous muscle fibers it innervates constitute a motor unit. Although the spatial domains of different units are intermingled, when an alpha motoneuron is excited, all fibers in the motor unit are triggered to contract together. Functional properties, such as speed and susceptibility to fatigue, vary with the dynamic requirements set by the neuronal firing pattern and the mechanical load, but they are homogeneous within

a motor unit. The level of muscle activity is controlled by varying the firing rate of twitches and the number of active motor units. As activity increases, more and larger units are recruited.

A second class of fibers, the intrafusal fibers, are inner-vated by gamma motoneurons and control the sensitivity of the Golgi tendon organs and spindle receptors that provide local feedback to the spinal cord regarding muscle length and force. Afferent feedback pathways modulate activity to control the desired movement. The same feedback system generates the monosynaptic stretch reflex.

NEUROMUSCULAR TRANSMISSION

At the neuromuscular junction, the axon tapers and loses its myelin sheath. The postsynaptic sarcolemmal mem-brane (the motor end plate) is indented into folds that increase its surface area (Fig. 5-3). Mitochondria and nuclei are concentrated in this region. The junctional cleft is a 50-nm-wide space between the presynaptic axonal mem-brane and the sarcolemma.

When the nerve action potential reaches the presynaptic terminal, local Ca2+ channels are gated open for the influx of Ca2+. This triggers the fusion of acetylcholine-loaded membrane vesicles with the neuronal presynaptic mem-brane.27 Exocytosed acetylcholine rapidly diffuses across the junctional cleft and binds to nicotinic acetylcholine-gated ion channels in the crests of the postsynaptic membrane folds. Ligand gating of the acetylcholine receptors increases

Protein Molecular Weight (kD) Subunits (kD) Location Function

Acetylcholine receptor 250 5 × 50 Postsynaptic membrane of neuromuscular junction

Neuromuscular signal trans-mission

Annexins 38 — F-actin binding protein Membrane repair

Dihydropyridine receptor 380 1 × 1601 × 1301 × 601 × 30

T tubule membrane Voltage sensor

Dysferlin 230 — Periphery of myofibers Membrane repair

ryanodine receptor 1800 4 × 450 Terminal cisternae of sr sr ca2+ release channel

ca2+-ATPase 110 — longitudinal sr uptake of ca2+ into sr

calsequestrin 63 — sr terminal cisternae lumen Binding and storage of ca2+

Troponin 78 1 × 181 × 211 × 31

Thin filament regulation of contraction

Tropomyosin 70 2 × 35 Thin filament regulation of contraction

Myosin 500 2 × 2202 × 152 × 20

Thick filament chemomechanical energy transduction

Actin 42 — Thin filament chemomechanical energy transduction

MM creatine phosphokinase

40 — M line ATP buffer, structural protein

α-Actinin 190 2 × 95 Z line structural protein

Titin 3000 — From Z line to M line structural protein

Nebulin 600 — Thin filaments, in the I band structural protein

Dystrophin 400 — subsarcolemma structural integrity of sarco-lemma

ATP, adenosine triphosphate; SR, sarcoplasmic reticulum.

Table 5-1 signaling and contractile Proteins of skeletal Muscle


their cation permeability, locally depolarizing the muscle cell. This change of membrane potential initiates a regen-erative action potential, mediated by voltage-gated sodium (Na+) and potassium (K+) channels. The action potential propagates at velocities up to 5 m/sec from the motor end plate throughout the sarcolemma. Thus, the entire fiber and motor unit contract at the same time.

EXCITATION-CONTRACTION COUPLING

Invaginations of the sarcolemma at regular intervals consti-tute the transverse tubule (T tubule) network, which per-vades the fiber and surrounds the contractile apparatus with connected longitudinal and lateral segments (Fig. 5-4). The lumen of this network is open to the extracellular space, and it contains the high-Na+ and low-K+ concentrations of interstitial fluid.28 Action potentials at the surface mem-brane invade the entire T tubular system. A specialized type of endoplasmic reticulum forms the entirely intracellular SR. Prevalent structures termed triads contain a T tubule flanked by two terminal cisternae of the SR to form junc-tional complexes (see Fig 5-4). Terminal cisternae contain the oligomers of the Ca2+-binding protein calsequestrin, which provide the fiber with an internal reservoir of Ca2+. Dihydropyridine receptors (DHPRs) are Ca2+ channels localized in the T tubule membranes facing the cytoplasmic domain of SR Ca2+-release channels (the ryanodine recep-tors [RyRs], or foot proteins) in the terminal cisternae mem-branes.29 These membrane proteins are further characterized in Table 5-1.

When an action potential depolarizes the T tubular membrane, the DHPRs, which are primarily voltage sensors

in skeletal muscle (but not in myocardium), transfer a sig-nal from the T tubules to the RyRs by direct interprotein coupling. Ca2+ is then released cooperatively through the RyRs from the SR into the myoplasm, where it activates the contractile machinery.30 This sequence of events is termed excitation-contraction coupling.

Mutations in the α-subunit of the DHPR in dysgenic mice lead to paralysis because, in these mutants, depolar-ization of the skeletal muscle membrane does not initiate the release of Ca2+ from the SR. Excitation-contraction coupling can be restored in cultured cells from these mice by transfection with the complementary DNA encoding for the DHPR,31 and transfections using chimeric con-structs32 have pinpointed the domain within the DHPR that specifies skeletal- or cardiac-type excitation-contraction coupling.33 Isoforms of the RyRs also help determine the characteristics of the coupling between T tubules and the SR.34 Channelopathies in human skeletal and heart muscle have been linked to DHPR mutations.35,36 Human malignant hyperthermia occurs in individuals with mutant RyRs that become trapped in the open state after exposure to halo-thane anesthetic agents.37

CONTRACTILE APPARATUS

The specific locations and functions of the contrac-tile proteins are listed in Table 5-1. Myofibrils are long, 1-μm-diameter cylindrical organelles that contain the con-tractile protein arrays responsible for work production, force generation, and shortening (Fig. 5-5). Each myofibril is a column of sarcomeres, the basic contractile units, which are approximately 2.5 μm long and delimited by Z lines (see

General Features I IIA IIB IIC

size Moderate small large small

Mitochondria Many Intermediate Few Intermediate

capillary blood supply extensive sparse sparse sparse

sr membrane sparse extensive extensive extensive

Z line Wide Wide Narrow Narrow

Protein isoforms Myosin heavy chain (Mhc) MhcI MhcIIA MhcIIB MhcI, MhcIIAMyosin essential light chain

(elc)elc1s elc1f, elc3f elc1f, elc3f elc1f, elc3f, elc1s

Myosin regulatory light chain (rlc)

rlc2s rlc2f rlc2f rlc2f, rlc2s

regulatory proteins slow Fast Fast Fast

Mechanical propertiescontraction time slow/sustained Fast twitch Fast twitch Moderate twitchsr calcium ATPase rate low high high highActomyosin ATPase rate low high high Moderateshortening velocity slow Fast Fast Moderateresistance to fatigue high Moderate low Moderate

Metabolic profileoxidative capacity high Intermediate low highGlycolytic capacity Moderate high high highNADh-Tr/sDh/MDh high Moderate low ModeratelDh and phosphorylase low Medium high ModerateGlycogen low high high VariableMyoglobin high Medium low high

f, fast; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; NADH-TR, nicotinamide adenine dinucleotide tetrazolium reductase; s, slow; SDH, succinate dehydrogenase; SR, sarcoplasmic reticulum.

Table 5-2 classification of Muscle Fiber Types


r

Fig. 5-5D and E) containing the densely packed structural protein α-actinin. The contractile and structural proteins within each sarcomere form a highly ordered, nearly crystal-line lattice of interdigitating thick and thin myofilaments (see Fig. 5-5E, I, and J).38 Myofilaments are remarkably uni-form in both length and lateral registration, even during

contraction,39 resulting in the cross-striated histological appearance of skeletal and cardiac muscles. This highly periodic organization has facilitated biophysical studies of muscle by sophisticated structural38 and spectroscopic tech-niques.40,41

Thick filaments (1.6 μm long) containing the motor pro-tein myosin are located in the center of the sarcomere in the optically anisotropic A band (see Fig. 5-5D). The thick filaments are organized into a hexagonal lattice stabilized by M protein42 and muscle-specific creatine phosphokinase43 in the M line (see Fig. 5-5D and E). Myosin (see Fig. 5-5K) is a highly asymmetric 470-kD protein containing two 120-kD globular NH2-terminal heads, termed cross-bridges or sub-fragment-1 (S-1; see Fig. 5-5L), and an α-helical coiled-coil rod. Two light chains—essential and regulatory, ranging from 15 to 22 kD—are associated with the heavy chain in each S-1 (see Fig. 5-5L). The rod portions of approximately 300 myosin molecules polymerize in a three-stranded helix to form the backbone of each thick filament (see Fig. 5-5K). The cross-bridges, protruding from these backbones, con-tain adenosine triphosphatase (ATPase) and actin-binding sites responsible for the conversion of chemical energy into mechanical work. Besides their role in muscle contraction, at least 20 classes of nonmuscle myosins accomplish diverse tasks in cell motility such as chemotaxis, cytokinesis, pino-cytosis, targeted vesicle transport, and signal transduction.44 Thus, myosin is the target for mutations leading to a number of inherited muscular and neurologic diseases.45,46

Thin filaments (see Fig. 5-5I) are double-stranded heli-cal polymers of actin that extend 1.1 μm from each side of the Z line and occupy the optically isotropic I band (see Fig. 5-5D and E). A regulatory complex containing one tropomyosin molecule and three troponin subunits (TnC, TnT, and TnI) is associated with each succes-sive group of seven actin monomers along the thin fila-ment (see Fig. 5-5J).38 In the region where the thick and thin filaments overlap, the thin filaments are positioned within the hexagonal lattice equidistant from three thick filaments (see Fig. 5-5F). Both sets of filaments are polar-ized. In an active muscle, an interaction between the two filaments causes a concerted translation of the thin

Myofibril

Ca2+-releasechannels

Transverse (T)tubules formedfrom invaginationsof plasmamembrane

Sarcoplasmicreticulum

0.5µm

Figure 5-4 Membrane systems that relay the excitation signal from the sarcolemma to the cell interior. In the electron micrograph, two T tubules are cut in cross section. The electron densities spanning the gap between the T tubules and the sarcoplasmic reticulum membranes are the ryanodine eceptors, channels that release calcium into the myoplasm. (From Alberts B, Bray D, Lewish J, et al: Molecular Biology of the Cell, 2nd ed. New York, Garland,

1989. Micrograph courtesy of Dr. Clara Franzini-Armstrong, University of Pennsylvania, Philadelphia.)

A

Muscle fiber

Motorend plate

Myofibrils

PostsynapticMembrane

SynapticVesicles

SynapticCleft

Nerve

PresynapticTerminal

10µm

1µmB

Figure 5-3 Neuromuscular junction. A, scanning electron micrograph of an alpha motoneuron innervating several muscle fibers in its motor unit. B, Transmission electron micrograph. (A, From Bloom W, Fawcett DW: A Textbook of Histology, 10th ed. Philadelphia, WB Saunders, 1975. B, Courtesy of Dr. Clara Franzini-Armstrong, University of Pennsylvania, Philadelphia.)


filaments toward the M line, which shortens the sarco-mere and thus the whole muscle. Actin is ubiquitous in the cytoskeleton of eukaryotic cells and, like myosin, ful-fills many roles in determining cell shape and motion.47,48 Control of the actin cytoskeleton and diseases due to mutations in actin-binding proteins are being intensively investigated.49

Two of the largest identified proteins, titin and nebulin, function in the assembly and maintenance of the sarcomeric structure. Individual titin molecules (∼3000 kD) are associ-ated with the thick filament and extend from the M line to the Z line.50 Titin contains repeating fibronectin-like immu-noglobulin and unusual proline-rich domains that confer molecular elasticity on the resting sarcomere.51 Nebulin

Muscle

Muscle fasciculus

Muscle fiber

M line Z Sarcomere Z Myofibril

Hband

Zline

Aband

Iband

Troponincomplex

Tnl Tnc TnT

Actin Tropomyosin

Actin thin filament

Single myosinmolecule Subfragment 1

Tail

Myosin filament

Light meromyosin

Sarcomere

ATP pocket

Actinbinding

site

Motor domainMyosin subfragment 1

S1

S2

Myosin molecule

A

B

C

D

E

F G H

I

J

KL

RLC

ELC

H

Light chain domain

Figure 5-5 Components of the contractile apparatus. components are shown at successively increasing magnifications from the whole muscle (A) to the molecular level (I-L). The myofibril (D) shows the banding pattern created by the lateral alignment of the myofilaments (I, J) in the sarcomeres (D, E). Parts F to H show the cross-sectional structure of the filament lattice at various points within the sarcomere. Myosin is shown at the single two-headed molecule level (K), and the crystal structure of the globular motor domain is shown (L, s-1) with the essential and regulatory light chains. (From Juanqueira LC, Carneiro J, Long JA: Basic Histology, 5th ed. Norwalk, Conn, Appleton-Lange, 1986. Modified from Bloom W, Fawcett DW: A Textbook of Histol-ogy, 10th ed. Philadelphia, WB Saunders, 1975; and Rayment I, Rypniewski WR, Schmidt-Base K, et al: Three-dimensional structure of myosin subfragment-1: A molecular motor. Science 261:50, 1993.)


(∼800 kD) is associated with the Z line and thin filaments.50 Protein connections from the contractile apparatus through the sarcolemma to the extracellular matrix are described later in this chapter. The cytoskeleton of muscle fibers also contains cytoplasmic actin, microtubules, and intermediate filaments.52

FORCE GENERATION AND SHORTENING

At rest, the thin-filament regulatory proteins troponin and tropomyosin inhibit contraction (see Fig. 5-5I). During a twitch, Ca2+ released from the SR binds to TnC, relieving this inhibition and thus allowing cross-bridges to attach to actin. A contraction results from a cyclic interaction between actin and myosin (the cross-bridge cycle) that pro-duces a relative sliding force between the thin and thick filaments.53 The energy source is the hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and orthophosphate (Pi).

A simplified model of the chemomechanical events in the cross-bridge cycle is illustrated in Figure 5-6. Motor proteins, including myosin, can now be studied by single-molecule biophysical techniques, which provide unprec-edented details of their dynamics.54 When Ca2+ is present, a complex of myosin, ADP, and Pi attaches to the thin fila-ment (step a in Fig. 5-6), and a structural change within the myosin S-1 initiates force production and the release of Pi (steps b and c).55,56 The conformational change in the cross-bridge that leads to force generation is a tilting motion of the light-chain region.41,57,58 The filament sliding that leads to shortening of the sarcomere occurs during a strain-dependent transition between two ADP states (step d). After ADP is released (step e), ATP binds to the active

site and dissociates myosin from actin (step f). Myosin then hydrolyzes ATP (step g) to form the ternary myosin-ADP-Pi complex, which can reattach to actin for the next cycle.

If the mechanical load on the muscle is high, the con-tractile apparatus produces a force without changing length (an isometric contraction). If the load is moderate, the thin filaments slide actively toward the center of the sarcomere, resulting in shortening of the whole muscle. The width of the muscle increases during shortening, so the volume stays constant. Work production (concomitant force and sliding) is associated with an increase in the ATPase rate. The thermodynamic efficiency (mechanical power divided by energy liberated by ATPase activity) approaches 50%—a remarkable figure, considering that manufactured com-bustion engines seldom achieve efficiencies greater than 20%.

RELAXATION

The twitch is terminated by reversal of all the steps in acti-vation. Ca2+ released from the SR is taken up again by Ca2+-ATPase pumps located in longitudinal membranes of the SR. The myoplasmic Ca2+ concentration then decreases, and Ca2+ dissociates from TnC, deactivating the thin fila-ment. When the number of attached cross-bridges declines below a certain threshold, tropomyosin inhibits further cross-bridge attachment, and tension declines to the rest-ing level. Ca2+ diffuses within the longitudinal SR to the calsequestrin sites in terminal cisternae, ready to be released in the next twitch. Myosin continues to hydrolyze ATP at a low rate in relaxed muscle, accounting for a sizable propor-tion of basal metabolism.

M-line

ADPPi

ADPPi

ADPPi

ADPPi

Z-line

(b)

(c) (d)

Force generation andfilament sliding

Atached

(a)

(g)Actin

thin filament

PrimedHydrolysis

ATP

Detached

Myosinthick filament

(f)Rigor

ATP

(e) ADP

ADP

Figure 5-6 The actomyosin cross-bridge cycle. Myo-sin molecules normally have two globular head regions (cross-bridges), but for clarity, only one is shown. The ⊗ within the globular domain of myosin represents a hinge point with maximal flexibility. each head binds with two actin monomers. The sequence of reactions is attachment (a); the force-generating transition (b); orthophosphate (Pi) release (c); force generation and filament sliding (d); adenosine diphosphate (ADP) re-lease (e); adenosine triphosphate (ATP) binding and detachment (f); and ATP hydrolysis (g). The shadowed heads near the detached and force-generating myosin heads indicate high mobility of the cross-bridges in these states.


TRANSMISSION OF FORCE TO THE EXTERIOR

CELL MATRIX ADHESIONS

The muscle cell is closely connected to the basal lamina along its entire surface. Several transmembrane macromo-lecular complexes link the myofibrils and actin cytoskel-eton to laminins and collagen in the extracellular matrix. Attachment complexes for muscle, analogous to the focal adhesions of motile and epithelial cells and to the adhesion plaques and intercalated disks in cardiac muscle, contain fil-amentous actin, vinculin, talin, and integrin (primarily the α7β1 isoform), which is the transmembrane link to laminin (Fig. 5-7). In muscle, the main laminin isoforms are lam-inin-2 (α2β1γ1) and laminin-4 (α2β2γ1), which are collec-tively termed merosin. In addition to providing mechanical coupling between the cytoskeleton and the extracellular matrix, the laminin-integrin system may provide a signaling pathway to regulate localized protein expression.59 Defects in the expression of many of the cytoskeletal proteins lead to various forms of muscular dystrophy (Table 5-3).60

A specialized linkage between the cytoskeleton and basal lamina in muscle, complementary to the integrin focal adhesion system, is the dystrophin-glycoprotein complex (see Fig. 5-7). Dystrophin, a 427-kD peripheral cytoskeletal protein, has been postulated to function as a mechanical link between the cytoskeleton and the cell membrane or as a shock absorber, or to contribute mechanical strength to

the membrane. Its absence or truncation causes Duchenne’s and Becker’s muscular dystrophies.61 The N-terminus of dys-trophin binds to actin via a region with sequence homology to the actin-binding domain of α-actinin. This end of the dystrophin molecule may be linked to the basal lamina via the same proteins described previously for the focal adhe-sion-like complexes (see Fig. 5-7). The C-terminus binds to a transmembrane dystroglycan-sarcoglycan complex, which in turn binds to the laminins. Muscular dystrophies of var-ied severity are associated with a loss of these components (see Table 5-3).62 Dystroglycans are also required for early embryonic development of muscle, possibly organizing lam-inin localization and assembly.63,64 Utrophin, a smaller dys-trophin-related protein (395 kD), may also link the actin cytoskeleton to the dystroglycans, especially near the neu-romuscular junction and in nonmuscle cells. Overexpres-sion of this protein or truncated dystrophin constructs are promising avenues for gene therapy in Duchenne’s muscular dystrophy.65 The unusual intricacy of cell matrix connection systems in muscle may relate to the high forces generated during contraction.

MYOTENDINOUS JUNCTION

The force of muscle contraction is transmitted to the skel-eton via the tendons, which are composed of type I and III collagens, blood vessels, lymphatic ducts, and fibroblasts. At the ends of the muscle fibers, myofibrils are separated by invaginations of sarcolemma filled with long bundles of

εβ

β βα

α

α

γζ δ

Cytoplasm

Syncoilin

C

α1

α-dystrobrevin

α1nNOS

Syntrophins

Sarcoglycans

Dystrophin

Dysferlin

Laminin

Dystroglycans

Basal lamina

Sarcospan

Sarcolemma

Vinculin

N

Actin

Talin

Caveolin 3

IntegrinsLaminin

Collagen

Annexins

A1 A2

Figure 5-7 Connections between the muscle cytoskeleton and extracellular matrix. Actin is linked through integrins to the matrix, as in many cell types. Dystrophin forms an extra link through the dystroglycan-sarcoglycan complex of glycosylated proteins. The helical section of dystrophin is ho-mologous to spectrin and may form homodimers or oligomers. Dystrophin links two intricate systems connecting the sarcolemma to the basal lamina. The cooh-terminus of dystrophin (N) is associated with the sarcoglycans, dystroglycans, dystrobrevin, syncoilin, neuronal nitric oxide synthase (nNos), and syntrophins. The Nh3-terminus links actin, vinculin, and the integrins with laminin and the basal lamina. These two adhesion systems provide a sup-portive substructure to maintain the integrity of the sarcolemma. The annexins and dysferlin have a role in muscle regeneration.


collagen arising from the tendon. These membrane folds increase the surface area for bearing the mechanical load by approximately 30-fold. Instead of terminating in the Z disks, actin filaments insert into a subsarcolemmal matrix containing α-actinin, vinculin, talin, and integrin. The force is transmitted through laminin to the collagen of the tendon.

ENERGETICS

Metabolic pathways in muscle cells are specialized for the variable, and at times extreme, rates of ATP splitting by the contractile apparatus and membrane ionic pumps. Because the muscle tissue compartment is large, and because certain protein isoforms are specific to muscle tissue, circulating

levels of some metabolic enzymes (e.g., lactate dehydroge-nase, creatine phosphokinase) are useful in the diagnosis of sarcolemmal disruption. Among the dozens of enzymes pres-ent, only the most important ones related to normal muscle function are mentioned here.

BUFFERING OF ADENOSINE TRIPHOSPHATE CONCENTRATION

The ATP content (∼5 mM) is sufficient for only a few seconds of contraction, so rapid and effective buffering of ATP during contraction is essential for the maintenance of activity. ADP formed by the hydrolysis of ATP is rephosphorylated by trans-fer of a phosphate group from creatine phosphate (20 mM in a resting cell), by creatine phosphokinase located within the

Disease Genetic Locus Inheritance Protein Outcome

Duchenne’s, Becker’s Xr Dystrophin lethal

emery-Dreifuss Xr emerin, laminins A and c 40% lethality

limb-girdle muscular dystrophies (lGMDs)

less severe forms can emerge during first 3 decades of life, leading to loss of ambulation after age 30 yr; most severe forms start at age 3-5 yr and progress rapidly

lGMD 1A 5q31 AD MyotilinlGMD 1B 1q11-q21 AD laminin A or clGMD 1c 3p35 AD caveolinlGMD 1D 6q23 AD —lGMD 1e 7q AD —lGMD 1F 7q32 AD —lGMD 1G 4p21 AD —lGMD 2A 15q15.1-q21.1 Ar calpain 3lGMD 2B 2p13 Ar DysferlinlGMD 2c 13q12 Ar γ-sarcoglycanlGMD 2D 17q12-q21.33 Ar α-sarcoglycanlGMD 2e 4q12 Ar β-sarcoglycanlGMD 2F 5q33-q34 Ar δ-sarcoglycanlGMD 2G 17q11-q12 Ar TelethoninlGMD 2h 9q31-q34.1 Ar e3 ubiquitin ligase (TrIM32)lGMD 2I 19q13.3 Ar Fukutin-related proteinlGMD 2J 2q24.3 Ar TitinlGMD 2K 9q34 Ar Protein

o-mannoslytransferase

cMDs with cNs involvementFukuyama cMD 9q31 Ar Fukutin le, 11-16 yrWalker-Warburg cMD 1p32 Ar o-Mannosyltransferas le, <3 yrMuscle-eye-brain cMD 1p32-34 Ar o-MNAGAT le, 10-30 yr

cMDs without cNs involvementMerosin-deficient classic type 6q2 Ar Merosin (laminin A2) Many patients never walk;

others have lGMD pat-tern

Merosin-positive classic type 4p16.3 Ar selenoprotein N1, collagen VI α2

course stabilizes in late childhood; many patients continue to walk into adulthood

Integrin-deficient cMD 12q13 Ar Integrin α7 Presents early in infancy with hypotonia and delayed milestones

other dystrophiesFacioscapulohumeral 4q35 AD — 20% wheelchair boundoculopharyngeal 14q11.2-q13 AD/Ar Polyadenylate binding

protein nuclear 1onset ∼48 yr; 100% symp-

tomatic by age 70 yrMyotonic dystrophy 19q13.3 AD DMPK, cchc-type zinc

finger, cNBP50% show signs by age

20 yr; variable severity

AD, autosomal dominant; AR, autosomal recessive; CCHC, cysteine and histidine amino acid sequence; CMD, congenital muscular dystrophy; CNBP, cellular nucleic acid binding protein; CNS, central nervous system; DMPK, dystrophia myotonica protein kinase; LE, life expectancy; O-MNAGAT, O-mannose β-1,2-N acetylglucosaminyl transferase; XR, X chromosome related.

Table 5-3 classification of Muscular Dystrophies


M line of the sarcomere, in the myoplasm, and between the inner and outer membranes of the mitochondria. Adenyl-ate kinase, known in muscle as myokinase, catalyzes the transfer of a phosphate group between two ADP molecules, forming ATP and adenosine monophosphate (AMP). The by-products of the rapid enzymatic reactions that maintain ATP concentration are, therefore, creatine, Pi, and AMP. Some of the AMP is converted to inosine monophosphate by adenylate deaminase. A creatine phosphate shuttle has been proposed to enhance energy flux.66 According to this hypothesis, creatine phosphate is split within the contractile apparatus, and creatine is predominantly rephosphorylated in the mitochondria.

GLYCOLYSIS

Muscles use glucose as fuel when possible; otherwise, they use fatty acids and ketone bodies (acetoacetate and 3-hydroxybutyrate). The muscle compartment contains most of the body storage of glycogen, which is converted to glucose 6-phosphate for local use. Muscle fibers lack glucose-6-phosphatase and thus do not export glucose. During intense activity, especially in anaerobic condi-tions, the rate of glycolysis and the production of pyru-vate exceed the rate of pyruvate consumption by the citric acid cycle. The excess pyruvate is reduced to lactate by lactate dehydrogenase, which has tissue-specific isoforms. The lactate dehydrogenase reaction also produces nicotin-amide adenine dinucleotide (NAD+), which is necessary for glycolysis, but otherwise, lactate is not useful within the muscle. Lactate is freely permeable through the sar-colemma, and a local increase in the extracellular lactate concentration or acidification produces exertional pain (“the burn”). Lactate is transported through the blood to the liver, where it is converted back to pyruvate and then glucose, which is released into the blood for use by other tissues, such as muscle and brain. This sequence of steps, termed the Cori cycle, transfers some of the high metabolic load to the liver and “buys time” until oxidative metabolism is available.

OXIDATIVE PHOSPHORYLATION

In aerobic conditions, pyruvate enters the mitochondria, where it is oxidized to carbon dioxide and water, gener-ating reduced NAD (NADH). A hydrogen ion (H+) gra-dient across the mitochondrial membrane is produced by the electron transport chain, and, finally, ADP is phos-phorylated to ATP by the mitochondrial ATP synthase. This remarkable rotary motor generator has been studied extensively using single-molecule biophysical techniques.67 By combining glycolysis and oxidative phosphorylation, up to 38 ATP molecules can be generated by the oxidation of each molecule of glucose. This process is energetically much more favorable than the production of lactate, but it can occur only when molecular oxygen is available. Myoglobin is an iron-heme complex protein that facili-tates oxygen transport within the muscle cells. The tissue hydrostatic pressure in a contracting muscle often exceeds arterial perfusion pressure, so the strongest contractions are anaerobic. The content of oxidative enzymes, myoglo-bin, and mitochondria determines the predominant type of

energy metabolism and varies in different muscle types, as discussed previously (see Table 5-2).

FATIGUE AND RECOVERY

During intense or prolonged activity, muscle fatigue is caused by alterations of metabolite levels that suppress force generation at the contractile apparatus,68 in excitation-con-traction coupling, or both.69 Markedly increased myoplasmic Pi and H+ concentrations and decreased creatine phosphate levels have been detected by magnetic resonance spectros-copy.70 When the creatine phosphate level declines, main-tained activity depends on glycogenolysis until glycogen stores are depleted. During prolonged intense activity, the respiratory and circulatory systems are unable to meet the oxygen demands of tissues. Force production declines well before the ATP concentration is compromised.

The chemomechanical link between Pi release and force generation (see Fig. 5-6) implies that the increase of myoplas-mic Pi in fatigued muscle reduces the magnitude of force sim-ply by mass action.56 Decreased muscle pH, partly from lactate accumulation, and insufficient acetylcholine at the neuro-muscular junction, leading to failure of synaptic transmission, also contribute to decreased work production. Because the respiratory and circulatory systems do not supply sufficient oxygen to support the metabolism during intense activity, an oxygen debt is incurred. Blood flow and oxygen uptake continue at an enhanced level after the period of exercise to reclaim this energy. Rephosphorylation of creatine can take place within a few minutes, but glycogen resynthesis requires several hours. Recovery processes also involve the restoration of ionic gradients across the membrane-bound compartments and require the consumption of additional energy.

PLASTICITY

The strength and endurance of a muscle are altered dramati-cally within weeks after changes in the demand for its use, its mobility, or its hormonal or metabolic environment. The effects of this adaptive response should be considered in any clinical situation that causes a substantial shift in these fac-tors and in terms of the patient’s long-term quality of life.

ADAPTATION TO EXERCISE

Exercise leads to adaptations in the muscle fibers, includ-ing alterations in specific contractile, regulatory, structural, and metabolic proteins, as well as optimization of motor unit recruitment. The frequency, intensity, and duration of a training stimulus and the external load influence the adaptive response.71 Trophic factors liberated from the nerve play a minor role, if any. Strength training causes cross-sectional hypertrophy of fast type IIB fibers (see Table 5-2), the expression of fast myosin isoforms, an increase in amino acid uptake, and decreased synthesis of mitochondrial proteins. Endurance training enhances the oxidative capac-ity and volume density of mitochondria in oxidative type I and IIA fibers, redistributes blood flow to these motor units, and increases the synthesis of contractile proteins. Long-term hyperplasia does not usually occur after training in humans. Although hypertrophy of preexisting fiber types is observed, training also induces changes in myosin isoform distribution,


N

especially in fibers already containing more than one isoform. There is little evidence that voluntary training regimens can switch fibers between the major categories.

When physical activity is reduced, such as during limb immobilization, the cross section of the fibers decreases, and endurance is reduced as a result of a decreased metabolic reserve. Acute exercise hypertrophy and disuse atrophy are both reversible, but after extensive alterations, the restora-tion may be incomplete.72

ENDOCRINE CONTROL

Endocrine factors also participate in adaptation. For example, normal circulating levels of thyroid hormones are required during muscle development and differentiation.73 Experi-mental alterations of thyroid hormone concentrations pro-mote changes in the relative levels of myosin and regulatory protein isoforms, as well as changes in the activity of some metabolic enzymes.74 An intact nerve supply is required for these thyroid effects. Myogenesis is partly controlled by the release of growth hormone from the pituitary via the production of insulin-like growth factor 1 (IGF-1) in the liver. IGF-1 activates satellite cells for muscle regeneration and stimulates the hypertrophy of mature muscle cells.75,76 Abuse of growth factors, drugs, steroids, and metabolites by athletes (primarily young men) to increase muscle mass and strength is a significant medical and societal problem.77,78 The use of androgenic-anabolic steroids (e.g., synthetic testosterone) to increase muscle mass and improve body image induces an increase in lean muscle mass as a result of skeletal muscle hypertrophy. Along with the increase in cross-sectional area of the muscle comes strength enhance-ment, with or without concurrent exercise training. Side effects of these performance-enhancing drugs include acne, erratic aggression, hirsutism, and male pattern baldness. These compounds also decrease high-density lipoprotein levels, increase erythropoiesis, increase bone density, and elevate liver enzymes. Most of the physiological changes are reversible. Acute cessation can lead to severe depression in many individuals.

As in other tissues, insulin regulates the entry of glucose through the cell membrane. In muscle, it also promotes the uptake of branched-chain amino acids (valine, leucine, and isoleucine), which are necessary for rapid protein synthesis, and it inhibits protein degradation. Muscle weakness and fatigue may also be symptoms of changes in serum insulin and glucose levels.

AGING

ormal individuals exhibit an approximately 30% decrease in total muscle mass between the ages of 30 and 80 years. The cross-sectional area of the fibers decreases, leading to a significant loss of strength. Gradual decreases in muscle activ-ity, as well as neuronal changes, contribute to this muscle atrophy, which can exacerbate age-related joint degeneration and make joints less stable and more prone to injury.

A decline in estrogen and testosterone levels in aging individuals has been hypothesized to affect muscle strength. Aging also produces a marked decrease in the level of IGF-1 and the subsequent ability to repair or build new muscle. Recombinant IGF-1 delivered via virus-mediated gene

transfer has been shown to increase the muscle mass and strength of the aged mouse.79 Further manipulations of specific growth factors may ameliorate muscle loss due to muscular dystrophies, other chronic illnesses, and aging.80,81 Muscle mass, strength, and flexibility are major factors in the maintenance of an independent and productive lifestyle among the elderly.

SUMMARY

The complex functional capacity of muscle to produce finely tuned and coordinated movements is ultimately expressed as the transduction of chemical to mechanical energy by actomyosin. A twitch is initiated by an action potential propagated from the central nervous system along an alpha motoneuron, neuromuscular chemical transmission, direct protein-protein communication at the T tubule–SR junc-tion, Ca2+ diffusion in the myoplasm, and Ca2+ binding to thin-filament regulatory proteins. Because the central ner-vous system controls activity through the recruitment of motor units, the gradation and coordination of movement depend on the pattern of connections between the alpha motoneurons and the muscle fibers and on the variation of properties among motor units. The development, mainte-nance, and aging of the muscular system involve a complex series of genetic programs and cellular interactions that are beginning to be understood at the molecular level. Adapta-tion of motor unit properties is evident not only in training regimens but also in reduced activity caused by pain or joint immobilization and in compromised metabolic, hormonal, or nutritional conditions. Hence, the plasticity of muscle affects the clinical course of many diseases. In addition to its importance in pathophysiology, muscle serves as an excel-lent substrate for understanding the molecular basis of cell development, protein structure-function relationships, cell signaling, and energy transduction processes.

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