Contractile Mechanisms in Skeletal Muscle Joseph Feher, Ph.D.

LECTURE OUTLINE:

I. Introduction

A. How can we explain overall behavior of muscle?

II. Muscle Cells have a highly organized structure

A. Large multinucleated muscle cells are striated.

B. Striations are the most prominent feature of skeletal muscle.

C. Striations are organized longitudinally into myofibrils.

D. Striations are due to overlap of thin and thick filaments.

E. Thick and thin filaments form interdigitating hexagonal arrays.

F. The functional unit of muscle is the sarcomere.

G. Excitation is carried into the muscle by transverse tubules.

H. Myofibrils are covered by an internal membrane network, the sarcoplasmic reticulum.

III. The sliding filament hypothesis explains the length-tension curve

A. I bands shorten while A band stay the same length during muscle shortening.

B. The sliding filament hypothesis predicts that force depends on overlap of the filaments.

IV. Force is produced by an interaction between thick and thin filament proteins

A. The thick filament consists primarily of myosin.

1. Myosin consist of 6 polypeptides

2. The giant protein titin is a template for thick filament assembly

3. Myosin forms a regular array of head groups on the thick filament.

4. The thick filament contains a “bare zone” in its middle with no head groups.

B. The thin filament consists primarily of actin.

1. Globular actin polymerizes to form filamentous actin.

2. The giant protein, nebulin, sets the length of the thin filament.

3. Thin filaments also contain tropomyosin.

4. Troponin is a complex of three proteins on the thin filament.

5. Thin filaments are anchored at the Z-disk by actinin.

C. Cross bridges from the thick filament split ATP and cause shortening.

1. Myosin heads bind to actin filaments

2. Myosin is an actin-activated ATPase

3. Actomyosin ATPase activity occurs in the cross-bridge cycle.

4. ATP hydrolysis allows the myosin to "walk" along the actin filament

V. Cross-bridge cycling rate explains the fiber type dependence of the force-velocity curve

A. Force of contraction is related to the number of myofibrils in parallel

B. Speed of contraction is related to the number of sarcomeres in series

C. Rate of shortening in a sarcomere depends directly on the turnover rate of the cross-bridges

D. Slow and fast twitch muscles have different myosin isoforms.

E. Muscles can be classified on the basis of their myosin staining.

VI. Force is transmitted outside the cell through the cytoskeleton and special transmembrane proteins

A. Costameres are located at the Z-disk.

B. Costameres are a complex assembly of proteins.

C. Costameres may have multiple functions

VII. Practice Questions OBJECTIVES:

1. In a drawing or electron micrograph, identify: myofibril, A band, I band, H band, M line, Z line

2. Define the sarcomere and give its approximate resting length

3. List the major protein components of the thick filaments and the overall length of thick filaments

4. List the major protein components of the thin filaments and the overall length of thin filaments

5. Describe the origin of the length-tension curve on the basis of overlap of thin filaments and force generators on the thick filaments

6. Indicate the part of the myosin molecule that hydrolyzes ATP

7. Indicate why cross-bridge cycling rate determines (in part) muscle speed

8. Describe how muscles can be classified on the basis of myosin isotype staining

9. Describe how force is thought to be transmitted outside of the muscle

Suggested Reading: Berne and Levy, pp. 223-227; 231-233

I. INTRODUCTION: from the last lecture we learned that...

• muscles are heterogeneous with respect to contractile properties

• muscle force can be graded by recruitment of motor units

• muscle force can be graded by increasing the frequency of motor neuron firing

• muscle force can be graded by changing the length of the muscle

• muscle velocity is inversely related to force of shortening

• the power of a muscle peaks at about 1/3 maximal force and at about 1/3 maximal velocity

We seek an explanation of these overall behaviors in the sub-cellular and molecular description of muscle.

II. MUSCLE FIBERS have a highly organized structure.

A. Large, multinucleated muscle fibers are striated Muscle cells are large multinucleated fibers some 10-100 μm in diameter and as long as 12 cm. In some muscles, they can span the distance between insertion and origin. They need many nuclei to govern protein synthesis and degradation in these large fibers. The nuclei are located near the periphery of the cell and often are more highly concentrated near the neuromuscular junction.

B. The striations are the most prominent feature of muscle cells. Viewed under light microscopy, the most striking feature of muscle cells is their stripes. These stripes, or striations, result from the highly organized arrangement of proteins in the muscle fiber. The striations consist of alternating A-bands and I-bands, named because the I-bands are isotropic to polarized light (meaning that they appear the same from all directions) whereas the A-bands are anisotropic to polarized light.

Figure 1. Microscopic appearance of skeletal muscle fibers. A bundle of frog sartorius muscle fibers was teased out and viewed under phase contrast microscopy. Cross-striations are readily apparent in these unstained muscle fibers.

C. The striations are organized longitudinally into myofibrils. Muscle cells are also organized longitudinally into tiny threads called myofibrils. These cylinders are composed of two kinds of filaments. The

thin filament contains actin and the thick filament contains myosin. These myofibrils also show cross-striations that are due to the way in which the filaments overlap each other. The myofibrils are kept in register across the entire cell to give rise to the striated appearance of the fiber.

Figure 2. Electron micrograph of striated muscle. The spaces between myofibrils are filled with membranes of the sarcoplasmic reticulum, mitochondria, and glycogen granules. The myofibrils are bundles of myofilaments arranged longitudinally parallel to the long axis of the muscle fiber. The various bands are named according to their position, appearance, or how they rotate the plane of polarized light.

D. The striated appearance is due to overlap of thick and thin filaments The anisotropy of the A bands is due to myosin in the thick filaments. The thick filaments are 1.6 μm long, so the A-band is also 1.6 μm wide. The thin filaments are about 1.0 μm long. Opposite thin filaments are connected at the Z-line (from the German zwischen, meaning “between”). Because the myofibrils are cylindrical, the Z-line is actually a disk and it is also called the Z-disk. Thin and thick filaments typically overlap and the distance between successive Z-disks is less than the A-band plus the length of two thin filaments (= 1.6 + 2 x 1.0 = 3.6 μm). The H-zone (from the German helles, meaning “clear”) is a clearer area in the middle of the A band that shows where the thin filaments do not overlap the thick filaments. Proteins at the M-line (from the German mittel, meaning “middle”) connect the thick filaments and keep them in register.

E. The thick and thin filament form interdigitating hexagonal arrays (Fig. 3) Both thick and thin filaments form hexagonal arrays. The array of the thin filament is rotated 30° from the thick filaments. Each thick filament is in the center of a hexagon of thin filaments, whereas each thin filament is in the center of a triangle of thick filaments. Thus, there are 2 thin filaments

for every thick filament. Electron micrographs show cross-bridges between the thick and thin filaments. The interaction of the filaments through these cross-bridges produces either shortening or force.

F. The functional unit of contraction is the sarcomere. The material found between successive Z-disks is called the sarcomere. This is the functional unit of contraction or force production. The myofibrils consist of thousands of these sarcomeres strung end to end. At rest, the sarcomere is about 2.2 μm long.

Figure 3. Structure of the muscle fiber and myofibrils. G. Excitation is carried into the muscle fiber by transverse tubules.

Long tubules come off the sarcolemma at regular intervals perpendicular to the long axis of the muscle fiber. These are the transverse tubules or T-tubules. They bring the excitation of the action potential on the sarcolemma into the deepest parts of the interior of the cell.

H. The myofibrils are covered with an internal membrane network called the sarcoplasmic reticulum (see Fig. 4). The sarcoplasmic reticulum, or SR, forms an internal compartment separate from the cytoplasm. It is functionally divided into a longitudinal SR and terminal cisternae. The terminal cisternae form sacs that closely appose the T-tubules, whereas the longitudinal SR are thin tubes of membrane that connect terminal cisternae from one side of the sarcomere to the other. The longitudinal SR and terminal cisternae form a single enclosed space. In skeletal muscle the junction of one T-tubule and two terminal cisternae forms a triad at the junction of the A-band and I-band, so there are two triads per sarcomere.

Figure 4. Structure of the SR around the myofibril. III. THE SLIDING FILAMENT HYPOTHESIS explains the length-tension

curve.

A. I bands shorten while A bands stay the same length during muscle shortening. Muscle shorten because the sarcomeres shorten. The Z-disks move closer together but there is no change in the length of the A band: all of the shortening appears to occur in the I-bands, which are the part of the thin filaments that don’t overlap the thick filaments. A.F. Huxley and R. Niedegerke first proposed that the thin (I-band) filaments slide past the thick (A-band) filaments and that force arises from the interaction between these filaments.

B. The sliding filament hypothesis predicts that force depends on the overlap of thick and thin filaments. At a sarcomere length of 3.65 μm, there is no overlap of the filaments because the A-band is 1.6 μm and each of the two I-band filaments is 1.0 μm, At no overlap, there is no force. At progressively shorter lengths, the A-band and I-band filaments overlap increasingly so the force proportionately increases. At 2.25 μm there is maximum overlap and so there is maximum force. This force does not decrease until the sarcomere shortens to less than 1.95 μm. The reason for this is that there is still maximal overlap of the force generators on the thick filament. The thick filament has a bare central region (about 0.3 μm) that lacks the force generators. When the sarcomeres shorten still further (below 1.95 μm), the thin filaments begin to run into each other. Although the thin filaments can slide past each other, they encounter resistance and so the active force falls. At still shorter lengths, the thick filament begins to butt up against the Z-disk. This occurs at about the length of the A-band, or 1.6 μm. Further shortening seriously distorts the thick filament and force falls precipitously with further shortening.

Figure 5. Dependence of tension on the degree of overlap of the thin and thick filaments.

IV. FORCE IS PRODUCED by an interaction between thick filament proteins and thin filament proteins.

A. The thick filament consists primarily of myosin

1. Myosin consists of 6 polypeptides. Myosin is a complex of 6 proteins having a combined molecular weight of 480,000 Da. It consists of two heavy chains of 200,000 Da each, and a total of 4 myosin light chains. The two heavy chains have a long, rod-shaped tail and a globular “head” region that contains the active site. An arm section attaches the head to the tail. The arm and heads form the cross-bridges that interact with the thin filament to produce force.

2. The giant protein, titin, serves as a template for the aggregation of myosin to form the thick filament. The giant protein titin, also called connectin, is the largest protein known to date with a molecular weight of about 3.7 million Da. At 8-10% of the myofibrillar protein, it is the third most abundant skeletal muscle protein. It spans the distance from Z-disk to M-line and binds α-actinin, myosin and M-protein, the one responsible for tying together the thick filaments at the M-line. Myosin binds to titin to form a large aggregate, the thick filament.

3. Myosin forms a regular array of head groups on the thick filament. Myosin binds to titin and to other myosin molecules along the myosin tail region. The myosin heads project out of the thick filaments at regular intervals of 14.3 nm. There appear to be 3 myosin molecules (6 heads) at each plane oriented at right angles to the long axis of the filament. There is an identical repeat every 43 nm. Each thick filament has about 300 myosin heads projecting from its surface. Fig. 6 shows the structure of the thick filament.

4. The thick filament is polarized, with head groups at each end and a “bare zone” in the middle. The myosin molecules lay themselves down along the titin template so that the tail regions point toward the middle of the filament. Because of this arrangement, the middle of the thick

filament contains only tails, and the heads are located at the ends of the filament.

Figure 6. Structure of the thick filament. B. The thin filament consists primarily of actin.

1. Globular actin (G-actin) polymerizes to form filamentous, or F-actin. Globular actin, or G-actin, is a 41.8 KDa monomer with a diameter of about 5.5 nm. It aggregates to form a filament, F-actin, consisting of two strands of actin molecules wound around each other. About 7 G-actin molecules forms each half-turn, giving a half-turn distance of about 38.5 nm.

2. The giant protein, nebulin, sets the length of the actin filament. Nebulin is another giant protein, with a molecular weight between 600,000 and 900,000 Da in different muscles. It spans the whole length of the thin filaments and is anchored at the Z-disk. It contains a string of about 200 actin binding domains. Nebulin may set the length of the thin filament (normally about 1.0 μm in skeletal muscle).

3. Thin filaments contain tropomyosin. Tropomyosin consists of two non-identical polypeptide chains, each with a molecular weight of about 33,000 Da. They are long, rod-shaped molecules, and the tropomyosin α and β wrap around each

other to form a supercoil. The tropomyosin complex is 38.5 nm long, the same size as a half-turn of the F-actin helix. Along with troponin, tropomyosin participates in the regulation of the active state of muscle.

4. Troponin is a complex of three proteins on the thin filament. Troponin consists of troponin T, a 37,000 Da protein; troponin C of 18,000 Da and troponin I of 21,000 Da. These three derive their names from their functions: TnT binds to tropomyosin; TnC binds calcium ions (and thereby confers Ca2+ sensitivity onto the myofilaments), and TnI inhibits the interaction between the thick and thin filaments that cause force development or shortening. These three proteins form a complex at one end of each tropomyosin molecule. The troponin complex is responsible for the final regulation of the contractile state of the myofilaments.

Figure 7. Structure of the thin filament. 5. Actin filaments of adjacent sarcomeres are joined at the Z-disk by α

actinin. The Z-disk contains a number of proteins that can bind F-actin. The precise way that these proteins align the thin filaments is not yet known. One of these proteins, α actinin, consists of two subunits of 95,000 Da that is located in the Z-disk and anchors the thin filaments there. A proposed arrangement of the Z-disks shown in Fig. 8 illustrates the complexity of this structure. Many forms of muscular dystrophy are linked to mutations in one or another of these proteins.

Figure 8. Postulated linkage of thin and thick filaments at the Z-disk. The thin filaments have opposite polarity at the Z disk. The Z-disk contains a variety of proteins that bind to other proteins in the disk. Gamma filamin is a cytoskeletal protein that links the Z-disk to the outside of the cell.

6. The actin filaments at the Z-disks have opposite polarity. The thin filaments to the right of a Z-disk project towards a thick filament to the right, whereas the thin filaments to the left of a Z disk projects towards a thick filament to the left. Thus, the polarity of the thin filaments at the Z-disk are opposite. The plus end, the end where the filament grows, is capped by CapZ and anchored by it at the Z disk.

C. Cross-bridges from the thick filament split ATP and generate force.

1. Myosin heads binds to actin filaments. Proteolytic cleavage of myosin with papain separates myosin into the long rod-shaped tail and two myosin subfragments called S1. These S1 fragments bind to actin filaments and can be seen as a lateral projection on the thin filament that resembles an arrowhead in negative stained electron micrographs. These arrowheads all point the same way, revealing the F-actin polarity.

2. Myosin is an actin-activated ATPase. Although myosin can split ATP all by itself, actin binding speeds it up

some 200-300 fold. The hydrolysis of ATP takes several steps: ATP binds to the myosin head (the “business” end of the molecule), then it is hydrolyzed to ADP and Pi, which remain non-covalently bound. Then the myosin must release the products as free ADP and Pi. The step that limits the speed of this reaction is the release of ADP and Pi.

3. Actomyosin ATPase activity occurs in the cross-bridge cycle. Myosin splits ATP according to the simplified reaction cycle shown in Fig. 9. The cycle involves sequential binding of ATP and conversion of myosin into a form having low actin affinity, hydrolysis of ATP without release of the product ADP and Pi, conversion of myosin into a form having a high affinity for actin, ejection of the ADP and Pi and subsequent change in conformation of the myosin head, and binding of ATP again.

4. ATP hydrolysis allows the myosin head to “walk” along the actin filament. The main idea here is that ATP hydrolysis is linked to a sequential change in the conformational state of the myosin head that allows myosin to walk along the actin filament. Since myosin is connected to the thick filament, this movement of the myosin head corresponds to a movement of the thick filament relative to the thin filament.

5. Many cross-bridges produce macroscopic force. The cross-bridges have been likened to so many oarsmen rowing a large boat. The problem with the analogy is that usually oarsmen row in concert, and the medium they pull against is completely fluid, as opposed to the thin filament. Perhaps a better analogy is a group of sailors pulling in a rope hand-over-hand. In our everyday experience, this is not highly coordinated unless the load on the rope is great.

Figure 9. The acto-myosin cross-bridge cycle.

V. CROSS-BRIDGE CYCLING RATE explains the fiber type dependence of the force-velocity curve

A. Force of contraction is related to the number of myofibrils (sarcomeres) in parallel The myosin heads tug on the actin filaments. These actin filaments are connected at the Z-disk to titin, which is in turn connected to the thick filament on the adjacent sarcomere. Thus the force developed in each sarcomere is transmitted through the Z-disk to the adjacent sarcomere. It is clear from this description that the tension in a single myofibril is the same everywhere in the myofibril: the developed force is equal to the force developed by each sarcomere. Each myofibril contributes force in relation to the number of force generators, which is proportional to the number of filaments in the myofibril. The total force developed by the muscle depends on the number of its myofibrils. Taken to its final conclusion, we should expect muscle force to be proportional to its cross-sectional area.

B. Speed of contraction is related to the number of sarcomeres in series. The shortening of a muscle is the result of the shortening of its composite sarcomeres. If one sarcomere shortens a distance Δx, and another sarcomere in series with it shortens the same amount, the total shortening is 2 x Δx. Since both of these sarcomeres shorten in the same time interval, Δt, the velocity of shortening is Δx/Δt for one sarcomere and 2 x

Δx/Δt for two sarcomeres in series. Thus, the velocity of shortening is directly related to the number of sarcomeres in series and the rate of sacromere shortening.

Figure 10. Relation between sarcomere and muscle shortening.

C. Rate of shortening in a sarcomere depends directly on the turnover rate of the cross-bridges. Each cross-bridge cycle slides the thin filament about 7-10 nm past the thick filament. Rapid turnover of the cross-bridge means that more of these cycles occur per second, and therefore the thin filament slides past the thick filament more quickly. Thus, the rate of shortening of each sarcomere, and therefore the entire muscle, depends on the turnover rate of the cross-bridges.

D. Slow and fast twitch muscles have different myosin isoforms. Muscles make a variety of different isoforms of myosin which are encoded by separate genes. In the adult there are two basic varieties: slow myosin and fast myosin. The catalytic mechanisms for the two types are similar, but the turnover number (the number of completed reactions per second) is about 10 s-1 for the fast myosin and 3 s-1 for slow myosin.

E. Muscles can be classified on the basis of their myosin staining. It is possible to stain for the different myosin isoform ATPase activity based on the incubation conditions such as the pH. On this basis, a variety of different staining patterns can be observed. Brooke has classified muscles as type I, type IIA, type IIB and type IIC on the basis of myosin staining. The type I is the slow type of fiber and type II refer to different fast twitch fiber types. Figure 11 illustrates muscle heterogeneity as evidenced by myosin staining.

Figure 11. Histological staining of myosin reveals muscle heterogeneity. Myosin staining differentiates among various muscle types. This histological section shows three well-defined classes of staining but several intermediate fibers are also evident. VI. FORCE IS TRANSMITTED outside the cell through the cytoskeleton and

special transmembrane proteins.

A. Costameres are located at the Z-disk. The cytoplasmic face of the sarcolemmal membrane possesses a cytoskeletal assembly of proteins in a discrete, rib-like lattice. These are the costameres. They are located at the Z-disk where they bind to actin filaments through γ-actin filaments.

B. Costameres are a complex assembly of proteins. The structure of costameres is still being worked out, but many proteins

participate in it. These include dystrophin, a 427 KDa protein that is absent in Duchenne muscular dystrophy. This protein is localized to the periphery of the muscle fibers, on the cytosolic side of the sarcolemma, and binds to cytoskeletal elements. It is concentrated at the costameres but is not restricted to this location.

C. Costameres may have multiple functions. The function of the costameres is not yet established. The possibilities include: (1) to transmit force from the contractile filaments to the outside of the cell; (2) to mechanically support the sarcolemma to protect it against damage, particularly during eccentric contractions; (3) to maintain uniform sarcomeric spacing between resting and active fibers of different motor units.

Figure 12. Proposed arrangement of proteins in costameres of skeletal muscle. VII. PRACTICE QUESTIONS

1. The Z-disk lies in the middle of the

A. A band

B. H zone

C. I band

D. Terminal cisternae

E. T-tubule

2. Which protein is not located in the Z disk

A. Actinin

B. Actin

C. Nebulin

D. Titin

E. Myomesin

3. The top of the length-tension curve is flat because

A. The actin filaments begin to interfere with each other, dropping force

B. The myosin filament is longer than the actin filament

C. All of the muscle fibers are already recruited

D. The middle of the myosin filament contains a bare zone with no force generators

E. Thin and thick filaments are maximally overlapped.

4. Troponin is located

A. On the thick filament

B. On the thin filament

C. Mainly in the Z-disk

D. In the M line

E. In the lumen of the SR

5. During concentric contractions

A. The A bands shorten and so does the H zone

B. The I bands shorten and so does the H zone

C. A and I bands shorten and so does the H zone

D. A bands and I bands shorten but H zone stays constant

E. The I bands shorten but the H zone stays constant

6. Costameres

A. Are sarcomeres in the intercostal muscles

B. Link sarcomeres together

C. Form a scaffold for the assembly of myosin

D. Link the myofilaments to the extracellular matrix

E. Are the units of force in muscle

Answers: 1C; 2E; 3D; 4B; 5B; 6D