On which skeletal muscle filament is troponin located? What is the function of the sarcoplasmic reticulum (SR)?

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1 CASE 6 A 21-year-old man presents to a rural emergency center with a 1-day history of progressive stiffness of the neck and jaw, difficulty swallowing, stiff shoulders and back, and a rigid abdomen. Upon further questioning, the patient reports that the stiff jaw was the first symptom, followed by the stiff neck and dysphagia. On examination he is noted to have stiffness in the neck, shoulder, and arm muscles. He has a grimace on his face that he cannot stop voluntarily and an arched back from contracted back muscles. The physician concludes that the patient has tetanic skeletal muscle contractions. A 3-cm laceration is noted on his left foot. The patient reports sustaining the laceration about 7 days ago while he was plowing the fields on his farm. He has not had a tetanus booster. He is diagnosed with a tetanus infection, and an injection of the tetanus antitoxin is given. On which skeletal muscle filament is troponin located? What is the function of the sarcoplasmic reticulum (SR)? What is the molecular basis for initiation of contraction in skeletal muscle?

2 54 CASE FILES: PHYSIOLOGY ANSWERS TO CASE 6: SKELETAL MUSCLE Summary: A 21-year-old man with acute tetanus presents with muscle rigidity in the face, jaw, shoulders, back, and upper extremities 7 days after sustaining a puncture wound while working on his farm. He is diagnosed with tetanus. Troponin location: Thin filaments Sarcoplasmic reticulum function: Storage and release of calcium Molecular basis of contraction: Calcium-troponin-C binding CLINICAL CORRELATION Tetanus is a neurologic disorder caused by the toxin produced in the bacterium Clostridium tetani. Clostridium tetani is an anaerobic gram-positive motile rod that is found worldwide in soil, inanimate environments, animal feces, and occasionally human feces. Contamination in wounds with spores of C. tetani is seen commonly, but germination and toxin production occur only in devitalized tissue, areas with foreign bodies, and active infection. The toxin that is released blocks the release of several inhibitory neurotransmitters, including γ-aminobutyric acid (GABA), altering the synaptic vesicle release apparatus. With diminished inhibition, the resting firing rate of motor neurons increases. Because of the increased repetitive stimulation of the motor neuron, the calcium released from the SR remains bound to troponin and extends the time for cross-bridge cycling, resulting in muscles that do not relax. Symptoms of tetanus often begin in facial muscles such as those in the jaw ( lockjaw ) and then progress down the neck, shoulder, back, and upper and lower extremities. Generalized spasms may jeopardize breathing. Antitoxin is administered to bind and neutralize circulating and unbound toxin. Wounds should be explored, cleaned, and debrided. Muscle spasm can be controlled with medications such as diazepam (GABA agonist). Protection of the airway is essential. APPROACH TO MUSCLE PHYSIOLOGY Objectives 1. Describe striated muscle structure and arrangement. 2. List the steps in excitation contraction. 3. Understand force velocity relationships. 4. Describe summation and tetanus. 5. Describe motor unit recruitment. Definitions Sarcomere: The basic contractile unit comprising striated muscle. Excitation contraction (E C) coupling: The events that describe the calcium movements within the muscle fiber.

3 CLINICAL CASES 55 DISCUSSION All muscle cells can be divided into two groups striated and smooth on the basis of their microscopic structure. Striated muscle can be subclassified on the basis of location into three subgroups: skeletal, cardiac, and visceral. In addition, skeletal muscle can be classified on the basis of contractile behavior as fast-twitch or slow-twitch and on the basis of biochemical activities as oxidative or glycolytic. Actin and myosin are proteins that form the basic structural characteristic of striated muscle and are arranged in filaments: actin in thin filaments and myosin in thick filaments (see Figure 6-1). In the thin filaments, the monomers of actin are polymerized together like two strands of pearls that are twisted in an α helix to form F-actin (filamentous). In the thick filaments, complex myosin molecules are arranged so that most of their filamentous tails intertwine to form the backbone of the thick filaments and parts of Z disk Thin filament Thick filament Z disk I Band A Band RELAXED Z disk Z disk I Band A Band CONTRACTED Figure 6-1. Relationships of thick and thin filaments and adjacent Z disks of a sarcomere in a relaxed and a contracted state.

4 56 CASE FILES: PHYSIOLOGY their tails and their globular heads protrude from the backbone to form structures called cross-bridges. These thick and thin filaments are very ordered in their anatomic arrangement within the striated muscle cell. Thin filaments extend in opposite directions from protein structures called Z disks. In relaxed muscle, the thin filaments from two opposing Z disks extend toward each other but do not touch or overlap. Bridging the gap between the thin filaments, and overlapping with them, are the thick filaments. This arrangement Z disk, thin filament, thick filament, thin filament, Z disk defines the functional unit called a sarcomere. In striated muscle, sarcomeres are arranged in transverse registry, accounting for the characteristic banding pattern or striations. Arrangement of the contractile proteins in sarcomeres gives striated muscle cells the ability to shorten. When striated muscles contract, cross-bridges from the thick filaments attach to specific regions on the actin molecules. The cross-bridge heads then change angles, causing the thick and the thin filaments to slide over one another. The cross-bridges then release, and their angles assume the resting positions. They now are ready to attach to a different actin molecule, thus repeating the cycle until the stimulus to contract ceases. Because two opposing sets of thin filaments are associated with a single set of thick filaments, filament sliding results in movement of the Z disks toward one another without either the thick filament or the thin filament changing length (see Figure 6-1). Also, because the Z disks and the thin filaments are linked with other cytoskeletal elements, movement of the Z disks toward one another results in shortening of the muscle cell. Skeletal muscle cells are among the largest cells and are formed by the fusion of many precursor cells. Thus, these multinucleated cells often are referred to as fibers rather than cells. A single muscle, such as the gastrocnemius, is composed of thousands of muscle fibers arranged parallel to one another. Although the fibers are bound together by connective tissue sheaths and are connected to the same tendons at each end of the muscle, they are not coupled electrically to one another. Thus, any muscle fiber can contract independently of its neighboring fibers. The tendons of a skeletal muscle are attached to bones in such a way that contractions bring about movement or stabilization of the skeleton. Attachment to the bone that is being moved most often is near the joint so that large movements of the bone can be accomplished by small changes in the length of the muscle. Whether a muscle is contracting or relaxed depends on the level of cytosolic calcium available to interact with a regulatory protein complex, troponin, which is located on the thin filament with actin. In relaxed muscle, the level of free cytosolic calcium (calcium that is not bound to other structures) is low. Upon stimulation of the muscle, free calcium levels increase to initiate contraction by binding directly to a component of the troponin complex to bring about a conformational change in the complex. Once the stimulus for muscle contraction ceases, free calcium levels decrease and calcium dissociates from the regulatory proteins. The muscle then relaxes. Because calcium is the mediator between the events in the cell membrane that indicate excitation and the

5 CLINICAL CASES 57 protein interactions that result in contraction, the events that describe calcium movements in muscle cells often are referred to as excitation contraction (E C) coupling. The calcium that normally takes part in E C coupling in skeletal muscle is stored inside the cell in the SR. The SR is highly developed and extensive in skeletal muscle and functionally serves as a storage place for calcium during muscle relaxation. Upon muscle excitation, calcium moves out of the SR and into the cytoplasm down a large concentration gradient. Once in the cytoplasm, calcium interacts with the tropomyosin-troponin complex to allow full activation of the contractile proteins. Calcium then is taken up by the SR by an active process that involves a calcium (adenosine triphosphatase) ATPase. This pump has a high affinity for calcium and can lower cytosolic calcium quickly to levels that do not support contraction. Calcium is released by the SR in response to excitation of the cell membrane (sarcolemma). Each skeletal muscle fiber is innervated by a motor nerve. These nerves release acetylcholine (ACh) at their junctions with the muscle cell (neuromuscular junction). The ACh induces an increase in permeability of that portion of the cell membrane to Na + and K +. This results in depolarization of adjacent areas of the membrane to threshold, at which point an action potential ensues. When a muscle contracts, it develops force and usually shortens. A contraction that generates only force, with no muscle shortening, is called an isometric contraction. One that results in shortening against a constant force is called an isotonic contraction. Contractions of skeletal muscles are graded in force and in duration through activity of the central nervous system. Each skeletal muscle is innervated by a somatic nerve that is comprised of many axons of a-motor neurons. Each of these axons branches to innervate a number of fibers in the muscle. An a-motor neuron and the muscle fibers it innervates are called a motor unit. The force generated by a whole muscle depends on the number of its motor units that are active at any one time because the muscle fibers are arranged in parallel and parallel forces are additive. Thus, the central nervous system can regulate contraction force by regulating the number of motor units activated at any one time; this is called recruitment. Muscle force also can be regulated by the frequency at which the motor units are activated. A single activation to produce a single action potential of a muscle fiber will elicit a small contraction called a twitch. If the frequency of activation is increased, contraction duration and force increase up to a maximum. This process is called summation and tetanus. Force increases because, before the muscle relaxes from the previous excitation, the contractile proteins are activated again and again to add to the force. During summation and tetanus, each excitation releases calcium. The maximum calcium level is no higher than it is with a single isolated action potential, but it is maintained for a longer time. The continued elevation of calcium allows for continual activation of the contractile proteins, and the full force of cross-bridge cycling can be realized at the ends of the muscle. If a muscle contracts isotonically, it will shorten, and the velocity of shortening will depend on the load being moved (often called the afterload).

6 58 CASE FILES: PHYSIOLOGY By using different afterloads, which really are equal to the forces that the muscle must develop in order to shorten, a force velocity relationship can be determined for a specific muscle. If the muscle is lifting no afterload, the maximal velocity (V max ) is obtained. With increasing afterload, velocity decreases until an afterload is reached against which the muscle cannot shorten. Now the muscle is contracting isometrically. Skeletal muscles differ from one another in their force velocity relationships. Some, such as the extensor digitorum longus, contract more quickly than do others, such as the soleus. This difference is because of variations in the number and types of muscle fibers that make up the various muscles in the body. Although there is a spectrum of velocities among various muscle fibers, they have been divided into two main groups: fast-twitch and slow-twitch. Fast-twitch muscle fibers generally are found in muscles associated with rapid movement; slow-twitch fibers are found in muscles associated more with endurance and posture. Many muscles are composed of a mixture of fast- and slow-twitch fibers. Fastand slow-twitch muscle fibers differ in the contractile protein isoforms that are present and in the ATPase activities of the myosin isoforms. COMPREHENSION QUESTIONS [6.1] A researcher was examining some arrows sent from South America. He accidentally pierced his hand with one of the arrows. After a while he started to notice muscle weakness. He went to the hospital immediately. Electrical recordings from nerves innervating muscles in his arm indicated normal frequencies and amplitudes of impulses when stimulated; however, nerve-induced contractions of the muscles were weak. When the muscles were stimulated directly, normal contractions occurred. Which of the following is the most likely reason for the muscle weakness? A. Decreased ability of ACh to stimulate the muscle fibers B. Decreased ability of calcium to bind to troponin in the muscle fibers C. Decreased ability of the muscle to produce adenosine triphosphatase (ATP) D. Decreased ability of the muscle to undergo summation and tetanus E. Depletion of intracellular calcium [6.2] If a person lifts weights routinely, the muscles involved in the lifting undergo hypertrophy and become capable of generating greater force. Which of the following is the best explanation for the basis for these adaptations? A. Increased length of the muscle fibers B. Increased maximal velocity (V max ) of contraction C. Increased number of fast-twitch fibers in the muscle D. Increased number of sarcomeres arranged in parallel E. Greater specific activity of the myosin ATPase

7 CLINICAL CASES 59 [6.3] While you are standing, holding a tray piled with dishes, an additional 5 lb of dishes is placed on your tray. Your muscles that are holding the dishes increase their force of contraction through an increase in which of the following? A. Length of the muscle B. Number of motor units activated and the frequency of their activation C. Peak intracellular calcium concentration in the muscle D. Strength of each individual cross-bridge interaction with actin E. V max of the muscles Answers [6.1] A. Because the muscle responded normally to direct stimulation, the defect was not in the muscle itself. Therefore, the weakness was not because of decreased ability of calcium to bind to troponin in the muscle fibers, depletion of intracellular calcium, decreased ability of the muscle to undergo summation and tetanus, or decreased ability of the muscle to produce ATP. The lack of response also could not be because of a failure of action potentials in the motor nerves. Thus, the most likely explanation is a defect at the neuromuscular junction caused by a decreased ability of ACh to stimulate the muscle fibers. Curare, which is used by the inhabitants of South America as an arrow poison, is a drug that binds to ACh receptors, blocking access by ACh, and thus decreases the activation of skeletal muscles by motor nerves. [6.2] D. When a skeletal muscle undergoes hypertrophy, this is due mainly to an increase in the number of sarcomeres in existing muscle fibers and perhaps also to an increase in muscle fibers. Either way, the increased contractile units are added in parallel to existing units. This increases the force with which the muscle can contract. The length of the muscle, which is limited by its origin and insertion, will not change. The sarcomeres being added will be similar to the ones already present or more likely will have lower ATPase activity. Thus, there will not be increases in the maximal myosin ATPase activity, the maximal velocity of contraction, or the number of fast-twitch fibers in the muscle. [6.3] B. The increase in the force of contraction of skeletal muscle is regulated by the number of motor units recruited by the central nervous system (CNS) and by their frequency of activation (summation and tetanus). V max does not come into play because the muscle is contracting isometrically and V max is determined by the maximal ATPase activity of the myosin, which is not changing. The length of the muscle is not changing because this is an isometric contraction and because even during an isotonic contraction, the length of a skeletal muscle does not change appreciably. With each contraction, the amount of calcium released from internal stores is about the same, and so peak intracellular

8 60 CASE FILES: PHYSIOLOGY calcium concentrations will not rise to higher levels. Finally, during an isometric contraction each individual cross-bridge interaction with actin will generate the same amount of force. The increase in total force is because of an increase in the number of actin myosin interactions taking place at the same time. PHYSIOLOGY PEARLS Contraction of skeletal muscle is due to interaction of the proteins actin and myosin, which constitute thin and thick filaments, respectively. ATP is consumed in the process. Upon stimulation, calcium released from the SR binds to troponin to initiate contraction. The force of muscle contraction is regulated by the number of motor units activated (recruitment) and by the frequency with which they are being activated (summation and tetanus). Contractions can be isometric (force generation but no change in length) or isotonic (force generation and changes in length). REFERENCES Watras J. Muscle. In: Levy MN, Koeppen BM, Stanton BA, eds. Berne & Levy, Principles of Physiology. 4th ed. Philadelphia, PA: Mosby; 2006: Weisbrodt NW. Striated muscle. In: Johnson LR, ed. Essential Medical Physiology. 3rd ed. San Diego, CA: Elsevier Academic Press; 2003: