NROSCI/BIOSC 1070 and MSNBIO 2070 September 1, 2017 Mechanisms of Skeletal Muscle Contraction

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1 NROSCI/BIOSC 1070 and MSNBIO 2070 September 1, 2017 Mechanisms of Skeletal Muscle Contraction Anatomy of Skeletal Muscle Fibers Most skeletal muscle fibers extend the entire length of a muscle, and are typically innervated by only one nerve ending. The cell membrane of a muscle fiber is called the sarcolemma. It is composed of a plasma membrane in combination with a thin layer of polysaccharide material similar to that of the basement membrane surrounding capillaries. Tendon fibers fuse to the sarcolemma. Within the sarcolemma are hundreds of elements, called myofibrils. Each myofibril, in turn, contains about 1500 myosin filaments and about 3000 actin filaments. The myosin filaments are thick whereas the actin filaments are thin; the two types of filaments partly overlap, which results in bands in microscopic sections of myofibrils. Areas that contain only actin fibers are light in color (isotropic to polarized light), and form the I bands. In contrast, areas that contain overlapping myosin and actin fibers are dark in color (anisotropic to polarized light), and form the A bands. Small projections, called cross bridges, extend from the sides of the myosin filaments in the A bands. These cross bridges interact with the actin filaments, and are responsible for the contraction of muscle. The banding pattern produced by the overlapping actin and myosin fibers gives skeletal muscle its striated appearance. Actin filaments are secured to the Z membrane; the Z membrane also passes from myofibril to myofibril, and holds the fibers together. This insures that the I and A bands will line up throughout muscle fiber. The portion of a myofibril (or of the whole muscle fiber) between two successive Z membranes is called a sarcomere. When the muscle fiber is at rest (and thus is fully stretched), the length of a sarcomere is approximately 2 µm. In resting muscle, when the actin fibers are maximally separated, an area containing only myosin fibers appears in the center of the sarcomere. This region is called the H zone. In normally functioning muscle, it is rare to see an H zone, because contraction brings the actin fibers together in the middle of the sarcomere. The myofibrils are suspended inside the muscle fiber in a matrix called the sarcoplasm, which is composed of usual intracellular constituents. The fluid of the sarcoplasm contains large quantities of potassium, magnesium, phosphate, and enzymes. Numerous mitochondria lie between and parallel to the myofibrils. The sarcoplasm also contains an extensive endoplasmic reticulum, called the sarcoplasmic reticulum. This reticulum has a special organization that is extremely important in the control of muscle contraction. Muscle Contraction Page 1 September 1, 2017

You should be able to label I bands, A bands, Z membranes, and H zone.

2 Schematic diagram of anatomy of muscle fiber. Electron micrograph of muscle myofibrils. You should be able to label I bands, A bands, Z membranes, and H zone. You should also be able to differentiate a sarcomere. Note the numerous mitochondria. Muscle Contraction Page 2 September 1, 2017

3 Molecular Mechanism of Muscle Contraction The basic mechanism of muscle contraction is illustrated below. In the relaxed state of a sarcomere, the ends of actin filaments derived from two successive Z membranes barely overlap with each other, so that an H zone can be present. In contrast, in the contracted state, these actin filaments are pulled inward so that they overlap considerably with each other. As a result, the Z membranes are pulled closer together, and both the I bands and sarcomeres decrease in width. This mechanism of muscle contraction is called the sliding filament mechanism. What causes the actin filaments to slide along the myosin filaments? Although the answer is not completely known, almost certainly it is related to attractive forces between the actin and myosin filaments. Presumably, these attractive forces are the result of mechanical, chemical, or electrostatic forces generated by the interaction of the cross bridges of the myosin filaments with the actin filaments. Under resting conditions, the attractive forces between actin and myosin molecules are inhibited. However, when an action potential travels over the muscle fiber membrane, the release of large quantities of calcium ions from the sarcoplasmic reticulum activates the attractive forces. It is known that energy is required for a muscle contraction to occur; this energy is derived from degrading ATP to ADP. Molecular Characteristics of the Contractile Filaments The myosin filament is composed of multiple myosin molecules. Each myosin molecule is composed of 6 polypeptide chains, two heavy chains and four light chains. The two heavy chains wrap spirally around each other to form a double helix, which is called the tail of the myosin molecule. One end of each of the heavy chains is folded into a globular polypeptide structure that forms the myosin head. The four light chains are also parts of the myosin head. These light chains control the function of the head during muscle contraction. The myosin filament is made up of 200 or more individual myosin molecules. At the Muscle Contraction Page 3 September 1, 2017

4 core of the myosin filament are the tails of myosin molecules, which are bundled together to form the body of the filament. However, part of the tails of the myosin molecules as well as the heads hang from the sides of the body; the protruding portions of the myosin molecule are called cross-bridges. Each cross-bridge is flexible at two points called hinges, one where the cross-bridge leaves the body and another where the head attaches to the tail part of the myosin molecule. The hinged arms allow the heads either to be extended far outward from the body or to be brought close to the body. The actin filament is also complex, and is composed of actin, tropomyosin, and troponin. The backbone of the actin filament is a double helix of the F-actin protein molecule. At regular intervals along the F-actin helix are the active sites where myosin interacts with actin. The actin filament also contains two additional protein strands that are polymers of tropomyosin molecules. The tropomyosin strands are believed to cover the active sites along the F-actin strands. The third components of the actin molecule are collections of three globular proteins called troponin. One of the globular proteins has affinity for actin, another for tropomyosin, and a third for calcium. It is believed that troponin holds the actin and tropomyosin together, but the binding of calcium to troponin causes the configuration between actin and tropomyosin to change, thereby exposing the active sites. Interaction of Actin and Myosin to Produce Muscle Contraction If actin and myosin molecules are placed together in the presence of ATP and magnesium, they bind together strongly. However, the addition of troponin and tropomyosin inhibits this binding. As stated above, it is believed that tropomyosin strands normally cover the active sites on the actin strands, but that the binding of calcium to troponin causes the tropomyosin strands to be pulled away, uncovering the active sites. As soon as the active sites are uncovered, the heads of the cross bridges immediately attach to the actin molecules at these points. It is postulated that when attachment occurs, profound changes take place in the intermolecular forces in the head and arm of the cross bridge. A configuration change then takes place in the cross bridge, causing the head to tilt while pulling the actin filament along with it. This tilt of the head of the cross bridge is called the power stroke, and is the major mechanism in muscle contraction. After the power stroke is completed (and the configuration of the myosin molecule has changed), the myosin is no longer attracted to actin and the two molecules separate. The myosin then reverts to its original configuration, and its attraction for actin returns. The myosin head then attaches to the next available active site. Muscle Contraction Page 4 September 1, 2017

5 Each of the cross bridges is believed to operate independently of all others, each attaching and pulling in a continuous, alternating ratchet cycle. Role of ATP in Producing Muscle Contraction Most of the muscle contraction proceeds without ATP. The attachment of myosin to actin is not ATP dependent; neither is the resulting change in confirmation in the myosin molecule It is believed that this change in shape of the myosin molecule exposes an ATP binding site. The binding of ATP to this site causes the myosin to be released from the actin. The ATP molecule then degrades to ADP, and the energy released causes the myosin molecule to return to its original confirmation. The myosin is then ready to bind to the next actin binding site. If ATP were to be depleted, as occurs following death, then the actin and myosin molecules would not separate, and would be permanently fixed together. This would cause the muscle length to become fixed, and the muscle would appear to be very stiff. In fact, this phenomenon explains rigor mortis. Obviously, large amounts of ATP are converted to ADP during muscle contraction. ATP must be replenished in order for the muscle contraction to continue. Muscle cells, like other cells, can generate ATP from both glycolysis and oxidative phosphorylation. In addition, muscle cells have an immediate precursor for the generation of ATP: creatine phosphate. Creatine phosphate is a high-energy molecule that can re-phosphorylate ADP to ATP. Creatine phosphate levels drop during muscle contraction; some athletes consume this chemical as a performance enhancer. Initiation of Muscle Contraction Initiation of contraction in skeletal muscle begins with action potentials in the muscle fibers. These elicit electrical currents that spread to the interior of the fiber where they cause release of calcium ions from the sarcoplasmic reticulum. Muscle action potentials are very similar to nerve action potentials, and are triggered by the release of neurotransmitter at a neuromuscular junction between the muscle fiber and the axon of a motoneuron. Typically, there is only one neuromuscular junction per muscle fiber, which is located near the middle of the fiber. The action potential must then propagate from the middle of the muscle fiber to the ends. Skeletal muscle fibers are so large that specialized mechanisms are required for action potentials to spread rapidly throughout all parts of the fiber. There are numerous invaginations in the cell membrane of the muscle fiber, which are called transverse tubules or T tubules. These T tubules allow an action potential to propagate deep within the muscle fiber. The sarcoplasmic reticulum conducts the action potential to all sarcomeres. The sarcoplasmic reticulum is composed of terminal cisternae that abut the T tubule, and longitudinal tubules that surround all of the myofibrils. Muscle Contraction Page 5 September 1, 2017

6 Muscle Contraction Page 6 September 1, 2017

Once the sarcoplasmic reticulum membrane is depolarized by the action potential, a release of calcium occurs due to the opening of voltage-gated calcium channels.

7 Once the sarcoplasmic reticulum membrane is depolarized by the action potential, a release of calcium occurs due to the opening of voltage-gated calcium channels. This released calcium binds to the troponin molecules, thereby causing the tropomyosin filaments to be pulled away from the actin active sites, and starting the muscle contraction. Termination of Muscle Contraction The calcium channels in the sarcoplasmic reticulum are only open for a short period after an action potential invades. However, the muscle contraction will persist as long as calcium is present in the myofibril. To eliminate the calcium, an active calcium pump is present in the sarcoplasmic reticulum. This calcium pump ensures that the muscle contraction elicited by a single release of neurotransmitter at the neuromuscular junction is short on the order of 300 ms in duration. If the contraction is to persist for longer periods of time, a series of action potentials must pass through the muscle fiber (see below). Muscle Contraction Page 7 September 1, 2017

8 Length-Tension Relationship in Muscle A skeletal muscle fiber will only contract efficiently if its resting length is near normal. If a muscle is stretched extensively, then there is little overlap between actin and myosin, and no contraction can take place. Similarly, if a muscle is compressed extensively, the relationship between actin and myosin is altered such that contraction cannot take place. This concept can also be applied to a whole muscle. If the muscle is stretched, tension develops due to elastic components in the muscle. However, if contraction is induced in a stretched muscle, the tension produced by the contraction will be small due to minimal overlap between actin and myosin at the onset of contraction. Similarly, if a muscle is compressed, very little tension will be produced during contraction because of the altered relationship between actin and myosin. Muscle Contraction Page 8 September 1, 2017

Activation of Muscle Contraction by Motoneurons Motoneurons are amongst the largest neurons in the central nervous system, and have been extremely well studied.

9 Activation of Muscle Contraction by Motoneurons Motoneurons are amongst the largest neurons in the central nervous system, and have been extremely well studied. Motoneurons send their axons out of the spinal cord through the ventral root, and into peripheral nerves. A motoneuron innervates a collection of muscle fibers in a particular muscle; motoneurons almost never innervate more than one muscle. A motoneuron and the contingent of muscle fibers it innervates is called a motor unit. The term muscle unit refers to all of the muscle fibers innervated by a particular motoneuron. A motoneuron innervates anywhere from 10 to > 1000 muscle fibers. The innervation ratio depends on the precision of motor control needed. In the hand, where fine motor control is necessary, a motoneuron innervates only a few muscle fibers. In axial muscles such as the back muscles, the innervation ratio is larger. A muscle fiber receives input from only one motoneuron. Chemical neurotransmission from the motoneuron end plate to the muscle fiber occurs at a special synapse called the neuromuscular junction. The neurotransmitter at this synapse is well established: acetylcholine. The acetylcholine receptors are found in many enfoldings in the muscle fiber membrane, called junctional folds. The junctional folds also contain a high concentration of acetylcholinesterase, to quickly degrade the transmitter after it has been released, to assure that the muscle contraction is brief. Acetylcholine acts at nicotinic receptors at the neuromuscular junction, and thus drugs can differentiate this synapse from the terminal synapse in the parasympathetic nervous system which utilizes muscarinic acetylcholine receptors. Since there is no blood-brain barrier in the periphery, transmission at the neuromuscular junction can be influenced by more drugs/ chemicals and diseases than synapses in the central nervous system. Some of the factors are listed in the table below. Synaptic transmission at the neuromuscular junction is very secure. If a motoneuron generates an action potential, then there is virtually a 100% chance that the associated muscle fibers will contract. Muscle Contraction Page 9 September 1, 2017

General Information: Receptor Subtypes Receptors that bind a particular neurotransmitter such as acetylcholine or norepinephrine are not all the same.

10 General Information: Receptor Subtypes Receptors that bind a particular neurotransmitter such as acetylcholine or norepinephrine are not all the same. In fact, binding of a neurotransmitter at one site can have vastly different effects than at another. Typically, receptors with differing responses to the binding of a particular neurotransmitter also have different configurations, and affinities for that neurotransmitter. It thus may be possible for a particular drug to bind to one neurotransmitter receptor subtype and not another. This is how neurotransmitter subtypes are differentiated. The acetylcholine receptor has two major subtypes: nicotinic and muscarinic receptors. The nicotinic receptors bind the plant alkaloid nicotine, whereas the muscarinic receptors bind the toadstool toxin muscarine. The agonist for both receptors in the body is the same (acetylcholine), but the selective affinity of the subtypes for one drug can be exploited by pharmacologists. Often, after receptor subtypes are discovered, it is found that these subtypes can be subdivided into additional groups. For example, 17 different types of nicotinic receptors have been identified. Each is composed of 5 protein building blocks, and 12 different building blocks have been discovered. All the building blocks are similar in their amino acid sequence, but not identical. Thus, the affinity of a nicotinic receptor for drugs depends on exactly which combination of building blocks it contains. The table below shows the affinity of three common subtypes of nicotinic receptors for drugs. Interestingly, the nicotinic receptors at the neuromuscular junction are relatively insensitive to nicotine, but are still classified as nicotinic receptors because their structure is so similar to others that bind nicotine well. Location Agonists Antagonists Neuromusclar Junction Autonomic Ganglia Brain acetylcholine carbachol suxamethonium acetylcholine carbachol nicotine epibatidine acetylcholine nicotine epibatidine curare pancuronium α-conotoxin α-bungarotoxin mecamylamine α-bungarotoxin hexamethonium α-conotoxin mecamylamine Structure of the nicotinic receptor Muscle Contraction Page 10 September 1, 2017

11 Development of Muscle Force Muscle force can be increased by two mechanisms: frequency modulation (increasing the discharge rate of the motor unit) or recruitment (activation of inactive motor units). If a motoneuron fires before the tension produced by the previous contraction has dissipated, then the force of the second contraction will add to the first. Obviously, the faster the firing rate of the motoneuron, the more cumulative force will be produced until the maximal contractile ability of the muscle is reached. If a motoneuron fires rapidly enough, a plateau of muscle tension will occur. This plateau is referred to as a tetanus. However, recruitment of new motor units is required for a muscle to develop a reasonable amount of force; there is a limit as to how much force a single motor unit can produce. Muscle Contraction Page 11 September 1, 2017

Properties of Muscle Units It is possible to recognize the muscle fibers innervated by a single motoneuron through glycogen depletion experiments.

12 Properties of Muscle Units It is possible to recognize the muscle fibers innervated by a single motoneuron through glycogen depletion experiments. Prolonged, repetitive stimulation of a single motoneuron via an intracellular electrode results in an eventual depletion of glycogen from the muscle fibers innervated by that motoneuron. Glycogen-free muscle fibers are rare in normal muscle. Thus, to recognize the muscle fibers innervated by the stimulated motoneuron, one must remove the muscle, stain for glycogen, and determine the location of the glycogen-free muscle fibers. Such studies have shown that muscle fibers innervated by a particular motoneuron are scattered fairly uniformly across an extensive territory, and that only occasionally are two adjacent muscle fibers innervated by the same motoneuron. It was also determined that all of the muscle fibers innervated by the same motoneuron have the same histochemical profile. In 1874, Ranvier noted that mammalian muscle fibers have a range of colors: some are red and some are white (you have probably noticed this yourself when eating muscle tissue from birds). It was later discovered that red muscle fibers have a smaller diameter than white muscle fibers. Most mammalian muscles (unlike the muscles of birds) are heterogeneous and contain both red and white fibers. However, there are exceptions. For example, the soleus muscle of the cat (a muscle that acts to extend the foot) is comprised almost entirely of red muscle fibers. Of course, red and white muscle fibers have a number of different characteristics in addition to diameter. The largest white muscle fibers tend to have few capillaries bordering them, little myoglobin (an oxygen-carrying protein), few mitochondria and low levels of oxidative enzymes. In contrast, they are richly supplied with glycogen, glycolytic enzymes and phosphorylase. At the other extreme are the small, red fibers. These fibers are adjacent to a rich capillary supply, and contain a great deal of myoglobin, and have many mitochondria. They are richly supplied with oxidative enzymes, but are relatively incapable of glycolytic metabolism. Another histochemical group exists that is intermediate between these extremes (the muscle fibers have both oxidative and glycolytic capabilities). As you might guess, the largest white fibers are capable of providing a very strong contraction for a short time, whereas the small red fibers are involved in less powerful but sustained contractions. The properties of muscle fibers are summarized in the following table: Muscle Contraction Page 12 September 1, 2017

13 In the 1960s and 1970s, Robert Burke developed a classification scheme for motor units. He stimulated intracellularly in motoneurons, and determined the contraction time for the stimulated motor unit. At the end of the experiment, he did glycogen depletion and determined the histochemical profile of the muscle fibers innervated by the stimulated motoneuron. Burke separated motor units into type S (slow contracting) and F (fast contracting); a 55 ms contraction time was the separation between the two groups. Burke also delivered repetitive stimuli and looked at the force of the contraction at the onset of muscle contraction and 120 sec later. He found that all of the type S motor units had the same contraction tension at the two testing times, and hence had a fatigue index (final contraction tension/initial contraction tension) of 1. In contrast, fast contacting muscles had a variety of fatigue indexes. Some of the fast contracting motor units produced less than 25% of the force at the end of the stimulus train than at the beginning; others could maintain up to 75% of their contraction tension throughout the test. Burke thus divided the type F motor units into type FF (fatigable), FR (fatigue resistant) and FI (intermediate fatigability). Burke confirmed using glycogen depletion and histochemical techniques that all of the muscle fibers belonging to a particular motor unit had the same properties. Question for discussion: Would it be possible for FF motor fibers to perform their job if they relied on aerobic metabolism? Why or why not? Relaxation of Muscle Thus far, we have only concentrated on the contraction of skeletal muscle. However, the mechanisms involved in relaxation also deserve comment. By definition, relaxation occurs when there is no contraction, and the muscle returns to its normal resting length. This must be done smoothly, but requires no active expenditure of energy. When the myosin heads disengage from actin, relaxation occurs. This process is smoothed by the elastic elements in the body, both in the muscle itself and structures to which it is attached. Muscle Contraction Page 13 September 1, 2017

14 Summary of Motor Unit Types: Muscle Contraction Page 14 September 1, 2017

*The plasma membrane of a muscle fiber

Skeletal Muscle Contraction: Histology: Sarcolemma: *The plasma membrane of a muscle fiber -T- tubules - Terminal Cisternae - Sarcoplasmic Reticulum *Transverse tubules- tube like invaginations or inward