Muscle

Learning Objectives

• Students should be able to contrast the architecture of muscle cells in skeletal, cardiac and smooth muscle.
• Students should be able to contrast the organization of myosin and actin filaments in skeletal, cardiac and smooth muscle.
• Students should be able to list the organization of extracellular matrix in skeletal muscle.
• Students should be able to describe the importance of dystrophin to the viability of skeletal muscle cells .
• Students should be able to describe the structure and events at neuromuscular junction that lead to contraction of skeletal muscle cells.

Types of Muscle

Skeletal

Skeletal muscle is involved most prominently in the movement of limbs but is also responsible for movement of the eyes. It can generate a range of forces from rapid and powerful to slow and delicate. Skeletal muscle is activated by voluntary and reflex signals. Skeletal muscle is made of a collection of cells and connective tissue. The cells of skeletal muscle span the length of entire muscle. So if a muscle is 5 cm, the muscle cells are 5 cm in length. To support the large volume of cytoplasm and all the proteins needed, skeletal muscle cells are multinucleated. Note that the cells are arranged in parallel arrays to generate contraction in one direction. Connective tissue envelopes each muscle cell to provide mechanical support, in the form of collagen fibers, and metabolic support via capillaries.

Cardiac

Cardiac muscle cells are responsible for pumping blood from the heart. They generate forceful contractions and are under involuntary control. Cardiac muscle cells are much smaller than skeletal muscle cells and are connected in series to span the length of cardiac muscle. Individual cells are linked and communicate via gap junctions which allows action potentials to pass from one cell to the next. Note that the cells are arranged in parallel arrays to generate contraction in one direction.

Smooth

Smooth muscle surrounds most of the internal organs: GI tract, respiratory tract, bladder. It is also surrounds most blood vessels and veins. It provides tone and shape but can also generate slow and powerful contractions to change the size and shape of an organ. Smooth muscle cells are under control of the autonomous nervous system. Smooth muscle is composed of numerous spindled shaped cells. Gap junctions between cells allows coordination of contraction. Note that the smooth muscle cells are often arranged in layers that are orthagonal to each other. Smooth muscle often contracts an organ in multiple directions.

To get a better sense of how smooth muscle cells control the shape of an organ, one can look at blood vessels and bronchioles. Here, the smooth muscle cells are arranged circumferentially around the vessels and bronchioles. Contraction of the cells, decreases the diameter of the lumen of the vessel to restrict the volume of blood that can flow through vessel. The cardiovascular system uses smooth muscle to control the distribution of blood to different capillary beds. Smooth muscle cells perform a similar function in the respiratory system. Smooth muscle cells contract to narrow the lumen of the bronchioles. This restricts the amount of air that flows through the bronchiole and is important for preventing access of foreign particles and microorganisms to the deeper aveoli. However, over stimulation or proliferation of smooth muscle cells can lead to pulmonary diseases such as asthma.

The Molecular Basis of Muscle Contraction

All three types of muscle cells share some features. All generate movement through contraction. All use force from myosin pulling on actin filaments to generate force for contraction. Myosin is arranged into bipolar filaments with motors oriented in opposite directions. Actin filaments are anchored to sites within cells, so that when myosin pulls on actin filaments, the cell contracts. All use calcium as trigger for contraction. The differences is in the inner architecture of the cells and how myosin is activated to generate contraction.

Structure of Myosin

The structure of myosin in all three muscle types is also similar. Muscle myosin is a homodimer of two polypeptides. The protein contains a motor domain at one end and form a long coiled-coil interaction at the other end. These muscle myosin dimers polymerize to form a bipolar filament with hundreds of motors on each side of the filament. A region in the middle of the filament lacks motors and is called the bare zone. Recall that myosin motors want to move toward the plus ends of actin filaments and in the bipolar filament the motors on the right side are oriented to move in the opposite direction from those on the left side of the filament.

Muscle Express Different Types of Myosin

The different types of muscle express different types of myosin which to a large extent determines the contractile properties of the muscle. There are several different types of skeletal muscle cell (e.g. slow-twitch and fast-twitch) and each expressed a specific type of myosin. In addition, the type of skeletal muscle cell exhibit differ metabolic programs with some relying on anaerobic metabolism and other aerobic.

Skeletal Muscle Cell

As mentioned above, skeletal muscle in humans is composed of linear arrays of muscle cells with each cell spanning the length of the muscle. If we examine a muscle stained with H&E in longitudinal section, we observe a series of alternating light and dark bands. This pattern gives skeletal muscle its other name: striated muscle. The light and dark bands reflect the pattern of actin and myosin filaments in the cytoplasm of the cells. The light bands are regions where there are only actin filaments and the dark bands contain both actin and myosin filaments. The light bands are called I-bands and the dark bands are called A-bands.

An electron micrograph of skeletal muscle reveals the arrangement and structure of the myosin and actin filaments. First notice the alternating regions of electron dense material (dark) and less dense (light), with correspond to the A-band and I-band, respectively, that was seen by light microscopy. Thus, the electron dense region contains actin and myosin filaments whereas the light region contains only actin filaments.

The arrangement of filaments is highly ordered. First note the thin dark link within the I-band. This is called the Z-disc and contains proteins that bind the plus ends of actin filaments. The Z-discs define the base longitudinal unit in skeletal muscle called a sarcomere. A sarcomere starts at one disc and ends at the next disc, a distance of 2.4 µm in relaxed muscle. Within a sarcomere actin filaments extend from each Z-disc toward the center of the sarcomere. Recall that the plus-ends of the filaments are at the Z-disc and the minus ends in the center of the sarcomere. In the center of the sarcomere is a bipolar myosin filament. Note within the A-band a light region called the H-zone. The H-zone lacks actin filaments. Thus, the actin filaments extend from Z-discs toward the center of the cell but end before reaching the exact center of the sarcomere.

Contraction of the sarcomere is initiated when the myosin motors on the filament contact actin filaments and generate a pulling force. Recall that the myosin filament is bipolar with motors on one end of the filament all wanting to walk toward the nearest Z-disc while those on the opposite end of the myosin filament want to walk toward their nearest Z-disc. Consequently, the myosin filament pulls the Z-discs close to each other.

Compare the sarcomere from a relaxed muscle to a contracted muscle. In the contracted sarcomere, the I-band has become smaller, indicating that the actin filaments have been pulled over the myosin filaments in the A-band. Note that in the contracted muscle the H-zone is darker than in the relaxed sarcomere. This reflects the movement of actin filaments into the H-zone. Contraction shortens the distance between Z-discs from 2.4 µm to 2 µm. This may seem like small distance considering that a typical muscle must shorten by centimeters. However, a skeletal muscle cells contains thousands of sarcomeres arranged in series.. For example, the bicep muscle is about 15 cm in length and each muscle cell has about 62500 sarcomeres. If each sarcomere contracts by 0.4 µm, then the total muscle will have contracted by 2.5 cm.

Other Components of Sarcomeres

Sarcomeres contain several other proteins that determine the architecture of the sarcomere and its contractile properties. Nebulin is a filamentous protein that wraps actin filaments. It extends from the Z-disk to the minus end of actin filaments. It contacts the actin monomers in the filaments and may function as a molecular ruler to determine the length of the filaments in the sarcomere.

Titan is probably the largest protein in the human genome and has multiple functions in the sarcomere. Titan is 1.2 µm and extends from both Z-disks into the M-line. Titan contacts the myosin filament at numerous points. Titan is thought to function as a molecular spring to prevent sarcomeres from overextending. If sarcomeres become too long, then some of the motors on the myosin filament become out of register with the actin filament, and the sarcomere generates less force because it has fewer motors to call upon.

Skeletal Muscle in Cross-Section

A cross section of a skeletal muscle cell reveals a cytoplasm that is filled with myofibrils (parallel arrays of actin and myosin filaments). Mitochondria are abundant in some skeletal muscle cells to provide ATP via oxidative phosphorylation. The nucleus of skeletal muscle cells is pushed to the periphery to accommodate the myofibrils.

A cross section of myofibril reveals precise arrangement of actin and myosin filaments in a myofibril. Six actin filaments (small dots) are arranged radially around one myosin filament (large dots). Motors in one myosin filament contact 6 different actin filaments.

Skeletal Muscle Architecture

Skeletal muscle is a multicellular structure with a specific organization. Recall that each cell in skeletal muscle spans the entire length of the muscle. Individual muscle cells are grouped together into a multicellular unit.

Three levels of connective tissue organized the skeletal muscle cells into an integrated functional unit. Individual muscle cells or fibers are wrapped by a layer of connective tissue called endomysium. Endomysium consists of a basement membrane that interacts directly with proteins in the cell membrane of skeletal muscle cells and a layer that contains more mechanically robust components such as type I collagen. Endomysium also contains capillaries to deliver oxygenated blood and nutrients to skeletal muscle cells.

Several skeletal muscle cells and their endomyseum are wrapped in another layer of connective tissue called the perimyseum. Perimyseum has more collagen fibers than endomysium and larger blood vessels that distribute blood to the capillaries in the endomysium.

The third layer of connective tissue is the epimysium which surrounds the entire muscle. Epimysium contains many collagen fibers and is classified as dense, irregular connective tissue.

Attachment to Bone

To generate movement of our limbs, muscles must pull on bone and therefore need a way to attach to bone. Tendon is the most common structure that links skeletal muscle to bone. Recall that tendon is a dense regular connective tissue which is very resistant to tension. At the muscle-tendon junction, the connective tissue layers of the muscle transition into the connective tissue of the tendon. The tendon connects to the outer (periosteal) surface of bone. Thus, as skeletal muscle contracts, it pulls on tendon which in turn pulls on bone.

Connections to Connective Tissue

Like other cells, skeletal muscle cells connect to the protein components in the extracellular matrix. Skeletal muscle cells connect to the endomyseum along the entire their entire length. Several sets of proteins link the myofibrils near the cell membrane to proteins in the endomyseum. One interaction utilizes dystrophin and a complex of proteins called the dystroglycan complex. Proteins in the dystroglycan complex interact with protein components in the extracellular matrix. Dystrophin links the dystroglycan complex to the Z-disks in the myofibrils nearest the cell membrane. The other myofibrils are linked at their Z-disks via an intermediate filament protein called desmin. Thus, all of the myofibrils are cross-linked into a large network of myofibrils and this network is linked to the extracellular matrix.

Transmission of Force Generation

The interconnectedness of Z-discs and endomysium raises the question of whether the endomysium and other connective tissue layers have a role in transmitting the contractile force generated by actin and myosin filaments. Current experimental evidence suggests that over half the force generated by contraction of sarcomeres is transmitted laterally to the extracellular matrix in the endomysium, suggesting that tension within the endomysium is responsible for transmitting a significant amount of contractile force to the tendons that connect skeletal muscle to bone. As sarcomeres shorten, the connections at Z-discs between myofibrils and between myofibrils and the ECM, generate tension in the ECM which pulls on the bones to which the muscle is attached.

The role of the connective tissue layers in force transmission makes sense when one considers the architecture of skeletal muscle. First, the connective tissue layers contain a lot of collagen fibers with makes them stiffer than the cytoplasm of muscle cells. Thus, the connective tissue layers are structurally more efficient transmitters of force. Second, the collagen fibers in the connective tissue layers of skeletal muscle transition into the collagen fibers in tendons.

Triggering Contraction

Muscle cells innervated by motor neurons that arise from spinal cord. Activation of motor neuron triggers contraction of muscle cell. Motor neurons contact muscle cells at a structure called the neuromuscular junction.

The axon of motor neurons will often branch to allow a single motor neuron to generate neuromuscular junctions (NMJ) with multiple skeletal muscle cells. However, each muscle cells is innervated by only one neuron.

At the neuromuscular junction, motor neurons form a synapse with the skeletal muscle cell. At the synapse, motor neurons contain high concentration of vesicles, called synaptic vesicles, that are filled with acetylcholine. Acetylcholine is the neurotransmitter that triggers contraction of skeletal muscle cells. At the neuromuscular junction, the cell membrane of the skeletal muscle is arranged into a fold. This increases the surface area of cell membrane to accommodate more acetylcholine receptors and voltage gated sodium channels. The acetylcholine receptor concentrates on the outer surface of the fold, whereas the sodium channels localize deeper in the fold. Basal lamina in folds contains acetycholinesterase that degrades acetylcholine.

When an action potential in a motor neuron reaches the axon terminus, it opens voltage-gated calcium channels. A rise in the concentration of cytosolic calcium triggers fusion of synaptic vesicles with the cell membrane, releasing acetylcholine onto the surface of the skeletal muscle cell. Ligand-gated ion channels in the cell membrane of the skeletal muscle cells bind acetylcholine. When bound to acetylcholine. the ion channels open allowing primarily sodium to enter the cell. The influx of sodium depolarizes membrane and leads to the opening of voltage-gated ion channels. The opening of voltage-gated ion channels initiate an action potential along muscle cell membrane.

An action potential starts on the surface of skeletal muscle cells but many of the sarcomeres lie deep within the muscle cell far from the cell membrane. How does the action potential trigger contraction of sarcomeres in the center of the cell?

The cell membrane of skeletal muscle cells invaginates in structures called T-tubules. The T-tubules penetrate into the center of the cell to carry the action potential that starts at the surface into the deepest regions of the skeletal muscle cell. Within skeletal muscle cells, t-tubules interact with the endoplasmic reticulum which in skeletal muscle is called the sarcoplasmic reticulum.

The t-tubules and sarcoplasmic reticulum are connected via a set of calcium channels. T-tubules contain the dihydropyridine or DHP calcium channel and the sarcoplasmic reticulum contain the ryanodine calcium channel. When an action potential travel down a T-tubule, the membrane depolarization open the DHP channel allowing calcium to flow down its electrochemical gradient into the cell. The opening of the DHP channel triggers the ryanodine channel in the sarcoplasmic reticulum to open. In skeletal muscle cells, the physical connection between DHP and ryanodine channels allows DHP channels to open ryanodine channels during an action potential. The sarcoplasm reticulum stores a high concentration of calcium which enters the cytosol through open ryanodine channels.

Tropomyosin and Calcium-Mediated Contraction

Actin filaments are wrapped by another filament called tropomyosin. Each tropomyosin covers 7 actin monomers and adjacent tropomyosins are linked to cover an entire actin filament. The position of tropomyosin on actin occludes the myosin binding site and prevents myosin from binding to actin. So, even though the myosin motors are active, they can’t bind actin to generate force and contraction. Cytosolic calcium causes tropomyosin to shift, exposing myosin-binding site. Troponin complex is calcium sensing protein along actin filaments. It binds calcium induces shift in tropomyosin. When calcium levels fall, the tropomyosin shifts back to occlude the myosin-binding site and knock myosin off the filament.

When an action potential ends, skeletal muscle cells remove cytosolic calcium by pumping calcium back into the sarcoplasmic reticulum or out of the cell. The fall in cytosolic calcium relaxes the muscle cell.

Skeletal muscle cells also have ways to reduce the amount of acetylcholine at synapses. Skeletal muscle cells secrete an enzyme called acetylcholinesterase which binds to the basement membrane at the synapse. Acetylcholinesterase digests acetylcholine and limits the amount of neurotransmitter that can trigger an action potential in the skeletal muscle cell.

Because the action potential along a skeletal muscle cell is transient, contraction is quickly relaxed. Thus, when a motor neuron releases acetylcholine, it causes a twitch in the skeletal muscle cell. At low frequencies of stimulation by the motor neuron, the tension in the skeletal muscle cell falls to the resting level between individual stimuli ( Panel A ). Single skeletal muscle twitches last between 25 and 200  ms, depending on the type of muscle. Although each twitch is elicited by a single muscle action potential, the duration of contraction is long compared with the duration of the exciting action potential, which lasts only several milliseconds. Because the muscle twitch far exceeds the duration of the action potential, it is possible to initiate a second action potential before the first contraction has fully relaxed. When this situation occurs, the second action potential stimulates a twitch that is superimposed on the residual tension of the first twitch and thereby achieves greater isometric tension than the first (compare Panels A and B ). This effect is known as summation.

If multiple action potentials occur close enough in time, the multiple twitches can summate and thus greatly increase the tension developed. Summation is more effective at increasing tension when the action potentials are grouped more closely in time, as in Panel C . In other words, tension is higher when action potentials are evoked at higher frequency. Because this type of tension enhancement depends on the frequency of muscle stimulation, it is referred to as frequency summation.

When the stimulation frequency is increased sufficiently, the individual twitches occur so close together in time that they fuse (see Panel D ) and cause the muscle tension to remain at a steady plateau. The state in which the individual twitches are no longer distinguishable from each other is referred to as tetanus. Tetanus arises when the time between successive action potentials is insufficient to return enough Ca 2+ to the SR to lower cytosolic calcium below a level that initiates relaxation. In fact, a sustained increase in cytosolic calcium persists until the tetanic stimulus ceases. At stimulation frequencies above the fusion frequency that causes tetanus, muscle fiber tension increases very little.

Repair of Skeletal Muscle

Because of the repeated and strong contractile forces generated in skeletal muscle, damage and cell death are a common problem. Skeletal muscle has the ability to regenerate skeletal muscle cells from the division and differentiation of stem cells. These stem cells are called satellite cells and are located on the surface of skeletal muscle cells just beneath the basement membrane.

Damaged muscle cells release signals that trigger satellite cells to undergo cell division to produce a cell that will remain a satellite cell and a second cell that will differentiate into a myocyte. Myocytes can fuse with existing skeletal muscle cells to repair sites of damage or with each other to form a new muscle cell.

Cardiac Muscle

Cardiac muscle consists of thousands of cells in series. Similar to skeletal muscle cells, the actin and myosin filaments in cardiac muscle cells are also arranged in sarcomeres. However, the myofibrils are not as ordered as in skeletal muscle. In addition, the nuclei in cardiac muscle cells resides in the center rather than at the periphery as in skeletal muscle cells.

Because many cardiac muscle cells must be linked in series to span the entire length of the cardiac muscle. The signal to initiate contraction must pass from cell to cell as direct innervation of each cell would be too inefficient. Instead cardiac muscle cells use gap junctions to allow ions from one cell to enter an adjacent cell. In this way, an action potential that starts in one cell can be transmitted to adjacent cells via the flow of ions through gap junctions. The gap junctions localize to a structure called the intercalated disc.

Intercalated discs appear as pinkish lines in H&E-stained samples. These are the junctions between adjacent cardiac muscle cells. In addition to gap junctions, intercalated discs contain desmosomes to link adjacent cardiac muscle cells and sites for where actin filaments from the adjacent sarcomere attach.

Propagation of Action Potentials in Cardiac Myocytes

Intercalated discs contain gap junctions that allow current to pass between cells. An action potential in one cell leads to an influx of cation that diffuses through the gap junctions to the neighboring cell. The amount of positive charge is sufficient to open voltage-gated channels in the second cell, leading to an action potential. Similar to skeletal muscle, the action potential and depolarization of the cell membrane leads to an increase in cytosolic calcium. The increase in calcium causes tropomyosin to shift, allowing myosin to bind and pull on actin filaments to generate contraction. This process continues down the muscle to generate contraction along the entire length of the muscle.

Smooth Muscle

In contrast to skeletal and cardiac muscle cells, smooth muscle cells lack sarcomeres. Instead, the actin and myosin filaments in smooth muscle cells are arranged in several directions. Actin filaments are anchored at the cell membrane and in the cytosol at structures called dense bodies. Dense bodies are the functional equivalent of Z-discs. Bipolar myosin filaments interdigitate between actin filaments. Activation of myosin pulls on the cell membrane to shrink the cell.

The mechanism by which calcium stimulate contraction also differs in smooth muscle cells. Instead of shifting tropomyosin on actin filaments to expose myosin-binding sites, calcium leads to the activation of myosin. When cytosolic calcium levels are low, muscle myosin is in an inactive state. A rise in cytosolic calcium bind to a protein called calmodulin. Calmodulin then activates an enzyme called myosin-light chain kinase or MLCK. MLCK phosphorylates light chains in myosin that activate the motor domain and lead to contraction.

One consequence of this mechanism to active myosin is that smooth muscle cells take longer to generate contraction and contractile force increases slowly over time. One benefit is that smooth muscle cells can maintain contraction long after the stimulus is removed. This is due in part to the length of time it takes to dephosphorylate myosin light chain and thereby inactive muscle myosin. Another contributing factor is the length of time it takes muscle myosin to release from actin filaments. Once bound to actin filaments, muscle myosin in smooth muscle cells very slowly releases from actin filaments. After a contractile event, it takes a considerable amount of time for all of the myosin motors to release from actin filaments and allow complete relaxation of the muscle cell. This allows smooth muscle to generate and maintain contraction without consuming a lot of ATP.