Muscle Lab

Introduction

Muscles are multicellular contractile units. They are divided into three types:

• skeletal muscle
• smooth muscle
• cardiac muscle

As you read about each type of muscle, think about the similarities and differences betweenthem in terms of structure and function.

Skeletal Muscle

Skeletal muscle is mainly responsible for the movement of the skeleton, but is also found in organs such as the globe of the eye and the tongue. It is a voluntary muscle, and therefore under conscious control. Skeletal muscle is specialized for rapid and forceful contraction of short duration.

Each muscle cell is defined by a cell membrane (sarcolemma) and contains many nuclei along its length. The nuclei are displaced peripherally within a cross section of the cytoplasm (sarcoplasm) while a large number of longitudinal myofibrils, groups of arranged contractile proteins, occupy most of the center space. The myofibril contains several important histological landmarks:

• The myofibril is composed of alternating bands. The I-bands (isotropic in polarized light) appear light in color and the A-bands (anisotropic in polarized light) appear dark in color. The alternating pattern of these bands results in the striated appearance of skeletal muscle.
• The Z-lines (Zwischenschieben) bisect the I-bands.
• A light band called the H-band (Heller) sits within each A-band.
• The M-line (Mittelschiebe) bisects each A-band (and, in doing so, bisects each H-band).

Each myofibril can be understood as a series of contractile units called sarcomeres that contains two types of filaments: thick filaments, composed of myosin, and thin filaments, composed of actin. The individual filaments do not change in length during muscle contraction; rather the thin filaments slide over the thick filaments to shorten the sarcomere. The nature of these filaments can be understood in the context of the histological landmarks of the myofibril.

• The thick filaments are a bipolar array of polymerized myosin motors. The motors on one side of the filament are oriented in the same direction whereas the motors on the other side of the filament are oriented in the opposite direction. The center of the filament lacks motors; it contains only the coiled-coil region of the myosins. A set of proteins crosslinks each myosin filament to its neighbors at the center of the filament. These proteins make up the M-line.
• The thin filaments are attached to a disc-like zone that appears histologically as the Z-line. The Z-lines contain proteins that bind and stabilize the plus ends of actin filaments. Z-lines also define the borders of each sarcomere.
• The I- and H-bands are areas where thick and thin filaments do not overlap (this is why these bands appear paler under the microscope). The I-band exclusively contains thin filaments whereas the H-band contains exclusively thick filaments.

Skeletal muscles are divided into two muscle fiber types:

• Slow-twitch (type I) muscle fibers contract more slowly and rely on aerobic metabolism. They contain large amounts of mitochondria and myoglobin, an oxygen-storage molecule. The reddish color of myoglobin is why these fibers may be referred to as red fibers. These muscles can maintain continuous contraction and are useful in activities such as the maintenance of posture.
• Fast-twitch (type II) muscle fibers contract more rapidly due to the presence of a faster myosin. Type II fibers can be subdivided into those that have large amounts of mitochondria and myoglobin and those that have few mitochondria and little myoglobin. The former primarily utilize aerobic respiration to generate energy, whereas the latter rely on glycolysis. The lack of myoglobin results in a paler color than the slow-twitch muscles, and fast-twitch fibers may therefore be referred to as white fibers. These muscles are important for intense but sporadic contractions; for example, those that take place in the biceps.

Most muscles contain a mixture of these extreme fiber types. In humans, the fiber types cannot be distinguished based on gross examination, but require specific stains or treatments to differentiate the fibers.

Neuromuscular Junction and Activation of Skeletal Muscle Cells

Skeletal muscle cells are innervated by motor neurons. A motor unit is defined as the neuron and the fibers it supplies. Some motor neurons innervate one or a few muscle cells whereas other motor neurons can innervate hundreds of muscle cells. Muscles that require fine control have motor neurons that innervate fewer muscle cells; muscles that participate in less controlled movements may have many fibers innervated by each neuron. Motor axons terminate in a neuromuscular junction on the surface of skeletal muscle fibers. The neuromuscular junction is composed of a pre-synaptic nerve terminal and a post-synaptic muscle fiber. Upon depolarization, the pre-synaptic vesicles containing the neurotransmitter acetylcholine fuse with the membrane, releasing acetylcholine into the cleft. Acetylcholine binds to receptors on the post-synaptic membrane and causes depolarization of the muscle fiber, which leads to its contraction. Typically, one action potential in the neuron releases enough neurotransmitter to cause one contraction in the muscle fiber.

In muscle cells, the sarcolemma or plasma membrane extends transversely into the sarcoplasm to surround each myofibril, establishing the T-tubule system. These T-tubules allow for the synchronous contraction of all sarcomeres in the myofibril. The T-tubules are found at the junction of the A- and I- bands and their lumina are continuous with the extracellular space. At such junctions, the T-tubules are in close contact with the sarcoplasmic reticulum, which forms a network surrounding each myofibril. The part of the sarcoplasmic reticulum associated with the T-tubules is termed the terminal cisternae because of its flattened cisternal arrangement. When an excitation signal arrives at the neuromuscular junction, the depolarization of the sarcolemma quickly travels through the T-tubule system and comes in contact with the sarcoplasmic reticulum, causing the release of calcium and resulting in muscle contraction.

Smooth Muscle

Smooth muscle forms the contractile portion of the wall of the digestive tract from the middle portion of the esophagus to the internal sphincter of the anus. It is found in the walls of the ducts in the glands associated with the alimentary tract, in the walls of the respiratory passages from the trachea to the alveolar ducts, and in the urinary and genital ducts. The walls of the arteries, veins, and large lymph vessels contain smooth muscle as well.

Smooth muscle is specialized for slow and sustained contractions of low force. Instead of having motor units, all cells within a whole smooth muscle mass contract together. Smooth muscle has inherent contractility, and the autonomic nervous system, hormones and local metabolites can influence its contraction. Since it is not under conscious control, smooth muscle is involuntary muscle.

Smooth muscle fibers are elongated spindle-shaped cells with a single nucleus. In general, they are much shorter than skeletal muscle cells. The nucleus is located centrally and the sarcoplasm is filled with fibrils. The thick (myosin) and thin (actin) filaments are scattered throughout the sarcoplasm and are attached to adhesion densities on the cell membrane and focal densities within the cytoplasm. Since the contractile proteins of these cells are not arranged into myofibrils like those of skeletal and cardiac muscle, they appear smooth rather than striated.

Smooth muscle fibers are bound together in irregular branching fasciculi that vary in arrangement from organ to organ. These fasciculi are the functional contractile units. There is also a network of supporting collagenous tissues between the fibers and the fasciculi.

Cardiac Muscle

Cardiac muscle shares important characteristics with both skeletal and smooth muscle. Functionally, cardiac muscle produces strong contractions like skeletal muscle. However, it has inherent mechanisms to initiate continuous contraction like smooth muscle. The rate and force of contraction is not subject to voluntary control, but is influenced by the autonomic nervous system and hormones.

Histologically, cardiac muscle appears striated like the skeletal muscle due to arrangement of contractile proteins. It also has several unique structural characteristics:

• The fibers of cardiac muscle are not arranged in a simple parallel fashion. Instead, they branch at the ends to form connections with multiple adjacent cells, resulting in a complex, three-dimensional network.
• Cardiac muscle fibers are long cylindrical cells with one or two nuclei. The nuclei are centrally situated like that of smooth muscle.
• Cardiac muscle sarcoplasm has a great amount of mitochondria to meet the energy demands.

Collagenous tissues are found surrounding individual cardiac muscle fibers. There is abundance vascularization within this supporting tissue, which is required to meet the high metabolic demands of cardiac muscle.

The cardiac muscle fibers are joined end to end by specialized junctional regions called the intercalated discs. The intercalated discs provide anchorage for myofibrils and allow rapid spread of contractile stimuli between cells. Such rapid spread of contraction allows the cardiac muscles to act as a functional syncytium. The intercalated discs contain three types of membrane-to-membrane contact:

• fascia adherens, which are connected to actin filaments to transmit contraction
• desmosomes, which connect to intermediate filaments of the cytoskeleton
• gap junctions, which are sites of low electrical resistance that allow the spread of excitation

In addition to the contractile cells, there is a specialized system made up of modified muscle cells whose function is to generate the stimulus for heartbeat and conduct the impulse to various parts of the myocardium. This system consists of sinoatrial node, atrioventricular node, bundle of His, and Purkinje fibers.

Virtual Microscope Slides

1. Skeletal Muscle
This is a light microscope slide of skeletal muscle stained by H&E. With this dye, the A-bands are stained dark and the I-bands light. Since both cardiac muscle fibers and skeletal muscle fibers are striated, how would you differentiate between them in a histological slide?
2. Cardiac Muscle
As you zoom in on the image, note the abundance of capillaries in between the cardiac muscle cells. Why are so many capillaries necessary? Note how the cardiac muscle cells are striated, like skeletal muscle cells. However, unlike skeletal muscle, note how the end of each cells splits into branches. In addition, the single nuclei of cardiac muscle cells are located centrally. Identify the intercalated discs. What are the functions of the intercalated discs?
3. Smooth Muscle
This is a transverse section of the gastrointestinal tract. Locate the two layers of smooth muscle and zoom in on them. In which direction do the fibers of each layer run? How would you describe the morphology of each smooth muscle cell? How many nuclei are there per cell and where are they located within the sarcoplasm?

1. What type of muscle tissue is this?

Answer: Skeletal muscle
2. What gives this muscle type its characteristic striated pattern?

Answer: Arrangement of thick (myosin) and thin (actin) filaments
3. Identify the type of muscle.

Answer: Skeletal muscle
4. What type of muscle tissue is this?

Answer: Cardiac muscle
5. What is the function of this structure? What protein complexes mediate those functions>

Answer: Physical connection of the cells and rapid spread of excitation impulse via its high electrical conductance. Desmosomes mediate physical connections and gap junctions mediate spread of action potentials.
6. Name the structure. Which neurotransmitter would you find here?

Answer: Neuromuscular junction. Acetylcholine.
7. What type of muscle tissue is this?

Answer: Smooth muscle
8. Both cardiac and skeletal muscle are striated. What other histological characteristics could you use to distinguish them under the light microscope?
Answer: Skeletal muscle contains many nuclei that are located peripherally; cardiac muscle has just a few located more centrally.
9. Which histological landmarks of the sarcomere shrink when a muscle contracts?
Answer: The I-band and the H-band decrease in size; the A-band remains the same.