Dr. P.C. Nahirney Muscle Lab

Histology Laboratory October 28, 2014

Skeletal Muscle and

Readings: Netter’s Essential (2nd Ed): Ch.4, pp.71-100; Ch.8, pp.178-186; Ch.12, pp.281, 283; Ch.20, pp.466-468 Basic Histology Text & Atlas (12th Ed): Ch.10, pp.167-184 & pp.187-194 Histology: A Text & Atlas (6th Ed): Ch.11, pp.310-327 & pp.331-338

Objective: To describe the essential histological features of skeletal and smooth muscle, comparing similarities and differences in their structure and function. Specific aims are to:

1. Describe the histology of and the . 2. Describe the fine structure of the skeletal muscle fiber with reference to , , mitochondria and the sarcotubular system. 3. Explain the ultrastructural basis of excitation-contraction (E-C) coupling and the sliding filament mechanism of . 4. Describe the key ultrastructural features of the . 5. Describe the histological organization of smooth muscle in visceral and vascular structures of the body, and comment on salient ultrastructure.

Slides for study: Electron micrographs for study: 77 (Tongue, Anterior) Skeletal muscle fiber nucleus 29 (Muscle-Tendon) Skeletal muscle, longitudinal section 27 (Muscle, Skeletal [xs]) Transverse section of skeletal muscle 28 (Muscle, Skeletal [ls]) Myofibrils in transverse section 116 (Gallbladder) Capillary and skeletal muscle fibers in transverse section 109 (Appendix) HRSEM of part of a type IIa skeletal muscle fiber Satellite cell in fetal skeletal muscle Neuromuscular junction in skeletal muscle (low power) Neuromuscular junction in skeletal muscle (high power) Muscle spindle in equatorial region Sensory nerve terminal in equatorial region of muscle spindle Transverse section of smooth muscle cells and smooth muscle cells in arteriolar wall

Skeletal Muscle

#77: Tongue (H&E): The core of the tongue is filled with skeletal muscle. The muscle fibers are arranged into small bundles called fascicles, that course in various directions. This design makes the tongue a highly maneuverable structure. Note that the fibers are cut in transverse, oblique and longitudinal section. Muscle fibers stain intensely eosinophilic due to their high protein content. Between fascicles is loose () which supports the blood vessels and nerves that supply the muscle fibers.

#29: Muscle-tendon, Lumbrical muscle cross section (H&E): Slide 29 is a cross- sectional view of skeletal muscle in an intrinsic muscle of the hand. At the bottom of the slide are two cross-sectional profiles of adjacent tendons. Skeletal muscle fibers Dr. P.C. Nahirney Muscle Lab

(also called extrafusal muscle fibers) are round or polyhedral in shape and are grouped into fascicles. Note that there is dehydration artifact and the spaces between muscle fibers are exaggerated. Zoom up on the muscle fibers and notice that the nuclei are small and peripherally located underneath the . Muscle fibers contain hundreds of nuclei along their length since they form as a fusion of many immature muscle cells (myoblasts) during development and regeneration. Between the muscle fibers is a rich network of capillaries embedded within the delicate . Larger blood vessels and nerve fascicles are present in the perimysium. See also slide #27 for good examples of muscle fascicles and muscle fibers sectioned in the transverse plane. Deep within this muscle are examples of sensory receptors known as muscle spindles. These are small encapsulated structures embedded within the perimysial connective tissue - they are typically situated near the major neurovascular bundles in the muscle. Each spindle is surrounded by a thin capsule - within the spindle capsule are specialized, small-diameter skeletal muscle fibers called intrafusal muscle fibers that have both a sensory and motor supply. Muscle spindles monitor the length and change in length of the muscle, and are primarily responsible for initiating the stretch reflex.

#28: Pectoralis minor (H&E): This is a longitudinal section of skeletal muscle stained with H&E. Fibers are elongated and cylindrical, and contain multiple nuclei arranged periodically along the periphery of their under the sarcolemma. At high magnification, look for regions where striations in the fibers can be seen. These are due to the parallel overlapping arrangement of the thick and thin .

Muscle satellite (myosatellite) cells are myogenic cells situated under the basal (external) lamina in adult skeletal muscle fibers and serve as a reserve stem cell population for regeneration of damaged muscle. When stimulated, they will divide, fuse and regenerate the site of damage, although only have limited regenerative potential and decline with age.

Electron Micrographs: Skeletal Muscle

1. This TEM shows a portion of a skeletal muscle fiber sectioned in the longitudinal plane. The sarcolemma (plasma membrane) is seen in the upper left. A subsarcolemmal nucleus containing a prominent nucleolus is situated immediately underneath. The sarcoplasm of striated muscle is filled with bundles of contractile filaments called myofibrils that are oriented parallel to the long axis of the fiber. Between myofibrils are elements of the sarcotubular system and mitochondria.

Clinical point: Duchenne muscular dystrophy is an X-linked myodegenerative disorder that affects young males. Muscle fibers are weakened due to the lack the subsarcolemmal protein, , which anchors and cytoskeletal elements to the sarcolemma.

2. Myofibrils consist of repeating units called sarcomeres and have a regular banding pattern that aligns with neighbouring myofibrils. There are three bands in myofibrils (A band, I band and Z band). The A band consists of the thick filaments () whereas the lighter I band consists of thin Dr. P.C. Nahirney Muscle Lab

filaments (actin). Thin electron-dense Z bands bisect the I bands and delineate the . Depending on the state of contraction, the width of the I band will vary. As a general note, thick filaments of A bands are always a constant length of 1.6 µm and serve as a ‘molecular ruler’ in striated muscle. Within the A band is a lighter region called the H zone which is bisected by a dense M line. The width of the H zone is also variable and represents the ends of the thin filaments in the overlap zone of the sarcomere. In the sliding filament model, the interdigitating thick and thin filaments slide past one another using ATP-dependent myosin motors (cross- bridges) on the surface of thick filaments.

3. The sarcotubular system consists of two distinct components: the (SR), an internal lace-like membrane network that surrounds each , and transverse (T)-tubules, which are tubular invaginations of the plasma membrane (sarcolemma). Two T- tubules per sarcomere run perpendicular to the long axis of the muscle fiber and make periodic contacts with paired flattened sacs (cisternae) of the SR. These sites are characterized by a central T-tubule and two flanking SR cisternae, and are thus called triads. When a nerve signal depolarizes the muscle plasma membrane, the action potential is carried to the interior of the muscle fiber along T- tubules which, in turn, triggers the release of calcium ions from the SR into the surrounding myofibrils. A calcium binding protein called C, which is situated on the thin filament, responds to calcium by shifting its conformation on the thin filament and exposing an actin binding site so that a myosin head on the thick filament can bind and pull on the thin filament, resulting in shortening of the sarcomere.

4. Neuromuscular junctions are sites where a nerve terminal makes contact with a muscle fiber. The axon terminal sits in a small depression on the muscle fiber and contains numerous uniformly-sized clear synaptic vesicles (~50 nm in diameter) which store the neurotransmitter, acetylcholine. These vesicles are concentrated near the nerve-muscle interface, and upon axon depolarization, are released by the process of exocytosis. Mitochondria, smooth ER, microtubules and neurofilaments are also seen within the terminal. A Schwann cell covers the external surface of the axon and the terminals. Between the nerve terminal and muscle fiber is a narrow gap, termed the synaptic cleft, where acetylcholine is released. The postsynaptic membrane (motor end plate) contains numerous postjunctional folds that increase the surface area for the acetylcholine receptors. Within the sarcoplasm beneath the motor end-plate are numerous mitochondria and myofibrils.

Clinical point: Myasthenia gravis is an autoimmune disorder that results in weakening of muscles due to circulating antibodies that block nicotinic acetylcholine receptors at muscle motor end-plates.

Smooth Muscle

#116: Gallbladder (H&E): The gallbladder contains a thin uniform layer of smooth muscle that sits beneath the luminal epithelial lining. This muscle functions to eject bile from the bladder to the duodenum. Smooth muscle cells are spindle-shaped, mononucleated contractile cells that are arranged into Dr. P.C. Nahirney Muscle Lab

parallel bundles within its wall. The cells are highly eosinophilic, they lack striations and their borders are indistinct. Between smooth muscle bundles are delicate layers of connective tissue - note small blood vessels distributed throughout these layers.

#109: Appendix (H&E): Smooth muscle in the appendix is organized into sheets oriented perpendicular to one another (i.e. longitudinal and circular). The muscle cells are bundled into groups, or fascicles. Individual smooth muscle fibers are difficult to discern with light microscopy due to their small size and tightly-packed arrangement. Between the circular and longitudinal layers of smooth muscle are elements of the autonomic nervous system known as the myenteric plexus. These can be recognized by their slightly washed out basophilic appearance and the occasional presence of nerve cell bodies.

There are two general types of smooth muscle in the body. Smooth muscle found in the walls of the gastrointestinal (GI) tract, airways of the respiratory system, urinary and gall bladder is termed visceral smooth muscle. It regulates the luminal caliber of organs and functions to propel food through the GI tract (peristalsis). In blood vessels, it is termed vascular smooth muscle. Contraction of smooth muscle in blood vessels reduces the diameter of the lumen, decreasing blood flow and redirecting blood within the body. The smallest , known as , have smooth muscle that plays an important role in regulation of systemic . Excessive can lead to .

Electron Micrograph: Smooth Muscle

This is an EM view of vascular smooth muscle in an . Seen in the upper left is a portion of the lining endothelium. Sitting beneath the endothelium in the wall of the arteriole are several layers of smooth muscle cells arranged in a staggered fashion. An elongated nucleus occupies the center of one of the muscle cells - in the surrounding cytoplasm are a scattered mitochondria and elements of the SR. Dense bodies (actin anchoring sites) and a rich network of thin (actin) and thick (myosin) filaments fill the cytoplasm, along with an array of intermediate filaments (/vimentin). Caveolae (small invaginations of the sarcolemma) are seen along the borders of the smooth muscle cells – they increase the cell surface area and are thought be similar in function to T-tubules of skeletal muscle. The intervening spaces contain a delicate connective tissue that is synthesized and secreted by smooth muscle cells.

Clinical point: In conditions such as asthma and hypertension, smooth muscle cells can undergo hyperplasia and hypertrophy resulting in abnormalities in proliferative and contractile properties.