“Introduction to Skeletal Muscle - Histology” Laboratory Exercise: “Introduction to Skeletal Muscle - Histology”

Skeletal Muscle – Introduction In this exercise, you will learn the functions of skeletal muscle, describe the organization of a skeletal muscle, describe the structure of a muscle fiber and label a slide and diagram and describe the structure of the neuromuscular junction and label a slide and diagram. These terms should be familiar to you: motor end plate, skeletal muscle, neuromuscular junction, tendon, fascicle, blood vessel, muscle fiber, epi-, peri-, endo-mysium, sarcolemma, I-, A-band, H-, M-line, Z- disc, Thick-, Thin-filament, transverse tubule, sarcoplasmic reticulum, mitochondria, myofibril, axon synaptic vesicle, motor end plate, synaptic cleft This laboratory exercise correlates with Chapter 10 of your textbook.

What procedures are we doing? Examine a prepared slide of a cross-section of skeletal muscle Examine a prepared slide of a longitudinal section of skeletal muscle Examine a skeletal muscle slide that is stained to show the neuromuscular junctions

Resources available… Anatomy and Physiology Laboratory Study Pages; submenu: Muscle http://ctle.hccs.edu/biologylabs/AP1/AP1index.html

Laboratory Lecture

An Introduction to Muscle Tissue Muscle Tissue is one of the four primary tissue types, and is divided into: Skeletal muscle tissue Cardiac muscle tissue Smooth muscle tissue

Functions of Skeletal Muscle Tissue Skeletal Muscles Are attached to the skeletal system (musculo-skeletal system) They facilitate our movement The muscular organ system includes only skeletal muscles (for locomotion) sidebar: smooth muscles are present in a variety of organ systems (digestive tract, cardiovascular system, etc…); cardiac muscle is only present in the cardiovascular system.

Skeletal Muscle performs six functions: Produce skeletal movement Maintain posture and body position Support soft tissues Guard entrances and exits Maintain body temperature Store nutrient reserves

Gross Organization of Skeletal Muscle Skeletal Muscles contain Skeletal muscle tissue (muscle cells or fibers) Connective tissues, that link the muscle tissue with other muscle or bone by tendonous bundle or aponeurosis sheet (lever system) Blood vessels, that feed muscle, deliver oxygen and remove wastes Nerves; the somatic nervous system commands voluntary skeletal muscle to contract – movement is a conscious act

Skeletal Muscle has three layers of connective tissue: Epimysium Perimysium Endomysium

Exterior collagen layer Epimysium Exterior collagen layer It is connected to deep fascia, a type of connective tissue Separates muscle from surrounding tissues 11

Perimysium Surrounds muscle fiber bundles (fascicles) within a muscle They contain blood vessels (arterioles and venuoles) and nerves that will feed and innervate the muscle cells (next slide)

Endomysium Surrounds individual muscle cells (muscle fibers or myofibers) It contains capillaries and nerve fibers that feed and contact muscle cells It also contains myosatellite cells (stem cells) that repair myofiber damage

Characteristics of Skeletal Muscle Fibers Skeletal Muscle Cells Skeletal muscle cells are very long and develop through the fusion of many precursor mesodermal cells, called myoblasts The immature muscle fiber can become very large and when mature, it contains hundreds of nuclei (multinucleated), becoming a myofiber (muscle cell or muscle fiber)

Sarcolemma The sarcolemma is the plasma membrane of a myofiber (muscle cell) It surrounds the sarcoplasm, the cytoplasm of a myofiber The sarcolemma is important, because a change in the transmembrane potential begins the contraction cycle

Transverse Tubules Transverse tubules (T tubules) are extensions of the sarcolemma into the interior of the cell – they transmit the action potential through the myofiber (which will act on contractile elements in the myofibril) Functionally, they allow the entire myofiber to contract at the same time. More on this in a couple of slides… #Truly Amazing!

Myofibrils A myofiber (cell) contains myofibrils, tubular structures which contain myofilaments The myofibrils are lengthwise subdivisions within the myofiber. Inside the myofibril are bundles of protein filaments called myofilaments Myofilaments are responsible for muscle contraction. There are two types of filamentous protein, thick and thin filaments Thin filament Thick filament

Sarcoplasmic Reticulum The Sarcoplasmic Reticulum is membranous structure surrounding each myofibril; it helps transmit the action potential to a myofibril It forms chambers (terminal cisternae) that are attached to T tubules A triad is formed by one T tubule and two terminal cisternae Cisternae concentrate Ca2+ (via ion pumps) and release Ca2+ into the sarcomeres to begin a muscle contraction Terminal cisterna

Sarcomeres Sarcomeres are the structural units of myofibrils and form visible, striped (striated) patterns. The patterns are created by alternating dark and light filaments (dark are thick filaments, A-bands) (light are thin filaments, I-bands) Due to their structure, sarcomeres are the contractile units of muscle

Sarcomere Structure, part I: Banding Patterns The A Band The M line is the center of the A band, at the midline of the sarcomere The H Band is the area around the M line. It only has thick filaments (no thin filaments) The zone of overlap is the most dense and darkest area on a light micrograph. This is where thick and thin filaments overlap The I Band Z lines are at the centers of the I bands. They define the two ends of a sarcomere Titin are alternating strands of protein that reach from tips of the thick filaments to the Z line. They stabilize the filaments and facilitate recoil after contraction

Sarcomere Structure, part II: 3-Dimensional Overlap The myofibril is a 3 dimensional cone Sarcomere Myofibril A superficial view of a sarcomere Thin filament Thick filament Actinin filaments Titin filament Attachment of titin Z line I band M line H band Zone of overlap Cross-sectional views of different portions of a sarcomere 21

amplification of structure = amplification of function Amplification is at every level of Functional Organization in Skeletal Muscle amplification of structure = amplification of function (Force and Rate)

Skeletal Muscle Fiber Contraction *3 Sarcomere Thin Filaments F-actin (filamentous actin) is composed of two twisted rows of G-actin (globular actin). Nebulin holds the two F-actin strands together. The active sites on G-actin bind to myosin Tropomyosin is a fibrous, double stranded protein. When the muscle is at rest, it prevents actin/myosin interaction by covering-up the active sites on actin Troponin is a globular protein that interacts with tropomyosin and regulates the binding affinity of tropomyosin for G-actin. Troponin’s structure is modified by binding to Ca2+, (binding to relaxes tropomyosin, exposing the active site of actin that interacts with myosin

Sarcomere Thick Filaments *3 Sarcomere Thick Filaments Thick filaments contain about 300 twisted myosin subunits that are associated (terminally) with strands of titin that recoil after stretching The myosin molecule tail binds to other myosin molecules (that are polarized on the other side of the M line). The myosin molecule head is made of two globular protein subunits that project from the tail on a hinge The myosin head will reach the nearest thin filament (actin) and apply a contractile force (this is referred to as the “Sliding Filament Theory”) M line

“Sliding Filament Theory” *3 Changes in the Appearance of a Sarcomere during Skeletal Muscle Fiber Contraction “Sliding Filament Theory”

*3 The Steps of Skeletal Muscle Contraction 1. 2. 3. Neural stimulation of the muscle cell sarcolemma This causes excitation- contraction coupling Muscle cell contraction Thick and thin filaments interact with each other Tension production 2. 3.

*3 Summary Skeletal muscle fibers shorten as thin filaments slide between thick filaments Free Ca2+ in the sarcoplasm triggers contraction SR releases Ca2+ when a motor neuron stimulates the muscle fiber Contraction is an active process Relaxation (return to rest) is a passive process What is Rigor Mortis? Cause of? Result of?

Steps Involved in Skeletal Muscle Contraction and Relaxation *3 Steps Involved in Skeletal Muscle Contraction and Relaxation Steps in Initiating Muscle Contraction (ACTIVE) Steps in Muscle Relaxation (PASSIVE) Synaptic terminal Motor end plate T tubule Sarcolemma Action potential reaches T tubule ACh released, binding to receptors ACh broken down by AChE Sarcoplasmic reticulum releases Ca2 Sarcoplasmic reticulum recaptures Ca2 Ca2 Actin Active site exposure, cross-bridge formation Active sites covered, no cross-bridge interaction Myosin Contraction ends Contraction begins Relaxation occurs, passive return to resting length 28

30 Myoblasts A muscle fiber forms by the fusion of myoblasts. Muscle fibers develop through the fusion of mesodermal cells called myoblasts. Myoblasts A muscle fiber forms by the fusion of myoblasts. Muscle fiber LM  612 Nuclei Sarcolemma Myofibrils Myosatellite cell Nuclei Mitochondria Immature muscle fiber A diagrammatic view and a micrograph of one muscle fiber. Myosatellite cell Up to 30 cm in length Mature muscle fiber 30

Thin Filaments F-actin (filamentous actin) is composed of two twisted rows of globular G-actin. The active sites on G-actin bind to myosin Nebulin holds the two F-actin strands together Tropomyosin is a fibrous, double stranded protein; it prevents actin–myosin interaction Troponin is a globular protein that binds tropomyosin to G-actin; it’s structure is modified by binding to Ca2+

Motor end plate Action potential ACh receptor site When the action potential reaches the neuron’s synaptic terminal, permeability changes in the membrane trigger the exocytosis of ACh into the synaptic cleft. Exocytosis occurs as vesicles fuse with the neuron’s plasma membrane. ACh molecules diffuse across the synatpic cleft and bind to ACh receptors on the surface of the motor end plate. ACh binding alters the membrane’s permeability to sodium ions. Because the extracellular fluid contains a high concentration of sodium ions, and sodium ion concentration inside the cell is very low, sodium ions rush into the sarcoplasm. The sudden inrush of sodium ions results in the generation of an action potential in the sarcolemma. AChE quickly breaks down the ACh on the motor end plate and in the synaptic cleft, thus inactivating the ACh receptor sites. Motor end plate Action potential AChE ACh receptor site 32

The contraction cycle

Figure 10-13 Shortening during a Contraction When both ends are free to move, the ends of a contracting muscle fiber move toward the center of the muscle fiber. When one end of a myofibril is fixed in position, and the other end free to move, the free end is pulled toward the fixed end. 34