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

2. 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.

3. 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

4. Resources available…
Anatomy and Physiology Laboratory Study Pages; submenu: Muscle

5. Laboratory Lecture

6. 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

7. 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.

8. 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

9. 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

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

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

12. 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)

13. 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

14. 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)

15. 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

16. 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!

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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)

23. 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

24. 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

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

26. *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.

27. *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?

28. 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. 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

31. 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+

32. 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

33. The contraction cycle

34. 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