67
Unit 4: Muscle Tissue
Skeletal, Cardiac, & Smooth
68
69
Introduction and Objectives
Muscle tissue consists of cells specialized for contraction. All muscle cells have filamentous
proteins composed of either actin or myosin present in thin filaments (actin) and thick filaments
(myosin). These filaments with the aid of intermediate filaments composed of desmin, are
highly ordered in skeletal and cardiac muscle resulting in refractile bands known as crossstriations. Smooth muscle cells also contain the myosin and actin contractile proteins but they
are not highly ordered and therfore, smooth muscle cells do not have cross-striations. Muscle
cells contract upon receiving a stimulus by a chemical directly or a chemical released at the end
of a nerve fiber at a specialized structure known as a neuromuscular junction. There are three
types of muscle: cardiac, smooth, and skeletal muscle. They can be distinguished by their
histological appearance. Skeletal muscle cells exhibit regularly spaced transverse bands through
their entire length, called cross-striations. The cells are syncytial (meaning the cells are
multinucleated as a result of the fusion of many myoblasts to make the adult skeletal muscle cell)
in nature and are under voluntary control. Because the skeletal muscle cell is long and
threadlike, the term “fiber” can be used interchangeably with the term “muscle cell”. Cardiac
muscle cells also exhibit the cross-striations, but each cell is a separate cellular unit that has an
inherent rhythmic contraction. Smooth muscle differs from both cardiac and skeletal muscle by
its lack of visible cross-striations. Smooth muscle cells are threadlike in appearance, and
therefore can be called fibers. Like that of cardiac muscle, the contraction of smooth muscle is
involuntary.
After completing this unit, you should be able to identify and distinguish between the following:
1. skeletal, cardiac, & smooth muscle.
2. bands, zones, and lines of striated muscle cells.
3. the connective tissue wrappings of skeletal muscle: endomysium, perimysium, &
epimysium.
3. cardiac muscle cell histology.
4. intercalated disks of cardiac muscle.
5. smooth muscle cell histology.
Also, after completing this unit in conjunction with reading a chapter on connective tissue in a
histology textbook and/or hearing a lecture on muscle tissue, you should understand:
1. the difference between a myofiber, myofibril, & myofilament.
2. the changes in the bands, lines, & zones of skeletal muscle when contracted and relaxed.
3. the importance of sarcoplasmic reticulum and the transverse tubular system in skeletal and
cardiac muscle.
4. thick filaments, thin filaments and intermediate filaments, especially in skeletal muscle
5. the importance of intercalated disks.
6. the importance of connecting and communicating junctions in cardiac and smooth muscle.
70
71
Learning to distinguish between the three types of muscle cells
Let’s begin our study of muscle histology by learning the histological appearance of each type.
We will look first at smooth muscle cells, then cardiac muscle cells, and finally, skeletal muscle
cells. Go to the general histology section and under muscle tissue, select:
Smooth Muscle (Esophagus) – Goldner
Scan the 5x image and note that there are bundles of muscle cells with intervening connective
tissue as you can see by the green staining collagen. Now scan the 40x image and observe the
difference between the smooth muscle cells and a fibroblast by studying the labeled cells. The
nucleus of the fibroblast is flatter and stains darker. Note also that smooth muscle cell nuclei are
longer than the fibroblast nuclei. Next choose and scan the first 100x image until you find the
smooth muscle cell that is outlined with a text-linked label. Read the text associated with the
label. Now you should understand that a smooth muscle cell is a spindle or fusiform shaped cell
with a long nucleus located in the center of the cell. Now toggle from the microscope screen
view to the full screen view and turn on the measurement tool. Measure the length and the width
at the widest point of this cell. The result should be that you determine that this smooth muscle
cell is about 7 times longer than it is wide. Note, however, that the nucleus is not as long as the
cell. Now imagine that you make a cross-section of this cell at one of the ends just beyond the
nucleus and another cross-section through the middle of the cell that includes the nucleus. Now
look around in the remaining areas of this image and note that the smooth muscle cells are
arranged so that the cells fit together in the smallest space; that is, adjacent cells fit one tapered
end against that of the adjacent cell so that if you made a cross-section of this entire image the
result would be some cells in which you would see nuclei whereas in other cells nearby you
would not see a nucleus. This, in fact is how smooth muscle cell tissue looks in cross-section.
Select the second 100x image which is cross-sectioned smooth muscle. Note the somewhat
circular lighter and darker red profiles. The lighter red profiles are smooth muscle cytoplasm
and the darker red profiles are the nuclei of smooth muscle cells. Now look carefully and you
will see that some smooth muscle cells have nuclei showing and others do not. Now you have
learned the histological appearance of smooth muscle tissue in cross-section. Smooth muscle
tissue is a collection of smooth muscle cells connected physically by fasica adherens and
physiologically by gap junctions. At various places in the body this organization with
connective tissue wrappings added to support the cells, allows smooth muscle to function in
blood vessels to change the diameter of the lumen, to propel food throughout the intestinal tract
72
by peristalsis, to empty the urinary bladder and gallbladder – all of which is done through the
influence of the autonomic system and is involuntary to our conscious will.
Now let’s study cardiac muscle cells and see how they are different. In the general histology
section under muscle tissue select:
Cardiac Muscle – Hematoxylin-Eosin-Methylene Blue stain
After a brief scan of the 5x image noting the general appearance of cardiac muscle tissue, select
the 20x image and find the cardiac muscle cell that is labeled and outlined. You can see
immediately that cardiac muscle cells are not shaped like smooth muscle cells. Read the text
linked to the label explaining the difference in the general histological appearance between
cardiac and smooth muscle cells. Cardiac muscle cells are connected end to end at special sites
called intercalated discs. Before leaving this image scan around until you have seen the
appearance of intercalated discs. Now select the100x image. One of the first observations that
should be made is that the cardiac muscle cells present in this image have striations running
across the cell at right angles to their long axes. These indeed are striations due to the way
certain filaments are arranged in these cells. For now it is only important to recognize the
presence of striations and to realize that smooth muscle cells do not have these striations. Read
the text related to the label ‘transverse striations’. Shortly, when we study skeletal muscle you
will get the opportunity to learn about these striations in more detail. Now find the labels
‘intercalated disk’. Observe these structures that connect cardiac muscle cells physically and
electronically noting that they appear as lines across the cells that are thicker and stain more
intensely. Read the related text. You will need to consult your text and/or lecture notes to fully
understand the structure and function of intercalated discs. Cardiac muscle cells are different
from smooth muscle cells in one more way. They are branched whereas smooth muscle cells are
not. Find the intercalated disk label that is situated at the lowest part of the image. Now look
immediately below and to the left of the label. There you will observe a strand of cardiac muscle
cell cytoplasm that is going from one cell towards another. This is a branch of a cardiac muscle
cell. If you now go back to the 20x image and look for branches you should be able to observe a
few.
73
Next we will study skeletal muscle cells. In the general histology section under muscle tissue
choose:
Skeletal Muscle, longitudinal – Hematoxylin-Eosin
First scan the 5x image and find the two muscle fiber labels. At this magnification you can see
no details, but it is important to realize that skeletal muscle cells are normally and routinely
much longer than either cardiac or smooth muscle cells. Skeletal muscle cells can reach lengths
of several hundred microns, even millimeters depending on the muscle in which they are located.
The cells connect at either end to the tendons of the muscle. Therefore, particularly in the case
of skeletal muscle cells, it is traditional to call these cells fibers. They do have the appearance of
fibers in that they are much longer then they are wide. However, they are not like collagen
fibers, but are living cells. It is certainly not incorrect to refer to smooth muscle and cardiac
muscle cells also as fibers as they are longer than they are wide, but routinely skeletal muscle
cells are the ones normally called fibers. Now scan the 20x image and observe the labeled
muscle fibers. Read the text commenting on the fact that skeletal muscle fibers have striations
like cardiac muscle cells. You will now need to go to the 100x image to observe the striations
and more histological features of skeletal muscle fibers. Find the labels ‘muscle fiber nucleus’
and ‘muscle fiber’. After reading this you now aware that skeletal muscle nuclei are located just
under the cell membrane on the sides of the fiber. What the label text does not tell you is that
each skeletal muscle cell has hundreds of nuclei. Skeletal muscle fibers are so-called ‘multinucleated masses of cytoplasm’ which is called a structural syncytium (in development many
myoblasts fuse to form a single long cylindrical skeletal muscle fiber resulting in a large amount
of cyotplasm containing the nuclei of all of the contributing myoblasts). Each nucleus within the
skeletal muscle fiber has its own unique domain of influence over a specific portion of the
cytoplasm. The DNA and RNA in each nucleus provide the genetic information, messages, and
instructions for maintaining and renewing the filaments of actin, myosin, desmin, titin, and
nexin. You should consult your text or lecture notes for more details. In this image, you can
begin to appreciate the repeating unit in a skeletal muscle, the sarcomere. A sarcomere extends
from one Z line to another. After you have read the text related to one of the ‘Z line’ labels, find
two Z lines that you can clearly see and measure the distance between them. You will discover
that the distance is somewhere near 3.5 microns in this specimen. If you now measure the A
band you should get something close to 1.5 microns which is the length of the thick myosin
filaments. Thin filaments composed of actin are anchored in the Z disks and extend into each
sarcomere about 1 micron. Thick filaments which make up the basic structure of the A band are
74
1.5 microns long (thick myosin filaments are always present in the A band but thin filaments
are present to the extent that the muscle is contracted). Therefore, a fully relaxed skeletal muscle
cell should have a sarcomere length on the order of 3.5 microns if it is not over stretched- two 1
micron long actin filaments plus one 1.5 micron long myosin filament = 3.5 microns. We will
revisit the details of the various bands and lines making up the striations of skeletal muscle in
another specimen. However, to complete our study of the contrast between cardiac, smooth, and
skeletal muscle cells, let’s take a look at a cross-section of skeletal muscle cells. In the general
histology section under muscle tissue choose:
Skeletal Muscle, transverse-Hematoxylin-Eosin
Briefly scan the 5x and the 20x images. In the 20x image you can observe the peripherally
located nuclei in each skeletal muscle cell. Now select the 40x image for a closer look at this
arrangement. Find the muscle fiber that is outlined with the nucleus labeled that has text related.
Observe the position of the nucleus and read the text comment. Note also that in this crosssectional view you cannot observe any striations. The resolution in the light microscope is not
great enough to resolve the content and arrangements of the different zones, bands, and lines in
skeletal muscle (and the sections are also too thick resulting in the viewing of several sarcomeres
superimposed). However, using electron microscopy, in which the sections are on the order of
1/50th micron, it is possible to know whether you cross-sectioned the cell in the A band, or I
band, or Z line etc, by looking for only thin filaments (I band), mixture of thin and thick
filaments (A band), and Z disk (dark smudgy profiles compared to circular profiles).
This concludes our effort to learn the distinguishing histological features of cardiac muscle cells,
smooth muscle cells, and skeletal muscle fibers. In summary, cardiac and skeletal muscle cells
have striations but are distinguished from one another by the location of their nuclei. Smooth
muscle and cardiac muscle cells have nuclei located in the center but the shape of the nuclei are
different -long and tapered in smooth muscle cells, but short and rectangular in cardiac muscle
cells. Additionally, cardiac muscle cells have striations. Now, before we study the connective
tissue wrappings of muscle, let’s study a specimen that will give us a better look at the striations
in skeletal muscle. In the general histology section under muscle tissue choose:
Skeletal Muscle, longitudinal – Iron Hematoxylin
In this specimen the striations are nicely demonstrated due to the iron added to the hematoxylin
in staining. Scan the 20x specimen and observe the light and dark bands. The light ones are I
bands where thin filaments composed of actin are located. The dark ones are the A bands where
75
both thick (myosin) and thin (actin) filaments can be located. Let’s take a closer look at these
bands. Select and scan the 100x image. In this image you can clearly see I and A bands. Note
that A bands have a light zone in their middles, the H-band (zone). The H zone is a transient
zone visible only when sarcomeres are fully relaxed. Even a slight contraction will cause this
zone to disappear due to the meeting of the actin filaments from the Z disks at either end of the
sarcomere. Read the text related to all of the labels. Note that Z-lines cannot be observed in this
specimen. The stain did not demonstrate them. Recall that they are located in the middle of each
I band. Measure the width of the I band in this specimen on the 100x image. You should arrive
at a measurement close to 2 microns. Thin actin filaments that are 1 micron long project toward
the center of adjacent sarcomeres. Therefore in relaxed (stretched) sarcomeres when there is
minimal overlap of thin and thick filaments, the I band width should approach the length of 2
actin filaments which would be 2 microns. At the time of fixation the cells in this muscle sample
were fully relaxed.
Connective Tissue Wrappings of Muscle
To conclude our study of muscle tissue let’s turn our attention to the organization of muscle as a
tissue/organ which involves understanding the role of connective tissue in ‘packaging’ muscle
tissue. To begin our study of the connective tissue wrappings of muscle go to the general
histology section and under muscle tissue choose:
Skeletal Muscle, transverse – Hematoxylin-Methylene Blue
In this specimen the main fiber component of the connective tissue in muscle, collagen, is
stained blue. As you scan the 5x image you can easily observe the blue stained connective tissue
and the red stained muscle tissue. Look for the label ‘primary bundle’. This is the term used for
a bundle of collagen fibers in tendon as you may recall when you studied dense regular
connective tissue. In this case the term is used to help you compare the arrangement of skeletal
muscle cells and how they are organized similarly to the arrangement in a tendon. The correct
and appropriate term is not ‘primary bundle’, but a ‘fascicle’. Before leaving this image note the
terms perimysium and epimysium. These are the terms that denote the connective tissue
surrounding a small bundle of skeletal muscle fibers, the fascicle, compared to the connective
tissue wrapping a large collection of fascicles, the epimysium. Now choose a specimen stained
only with Hematoxylin and Eosin that does not demonstrate the connective tissue well. Choose
in the general histology section under muscle tissue:
76
Skeletal Muscle, transverse – Hematoxylin-Eosin
Scan the 5x image and find the label ‘muscle fascicle’. A muscle fascicle a component of a
muscle consisting of a group of muscle fibers wrapped by a special connective tissue. You can
also see that the fascicles are separated by boundaries that are not stained because in this
specimen stained with hematoxylin and eosin, the collagen did not take up the eosin stain as it
normally would. Now return in the same list of specimens to:
Skeletal Muscle, transverse – Hematoxylin-Methylene Blue
Review again the connective tissue of the perimysium and read the text related to the label,
‘epimysium’. Scan the 20x and 100x images looking for endomysium. In the 20x image you
will note that several nerve bundles are present, two of which are labeled. Nerve fibers, similar
to muscle fibers, have three named connective tissue wrappings: 1) individually, nerve fibers are
surrounded by endoneurium, 2) bundles called nerve fascicles, are surrounded by perineurium,
and 3) groups of bundles, fascicles are surrounded by epimysium. (You will learn about the
wrappings of nerve tissue in more detail when you study the chapter on nerve tissue. It is helpful
at this point when you are learning the connective tissue wrappings of muscle, to realize that the
same arrangement exists for nerve fibers.) Study and observe the endomysium in the 100x image
and see that it is composed of a few collagen fibers. This is a loose connective tissue containing
fibroblasts and mast cells arranged along the blood vessels supplying the muscle fibers. Now
return to the 5x image and, as you scan it with the labels turned on, review the organization of
muscle fibers in skeletal muscle. The wrappings of endomysium, perimysium, and epimysium
organize the muscle fibers into a gross anatomical structure, like, for example, the biceps muscle.
All three wrappings of a biceps muscle converge at either end to form the origin and insertion
tendons. The epimysium of a biceps muscle is the same as the deep fascia that one finds as the
skin and superficial fascia are removed in the dissection of a cadaver. Larger nerve bundles and
blood vessels travel in and are encased in the perimysium. Capillaries and single nerve fibers
travel and are encased in the endomysium. It is important to note that the skeletal muscle fibers
themselves constitute skeletal muscle tissue. In reality skeletal muscle cells without their
connective tissue wrappings do not exist. The connective tissue wrappings we have just learned
about along with the nerve tissue and connective tissue, and even epithelium lining the blood
vessels present, constitute an organ such as a biceps muscle. An organ is defined as a structure
that consists of the four basic tissues organized in such as way as to carry out a unique function
or functions. The biceps muscle functions to flex the arm at the elbow joint.
77
Before concluding your study of muscle tissue it will be helpful to observe again the
specimens of smooth and cardiac muscle. As you revisit and scan the images of these specimens
look for the connective tissue. Traditionally, histologists do not describe endomysium,
perimysium and epimysium in smooth and cardiac muscle. However, as you will observe, both
cardiac muscle and smooth muscle cells, as was pointed out for skeletal muscle, do not exist in
nature by themselves. Smooth and cardiac muscle also have connective tissue wrappings which
organize the respective cells into organs like the heart, urinary bladder muscle, gall bladder
muscle, muscle of the gastrointestinal tract, etc. So, while you will be able to observe the
connective tissue in cardiac and smooth muscle it is not common practice to apply the naming
scheme as in skeletal muscle.
This concludes the exercise on the histology of muscle tissue. You have learned that there are
three types of muscle cells. Cardiac and smooth muscle are similar in that their cells have nuclei
located in the center of each cell. Cardiac and smooth muscle cells have two main differences:
1) nuclei of cardiac muscle cells are box (rectangular) shaped whereas the nuclei of smooth
muscle cells are fusiform (wide in the middle and tapered at both ends), and 2) cardiac muscle
cells have striations while striations are absent from smooth muscle cells. Skeletal muscle cells
are different from both smooth and cardiac muscle cells because each cell has many nuclei and
all the nuclei are located at the peripheral aspect of the cell just beneath the cell membrane.
While skeletal muscle cells and cardiac muscle cells both have striations, the striations of
skeletal muscle are more distinct than cardiac muscle cells. You have also learned that cardiac
muscle cells are branched and that they are joined together by special junctions called
intercalated disks. Similarly, smooth muscle cells are joined together but not by intercalalated
disks. Smooth muscle cells are connected by fascia adherens to form a physical unit of many
cells and by gap junctions that provide chemical and electrical communication between the cells
in the unit. Finally, you have learned that all muscle tissue is organized into bundles and
ultimately in large groups of bundles, especially evident in skeletal muscle. This organization is
due to the wrappings of individual muscle fibers (endomysium), groups of muscle fibers
(perimysium), and multiple fascicles bound together to make a gross anatomical muscle
(epimysium). This connective tissue is delicate in the endomysium, denser in the perimysium,
and the most dense in the epimysium. The connective tissue wrappings enclose blood vessels
and nerves supplying the muscle tissue.
78
Sample Practice Questions:
Which of the following tissues is represented by the above image?
A. dense regular connective tissue
B. smooth muscle tissue
C. skeletal muscle tissue
D. cardiac muscle tissue
E. stratified squamous keratinized epithelium
Structure labeled 2 is which of the following?
A. intercalated disk
B. A band
C. I band
D. Z line
E. H zone
79
Muscle Section Labeled Structures
Viewing the structures you have studied in this lesson in sections stained with special stains
other than Hematoxylin and Eosin could be helpful in making your learning complete. In this
unit and all others the specimens under each heading will be listed in this way that will help also
when you are reviewing to know which specimen and which magnification that certain structures
are labeled.
Smooth Muscle (Esophagus)-Staining: Goldner
20X
160X
Smooth muscle cell bundle Fibroblast
Connective tissue
400X-A
400X-B
Smooth muscle cell - nucleus
Smooth muscle cell - nucleus
Smooth muscle cell Endomysium - collagenous fiber Smooth muscle cell - cytoplasm
Smooth muscle cell Smooth muscle cell
Endomysium
Collagenous fiber
Fibroblast
Smooth muscle cell
Skeletal Muscle, longitudinal-Staining: Iron Hematoxylin
80X
400X
Muscle fiber
I band
Endomysium
A band
H band
Nucleus
Skeletal Muscle, longitudinal-Staining: Hematoxylin-Eosin
20X
80X
400X
Muscle fiber
Muscle cell nucleus
Muscle fiber nucleus
Endomysium
Endomysium
Muscle fiber
Artery
Fibroblast nucleus
Z line
Muscle fiber
A band
I band
Myofibril
80
Skeletal Muscle, longitudinal-Staining: Hematoxylin-Eosin
20X
80X
400X
Muscle fiber
Muscle cell nucleus
Muscle fiber nucleus
Endomysium
Endomysium
Muscle fiber
Artery
Fibroblast nucleus
Z line
Muscle fiber
A band
I band
Myofibril
Skeletal Muscle, transverse-Staining: Hematoxylin-Eosin
20X
80X
160X
Muscle fascicle
Perimysium
Muscle fiber nucleus
Nerve fiber bundle
Muscle fiber
Muscle fiber
Artery
Endomysium
Endomysium
Perimysium
Muscle fiber nucleus
Fibroblast nucleus
Epimysium
Capillary
Cardiac Muscle- Staining: Hematoxylin-Eosin-Methylene Blue
20X
80X
400X
Endomysium
Endomysium
Cardiac muscle cell nucleus
Cardiac muscle cell
Intercalated disk
Fibroblast
Cardiac muscle cell nucleus
Intercalated disk
Endomysium
Transverse striations
Nucleus