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Through the Looking Glass - From Stem Cells to Tissues and Organs

Learning Resources The University of Michigan Medical School

Muscle

Atlas   Wheater's, pgs. 101-121, Muscle
Text Ross and Pawlina (6th ed), Chapter 11 Muscle Tissue, pgs. 310-351
Lab Resources

OBJECTIVES:

  1. Be able to name and to identify the three types of muscle at the light and electron microscope levels, including distinctive features of each, such as the intercalated discs of cardiac muscle.
  2. Be able to describe and understand the structural basis of muscle striation at the light microscope and EM levels and the molecular level.
  3. Know the structural elements that harness muscle contraction (i.e., the shortening of myofibrils) to the movement of a body part (i.e., via connection to bone) as well as the mechanism by which muscle cells (skeletal, cardiac and smooth muscle) contract.
  4. Understand where stem cells are located in skeletal muscle and be able to identify their location at both the light and EM levels.
  5. Be familiar with the regenerative potential of each muscle type.

There are three major types of muscle, and their structure reflects their function.  Skeletal and cardiac muscle cells are called striated muscle because of the very regular arrangement of their intracellular contractile units, sarcomeres, at the light microscope (LM) and electron microscope (EM) levels.  This regular arrangement imparts a cross-striated (or striped) appearance.  Such an arrangement is not seen in smooth muscle cells.  Skeletal muscle is also called voluntary muscle, because its contraction is under conscious neural control.  In contrast, cardiac and smooth muscles are called involuntary muscles because their contractions are either spontaneously generated or are under the control of the autonomic nervous system.

Each of these three types of muscle has a characteristic appearance in both cross and longitudinal sections.  You should be able to recognize each type of muscle in both planes of section.

I. SKELETAL MUSCLE

A. Cytology of skeletal muscle cells

058L skeletal muscle H&E longitudinal Webscope Imagescope
058Lex Skeletal muscle H&E longitudinal Webscope Imagescope
058Thin skeletal muscle H&E longitudinal Webscope Imagescope
058T Skeletal muscle H&E cross Webscope Imagescope

In longitudinal sections of skeletal muscle (Slide 58, odd-numbered slide boxes), observe the following :

  1. Nonbranching, cylindrical shape of the cell (also referred as muscle fiber). These cells are very long; you cannot see their ends.
  2. Be sure you can identify the borders of the muscle cell. You might see occasional nuclei which appear to be centrally located, but aren't. Why?  answerAgain the answer to the question is in the plane of section. Cells with peripheral nuclei that are cut at an angle can appear to have centrally located nuclei. This is why it is important to look at other features of the cell to determine what exactly it is.
  3. Peripheral position of the elongate nuclei just inside of the sarcolemma (plasma membrane). Note that each cell contains large numbers of nuclei. What exactly is a muscle cell and what's the difference between a muscle fiber and a muscle fibril?  answerThe basic units of muscle are the contractile proteins actin and myosin arranged in sarcomeres.  Placed end to end, these sarcomeres form long bands called myofibrils.  Within a skeletal muscle cell, the numerous myofibrils are separated by glycogen, mitochondria, and muscle triads (two terminal cisternae and a T tubule) and other organelles.  Chances are, if you are looking at an electron micrograph of muscle tissue, you are looking at myofibrils.  Groups of these fibrils form a muscle fiber, which is surrounded by endomysium.  Muscle fiber and muscle cell are synonymous.  Groups of individual cells that are surrounded by perimysium are known as fascicles and groups of these fascicles, surrounded by epimysium make up a muscle.
  4. Cross striations can be seen, and are due to the structure of the sarcomere. A sarcomere consists of the structures between two Z lines. You should recognize: (Using your microscope and glass slides may help to see these fine structures)
    1. The dark A band
    2. The lighter H zone which bisects the A band
    3. The light I band d. The dark Z line which bisects the I band.
    It is admittedly difficult to see the Z line and especially the H zone with the light microscope. However, they can be seen clearly on some areas of almost all slides, and it is just necessary to do some looking around for a favorable area on your slide. Here are some good examples showing cross striations: #058L   Webscope #058L   Webscope Electron Micrograph 16 Epithelium Webscope Imagescope
  5. A less regular longitudinal striping can sometimes be seen within the muscle fibers and is due to the bundling together of the thick and thin filaments into myofibrils, which are arranged parallel to the long axis of the cell. Note that sometimes the cross striations are not aligned all the way across the cell. This is because different myofibrils may not be aligned.

    In transverse sections of skeletal muscle cells (slide 58, even slide boxes), observe the cylindrical shape of the cells (fibers) and the peripherally-located nuclei. Note also the the cytoplasm of the muscle cells has a stippled, punctate appearance which is due to the bundling of thick and thin filaments into myofibrils as mentioned above. What exactly is a muscle cell and what's the difference between a muscle fiber and a muscle fibril?  answerThe basic units of muscle are the contractile proteins actin and myosin arranged in sarcomeres.  Placed end to end, these sarcomeres form long bands called myofibrils.  Within a skeletal muscle cell, the numerous myofibrils are separated by glycogen, mitochondria, and muscle triads (two terminal cisternae and a T tubule) and other organelles.  Chances are, if you are looking at an electron micrograph of muscle tissue, you are looking at myofibrils.  Groups of these fibrils form a muscle fiber, which is surrounded by endomysium.  Muscle fiber and muscle cell are synonymous.  Groups of individual cells that are surrounded by perimysium are known as fascicles and groups of these fascicles, surrounded by epimysium make up a muscle.

B. Perimuscular Connective Tissue

059-3 forearm muscle Masson cross fetal Webscope Imagescope Image
059-1 forearm muscle H&E cross fetal Webscope Imagescope Image

In slide 59, stained with trichrome (even slide boxes) or H&E (odd slide boxes), three layers of connective tissue sheaths are visible. This is a transverse section of an entire fetal forearm, and contains many elements in addition to the muscle, such as nerves and tendons, both of which are, unfortunately, simlarly bundled into fascicles and therefore easily confused with muscle. So, make sure you're looking at the muscles Image Image

  1. Endomysium - thin connective tissue sheath, consisting of basal lamina, some reticular fibers and capillaries around each muscle fiber. In fetal muscle, endomysium is not always clearly defined, so it is best to use the trichrome-stained slide (slide 59-3) to this --it is the connective tissue immediately surrounding the individual cells and very delicate perhaps visible only as a bluish tinge.
  2. Perimysium - loose connective tissue sheath consisting of type I collagen fibers found around fascicles (bundles of muscle fibers). Note that the tissue in these specimens underwent some shrinkage during preparation so the separation between fascicles is somewhat exaggerated. Similarly, much of the perimysium has been damaged; however there are still some areas where it can be clearly seen (e.g. around many of the blood vessels found within the belly of the muscles).
  3. Epimysium - the dense irregular connective tissue sheath around the entire muscle = deep fascia in gross anatomy; contains even larger blood vessels and nerves.

Three layers can be seen in #059-3   Webscope Image

II. CARDIAC MUSCLE

057 ventricle H&E Webscope Imagescope
098-1 Heart ventricle H&E Webscope Imagescope
098N right wall Masson Webscope Imagescope
305 heart ventricle H&E Webscope Imagescope (note: this slide NOT in glass slide collection)

Cardiac muscle will be studied in the wall of the ventricle of the heart. In comparison with skeletal muscle, note the following differences. Cardiac muscle cells branch and form a three-dimensional network. These branch points can sometimes be seen in your sections, and you should also note that the muscle fibers are less parallel than in skeletal muscle.

A. In longitudinal sections, observe:

  1. Intercalated discs which are dark lines which cross the cell transversely. Look around inslide 57 #057   Webscope or slide 305 INDO 390 for lightly stained regions of the slide. There are artifactual transverse breaks in some of the muscle fibers, which are NOT intercalated discs. Intercalated discs are also fairly easy to find in the areas where muscle fibers are longitudinally oriented in slide 98-1 #098-1   Webscope and/or slide 98-N #098N   Webscope (be sure to look at both H&E and trichrome-stained sections).
  2. The nucleus is centrally located in a fiber.
  3. Longitudinal striping due to myofibrils can sometimes be seen.

B. In transverse sections, observe:

  1. The myofibrils are coarse and give rise to the nonhomogeneous, punctate appearance of the sarcoplasm.
  2. The nuclei are centrally-located. You won't see a nucleus in every fiber cross-section.
  3. The extensive network of capillaries in the endomysium --heart muscle is ALWAYS beating and therefore always in demand of oxygen and nutrients delivered via the blood.

MAKE SURE YOU CAN DIFFERENTIATE BETWEEN CARDIAC AND SKELETAL MUSCLE IN BOTH LONGITUDINAL AND TRANSVERSE SECTIONS!

III. SMOOTH MUSCLE

029-1 Small Intestine (simple columnar epithelium, simple squamous epithelium) H&E cross Webscope Imagescope
169 jejunum H&E cross Webscope Imagescope
155 gastro-esophageal junction H&E longitudinal Webscope Imagescope
250-2 vagina Masson Webscope Imagescope
250-1 vagina H&E Webscope Imagescope

Smooth muscle may be studied using slide #29 #029-1 smooth muscle  Webscope again in section of intestine or slide #169, #169   Webscope also in the intestine.  To find the muscle layer, look at the at slide at the lowest power (this is about the same as looking at the glass slide with the naked eye).  The purple layer is largely the epithelium and the lamina propria filled with plasma cell and lymphocytes.  Next to that you see a lighter region of connective tissue (the submucosa you looked at to see loose connective tissue and fibroblasts), then a darker pink region which is made up of the two layers of smooth muscle you want to look at.  Slide 29 is a cross section of the intestine, so the inner, circular layer of muscle will have cells oriented longitudinally (or, in places, the cells may appear to be oriented more obliquely).  Move further out to see the outer sheet of smooth muscle which runs longitudinally along the intestine, and will therefore be seen in cross section.

Look at slide #155 which is a longitudinal section of the GI tract at the gastro-esophageal junction to see more smooth muscle in various planes of section.  The smooth muscle in the esophagus (the part lined with a stratified, non-keratinizing squamous epithelium) #155   Webscope is organized in the "classic" inner circular and outer longitudinal arrangement.  This stomach (the part lined by a columnar epithelium) #155   Webscope , however, has an inner oblique layer (seen mostly as longitudinal here), a very prominent middle circular layer, and a sometimes less obvious outer longitudinal layer. Don't worry knowing about the specific layers or being able to tell esophagus from stomach, but you should definitely be able to identify smooth muscle in any plane of section (tranverse, longitudinal, or even oblique).   In this particular slide, both the hematoxylin and eosin staining are quite intense which should help you see the cytoplasm more clearly especially when the muscle is cut in cross section.

A. In longitudinally cut smooth muscle cells, observe the following points:

1. Cells are small and spindle-shaped (fusiform); this may be hard to appreciate because the cell membrane is indistinct.
2. Myofibirils and cross striation cannot be seen.
3. Nuclei are narrow, elongated and sometimes kinked or spiraling. They are centrally located.

B. In transversely cut smooth muscle cells, observe the following points:

1. The cell has a small diameter.
2. The nucleus is located centrally, but will not be seen in every cross section.
3. Myofibrils cannot be seen.
4. Cross-sectional diameters vary due to the spindle shape of the cells.

Now, look at slide #250 and see if you can distinguish between small fascicles of smooth muscle and collagen fibers in the lamina propria (it's easier if you look first at the trichrome-stained section, which stains the muscle pink(ish) and the collagen blue) #250-2   Webscope . It's more challenging to make this distinction in the H&E-stained section #250-1   Webscope , but you should note that smooth muscle is pink wheras collagen is a bit more orange-red. Also, smooth muscle tissue is mostly cellular (and therefore more nuclei are present) whereas the connective tissue is mostly extracellular collagen fibers with many fewer cells. The table below compares the differences in the morphology of the three types of muscle.

Major Histological Characteristics Of The Three Types Of Muscle As Seen With The Light Microscope
  Skeletal Muscle Cardiac Muscle Smooth Muscle
  Prominet cross- striations Prominet cross- striations No cross- striations
  Fiber diameter large Fiber diameter medium Fiber diameter small
  Nuclei usually peripheral Central nucleus Central nucleus
  Unbranched Branched Unbranched
  No intercalated discs Intercalated discs No intercalated discs
  Longitudinal striation (myofibrils) Longitudinal striation No longitudinal striation

Electron Micrograph Wall Charts

#41 SKELETAL MUSCLE (longitudinal secion) WebScope ImageScope Find the skeletal muscle nuclei and note their peripheral location. Note the intimate contact between capillaries and muscle cells, and be sure you can tell where one muscle cell or fiber stops and another begins (you can see parts of four fibers in this picture). Make sure you known which is the longitudinal axis of the cell. Identify sarcomeres, A bands, I bands, Z lines and H zones. Note that, as you saw at the LM level, the individual myofibrils do not line up perfectly across the fiber.

#42 SKELETAL MUSCLE (cross section) WebScope ImageScope Note location of muscle fiber nuclei. You can see cross sections of A bands (darker) and I bands (lighter) side by side in the same cell because of the fact that the myofibrils don't line up perfectly. Identify the approximate outline of a single myofibril.

#43 SKELETAL MUSCLE (longitudinal section) WebScope ImageScope Identify a sarcomere. Relate the sarcomeric structure seen in the LM to the structure seen here. Note that there is also lots of glycogen in the region between the two myofibrils in this picture, a storage form for glucose (which is metabolized to provide energy for muscle contraction). At the border of the I and A-bands, note the triads consisting of a central T (transverse) tubule and flanking cisternae of the sarcoplasmic reticulum.

#44 SKELETAL MUSCLE (cross section) WebScope ImageScope Review the reasons for the fact that you can see only cross-sectioned thin filaments in the I bands, whereas the A bands may contain both thin and thick filaments. Thus, you should be able to tell at which level each of these myofibrils was cut. Note the abundance of the sarcoplasmic reticulum.

#45 CARDIAC MUSCLE (longitudinal section) WebScope ImageScope Note central location of muscle nuclei. Note the "stacks" of mitochondria between myofibrils. Cardiac muscle is even richer than skeletal muscle in mitochondria (again, important for energy production). An intercalated disc is present in the upper left region of the picture.

#46 CARDIAC MUSCLE (intercalated disc) WebScope ImageScope Note the irregular course of the intercalated disc. In this preparation, the I bands are very short, indicating that the sarcomere is in a contracted state. Review the types of junctions present in an intercalated disc and their functions.

#48 SMOOTH MUSCLE WebScope ImageScope Study the orientation of the smooth muscle layers in the intestine. The diagram will help you understand the patterns which arise from longitudinal and cross-sections.

#50 SMOOTH MUSCLE WebScope ImageScope Here you can see the filaments in cross-section, appearing as dots. Also, the dark areas which are membrane-associated are called dense plaques and are sites of filament attachment.

Practice Questions

  1. Caveolae in smooth muscle are functionally analogous to:
    1. transverse portion of intercalated disks in cardiac muscle.
    2. lateral portion of intercalated disks in cardiac muscle.
    3. Z lines in skeletal muscle.
    4. sarcoplasmic reticulum in skeletal muscle.
    5. T tubules in skeletal muscle.
    6. AnswerD -caveolae sequester and release Ca+ ions
  2. The predominant tissue in this slide: [WebScope or ImageScope]
    1. contracts rapidly and voluntarily.
    2. interacts directly with nerves via motor end plates.
    3. consists of regularly arranged sarcomeres.
    4. can readily regenerate and/or proliferate via cell division.
    5. ALL of the above
    6. AnswerD -the tissue shown is smooth muscle; all of the other responses apply to skeletal muscle (well, choice C actually applies to both cardiac AND skeletal muscle).
  3. Identify the tissue [WebScope or ImageScope].
    1. skeletal muscle
    2. cardiac muscle
    3. smooth muscle
    4. epimysium
    5. dense regular connective tissue (tendon)
    6. AnswerB -the tissue is CARDIAC muscle; note the striations, branched fibers, intercalated discs, and centrally located nuclei.
  4. Which of the lines in the electron micrograph indicates a single sarcomere?
    1. A
    2. B
    3. C
    4. D
    5. E
    6. AnswerC -a sarcomere is the region spanning from one Z line to another.