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MUSCLE
Motion, as a reaction of multicellular organisms to changes in the internal and
external environment, is mediated by muscle cells.
The basis for motion mediated by muscle cells is the conversion of chemical energy
(ATP) into mechanical energy by the contractile apparatus of muscle cells. The
proteins actin and myosin are part of the contractile apparatus. The interaction of
these two proteins mediates the contraction of muscle cells. Actin and myosin
filaments, each composed of many action and myosin molecules, form myofibrils
arranged parallel to the direction of cellular contraction.
A further specialisation of muscle cells is an excitable cell membrane which
propagates the stimuli which initiate cellular contraction.
Three structurally and functionally distinct types of muscle are found in
vertebrates:
1 smooth muscle,
2 skeletal muscle and
3 cardiac muscle.

Smooth Muscle
• Smooth muscle consists of spindle shaped cells of variable size.
• The largest smooth muscle cells occur in the uterus during pregnancy (12x600
µm). The smallest are found around small arterioles (1x10 µm).
• Smooth muscle cells contain one centrally placed nucleus.
• The chromatin is finely granular and the nucleus contains 2-5 nucleoli.
• The innervation of smooth muscle is provided by the autonomic nervous
system.
• Smooth muscle makes up the visceral or involuntary muscle.
Structure of smooth muscle
In the cytoplasm, we find longitudinally oriented bundles of the myofilaments actin
and myosin. Actin filaments insert into attachment plaques located on the
cytoplasmic surface of the plasma membrane. From here, they extend into the
cytoplasm and interact with myosin filaments. The myosin filaments interact with a
second set of actin filaments which insert into intracytoplasmatic dense bodies.
From these dense bodies further actin filaments extend to interact with yet another
set of myosin filaments. This sequence is repeated until the last actin filaments of the
bundle again insert into attachment plaques .
In principle, this organisation of bundles of myofilaments, or myofibrils, into repeating
units corresponds to that in other muscle types. The repeating units of different
myofibrils are however not aligned with each other, and myofibrils do not run exactly
longitudinally or parallel to each other through the smooth muscle cells. Striations,
which reflect the alignment of myofibrils in other muscle types, are therefore not
visible in smooth muscle.
Smooth endoplasmatic reticulum is found close to the cytoplasmatic surface of the
plasma membrane. Most of the other organelles tend to accumulate in the
cytoplasmic regions around the poles of the nucleus. The plasma membrane,
cytoplasm and endoplasmatic reticulum of muscle cells are often referred to
as sarcolemma, sarcoplasm, and sarcoplasmatic reticulum.
During contraction, the tensile force generated by individual smooth muscle cells is
conveyed to the surrounding connective tissue by the sheath of reticular fibres.
These fibres are part of a basal lamina which surrounds muscle cells of all muscle
types. Smooth muscle cells can remain in a state of contraction for long
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periods. Contraction is usually slow and may take minutes to develop.

Origin of smooth muscle

Smooth muscle cells arise from undifferentiated mesenchymal cells. These cells
differentiate first into mitotically active cells, myoblasts, which contain a few
myofilaments. Myoblasts give rise to the cells which will differentiate into mature
smooth muscle cells.
Types of smooth muscle
Two broad types of smooth muscle can be distinguished on the basis of the type of
stimulus which results in contraction and the specificity with which individual smooth
muscle cells react to the stimulus:
1 The multiunit type represents functionally independent smooth muscle cells
which are often innervated by a single nerve terminal and which never
contract spontaneously (e.g. smooth muscle in the walls of blood
vessels).
2 The visceral type represents bundles of smooth muscle cells connected by GAP
junctions, which contract spontaneously if stretched beyond a certain
limit (e.g. smooth muscle in the walls of the intestines).

Suitable Slides
Sections of the intestines (duodenum, jejunum, ileum or colon) - H&E
Jejunum, baboon - H&E
The outer part of the tube forming the intestines consists of two layers of
smooth muscle - one circular layer and one longitudinal layer. If you look at the
tissue close to the border between the two layers of smooth muscle, you will be able
to see both longitudinally sectioned smooth muscle cells and transversely sectioned
smooth muscle cells. The smooth muscle cells are much longer than their nuclei.
Transversely sectioned smooth muscle cells may not have their nuclei in the plane of
the section.
Occasionally you will find small nerves between the two muscle layers, and, if you
are lucky and/or patient, you will also see some very large nuclei in this region.
These nuclei belong to peripheral nerve cells (ganglion cells of the myenteric
plexus), which regulate the contraction of the muscle around the gastrointestinal
tract.
Draw a small area which contains both longitudinally sectioned and
transversely sectioned smooth muscle at high magnification.
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? The only tissues which perhaps could be confused with smooth muscle are dense
regular connective tissues and peripheral nerves. Both the number of nuclei and
their shapes clearly distinguish smooth muscle from dense regular connective
tissues. Nuclei are much more frequent and larger in smooth muscle, and they are
very elongated if cut longitudinally. Peripheral nerves will be surrounded by a
capsule of cells and connective tissue - the perineurium. The thickness of
longitudinally cut nerve fibres is constant while smooth muscle cells are spindle
shaped. Also, axon and nodes of Ranvier should be visible in peripheral nerves

Skeletal Muscle
• Skeletal muscle consists of very long tubular cells, which are also called muscle
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fibres.
• The average length of skeletal muscle cells in humans is about 3 cm (sartorius
muscle up to 30 cm, stapedius muscle only about 1 mm). Their diameters
vary from 10 to 100 µm.
• Skeletal muscle fibres contain many peripherally placed nuclei.
• Up to several hundred rather small nuclei with 1 or 2 nucleoli are located just
beneath the plasma membrane.
• Skeletal muscle fibres show in many preparations characteristic cross-
striations. It is therefore also called striated muscle.
• Skeletal muscle is innervated by the somatic nervous system.
• Skeletal muscle makes up the voluntary muscle.
Structure of skeletal muscle
Muscle fibres in skeletal muscle occur in bundles, fascicles, which make up the
muscle. The muscle is surrounded by a layer of connective tissue, the epimysium,
which is continuous with the muscle fascia. Connective tissue from the epimysium
extends into the muscle to surround individual fascicles (perimysium). A delicate
network of loose connective tissue composed of fine collagenous and reticular fibres
(endomysium) is found between the muscle fibres of a fascicle. Finally, each
muscle fibre is surrounded by a basement membrane.
Origin of skeletal muscle
The myoblasts of all skeletal muscle fibres originate from the paraxial mesoderm.
Myoblasts undergo frequent divisions and coalesce with the formation of a
multinucleated, syncytial muscle fibre or myotube. The nuclei of the myotube are still
located centrally in the muscle fibre. In the course of the synthesis of the
myofilaments/myofibrils, the nuclei are gradually displaced to the periphery of the
cell.
Satellite cells are small cells which are closely apposed to muscle fibres within the
basal lamina which surrounds the muscle fibre. Their nuclei are slightly darker than
those of the muscle fibre. Satellite cells are believed to represent persistent
myoblasts. They may regenerate muscle fibres in case of damage.

Suitable Slides
Sections of skeletal muscle, tongue or upper oesophagus - H&E
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Tongue, Skeletal Muscle, human - H&E

Skeletal muscle in the tongue is arranged in bundles which typically run at
right angles to each other. Both longitudinally and transversely cut skeletal
muscle fibres are present. In both section planes you can see that the nuclei
are located in the periphery of the muscle fibre. Myofibrils may be visible as
very fine dots in some of the transversely muscle fibres. Striations formed by
the A- and I-Bands of the sarcomeres are visible in longitudinally cut
fibres. Z-lines and H-bands can be identified in well-preserved tissue.
Details of the sarcomeres stand out more clearly if you close the iris
diaphragm of the microscope. Remember to open the diaphragm after you
have seen the striations clearly !
In the connective tissue between the muscle fibres, the endomysium,
numerous capillaries supply the muscle with oxygen and nutrients.
Draw a small section of longitudinal and transversely cut skeletal
muscle at high magnification.
The muscle surrounding the upper one-third of the oesophagus is
skeletal muscle. Smooth muscle surrounds its lower one-third. In section of
the middle of the esophagus it is usually possible to identify both muscle types
and their appearances can be compared.
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The Contractile Apparatus of Skeletal Muscle

The spatial relation between the filaments that make up the myofibrils within skeletal
muscle fibres is highly regular. This regular organisation of the myofilaments gives
rise to the cross-striation, which characterises skeletal and cardiac muscle. Sets of
individual "stria" correspond to the smallest contractile units of skeletal
muscle, the sarcomeres. Rows of sarcomeres form the myofibrils (), which extend
throughout the length of the skeletal muscle fibre.

Depending on the distribution and interconnection of myofilaments a number of

"bands" and "lines" can be distinguished in the sarcomeres :
The THIN bands = actin
The THICK bands = myosin

I-band - actin filaments, “I band” = L“I”ght

A-band - myosin filaments which may overlap with actin filaments,
“A band = d“A”rk
H-band - zone of myosin filaments only (no overlap with actin
filaments) within the A-band,
Z-line - zone of apposition of actin filaments belonging to two
neighbouring sarcomeres (mediated by a protein called alpha-
actinin),
M-line - band of connections between myosin filaments (mediated by
proteins, e.g. myomesin, M-protein).

So at contraction:
1. The two Z lines move closer together
2. The H band becomes narrower
3. The I band becomes narrower
4. The A band grows (more overlap between actin and myosin)

The average length of a sarcomere is about 2.5 µm (contracted ~1.5 µm, stretched
~3 µm).
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The protein titin extends from the Z-line to the M-line. It is attached to the Z-line and
the myosin filaments. Titin has an elastic part which is located between the Z-line
and the border between the I- and A-bands. Titin contributes to keeping the filaments
of the contractile apparatus in alignment and to the passive stretch resistance of
muscle fibres.
Other cytoskeletal proteins interconnect the Z-lines of neighbouring myofibrils.
Because of this connection, the A- and I-bands of neighbouring myofibrils lie side-by-
side in the muscle fibre. These cytoskeletal proteins also connect the Z-lines of the
peripheral myofibrils to the sarcolemma.
Muscle-Tendon Junction
At the muscle-tendon junction, the end of a muscle fibre forms deep invaginations,
which increase its surface area. The basement membrane of the muscle fibre
extends into these invagination and, so do the collagen fibrils of the tendons. The
actin filaments of the last sarcomeres extend into cytoplasmic specialisations
associated with zonula adherens-like membrane specialisations. Instead of
interconnecting two cells, the cell membrane is here anchored to the basement
membrane of the muscle cell. The basement membrane is, in turn, connected to the
collagen fibrils of the tendons.

Excitation and Contraction of Skeletal Muscle

The area of contact between the end of a motor nerve and a skeletal
muscle cell is called the motor end plate. Small branches of the motor
nerve form contacts (boutons) with the muscle cell in a roughly eliptical area.
The excitatory transmitter at the motor end plate is acetylcholine. The
space between the boutons and the muscle fibre is called primary synaptic
cleft. Numerous infoldings of the sarcolemma in the area of the motor
end plate form secondary synaptic clefts. Motor end plates typically
concentrate in a narrow zone close to the middle of the belly of a muscle.
The excitable sarcolemma of skeletal muscle cells will allow the stimulus to
spread, from this zone, over the entire muscle cell.
The spread of excitation over the sarcolemma is mediated by voltage-gated ion
channels.
Invaginations of the sarcolemma form the T-tubule system which "leads" the
excitation into the muscle fibre, close to the border between the A- and I-bands
of the myofibrils. Here, the T-tubules are in close apposition with cisternae
formed by the sarcoplasmatic reticulum. This association is called a triad. The
narrow gap between the T-tubule and the cisternae of the sarcoplasmatic reticulum
is spanned by proteins which mediate the excitation-contraction coupling.
Proteins in the sarcolemma which forms the wall of the T-tubule (dihydropyridine
(DHP) receptors) change conformation, i.e. they change their shape, in response to
the excitation travelling over the sarcolemma. These proteins are in touch with
calcium channels (ryanodine receptors) which are embedded in the membrane
of the cisternae of the sarcoplasmatic reticulum. The change in the shape of
the proteins belonging to the T-tubule opens the calcium channels of the
sarcoplasmatic reticulum. Calcium can now move from stores in the
sarcoplasmatic reticulum into the cytoplasm surrounding the myofilaments.
Sites of interaction between actin and myosin are in resting muscle cells "hidden" by
tropomyosin. Tropomyosin is kept in place by a complex of proteins collectively
called troponin. The binding of calcium to troponin-C induces a conformational
change in the troponin-tropomyosin complex which permits the interaction between
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myosin and actin and, as a consequence of this interaction, contraction.

ATP-dependent calcium pumps in the membrane of the sarcoplasmatic
reticulum typically restore the concentration of Ca to resting levels within 30
milliseconds after the activation of the muscle fibre.

Types of Skeletal Muscle

Skeletal muscle cells respond to stimulation with a brief maximal contraction - they
are of the twitch type. Individual muscles fibres cannot maintain their contraction
over longer periods. The sustained contraction of a muscle depends on the
"averaged" activity of often many muscles fibres, which individually only contract for
a brief period of time.
The force generated by the muscle fibre does depend on its state of contraction at
the time of excitation. Excitation frequency and the mechanical summation of the
force generated is one way to graduate the force generated by the entire muscle.
Another way is the regulation of the number of muscle fibres which contract in the
muscle. Additional motor units, i.e. groups of muscle fibres innervated by one motor
neurone and its branches, are recruited if their force is required. The functional
properties of the muscle can be "fine-tuned" further to the tasks the muscle performs
by blending functionally different types of muscle fibres:
Type I fibres (red fibres)
Red muscles contain predominantly (but not exclusively) red muscle cells. Red
muscle fibres are comparatively thin and contain large amounts of myoglobin and
mitochondria. Red fibres contain an isoform of myosin with low ATPase activity, i.e.
the speed with which myosin is able to use up ATP. Contraction is therefore slow.
Red muscles are used when sustained production of force is necessary, e.g. in the
control of posture.
Type II fibres
White muscle cells, which are predominantly found in white muscles, are
thicker and contain less myoglobin. ATPase activity of the myosin isoform in
white fibres is high, and contraction is fast. Type IIA fibres (red) contain
many mitochondria and are available for both sustained activity and short-
lasting, intense contractions. Type IIB/IIX fibres (white) contain only few
mitochondria. They are recruited in the case of rapid accelerations and
short lasting maximal contraction. Type IIB/IIX fibres rely on anaerobic
glycolysis to generate the ATP needed for contraction.
Skeletal muscle fibres do not contract spontaneously. Skeletal muscle fibres are not
interconnected via GAP junctions but depend on nervous stimulation for contraction.
All muscle fibres of a motor unit are of the same type.
Fibre type is determined by the pattern of stimulation of the fibre, which, in turn, is
determined by the type of neuron which innervates the muscle. If the stimulation
pattern is changed experimentally, fibre type will change accordingly. This is of some
clinical / pathological importance. Nerve fibres have the capacity to form new
branches, i.e. to "sprout", and to re-innervate muscle fibres, which may have lost
their innervation as a consequence of an acute lesion to the nerve or a
neurodegenerative disorder. The type of the muscle fibre will change if the type of
stimulation provided by the sprouting nerve fibre does not match with the type of
muscle. The process of reinnervation and type adjustment may result in fibre type
grouping within the muscle, i.e. large areas of the muscle are populated by muscle
fibres of one type.
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Muscle Spindles
Muscle spindles are sensory specialization of the muscular tissue. A
number of small specialised intrafusal muscle fibres (nuclear bag fibres
and nuclear chain fibres) are surrounded by a capsule of connective
tissue. The intrafusal fibres are innervated by efferent motor nerve fibres.
Afferent sensory nerve fibres surround the intrafusal muscle fibres.
If the muscle is stretched, the muscle fibres in the muscle spindle are
stretched, sensory nerves are stimulated, and a change in contraction of
the muscle is perceived. Different types of intrafusal fibres and nerve
endings allow the perception of position, velocity and acceleration of the
contraction of the muscle.
The contraction of the intrafusal fibres, after stimulation by the efferent
nerve fibres, may counteract or magnify the changes imposed on the
muscle spindle by the surrounding muscle. The intrafusal fibres and the
efferent nerves can in this way set the sensitivity for the sensory nerve
ending in the muscle spindle.

Cardiac Muscle
• Cardiac muscle, the myocardium, consists of muscle cells, cardiomyocytes,
with one centrally placed nucleus.
• Nuclei are oval, rather pale and located centrally in the muscle cell which is 10 - 15
µm wide.
• Cardiac muscle cells exhibit cross-striations.
• Cardiac muscle cells excitation is mediated by rythmically active modified
cardiac muscle cells.
• Cardiac muscle is innervated by the autonomic nervous system, which
adjusts the force generated by the muscle cells and the frequency of the
heart beat.
• Cardiac muscle is for these reasons also called involuntary striated muscle.
Structure of cardiac muscle
The ultrastructure of the contractile apparatus and the mechanism of contraction
largely correspond to that seen in skeletal muscle cells. Although equal in
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ultrastructure to skeletal muscle, the cross-striations in cardiac muscle are less

distinct, in part because rows of mitochondria and many lipid and glycogen
droplets are found between myofibrils.
In contrast to skeletal muscle cells, cardiac muscle cells often branch at acute angles
and are connected to each other by specialisations of the cell membrane in the
region of the intercalated discs. Intercalated discs invariably occur at the ends of
cardiac muscle cells in a region corresponding to the Z-line of the myofibrils (the last
Z-line of the myofibril within the cell is "replaced" by the intercalated disk of the cell
membrane). In the longitudinal part of the cell membrane, between the "steps"
typically formed by the intercalated disk, we find extensive GAP junctions.
T-tubules are typically wider than in skeletal muscle, but there is only one T-
tubule set for each sarcomere, which is located close to the Z-line. The
associated sarcoplasmatic reticulum is organised somewhat simpler than in skeletal
muscle. It does not form continuous cisternae but instead an irregular tubular
network around the sarcomere with only small isolated dilations in association with
the T-tubules.
Cardiac muscle does not contain cells equivalent to the satellite cells of skeletal
muscle. Therefore cardiac muscle cannot regenerate.

Suitable Slides
Sections of cardiac muscle - Alizarin Blue, Whipf's polychrome, iron haematoxylin,
H&E
Cardiac Muscle, human - H&E
Use a low magnification to find a part of the tissue in which the cardiac
muscle cells are cut longitudinally. At high magnification you should see
striations and the large nuclei of the cardiac muscle cells. If you follow the
course of individual cardiac muscle cells you will note fine, darker lines
which seem to cross the fibres. These are the intercalated discs which
connect the individual muscle cells mechanically and permit the
conduction of electrical impulses between the cells ... how?
A light streak of cytoplasm is often visible extending from the poles of the
nucleus. This part of the cytoplasm does not contain myofibrils, and it
appears very light in transversely cut cardiac muscle cells. Myofibrils are
often visible in transversely cut cells. Their visible separation reflects the
large numbers of mitochondria located between them. Also, the large
number capillaries reflect the need of a good blood supply to the
constantly active muscle cells.
Draw longitudinally cut cardiac muscle cells which show all the features
mentioned. Label the features in your drawing, and include an suitable
scale.
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Excitation in cardiac muscle

A number of specialised structures, which are composed of modified cardiac muscle
cells, ensure that the contraction of the atria and ventricles takes place in the order
that is most appropriate to the pumping function of the heart. The excitation of the
myocardium originates from the sinuatrial node, which is located in the wall of
the right atrium lateral to the opening of the superior vena cava into the atrium.
The sinuatrial node initiates the contraction of atrial myocardium. Excitation also
reaches the atrioventricular node at the base of the interatrial septum. The
myocardium of the atria and ventricles are separated from each other by a zone of
connective tissue, the fibrous body of the heart. The fibrous body prevents the
spread of excitation from atrial muscle cells to those of the ventricles.
A system of modified cardiac muscle cells, Purkinje fibres, has developed, which
conduct stimuli faster than ordinary cardiac muscle cells (2-3 m/s vs. 0.6 m/s). A
bundle of Purkinje fibres extends from the atrioventricular node, pierces the
fibrous body, divides into left and right bundles, and travels, beneath the
endocardium, towards the tip (apex) of the heart. Branches of the bundle contact
ordinary cardiac muscle cells by way of specialisations similar to intercalated discs.
Purkinje fibres contain large amounts of glycogen but fewer myofibrils than ordinary
cardiac muscle cells. Myofibrils are mainly located in the periphery of the cell.
Purkinje fibres are also thicker than ordinary cardiac muscle cells.
Modified muscle cells in nodal tissue (nodal muscle cells or P cells; P ~ pacemaker
or pale-staining) of the heart exert the pacemaker function that drives the Purkinje
cells. The rhythm generated by the nodal muscle cells can be modified by the
autonomic nervous system, which innervates the nodal tissue and accelerates
(sympathetic) or decelerates (parasympathetic) heart rate.

Suitable Slides
Sections of cardiac muscle (interventricular septum) - Whipf's polychrome, iron
haematoxylin, H&E
Purkinje Fibre, sheep - Whipf's polychromeCardiac muscle cells in this
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preparation have a red-violet appearance. Much of the connective tissue looks light
blue, striations of cardiac muscle cells are visible. Intercalated discs may be more
difficult to find, but nuclei stand out very clearly. Bundles of Purkinje fibres are
present in areas of connective tissue between areas of "normal" cardiac muscle
tissue and beneath the endocardium. Purkinje fibres appear as a chain of light blue
profiles with a red rim. Browse through the tissue at low magnification and change to
high magnification when you suspect the presence of Purkinje fibres. The red rim is
formed by the contractile filaments. They are displaced to the periphery of the cells
and can sometimes be used to define the outline of individual cells. The nuclei are
large, but the cells are even larger and you will not see a nucleus in each cell.
Draw a Purkinje fibre at high magnification. Try to include a bit of "normal"
cardiac muscle and a suitable scale.