Download The Proteins themselves

MUSCLE
Roger J. Bick, PhD, MMEd
Learning Objectives: From lecture and lab you should be able to:
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Describe ultrastructural and histological characteristics of skeletal, cardiac and smooth muscle.
Understand cellular and macromolecular mechanisms governing regulated muscle contraction.
Understand basic adaptive and regenerative capacities of each muscle type.
Name specialized structures – spindle, etc
Key Words: Skeletal, smooth, cardiac, striated, intercalated disc, t-tubule, sarcoplasmic reticulum,
sarcolemma, epi-, peri-, and endomysium. Fast twitch v slow twitch. Glycolytic, oxidative. Z, I, A, H and M
bands. Actin, myosin, actinin. Intermediate filaments, sarcomere, calmodulin
There are three human muscle types:
Skeletal:
striated, usually voluntary contractions
Cardiac:
striated, involuntary
Smooth:
nonstriated, involuntary
Point 1- Skeletal and cardiac muscles are BOTH striated, BUT cardiac fibers branch
Point 2- position of nuclei - Smooth and cardiac centrally located, skeletal on the periphery as shown below
Point 3 for comparison – Skeletal and cardiac have long fibers; smooth muscle has individual, distinct
cells. Both cardiac and skeletal are striped (but not smooth); both cardiac and smooth muscle have centrally located
nuclei (not skeletal).
Macroscopic Features of muscle.
1. Skeletal Muscle (striated, voluntary)
Fiber: Each fiber is an elongate, multi-nucleate cell; from embryonic fusion of 100's of myoblasts.
Individual fibers (cells) are 10-120 M in diameter and up to 30 cm long (long muscles of upper
and lower extremities). Each fiber is surrounded by thin layer of connective tissue, the
endomysium (Gr. endo, within; mys, muscle), synthesized primarily by fibroblasts.
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Muscle fascicle: (L. fasces, bundle) A group of fibers surrounded by a thicker connective tissue
sheath, the perimysium (Gr. peri, around).
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Muscle: Bundle of fascicles surrounded by a thick, connective tissue epimysium (Gr. epi, upon).
Muscle grouped as broad sheets (rectus abdominis), or in bundles (biceps), or in circular arrays
(obiscularis oris, anal and urinary sphincters).
And here is the whole thing put together,
Contraction is parallel to orientation of fibers. Strength, endurance and fine control dependent on
number, size and type of innervation (see below). Pink-red color of muscle due to blood supply and
myoglobin content. Contractile force transmitted to bone or other muscle via myotendonal
junctions, then connective tissue tendons. Unlike other skeletal muscles, contraction of the
diaphragm is involuntary, e.g. during sleep.
Skeletal muscle types: Most mammalian skeletal muscle is a mixture of three fiber types: fast
twitch, glycolytic (FG; white like chicken breast meat), slow twitch, oxidative (SO; red, like a
drumstick) and fast twitch, oxidative-glycolytic (FOG; intermediate type, possessing
characteristics of both FG and SO (Sometimes referred to as mixed muscles).
Innervation (fast or slow acting) determines fiber type.
Note such things as myoglobin content, number of mitochondria and substrate utilized.in this Table
FG
SO
“Color"
White
Red
Contraction
Fast
Slow
Metabolism
Glycolytic
Oxidative (aerobic)
(anaerobic)
Substrate
Glucose
Fatty acids
Endurance
Low
High
Blood supply
Low
High
Fiber diameter
Mitochondria
Lipid Droplets
Sarcoplasmic reticulum
Myoglobin
Glycogen
Myosin ATPase
Large
Few
Few
Extensive
Low
High
High
Small
Many
Many
Sparse
High
Low
Low
Cellular Structure - Overall regular striated appearance due to registered myofibrils, in each cell or
fiber (imagine a bundle of spaghetti!); contractile elements are repeating units - sarcomeres (dark line
to dark line in above image); approx. 2.2 m in width (a most useful internal ruler!); the fundamental
contractile unit.
Each sarcomere comprised of:
Z-line: ..at each end of sarcomere
..contains actin binding protein, actinin
.. bisects each I band
I-band: ..mostly actin thin filaments
..also regularly spaced regulatory proteins, tropomyosin and troponin.
..troponin - three subunits, I, T and C
.."isotropic", does not bend polarized light, so shows up bright
..width decreases during contraction
A-band: ..mostly myosin, but some actin-myosin overlap
.."anisotropic", bends polarized light, shows up as dark band, which gives striated
muscle its characteristic appearance
..width remains constant during contractile cycle
H-band:..central region of A band which contains only myosin thick filaments
.."Heller", light
..width decreases during contraction
M-line:..middle of H band
..each myosin joined laterally to its myosin neighbor
.."Mittel" line = middle line
The Proteins themselves
Numerous proteins in muscle, but four major ones comprising myofibrils that we need to know
about at this point:
Myosin:
major component of thick filaments;
shaped like golf club with two heads on one end.
head region contains ATPase and several "light chain" proteins that help
regulate interaction with actin during contractile cycle.
Actin:
major component of thin filaments: globular monomeric actin molecules (Gactin) linked end to end (like a pearl necklace) to form an actin filament (Factin).
Two actin "strands" entwine to form a thin filament.
Tropomyosin: long protein molecule; lies in the groove of the two entwined actin strands.
Troponin:
three subunits
TnT- binds to tropomyosin
TnC- binds Ca2+
TnI- inhibits actin-myosin interaction
One troponin complex binds to one tropomyosin molecule, which in turn
spans seven G-actin molecules.
Page 112 Gartner and Hiatt
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Membrane Systems/microstructures
Sarcolemma [SL] (Gr. sarco, flesh; lemma, husk) is the plasma membrane (outer-covering) of
muscle cells; contains many ion transport proteins, ion channels and receptors; regulates flow of
ions and metabolism; Invaginates in striated muscle, at each Z-band in cardiac, and at A-I band
junction in skeletal muscle, to form transverse tubule system (T-tubules;TT); TT brings
depolarization signal deep into the muscle cells.
 Note. There is a specialized region in each skeletal muscle fiber that forms the post
synaptic membrane portion of the motor end plate. No such structure in cardiac
or smooth muscle.
Sarcoplasmic reticulum [SR]; internal membrane network; no connection with the extracellular
space; does make junctional complexes with T-tubules forming triads in skeletal, dyads in cardiac;
takes up and releases Ca2+ to regulate contraction (More on this later).
Mitochondria. Very elongate, branch extensively through and around myofibrils; Produce ATP for
contractile proteins and membrane ion pumps; Smaller mito often found under the sarcolemma,
around the nuclei; mito may exist as a "reticulum" (Continuous complex).
Nuclei. Many, often end-to-end, and located just beneath the sarcolemma
Other things present in the cells (fibers); Compliment of glycogen particles, Golgi apparatus,
lysosomes, peroxisomes, etc.
Putting all these bits and pieces together, t-tubules, mitochondria, nuclei, etc, we end up with structures that
are represented in the following two diagrams. In skeletal muscle we mentioned the triad, where two
terminal elements of SR abut the invaginating t-tubule and form a 3-component complex. Not in cardiac
muscle, where only one terminal element of the SR meets the t-tubule, a 2-component complex or a dyad.
NOTE that that t-tubules in skeletal muscle are narrow, (4 to 5 times wider in cardiac muscle), and
invaginate at the A-I band junction (at Z-line in cardiac muscle).
2. Cardiac muscle (striated, involuntary)
A. Macrostructure.
Cells joined by specialized electro-mechanical junctions the intercalated disks;
Branching; Connective tissue is interspersed throughout, but no distinct fascicular structure;
No direct innervation, although autonomic nerves do travel in myocardium. (Some further
discussion on the specialized conductive regions will be dealt with in the lecture and labs on the
Cardiovascular System); Dense vascularization due to substrate and oxidative demands of the
myocardium (What are they?).
B. Cellular organization
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Striated appearance; 1-2 centrally located nuclei; Cells 15-25 m wide and 85-120 m long.
Remember considerable branching.
Fibers contain basically the same organellar complement as skeletal muscle with some subtle (but
important!) differences;
o Myofibrils - not as organized as in skeletal muscle, so striations not as apparent; T-tubules
larger in diameter and invaginate at Z-band; SR: t-tubule junction is dyad not triad; SR less
extensive;
o Higher mitochondrial density.
Intercalated disks connect cardiac muscle cells at membranes of longitudinal ends.
Three distinct types of membrane specialization:
 Macula adherens (desmosomes) usually at perpendicular region of the intercalated disc.
 Fascia adherens: ends of actin filaments anchor into "felt-like" mat on membrane.
 These two, a and b, are mechanical junctions
 Gap junction (nexus). Low resistance electrical coupling between two cardiac cells, where the wave
of depolarization is transmitted from cell to cell in a cable fashion.
3. Smooth Muscle (nonstriated, involuntary)
A. Macrostructure
Depending on location, smooth muscle can be in bundles or sheets, longitudinal or circular in array
(blood vessels or GI system); In subsequent lectures, you will see the diverse locations,
organization, and function of smooth muscle, but the GI system, blood vessels, vagina, bladder, iris
of eye, and respiratory system contain copious amounts of smooth muscle; paler than cardiac or
"slow twitch oxidative" skeletal muscle (less vascularization and lower myoglobin content);
Endomysium is sparse except between circumferential and longitudinal layers; Cells are spindle
shaped resulting in the nuclei having a staggered appearance
B. Cellular organization
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Spindle shaped cells, 10-15 m in diameter 25-300 m in length; a single central nucleus, which
appears wavy if the muscle is contracted; Cytoplasm is eosinophilic but difficult to see distinct cell
boundaries.
No striations or sarcomeres
Actin and myosin bundles anchor on SL membrane dense bodies. Organization is usually diagonal.
SR not as distinct or well organized.
No T-tubules (!!!!)
Gap junctions between cells
Autonomic nerve endings.
Part II. Cellular Control and Innervation of muscle types
A. Skeletal muscle
Each fiber receives electrical stimulation via branch of a motor neuron; each motor neuron can
innervate up to 100 muscle fibers; combination of the neuron and muscle fibers is termed the
motor unit; Stimulation of the motor nerve results in contraction in an all-or-none response of all
of its muscle fibers.
Strength, speed and duration of contraction are dependent on:
 Type of skeletal muscle
 Number of motor units stimulated
 Frequency of stimulation
Contractile Event of the skeletal muscle
Each skeletal muscle fiber innervated by one motor neuron via the motor end plate (a.k.a.,
neuromuscular junction); At nerve terminus, acetylcholine (ACH) is stored in numerous synaptic
vesicles; nerve impulse produces fusion of vesicles in nerve terminal with nerve presynaptic
membrane; ACH discharged into synaptic cleft (gap) and binds to receptor on muscle postsynaptic
membrane resulting in Na+ influx and depolarization.
Courtesy of Dr. J. Illingworth, Sheffield University, UK
Depolarization spreads outward over sarcolemma and down into T-tubules initiating following
cascade:
At the triad, depolarization is "sensed" by the SR via "dense feet"
Stored Ca2+ in SR is released through channels into sarcoplasm
Sarcoplasmic [Ca2+] increases from resting level of 10-7 to 10-5
Ca2+ binds to TnC causing conformational change
Troponin change alters tropomyosin to uncover actin
Myosin head-ATP interacts with actin
Myosin activated, hydrolyses ATP
Ratcheting of attached myosin head pulls actin into A band
Z-Z distance decreases as sarcomere shortens
I band decreases
A band constant
Cyclic repeats of Myosin-ATP formation, ATP hydrolysis, and contraction until full contraction of
sarcomeres
Uptake of Ca2+ by SR
Muscle relaxes
-----------------------------------------------------------------------------------------------------------------------B. Cardiac Muscle.
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Cardiac contraction (heartbeat) occurs without direct nerve stimulation, but VIA an initiating
wave of depolarization that originates at sino-atrial (SA) node and travels through the conductive
pathway cells (atrioventricular [AV] node, Bundle of His and Purkinje fibers) to the contractile
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cells of the myocardium.
o Nodal and conductive pathway cells differ structurally from those of the "working"
myocardium - they are shorter and paler staining [sparse myofibrils, abundant glycogen].
Heart rate affected by autonomic innervation of the SA node and of other regions by the
vagus
nerve, as well by hormones such as adrenaline.
Contractile activities dependent on neighboring cell depolarizations (Intercalated discs !!!!).
C. Smooth Muscle
Like cardiac muscle, smooth muscle contraction regulated by autonomic innervation; responds to
hormones; Contractile-activating depolarization passes from cell-to-cell - like cardiac muscle via
gap junctions; Contraction is slow ("wave-like" as in peristaltic contractions in the GI tract).
Depolarization of SL causes an increase in sarcoplasmic Ca2+ concentration but, unlike skeletal
muscle, actin-myosin interaction is mediated through calmodulin, which activates myosin light
chain kinase, which in turn phosphorylates the myosin light chain, permitting myosin to interact
with actin. No troponin regulation in smooth muscle!!!. Because of diagonal arrangement of
contractile apparatus in smooth muscle cells, and anchoring of fibers and intermediate filaments at
dense bodies, cells become shorter and fatter in contraction.
------------------------------------------------------------------------------------------------------------Specialized structures. There are sensory organs in skeletal
muscle (proprioreceptors), as well as in tendons and joints, which
provide feedback on the contractile state of the muscle, tendon
tension and position of the joint. Muscle spindle is found in all
human skeletal muscles. Consists of 2-20 intrafusal muscle fibers,
an afferent nerve ending enclosed in a connective tissue capsule.
Stretching of the muscle, and hence the enclosed spindle,
stimulates the nerve ending whose sensory action potential is
sensed by the spinal cord. The appropriate motor neuron is
stimulated and the muscle is stimulated to contract.
Golgi tendon organ is a sensory
ending in tendon, which inhibits
muscle contraction when it
excess tension placed on the
tendon by a muscle. There are
several types of joint receptors,
of which resemble Golgi organs
sense joint position.
nerve
senses
some
and
V. Development,
Adaptation
and
Regeneration (Underlined words ARE important for the histology exam)
 Skeletal Muscle - fusion of myoblasts form mulinucleate cell myotube syncytia (G. same cell),
which synthesize contractile proteins to become mature fibers; Once cell (fiber) is formed no
further division occurs; cell can enlarge (hypertrophy) in response to exercise or hormones, or
atrophy in response to inactivity, injury, loss of innervation (such as with polio), or malnutrition;
Undifferentiated progenitor cells (satellite cells) lie in connective tissue adjacent to mature fibers
and may proliferate and fuse to become new fibers.
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Cardiac Muscle - Myocardium develops from splanchnic mesodermal cells; once formed no further
division; no satellite cells so tissue has no capacity for replacement of damaged cells instead,
connective tissue (fibrotic scar) replaces dead myocardial cells; Myocardial cells will enlarge
(hypertrophy) in response to increased vascular resistance or exercise; they will atrophy in
response to decreased workload (a problem that has to be dealt with in prolonged zero gravity
conditions in outer space).
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Smooth muscle - In response to increased demand cells can hypertrophy (increase in the size of
cells, as with the other two muscles); smooth muscle can also undergo hyperplasia (increase in the
number of cells) by cell division.
SKELETAL AND CARDIAC MUSCLE LABORATORY
Skeletal muscle (longitudinal and cross section in slide #41)
First examine the cross section to see various layers of connective tissue (epimysium, perimysium and
endomysium). Muscle tissues shrink considerably during fixation, so learn to recognize shrinkage
artifact, which shows up "well" on this slide. You will also note some small nerves and blood vessels in
the connective tissue regions. What type of nerves would these probably be?
From the capillary density, can you make an educated guess (AKA, "diagnosis") as to what the majority
of (type) skeletal muscle fibers are in this sample?
Note the general peripheral location of the nuclei in cross-sectioned fibers. How does their position
differ from that in cardiac myocardial cells, which you'll see in the next slide?
Don't confuse skeletal muscle bundles with peripheral nerve. Why is muscle so eosinophilic?
Now look at the longitudinal section on the slide. Do you see striations across the myofibrils? (Hint:
You'd better!!!) Lower your scope's condenser to make them stand out. Look at a good section at high
magnification and while carefully focussing up and down you should make out A band, I band and Z
band.
Have a look at tongue, slide 38 too. Be sure you can easily identify striated muscle as it may turn up in
places you wouldn't expect as in your slide of tonsil.
Cardiac muscle (slide #37, heart)
Try to find an area of good longitudinal representations of cardiac cells. Note that the nuclei are
centrally located unlike that of skeletal muscle fibers. Myocardial cells abut each other at intercalated
disks. If you can't differentiate these well, look at the demonstration scope set up in the front of the lab.
You'll also note that the cell striations are not as easily seen in myocardial cells compared to skeletal.
Smooth muscle (#9, scalp; #5 and 6, small intestine)
Smooth muscle bundles form the arrector pili muscle that attaches to the hair shaft. It's hard to find the
muscle attaching directly to the hair apparatus. Hints: muscle is lighter pink than surrounding almost
"red" connective tissue; the nuclei in smooth muscle are usually staggered and oriented in rows rather
than the disorder of connective tissue fibroblast nuclei; the arrector pili muscle is usually seen near the
sebaceous gland of the hair.
In small intestine (5 and 6) look for the smooth muscle in outer layers of the cross section of the
intestine. Note that the two outer layers run in two different orientations.
You'll learn more of these bundles in the G.I. System lectures and labs. Again notice the more orderly
appearance of smooth muscle compared to the underlying connective tissue. Be thinking ahead as to
why intestine has these layers of muscle and why they are oriented as they are.