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A Review of the
Technologies and
Methodologies Used to
Quantify Muscle-
Tendon Structure and
6
Function
6.1
6.2
6.3
Structure
6.4
Functions of Specific Structures • Processes Involved in Energy
Supply • Processes Involved in Force Development and
Transmission • Factors Affecting Muscle-Tendon Performance
6.5
Function
David Hawkins
Muscle Mechanics and Energy Utilization • Force and Neural
Input • Force and Length • Force and Velocity • General
Performance and Muscle-Tendon Architecture • General
Performance and Muscle Composition • General Performance
and Contraction History • General Performance and Multiple
Muscle Systems
University of California at Davis
6.6
6.1 Introduction
Muscle-tendon units are complex biological actuators able to generate considerable force to stabilize
and/or move segments of the body and absorb energy imparted to the body. They are controlled through
neural inputs and generate their forces by converting chemical energy into mechanical energy. Their
mechanical behavior is directly linked to their macroscopic and microscopic structures and the properties
of the specific proteins constituting these structures. Muscle-tendon units are highly adaptable, modifying
their structure and protein forms in response to changes in environmental stimuli. Due to the integral
role skeletal muscle plays in human function, an understanding of its behavior has been of interest for
thousands of years. However, because of its complex organization of membranes, organelles, proteins,
© 2001 by CRC Press LLC
 nerves, and vessels, and its versatility and adaptability, increases in our understanding of the detailed
workings of skeletal muscle have often depended on the development of new technologies and method-
ologies. Much is still unknown about muscle-tendon structure and function and it is likely that further
knowledge in this area will be achieved through technological innovations.
The purpose of this chapter is to provide detailed descriptions of muscle-tendon structure and func-
tion, and to summarize many of the technologies and methodologies employed over the years to unravel
the intricate structures and functions of muscle-tendon units. While structure and function are directly
related, for the sake of simplicity, they will be discussed separately. Muscle-tendon structure will be
presented first, and a review of various approaches employed to study this structure will follow. Muscle-
tendon function will be presented next, followed by a review of the approaches employed to study
function.
6.2 Muscle-Tendon Structure
In this section, a detailed description of the structural organization of a muscle-tendon unit is presented.
The description of the structural organization of muscle begins at the level of the whole muscle and
proceeds to the smaller subunits, concluding with the proteins constituting the myofilaments. Membrane
systems, neural, vascular, and connective tissue networks are described. The variability in muscle fiber
structures and how this variability has led to various fiber-type naming schemes will then be discussed.
Skeletal muscle exists in a variety of shapes and sizes. It is composed of many subunits arranged in
an organized, but complex manner (see
. Additionally, muscles connect in series to tendons, are
innervated by nerves, and supplied with vascular networks. A whole muscle is surrounded by a strong
sheath called the epimysium, and divided into a variable number of subunits called fasciculi. Each
fasciculus is surrounded by a connective tissue sheath called the perimysium. Fascicles may be further
divided into bundles of fibers (or muscle cells) surrounded by a connective tissue sheath called the
endomysium.
8,26,51,54,88,91,108,109,110
Beneath the endomysium are two additional membranes, the basal lam-
The orientation of fibers relative to the line of action of the muscle-
tendon complex is referred to as the pinnation angle. In humans, the pinnation angle ranges from 0 to
25°.
26,88,96
Muscle may be classified as fusiform (or spindle), penniform, bipenniform, triangular, rectan-
gular (or strap), and rhomboidal. Fibers attach at both ends to tendon or other connective tissue. Muscle
fibers contain mitochondria, multiple nuclei, ribosomes, soluble proteins, lipids, glycogen, and satellite
cells. Fibers are cylindrical, with their diameter ranging from 10 micrometers (
µ
m) to 100
µ
m (smaller
They may be a few millimeters (mm) or many centimeters (cm) in
length. Fibers are subdivided radially into myofibrils having diameters of approximately 1
88
m. Myofibrils
are divided longitudinally into sarcomeres and radially into myofilaments. A saromere is defined as the
region between Z-lines (defined below). Sarcomeres have a rest length of about 2.0 to 3.0 µm. Myofila-
ments are often classified as either thick or thin filaments.
Thick filaments are composed primarily of myosin molecules. Myosin accounts for approximately 55%
of the myofibril volume. It is composed of two heavy chains and four light chains. Two light chains are
associated with each heavy chain. The two heavy chains are identical, whereas the light chains vary within
different fiber types. Each myosin molecule is rod shaped with two adjacent globular heads at one end.
The myosin molecule structure has been defined in terms of two general regions: the light meromyosin
(LMM), and the heavy meromyosin (HMM). The LMM represents part of the tail. The HMM contains
the two heads, and the remaining part of the tail not considered part of the LMM. HMM is further
divided into subfragment 1 (S1) and subfragment 2 (S2) (see
. Myosin molecules are about 160
nanometers (nm) long (myosin rod is 140 nm and head is 15 nm) and 2 nm in diameter.
µ
8,26,108,110
Myosin
There
are approximately 100 axial locations along the thick filament, separated by 14.3 nm where myosin heads
exist. The number of myosin molecules terminating at each axial repeat location is still controversial.
Most of the evidence has been interpreted as suggesting three myosin ends per axial repeat distance. Each
80
© 2001 by CRC Press LLC
ina and the plasmalemma.
88,121
than the size of a human hair).
molecules are arranged to give a total thick filament length of 1.55 µm and 12 to 15 nm diameter.
  Illustration of the strucutral organization of muscle. A whole muscle is shown in A, a muscle fiber in
B, a myofibril in C, a sarcomere in D, a thin filament in E, a thick filament in F, and a myosin molecule in G.
At least 8 proteins in addition to myosin are affiliated with the thick filament: C-
protein, H-protein, M-protein, myomesin, M-creatine kinase, adenosine monophosphate (AMP) deam-
inase, skelemin, and titin.
26
8,26,88,110
Thin filaments are composed primarily of actin, tropomyosin, and troponin. Thin filaments are
approximately 1
µ
8
Actin monomers polymerize to form a
Because of symmetry and the spherical shape
of the actin monomers, there exists a groove on either side of the helix chain. Each groove is filled by a
series of tropomyosin-troponin complexes, each spanning a length of seven actin monomers (41 nm in
length). There is one troponin molecule, approximately 26 nm long, for each tropomyosin molecule.
8,88
© 2001 by CRC Press LLC
FIGURE 6.1
thick filament contains approximately 300 myosin molecules (assuming three myosin ends per axial
repeat location).
m long and 8 nm in diameter. Each thin filament contains about 360 actin monomers.
Each actin monomer consists of a single polypeptide chain.
double helix pattern with a repeat spacing of 5.5 nm.
 The tropomyosin molecule forms an
α
-helical coiled coil structure. The troponin molecule can be further
divided into troponins C, I, and T.
88,108
Thick and thin filaments are oriented parallel to one another within a sarcomere and typically have a
zone of overlap (see
. The region containing the thick filaments is referred to as the anisotropic
or A-band, approximately 1.55
µ
µ
m region in the center of the A-
or H-zone. In the middle of the A-band is a
region called the middle or M-line. The M-line is composed of a connective tissue network binding the
thick filaments. At the end of each sarcomere is a dense protein zone called the Z-line
*
**
(also referred to
The Z-disk is composed of a connective tissue network binding the thin
filaments. It contains the proteins
42,91
Thin filaments are
attached at the Z-disk but are free to interdigitate with the thick filaments at their other ends. When
viewed in cross section through the zone of overlap between thin and thick filaments, a hexagonal lattice
appears with one thick filament surrounded by six thin filaments. The spacing between thick filaments
is 40 to 50 nm.
α
-actinin, desmin, filamin, and zeugmatin.
26
80
The spacing between thick and thin filaments is 20 to 30 nm.
8
The T-system is part of
the plasmalemma and makes a network of invaginations into the cell near the Z-line in amphibian muscle
and near the junction of the A- and I-bands in mammalian muscle.
8,26.80,88
26
No part of the contractile machinery
is further than 1.5
µ
m from a T-tubule.
72
Two terminal cisternae (part of the SR) run parallel to the T-
The T-system is separated from the terminal cisternae by a distance of about
16 nm but connects to the terminal cisternae via numerous feet.
96
72
The SR traverses longitudinally from
the terminal cisternae.
In addition to the structures mentioned above, vascular, neural, and connective tissues play important
roles in muscle function. Muscles have a rich supply of blood vessels that supplies the oxygen needed for
oxidative metabolism. Capillary networks are arranged around each fiber with the capillary densities
varying around different fiber types.
80
Fibers from
a given motor unit tend to be dispersed throughout the muscle cross section rather than clumped together
in one region. Oxidative fibers tend to occur in greater percentages deeper in the muscle compared to
glycolytic fibers which have higher percentages in the perphery.
23,80
The structure of the neuromuscular
junction can vary significantly between different species, between different fiber types of the same species,
and during the course of development. In general, the nerve terminal ending on a muscle fiber contains
vesicles 50 to 60 nm in diameter. These vesicles contain acetylcholine (Ach), adenosine triphosphate
(ATP), a vesicle-specific proteoglycan, and a membrane phosphoprotein, synapsin. Approximately 15%
of the nerve terminal volume is taken up by mitochondria. The nerve and muscle membranes are not
in direct contact. The synaptic space is approximately 50 to 70 nm wide and contains acetylcholinesterase
(AchE). The muscle membrane contains nicotinic Ach receptors.
89
The muscle membrane has several
folds in the regions of the nerve endings to increase the transmitter reception area eightfold to tenfold.
Muscles have extensive connective tissue networks located both in parallel and in series with the fibers.
Myofibrils appear to be attached transversely at periodic adhesion sites. The protein titin spans the
distance between Z-lines and the middles of the thick filaments.
26
Muscle fibers are connected in series
with tendons. The primary structural unit of tendon is the collagen molecule. Type I collagen consists
of three polypeptide chains coiled together in a right-handed triple helix held together by hydrogen and
covalent bonds.
8
Collagen molecules are organized into long, cross-striated fibrils that are arranged
into bundles to form fibers. Fibers are further grouped into bundles called fascicles, which group together
43,120
*
German for “light.”
**
From Zwischen-Scheibe, meaning “interimdisk.”
© 2001 by CRC Press LLC
m in length. The region containing the thin filaments with no overlap
with the thick filaments is termed the isotropic or I-band. The 0.16
band that has no thin filament overlap is called the Helle
as the Z-disk or Z-band).
The muscle fiber contains two distinct membranous systems: the transverse tubular system (T-system
or T-Tubule system) and the sarcoplasmic reticulum (SR) (see
.
system to form a triad.
The basic neuromuscular element is called the motor unit. It consists of a single alpha motoneuron
and all the muscle fibers it innervates. The number of fibers per motor unit is variable, ranging from
just a few in ocular muscles requiring fine control, to thousands in large limb muscles.
to form the gross tendon. Elastic and reticular fibers are also found in tendon along with ground substance
(a composition of glycosaminoglycans and tissue fluid). In an unstressed state, collagen fibers take on a
sinusoidal appearance, referred to as a crimp pattern.
Although the general structures (i.e., actin and myosin filament lengths and their lattice arrangement)
are similar among vertebrate muscle fibers, there are differences in the regulatory proteins of the myosin
and troponin, the extensiveness of membrane networks, and the number of mitochondria and other
organelles. These variations have functional consequences that led to the development of a variety of
naming schemes to identify fibers with specific structural and functional properties (e.g., red/white,
fast/slow, oxidative/glycolytic, types I/IIa,b,c, and SO/FOG/FG).
19,20,23-25,29,94,107
The myosin molecule
appears in various isoforms.
56,79,105
These isoforms exhibit different amino acid sequences, ATPase activity,
The troponin C protein may vary in its sensitivity to calcium. There are
differences in the membrane networks. The T-system may be twice as extensive in one fiber compared
to another. Mitochondrial density also varies among fibers.
99
26
6.3 Approaches Used to Study Muscle-Tendon Structure
However, most of the studies conducted prior to the 17th century, which contributed to our
understanding of muscle structure, were based on gross dissections and involved identifying muscles,
tendons, nerves, and the vascular network. Since then, advances in mathematics, chemistry, physics, and
genetics have played a major role in identifying and characterizing muscle-tendon structure.
Microscopy has been used extensively to study muscle. Lenses were first used to magnify objects around
1600 A.D.
Microscopes, in which various arrangements of flat, concave, and convex lenses are used to
magnify images, were introduced around the beginning of the 17th century. Microscopy has developed
into a highly technical field utilizing a variety of illuminating approaches.
Light microscopy was the first technique employed to study muscles and other biological tissues.
Leeuwenhoek (1632–1723) was one of the first great biological microscopists. He manufactured hundreds
of microscopes which he used to observe many biological tissues. Unfortunately, much of his expertise
in tissue preparation and illumination was lost throughout the 18th and 19th centuries. Much of the
work in light microscopy conducted then centered around correcting for artifacts and aberrations through
matching glass, refractive media, and improving lens manufacturing.
104
Muscle appears transparent when
viewed using normal light microscopy, and therefore it is often stained prior to viewing. A variety of
stains have been used to provide the contrast necessary to identify different organelles and gross struc-
tures.
104
In addition, the light used to illuminate the specimen has been manipulated in various ways to
cause refraction and interference patterns that allow different structures within muscle to be visible.
Dark-ground, phase contrast, interference, and polarization microscopy identify regions of different
refractive indices, but they accomplish this based on fundamentally different approaches. While most
living, non-stained biological tissue is transparent when investigated with normal light microscopy,
different regions of a cell have different refractive indices. In dark-ground microscopy, light is passed
through the specimen at rather oblique angles so that the direct light beam passes to the side of the
objective.
104
The only light entering the objective comes from refracted light. Regions of high refractive
index appear bright against a black background as they reflect the light to the eyepiece or viewing port.
Phase contrast microscopy makes use of the relative phase differences in light passing through different
regions of the tissue having different refractive indices. These phase differences are converted to changes
in light intensity in the image plane.
104,114
Interference microscopy splits the illuminating beam into two
beams. One beam passes through the specimen and the other beam passes around it.
114
The two beams
are recombined before the objective. Light passing through high refractive index tissue is slowed down,
phase shifted, relative to light passing around the tissue. The interference pattern that results indicates
different protein-dense zones. If the proteins within a region which give rise to its refraction index are
8
© 2001 by CRC Press LLC
and affinity for calcium.
Our understanding of the complex structural organization of muscle-tendon units described above has
come from keen observations and the development of a variety of technical tools and novel methodol-
ogies. The first recorded scientific medical studies were undertaken by the Greeks around the 6th century
B.C.
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