Sarcopenia Age-Related Muscle Wasting and Weakness: Mechanisms and Treatments P10 pptx

76 L.V. Thompson
The extent of age-related physiological and molecular changes is dependent on
many factors. Epidemiological studies suggest multiple contributing factors, includ-
ing neuronal and hormonal changes, inadequate nutrition, low-grade chronic inflam-
mation, and physical inactivity. Physiological studies indicate that protein structure
and function in addition to translational and transcriptional mechanisms are involved.
Therefore, understanding the aging process requires systematic, multidisciplinary
studies on physiological, biochemical, structural, and chemical changes in specific
muscles. The purpose of this chapter is to highlight cellular, molecular, and biochemi-
cal changes that contribute to age-related muscle dysfunction, specifically age-related
changes in the interaction between the contractile proteins myosin and actin.
2 Muscle Structure, Contraction, Plasticity Review
2.1 Muscle Structure
Skeletal muscles are composed of individual, multinucleated cells, containing
specialized structures for excitation–contraction coupling. Each individual skeletal
muscle cell, termed fiber, is more or less cylindrical, with diameters between 10
and 100 mm and up to a few centimeters in length. The fiber is composed of a
bundle of myofibrils, each being a linear array or a string of sarcomeres (1 mm in
diameter) from one end of the fiber to the other. The sarcomere, the primary
contractile unit, is composed of interdigitating thick and thin myofilaments.
Figure 1 is a schematic of a muscle, an individual fiber and one sarcomere.
The major components of the thick filament are myosin dimers, composed of
two myosin heavy chains (Fig. 2). Each of the myosin heavy chains (~220 kD) has
a globular “head” domain at the N-terminal and an alpha-helix at the C-terminal
“tail” domain. The tail region is periodically interspersed with hydrophobic
residues to give a “coiled-coil” type rod. The amino acids in the C-terminal are
non-helical, which provide filament backbone stabilization. The tail regions
aggregate into bipolar filaments to form the thick filament.
The N-terminal of the myosin heavy chain contains specific regions essential to
myosin’s contractile and enzymatic activity. The catalytic (ATP hydrolysis) and
force-generating (interaction with actin) functions of myosin are located in its

“head” region, specific to the subfragment-1 or S1 region (95 kD). In the catalytic
domain there is a SH1–SH2 helix consisting of two critical cysteines, SH1 (Cys707)
and SH2 (Cys697), both fast-reacting sulfhydryls. The myosin head region also
contains the light chain (LC) domain, which contains the essential (ELC) and
regulatory (RLC) light chains. The light chains of skeletal muscle myosin have
regulatory functions such as calcium regulation, shortening velocity and the extent
of actin-activation of myosin ATPase. Figure 2 describes the myosin filament, myo-
sin dimer and the components of the S1 region.
The thin filaments are composed of three different types of protein: actin,
tropomyosin, and troponin (Fig. 3). Actin is a globular protein (~40 kD), which
contains specific sites of interaction with myosin. The globular actin or G-actin
Fig. 1 Skeletal muscle structure. Skeletal muscles are composed of individual muscle fibers
arranged in parallel. In this example, the individual fiber is ~100 mm in diameter. An individual
muscle fiber is composed of many myofibrils arranged in parallel. The myofibril is made up of
the major contractile unit, the sarcomere. The sarcomere is ~1mm in diameter and 2.5 mm in
length. Individual sarcomeres are arranged in series to form a single myofibril. The sarcomere
contains interdigitating thick and thin filaments. The main components of the thick filaments are
myosin molecules. The major components of the thin filaments are actin monomers. With aging,
the individual fibers decrease in cross-sectional area by decreasing the number of myofibrils
Fig. 2
Structure of skeletal muscle myosin (myosin class II). The myosin filament is composed of
myosin molecules aggregated via their “tail” regions into a bipolar filament, ~2 mm in length. Skeletal
muscle myosin forms a dimer composed of two heavy chains (MHC) containing regions involved in
enzymatic and actin-binding functions of myosin (S1), two pairs of regulatory light chains (RLC, red)
and two pairs of essential light chains (ELC, green). The crystal structure of myosin head shows that
the catalytic and force-generating function of myosin is located in its “head” region, which contains the
catalytic domain (with sites for ATP hydrolysis and interaction with actin), and the light chain domain,
which contains the ECL and RLC light chains. In the catalytic domain there is a SH1–SH2 helix
consisting of two critical cysteines, SH1 (Cys707) and SH2 (Cys697), both fast-reacting sulfhydryls
78 L.V. Thompson

subunits assembles into long filamentous polymers called F-actin forming two
strands of an alpha helix. Interdigitated between the actin strands are rod-shaped
proteins termed tropomyosin. There are 6–7 actin molecules per tropomyosin.
Attached to the tropomyosin at regular intervals is the troponin complex (Fig. 3),
which is made up of three subunits: troponin-T (TN-T), which attaches to the
tropomyosin; troponin-C (TN-C), which serves as a binding site for Ca
2+
during
excitation–contraction coupling (four Ca
2+
can bind per TN-C); and troponin-I
(TN-I), which inhibits the myosin binding site on the actin. When Ca
2+
binds to
TN-C, there is a conformational change in the troponin complex such that TN-I
moves away from the myosin binding site on the actin, thereby making it assessable
to the myosin head. When Ca
2+
is removed from the TN-C, the troponin complex
resumes its inactivated position, thereby inhibiting myosin-actin binding.
2.2 Molecular Basis of Muscle Contraction
2.2.1 Force Generation, Structural States of Myosin, and Myosin ATPase
The basic process of muscle contraction is well understood and it is a result of cyclic
interactions between myosin and actin, driven by the chemical free energy released
from ATP hydrolysis. The interactions of the myosin head with actin, in the presence
Myosin
binding
domain
Actin
crystal structure

Actin
filament
G-actin monomer
Tropomyosin
Troponin complex
Fig. 3 Structure of skeletal muscle actin. The main component of the actin thin filaments is a
helical polymer of globular actin monomers. Interdigitated between the actin strands are rod-
shaped proteins termed tropomyosin. Attached to the tropomyosin at regular intervals is the
troponin complex. The troponin complex is made up of three subunits: troponin-T (TN-T), which
attaches to the tropomyosin; troponin-C (TN-C), which serves as a binding site for Ca
2+
during
excitation–contraction coupling (four Ca
2+
can bind per TN-C); and troponin-I (TN-I), which
inhibits the myosin binding site on the actin. The crystal structure of the actin monomer shows
specific sites of interaction with myosin
79Age-Related Decline in Actomyosin Structure and Function
of ATP during the actomyosin ATPase cycle, results in sliding of thin filaments past
thick filaments toward the center of the sarcomere (muscle contraction).
The biochemical steps of ATP hydrolysis during the cyclic interaction of actin
with myosin are accompanied by a sequence of structural transitions in both pro-
teins. Figure 4 is a schematic of structural and biochemical steps of ATP hydrolysis
by myosin in the presence of actin (actomyosin ATPase cycle). Force is generated
during transition of the myosin head from the states of weak binding (pre-power-
stroke) to the states of strong binding (post-powerstroke) with actin. Particularly, in
the absence of ATP and/or the presence of ADP, the myosin head forms a strong
and well-ordered complex with actin. Binding of ATP to myosin produces a weaker
complex where the catalytic domain and light chain domain of myosin are disor-
dered. The release of phosphate (P

i
) from myosin causes a structural transition in
the catalytic and light chain binding domains, generates force, and initiates a new
cycle (Prochniewicz et al. 2004; Thomas et al. 2009).
AM AM•ATP AM
P
i
ADP
M •ATP M •ADP•Pi
Weak binding
structural state
Strong binding
structural state
ATP
Myosin head
Actin
AM•ADP•Pi
AM•ADP
Fig. 4 Schematic of structural changes of the myosin head during muscle contraction, coupled to
the ATPase cycle. The primary molecular event responsible for force generation in muscle is a
global rotation of myosin heads (cross-bridges) coupled to myosin-catalyzed ATP hydrolysis.
Interaction of the myosin head with actin in the presence of ATP results in sliding of thin filaments
past thick filaments toward the center of the sarcomere, contracting the sarcomere. The biochemical
steps of ATP hydrolysis during this cyclic interaction of actin with myosin are accompanied by a
sequence of structural transition in both proteins. The biochemical step associated with the power
stroke is the release of the hydrolysis product phosphate (P
i
), while the release of ADP follows the
execution of the power stroke. In the absence of ATP and/or the presence of ADP, the myosin head
forms a strong and well-ordered complex with actin. Binding of ATP to myosin produces a weaker

complex where the catalytic domain and light chain domain of myosin are disordered and identified
as a weak binding structural state (red, pre-powerstroke). The release of phosphate (P
i
) from myosin
causes a structural transition (black, strong binding structural state) in the catalytic and light chain
binding domains, generates force (post-powerstroke), and initiates a new cycle
80 L.V. Thompson
The physiologically relevant step of force generation is a transition of the
actomyosin complex from the states of weak interaction (AM•ATP and AM•ADP•P
i
)
to the states of strong interaction (AM•ADP and AM). In other words, force genera-
tion occurs when myosin and actin are in the strong-binding structural state (Fig. 4).
Relevant to this chapter, force can decline if the system spends too much time in
the weak-binding states, or the strong-binding states are weakened. Velocity can
decline if the system spends too much time in the strong-binding states.
It is important to note that the hydrolysis of ATP requires myosin. Myosin is an
enzyme which catalyzes the hydrolysis of ATP in the presence of actin, providing
the source of free energy that drives muscle contraction. In the absence of actin,
myosin ATPase activity is low and requires Ca
2+
. Myosin ATPase activity is posi-
tively correlated with the myosin heavy chain isoform, with myosin heavy chain
type I (MHC type I) hydrolyzing ATP at a slower rate than myosin heavy chain type
II (MHC type II). Thus, any structural or chemical changes in myosin and actin that
affect actomyosin ATPase activity by affecting the weak-to-strong actomyosin
transition are likely to alter muscle function.
2.3 Two Parameters of Contractility: Unloaded Shortening
Velocity and Specific Force
The maximal unloaded shortening velocity (V

o
) is a mechanical parameter associated
with the rate of cross-bridge cycling. V
o
is directly related to, and dependent on,
the activity of myosin ATPase. ATPase is predominantly determined by the MHC
isoform because, as noted above, the MHC contains the catalytic site for ATPase
activity. For individual skeletal muscle fibers, V
o
varies with the MHC isoform with
the hierarchy for V
o
is type II > type I, with type IIb > IIx > IIa > I.
Muscle strength or the force-generating capacity varies directly with muscle (or
fiber) cross-sectional area, and thus the ratio of force to muscle (or fiber) size is
defined as specific force. Analysis of specific force allows for a comparison of the
intrinsic capacity of the contractile unit between samples or after an intervention,
even though muscle cross-sectional area may change.
2.4 Muscle Plasticity
Skeletal muscles and fibers are considered dynamic because they are capable of
changing contractile properties in response to altered functional demands or
changes in the pattern of recruitment. This plasticity is reflected by pronounced
changes in muscle and single fiber strength, endurance and contractility as a result
of an alteration in demand. Generally, increased contractile activities, e.g., chronic
stimulation, the removal of a synergist muscle, or progressive resistance exercise
training, are responsible for an increase in muscle strength. In contrast, decreased
81Age-Related Decline in Actomyosin Structure and Function
contractile activities, e.g., limb immobilization leads to a decrease in muscle
strength. The peripheral adaptation most often observed with a change in muscle
strength is an alteration in muscle mass.

3 Age-Related Changes in Contractility
Aging is associated with a progressive decline of muscle mass, strength, and qual-
ity, a condition often described as sarcopenia. The prevalence of sarcopenia in older
adults is about 25% under the age of 70 years, and increases to 40% in adults 80
years or older (Baumgartner et al. 1998). Sarcopenia is a risk factor for frailty, loss
of independence, and physical disability (Roubenoff 2000). The mechanisms
responsible for the age-dependent contractile dysfunction are multi-factorial,
resulting in altered cellular homeostasis (Prochniewicz et al. 2007; Thompson
2009). Having an understanding of the mechanisms contributing to altered cellular
homeostasis leading to muscle dysfunction (e.g., weakness) provides a foundation
for the testing of potential interventions (e.g., exercise).
In view of the many investigations elucidating the mechanisms responsible for
muscle dysfunction, several points require attention. (1) Age-related deterioration
of contractility is progressive, with the extent of changes being variable, depending
on the muscle and age of the subjects. (2) The multitude of experimental approaches
(e.g., permeabilized fiber preparation, intact fiber preparation, isolated proteins)
enables specific hypotheses to be tested about the underlying mechanisms. (3) The
experimental results from rodent studies, over the past 30 years, parallel the find-
ings from human biopsy studies.
3.1 Single Fiber Contractility
Single skeletal muscle fiber contractility includes an array of contractile parameters
(i.e., force-generating capacity, contraction velocity (V
o
), power output) that are
sensitive to the protein composition. It is possible to investigate single muscle fiber
contractility using the permeabilized or skinned muscle fiber preparation. The per-
meabilized fiber preparation is a fiber that does not have intact membranes, so force
generation and contraction velocity reflect directly the interactions of myosin and
actin, exclusive of other factors in excitation–contraction coupling (e.g., sarcoplasmic
reticulum Ca

2+
release).
Two principal contractile parameters, specific force (P
o
) and maximal unloaded
shortening velocity (V
o
), decrease progressively with age in studies using the per-
meabilized fiber preparation. The extent of deterioration depends on many factors,
such as the fiber type composition of the single fiber (i.e., myosin heavy chain
isoform type I or type II), the selected muscle (i.e., postural or phasic function), and
the age group (i.e., young adult, aged).
82 L.V. Thompson
Overall with progressive aging, skeletal fibers show a deficit in specific force
(P
o
) and in maximal unloaded shortening velocity (V
o
), independent of the myosin
heavy chain isoform (reviewed in Prochniewicz et al. 2007). It is important to note
that single skeletal fibers with myosin heavy chain type II show faster age-related
declines (or earlier) compared to single skeletal fibers with myosin heavy chain
type I, revealing an aging phenotype between the fiber types (Fig. 5). The reduction
in specific force (force generation normalized for cross-sectional area) with aging
suggests qualitative as well as quantitative deficiencies in myosin and/or actin
(defects in contractile protein). The decline in maximal unloaded shortening
velocity with aging suggests alterations in the ability of the myosin ATPase to
hydrolyze ATP.
The next questions to be answered, why do these occur? One possibility is that
the decline in specific force is due to an alteration in the number of cross-bridges

producing force, or in other words, it is possible that there is a reduction in the
fraction of myosin heads in the strong-binding structural state during contraction.
Thus, age-related structural or chemical changes in actin and myosin that affect
the weak-to-strong actomyosin transition are potential candidates contributing to
0
20
40
60
80
100
100% 50% 25%
type I
type II
Survival
Functional Performance (%)
Muscles and fibers
composed of MHC Type II
show age-related changes
at 50% survival rate.
Muscles and fibers composed
of either MHC Type II or MHC
Type I show age-related
changes at 25% survival rate.
Fig. 5 Skeletal muscle aging phenotype. Contractility or functional performance of muscles and
individual skeletal muscles, composed predominantly of type I (gray) or type II (black) myosin
heavy chain (MHC), show characteristic aging phenotypes across the lifespan (defined as percent-
age of survival). At 50% survival, the contractility (e.g., specific force, velocity of shortening) of
muscles and fibers composed of MHC type II show a greater reduction compared to muscle
(fibers) composed of MHC type I, demonstrating that type II fibers are very sensitive to the aging
process. At 25% survival, contractility is reduced in muscles (fibers) regardless of MHC isoform

composition, demonstrating a susceptibility of both MHC isoform types to aging
83Age-Related Decline in Actomyosin Structure and Function
age-related decline in specific force. In contrast to changes in structural properties
as a mechanism to explain force declines with aging, an age-related slowing of
myosin ATPase may explain the decline in unloaded shortening velocity.
The hypothesis that age-related deterioration of specific force involves structural
changes was suggested by a series of studies showing changes in myosin structure
can compromise muscle function. For example, oxidation of cysteine residues (thiols)
in myosin decreases force production, velocity, and ATPase activity (Perkins et al.
1997). Because thiols are not involved in the catalytic mechanism of myosin ATPase,
this strongly suggests that a structural perturbation in myosin occurs as a result of
oxidation, culminating in altered myosin function. Specific structural changes within
the myosin molecule have also been implicated in a variety of muscle disorders.
4 Age-Related Structural Changes in Muscle Protein Structure
4.1 EPR Spectroscopy
If age-related structural changes in myosin contribute to the decline in specific
force, it is important to be able to determine myosin structural states during con-
traction. The determination of protein structure requires experimental technology
with high resolution and specificity. Electron paramagnetic resonance (EPR) has
both high resolution and specificity needed to analyze purified proteins, as well as
large protein complexes and intact cells (reviewed in (Thomas et al. 2009). EPR is
a spectroscopic technique that detects and quantifies signals corresponding to dis-
tinct protein structures and motions (dynamics). Special extrinsic probes are used
to label proteins because EPR spectroscopy detects unpaired electrons, which are
not found on most stable proteins. The special extrinsic probes, termed spin-labels,
are stable nitroxide free radicals. The successful use of spin labels in the investiga-
tion of protein structure and dynamics usually requires the probe to be both small
and site-specific. Particularly, the attachment of the spin-labels to the protein of
interest is strategic and selective. Although the probes are attached to the protein,
the probes cause very little steric perturbations and do not perturb the function

of the protein.
In order for spectroscopic methods to test protein structure certain locations
within the protein complex (e.g., actin-myosin) must be probed specifically. Two
commonly used spin labels, maleimide and iodoacetamide derivatives, are specific
for cysteine residues (Cys). A very small number of Cys residues are reactive in a
given protein and Cys is a fairly uncommon amino acid. Recently, site-directed
mutagenesis makes it possible to label virtually any amino acid residue by site-
directed labeling. Site-directed spin labeling is also achieved by removing reactive
Cys residues by mutation and introducing single Cys at the site of choice.
An important advantage of spin-labels is that high-quality EPR data can be
obtained with these probes under physiological or near – physiological conditions,
84 L.V. Thompson
on small amounts of protein, and within a time frame of seconds (e.g., Lowe et al.
2001; Zhong et al. 2006). This is obviously advantageous for studying the relation-
ship between protein structural dynamics and physiological functions. Depending
on probe choice, sample preparation, labeling procedures, sample orientation, and
other conditions of the experiment, spin-label probes report protein orientation
(structural state), rotational motion, conformational changes, or information about
the local environment of the protein. The EPR spectrum offers the possibility of
high resolution, because different probe behaviors give rise to distinct spectral lines
that can be resolved and quantified.
In principle, EPR involves the absorption of light by the biological sample (e.g.,
muscle fibers; Fig. 6; Thompson et al. 2001). The light source is a microwave klys-
tron or diode in an EPR spectrometer. Microwaves (l) are sent through a waveguide
buffer
flow
+
waveguide
cavity
force

transducer
magnet
magnet
Spin-label
probe
Fiber
bundle
EPR
spectrometer
Spectrum
O
O
O
N
N
0.5 mm
5 cm
strong weak
Fig. 6 Schematic of the major components of a muscle fiber EPR experiment. EPR with site-
specific spin labeling is the only method that is able to directly determine the fraction of myosin
heads in the weak-binding and strong-binding structural states during a muscle contraction. EPR
has shown that force is produced when the myosin head changes from a weak-binding structure
(red), in which the catalytic domain is dynamically disordered, to a strong-binding structure
(black), in which the head is rigid. A nitroxide spin-label probe (far left) is reacted with a small
bundle of permeabilized muscle fibers under conditions in which the probe is specific for SH-1 on
the myosin head. A capillary tube containing the labeled fibers is fixed in a resonant cavity per-
pendicular to the magnetic field (located between two magnets), with one end of the fiber bundle
attached to a force transducer and the other end stabilized to hold the fibers isometrically. The
cavity is custom-designed to allow buffers to flow over the fibers at a designated flow rate that
allows for adequate diffusion of substances (e.g., ATP and Ca

2+
). During muscle rigor, relaxation,
or maximal isometric contraction microwave energy (~10 GHz) is delivered into the cavity
through a waveguide and is absorbed by the unpaired electrons in the labeled fibers. The result is
a high resolution spectrum
85Age-Related Decline in Actomyosin Structure and Function
into a resonator that contains the sample (cavity). Within the resonant cavity, a
strong magnetic field induces a magnetic moment in the unpaired electrons (•) and
results in the absorption of microwave radiation, termed “resonance”. Next, the
magnetic field is scanned and an EPR spectrum is obtained (the derivative of the
absorption spectrum is plotted).
4.2 Myosin and EPR
EPR spectroscopy and site-directed spin labeling methods have been used consid-
erably for investigating myosin because EPR is extremely sensitive to myosin’s
structural changes and EPR’s high resolution permits the quantitative analysis of
myosin’s distinct structural states. These methods have allowed researchers in the
field of skeletal muscle physiology to probe previously unexplored domains of
muscle proteins, and to test and refine molecular models of muscle structure and
function (reviewed in Thomas et al. 2009).
One method of specifically detecting the movements of myosin cross-bridges
(structural information about a specific domain with myosin) is by using EPR
spectroscopy in combination with site-specific spin-label probes that are intro-
duced directly into the acto-myosin assembly of the sarcomere (to a distinct resi-
due within myosin) in permeabilized muscle fibers (discussed in the previous
section). The SH1–SH2 helix was among the first sites labeled covalently with
probes on myosin, because SH1 (Cys707) is the most reactive Cys residue in the
catalytic domain of the myosin head and can therefore be labeled specifically
with a wide range of thiol reagents. Thus, SH1 (Cys707) is the site on the myosin
cross-bridge most commonly used for labeling with spectroscopic probes. The
mobility of the iodoacetamide spin labels (IASLs), attached to SH1, is sensitive

to conformational changes near the myosin active site that occurs with ATP
binding, hydrolysis, and P
i
release.
The spin-label probes have been used to resolve and quantify distinct structural
states of myosin that occur with muscle contraction in skeletal muscle fibers
(reviewed in Thomas et al. 2009). The advantage of investigating muscle fibers
with intact contractile units is the ability to directly correlate muscle function with
protein (myosin) structure. Specifically, EPR has been used to show that the
myosin head has two primary structures: a weak-binding (to actin) structural state
that is dynamically disordered, in which no force is produced, and a strong-
binding (to actin) structural state, in which force is produced (Figs. 4 and 6). The
quantitative resolution by EPR of the weak-binding and strong-binding (pre- and
post-powerstroke) structural states of myosin in active muscle, coupled with
simultaneous measurement of muscle force, enables analysis of the coupling
between thermodynamics and structural mechanics within the muscle filament
lattice. Thus, the resolved EPR lines are correlated directly with intermediates in
the myosin ATPase cycle.