Sarcopenia Age-Related Muscle Wasting and Weakness: Mechanisms and Treatments P6 ppt

37
G.S. Lynch (ed.), Sarcopenia – Age-Related Muscle Wasting and Weakness,
DOI 10.1007/978-90-481-9713-2_3, © Springer Science+Business Media B.V. 2011
Abstract Remodeling of neuromuscular junctions (NMJs) and ensuing structural
and functional plasticity occurs with aging. Age-related changes result from
reductions in physical activity, loss of motor neurons, and decreased muscle fiber
size (sarcopenia). The properties of motor neurons and muscle fibers are precisely
matched. In addition, motor unit recruitment in a selective manner is a primary
mechanism by which the nervous system controls muscle contraction. Thus, it is
essential to consider motor unit (and muscle fiber) type in any age-related plasticity.
The following chapter examines changes in motor unit properties associated with
aging and how these affect structural and functional remodeling at NMJs.
Keywords Aging • Morphological adaptations • Motor units • Muscle fiber type
• Plasticity • Recruitment • Skeletal muscle
1 Introduction
The neuromuscular junction provides the sole link between a motor neuron and
muscle fibers. Within a motor unit (Fig. 1), the mechanical and biochemical proper-
ties of muscle fibers are relatively uniform, and it is clear that the motor neuron plays
an important role in influencing these properties through the neuromuscular junction.
This influence is imparted either through activity levels or nerve-derived trophic
factors (Mantilla and Sieck 2008; Delbono 2003). As a result, the mechanical and
metabolic properties of muscle fibers and motor neurons are precisely matched
(Burke et al. 1971; Sieck et al. 1989) – an essential feature of neuromotor control and
functional performance of a skeletal muscle across a range of physiological behaviors.
C.B. Mantilla and G.C. Sieck (*)
Departments of Physiology and Biomedical Engineering and Anesthesiology,
College of Medicine, Mayo Clinic, St. Marys Hospital, Joseph 4W-184,
200 First Street SW, Rochester, MN 55905, USA
e-mail: ;
Age-Related Remodeling of Neuromuscular

Junctions
Carlos B. Mantilla and Gary C. Sieck
38 C.B. Mantilla and G.C. Sieck
In most skeletal muscles, motor units exhibit considerable functional diversity in
terms of size, mechanical and fatigue properties (Burke et al. 1971; Sieck et al. 1989).
Accordingly, recruitment of specific motor unit types is a major mechanism in neural
control of muscle force generation and fatigue resistance (Clamann 1993).
1.1 Synaptic Plasticity
More than 60 years ago, Donald Hebb introduced a conceptual framework (Hebbian
Theory) to describe the basic mechanisms for changes in synaptic efficacy (synaptic
plasticity). Central to his theory was the observation that synaptic efficacy improves
when the fidelity between pre- and post-synaptic activity increases. Conversely,
when fidelity between pre- and postsynaptic activity is disrupted, synaptic transmis-
sion worsens. Synaptic plasticity has both structural and functional correlates.
For examples, structurally, there may be axonal terminal sprouting or retraction,
changes in the size and distribution of synaptic vesicle pools, and/or changes in the
FF
Type IIb fibers
MyHC
2B
FInt
Type IIx fibers
MyHC
2X
FR
Type IIa fibers
MyHC
2A
S
Type I fibers

MyHC
Slow
Motor Unit Types
Fig. 1 Motor units (i.e., a motor neuron and the muscle fibers it innervates) are classified based
on the mechanical and fatigue properties of muscle fibers. Four types are commonly described:
(1) slow-twitch, fatigue resistant (type S), (2) fast-twitch, fatigue resistant (type FR), (3) fast-
twitch, fatigue-intermediate (type FInt), and (4) fast-twitch, fatigable (type FF), which generally
correspond to the expression of specific myosin heavy chain (MyHC) isoforms in the muscle
fibers (type I fibers - MyHC
Slow
, type IIa fibers - MyHC
2A
, type IIx fibers - MyHC
2X
and type IIb
fibers - MyHC
2B
). Motor unit recruitment order is generally matched to their mechanical and
fatigue properties; thus, type S and FR motor units are recruited first and more often than type FInt
and FF units
39Age-Related Remodeling of Neuromuscular Junctions
extent of pre- and postsynaptic apposition and overlap. Functionally, synaptic plas-
ticity is reflected by enhanced evoked postsynaptic potentials, persistent changes in
presynaptic neurotransmitter release or postsynaptic excitability (long-term facilita-
tion or depression), and changes in safety factor for neurotransmission resulting in
either improved neurotransmission fidelity or neurotransmission failure.
1.2 Aging and Synaptic Plasticity
With aging and senescence, there is a decrease in muscle activity often accompanied
by unloading of limb muscle fibers. However, inactivity alone may not drive
synaptic plasticity at the neuromuscular junction if fidelity of neuromuscular

transmission (i.e., extent of correlation between pre- and postsynaptic activity) is
maintained. Other age-related changes may drive synaptic plasticity. For example,
an age-related loss of motor neurons amounts to denervation of some muscle
fibers, consequently there may be axonal sprouting of spared motor neurons and
re- innervation of muscle fibers and an increase in motor unit innervation ratio
(Gordon et al. 2004; Balice-Gordon 1997). Age-related muscle fiber atrophy (i.e.,
sarcopenia) is also associated with concomitant changes in neuromuscular junction
morphology, which may relate to removal of shared trophic influences (Vandervoort
2002; Delbono 2003). The effects of age-related inactivity, motor neuron loss and
sarcopenia all depend on motor unit and/or muscle fiber type (Macaluso and
De Vito 2004). Thus, it is likely that synaptic plasticity is a part of the normal
aging process necessary to maintain muscle performance.
2 Motor Unit Properties and Recruitment
The concept of the motor unit was introduced by Charles Sherrington in 1925 and
forms the cornerstone of neuromotor control. A motor unit comprises a motor neu-
ron and the group of muscle fibers it innervates (Fig. 1). In adult mammals, each
muscle fiber is innervated by only a single motor neuron, while each motor neuron
can innervate multiple muscle fibers. The number of muscle fibers innervated by a
motor neuron (innervation ratio) varies widely from very small innervation ratios
in hand and eye muscles (<10 fibers per motor neuron) to very large innervation
ratios in trunk and proximal limb muscles (>500 fibers per motor neuron).
Innervation ratio is inversely related to the fine control of force gradation with
motor unit recruitment. Together with average muscle fiber cross-sectional area,
innervation ratio determines the size of a motor unit and maximal force contributed
by the motor unit. The level of force contributed by a motor unit is also dependent
on the frequency of motor neuron discharge rate (frequency coding of force).
Force-frequency properties of muscle fibers comprising motor units vary depending
on contractile protein composition, which forms the basis of muscle fiber type clas-
sification (Fig. 1; see below).
40 C.B. Mantilla and G.C. Sieck

2.1 Motor Unit and Muscle Fiber Type Classification
Motor unit and muscle fiber type classification are concordant since they both
relate to the mechanical and fatigue properties of muscle fibers. Different muscle
fiber type classification schemes have been proposed, but the most commonly
accepted scheme is based on the expression of different myosin heavy chain
(MyHC) isoforms. Accordingly, in adult mammals, four muscle fiber types are
classified: (1) type I (fibers expressing MyHC
Slow
), (2) type IIa (fibers expressing
MyHC
2A
), (3) type IIx (fibers expressing MyHC
2X
) and (4) type IIb (fibers express-
ing MyHC
2B
). In single fiber studies, MyHC isoform expression has been shown
to correlate with maximum isometric force, Ca
2+
sensitivity (related to force at
submaximal activation underlying the force-frequency relationship), maximum
velocity of shortening, cross-bridge cycling rate, ATP consumption rate, mito-
chondrial volume density, and fatigue resistance (Geiger et al. 1999, 2000; Han
et al. 2001, 2003; Sieck et al. 2003).
Since motor units comprise a relatively homogenous group of muscle fibers,
classification of four motor unit types is based on the mechanical and fatigue
properties of their constituent muscle fibers: (1) slow-twitch, fatigue resistant
(type S; comprising type I fibers), (2) fast-twitch, fatigue resistant (type FR;
comprising type IIa fibers), (3) fast-twitch, fatigue-intermediate (type FInt; com-
prising type IIx fibers), and (4) fast-twitch, fatigable (type FF; comprising type

IIb fibers) (Fig. 1). As mentioned above, innervation ratio varies across muscles,
but within a muscle, innervation ratio is generally greater for type FInt and FF
motor units compared to type S and FR units. Muscle fiber size also varies
across muscles, but within a muscle type IIx and IIb fibers are generally larger
than type I and IIa fibers. Thus, there are differences in motor unit size across
muscles and within a muscle, but generally type FInt and FF motor units are
larger than type S and FR motor units. There are also differences in specific
force (i.e., force per unit cross-sectional area) of different muscle fiber and
motor unit types. Generally, type IIx and IIb fibers (type FInt and FF motor
units) have greater specific force than type I and IIa fibers (type S and FR motor
units). Consequently, because of their greater innervation ratio, larger fiber size
and greater specific force, type FInt and FF motor units contribute greater forces
than type S and FR units.
2.2 Motor Unit Recruitment
In muscles of heterogeneous muscle fiber type composition, motor unit recruitment
order is generally matched to their mechanical and fatigue properties; thus, type S
and FR motor units are recruited first followed by type FInt and FF units. In models
41Age-Related Remodeling of Neuromuscular Junctions
where this recruitment order was assumed and where the force contributed by each
motor unit type was known, it was predicted that the forces required during most
sustained motor behaviors (e.g., standing in the medial gastrocnemius (Walmsley
et al. 1978) or quiet breathing in the diaphragm muscle (Sieck and Fournier 1989))
could be accomplished by recruitment of only type S and FR motor units (Fig. 2).
In these models, recruitment of type FInt and FF motor units was required only
during high force, short duration motor behaviors (e.g., jumping in the medial gas-
trocnemius and coughing/sneezing in the diaphragm).
Airway occlusion
Fictive sneezing
Eupnea
Hypercapnia & Hypoxia

0
10
20
30
40
50
60
70
80
90
100
Recruitment of motor unit pool (%)
Force (%)
Type S
Type FR
Type FInt
Type FF
0102030405060708090 100
Fig. 2 Model of motor unit recruitment for the rat diaphragm muscle. Motor units were assumed
to be recruited in order: type S ® type FR ® type FInt ® type FF with complete activation of
one motor unit type before the next type is recruited. Data is derived from previous studies
reporting diaphragm muscle fiber type composition, force generated by type-identified fibers,
and innervation ratio in adult male rats (Miyata et al. 1995; Zhan et al. 1997; Geiger et al. 2000;
Sieck 1994). The relative force developed during different ventilatory (e.g., eupnea and hyper-
capnia & hypoxia) and non-ventilatory tasks (e.g., airway occlusion and fictive sneezing). Based
on the model, the inspiratory effort necessary to accomplish ventilatory demands imposed during
eupnea requires recruitment of all of the type S motor units and some of the type FR motor units,
while chemical airway irritation (i.e., fictive sneezing) would result in recruitment of most
diaphragm motor units
42 C.B. Mantilla and G.C. Sieck

2.3 Aging Effects on Motor Unit Properties
Clearly, age affects the mechanical properties of muscle fibers and consequently motor
units. Generally muscles become weaker with age and this effect may reflect changes
in muscle fiber cross-sectional area, MyHC content per half-sarcomere, and/or specific
force. The cross-sectional area of type IIx and/or IIb fibers decreases with age
(Maxwell et al. 1973). This may be the result of motor neuron loss and consequent
denervation-induced atrophy (Xie et al. 2003). It may also reflect decreased neuromus-
cular activity, mechanical unloading or altered trophic influences (Delbono 2003).
MyHC content per half-sarcomere varies across muscle fiber types (Geiger et al. 2003,
2000), but does not appear to be affected by aging (Lowe et al. 2004b). However, with
aging there is an increase in the proportion of fibers co-expressing MyHC isoforms,
something that is relatively rare in young adults (Andersen et al. 1999). Specific force
decreases with age, and this effect is especially pronounced at type IIx and IIb muscle
fibers (i.e., type FInt and FF motor units) (Gosselin et al. 1994). Thus, muscle fiber
weakness appears to reflect the combined influence of decreased fiber cross-sectional
area and specific force. With respect to other mechanical properties of muscle fibers,
converging evidence indicates that maximum velocity of shortening, cross-bridge
cycling rate and ATP consumption rate are unaffected by aging across fiber types, but
there may be differences across muscles (Lowe et al. 2004a). Importantly, there
appears to be no age-related change in fatigability across muscle fiber types (Gonzalez
and Delbono 2001), although maximum oxidative capacity is reduced in type II fibers
of aged individuals (Proctor et al. 1995).
2.4 Aging Effects on Motor Unit Recruitment
Based on converging indirect evidence it appears that with aging, there is a decrease
in the number of type FInt and FF motor units due to the specific loss of these motor
neurons (Hashizume et al. 1988; Caccia et al. 1979; Ishihara et al. 1987; Hashizume
and Kanda 1995). This conclusion is based on the observation of a reduction in the
number of retrogradely labeled motor neurons which appears to be most pronounced
in fast-twitch hind limb muscles (Ishihara et al. 1987; Hashizume and Kanda 1995).
In the same studies, it was observed that there were fewer type II fibers (no distinction

was made between type IIa, IIx or IIb fibers) in hind limb muscles showing fewer
motor neurons. In separate studies that did not estimate the number of motor neurons,
selective reduction in the proportion of type IIx and IIb fibers was observed (Caccia
et al. 1979). Selective loss of type FF and FInt motor units is also indirectly supported
by the observation of an age-related increase in the proportion of type S and FR motor
units in the rat plantaris muscle (Pettigrew and Gardiner 1987; Pettigrew and Noble
1991). The underlying basis for a selective loss of motor neurons is not yet resolved,
but such an effect would definitely impact the ability to accomplish motor behaviors
that require generation of greater forces (Fig. 2). As a result of motor neuron loss,
some type IIx and IIb fibers would be denervated, and with subsequent reinnervation
43Age-Related Remodeling of Neuromuscular Junctions
by remaining motor axons (mostly those of type S and FR motor units), there may be
fiber type conversion as reflected by an increase in the proportion of fibers
co-expressing different MyHC isoforms (Larsson et al. 1991). Sprouting and rein-
nervation of adjacent muscle fibers should lead to an increase in motor unit innerva-
tion ratios. Indirect evidence for such an increase in innervation ratios stems from
analysis of changes in EMG during incremental force steps relative to the maximum
evoked EMG response (M-wave) (Galea 1996). Age-related changes in the specific
force of type IIx and IIb fibers together with the decrease in the overall proportion of
these motor unit types would tend to decrease the diversity of motor unit properties
within a muscle. An increase in the innervation ratio of type S and FR motor units
would result in increased force production by these units, but it is unclear whether this
increased force is required for the normal recruitment of these motor unit types (e.g.,
standing or quiet breathing). It is possible that an age-related increase in force genera-
tion by type S and FR motor units partially offsets any age-related effects on type FInt
and FF motor units, but it is unlikely that recruitment of type S and FR motor units
can completely compensate for the forces required during high-force generating
behaviors (e.g., jumping or coughing/sneezing). With aging, there appears to be a
selective preservation of mechanical properties of motor units required for low force,
sustained motor behaviors. In some cases, the advantage of such preservation is quite

obvious, e.g., recruitment of type S and FR motor units in the diaphragm muscle to
sustain ventilation or a similar recruitment of motor units in anti-gravity muscles to
sustain posture.
3 Structural Properties of Neuromuscular Junctions
The structural properties of neuromuscular junctions are matched to the functional
demands of muscle fibers such that within a motor unit type the structure of neuromus-
cular junctions is relatively uniform but there is considerable variability across differ-
ent muscle fiber types (Fig. 3). The matching of pre- and post-synaptic specializations
at the neuromuscular junction also depends on muscle fiber type. For example, presyn-
aptically, there are differences in the distribution and size of synaptic vesicle pools and
terminal surface area. Postsynaptically, there are differences in the number and depth
of junctional folds and apposition of subcellular organelles such as mitochondria.
Finally, the overlap of pre- and post-synaptic structures varies across fiber types.
3.1 Fiber Type Differences in Neuromuscular Junction
Structure
Within a muscle, neuromuscular junctions at type I and IIa fibers are smaller with less
complex branching patterns than those at type IIx and/or IIb fibers (Prakash and Sieck
1998; Mantilla et al. 2004; Prakash et al. 1995, 1996b; Sieck and Prakash 1997).
44 C.B. Mantilla and G.C. Sieck
However, it is difficult to extrapolate across muscles since neuromuscular junctions
at type I fibers in the soleus muscle are larger and more complex than neuromuscular
junctions at type IIx and/or IIb fibers in the extensor digitorum longus muscle (Reid
et al. 2003). Within a muscle, fiber size is an important determinant of neuromuscular
junction area and complexity. For example, in the rat diaphragm muscle, the area of
neuromuscular junctions among type I fibers varies directly with fiber cross-sectional
area (Prakash and Sieck 1998; Sieck and Prakash 1997).
Fiber type dependent differences in gross structural properties of neuromuscu-
lar junctions are also reflected at pre- and post-synaptic elements. For example,
both axon terminal and motor end-plate surface areas are ~75–90% greater at type
IIx and/or IIb fibers than at type I and IIa fibers in the rat diaphragm (Sieck and

Prakash 1997; Prakash et al. 1996b; Rowley et al. 2007; Mantilla et al. 2004). At
all muscle fibers, the surface area of axon terminals is smaller than their corre-
sponding motor end-plate and the extent of this difference varies across muscle
fiber types (Prakash et al. 1996b). For example, at type I diaphragm fibers, the
surface area of the presynaptic terminal more closely approximates that of
the motor end-plate, with nearly 95% overlap. By comparison, at type IIb fibers, the
presynaptic terminal only overlaps ~70% of the motor end-plate. These differ-
ences in the extent of overlap may reflect phenotypic differences in the ability of
nerve terminal branches to invade motor end-plate gutters during development
(Prakash et al. 1995) or remodeling (Prakash et al. 1996a, 1999). It is also possible
that the increased fragmentation of neuromuscular junctions at muscle fibers of
greater size results in greater branch termination limiting invagination of the axon
terminal into motor end-plate gutters. In either case, these differences in extent of
overlap may have significant physiological implications, impacting neuromuscular
transmission.
Fig. 3 Structural characteristics of a neuromuscular junction (NMJ) vary across muscle fiber
types. Pre-synaptic terminals and motor end-plates at the diaphragm muscle of young (6 months)
and old rats (24 months) were labeled with the neuronal ubiquitin decarboxylase PGP9.5 and
a-bungarotoxin, respectively (Prakash and Sieck 1998). Note the differences in size and complex-
ity (number and length of branches) across fiber types, with NMJs present at type I or IIa fibers
being smaller and less complex than those at type IIx and/or IIb fibers. With aging there is con-
siderable fragmentation and expansion of both pre- and post-synaptic elements
45Age-Related Remodeling of Neuromuscular Junctions
Fiber type differences in neuromuscular junction remodeling vary depending
on a number of factors including hormonal environment and activity. For
example, the areas of both pre- and postsynaptic elements of neuromuscular
junctions at type I diaphragm fibers decreased after 3 weeks of hypothyroidism
induced by propylthiouracil (Prakash et al. 1996a). In contrast, after 2 weeks of
diaphragm inactivity induced by either tetrodotoxin phrenic nerve blockade or
spinal cord hemisection at C

2
the areas of both pre- and postsynaptic elements
of neuromuscular junctions at type IIx and/or IIb diaphragm fibers increased
while those at type I fibers decreased (Prakash et al. 1999). At type IIx and/or
IIb fibers, the extent of overlap between pre- and postsynaptic elements of the
neuromuscular junction increased to ~90% after 2 weeks of diaphragm inactiv-
ity induced by tetrodotoxin phrenic nerve blockade or spinal cord hemisection
at C
2
. Surprisingly, the similar structural changes induced by tetrodotoxin
phrenic nerve blockade and spinal cord hemisection at C
2
yielded markedly dif-
ferent effects on neuromuscular transmission. Following inactivity induced by
spinal cord hemisection at C
2
neuromuscular transmission with repetitive acti-
vation was markedly improved, whereas there was substantially greater neuro-
muscular transmission failure following tetrodotoxin phrenic nerve blockade.
These functional differences are closely related to ultrastructural differences at
the neuromuscular junction that form the basis of neuromuscular transmission
(see below).
3.2 Ultrastructural Properties of Presynaptic Terminals
The total number of synaptic vesicles undergoing repeated cycles of endo- and
exocytosis (i.e., cycling) is greater at type IIx and/or IIb fibers compared to type I
and IIa fibers (Mantilla et al. 2004, 2007; Rowley et al. 2007). Ultrastructurally,
synaptic vesicles at presynaptic terminals segregate into a pool of vesicles docked
at specialized sites for neurotransmitter release – active zones – i.e., readily releas-
able, a pool immediately adjacent to active zones (within 200 nm) and a more
distant, reserve pool (Sudhof 2004). Consistent with greater overall size of the

cycling synaptic vesicle pool size, the densities of synaptic vesicles in both the
immediately adjacent pool and the reserve pool are greater at presynaptic termi-
nals of type I and IIa fibers compared to type IIx and/or IIb fibers. The size
(length) and distribution of individual active zones does not vary across presynap-
tic terminals at the different fibers types (Fig. 4). Similarly, the number of synaptic
vesicles docked at each active zone (i.e., readily releasable) is consistent across
fiber types (Rowley et al. 2007). However, fiber type differences in presynaptic
terminal surface area yield greater total number of active zones per presynaptic
terminal at type IIx and/or IIb fibers than at type I and IIa fibers, and thus, a greater
total number of synaptic vesicles in the readily releasable pool at type IIx and/or
IIb fibers compared to type I and IIa fibers (Mantilla et al. 2004; Rowley et al.
2007). Consistent with these ultrastructural properties, quantal release at type IIx