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

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

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: mantilla.carlos@mayo.edu; sieck.gary@mayo.edu

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

MyHC2B

FInt

Type IIx fibers

MyHC2X

FR

Type IIa fibers

MyHC2A

S

Type I fibers

MyHCSlow

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) twitch, fatigue resistant (type FR), (3) fast-twitch, fatigue-intermediate (type FInt), and (4) fast-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 - MyHCSlow, type IIa fibers - MyHC2A, type IIx fibers - MyHC2X and type IIb fibers - MyHC2B) 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

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 MyHCSlow), (2) type IIa (fibers expressing MyHC2A), (3) type IIx (fibers expressing MyHC2X) and (4) type IIb (fibers express-ing MyHC2B) In single fiber studies, MyHC isoform expression has been shown

to correlate with maximum isometric force, Ca2+ 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; comcom-prising 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

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 (%)

Type S

Type FR

Type FInt

Type FF

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

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 innervainnerva-tion 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 differdiffer-ences 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

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

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 C2 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 C2 Surprisingly, the similar structural changes induced by tetrodotoxin phrenic nerve blockade and spinal cord hemisection at C2 yielded markedly dif-ferent effects on neuromuscular transmission Following inactivity induced by spinal cord hemisection at C2 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