Ageing Research Reviews 67 (2021) 101305

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Ageing Research Reviews

journal homepage: www.elsevier.com/locate/arr

Review

The emerging role of the sympathetic nervous system in skeletal muscle

motor innervation and sarcopenia
Osvaldo Delbono a, b, c, d, *, Anna Carolina Zaia Rodrigues a, d, Henry Jacob Bonilla a,
Maria Laura Messi a
a
Department of Internal Medicine, Section on Gerontology and Geriatric Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA
b
Department of Internal Medicine, The Neuroscience Program, Wake Forest School of Medicine, Winston-Salem, NC, USA
c
Department of Internal Medicine, The Comprehensive Cancer Center, Wake Forest School of Medicine, Winston-Salem, NC, USA
d
Department of Internal Medicine, The Sticht Center for Healthy Aging and Alzheimer’s Prevention, Wake Forest School of Medicine, Winston-Salem, NC, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Examining neural etiologic factors’role in the decline of neuromuscular function with aging is essential to our
Skeletal muscle understanding of the mechanisms underlying sarcopenia, the age-dependent decline in muscle mass, force and
Sarcopenia power. Innervation of the skeletal muscle by both motor and sympathetic axons has been established, igniting
Motoneuron
interest in determining how the sympathetic nervous system (SNS) affect skeletal muscle composition and
Autonomic nervous system
Sympathetic nervous system
function throughout the lifetime. Selective expression of the heart and neural crest derivative 2 gene in peripheral
Neuromuscular junction SNs increases muscle mass and force regulating skeletal muscle sympathetic and motor innervation; improving
Denervation acetylcholine receptor stability and NMJ transmission; preventing inflammation and myofibrillar protein
Aging degradation; increasing autophagy; and probably enhancing protein synthesis. Elucidating the role of central SNs
will help to define the coordinated response of the visceral and neuromuscular system to physiological and
pathological challenges across ages.
This review discusses the following questions: (1) Does the SNS regulate skeletal muscle motor innervation?
(2) Does the SNS regulate presynaptic and postsynaptic neuromuscular junction (NMJ) structure and function?
(3) Does sympathetic neuron (SN) regulation of NMJ transmission decline with aging? (4) Does maintenance of
SNs attenuate aging sarcopenia? and (5) Do central SN group relays influence sympathetic and motor muscle
innervation?

Introduction
Age-dependent decline in muscle mass, force and power, termed
sarcopenia (Rosenberg, 1989), impairs mobility to increasing the risk of
falls, institutionalization, comorbidity, and premature death (Larsson
et al., 2019; Tieland et al., 2018). In addition to impairing quality of life
in the adult population, sarcopenia is also a major risk factor for many
age-associated diseases (Chin et al., 2013; DeAndrade et al., 2018; Landi
et al., 2012; Maeda and Akagi, 2016; Nishiguchi et al., 2016; Srikanthan
et al., 2010; Tournadre et al., 2019; Upadhya et al., 2015). Sedentary
people lose 0.5 percent of lean muscle mass every year between age 25
and 60; the rate doubles to about 1 percent after 60, and doubles again
each decade thereafter (Lynch et al., 1999). Once muscle mass in older
adults falls below two standard deviations of the mean of young controls
and gait speed falls below 0.8 m/s, a clinical diagnosis of sarcopenia is
reached (Studenski et al., 2011). Although, the average population un­
dergoing sarcopenia varies according to the nutritional status and
physical activity, more than 50 % of people over 80 undergo sarcopenia
(von Haehling et al., 2010), and all others exhibit some degree of muscle
atrophy and loss of muscle function (Larsson et al., 2019). Those with
sarcopenia subjects are not necessarily frail; sarcopenia is about twice as
common as frailty (von Haehling et al., 2010).
Physiological and pathological studies show that innervation and
systemic factors exert a strong influence on skeletal muscle mass,
composition and function (Delbono, 2003; Larsson et al., 2019). Sar­
copenia is associated with: (a) excitation-contraction uncoupling (Del­
bono, 2011a; Delbono et al., 1995; Wang et al., 2002), which diminishes
muscle specific force (force normalized to cross-sectional area)

* Corresponding author at: Wake Forest School of Medicine, Medical Center Boulevard, Department of Internal Medicine, Gerontology and Geriatric Medicine,
Winston-Salem, NC 27157, USA.
E-mail address: odelbono@wakehealth.edu (O. Delbono).

https://doi.org/10.1016/j.arr.2021.101305
Received 2 December 2020; Received in revised form 6 January 2021; Accepted 15 February 2021
Available online 18 February 2021
1568-1637/© 2021 Elsevier B.V. All rights reserved.
O. Delbono et al.
(Gonzalez et al., 2000a, b; Wang et al., 2000); (b) decreased number of
muscle fibers as well as fiber atrophy (Dutta, 1997; Lexell, 1995); (c)
changes in fiber type (Frontera et al., 2000; Larsson et al., 1991); (d)
diminished muscle power related to decreased maximal isometric force
and slower sliding of actin on myosin (Brooks and Faulkner, 1994; Hook
et al., 1999); (e) impaired recovery after eccentric contraction (Faulkner
et al., 1993); and (f) deposit of intra- and extra-myocellular lipids with
consequent changes in skeletal muscle quality (Choi et al., 2012). Motor
and/or sympathetic muscle denervation trigger and/or exacerbate some
of these processes.
Explanations for these age-dependent changes in muscle properties
include: alterations in myofiber calcium homeostasis (Delbono, 2011b;
Jimenez-Moreno et al., 2008; Tang et al., 2011), decreased muscle
loading associated with sedentary lifestyle, (Landi et al., 2014; Tseng
et al., 1995) oxidative damage (Jackson, 2016; Jang et al., 2010;
Leeuwenburgh et al., 1998; Vasilaki et al., 2017; Weindruch, 1995),
impaired mitochondrial quality control (Picca et al., 2019),
age-dependent decrease in insulin-like growth factor-1 (IGF-1) expres­
sion or tissue sensitivity (Owino et al., 2001; Renganathan et al., 1997;
Schiaffino et al., 2013), hormonal and metabolic alterations (Kamwa
et al., 2020; Vitale et al., 2016), impaired autophagy (Masiero et al.,
2009; Sakuma et al., 2016; White et al., 2016; Wokke et al., 1990),
accumulation of senescence cells (Tchkonia et al., 2013; Zhu et al.,
2014), inflammation (Ferrucci et al., 1999, 2002; Tchkonia et al., 2013),
decline in satellite cell proliferation (Garcia-Prat et al., 2016; Hwang
and Brack, 2018), and loss of neural control in skeletal muscle (Delbono,
2003). Unfortunately, some of these mechanisms are technically diffi­
cult to test in the whole organism, particularly in mammalian species.
Pharmacological interventions produce numerous side effects, have
yielded inconsistent results, and their rationales have been challenged
(Fry et al., 2015; Jackson, 2016; Larsson et al., 2019; Ristow, 2014;
Sakellariou et al., 2017; Wang and Hekimi, 2015). Still, the interaction
between nerve and skeletal muscle is clearly crucial for both tissues to
survive and function adequately throughout life (Delbono, 2003; Payne
et al., 2006b). Does muscle atrophy and weakness result from primary
neural or muscular etiologic factors or a combination? Does
age-dependent maintenance of sympathetic neuron transcription fac­
tors, particularly the heart and neural crest derivative 2 (Hand2) (Hen­
dershot et al., 2008; Rodrigues et al., 2020; Stanzel et al., 2016),
attenuate motor denervation? Examining neural etiologic factors’ role in
the decline of neuromuscular function with aging is essential to our under­
standing of the mechanisms underlying sarcopenia.
This review focuses on how aging influences skeletal muscle sym­
pathetic and motor innervation and how their alteration mediates sar­
copenia pathogenesis. It discusses basic aspects of muscle innervation,
even at a young age, that are incompletely understood. This review is
organized to consider the following questions: (1) Does the sympathetic
nervous system (SNS) regulate skeletal muscle motor innervation? (2)
Does the SNS regulate presynaptic and postsynaptic neuromuscular
junction (NMJ) structure and function? (3) Does sympathetic neuron
regulation of NMJ transmission decline with aging? (4) Does mainte­
nance of sympathetic neurons attenuate aging sarcopenia? and (5) Do
central sympathetic neuron group relays influence sympathetic and
motor muscle innervation?
1. Does the SNS regulate skeletal muscle motor innervation?
Relationship between sarcopenia and the dual muscle
innervation
Sarcopenia is characterized by heterogeneous muscle fiber size and
myosin heavy-chain composition (Schiaffino, 2018; Schiaffino and
Reggiani, 1994), selective myofiber atrophy, myofiber grouping, and
progressive NMJ degeneration, leading to complete or partial synapse
denervation, and Schwann cell depletion (Delbono, 2003; Li and
Thompson, 2011; Wang et al., 2005). Despite its relevance to the onset
and outcome of sarcopenia, the etiology of age-related skeletal muscle
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Ageing Research Reviews 67 (2021) 101305
denervation remains unknown (Delbono, 2011a). Several groups have
reported skeletal muscle denervation and reinnervation and motor unit
remodeling or loss in aging rodents or humans (Doherty et al., 1993;
Einsiedel and Luff, 1992; Hashizume et al., 1988; Johnson et al., 1995;
Kanda and Hashizume, 1989, 1992; Larsson and Ansved, 1995; Larsson
et al., 1991, 2019; Larsson and Edstrom, 1986; Zhang et al., 1996).
Motor-unit remodeling leads to changes in fiber-type composition (Pette
and Staron, 2001; Schiaffino and Reggiani, 1994, 1996).
During development, muscle fiber phenotype is determined by in­
teractions among subpopulations of ventral spinal cord motor neurons
that activate contraction at rates ranging from 10 (slow fibers) to 100
(fast-fatigue resistant) to 150 Hz (fast-fatigue sensitive) (Buller, 1960a,
b; Greensmith and Vrbova, 1996). During aging, motor-unit remodeling
appears to involve denervation of fast muscle fibers and their reinner­
vation by axonal sprouting from slow fibers (Frey et al., 2000; Kadhir­
esan et al., 1996; Larsson, 1995; Lexell, 1995). When denervation
exceeds reinnervation, that population of muscle fibers becomes atro­
phic and non-functional. Although denervation contributes to skeletal
muscle atrophy and functional impairment with aging (Larsson and
Ansved, 1995), its time course, and prevalence in human and animal
models of aging remain undetermined. Combining contraction force
recordings with muscle immunostaining for the neural cell-adhesion
molecule (NCAM) protein, a marker of fiber denervation and reinner­
vation in rat studies showed that the old have significantly more
denervated fibers than the young (Andersson et al., 1993; Gosztonyi
et al., 2001). Moreover, the area of denervated fibers, detected by pos­
itive staining with NCAM antibodies, accounts for a significant fraction
of the decline in specific force (Urbancheck et al., 2001). Human skeletal
muscle studies confirm NCAM’s usefulness as a denervation/ reinner­
vation marker (Biral et al., 2008; Messi et al., 2016; Sonjak et al., 2019).
We reported that muscle denervation in aging mice is more extensive
than previous functional and structural assays predicted. Two sodium
channel isoforms are expressed in skeletal muscle, the TTX-sensitive
Nav1.4 or SkM1 and the TTX-resistant Nav1.5 or SkM2 (Kallen et al.,
1990; Trimmer et al., 1989). The SkM2 mRNA level is highest in early
development, when TTX-insensitive channels predominate, but declines
rapidly with age as SkM1 mRNA increases; SkM2 mRNA is not detect­
able in normally innervated adult skeletal muscle but increases greater
than 100-fold after denervation; rat cardiac muscle has abundant SkM2
mRNA but no detectable SkM1 message (Kallen et al., 1990). To assess
the extent of denervation in aging rodents, we combined electrophysi­
ological and immunohistochemical assays to detect the expression of
TTX-resistant Nav1.5 in flexor digitorum brevis muscles from
young-adult and senescent mice. Sodium current density measured with
the macropatch cell-attached technique did not show significant dif­
ferences between muscle fibers from young and old mice. The TTX
dose-response curve, using the whole-cell voltage-clamp technique,
showed three populations of fibers in senescent mice: one similar to fi­
bers from young mice (TTX sensitive); another similar to fibers from
experimentally denervated muscle (TTX resistant); and an intermediate
group. Partially and fully denervated fibers made up approximately 50
percent of the total tested, which aligns with the percentage of fibers
positive for the Nav1.5 channel, as shown by specific immunostaining
(Wang et al., 2005) Discrepant results related to the time course, loca­
tion, and extent of denervation in rodents (Hendrickse et al., 2018) can
be accounted for by the age of the animals and the denervation and
immunofluorescence techniques used. These studies revealed that new
technical approaches are needed to accurately assess muscle denervation,
which, although subtle, can be devastating over time (Delbono, 2003).
Increased frequency of hybrid fiber types and quantification of myosin
heavy chain (MHC) composition in aging skeletal muscle
Since innervation regulates MHC expression (Schiaffino and
Reggiani, 1996), muscle morphometric analysis of MHC predominance
is critical to understanding the myofiber/motoneuron relationship in
O. Delbono et al.
health and disease (Lefeuvre et al., 1996). Determining fibers’ shapes,
sizes, and arrangements elucidates muscle tissue morphology and
overall health (Gropp, 2017), while immunohistochemistry (IHC) using
MHC antibodies is a widely accepted approach to examine skeletal
muscle fiber type composition (Bloemberg and Quadrilatero, 2012).
However useful for compositional studies of larger portions of the body,
computed tomography (CT) scan, magnetic resonance imaging (MRI),
and dual-energy X-ray absorptiometry cannot visualize the microscopic
structural features of skeletal muscle. Performed with the skill needed to
correctly interpret and generate reproducible results, IHC offers the best
combination of molecular and microscopic morphological information
for the analysis of skeletal muscle.
Both semiautomatic and automatic approaches have been developed
to aid IHC analysis (Bergmeister et al., 2016; Briguet et al., 2004; Lau
et al., 2018; Mayeuf-Louchart et al., 2018; Miazaki et al., 2015; Pertl
et al., 2013; Smith and Barton, 2014; Wen et al., 2018); but aging
skeletal muscle poses difficulties that young, healthy muscle tissue does
not. Failing autophagy leads to intracellular protein deposits (Jokl and
Blanco, 2016). Aging skeletal muscle usually displays some degree of
atrophy; more fibers express more than one MHC (hybrid); interstices
are infiltrated with fat cells, lipofuscin, and fibrous tissue, and angulated
small myofibers disperse heterogeneously throughout the tissue (Dis­
tefano and Goodpaster, 2018; Hepple, 2018; Purves-Smith et al., 2014).
These characteristics can affect the efficacy of muscle analysis programs
that rely on tissue homogeneity.
We designed a reliable, user-friendly, and accessible approach to
quantifying basic muscle morphometric parameters that does not
require computer experts or statisticians (Bonilla et al., 2020). It em­
phasizes image thresholding and editing capability to properly quantify
hybrid fibers and to identify muscle fibers, regardless of their degree of
atrophy, respectively (Bonilla et al., 2020).
Plasticity at the aging neuromuscular junction
NMJ formation involves a number of resident molecules, such as low-
density lipoprotein (LDL)-related receptor protein 4 (LRP4) (Messéant
et al., 2017; Weatherbee et al., 2006), agrin (differentiation) (Ruegg and
Bixby, 1998), Dok-7 (synaptogenesis) (Okada et al., 2006), and
muscle-specific kinase (MuSK) (synapse maintenance) (Koneczny et al.,
2014), and others. MuSK participates in three signaling pathways:
Agrin-LRP4, Wnt, and MuSK-BMP, via distinct domains. Although ge­
netic studies have elucidated the role these proteins play in NMJ
structure and function, how they contribute to its age-associated func­
tional decline remains ill-defined (Fish and Fallon, 2020).
Age-associated structural and functional alterations of the neuromus­
cular junction such as instability of neuromuscular junction have been
reported (Balice-Gordon, 1997; Khan et al., 2016; Payne and Delbono,
2004; Payne et al., 2006a; Rodrigues et al., 2018; Valdez et al., 2010).
The events leading to denervation in aging mammals are less understood
than the formation and activity-dependent editing of neuromuscular
synaptic connections (Personius and Balice-Gordon, 2000). Activity has
been shown to play a crucial role (Personius and Balice-Gordon, 2000),
prompting the interesting hypothesis that a similar mechanism affects
senescent mammals. While exercise and calorie restriction attenuate
NMJ spatial distortion in aging mice and humans (Messi et al., 2016;
Valdez et al., 2010), the levels required are highly variable and difficult
to sustain. Such interventions must be personalized and closely
monitored.
Nerve terminal remodeling during the period of target absence does
not adequately explain the subsequent changes to the NMJ. Mechanisms
for remodeling the synapse include failure of the regenerating muscle
fiber to contact the old basal lamina and nerve terminal, growth of the
nerve terminal and its glia toward the regenerating fiber, and remod­
eling of the initial contact as the motor nerve terminal becomes varicose
(Li and Thompson, 2011).
In skeletal muscles from aging rodents, motor denervation causes
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reorientation of costameres (rib-like structures formed by dystrophin
and β-dystroglycan) (Bezakova and Lomo, 2001), proliferation of triadic
membranes (Salvatori et al., 1988), reduction of voltage-gated calcium
channels, and alterations to the sarcoplasmic reticulum Ca2+ release
channel. (Delbono, 1992; Delbono et al., 1997; Delbono and Stefani,
1993; Wang et al., 2000). Costameric proteins transmit lateral forces and
provide structural integrity in contracting mechanically loaded muscle
fibers (Straub and Campbell, 1997). Along with muscle activity, muscle
agrin at a concentration two orders of magnitude lower than the effec­
tive concentration of neural agrin, regulates the organization of cyto­
skeletal proteins in skeletal muscle fibers (Bezakova and Lomo, 2001).
These molecular changes in aging muscle should be explored (Taetzsch
and Valdez, 2018). Compared to young and middle-aged mice, aging
C57BL/6 mice show an increase in agrin, calcitonin gene-related pep­
tide, growth associated protein 43, fibroblast growth factor binding
protein 1, and transforming growth factor-β1at the NMJs of the tibialis
anterior, soleus, extensor digitorum longus, and gracilis muscles as ev­
idence of increased development, plasticity, and maintenance of NMJ
signaling with aging (Blasco et al., 2020).
The studies reported above strongly associate neural alterations with
the onset and gradual decline of aging skeletal muscle function. In­
terventions focused on motor neurons, their axons, and associated non-
neuronal cells, and the NMJ may slow or even reverse age-related im­
pairments of skeletal muscle.
Dual innervation of the skeletal muscle
Unlike the skeletal motor system, which innervates striated muscles,
the autonomic nervous system (ANS) innervates the smooth muscle
organs and tissues (Powley, 2008). Although skeletal muscle sympa­
thetic muscle innervation was described over a century ago (Boeke,
1909); myofiber sympathetic innervation remained unexplored, prob­
ably due to technical limitations. Sympathetic axons travel through the
peripheral nervous system together with motor and sensory axons and
then surrounding blood vessels (Eichmann and Brunet, 2014; Wang
et al., 2020a). to reach the muscle fiber (Griffin and Thompson, 2008).
Sympathectomy and sympathetic neuron-labeling experiments using
the monosynaptic retrograde tracer β-subunit of cholera toxin (CTβ)
helped to elucidate the innervation of mouse hindlimb muscles by par­
avertebral sympathetic ganglia and ventral horn motoneurons (Card and
Enquist, 2012; Kerman et al., 2003; Rodrigues et al., 2018). For a long
time, sympathetic innervation of skeletal muscle was thought to be
restricted to the blood vessels until it was observed in the intra- and
extrafusal fibers of several muscles (Barker and Saito, 1981). This
finding sparked researchers’ interest in the role of postganglionic sym­
pathetic neurons and their neurotransmitter(s) in myofiber composition,
trophism, and function. Even at birth, sympathetic innervation and
interaction with NMJs in hindlimb, diaphragm, and levator auris longus
muscles is extensive and, in the extensor digitorum longus muscle, in­
creases until adulthood (Straka et al., 2018).
The ANS coordinates homeostasis through sympathetic and para­
sympathetic outflows and the enteric nervous system (Robertson et al.,
2012). The SNS regulates the function of many tissues, and its genetic,
autoimmune, or degenerative alteration results in a variety of clinical
disorders affecting the brain, spinal cord, and/or nerve structure and
function. While it also plays a role in skeletal muscle regeneration after
injury (Beitzel et al., 2007) and the ability to perform short intensive
tasks like the Wingate test (Le Panse et al., 2007), its deterioration with
age results in incapacitating symptoms (Delbono, 2003; Gonzalez-Freire
et al., 2014; Lipsitz and Novak, 2008). Its functional impairment and the
resulting muscle weakness are manifestations of Alzheimer’s disease
(Jensen-Daham et al., 2015) and such synucleinopathies as Parkinson’s
disease, dementia with Lewy bodies, multiple system atrophy, and pure
autonomic failure (Askmark et al., 2001; Cano-Jaimez et al., 2010;
Femminella et al., 2014; Janig, 2012; Kandinov et al., 2011; Low and
Bennarroch, 2008; Mu et al., 2013; Munver and Volfson, 2006; Okamoto
O. Delbono et al.
and Biaggioni, 2012; Ooi et al., 1997; Orimo et al., 2007; Tachi et al.,
1995). Muscle weakness associated with autonomic dysfunction is also
obvious in adrenal insufficiency, Lambert-Eaton myasthenic syndrome,
and chronic fatigue syndrome (Beitzel et al., 2007; Lindquist and Stan­
gel, 2011; O’Suilleabhain et al., 1998). Aging produces several changes
in the ANS that may impair adaptations to common physiologic stressors
and increase the risk of developing diseases that further harm autonomic
function.
We used a microsurgical approach to muscle sympathetic denerva­
tion to define the role of the SNS in regulating NMJ stability and func­
tion in adult mice (Khan et al., 2016; Rodrigues et al., 2019). A decrease
in synaptic vesicle release and quantum content indicates that motor
axon function is impaired. Next, to determine how sympathetic and
motor axons in the spinal nerve communicate under normal and path­
ological conditions, we examined whether sympathectomy alters mye­
lination, myelin thickness, and/or neurofilament (NF) phosphorylation
in large axons like those in motor neurons. We found increased variation
in myelin thickness, probably a result of Schwann cell reprogramming
(Jessen and Mirsky, 2016). Myelinating Schwann cells modulate NF
phosphorylation, axonal caliber, and slow axonal transport (de Waegh
et al., 1992). Although our analysis at the sciatic-tibio-peroneal nerve
showed no downregulation of genes encoding axonal myelination, we
found a reactive increase in neurotrophin gene transcription. NFs are
abundant in motor and other neurons with large-diameter axons (Ohara
et al., 1993; Sakaguchi et al., 1993). The mechanism underlying cross­
talk between sympathetic and motor axons remains unclear. The inter­
action of both axons with dephosphorylated NFs, revealed by
transmission electron microscopy, may interfere with microtubules’
transport of enzymes and peptide precursors (Purves et al., 2011).
The influence of the SNS on muscle motor innervation and
Fig. 1. Model of SNS regulation of the NMJ. The SNS regulates NMJ motor axon vesicle release and postsynaptic AChR stability through the Gαi2-Hdac4-myogenin-
MuRF1-miR-206 pathway. (Rodrigues et al., 2018) AChR: acetylcholine receptor; DRG: dorsal root ganglion; Hdac4: histone deacetylase-4; IML: intermediolateral
column; NA: noradrenaline.
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acetylcholine receptor stability is extensive. It regulates NMJ trans­
mission, maintains optimal Gαi2 expression, and prevents any increase
in histone deacetylase 4 (Hdac4), myogenin, the muscle RING-finger
protein-1 MuRF1 (a.k.a. TRIM63), and muscle-specific microRNA-206
(miR-206). The SNS also prevents elevations in MuRF1 levels, muscle
atrophy, and maintains postsynaptic membrane acetylcholine receptor
(AChR) levels (Rodrigues et al., 2018), as depicted in Fig. 1.
After sympathectomy, levels of the neurotransmitter noradrenaline
(NA) decreased 80 percent or more in soleus and EDL muscles, while
plasma catecholamines were not affected (Silveira et al., 2014), which
indicates that the surgical removal of lumbar paravertebral sympathetic
ganglia did not prevent the adrenal gland medulla from releasing
adrenaline into the bloodstream. Since the β2-AR (adrenergic receptor)
is a heterotrimeric guanine nucleotide-binding protein (G pro­
tein)-coupled receptor, and muscle NA levels and receptor activity
decrease with sympathectomy (Silveira et al., 2014), we examined
whether sympathectomy led to decreased Gαi2 activation, a mechanism
postulated to activate myogenin (Mei et al., 2011). Minetti et al. reported
that Gαi2 promotes skeletal muscle hypertrophy, myoblast differentia­
tion, and muscle regeneration, indicating that receptors that act through
Gα(i2) might be targets to prevent skeletal muscle wasting (Minetti
et al., 2014).
We found Gαi2 significantly decreased in hindlimb mouse muscles
after sympathectomy, and that restoring it using a human Gαi2 (Q205 L)
mutant, subcloned into an adeno-associated viral vector, prevented
decreases in membrane AChR levels and increases in MuRF1, Hdac4,
myogenin, and β2-AR expression. These results indicate that increasing
muscle levels of Gαi2 prevents skeletal muscle motor denervation (Rodrigues
et al., 2018).
O. Delbono et al.
The SNS regulates skeletal muscle motor innervation
To address whether the SNS regulates skeletal muscle motor inner­
vation, we studied the impact of regional and systemic sympathetic
ablation on muscle motor innervation using transcriptome analysis,
retrograde tracing of the sympathetic outflow to the skeletal muscle, and
electrophysiological, imaging, and protein analysis of NMJ trans­
mission. We and others also used state-of-the-art microsurgical tech­
niques in several wild-type and transgenic mice, including C57BL6,
enzyme E3 ubiquitin-protein ligase TRIM63/muscle specific RING finger
1 (MuRF1KO), and dopamine beta-hydroxylase (DBH)-tomato and Thy-
1 mice (Khan et al., 2016; Rodrigues et al., 2019).
After SNS ablation, we found elevated levels of MuRF1 transcript and
protein. MuRF1 is involved in muscle trophism and maintenance
(Moriscot et al., 2010). It is expressed in both type-I and type-II fibers,
preferentially the latter; highly enriched in the postterminal; and in­
teracts with AChR in endocytic structures (Rudolf et al., 2013). The
procedure evoked an increase in E3 ligase that led to an overall increase
in AChR but a decrease in sarcolemmal AChR, indicating that sympa­
thetic neurons influence AChR partition between the sarcolemmal and
subsarcolemmal compartments. Moreover, sympathectomized
MuRF1KO mice showed AChR partitioned much as it is in innervated
young mice (Rodrigues et al., 2018). These results support the conclu­
sion that MuRF1 mediates SNS regulation of the fraction of receptors
located at the postterminal NMJ sarcolemma.
Severing the sciatic nerve is considered a model of motor muscle
denervation since flaccid paralysis and massive atrophy ensue (Bodine
et al., 2001; Carlson et al., 2002; Machado et al., 2019). Rapidly,
following nerve transection, MuRF1 and F-Box Protein 32 (Fbxo32, a.k.a.
atrogin-1) increase (Bodine et al., 2001), while at day 14, MuRF1, but not
Fbxo32, was significantly increased after motor denervation in both
soleus and tibialis anterior muscles as determined by immunoblot
(Moriscot et al., 2010). Note that treatment with the β2-AR agonist
clenbuterol prevents denervation-induced increases in Fbxo32 levels
(Goncalves et al., 2012). These results indicate that maintaining sympa­
thetic activity or stimulating β2-AR prevents Fbxo32 levels from rising and
that muscle sympathetic ablation mimics, to some extent, muscle motor
denervation and may account for changes in muscle gene expression previ­
ously attributed primarily to motoneuron ablation.
Whether MuRF1 mediates muscle atrophy is controversial. Trans­
genic muscle-specific overexpression of MuRF1 does not cause muscle
atrophy (Hirner et al., 2008), but the same group demonstrated that
MuRF1 KO dramatically prevents denervation-induced atrophy in
mouse TA muscle (Moriscot et al., 2010). More recently, that induction
of genes associated with the NMJ is blunted in MuRF1 KO mice has been
reported (Furlow et al., 2013). Thus, we cannot rule out MuRF1
participation in denervation-induced muscle atrophy.
The increase in MuRF1 after sympathectomy suggests that the SNS
inhibits its expression under physiological conditions. We ruled out the
participation of several alternative pathways (Bonaldo and Sandri,
2013), and a potential effect of β2-AR (Rodrigues et al., 2018). We also
found an increase in protein kinase regulatory subunit-I alpha (PKA RIα)
but a decrease in the PKA(RIIα)/PKA(RIα) ratio, which we recently
proposed as a marker of the failure of postsynaptic AChR recycling and
membrane insertion (Xu et al., 2017). An alternative mechanism for the
elevation in MuRF1 levels after SNS ablation could be a decrease in
muscle autophagy. However, although lysosome degradation of pro­
teasomes has been demonstrated in rat liver (Cuervo et al., 1995) its role
in the skeletal muscle is not known.
The elevated Hdac4 concentration at the NMJ further supports the
concept that sympathetic ablation impairs motor axon function (Cohen
et al., 2007; Moresi et al., 2010). MuRF1 regulation by the canonical
Gαi2-myogenin cascade has been reported (Minetti et al., 2014), but
whether SNS dysfunction is involved is unknown. We found that sym­
pathectomy downregulates Gαi2, which has been shown to increase
Hdac4 (Minetti et al., 2014), and infecting the TA muscle with the
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constitutively activated form of Gαi2 (Moxham and Malbon, 1996) using
an adeno-associated virus, AAV-Gαi2(Q205 L), prevented both. Myoge­
nin induces muscle and serum miR-206, which suppresses Hdac4 and
promotes reinnervation (Moresi et al., 2010). SNS denervation of blood
vessels may affect NMJ molecular composition and function, but specific
Gαi2(Q205 L) expression in myofibers (Minetti et al., 2014) is consistent
with a direct sympathetic effect on NMJs. Also, muscle-flow recordings
have shown that epinephrine’s effect on neuromuscular transmission is
independent of concomitant vascular changes produced by the cate­
cholamine (Bowman and Raper, 1966).
2. Does the SNS regulate presynaptic and postsynaptic NMJ
structure and function? Sympathomimetics can be used to
answer this question
To explore sympathetic neuron-motoneuron cross-talk and its influ­
ence on NMJ transmission, we tested the effects of the sympathetic
postganglionic neurotransmitter, noradrenaline, and sympathomimetics
on such motoneuron-terminal targets as TRPV1 and P/Q- and N-type
voltage-activated Ca2+ channels and the resulting Ca2+ fluxes and syn­
aptic vesicle release dynamics (Khuzakhmetova and Bukharaeva, 2020;
Kuba, 1970; Rodrigues et al., 2018; Tsentsevitsky et al., 2019). The
impact of SNS ablation and sympathomimetics on the amplitude of the
compound muscle action potential has also been reported (Khan et al.,
2016).
Catecholamines act on specific receptors located on the cell surface
of target organs. These receptors, expressed throughout the body, are
classed in two groups. The α-subtype includes the Gq-coupled receptors
α1A, α1B, and α1D and Gi-coupled receptors α2A, α2B, and α2C. The
β-subtype comprises the Gs-coupled β1 and Gs/Gi-coupled β2 and β3
receptors. β1 and α2B are particularly important in understanding the
mechanisms by which the SNS regulates NMJ transmission (Rodrigues
et al., 2019; Wang et al., 2020a), and β2 directly regulates skeletal
muscle mass and metabolism (Lynch and Ryall, 2008).
Noradrenaline (NA), the main neurotransmitter of ganglionic sym­
pathetic neurons, modulates neuromuscular transmission (Kuba, 1970).
Regional surgical removal or systemic chemical ablation of mouse hin­
dlimb SNS, causes a dramatic decrease in muscle NA (Silveira et al.,
2014). and the release of synaptic vesicles (Rodrigues et al., 2018).
Administration of sympathomimetic agents prevents the effects of
chemical sympathectomy and reestablishes the compound muscle action
potential disrupted by SNS ablation, demonstrating that SNS innerva­
tion controls NMJ homoeostasis (Khan et al., 2016). Their use in vivo
and ex vivo helped us to define the acute and chronic effects of the SNS
on muscle function, and investigating how postganglionic sympathetic
axons and their neurotransmitter(s) affect myofiber composition and
function clarified the process of aging sarcopenia. β2-AR agonists in­
crease protein synthesis in denervated muscle and after injury (Gon­
calves et al., 2012), by suppressing proteolysis (Navegantes et al., 2009).
While the postsynaptic mechanism is well-established, these findings
reveal a presynaptic mechanism based on crosstalk between the motor and
sympathetic nervous systems at the NMJ.
The discovery prompted investigation of NMJ presynaptic vesicle
release in response to acute ex-vivo or chronic, repetitive in-vivo
administration of β-AR agonists. We focused on their effects on extra­
cellular Ca2+ concentration, TRPV1, and P/Q- and N-type voltage-
activated Ca2+ channels in young adult mice to determine how the
SNS influences NMJ transmission (Rodrigues et al., 2019).
If sympathomimetic agents enhance NMJ transmission and reme­
diate muscle wasting and strength in aged rodents (Carter et al., 1991;
Ryall and Lynch, 2008), why do endogenously secreted catecholamines
fail to maintain muscle mass and force in the absence of significant
changes in β2-AR levels? Recent studies support a novel mechanism by
which motor and sympathetic neurons interact at the NMJ presynapse
(Khan et al., 2016; Rodrigues et al., 2018, 2019; Wang et al., 2020a).
Impaired neuronal crosstalk can explain, at least partially, progressive
O. Delbono et al.
atrophy and weakness with aging (Rodrigues et al., 2019).
We examined whether the SNS regulates motor neuron axon function
and neuromuscular transmission in situ—without any exogenous
intervention, including delivery of catecholamines or sympathomimetic
agents. Using endogenous neurotransmitters to regulate motoneuron
synaptic vesicle release allowed fine-tuning of the acute stress response
under physiological conditions. Our comprehensive approach included
nerve immunolabeling co-registration and electrophysiological re­
cordings in a novel mouse ex-vivo preparation that preserves the
complexity of the interaction between motoneuron and SNS at the NMJ
and muscle innervation in the living mouse. Defining the mechanisms by
which the SNS regulates neuromuscular properties may have broad health
implications, particularly if we can learn how to counteract sarcopenia,
which impairs gait and mobility in older adults and people suffering from
neurodegenerative diseases (Wang et al., 2020a).
Fig. 2 shows how sympathetic axon depolarization leads to N-type
Ca2+ channel activation, Ca2+ influx, and NA release. This neurotrans­
mitter then acts on sympathetic axon α2B- and β1-AR to inhibit N-type
Ca2+ channels mainly through Gi and possibly exclusively via G(o)/G(i)
and activate them through Gαs. N-type VGCC Ca2+ influx levels may
result from the balance between yohimbine-sensitive α2-AR autor­
egulation and β1-AR activation. Other rapid pertussis toxin (PTX)-
insensitive G-protein pathways may involve NA-independent activating
mechanisms (G#) (Mirotznik et al., 2000). Propranolol, a β1/β2-AR
blocker, and atenolol, a β1-AR blocker, prevent β1-AR activation and
diminish NA release at the NMJ (Wang et al., 2020b).
Since NA has no effect on miniature end-plate potential (MEPP)
frequency, end-plate potential (EPP) amplitude, and quantal content in
the presence of propranolol (Rodrigues et al., 2019), we proposed the
motoneuron as a novel target of the SNS. We also proposed that Ca2+
influx through TRPV1 channels and P/Q-type Ca2+ channels (Cav2.1)
determines the axoplasmic Ca2+ concentration at rest and during motor
neuron depolarization, respectively. These data indicate that by acting on
β1-AR, NA modulates TRPV1 and P/Q-type VGCC and, therefore, moto­
neuron Ca2+ concentration and the likelihood of ACh synaptic vesicle release
(Fig. 2).
Fig. 2. Downregulation of preterminal adrenergic receptors and its impact on motoneuron synaptic vesicle release with aging. AP: axon potential. Red arrows
indicate decreased function or protein expression with aging. Dashed lines indicate proposed pathways. Adapted from (Rodrigues et al., 2019; Wang et al., 2020a).
AP: action potential; EPP: end-plate potential; NA: noradrenaline; QC: quantal content; tSC: terminal Schwann cell; VGCC: voltage-gated calcium channel.
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Ageing Research Reviews 67 (2021) 101305
3. Does sympathetic neuron regulation of NMJ transmission
decline with aging?
To address this question, we analyzed sciatic-peroneal nerve
immunolabeling co-registration and NMJ transmission recordings in a
novel mouse ex-vivo preparation. The sympathetic-peroneal nerve/
lumbricalis muscle preparation (SPNL) maintains the anatomical
complexity of the relationship between the sympathetic and motor
nervous systems. This approach unveils the molecular substrate that
accounts for the influence of endogenous sympathetic neurons on
motoneuron-muscle transmission in young mice and older rodents,
providing a unique opportunity to examine whether sympathetic neuron
regulation of NMJ transmission declines with age (Wang et al., 2020a).
Compared to young mice, geriatric mice show no significant change
in NMJ-transmission frequency but higher MEPP amplitude and lower
EPP amplitude and quantal content (Wang et al., 2020a). MEPP fre­
quency and amplitude vary according to the muscle examined. In
age-matched CBF-1 mice, MEPP frequency decreased in EDL, soleus, and
EDC but not gluteus maximus or diaphragm muscles, while MEPP
amplitude increased in all but diaphragm (Banker et al., 1983).
In aging lumbricalis NMJs, sympathetic neuron stimulation does not
increase MEPP frequency. Our nerve image co-registration analysis in­
dicates that the strong decline in motoneuron β1-AR expression with
aging is the factor limiting its synaptic vesicle release. In contrast to the
reported increase in β-AR in the cerebral cortex, hypothalamus (Meitzen
et al., 2013), and hippocampus with aging (Santulli and Iaccarino,
2013), our data show a significant decline in β1-AR in
choline-acetyltransferase and β1- and α2B-AR in tyrosine hydroxylase
immunoreactive axons. We proposed that the age-dependent decline in
β1-AR expression in sympathetic neurons contributes to diminished NA
release (Wang et al., 2020a).
For functional recordings, we used the SPNL technique to determine
whether sympathetic neuron regulation of motoneurons synaptic vesicle
release mediated by β1- and α2B-AR is diminished in geriatric mice.
However, this approach has some limitations. First, SPNL cannot assess
the influence of sympathetic relays from the brain and brainstem nuclei
O. Delbono et al.
on the intermediolateral column of the spinal cord. Second, it cannot
assess the systemic influence of the humoral milieu on the structures
involved in neuromuscular transmission. Third, repetitive stimulation
might exhaust catecholamine and/or acetylcholine neuron pools. Future
experiments in anaesthetized, live mice can overcome these limitations.
SNS activation may influence the activity of peripheral targets other
than skeletal muscle to modify neuromuscular transmission and muscle
innervation. Optogenetic studies targeting specific areas of the SNS
should address this question (see below).
Older adults younger than 75 show modest effects of aging on basal
SNS activity (Ng et al., 1994). Above 75, concentrations of basal plasma
NA and NA responses to a stressor were higher than did either younger
subjects (Barnes et al., 1982; Esler et al., 1995; Hoeldtke and Cilmi,
1985; Mazzeo et al., 1997; Palmer et al., 1978; Veith et al., 1986).
Exaggerated increased basal plasma NA suggests advanced age has
specific effects on the sympathoneural and sympathoadrenomedullary
components of the SNS (Pascualya et al., 1999), Both increased release
and decreased clearance would account for high NA levels with
advanced age (Pascualya et al., 1999). SNS hyperactivity with aging is
associated with ARs desensitization, downregulation, and changes in
subtypes expression patterns (Insel, 1993). Cell-signaling molecules,
such as GPRC kinases, are responsible for β-AR phosphorylation,
desensitization, and downregulation (Urasawa et al., 1991), which may
contribute to the dysfunction of the target (Pfleger et al., 2019); for
example, the age-dependent impairment of NMJ transmission in
response to sympathetic stimulation. β1-AR is among the most studied
AR subtypes, probably due to its role in excitation-contraction coupling
in normal and diseased hearts. Chronic ischemic heart failure, common
in older adults, is associated with decreased β1-AR expression after a
period of increased expression that also increases cAMP and
protein-kinase A. Phosphorylated GATA4 then mediates the upregula­
tion of miRNA let7, which directly targets ADRB1 mRNA 3’UTR to
repress β1-AR (Du et al., 2017). Whether this mechanism operates in
distal axons is unknown. Overall, regulation of β-AR expression in the
nervous system and especially the nerve is unclear.
Our data on the age-dependent decline in α2-AR levels are consistent
with their diminished responsiveness in prejunctional noradrenergic
nerve terminals reported in the cardiovascular system (Docherty, 2002).
However, the mechanism leading to the age-dependent decline in their
expression in the spinal nerve remains unknown.
In summary, the age-dependent decline in sympathetic axon β1 and
α2B and motoneuron β1-AR accounts for the decline in EPP amplitude
and synaptic vesicle quantum content (Fig. 2).
The pharmacological vs the physiological approach to restor SNS influence
on NMJ transmission
Maintaining or recovering sympathetic muscle innervation
throughout life is undoubtedly preferable to chronic use of AR phar­
macological agents. Drug administration cannot mimic the complex,
concerted tissue response to endogenous sympathetic neurotransmit­
ters, which depends on the AR subtype’s repertoire and location and the
intricate relationship between central and peripheral nervous system
relays. Loss of receptor selectivity, tolerance, and tachyphylaxis can also
trigger adverse effects. Moreover, pharmaceutical compounds reach
stable levels in blood, precluding any adaptation to physiological or
pathological challenges. Thus, we cannot prevent or reverse age-related
decline in skeletal muscle mass and force until we develop interventions
that target the molecular mechanisms governing NMJ instability and muscle
denervation.
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Ageing Research Reviews 67 (2021) 101305
4. Does maintenance of sympathetic neurons attenuate aging
sarcopenia?
The transcription factor Hand2 points to an intervention that will maintain
sympathetic neurons and attenuate sarcopenia with aging
The process by which sympathetic neurons are maintained during
development has been amply investigated (Rohrer, 2011). However, the
factors that sustain its integrity into old age are unknown. The differ­
entiation of neural crest cells into NA sympathetic neurons is complex
and requires a network of transcription factors. One of them, Hand2,
plays a prominent role in adult sympathetic neuron maintenance of NA
genes and cell survival as demonstrated by axotomy (Pellegrino et al.,
2011) and in conditional Hand2 KO sympathetic neurons (Stanzel et al.,
2016). The Hand2 gene encodes a protein in the basic helix-loop-helix
family of transcription factors. Eliminating it in adult sympathetic
neurons reduces expression of the genes involved in synapse develop­
ment and function (Stanzel et al., 2016). Moreover, in vitro knockdown
of Hand2 in differentiated, postmitotic neurons leads to a selective loss
of TH and DBH with no effect on generic neuronal genes such as
neuron-specific class III beta-tubulin (Tuj1), embryonic lethal, abnormal
vision-like RNA-binding protein level 3 (ELAVL3 or HuC) and superior
cervical ganglia (Schmidt et al., 2009). Conditional Hand2 knockout in
Hand2-DBHCre mouse embryos caused a selective reduction of TH and a
massive reduction of proliferating, DBH-expressing immature neurons
(Schmidt et al., 2009). Despite the detailed examination of Hand2’s
function in neurogenesis, and noradrenergic differentiation in embry­
onic, postnatal, and adult mice (Rohrer, 2011), analysis of its pivotal
role in sympathetic neuron genotype and phenotype in aged mammals is
missing and necessary for its potential to attenuate or reverse
sarcopenia.
Hand2 expression declines throughout the mouse’s life, but inducing it even
at advanced ages preserves the number and size of paravertebral
sympathetic ganglia neurons
Hand2 plays a prominent role in adult sympathetic neurons’ adren­
ergic differentiation and maintenance (Doxakis et al., 2008; Tsarovina
et al., 2010). Fig. 3 shows the convergence of a hierarchical network
comprised of the bone morphogenetic proteins (BMPs), paired-like ho­
meobox 2b (Phox2b), achaete-scute family BHLH transcription factor 1
(Ascl1), and the insulinoma-associated-1 (Insm1) that regulates its
expression. GATA2 and its downstream GATA3 transcription factors are
also required for the survival of embryonic and adult sympathetic neu­
rons (Tsarovina et al., 2010), but their role in old age has not been
explored.
Adult nerve injury suppresses Hand2, which directly regulates DBH
Fig. 3. The transcription factor Hand2 regulates noradrenergic differentiation
and sympathetic neuron maintenance. Relationship among the bone morpho­
genetic protein (BMP)-induced transcription factors paired-like homeobox 2b
(Phox2b), Achaete-Scute Family BHLH Transcription Factor 1 (Ascl1),
Insulinoma-associated 1 (Insm1), and Hand2 (black arrows). Adapted from
(Rohrer, 2011). Ascl1: Achaete-scute homolog 1; BMP: bone morphogenetic
protein; Hand2: heart and neural crest derivative 2; Insm1: insulinoma asso­
ciated 1; Phox2b: paired-like homeobox 2b.
O. Delbono et al.
expression (Rychlik et al., 2005) and, most important, the genes con­
trolling synaptic and neurotransmission functions in adult sympathetic
neurons (Stanzel et al., 2016). Based on the reported role of the tran­
scription factors supporting developing and adult autonomic neurons
(Stanzel et al., 2016), we examined whether inducing HAND2 expres­
sion exclusively in sympathetic neurons would attenuate (a) motor
denervation, (b) impaired NMJ transmission, and (c) loss of muscle mass
and function in old mice. We delivered a viral vector carrying Hand2
expression or an empty vector (EV) exclusively to sympathetic neurons.
Since Hand2 expression is restricted to the peripheral SNS (Hendershot
et al., 2008), we ruled out any influence of central sympathetic nuclei on
our measures.
Neurons from 22-month-old EV-treated mice were not immunore­
active to Hand2 antibody probably because they had less than 20
percent of the transcript recorded at postnatal day-5 (Rodrigues et al.,
2020). Sympathetic neuron hypermethylation may account, at least
partially, for this age-dependent decrease in Hand2 gene transcription,
since CpG islands, but not C combined with any other nucleotide (CH or
CHH) exhibit hypermethylation in paravertebral sympathetic ganglia
neuron with aging (Rodrigues et al., 2020). Epigenetic silencing of
Hand2 has been reported in other territories in older adults, for instance
in the development of endometrial cancer, which occurs in older women
(Jones et al., 2013). Hand2 undergoes age-associated DNA hyper­
methylation in epithelial tissues (Maegawa et al., 2010). The mouse
intestine, a tissue densely innervated by the SNS, shows a high rate of
Hand2 hypermethylation as a function of age, suggesting that Hand2 can
be hypermethylated in other cells as well (Maegawa et al., 2010).
Sustained Hand2 expression in sympathetic neurons in old mice
preserves ganglia sympathetic neurons size to a significant extent, which
indicates that Hand2 ameliorates the loss of ganglia neurons with aging
(Stanzel et al., 2016). Conditional deletion of Hand2 reveals its critical
functions in neurogenesis during development (Hendershot et al., 2008)
and adulthood (Stanzel et al., 2016). Preserving the number and size of
sympathetic neurons in Hand2-treated mice extends and enriches the
complexity of sympathetic muscle innervation with aging.
The increase in phosphorylated PKA RI and mechanistic target of
rapamycin complex 1 (mTORC1) with Hand2 increases phosphorylation
of the cAMP-response element binding protein CREB modulator- CREM,
which translocates to the nucleus. Both CREB and CREM, bind a specific
cis element - cAMP-response element (CRE) - in the promoter region of
skeletal muscle target genes (Zheng et al., 2002). In the Hand2-treated
mice, upregulation of Crtc3, a member of the CREB regulated tran­
scription co-activator gene family, is consistent with activation of
cAMP-responsive genes. CREM, a top upstream regulator of genes
controlled by sympathetic neurons expressing Hand2 in old age. CREM
targets include the TH and dHand, two genes essential for SN function
and maintenance (Rauen et al., 2013). In summary, low levels of PKA RI
phosphorylation (Xu et al., 2017), together with genomic hyper­
methylation may account for the age-dependent decrease in Hand2 gene
transcription.
Hand2 increases skeletal muscle mass and force and whole-body strength
but not spontaneous mobility or endurance
The increase in muscle mass in Hand2- compared to EV-treated mice
was associated with enhanced muscle strength, which we attribute to
the increase in cross-sectional area (CSA) of type-II fibers in extensor
digitorum longus (EDL) muscle, and type-I and type-II fibers in soleus
muscle (Rodrigues et al., 2020).
In the EV- compared to the Hand2-treated mice, electrical stimula­
tion of distal peroneal nerves resulted in unsustained and weaker lum­
bricalis muscle peak force. However, this effect was more pronounced in
response to muscle activation through the nerve. These data indicate
that in addition to muscle sympathetic innervation, aging impairs motor
innervation and NMJ transmission, and Hand2 expression in sympa­
thetic neurons rescues them significantly (Rodrigues et al., 2020).
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Ageing Research Reviews 67 (2021) 101305
Sympathetic denervation of various target organs
In old mice, we found that blood vessels were still surrounded by
sympathetic axons, but their projections toward myofibers and NMJs
were fewer, depriving the muscle of the dense sympathetic network
observed in young mammals (Rodrigues et al., 2018, 2020; Straka et al.,
2018). The skeletal muscle does not seem to be the only organ pro­
gressively losing sympathetic innervation density and complexity, since
blood vessels and the heart have been reported to exhibit marked
deprivation of sympathetic axons with aging (Brown et al., 2019; Li
et al., 2003; Rengo et al., 2016). The pulmonary nervous system consists
of afferent and efferent nerve fibers that help to maintain organ ho­
meostasis (Brandenberger and Mühlfeld, 2017). In the lower airways,
aging is associated with a reduction in noradrenergic innervation of the
bronchial tree (Ricci et al., 1997) and a decrease in vasoactive intestinal
peptide concentration and nerve fiber density (Geppetti et al., 1988).
Evidence for a decrease in sympathetic control of intestinal function in
aged rodents has been reported (Baker et al., 1991), and the timing
aligns with the onset and rate of myenteric cell loss (Cowen et al., 2000;
El-Salhy et al., 1999; Phillips and Powley, 2001, 2007). Noradrenergic
networks are abundant in young adult rats, but in aged rats, noradren­
ergic innervation is less dense, and splenic NA levels are significantly
lower. A relationship between diminished noradrenergic innervation
and reduced immune responsiveness in aging mammals has been pro­
posed (Felten et al., 1987).
The mechanism by which organs lose sympathetic axon terminals is
incompletely understood. During development, target tissues secrete
trophic factors to regulate axon branching at sympathetic nerve termi­
nals, an essential step in forming functional connections. Compart­
mentalized neuronal cultures revealed that the autocrine Wnt5a-
(retinoic acid-related orphan receptor [ROR]) signaling loop directs
sympathetic axon branching during target innervation (Ryu et al.,
2013). Although Wnt5a induced β-catenin nuclear translocation in
vascular smooth muscle cells (VSMCs) from young and old mice, CREB
phosphorylation and Wnt1-inducible signaling pathway protein-1
(WISP-1) upregulation did not occur in old VSMCs. Wnt5a-mediated
survival was lost with aging due to impaired CREB activation and
WISP-1 regulation. Better understanding of the effect of age on Wnt
signaling may identify targets to promote VSMC survival in elderly pa­
tients (Brown et al., 2019). Due to the relevance of skeletal muscle
sympathetic innervation, we tested whether maintaining postganglionic
sympathetic neurons by Hand2 expression prevents or attenuates NMJ
transmission and muscle innervation.
Hand2 expression enhances NMJ transmission and muscle innervation
Sympathetic neuron Hand2 enhances spontaneous and evoked NMJ
transmission and prevents myofiber motor and sympathetic denerva­
tion. It also preserves the complexity of muscle sympathetic innervation
and its proximity to the NMJ. Fragmentation and simplification of the
postterminals are hallmarks of age-dependent deterioration of skeletal
muscle innervation (Li and Thompson, 2011), while sustained Hand2
expression by sympathetic neurons preserves their molecular integrity
and morphology. Hand2-treated mice had fewer small postterminal
fragments than the EV group and showed an approximately five-fold
decrease when the number of fragments was normalized to the muscle
area. The wider range of postterminal fragment sizes in the Hand2 group
indicates that, to a significant degree, muscle sympathetic innervation
ameliorates fragmentation.
Hand2 significantly preserves skeletal muscle sympathetic innerva­
tion, as shown by (a) higher TH levels in nerve and muscle, detected by
immunoblot; (b) longer muscle sympathetic axon path; and the shorter
distance between sympathetic neurons and NMJs, confirmed by
immunohistochemistry. NMJ pre- and postterminal co-localization and
quantification of neurofilament (NF) path length per muscle area shows
that the preservation of sympathetic innervation enhances myofiber
O. Delbono et al.
motor innervation. The mechanism underlying preservation of muscle
sympathetic and motor innervation is uncertain; however, low protein
phosphatase-2 (PP2A), and protein phosphatase-1 (PP1) levels and more
NF-heavy chain (NFH-), NF-medium chain (NFM-), and NF-light chain
(NFL) subunit phosphorylation indicate that NF phosphorylation plays a
role in stabilizing muscle motor innervation in Hand2-treated mice. This
conclusion is further supported by confocal and electron microscopy
studies on muscle sympathetic ablation, which show compromised NF
phosphorylation and disorganized axon NF and microtubules (Rodri­
gues et al., 2020, 2019).
Additionally, Hand2 upregulates the genes encoding critical com­
ponents of NMJ and myofiber innervation, including Nrcam, a neural
cell-adhesion molecule, its product, NCAM, which is a marker of muscle
denervation/reinnervation (Messi et al., 2016), and Nrxn1, a presyn­
aptic membrane cell-adhesion molecule that primarily binds to neuro­
ligins, forming a complex required for efficient neurotransmission
(Hadley et al., 2006). Hand2 also upregulates the
microtubule-associated protein-2 (MAP2) gene, which may stabilize the
microtubules against depolimerization, synapse differentiation inducing
1 like (Sindig1l), which plays a role in the shaping of synaptic specificity
(Cadwell et al., 2016), and the GDNF receptor Gfra1, expressed at
myelinated nerves and NMJ. Both GDNF and Gfra1 play a crucial role in
muscle innervation and GDNF signaling (Hase et al., 1999; Henderson
et al., 1994; Rodrigues et al., 2020). These results indicate that Hand2
expression enhances NMJ transmission and muscle innervation in the old.
G protein-coupled receptor (GPCR) signaling plays a pivotal role in
sympathetic neuron-mediated myofiber maintenance
The GPCR constitute the largest family of membrane receptors. It is
responsible for cell signaling upon binding to hormones, neurotrans­
mitters, or ions. Notably, many pharmacological agents target these
receptors, stressing their involvement in pathophysiological conditions
and therapeutics (Rosenbaum et al., 2009). In contrast to stimulating Gs,
the inhibitory subunit Gαi2 isoform blocks adenylate cyclase activation
and cAMP production. Physiological or pathological circumstances that
decrease levels of sympathetic neurotransmitter or Gαi2 expression
prevents the myogenin- and MuRF1-mediated pro-cachectic action of
tumor necrosis factor alpha (TNF-α) and myofiber atrophy (Minetti
et al., 2014). Minetti and coworkers showed that Gαi2 induces muscle
hypertrophy by promoting mTOR and protein synthesis in an
Akt-independent mechanism, while inhibiting atrophy in response to
TNF-α. Ablation of muscle sympathetic innervation decreases muscle NA
(Silveira et al., 2014), and Gαi2 protein levels leading to muscle atrophy
(Rodrigues et al., 2018). The model of muscle sympathetic ablation
shows downregulation of Gαi2, which activates the
Hdac4-myogenin-MyoD cascade to raise MuRF1 levels and increase
AChR destabilization (Rodrigues et al., 2019). Note that both experi­
mental sympathetic ablation and aging can cause vasculature denerva­
tion, which might influence the deterioration of muscle structure and
function. However, muscle flow recordings have shown that the effect of
epinephrine on neuromuscular transmission is independent of
concomitant vascular changes produced by the catecholamine (Bowman
and Raper, 1966). These data indicate that catecholamines trigger intra­
myocellular signaling independently of their vasoactive function.
Sympathetic neuron Hand2 prevents Gαi2 downregulation with aging
We found that Hand2 expression in sympathetic neurons prevents
the decrease in Gαi2 levels with aging and activation of the signaling
cascade that has been associated with muscle motor denervation
(Minetti et al., 2014). It also lowers Hdac4 concentration to improve
motor-axon function (Cohen et al., 2007; Moresi et al., 2010). Hdac4
mediates skeletal muscle response to denervation, suggesting its inhi­
bition as a target for treating neurogenic muscle atrophy. However,
studies in mutant mice where HDAC4 is specifically deleted in the
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Ageing Research Reviews 67 (2021) 101305
skeletal muscle (Pigna et al., 2018), show that it preserves skeletal
muscle structure following long-term denervation, suggestging that its
role in muscle denervation is more complex than previously thought. It
might involve oxidative stress, the ubiquitin-proteasome system, and
autophagy (Pigna et al., 2018).
Hand2 also downregulates myogenin and myoD, both associated
with denervation in gastrocnemius and tibialis anterior muscles. Myo­
genin and myoD genes are regulated by electrical activity (Eftimie et al.,
1991; Tang and Goldman, 2006). Downregulating these genes reduces
MuRF1 and atrogin-1. MuRF1 is expressed in both type-I and type-II
fibers, but predominantly the latter; it is highly enriched in the post­
terminal; interacts with AChR in endocytic structures (Rudolf et al.,
2013) and participates in muscle trophism and maintenance (Moriscot
et al., 2010). Consistently, Hand2-induced preservation of muscle
sympathetic innervation downregulates both MuRF1 and atrogin and
prevents AChR endocytosis.
In old mice, the PKA RIα subunit is largely removed from the NMJ,
while PKA RIIα and RIIβ are enriched there (Xu et al., 2017). Induced
sustained levels of Hand2 in sympathetic neurons, like other in­
terventions to counter muscle denervation (Xu et al., 2017), upregulates
PKA RI in the TA muscle, and downregulates PKA RII and the PKA
RII/PKA RI ratio in both GA and TA muscles. These results support the
hypothesis that muscle sympathetic innervation prevents activation of the
signaling cascade reported in skeletal muscle denervation (Moresi et al.,
2010), by maintaining Gαi2 expression.
Mechanisms by which Hand2 prevents age-related decline in muscle mass
and force. Role of Akt, atrogins, inflammation, mTORC1, and autophagy
(Fig. 4)
The protein kinase B (a.k.a. Akt) promotes myotube hypertrophy by
stimulating protein synthesis through mTOR activity while inhibiting
the forkhead box FoxO to reduce MuRF1 abundance, preventing
myofibrillar protein turnover (Rommel et al., 2001). Hand2 treatment
increases muscle Akt phosphorylation, a critical step in the activation of
the noncanonical β-AR pathway, which leads to FoXO1 phosphorylation
and exclusion from the nucleus and downregulation of MuRF1 and
atrogin/MAFbx (Rodrigues et al., 2020). This mechanism, together with
downregulation of the Hdac4-myogenin-MyoD-atrogin/MuRF1
signaling cascade (see above), reduces atrogin expression to prevent
muscle atrophy (Ryall et al., 2008).
Activation of the transcription factor nuclear factor kappa B (NF-κB)
is a fulcrum in the processes that mediate muscle atrophy. The signifi­
cance of NF-κB as a key regulator of muscle atrophy has been empha­
sized by several in vivo studies, which have demonstrated that NF-κB-
targeted therapies can abrogate muscle atrophy (Thoma and Lightfoot,
2018). NFκB signaling plays a role in age-related skeletal muscle atrophy
probably by activating the senescence-associated secretory phenotype
(Salminen et al., 2009). Moreover, Hand2 decreased the inhibitor IκB
and NFκB phosphorylation (Rodrigues et al., 2020). These results sup­
port our RNA-sequencing analysis showing that Hand2-induced toll-like
receptor 5 (Tlr5) downregulation decreases IκB activation, leading to
nuclear exclusion of NFκB and preventing activation of the canonical
pro-inflammatory pathway. Consistently, downregulation of
TNF-related-weak-inducer-of-apoptosis (TWEAK) and Toll-like recep­
tor-1 (TLR-1) and interleukin (IL) 16, which play roles in the innate
immune system and the cell cycle, respectively, contribute to SNS
regulation of inflammation and immune-system homeostasis (Révész
et al., 2014). Cytokines downregulate Hand2 in cultured sympathetic
neurons, while Hand2 overexpression rescues the noradrenergic
phenotype. These observations support a third mechanism for decreased
Hand2 transcription with aging, in addition to reduced levels of PKA RI
(Xu et al., 2017) and Hand2 genomic DNA hypermethylation.
Rapamycin-induced mammalian target of rapamycin complex 1
(mTORC1) inhibition prevents age-related muscle loss, while its chronic
activation advances muscle damage and loss (Tang et al., 2019),
O. Delbono et al. Ageing Research Reviews 67 (2021) 101305

Fig. 4. Schematic representation of Hand2-induced expression in sympathetic neurons and its impact on skeletal muscle sympathetic and motor innervation in old
mice. Selective expression of Hand2 in the mouse peripheral sympathetic system from middle- through old-age increases muscle mass and force by (a) regulating
skeletal muscle sympathetic and motor innervation; (b) improving AChR stability and NMJ transmission; (c) preventing inflammation and myofibrillar protein
degradation; (d) increasing autophagy; and (e) probably enhancing protein synthesis. (Rodrigues et al., 2020). AChR: acetylcholine receptor; CREB: cAMP response
element-binding protein; CREM: cAMP Responsive Element Modulator; FoXO1: Forkhead box protein O1; IκB: inhibitor of nuclear factor kappa B; IML: inter­
mediolateral column; LC-I/LC-II: microtubule-associated protein light chain; mTOR: the mechanistic target of rapamycin; NA: noradrenaline; NFκB: nuclear factor
kappa-light-chain-enhancer of activated B cells; NFH: neurofilament heavy chain; NFL: neurofilament light polypeptide; NFM: neurofilament medium chain; Nrcam:
Neuronal cell adhesion molecule; Nrxn1: neurexin-1-alpha; p62: 62 kDa protein; PKA: protein kinase A; TH: tyrosine hydroxylase; TSC2: tuberous sclerosis complex
2; TLR5: Toll-like receptor 5.

inhibiting constitutive and starvation-induced autophagy in skeletal
muscle (Castets et al., 2013). We found in both gastrocnemius and
tibialis anterior muscles, that Hand2 treatment increases mTORC1
phosphorylation at serine 2448, fine-tuning its activity at the “repressor
domain (Chiang and Abraham, 2005; Figueiredo et al., 2017). Deleting
this site strongly activates mTORC1 (Sekulic et al., 2000). Increased
phosphorylation at the repressor domain prevents Unc-51-like kinase
(ULK) and autophagy-related-13 (ATG13) phosphorylation, activating
autophagy (Laplante and Sabatini, 2012).
Does increased macroautophagy improve skeletal muscle mass and/
or function in old mice? One common feature of all age-related changes
at the tissue level is the accumulation of damage and harmful modifi­
cations in DNA, proteins, lipids, and cellular organelles; autophagic ef­
ficiency declines over time, and intracellular waste products amass
(Salminen et al., 2009). Robust genetic evidence across species supports
the inhibition of autophagy in aging (Carnio et al.; Rubinsztein et al.,

10
O. Delbono et al.
2011). Analysis of muscle autophagy showed lower p62 and higher
ATG-7 levels and LC3-I lipidation in the Hand2 group than the EV group.
Atg7 ablation induces NMJ degeneration and precocious aging (Carnio
et al., 2014), which suggests that high ATG-7 levels are required to
improve muscle autophagy. Autophagy selectively degrades the
LC3-binding adaptor p62 protein (sequestosome-1 [SQSTM1]) (Ichi­
mura et al., 2008), and since a drop in p62 is associated with activation
of the autophagic process, it serves as a readout of autophagic flux,
together with LC3-I conversion to LC3-II (Bjorkoy et al., 2005; Sei­
benhener et al., 2004). Non-significant changes in lysosome-associated
membrane protein 2 (Lamp2) and heat-shock cognate protein of 70
kDa (HSC70) levels indicate that macroautophagy, but not
chaperone-mediated autophagy, is activated by sustained Hand2
expression in sympathetic neurons with aging. Note that direct stimu­
lation of sympathetic neurons activates muscle postsynaptic β2-AR,
cAMP production, and import of the transcriptional co-activator
peroxisome proliferator-activated receptor γ-coactivator 1α
(PPARGC1A) into the myonucleus (Khan et al., 2016), which increases
autophagy (Vainshtein et al., 2015).
In summary, preventing decline in peripheral sympathetic neuron
Hand2 expression with aging attenuates sympathetic and motor muscle
denervation and aging sarcopenia (Rodrigues et al., 2020).
5. Do central sympathetic neuron relays influence sympathetic
and motor muscle innervation?
The studies reviewed above indicate that activation of peripheral
sympathetic neurons increase NMJ transmission. However, we must still
define the specific neuronal groups in the peripheral and central nervous
system that account for SNS influence on NMJ transmission; for
example, whether or how postganglionic sympathetic neurons regulate
synaptic vesicle release in vivo or central autonomic relays regulate NMJ
transmission. Previous studies have associated the pontine locus
coeruleus (LC) (a.k.a. A6 nucleus) with human mobility, motility, and
skeletal muscle physiology (Buchman et al., 2012; Del Tredici and
Braak, 2013; Dobbins and Feldman, 1994; Jacobs et al., 1991; Robinson,
1978; Xiang et al., 2014). A series of experiments in our laboratory
focused on lumbar postganglionic sympathetic neurons, and the LC, a
subdivision of which projects to preganglionic neurons in the spinal cord
intermediolateral (IML) column to address this issue. Combining opto­
genetics (a technique that involves the use of light to control neuron
activity that have been genetically modified to express light-sensitive
ion channels) with NMJ transmission recordings provided a unique
opportunity to determine the precise role of postganglionic sympathetic
neurons and LC neurons in NMJ transmission in adult living mice
(Rodrigues et al., 2020).
We crossed Ai32(RCL-ChR2(H134R/EYFP)) (Madisen et al., 2012)
and TH-Cre (Savitt et al., 2005) to create a mouse model that would
express channelrodhopsin-2 (ChR2) in the central and peripheral
noradrenergic neurons. We concluded that in vitro and in living mice
sympathetic axon optostimulation, mediated by β1-ARs, enhances NMJ
transmission and LC does not, despite its projections to the IML
(Rodrigues et al., 2020), perhaps because they do not establish en passant
synapse with preganglionic sympathetic neurons. Confocal z-stack pro­
jections of sections through the spinal cord showed that the injected
AAV2/rh8-PRSx8-EGFP-W viral vector appears at both the dorsal horn
and the IML (Bruinstroop et al., 2012). Also, we stimulated LC at fre­
quencies below and above 5 Hz with no obvious response, which in­
dicates that by innervating the muscle spindle (Radovanovic et al.,
2015), LC modulates posture (Carter et al., 2010), but not NMJ
transmission.
Detailed analysis of central neuroanatomical circuitries show that
several brainstem noradrenergic regions project to the IML, including
the noradrenergic bulbospinal A1 and A2 areas (Rodovalho et al., 2020)
and pontine A5, A6, and A7 (Bruinstroop et al., 2012). The functional
relevance of these CNS projections for NMJ transmission and muscle
11
Ageing Research Reviews 67 (2021) 101305
motor innervation remains unknown. Elucidating the functional role of
brainstem sympathetic neuron projections to preganglionic noradrenergic
neurons will help to define the coordinated response of the visceral and
neuromuscular system to physiological and pathological challenges.
6. Conclusions and future directions
The SNS regulates several primary synaptic and non-synaptic events.
Inducing Hand2 expression in sympathetic neurons from middle-aged
mice improves their NMJ transmission, myofiber innervation, muscle
mass, and function maintenance as they age. Despite recent progress in
our understanding of the process by which the SNS regulates skeletal
muscle structure and function with aging, several questions remain.
Hand2 is critical for sympathetic neurons maintenance in adult mice,
but its expression is regulated by transcription factors, such as BMPs,
ASCL1, Phox2b, and Insm1 (Rohrer, 2011), and their expression levels
and function have not been examined in aging organisms. Also, despite
the beneficial impact of Hand2 induced expression on muscle innerva­
tion, we do not know whether Hand2 expression induced for a longer
time, and up to very old age (geriatric) will still ameliorate sarcopenia.
Treated middle-aged mice should be examined not only 6-months, but
10–12 months after Hand2- or EV-treatment.
Whether mechanisms other than hypermethylation influence Hand2
expression in aging sympathetic neurons remains unknown. Although
some of these mechanisms are discussed above, the potential role of the
recently identified enhancer RNAs (eRNAs) should be investigated.
eRNAs are a novel class of short, noncoding RNAs transcribed from
active enhancer regions in the genome. They regulate downstream genes
promoting DNA looping/interactions, regulating chromatin accessi­
bility, and interfering in transcription, which is clinically relevant (Li
et al., 2014, 2016; Mora et al., 2016).
We do not know whether the age-dependent decline in central
autonomic nucleus composition and/or function accounts for skeletal
motor denervation and sarcopenia. A decrease in LC neuron density is
associated with impaired mobility in older adults, which indicates that
central sympathetic relays may play a critical role in physical activity
(Buchman et al., 2012). Our research shows that postganglionic, but not
LC neurons regulate NMJ transmission. Whether other brainstem
noradrenergic neuron groups more densely projected to the IML influ­
ence mammalians’ motility is an open question for future research on
the mechanisms of aging sarcopenia.
Recent findings can be translated to human sarcopenia. Non-
pathogenic viral vectors with long-lasting expression, recently tested
in rodents, could be used for safe, efficient gene delivery in humans in
the near future (Rodrigues et al., 2020). Nanomaterials or lipid micelles
are alternative approaches to deliver Hand2 to ganglionic sympathetic
neurons.
Declaration of Competing Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
The National Institutes of Health grants R01AG057013 and
R01AG057013-02S1, and the Wake Forest Claude D. Pepper Older
Americans Independence Center (P30-AG21332) supported this work
and the original research published by the Delbono laboratory.
References
Andersson, A.M., Olsen, M., Zhernosekov, D., Gaardsvoll, H., Krog, L., Linnemann, D.,
Bock, E., 1993. Age-related changes in expression of the neural cell adhesion
molecule in skeletal muscle: a comparative study of newborn, adult and aged rats.
Biochem. J. 290 (Pt 3), 641–648.
O. Delbono et al.
Askmark, H., Eeg-Olofsson, K., Johansson, A., Nilsson, P., Olsson, Y., Aquilonius, S.,
2001. Parkinsonism and neck extensor myopathy: A new syndrome or coincidental
findings? Arch. Neurol. 58, 232–237.
Baker, D.M., Watson, S.P., Santer, R.M., 1991. Evidence for a decrease in sympathetic
control of intestinal function in the aged rat. Neurobiol. Aging 12, 363–365.
Balice-Gordon, R.J., 1997. Age-related changes in neuromuscular innervation. Muscle
Nerve S5, S83–S87.
Banker, B.Q., Kelly, S.S., Robbins, N., 1983. Neuromuscular transmission and correlative
morphology in young and old mice. J. Physiol. 339, 355–377.
Barker, D., Saito, M., 1981. Autonomic innervation of receptors and muscle fibres in cat
skeletal muscle. Proc. R. Soc. Lond. B Biol. Sci. 212, 317–332.
Barnes, R.F., Raskind, M., Gumbretch, G., Halter, J.B., 1982. The effects of age on the
plasma catecholamine response to mental stress in man. J. Clin. Endocrinol. Metab.
54, 64–69.
Beitzel, F., Sillence, M.N., Lynch, G.S., 2007. Beta-Adrenoceptor signaling in
regenerating skeletal muscle after beta-agonist administration. Am. J. Physiol.
Endocrinol. Metab. 293, E932–940.
Bergmeister, K.D., Gröger, M., Aman, M., Willensdorfer, A., Manzano-Szalai, K.,
Salminger, S., Aszmann, O.C., 2016. Automated muscle fiber type population
analysis with ImageJ of whole rat muscles using rapid myosin heavy chain
immunohistochemistry. Muscle Nerve 54, 292–299.
Bezakova, G., Lomo, T., 2001. Muscle activity and muscle agrin regulate the organization
of cytoskeletal proteins and attached acetylcholine receptor (AchR) aggregates in
skeletal muscle fibers. J. Cell Biol. 153, 1453–1463.
Biral, D., Kern, H., Adami, N., Boncompagni, S., Protasi, F., Carraro, U., 2008. Atrophy-
resistant fibers in permanent peripheral denervation of human skeletal muscle.
Neurol. Res. 30, 137–144.
Bjorkoy, G., Lamark, T., Brech, A., Outzen, H., Perander, M., Overvatn, A., Stenmark, H.,
Johansen, T., 2005. p62/SQSTM1 forms protein aggregates degraded by autophagy
and has a protective effect on huntingtin-induced cell death. J. Cell Biol. 171,
603–614.
Blasco, A., Gras, S., Mòdol-Caballero, G., Tarabal, O., Casanovas, A., Piedrafita, L.,
Barranco, A., Das, T., Pereira, S.L., Navarro, X., Rueda, R., Esquerda, J.E.,
Calderó, J., 2020. Motoneuron deafferentation and gliosis occur in association with
neuromuscular regressive changes during ageing in mice. J. Cachexia Sarcopenia
Muscle 6, 1628–1660.
Bloemberg, D., Quadrilatero, J., 2012. Rapid determination of myosin heavy chain
expression in rat, mouse, and human skeletal muscle using multicolor
immunofluorescence analysis. PLoS One 7, e35273.
Bodine, S.C., Latres, E., Baumhueter, S., Lai, V.K., Nunez, L., Clarke, B.A.,
Poueymirou, W.T., Panaro, F.J., Na, E., Dharmarajan, K., Pan, Z.Q., Valenzuela, D.
M., DeChiara, T.M., Stitt, T.N., Yancopoulos, G.D., Glass, D.J., 2001. Identification of
ubiquitin ligases required for skeletal muscle atrophy. Science 294, 1704–1708.
Boeke, J., 1909. Ueber eine aus marklosen Fasern hervorgehende zweite Art von
hypolemmalen Nervenendplatten bei den quergestreiften Muskelfasern der
Vertebraten. Anat. Anz. 35, 481–484.
Bonaldo, P., Sandri, M., 2013. Cellular and molecular mechanisms of muscle atrophy.
Dis. Model. Mech. 6, 25–39.
Bonilla, H.J., Messi, M.L., Sadieva, K.A., Hamilton, C.A., Buchman, A.S., Delbono, O.,
2020. Semiautomatic morphometric analysis of skeletal muscle obtained by needle
biopsy in older adults. GeroScience 6, 1431–1443.
Bowman, W.C., Raper, C., 1966. Effects of sympathomimetic amines on neuromuscular
transmission. Br. J. Pharmacol. Chemother. 27, 313–331.
Brandenberger, C., Mühlfeld, C., 2017. Mechanisms of lung aging. Cell Tissue Res. 367,
469–480.
Briguet, A., Courdier-Fruh, I., Foster, M., Meier, T., Magyar, J.P., 2004. Histological
parameters for the quantitative assessment of muscular dystrophy in the mdx-mouse.
Neuromuscul. Disord. 14, 675–682.
Brooks, S.V., Faulkner, J.A., 1994. Skeletal muscle weakness in old age: underlying
mechanisms. Med. Sci. Sports Exerc. 26, 432–439.
Brown, B.A., Connolly, G.M., Mill, C.E.J., Williams, H., Angelini, G.D., Johnson, J.L.,
George, S.J., 2019. Aging differentially modulates the Wnt pro-survival signalling
pathways in vascular smooth muscle cells. Aging Cell 18, e12844.
Bruinstroop, E., Cano, G., Vanderhorst, V.G.J.M., Cavalcante, J.C., Wirth, J., Sena-
Esteves, M., Saper, C.B., 2012. Spinal projections of the A5, A6 (locus coeruleus), and
A7 noradrenergic cell groups in rats. J. Comp. Neurol. 520, 1985–2001.
Buchman, A.S., Nag, S., Shulman, J.M., Lim, A.S., VanderHorst, V.G., Leurgans, S.E.,
Schneider, J.A., Bennett, D.A., 2012. Locus coeruleus neuron density and
parkinsonism in older adults without Parkinson’s disease. Mov. Disord. 27,
1625–1631.
Buller, A.J., Eccles, J.C., Eccles, R.M., 1960a. Differentiation of fast and slow muscles in
the cat hind limb. J. Physiol. 150, 399–416.
Buller, A.J., Eccles, J.C., Eccles, R.M., 1960b. Interactions between motoneurones and
muscles in respect of the characteristic speeds of their responses. J. Physiol. 150,
417–439.
Cadwell, C.R., Palasantza, A., Jiang, X., Berens, P., Deng, Q., Yilmaz, M., Reimer, J.,
Shen, S., Bethge, M., Tolias, K.F., Sandberg, R., Tolias, A.S., 2016.
Electrophysiological, transcriptomic and morphologic profiling of single neurons
using Patch-seq. Nat. Biotechnol. 34, 199–203.
Cano-Jaimez, M., Pérez-Sánchez, F., Milán, M., Buendía, P., Ambrosio, S., Fariñas, I.,
2010. Vulnerability of peripheral catecholaminergic neurons to MPTP is not
regulated by α-synuclein. Neurobiol. Dis. 38, 92–103.
Card, J.P., Enquist, L.W., 2012. Use and visualization of neuroanatomical viral
transneuronal tracers. In: Badoer, E. (Ed.), Visualization Techniques: From
Immunohistochemistry to Magnetic Resonance Imaging. Humana Press, Totowa, NJ,
pp. 225–268.
12
Ageing Research Reviews 67 (2021) 101305
Carlson, B.M., Borisov, A.B., Dedkov, E.I., Khalyfa, A., Kostrominova, T.Y.,
Macpherson, P.C., Wang, E., Faulkner, J.A., 2002. Effects of long-term denervation
on skeletal muscle in old rats. J. Gerontol. A Biol. Sci. Med. Sci. 57, B366–374.
Carnio, S., LoVerso, F., Baraibar, Martin A., Longa, E., Khan, Muzamil M., Maffei, M.,
Reischl, M., Canepari, M., Loefler, S., Kern, H., Blaauw, B., Friguet, B., Bottinelli, R.,
Rudolf, R., Sandri, M., 2014. Autophagy impairment in muscle induces
neuromuscular junction degeneration and precocious aging. Cell Rep. 8 (5),
1509–1521.
Carter, W.J., Dang, A.Q., Faas, F.H., Lynch, M.E., 1991. Effects of clenbuterol on skeletal
muscle mass, body composition, and recovery from surgical stress in senescent rats.
Metabolism 40, 855–860.
Carter, M.E., Yizhar, O., Chikahisa, S., Nguyen, H., Adamantidis, A., Nishino, S.,
Deisseroth, K., de Lecea, L., 2010. Tuning arousal with optogenetic modulation of
locus coeruleus neurons. Nat. Neurosci. 13, 1526–1533.
Castets, P., Lin, S., Rion, N., Fulvio, S., Romanino, K., Guridi, M., 2013. Sustained
activation of mTORC1 in skeletal muscle inhibits constitutive and starvation-induced
autophagy and causes a severe, late-onset myopathy. Cell Metab. 17, 731–744.
Chiang, G.G., Abraham, R.T., 2005. Phosphorylation of mammalian target of rapamycin
(mTOR) at Ser-2448 is mediated by p70S6 kinase. J. Biol. Chem. 280, 25485–25490.
Chin, S.O., Rhee, S.Y., Chon, S., Hwang, Y.-C., Jeong, I.-K., Oh, S., Ahn, K.J., Chung, H.Y.,
Woo, J.-T., Kim, S.-W., Kim, J.-W., Kim, Y.S., Ahn, H.-Y., 2013. Sarcopenia is
independently associated with cardiovascular disease in older Korean adults: the
korea national health and nutrition examination survey (KNHANES) from 2009.
PLoS One 8, e60119.
Choi, S.J., Shively, C.A., Register, T.C., Stehle, J., High, K., Ip, E., Feng, X.,
Kritchevsky, S.B., Nicklas, B.J., Delbono, O., 2012. Force generation capacity of
single muscle fibers of vastus lateralis declines with age and correlates with physical
function in african green vervet monkeys. J. Gerontol. Biol. Sci. 68 (3), 258–267.
Cohen, T.J., Waddell, D.S., Barrientos, T., Lu, Z., Feng, G., Cox, G.A., Bodine, S.C., Yao, T.
P., 2007. The histone deacetylase HDAC4 connects neural activity to muscle
transcriptional reprogramming. J. Biol. Chem. 282, 33752–33759.
Cowen, T., Johnson, R., Soubeyre, V., Santer, R.M., 2000. Restricted diet rescues rat
enteric motor neurones from age related cell death. Gut 47, 653–660.
Cuervo, A.M., Palmer, A., Rivett, A.J., Knecht, E., 1995. Degradation of proteasomes by
lysosomes in rat liver. Eur. J. Biochem. 227, 792–800.
de Waegh, S.M., Lee, V.M., Brady, S.T., 1992. Local modulation of neurofilament
phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann
cells. Cell 68, 451–463.
DeAndrade, J., Pedersen, M., Garcia, L., Nau, P., 2018. Sarcopenia is a risk factor for
complications and an independent predictor of hospital length of stay in trauma
patients. J. Surg. Res. 221, 161–166.
Del Tredici, K., Braak, H., 2013. Dysfunction of the locus coeruleus-norepinephrine
system and related circuitry in Parkinson’s disease-related dementia. J. Neurol.
Neurosurg. Psychiatry 84, 774–783.
Delbono, O., 1992. Calcium current activation and charge movement in denervated
mammalian skeletal muscle fibres. J. Physiol. 451, 187–203.
Delbono, O., 2003. Neural control of aging skeletal muscle. Aging Cell 2, 21–29.
Delbono, O., 2011a. Excitation-contraction coupling regulation in aging skeletal muscle.
In: Lynch, G.S. (Ed.), Sarcopenia-Age-Related Muscle Wasting and Weakness.
Mechanisms and Treatment. Springer, Victoria, pp. 113–134.
Delbono, O., 2011b. Expression and regulation of excitation-contraction coupling
proteins in aging skeletal muscle. Curr. Aging Sci. 4 (3), 248–259.
Delbono, O., Stefani, E., 1993. Calcium current inactivation in denervated rat skeletal
muscle fibres. J. Physiol. 460, 173–183.
Delbono, O., O’Rourke, K.S., Ettinger, W.H., 1995. Excitation-calcium release uncoupling
in aged single human skeletal muscle fibers. J. Membr. Biol. 148, 211–222.
Delbono, O., Renganathan, M., Messi, M.L., 1997. Regulation of mouse skeletal muscle L-
type Ca2+ channel by activation of the insulin-like growth factor-1 receptor.
J. Neurosci. 17, 6918–6928.
Distefano, G., Goodpaster, B.H., 2018. Effects of exercise and aging on skeletal muscle.
Cold Spring Harb. Perspect. Med. 8 (3), a029785.
Dobbins, E.G., Feldman, J.L., 1994. Brainstem network controlling descending drive to
phrenic motoneurons in rat. J. Comp. Neurol. 347, 64–86.
Docherty, J.R., 2002. Age-related changes in adrenergic neuroeffector transmission.
Auton. Neurosci. 96, 8–12.
Doherty, T.J., Vandervoort, A.A., Taylor, A.W., Brown, W.F., 1993. Effects of motor unit
losses on strength in older men and women. J. Appl. Physiol. 74, 868–874.
Doxakis, E., Howard, L., Rohrer, H., Davies, A.M., 2008. HAND transcription factors are
required for neonatal sympathetic neuron survival. EMBO Rep. 9, 1041–1047.
Du, Y., Zhang, M., Zhao, W., Shu, Y., Gao, M., Zhuang, Y., Yang, T., Mu, W., Li, T., Li, X.,
Sun, F., Pan, Z., Lu, Y., 2017. Let-7a regulates expression of β1-adrenoceptors and
forms a negative feedback circuit with the β1-adrenoceptor signaling pathway in
chronic ischemic heart failure. Oncotarget 8, 8752–8764.
Dutta, C., 1997. Significance of sarcopenia in the elderly. J. Nutr. 127, 992S–993S.
Eftimie, R., Brenner, H.R., Buonanno, A., 1991. Myogenin and MyoD join a family of
skeletal muscle genes regulated by electrical activity. Proc. Natl. Acad. Sci. U. S. A.
88, 1349–1353.
Eichmann, A., Brunet, I., 2014. Arterial innervation in development and disease. Sci.
Transl. Med. 6, 252.
Einsiedel, L.J., Luff, A.R., 1992. Effect of partial denervation on motor units in the ageing
rat medial gastrocnemius. J. Neurol. Sci. 112, 178–184.
El-Salhy, M., Sandström, O., Holmlund, F., 1999. Age-induced changes in the enteric
nervous system in the mouse. Mech. Ageing Dev. 107, 93–103.
Esler, M.D., Thompson, J.M., Kaye, D.M., Turner, A.G., Jennings, G.L., Cox, H.S.,
Lambert, G.W., Seals, D.R., 1995. Effects of aging on the responsiveness of the
human cardiac sympathetic nerves to stressors. Circulation 91, 351–358.
O. Delbono et al.
Faulkner, J.A., Brooks, S.V., Opiteck, J.A., 1993. Injury to skeletal muscle fibers during
contractions: conditions of occurrence and prevention. Phys. Ther. 73, 911–921.
Felten, S.Y., Bellinger, D.L., Collier, T.J., Coleman, P.D., Felten, D.L., 1987. Decreased
sympathetic innervation of spleen in aged fischer 344 rats. Neurobiol. Aging 8,
159–165.
Femminella, G.D., Rengo, G., Komici, K., Iacotucci, P., Petraglia, L., Pagano, G., de
Lucia, C., Canonico, V., Bonaduce, D., Leosco, D., Ferrara, N., 2014. Autonomic
dysfunction in Alzheimer’s disease: tools for assessment and review of the literature.
J. Alzheimers Dis. 42, 369–377.
Ferrucci, L., Harris, T.B., Guralnik, J.M., Tracy, R.P., Corti, M.C., Cohen, H.J.,
Penninx, B., Pahor, M., Wallace, R., Havlik, R.J., 1999. Serum IL-6 level and the
development of disability in older persons. J. Am. Geriatr. Soc. 47, 639–646.
Ferrucci, L., Penninx, B.W., Volpato, S., Harris, T.B., Bandeen-Roche, K., Balfour, J.,
Leveille, S.G., Fried, L.P., Md, J.M., 2002. Change in muscle strength explains
accelerated decline of physical function in older women with high interleukin-6
serum levels. J. Am. Geriatr. Soc. 50, 1947–1954.
Figueiredo, V.C., Markworth, J.F., Cameron-Smith, D., 2017. Considerations on mTOR
regulation at serine 2448: implications for muscle metabolism studies. Cell. Mol. Life
Sci. 74, 2537–2545.
Fish, L.A., Fallon, J.R., 2020. Multiple MuSK signaling pathways and the aging
neuromuscular junction. Neurosci. Lett. 731, 135014.
Frey, D., Schneider, C., Xu, L., Borg, J., Spooren, W., Caroni, P., 2000. Early and selective
loss of neuromuscular synapse subtypes with low sprouting competence in
motoneuron diseases. J. Neurosci. 20, 2534–2542.
Frontera, W.R., Suh, D., Krivickas, L.S., Hughes, V.A., Goldstein, R., Roubenoff, R., 2000.
Skeletal muscle fiber quality in older men and women. Am. J. Physiol. Cell Physiol.
279, C611–618.
Fry, C.S., Lee, J.D., Mula, J., Kirby, T.J., Jackson, J.R., Liu, F., Yang, L., Mendias, C.L.,
Dupont-Versteegden, E.E., McCarthy, J.J., Peterson, C.A., 2015. Inducible depletion
of satellite cells in adult, sedentary mice impairs muscle regenerative capacity
without affecting sarcopenia. Nat. Med. 21, 76–80.
Furlow, J.D., Watson, M.L., Waddell, D.S., Neff, E.S., Baehr, L.M., Ross, A.P., Bodine, S.
C., 2013. Altered gene expression patterns in muscle ring finger 1 null mice during
denervation- and dexamethasone-induced muscle atrophy. Physiol. Genomics 45,
1168–1185.
Garcia-Prat, L., Martinez-Vicente, M., Perdiguero, E., Ortet, L., Rodriguez-Ubreva, J.,
Rebollo, E., Ruiz-Bonilla, V., Gutarra, S., Ballestar, E., Serrano, A.L., Sandri, M.,
Munoz-Canoves, P., 2016. Autophagy maintains stemness by preventing senescence.
Nature 529, 37–42.
Geppetti, P., De Rossi, M., Mione, M., Renzi, D., Amenta, F., 1988. Age-related changes in
vasoactive intestinal polypeptide levels and distribution in the rat lung. J. Neural
Transm. 74, 1–10.
Goncalves, D.A., Silveira, W.A., Lira, E.C., Graca, F.A., Paula-Gomes, S., Zanon, N.M.,
Kettelhut, I.C., Navegantes, L.C., 2012. Clenbuterol suppresses proteasomal and
lysosomal proteolysis and atrophy-related genes in denervated rat soleus muscles
independently of Akt. Am. J. Physiol. Endocrinol. Metab. 302, E123–133.
Gonzalez, A., Kirsch, W.G., Shirokova, N., Pizarro, G., Brum, G., Pessah, I.N., Stern, M.D.,
Cheng, H., Rios, E., 2000a. Involvement of multiple intracellular release channels in
calcium sparks of skeletal muscle. Proc. Natl. Acad. Sci. U. S. A. 97, 4380–4385.
Gonzalez, E., Messi, M.L., Delbono, O., 2000b. The specific force of single intact extensor
digitorum longus and soleus mouse muscle fibers declines with aging. J. Membr.
Biol. 178, 175–183.
Gonzalez-Freire, M., de Cabo, R., Studenski, S., Ferrucci, L., 2014. The Neuromuscular
Junction (NMJ): aging at the crossroad between nerves and muscle. Front. Aging
Neurosci. 6, 208.
Gosztonyi, G., Naschold, U., Grozdanovic, Z., Stoltenburg-Didinger, G., Gossrau, R.,
2001. Expression of Leu-19 (CD56, N-CAM) and nitric oxide synthase (NOS) I in
denervated and reinnervated human skeletal muscle. Microsc. Res. Tech. 55,
187–197.
Greensmith, L., Vrbova, G., 1996. Motoneurone survival: a functional approach. Trends
Neurosci. 19, 450–455.
Griffin, J.W., Thompson, W.J., 2008. Biology and pathology of nonmyelinating Schwann
cells. Glia 56, 1518–1531.
Gropp, K.E., 2017. Skeletal muscle toolbox. Toxicol. Pathol. 45, 939–942.
Hadley, D., Murphy, T., Valladares, O., Hannenhalli, S., Ungar, L., Kim, J., Bucan, M.,
2006. Patterns of sequence conservation in presynaptic neural genes. Genome Biol.
7, R105.
Hase, A., Suzuki, H., Arahata, K., Akazawa, C., 1999. Expression of human GFR alpha-1
(GDNF receptor) at the neuromuscular junction and myelinated nerves. Neurosci.
Lett. 269, 55–57.
Hashizume, K., Kanda, K., Burke, R., 1988. Medial gastrocnemius motor nucleus in the
rat: age-related changes in the number and size of motoneurons. J. Comp. Neurol.
269, 425–430.
Hendershot, T.J., Liu, H., Clouthier, D.E., Shepherd, I.T., Coppola, E., Studer, M.,
Firulli, A.B., Pittman, D.L., Howard, M.J., 2008. Conditional deletion of Hand2
reveals critical functions in neurogenesis and cell type-specific gene expression for
development of neural crest-derived noradrenergic sympathetic ganglion neurons.
Dev. Biol. 319, 179–191.
Henderson, C.E., Phillips, H.S., Pollock, R.A., Davies, A.M., Lemeulle, C., Armanini, M.,
Simmons, L., Moffet, B., Vandlen, R.A., Simpson, L., Koliatsos, V.E., Rosenthal, A.,
et al., 1994. GDNF: a potent survival factor for motoneurons present in peripheral
nerve and muscle. Science 266, 1062–1064.
Hendrickse, P., Galinska, M., Hodson-Tole, E., Degens, H., 2018. An evaluation of
common markers of muscle denervation in denervated young-adult and old rat
gastrocnemius muscle. Exp. Gerontol. 106, 159–164.
13
Ageing Research Reviews 67 (2021) 101305
Hepple, R.T., 2018. When motor unit expansion in ageing muscle fails, atrophy ensues.
J. Physiol. 596, 1545–1546.
Hirner, S., Krohne, C., Schuster, A., Hoffmann, S., Witt, S., Erber, R., Sticht, C., Gasch, A.,
Labeit, S., Labeit, D., 2008. MuRF1-dependent regulation of systemic carbohydrate
metabolism as revealed from transgenic mouse studies. J. Mol. Biol. 379, 666–677.
Hoeldtke, R.D., Cilmi, K.M., 1985. Effects of aging on catecholamine metabolism. J. Clin.
Endocrinol. Metab. 60, 479–484.
Hook, P., Li, X., Sleep, J., Hughes, S., Larsson, L., 1999. In vitro motility speed of slow
myosin extracted from single soleus fibres from young and old rats. J. Physiol. 520,
463–471.
Hwang, A.B., Brack, A.S., 2018. Chapter Ten - muscle stem cells and aging. In:
Sassoon, D. (Ed.), Current Topics in Developmental Biology. Academic Press,
pp. 299–322.
Ichimura, Y., Kominami, E., Tanaka, K., Komatsu, M., 2008. Selective turnover of p62/
A170/SQSTM1 by autophagy. Autophagy 4, 1063–1066.
Insel, P.A., 1993. Adrenergic receptors, G proteins, and cell regulation: implications for
aging research. Exp. Gerontol. 28, 341–348.
Jackson, M.J., 2016. Reactive oxygen species in sarcopenia: Should we focus on excess
oxidative damage or defective redox signaling? Mol. Aspects Med. 50, 33–40.
Jacobs, B.L., Abercrombie, E.D., Fornal, C.A., Levine, E.S., Morilak, D.A., Stafford, I.L.,
1991. Single-unit and physiological analyses of brain norepinephrine function in
behaving animals. Prog. Brain Res. 88, 159–165.
Jang, Y.C., Lustgarten, M.S., Liu, Y., Muller, F.L., Bhattacharya, A., Liang, H., Salmon, A.
B., Brooks, S.V., Larkin, L., Hayworth, C.R., Richardson, A., Van Remmen, H., 2010.
Increased superoxide in vivo accelerates age-associated muscle atrophy through
mitochondrial dysfunction and neuromuscular junction degeneration. FASEB J. 24,
1376–1390.
Janig, W., 2012. Complex Regional Pain Syndrome, 3rd ed. Academic, Amsterdam.
Jensen-Daham, C., Waldemar, G., Jensen, T.S., Malmqvist, L., Moeller, M.M.,
Andersen, B.B., Høgh, P., Ballegaard, M., 2015. Autonomic dysfunction in patients
with mild to moderate Alzheimer’s disease. J. Alzheimer Dis. 47, 681–689.
Jessen, K.R., Mirsky, R., 2016. The repair Schwann cell and its function in regenerating
nerves. J. Physiol. 594, 3521–3531.
Jimenez-Moreno, R., Wang, Z.M., Guerring, R.C., Delbono, O., 2008. Sarcoplasmic
reticulum Ca2+ release declines in muscle fibers from aging mice. Biophys. J. 94 (8),
3178–3188.
Johnson, H., Mossberg, K., Arvidsson, U., Piehl, F., Hokfelt, T., Ulfhake, B., 1995.
Increase in alpha-CGRP and GAP-43 in aged motoneurons: a study of peptides,
growth factors, and ChAT mRNA in the lumbar spinal cord of senescent rats with
symptoms of hindlimb incapacities. J. Comp. Neurol. 359, 69–89.
Jokl, E.J., Blanco, G., 2016. Disrupted autophagy undermines skeletal muscle adaptation
and integrity. Mamm. Genome 27, 525–537.
Jones, A., Teschendorff, A.E., Li, Q., Hayward, J.D., Kannan, A., Mould, T., West, J.,
Zikan, M., Cibula, D., Fiegl, H., Lee, S.-H., Wik, E., Hadwin, R., Arora, R., Lemech, C.,
Turunen, H., Pakarinen, P., Jacobs, I.J., Salvesen, H.B., Bagchi, M.K., Bagchi, I.C.,
Widschwendter, M., 2013. Role of DNA methylation and epigenetic silencing of
HAND2 in endometrial Cancer development. PLoS Med. 10, e1001551.
Kadhiresan, V.A., Hassett, C.A., Faulkner, J.A., 1996. Properties of single motor units in
medial gastrocnemius muscles of adult and old rats. J. Physiol. 493, 543–552.
Kallen, R.G., Sheng, Z.H., Yang, J., Chen, L.Q., Rogart, R.B., Barchi, R.L., 1990. Primary
structure and expression of a sodium channel characteristic of denervated and
immature rat skeletal muscle. Neuron 4, 233–242.
Kamwa, V., Welch, C., Hassan-Smith, Z.K., 2020. The endocrinology of sarcopenia and
frailty. Minerva Endocrinol. Online ahead of print.
Kanda, K., Hashizume, K., 1989. Changes in properties of the medial gastrocnemius
motor units in aging rats. J. Neurophysiol. 737–746.
Kanda, K., Hashizume, K., 1992. Factors causing difference in force output among motor
units in the rat medial gastrocnemius muscle. J. Physiol. 448, 677–695.
Kandinov, B., Korczyn, A.D., Rabinowitz, R., Nefussy, B., Drory, V.E., 2011. Autonomic
impairment in a transgenic mouse model of amyotrophic lateral sclerosis. Auton.
Neurosci. 159, 84–89.
Kerman, I.A., Enquist, L.W., Watson, S.J., Yates, B.J., 2003. Brainstem substrates of
sympatho-motor circuitry identified using trans-synaptic tracing with pseudorabies
virus recombinants. J. Neurosci. 23, 4657–4666.
Khan, M.M., Lustrino, D., Silveira, W.A., Wild, F., Straka, T., Issop, Y., O’Connor, E.,
Cox, D., Reischl, M., Marquardt, T., Labeit, D., Labeit, S., Benoit, E., Molgó, J.,
Lochmüller, H., Witzemann, V., Kettelhut, I.C., Navegantes, L.C.C., Pozzan, T.,
Rudolf, R., 2016. Sympathetic innervation controls homeostasis of neuromuscular
junctions in health and disease. Proc. Natl. Acad. Sci. 113, 746–750.
Khuzakhmetova, V., Bukharaeva, E., 2020. Adrenaline facilitates synaptic transmission
by synchronizing release of acetylcholine quanta from motor nerve endings. Cell.
Mol. Neurobiol. Online ahead of print.
Koneczny, I., Cossins, J., Vincent, A., 2014. The role of muscle-specific tyrosine kinase
(MuSK) and mystery of MuSK myasthenia gravis. J. Anat. 224, 29–35.
Kuba, K., 1970. Effects of catecholamines on the neuromuscular junction in the rat
diaphragm. J. Physiol. 211, 551–570.
Landi, F., Liperoti, R., Russo, A., Giovannini, S., Tosato, M., Capoluongo, E., Bernabei, R.,
Onder, G., 2012. Sarcopenia as a risk factor for falls in elderly individuals: results
from the ilSIRENTE study. Clin. Nutr. 31, 652–658.
Landi, F., Marzetti, E., Martone, A.M., Bernabei, R., Onder, G., 2014. Exercise as a
remedy for sarcopenia. Curr. Opin. Clin. Nutr. Metab. Care 17 (1), 25–31.
Laplante, M., Sabatini, D.M., 2012. mTOR signaling in growth control and disease. Cell
149, 274–293.
Larsson, L., 1995. Motor units: remodeling in aged animals. J. Gerontol. Ser. A Biol. Sci.
Med. Sci. 50, 91–95.
O. Delbono et al.
Larsson, L., Ansved, T., 1995. Effects of ageing on the motor unit. Prog. Neurobiol. 45,
397–458.
Larsson, L., Edstrom, L., 1986. Effects of age on enzyme-histochemical fibre spectra and
contractile properties of fast- and slow-twitch skeletal muscles in the rat. J. Neurol.
Sci. 76, 69–89.
Larsson, L., Ansved, T., Edstrom, L., Gorza, L., Schiaffino, S., 1991. Effects of age on
physiological, immunohistochemical and biochemical properties of fast-twitch single
motor units in the rat. J. Physiol. 443, 257–275.
Larsson, L., Degens, H., Li, M., Salviati, L., Lee, Yi., Thompson, W., Kirkland, J.L.,
Sandri, M., 2019. Sarcopenia: aging-related loss of muscle mass and function.
Physiol. Rev. 99, 427–511.
Lau, Y.S., Xu, L., Gao, Y., Han, R., 2018. Automated muscle histopathology analysis using
CellProfiler. Skelet. Muscle 8 (1), 32.
Le Panse, B., Arlettaz, A., Portier, H., Lecoq, A.M., De Ceaurriz, J., Collomp, K., 2007.
Effects of acute salbutamol intake during supramaximal exercise in women. Br. J.
Sports Med. 41, 430–434.
Leeuwenburgh, C., Hansen, P., Shaish, A., Holloszy, J.O., Heinecke, J.W., 1998. Markers
of protein oxidation by hydroxyl radical and reactive nitrogen species in tissues of
aging rats. Am. J. Physiol. 274, R453–461.
Lefeuvre, B., Crossin, F., Fontaine-Perus, J., Bandman, E., Gardahaut, M.F., 1996.
Innervation regulates myosin heavy chain isoform expression in developing skeletal
muscle fibers. Mech. Dev. 58, 115–127.
Lexell, J., 1995. Human aging, muscle mass, and fiber type composition. J. Gerontol. Ser.
A Biol. Sci. Med. Sci. 50, 11–16.
Li, Y., Thompson, W.J., 2011. Nerve terminal growth remodels neuromuscular synapses
in mice following regeneration of the postsynaptic muscle fiber. J. Neurosci. 31,
13191–13203.
Li, S.T., Holmes, C., Kopin, I.J., Goldstein, D.S., 2003. Aging-related changes in cardiac
sympathetic function in humans, assessed by 6-18F-fluorodopamine PET scanning.
J. Nucl. Med. 44, 1599–1603.
Li, W., Lam, M.T., Notani, D., 2014. Enhancer RNAs. Cell Cycle 13, 3151–3152.
Li, W., Notani, D., Rosenfeld, M.G., 2016. Enhancers as non-coding RNA transcription
units: recent insights and future perspectives. Nat. Rev. Genet. 17, 207–223.
Lindquist, S., Stangel, M., 2011. Update on treatment options for Lambert-Eaton
myasthenic syndrome: focus on use of amifampridine. Neuropsychiatr. Dis. Treat. 7,
341–349.
Lipsitz, L.A., Novak, V., 2008. Aging and Autonomic Function, third ed. Mayo
Foundation, Rochester.
Low, P.A., Bennarroch, E.E., 2008. Clinical Autonomic Disorders, 3rd ed. Lippincott
Williams & Wilkins.
Lynch, G.S., Ryall, J.G., 2008. Role of beta-adrenoceptor signaling in skeletal muscle:
implications for muscle wasting and disease. Physiol. Rev. 88, 729–767.
Lynch, N.A., Metter, E.J., Lindle, R.S., Fozard, J.L., Tobin, J.D., Roy, T.A., Fleg, J.L.,
Hurley, B.F., 1999. Muscle quality. I. Age-associated differences between arm and
leg muscle groups. J. Appl. Physiol. 86, 188–194.
Machado, J., Silveira, W.A., Gonçalves, D.A., Schavinski, A.Z., Khan, M.M., Zanon, N.M.,
Diaz, M.B., Rudolf, R., Kettelhut, I.C., Navegantes, L.C., 2019. А− Calcitonin gene-
related peptide inhibits autophagy and calpain systems and maintains the stability of
neuromuscular junction in denervated muscles. Mol. Metab. 28, 91–106.
Madisen, L., Mao, T., Koch, H., Zhuo, J.-m., Berenyi, A., Fujisawa, S., Hsu, Y.-W.A.,
Garcia, Iii, A.J., Gu, X., Zanella, S., Kidney, J., Gu, H., Mao, Y., Hooks, B.M.,
Boyden, E.S., Buzsáki, G., Ramirez, J.M., Jones, A.R., Svoboda, K., Han, X.,
Turner, E.E., Zeng, H., 2012. A toolbox of Cre-dependent optogenetic transgenic
mice for light-induced activation and silencing. Nat. Neurosci. 15 (5), 793–802.
Maeda, K., Akagi, J., 2016. Sarcopenia is an independent risk factor of dysphagia in
hospitalized older people. Geriatr. Gerontol. Int. 16, 515–521.
Maegawa, S., Hinkal, G., Kim, H.S., Shen, L., Zhang, L., Zhang, J., Zhang, N., Liang, S.,
Donehower, L.A., Issa, J.P., 2010. Widespread and tissue specific age-related DNA
methylation changes in mice. Genome Res. 20, 332–340.
Masiero, E., Agatea, L., Mammucari, C., Blaauw, B., Loro, E., Komatsu, M., Metzger, D.,
Reggiani, C., Schiaffino, S., Sandri, M., 2009. Autophagy is required to maintain
muscle mass. Cell Metab. 10, 507–515.
Mayeuf-Louchart, A., Hardy, D., Thorel, Q., Roux, P., Gueniot, L., Briand, D.,
Mazeraud, A., Bougle, A., Shorte, S.L., Staels, B., Chretien, F., Duez, H.,
Danckaert, A., 2018. MuscleJ: a high-content analysis method to study skeletal
muscle with a new Fiji tool. Skelet. Muscle 8 (1), 25.
Mazzeo, R.S., Rajkumar, C., Jennings, G., Esler, M., 1997. Norepinephrine spillover at
rest and during submaximal exercise in young and old subjects. J. Appl. Physiol. 82,
1869–1874.
Mei, H., Ho, M.K.C., Yung, L.Y., Wu, Z., Ip, N.Y., Wong, Y.H., 2011. Expression of Gαz in
C2C12 cells restrains myogenic differentiation. Cell. Signal. 23, 389–397.
Meitzen, J., Perry, A.N., Westenbroek, C., Hedges, V.L., Becker, J.B., Mermelstein, P.G.,
2013. Enhanced striatal β1-Adrenergic receptor expression following hormone loss
in adulthood is programmed by both early sexual differentiation and puberty: a
study of humans and rats. Endocrinology 154, 1820–1831.
Messéant, J., Ezan, J., Delers, P., Glebov, K., Marchiol, C., Lager, F., Renault, G.,
Tissir, F., Montcouquiol, M., Sans, N., Legay, C., Strochlic, L., 2017. Wnt proteins
contribute to neuromuscular junction formation through distinct signaling
pathways. Development 144, 1712–1724.
Messi, M.L., Li, T., Wang, Z.M., Marsh, A.P., Nicklas, B., Delbono, O., 2016. Resistance
training enhances skeletal muscle innervation without modifying the number of
satellite cells or their myofiber association in obese older adults. J. Gerontol. A Biol.
Sci. Med. Sci. 71, 1273–1280.
Miazaki, M., Viana, M.P., Yang, Z., Comin, C.H., Wang, Y., da, F.C.L., Xu, X., 2015.
Automated high-content morphological analysis of muscle fiber histology. Comput.
Biol. Med. 63, 28–35.
14
Ageing Research Reviews 67 (2021) 101305
Minetti, G.C., Feige, J.N., Bombard, F., Heier, A., Morvan, F., Nürnberg, B., Leiss, V.,
Birnbaumer, L., Glass, D.J., Fornaro, M., 2014. Gαi2 signaling is required for skeletal
muscle growth, regeneration, and satellite cell proliferation and differentiation. Mol.
Cell. Biol. 34, 619–630.
Mirotznik, R.R., Zheng, X., Stanley, E.F., 2000. G-protein types involved in calcium
channel inhibition at a presynaptic nerve terminal. J. Neurosci. 20, 7614–7621.
Mora, A., Sandve, G.K., Gabrielsen, O.S., Eskeland, R., 2016. In the loop: promoter-
enhancer interactions and bioinformatics. Brief Bioinform 17, 980–995.
Moresi, V., Williams, A.H., Meadows, E., Flynn, J.M., Potthoff, M.J., McAnally, J.,
Shelton, J.M., Backs, J., Klein, W.H., Richardson, J.A., Bassel-Duby, R., Olson, E.N.,
2010. Myogenin and class II HDACs control neurogenic muscle atrophy by inducing
E3 ubiquitin ligases. Cell 143, 35–45.
Moriscot, A.S., Baptista, I.L., Bogomolovas, J., Witt, C., Hirner, S., Granzier, H.,
Labeit, S., 2010. MuRF1 is a muscle fiber-type II associated factor and together with
MuRF2 regulates type-II fiber trophicity and maintenance. J. Struct. Biol. 170,
344–353.
Moxham, C.M., Malbon, C.C., 1996. Insulin action impaired by deficiency of the G-
protein subunit Gi[alpha]2. Nature 379, 840–844.
Mu, L., Sobotka, S., Chen, J., Su, H., Sanders, I., Adler, C.H., Shill, H.A., Caviness, J.N.,
Samanta, J.E., Beach, T.G., Arizona Parkinson’s Disease, C, 2013. Alpha-synuclein
pathology and axonal degeneration of the peripheral motor nerves innervating
pharyngeal muscles in Parkinson disease. J. Neuropathol. Exp. Neurol. 72, 119–129.
Munver, R., Volfson, I.A., 2006. Adrenal insufficiency: diagnosis and management. Curr.
Urol. Rep. 7, 80–85.
Navegantes, L.C., Baviera, A.M., Kettelhut, I.C., 2009. The inhibitory role of sympathetic
nervous system in the Ca2+-dependent proteolysis of skeletal muscle. Braz. J. Med.
Biol. Res. 42, 21–28.
Ng, A.V., Callister, R., Johnson, D.G., Seals, D.R., 1994. Sympathetic neural reactivity to
stress does not increase with age in healthy humans. Am. J. Physiol. Heart Circ.
Physiol. 267, H344–H353.
Nishiguchi, S., Yamada, M., Shirooka, H., Nozaki, Y., Fukutani, N., Tashiro, Y.,
Hirata, H., Yamaguchi, M., Tasaka, S., Matsushita, T., Matsubara, K., Tsuboyama, T.,
Aoyama, T., 2016. Sarcopenia as a risk factor for cognitive deterioration in
community-dwelling older adults: a 1-Year prospective study. J. Am. Med. Dir.
Assoc. 17, 372.e375-372.e378.
O’Suilleabhain, P., Low, P.A., Lennon, V.A., 1998. Autonomic dysfunction in the
Lambert-Eaton myasthenic syndrome: serologic and clinical correlates. Neurology
50, 88–93.
Ohara, O., Gahara, Y., Miyake, T., Teraoka, H., Kitamura, T., 1993. Neurofilament
deficiency in quail caused by nonsense mutation in neurofilament-L gene. J. Cell
Biol. 121, 387–395.
Okada, K., Inoue, A., Okada, M., Murata, Y., Kakuta, S., Jigami, T., Kubo, S., Shiraishi, H.,
Eguchi, K., Motomura, M., Akiyama, T., Iwakura, Y., Higuchi, O., Yamanashi, Y.,
2006. The muscle protein Dok-7 is essential for neuromuscular synaptogenesis.
Science 312, 1802–1805.
Okamoto, L.E., Biaggioni, I., 2012. Chronic fatigue syndrome and the autonomic nervous
system. In: Robertson, D., Biaggioni, I., Burnstock, G., Low, P.A., Paton, J.F.R. (Eds.),
Primer on the Autonomic Nervous System, 3rd ed. Academic, Amsterdam,
pp. 531–534.
Ooi, W., Barrett, S., Hossain, M., Kelley-Gagnon, M., Lipsitz, L.A., 1997. PAtterns of
orthostatic blood pressure change and their clinical correlates in a frail, elderly
population. JAMA 277, 1299–1304.
Orimo, S., Kanazawa, T., Nakamura, A., Uchihara, T., Mori, F., Kakita, A.,
Wakabayashi, K., Takahashi, H., 2007. Degeneration of cardiac sympathetic nerve
can occur in multiple system atrophy. Acta Neuropathol. 113, 81–86.
Owino, W., Yang, S.Y., Goldspink, G., 2001. Age-related loss of skeletal muscle function
and the inability to express the autocrine form of insulin-like growth factor-1 (MGF)
in response to mechanical overload. FEBS Lett. 505, 259–263.
Palmer, G.J., Ziegler, M.G., Lake, C.R., 1978. Response of norepinephrine and blood
pressure to stress increases with age. J. Gerontol. 33, 482–487.
Pascualya, M., Petrie, E.C., Brodkin, K., Peskind, E.R., Veith, R.C., Raskind, M.A., 1999.
Effects of advanced aging on plasma catecholamine responses to the cold pressor
test. Neurobiol. Aging 20, 637–642.
Payne, A.M., Delbono, O., 2004. Neurogenesis of excitation-contraction uncoupling in
aging skeletal muscle. Exerc. Sport Sci. Rev. 32, 36–40.
Payne, A.M., Messi, M.L., Zheng, Z., Delbono, O., 2006a. Motor neuron targeting of IGF-1
attenuates age-related external Ca2+-dependent skeletal muscle contraction in
senescent mice. J. Physiol. 570, 283–294.
Payne, A.M., Zheng, Z., Messi, M.L., Milligan, C.E., Gonzalez, E., Delbono, O., 2006b.
Motor neurone targeting of IGF-1 prevents specific force decline in ageing mouse
muscle. J. Physiol. 570, 283–294.
Pellegrino, M.J., Parrish, D.C., Zigmond, R.E., Habecker, B.A., 2011. Cytokines inhibit
norepinephrine transporter expression by decreasing Hand2. Mol. Cell. Neurosci. 46,
671–680.
Personius, K.E., Balice-Gordon, R.J., 2000. Activity-dependent editing of neuromuscular
synaptic connections. Brain Res. Bull. 53, 513–522.
Pertl, C., Eblenkamp, M., Pertl, A., Pfeifer, S., Wintermantel, E., Lochmuller, H.,
Walter, M.C., Krause, S., Thirion, C., 2013. A new web-based method for automated
analysis of muscle histology. BMC Musculoskelet. Disord. 14, 26.
Pette, D., Staron, R.S., 2001. Transitions of muscle fiber phenotypic profiles. Histochem.
Cell Biol. 115, 359–372.
Pfleger, J., Gresham, K., Koch, W.J., 2019. G protein-coupled receptor kinases as
therapeutic targets in the heart. Nat. Rev. Cardiol. 16, 612–622.
Phillips, R.J., Powley, T.L., 2001. As the gut ages: timetables for aging of innervation
vary by organ in the Fischer 344 rat. J. Comp. Neurol. 434, 358–377.
O. Delbono et al.
Phillips, R.J., Powley, T.L., 2007. Innervation of the gastrointestinal tract: patterns of
aging. Auton. Neurosci. 136, 1–19.
Picca, A., Calvani, R., Leeuwenburgh, C., Coelho-Junior, H.J., Bernabei, R., Landi, F.,
Marzetti, E., 2019. Targeting mitochondrial quality control for treating sarcopenia:
lessons from physical exercise. Expert Opin. Ther. Targets 23, 153–160.
Pigna, E., Renzini, A., Greco, E., Simonazzi, E., Fulle, S., Mancinelli, R., Moresi, V.,
Adamo, S., 2018. HDAC4 preserves skeletal muscle structure following long-term
denervation by mediating distinct cellular responses. Skelet. Muscle 8, 6.
Powley, T.L., 2008. Central control of autonomic functions: organization of the
autonomic nervous system. In: Squire, L., Berg, D., Bloom, F., du Lac, S., Gosh, A.,
Spitzer, N. (Eds.), Fundamental Neuroscience, third ed. Elsevier Inc., Burlington,
MA. USA, pp. 807–928.
Purves, D., Augustine, G.J., Fitzpatrick, D., Hall, W.C., LaMantia, A.S., White, L.E., 2011.
Neuroscience, 5th ed. Sinauer Associates, Inc., Massachusetts, USA.
Purves-Smith, F.M., Sgarioto, N., Hepple, R.T., 2014. Fiber typing in aging muscle. Exerc.
Sport Sci. Rev. 42, 45–52.
Radovanovic, D., Peikert, K., Lindström, M., Domellöf, F.P., 2015. Sympathetic
innervation of human muscle spindles. J. Anat. 226, 542–548.
Rauen, T., Hedrich, C.M., Tenbrock, K., Tsokos, G.C., 2013. cAMP responsive element
modulator: a critical regulator of cytokine production. Trends Mol. Med. 19,
262–269.
Renganathan, M., Sonntag, W.E., Delbono, O., 1997. L-type Ca2+ channel-insulin-like
growth factor-1 receptor signaling impairment in aging rat skeletal muscle. Biochem.
Biophys. Res. Commun. 235, 784–789.
Rengo, G., Pagano, G., Vitale, D.F., Formisano, R., Komici, K., Petraglia, L., Parisi, V.,
Femminella, G.D., de Lucia, C., Paolillo, S., Cannavo, A., Attena, E., Pellegrino, T.,
Dellegrottaglie, S., Memmi, A., Trimarco, B., Cuocolo, A., Filardi, P.P., Leosco, D.,
Ferrara, N., 2016. Impact of aging on cardiac sympathetic innervation measured by
123I-mIBG imaging in patients with systolic heart failure. Eur. J. Nucl. Med. Mol.
Imaging 43, 2392–2400.
Révész, D., Verhoeven, J.E., Milaneschi, Y., de Geus, E.J.C.N., Wolkowitz, O.M.,
Penninx, B.W.J.H., 2014. Dysregulated physiological stress systems and accelerated
cellular aging. Neurobiol. Aging 35, 1422–1430.
Ricci, A., Mariotta, S., Greco, S., Pallone, G., Papale, M., Bisetti, A., 1997. Age-related
changes of the noradrenergic innervation of rat tracheo-bronchial tree and
pulmonary vasculature. Mech. Ageing Dev. 99, 245–255.
Ristow, M., 2014. Unraveling the truth about antioxidants: mitohormesis explains ROS-
induced health benefits. Nat. Med. 20, 709–711.
Robertson, D., Biaggioni, I., Burnstock, G., Low, P.A., Paton, J.F.R., 2012. Primer on the
Autonomic Nervous System, third ed. Elsevier Inc., London, UK.
Robinson, T.E., 1978. Electrical stimulation of the brain stem in freely moving rats: I.
Effects on behavior. Physiol. Behav. 21, 223–231.
Rodovalho, G.V., Drummond, L.R., Coimbra, C.C., 2020. Involvement of brainstem
noradrenergic system in cutaneous heat loss during exercise. Brain Res. Bull. 164,
372–379.
Rodrigues, A.C.Z., Messi, M.L., Wang, Z.-M., Abba, M.C., Pereyra, A., Birbrair, A.,
Zhang, T., O’Meara, M., Kwan, P., Lopez, E.I.S., Willis, M.S., Mintz, A., Files, D.C.,
Furdui, C., Oppenheim, R.W., Delbono, O., 2018. The sympathetic nervous system
regulates skeletal muscle motor innervation and acetylcholine receptor stability.
Acta Physiol. 225, e13195.
Rodrigues, A.Z.C., Wang, Z.-M., Messi, M.L., Delbono, O., 2019. Sympathomimetics
regulate neuromuscular junction transmission through TRPV1, P/Q- and N-type Ca2
+ channels. Mol. Cell. Neurosci. 95, 59–70.
Rodrigues, A.C.Z., Wang, Z.-M., Messi, M.L., Bonilla, H.J., Liu, L., Freeman, W.M.,
Delbono, O., 2020. Heart and neural crest derivative 2-induced preservation of
sympathetic neurons attenuates sarcopenia with aging. J. Cachexia Sarcopenia
Muscle 11.
Rohrer, H., 2011. Transcriptional control of differentiation and neurogenesis in
autonomic ganglia. Eur. J. Neurosci. 34, 1563–1573.
Rommel, C., Bodine, S.C., Clarke, B.A., Rossman, R., Nunez, L., Stitt, T.N.,
Yancopoulos, G.D., Glass, D.J., 2001. Mediation of IGF-1-induced skeletal myotube
hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat. Cell Biol.
3, 1009–1013.
Rosenbaum, D.M., Rasmussen, S.G., Kobilka, B.K., 2009. The structure and function of G-
protein-coupled receptors. Nature 459, 356–363.
Rosenberg, 1989. Summary comments. Am. J. Clin. Nutr. 50, 1231–1233.
Rubinsztein, D.C., Marino, G., Kroemer, G., 2011. Autophagy and aging. Cell 146,
682–695.
Rudolf, R., Bogomolovas, J., Strack, S., Choi, K.R., Khan, M.M., Wagner, A., Brohm, K.,
Hanashima, A., Gasch, A., Labeit, D., Labeit, S., 2013. Regulation of nicotinic
acetylcholine receptor turnover by MuRF1 connects muscle activity to endo/
lysosomal and atrophy pathways. Age (Dordr) 35, 1663–1674.
Ruegg, M.A., Bixby, J.L., 1998. Agrin orchestrates synaptic differentiation at the
vertebrate neuromuscular junction. Trends Neurosci. 21, 22–27.
Ryall, J.G., Lynch, G.S., 2008. The potential and the pitfalls of beta-adrenoceptor
agonists for the management of skeletal muscle wasting. Pharmacol. Ther. 120,
219–232.
Ryall, J.G., Schertzer, J.D., Alabakis, T.M., Gehrig, S.M., Plant, D.R., Lynch, G.S., 2008.
Intramuscular {beta}2-agonist administration enhances early regeneration and
functional repair in rat skeletal muscle after myotoxic injury. J. Appl. Physiol. 105
(1), 165–172.
Rychlik, J.L., Hsieh, M., Eiden, L.E., Lewis, E.J., 2005. Phox2 and dHAND transcription
factors select shared and unique target genes in the noradrenergic cell type. J. Mol.
Neurosci. 27, 281–292.
Ryu, Y.K., Collins, S.E., Ho, H.-Y.H., Zhao, H., Kuruvilla, R., 2013. An autocrine Wnt5a-
Ror signaling loop mediates sympathetic target innervation. Dev. Biol. 377, 79–89.
15
Ageing Research Reviews 67 (2021) 101305
Sakaguchi, T., Okada, M., Kitamura, T., Kawasaki, K., 1993. Reduced diameter and
conduction velocity of myelinated fibers in the sciatic nerve of a neurofilament-
deficient mutant quail. Neurosci. Lett. 153, 65–68.
Sakellariou, G.K., Lightfoot, A.P., Earl, K.E., Stofanko, M., McDonagh, B., 2017. Redox
homeostasis and age-related deficits in neuromuscular integrity and function.
J. Cachexia Sarcopenia Muscle 8, 881–906.
Sakuma, K., Kinoshita, M., Ito, Y., Aizawa, M., Aoi, W., Yamaguchi, A., 2016. p62/
SQSTM1 but not LC3 is accumulated in sarcopenic muscle of mice. J. Cachexia
Sarcopenia Muscle 7, 204–212.
Salminen, A.L., Brandt, A., Samuelsson, K., Toytari, O., Malmivaara, A., 2009. Mobility
devices to promote activity and participation: a systematic review. J. Rehabil. Med.
41, 697–706.
Salvatori, S., Damiani, E., Zorzato, F., Volpe, P., Pierobon, S., Quaglino, D., Salviati, G.,
Margreth, A., 1988. Denervation-induced proliferative changes of triads in rabbit
skeletal muscle. Muscle Nerve 11, 1246–1259.
Santulli, G., Iaccarino, G., 2013. Pinpointing beta adrenergic receptor in ageing
pathophysiology: victim or executioner? Evidence from crime scenes. Immun.
Ageing 10 (1), 10.
Savitt, J.M., Jang, S.S., Mu, W., Dawson, V.L., Dawson, T.M., 2005. Bcl-x is required for
proper development of the mouse substantia nigra. J. Neurosci. 25, 6721–6728.
Schiaffino, S., 2018. Muscle fiber type diversity revealed by anti-myosin heavy chain
antibodies. FEBS J. 285, 3688–3694.
Schiaffino, S., Reggiani, C., 1994. Myosin isoforms in mammalian skeletal muscle.
J. Appl. Physiol. 77, 493–501.
Schiaffino, S., Reggiani, C., 1996. Molecular diversity of myofibrillar proteins: gene
regulation and functional significance. Physiol. Rev. 76, 371–423.
Schiaffino, S., Dyar, K.A., Ciciliot, S., Blaauw, B., Sandri, M., 2013. Mechanisms
regulating skeletal muscle growth and atrophy. FEBS J. 280, 4294–4314.
Schmidt, M., Lin, S., Pape, M., Ernsberger, U., Stanke, M., Kobayashi, K., Howard, M.J.,
Rohrer, H., 2009. The bHLH transcription factor Hand2 is essential for the
maintenance of noradrenergic properties in differentiated sympathetic neurons. Dev.
Biol. 329, 191–200.
Seibenhener, M.L., Babu, J.R., Geetha, T., Wong, H.C., Krishna, N.R., Wooten, M.W.,
2004. Sequestosome 1/p62 is a polyubiquitin chain binding protein involved in
ubiquitin proteasome degradation. Mol. Cell. Biol. 24, 8055–8068.
Sekulic, A., Hudson, C.C., Homme, J.L., Yin, P., Otterness, D.M., Karnitz, L.M.,
Abraham, R.T., 2000. A direct linkage between the phosphoinositide 3-kinase-AKT
signaling pathway and the mammalian target of rapamycin in mitogen-stimulated
and transformed cells. Cancer Res. 60, 3504–3513.
Silveira, W.A., Goncalves, D.A., Graca, F.A., Andrade-Lopes, A.L., Bergantin, L.B.,
Zanon, N.M., Godinho, R.O., Kettelhut, I.C., Navegantes, L.C., 2014. Activating
cAMP/PKA signaling in skeletal muscle suppresses the ubiquitin-proteasome-
dependent proteolysis: implications for sympathetic regulation. J. Appl. Physiol. 117
(1985), 11–19.
Smith, L.R., Barton, E.R., 2014. SMASH - semi-automatic muscle analysis using
segmentation of histology: a MATLAB application. Skelet. Muscle 4, 21.
Sonjak, V., Jacob, K., Morais, J.A., Rivera-Zengotita, M., Spendiff, S., Spake, C.,
Taivassalo, T., Chevalier, S., Hepple, R.T., 2019. Fidelity of muscle fibre
reinnervation modulates ageing muscle impact in elderly women. J. Physiol. 597
(19), 5009–5023.
Srikanthan, P., Hevener, A.L., Karlamangla, A.S., 2010. Sarcopenia exacerbates obesity-
associated insulin resistance and dysglycemia: findings from the national health and
nutrition examination survey III. PLoS One 5, e10805.
Stanzel, S., Stubbusch, J., Pataskar, A., Howard, M.J., Deller, T., Ernsberger, U.,
Tiwari, V.K., Rohrer, H., Tsarovina, K., 2016. Distinct roles of Hand2 in developing
and adult autonomic neurons. Dev. Neurobiol. 76 (10), 1111–1124.
Straka, T., Vita, V., Prokshi, K., Horner, S.J., Khan, M.M., Pirazzini, M., Williams, M.P.I.,
Hafner, M., Zaglia, T., Rudolf, R., 2018. Postnatal development and distribution of
sympathetic innervation in mouse skeletal muscle. Int. J. Mol. Sci. 19.
Straub, V., Campbell, K.P., 1997. Muscular dystrophies and the dystrophin-glycoprotein
complex. Curr. Opin. Neurol. 10, 168–175.
Studenski, S., Perera, S., Patel, K., Rosano, C., Faulkner, K., Inzitari, M., Brach, J.,
Chandler, J., Cawthon, P., Connor, E.B., Nevitt, M., Visser, M., Kritchevsky, S.,
Badinelli, S., Harris, T., Newman, A.B., Cauley, J., Ferrucci, L., Guralnik, J., 2011.
Gait speed and survival in older adults. JAMA 305, 50–58.
Tachi, N., Ohya, K., Chiba, S., Nihira, H., Minagawa, K., 1995. Muscle involvement in
congenital insensitivity to pain with anhidrosis. Pediatr. Neurol. 12, 264–266.
Taetzsch, T., Valdez, G., 2018. NMJ maintenance and repair in aging. Curr. Opin.
Physiol. 4, 57–64.
Tang, H., Goldman, D., 2006. Activity-dependent gene regulation in skeletal muscle is
mediated by a histone deacetylase (HDAC)-Dach2-myogenin signal transduction
cascade. Proc. Natl. Acad. Sci. U. S. A. 103, 16977–16982.
Tang, S., Wong, H.-C., Wang, Z.M., Huang, Y., Zou, J., Zhuo, Y., Pennati, A., Gadda, G.,
Delbono, O., Yang, J.J., 2011. Design and application of a class of sensors to monitor
Ca2+ dynamics in high Ca2+ concentration cellular compartments. Proc. Natl.
Acad. Sci. 16265–16270, 2011 Sep 27. Epub 2011 Sep 13.
Tang, H., Shrager, J.B., Goldman, D., 2019. Rapamycin protects aging muscle. Aging
(Albany NY) 11, 5868–5870.
Tchkonia, T., Zhu, Y., van Deursen, J., Campisi, J., Kirkland, J.L., 2013. Cellular
senescence and the senescent secretory phenotype: therapeutic opportunities.
J. Clin. Invest. 123, 966–972.
Thoma, A., Lightfoot, A.P., 2018. NF-kB and inflammatory cytokine signalling: role in
skeletal muscle atrophy. Adv. Exp. Med. Biol. 1088, 267–279.
Tieland, M., Trouwborst, I., Clark, B.C., 2018. Skeletal muscle performance and ageing.
J. Cachexia Sarcopenia Muscle 9, 3–19.
O. Delbono et al.
Tournadre, A., Vial, G., Capel, F., Soubrier, M., Boirie, Y., 2019. Sarcopenia. Joint Bone
Spine 86, 309–314.
Trimmer, J.S., Cooperman, S.S., Tomiko, S.A., Zhou, J.Y., Crean, S.M., Boyle, M.B.,
Kallen, R.G., Sheng, Z.H., Barchi, R.L., Sigworth, F.J., et al., 1989. Primary structure
and functional expression of a mammalian skeletal muscle sodium channel. Neuron
3, 33–49.
Tsarovina, K., Reiff, T., Stubbusch, J., Kurek, D., Grosveld, F.G., Parlato, R., Schütz, G.,
Rohrer, H., 2010. The Gata3 transcription factor is required for the survival of
embryonic and adult sympathetic neurons. J. Neurosci. 30, 10833–10843.
Tseng, B.S., Marsh, D.R., Hamilton, M.T., Booth, F.W., 1995. Strength and aerobic
training attenuate muscle wasting and improve resistance to the development of
disability with aging. J. Gerontol. A Biol. Sci. Med. Sci. 50, 113–119. Spec No.
Tsentsevitsky, A.N., Kovyazina, I.V., Bukharaeva, E.A., 2019. Diverse effects of
noradrenaline and adrenaline on the quantal secretion of acetylcholine at the mouse
neuromuscular junction. Neuroscience 423, 162–171.
Upadhya, B., Haykowsky, M.J., Eggebeen, J., Kitzman, D.W., 2015. Exercise intolerance
in heart failure with preserved ejection fraction: more than a heart problem.
J. Geriatr. Cardiol. 12, 294–304.
Urasawa, K., Murakami, T., Yasuda, H., 1991. Age-related alterations in adenylyl cyclase
system of rat hearts. Jpn. Circ. J. 55, 676–684.
Urbancheck, M.G., Picken, E.B., Kalliainen, L.K., Kuzon, W.M., 2001. Specific force
deficit in skeletal muscles of old rats is partially explained by the existence of
denervated muscle fibers. J. Gerontol. Biol. Sci. 56A, B191–B198.
Vainshtein, A., Tryon, L.D., Pauly, M., Hood, D.A., 2015. Role of PGC-1alpha during
acute exercise-induced autophagy and mitophagy in skeletal muscle. Am. J. Physiol.
Cell Physiol. 308, C710–719.
Valdez, G., Tapia, J.C., Kang, H., Clemenson Jr., G.D., Gage, F.H., Lichtman, J.W.,
Sanes, J.R., 2010. Attenuation of age-related changes in mouse neuromuscular
synapses by caloric restriction and exercise. Proc. Natl. Acad. Sci. U. S. A. 107,
14863–14868.
Vasilaki, A., Richardson, A., Van Remmen, H., Brooks, S.V., Larkin, L., McArdle, A.,
Jackson, M.J., 2017. Role of nerve-muscle interactions and ROS in regulation of
muscle proteostasis with aging. J. Physiol. 595, 6409–6415.
Veith, R.C., Featherstone, J.A., Linares, O.A., Halter, J.B., 1986. Age differences in
plasma norepinephrine kinetics in humans. J. Gerontol. 41, 319–324.
Vitale, G., Cesari, M., Mari, D., 2016. Aging of the endocrine system and its potential
impact on sarcopenia. Eur. J. Intern. Med. 35, 10–15.
von Haehling, S., Morley, J.E., Anker, S.D., 2010. An overview of sarcopenia: facts and
numbers on prevalence and clinical impact. J. Cachexia Sarcopenia Muscle 1,
129–133.
Wang, Y., Hekimi, S., 2015. Mitochondrial dysfunction and longevity in animals:
untangling the knot. Science 350, 1204–1207.
Wang, Z.-M., Messi, M.L., Delbono, O., 2000. L-type Ca2+ channel charge movement and
intracellular Ca2+ in skeletal muscle fibers from aging mice. Biophys. J. 78,
1947–1954.
16
Ageing Research Reviews 67 (2021) 101305
Wang, Z.-M., Messi, M.L., Delbono, O., 2002. Sustained overexpression of IGF-1 prevents
age-dependent decrease in charge movement and intracellular calcium in mouse
skeletal muscle. Biophys. J. 82, 1338–1344.
Wang, Z.M., Zheng, Z., Messi, M.L., Delbono, O., 2005. Extension and magnitude of
denervation in skeletal muscle from ageing mice. J. Physiol. 565, 757–764.
Wang, Z.-M., Rodrigues, A.C.Z., Messi, M.L., Delbono, O., 2020a. Aging blunts
sympathetic neuron regulation of motoneurons synaptic vesicle release mediated by
β1- and α2B-Adrenergic receptors in geriatric mice. J. Gerontol. Ser. A 75,
1473–1480.
Wang, Z.M., Messi, M.L., Grinevich, V., Budygin, E., Delbono, O., 2020b. Postganglionic
sympathetic neurons, but not locus coeruleus optostimulation, activates
neuromuscular transmission in the adult mouse in vivo. Mol. Cell. Neurosci. 109,
103563.
Weatherbee, S.D., Anderson, K.V., Niswander, L.A., 2006. LDL-receptor-related protein 4
is crucial for formation of the neuromuscular junction. Development 133,
4993–5000.
Weindruch, R., 1995. Interventions based on the possibility that oxidative stress
contributes to sarcopenia. J. Gerontol. A Biol. Sci. Med. Sci. 50, 157–161.
Wen, Y., Murach, K.A., Vechetti Jr., I.J., Fry, C.S., Vickery, C., Peterson, C.A.,
McCarthy, J.J., Campbell, K.S., 2018. MyoVision: software for automated high-
content analysis of skeletal muscle immunohistochemistry. J. Appl. Physiol. 124
(1985), 40–51.
White, Z., White, R.B., McMahon, C., Grounds, M.D., Shavlakadze, T., 2016. High
mTORC1 signaling is maintained, while protein degradation pathways are perturbed
in old murine skeletal muscles in the fasted state. Int. J. Biochem. Cell Biol. 78,
10–21.
Wokke, J.H., Jennekens, F.G., van den Oord, C.J., Veldman, H., Smit, L.M., Leppink, G.J.,
1990. Morphological changes in the human end plate with age. J. Neurol. Sci. 95,
291–310.
Xiang, H.-B., Liu, C., Liu, T.-T., Xiong, J., 2014. Central circuits regulating the
sympathetic outflow to lumbar muscles in spinally transected mice by retrograde
transsynaptic transport. Int. J. Clin. Exp. Pathol. 7, 2987–2997.
Xu, Z., Feng, X., Dong, J., Wang, Z.-M., Lee, J., Furdui, C., Clark Files, D., Beavers, K.M.,
Kritchevsky, S., Milligan, C., Jin, J.-P., Delbono, O., Zhang, T., 2017. Cardiac
troponin T and fast skeletal muscle denervation in ageing. J. Cachexia Sarcopenia
Muscle 8, 808–823.
Zhang, C., Goto, N., Suzuki, M., Ke, M., 1996. Age-related reductions in number and size
of anterior horn cells at C6 level of the human spinal cord. Okajimas Folia Anatomica
Japonica 73, 171–177.
Zheng, Z., Wang, Z.M., Delbono, O., 2002. Insulin-like growth factor-1 increases skeletal
muscle DHPR alpha 1S transcriptional activity by acting on the cAMP-response
element-binding protein element of the promoter region. J. Biol. Chem. 277,
50535–50542.
Zhu, Y., Armstrong, J.L., Tchkonia, T., Kirkland, J.L., 2014. Cellular senescence and the
senescent secretory phenotype in age-related chronic diseases. Curr. Opin. Clin.
Nutr. Metab. Care 17, 324–328.