The Emerging Role of the Sympathetic Nervous System in Skeletal Muscle Motor Innerfvation and Sarcopenia

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 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.(Distefano 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 emphasizes 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 genetic studies have elucidated the role these proteins play in NMJ structure and function, how they contribute to its age-associated functional decline remains ill-defined.(Fish and Fallon, 2020) Age-associated structural and functional alterations of the neuromuscular 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 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 remodeling of the initial contact as the motor nerve terminal becomes varicose.(Li and Thompson, 2011)

In skeletal muscles from aging rodents, motor denervation causes 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 effective concentration of neural agrin, regulates the organization of cytoskeletal 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 peptide, 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 evidence 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. Interventions focused on motor neurons, their axons, and associated non‐ neuronal cells, and the NMJ may slow or even reverse age‐ related impairments 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 sympathetic muscle innervation was described over a century ago;(Boeke, 1909) myofiber sympathetic innervation remained unexplored, probably 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 paravertebral 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 sympathetic 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, increases until adulthood.(Straka et al., 2018)

The ANS coordinates homeostasis through sympathetic and parasympathetic outflows and the enteric nervous system.(Robertson, 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 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 Stangel, 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 denervation to define the role of the SNS in regulating NMJ stability and function 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 pathological conditions, we examined whether sympathectomy alters myelination, 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 crosstalk between sympathetic and motor axons remains unclear. The interaction 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 acetylcholine receptor stability is extensive. It regulates NMJ transmission, maintains optimal Gα_i2 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 Figure 1.

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Figure 1.

Model of SNS regulation of the NMJ. The SNS regulates NMJ motor axon vesicle release and postsynaptic AChR stability through the G_αi2-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.

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 protein)‐ coupled receptor, and muscle NA levels and receptor activity decrease with sympathectomy,(Silveira et al., 2014) we examined whether sympathectomy led to decreased Gα_i2 activation, a mechanism postulated to activate myogenin.(Mei et al., 2011) Minetti et al. reported that Gα_i2 promotes skeletal muscle hypertrophy, myoblast differentiation, 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 Gα_i2 significantly decreased in hindlimb mouse muscles after sympathectomy, and that restoring it using a human Gα_i2 (Q205L) 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 Gα_i2 prevents skeletal muscle motor denervation.(Rodrigues et al., 2018)

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’ adrenergic differentiation and maintenance. (Doxakis et al., 2008; Tsarovina et al., 2010) Figure 3 shows the convergence of a hierarchical network comprised of the bone morphogenetic proteins (BMPs), paired-like homeobox 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 neurons,(Tsarovina et al., 2010) but their role in old age has not been explored.

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Figure 3.

The transcription factor Hand2 regulates noradrenergic differentiation and sympathetic neuron maintenance.

Relationship among the bone morphogenetic 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 associated 1; Phox2b: paired-like homeobox 2b.

Adult nerve injury suppresses Hand2, which directly regulates DBH expression (Rychlik et al., 2005) and, most important, the genes controlling synaptic and neurotransmission functions in adult sympathetic neurons. (Stanzel et al., 2016) Based on the reported role of the transcription factors supporting developing and adult autonomic neurons,(Stanzel et al., 2016) we examined whether inducing HAND2 expression 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 immunoreactive 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 hypermethylation 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 transcription 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 hypermethylation 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 stimulation of distal peroneal nerves resulted in unsustained and weaker lumbricalis 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 sympathetic neurons rescues them significantly. (Rodrigues et al., 2020)

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; Rodrigues et al., 2020; Straka et al., 2018) The skeletal muscle does not seem to be the only organ progressively 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 homeostasis.(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; Phillips and Powley, 2007) Noradrenergic networks are abundant in young adult rats, but in aged rats, noradrenergic 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 proposed.(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 terminals, an essential step in forming functional connections. Compartmentalized 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 patients.(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 denervation. 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 innervation, 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 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.(Rodrigues et al., 2020; Rodrigues et al., 2019)

Additionally, Hand2 upregulates the genes encoding critical components 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 presynaptic membrane cell-adhesion molecule that primarily binds to neuroligins, 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, neurotransmitters, 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 G_s, the inhibitory subunit Gα_i2 isoform blocks adenylate cyclase activation and cAMP production. Physiological or pathological circumstances that decrease levels of sympathetic neurotransmitter or Gα_i2 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 Gα_i2 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 Gα_i2 protein levels leading to muscle atrophy. (Rodrigues et al., 2018) The model of muscle sympathetic ablation shows downregulation of Gα_i2, which activates the Hdac4-myogenin-MyoD cascade to raise MuRF1 levels and increase AChR destabilization.(Rodrigues et al., 2019) Note that both experimental sympathetic ablation and aging can cause vasculature denervation, 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 intramyocellular signaling independently of their vasoactive function.

Sympathetic neuron Hand2 prevents Gα_i2 downregulation with aging

We found that Hand2 expression in sympathetic neurons prevents the decrease in Gα_i2 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 inhibition as a target for treating neurogenic muscle atrophy. However, studies in mutant mice where HDAC4 is specifically deleted in the 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. Myogenin 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 postterminal; 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 interventions 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 Gα_i2 expression.

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.