Age-related Denervation Research Articles

Alterations in neuromuscular junction morphology with ageing and endurance training modulate neuromuscular transmission and myofibre composition.

Both ageing and exercise training affect the neuromuscular junction (NMJ) structure. Morphological alterations in the NMJ have been considered to influence neuromuscular transmission and myofibre properties, but the direct link between the morphology and function has yet to be established. We measured the neuromuscular transmission, myofibre composition and NMJ structure of 5-month-old (young) and 24-month-old untrained (aged control) and trained (aged trained) mice. Aged trained mice were subjected to 2 months of endurance training before the measurement. Neuromuscular transmission was evaluated in vivo as the ratio of ankle plantar flexion torque evoked by the sciatic nerve stimulation to that by direct muscle stimulation. The torque ratio was significantly lower in aged mice than in young and aged trained mice at high-frequency stimulations, showing a significant positive correlation with voluntary grip strength. The degree of pre- to post-synaptic overlap of the NMJ was also significantly lower in aged mice and positively correlated with the torque ratio. We also found that the proportion of fast-twitch fibres in the soleus muscle decreased with age, and that age-related denervation occurred preferentially in fast-twitch fibres. Age-related denervation and a shift in myofibre composition were partially prevented by endurance training. These results suggest that age-related deterioration of the NMJ structure impairs neuromuscular transmission and alters myofibre composition, but these alterations can be prevented by structural amelioration of NMJ with endurance training. Our findings highlight the importance of the NMJ as a major determinant of age-related deterioration of skeletal muscles and the clinical significance of endurance training as a countermeasure. KEY POINTS: The neuromuscular junction (NMJ) plays an essential role in neuromuscular transmission and the maintenance of myofibre properties. We show that neuromuscular transmission is impaired with ageing but recovered by endurance training, which contributes to alterations in voluntary strength. Neuromuscular transmission is associated with the degree of pre- to post-synaptic overlap of the NMJ. Age-related denervation of fast-twitch fibres and a shift in myofibre composition toward a slower phenotype are partially prevented by endurance training. Our study provides substantial evidence that age-related and exercise-induced alterations in neuromuscular transmission and myofibre properties are associated with morphological changes in the NMJ.

Use of a novel technique to assess impact of age-related denervation on mouse soleus muscle function

Denervation contributes to loss of force-generating capacity in aged skeletal muscles, but problems with quantification of denervated fibers mean the precise impact of denervation on muscle function remains unclear. This study therefore looked to develop a reliable assay for identifying denervated muscle fibers, and used this to explore the impact of denervation on age-related force-generation in mouse skeletal muscle. Thirteen young (6-month-old) and 10 old (24-months-old) C57Bl/6 J female mice were utilized. Anaesthetized mice were infused with the fluorescent deoxyglucose analog 2[N-(7-nitrobenz-2-oxa-1,2-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG) and the tibial nerve was repeatedly stimulated to label active skeletal muscle fibers by activity-dependent uptake of 2-NBDG. Data on muscle force generation were acquired as part of the stimulation routine. Labeled muscles were removed, snap frozen, sectioned, and slide mounted. Sections were imaged to show accumulation of 2-NBDG in activated fibers and lack of 2-NBDG accumulation in quiescent (denervated) fibers, then processed using immunohistochemistry to allow collection of data on fiber number and morphology. Soleus muscles from older mice had nine times as many denervated fibers as those from young mice (average n = 36 vs 4, old vs young). Older muscles developed significantly more passive force and less specific force, but denervation only partly accounted for age-related deficits in specific force. Further investigations are required to definitively identify contributors to the decrease in force generation that remain unaccounted for.

Reinnervation of Vastus lateralis is increased significantly in seniors (70-years old) with a lifelong history of high-level exercise (2013, revisited here in 2022).

In 2013 we presented results showing that at the histological level lifelong increased physical activity promotes reinnervation of muscle fibers in aging muscles. Indeed, in muscle biopsies from 70-year old men with a lifelong history of high-level physical activity, we observed a considerable increase in fiber-type groupings (F-TG), almost exclusively of the slow type. Slow-type transformation by denervation-reinnervation in senior sportsmen seems to fluctuate from those with scarce fiber-type transformation and groupings to almost fully transformed muscle, going through a process in which isolated fibers co-expressing fast and slow Myosin Heavy Chains (MHCs) seems to fill the gaps. Taken together, our results suggest that, beyond the direct effects of aging on the muscle fibers, changes occurring in skeletal muscle tissue appear to be largely, although not solely, a result of sparse denervation-reinnervation. The lifelong exercise allows the body to adapt to the consequences of the age-related denervation and to preserve muscle structure and function by saving otherwise lost muscle fibers through recruitment to different, mainly slow, motor units. These beneficial effects of high-level life-long exercise on motoneurons, specifically on the slow type motoneurones that are those with higher daily activity, and on muscle fibers, serve to maintain size, structure and function of muscles, delaying the functional decline and loss of independence that are commonly seen in late aging. Several studies of independent reserchers with independent analyses confirmed and cited our 2013 results. Thus, the results we presented in our paper in 2013 seem to have held up rather well. Trial Registration: ClinicalTrials.gov: NCT01679977

Increased MFG-E8 at neuromuscular junctions is an exacerbating factor for sarcopenia-associated denervation.

Sarcopenia is an important health problem associated with adverse outcomes. Although the etiology of sarcopenia remains poorly understood, factors apart from muscle fibers, including humoral factors, might be involved. Here, we used cytokine antibody arrays to identify humoral factors involved in sarcopenia and found a significant increase in levels of milk fat globule epidermal growth factor 8 (MFG‐E8) in skeletal muscle of aged mice, compared with young mice. We found that the increase in MFG‐E8 protein at arterial walls and neuromuscular junctions (NMJs) in muscles of aged mice. High levels of MFG‐E8 at NMJs and an age‐related increase in arterial MFG‐E8 have also been identified in human skeletal muscle. In NMJs, MFG‐E8 is localized on the surface of terminal Schwann cells, which are important accessory cells for the maintenance of NMJs. We found that increased MFG‐E8 at NMJs precedes age‐related denervation and is more prominent in sarcopenia‐susceptible fast‐twitch than in sarcopenia‐resistant slow‐twitch muscle. Comparison between fast and slow muscles further revealed that arterial MFG‐E8 can be uncoupled from sarcopenic phenotype. A genetic deficiency in MFG‐E8 attenuated age‐related denervation of NMJs and muscle weakness, providing evidence of a pathogenic role of increased MFG‐E8. Thus, our study revealed a mechanism by which increased MFG‐E8 at NMJs leads to age‐related NMJ degeneration and suggests that targeting MFG‐E8 could be a promising therapeutic approach to prevent sarcopenia.

Phrenic motor neuron loss in aged rats.

Sarcopenia is the age-related reduction of muscle mass and specific force. In previous studies, we found that sarcopenia of the diaphragm muscle (DIAm) is evident by 24 mo of age in both rats and mice and is associated with selective atrophy of type IIx and IIb muscle fibers and a decrease in maximum specific force. These fiber type-specific effects of sarcopenia resemble those induced by DIAm denervation, leading us to hypothesize that sarcopenia is due to an age-related loss of phrenic motor neurons (PhMNs). To address this hypothesis, we determined the number of PhMNs in young (6 mo old) and old (24 mo old) Fischer 344 rats. Moreover, we determined age-related changes in the size of PhMNs, since larger PhMNs innervate type IIx and IIb DIAm fibers. The PhMN pool was retrogradely labeled and imaged with confocal microscopy to assess the number of PhMNs and the morphometry of PhMN soma and proximal dendrites. In older animals, there were 22% fewer PhMNs, a 19% decrease in somal surface area, and a 21% decrease in dendritic surface area compared with young Fischer 344 rats. The age-associated loss of PhMNs involved predominantly larger PhMNs. These results are consistent with an age-related denervation of larger, more fatigable DIAm motor units, which are required primarily for high-force airway clearance behaviors. NEW & NOTEWORTHY Diaphragm muscle sarcopenia in rodent models is well described in the literature; however, the relationship between sarcopenia and frank phrenic motor neuron (MN) loss is unexplored in these models. We quantify a 22% loss of phrenic MNs in old (24 mo) compared with young (6 mo) Fischer 344 rats. We also report reductions in phrenic MN somal and proximal dendritic morphology that relate to decreased MN heterogeneity in old compared with young Fischer 344 rats.

Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles.

Laryngeal dysfunction in the elderly is a major cause of disability, from voice disorders to dysphagia and loss of airway protective reflexes. Few, if any, therapies exist that target age-related laryngeal muscle dysfunction. Neurotrophins are involved in muscle innervation and differentiation of neuromuscular junctions (NMJs). It is thought that neurotrophins enhance neuromuscular transmission by increasing neurotransmitter release. The neuromuscular junctions (NMJs) become smaller and less abundant in aging rat laryngeal muscles, with evidence of functional denervation. We explored the effects of NTF4 for future clinical use as a therapeutic to improve function in aging human laryngeal muscles. Here, we provide the detailed protocol for systemic application and direct injection of NTF4 to investigate the ability of aging rat laryngeal muscle to remodel in response to NTF4 application. In this method, rats either received NTF4 either systemically via osmotic pump or by direct injection through the vocal folds. Laryngeal muscles were then dissected and used for histological examination of morphology and age-related denervation.

Subcutaneous Neurotrophin 4 Infusion Using Osmotic Pumps or Direct Muscular Injection Enhances Aging Rat Laryngeal Muscles

Laryngeal dysfunction in the elderly is a major cause of disability, from voice disorders to dysphagia and loss of airway protective reflexes. Few, if any, therapies exist that target age-related laryngeal muscle dysfunction. Neurotrophins are involved in muscle innervation and differentiation of neuromuscular junctions (NMJs). It is thought that neurotrophins enhance neuromuscular transmission by increasing neurotransmitter release. The neuromuscular junctions (NMJs) become smaller and less abundant in aging rat laryngeal muscles, with evidence of functional denervation. We explored the effects of NTF4 for future clinical use as a therapeutic to improve function in aging human laryngeal muscles. Here, we provide the detailed protocol for systemic application and direct injection of NTF4 to investigate the ability of aging rat laryngeal muscle to remodel in response to NTF4 application. In this method, rats either received NTF4 either systemically via osmotic pump or by direct injection through the vocal folds. Laryngeal muscles were then dissected and used for histological examination of morphology and age-related denervation.

Use it or Lose It: Tonic Activity of Slow Motoneurons Promotes Their Survival and Preferentially Increases Slow Fiber-Type Groupings in Muscles of Old Lifelong Recreational Sportsmen.

Histochemistry, immuno-histochemistry, gel electrophoresis of single muscle fibers and electromyography of aging muscles and nerves suggest that: i) denervation contributes to muscle atrophy, ii) impaired mobility accelerates the process, and iii) lifelong running protects against loss of motor units. Recent corroborating results on the muscle effects of Functional Electrical Stimulation (FES) of aged muscles will be also mentioned, but we will in particular discuss how and why a lifelong increased physical activity sustains reinnervation of muscle fibers. By analyzing distribution and density of muscle fibers co-expressing fast and slow Myosin Heavy Chains (MHC) we are able to distinguish the transforming muscle fibers due to activity related plasticity, to those that adapt muscle fiber properties to denervation and reinnervation. In muscle biopsies from septuagenarians with a history of lifelong high-level recreational activity we recently observed in comparison to sedentary seniors: 1. decreased proportion of small-size angular myofibers (denervated muscle fibers); 2. considerable increase of fiber-type groupings of the slow type (reinnervated muscle fibers); 3. sparse presence of muscle fibers co-expressing fast and slow MHC. Immuno-histochemical characteristics fluctuate from those with scarce fiber-type modulation and groupings to almost complete transformed muscles, going through a process in which isolated fibers co-expressing fast and slow MHC fill the gaps among fiber groupings. Data suggest that lifelong high-level exercise allows the body to adapt to the consequences of the age-related denervation and that it preserves muscle structure and function by saving otherwise lost muscle fibers through recruitment to different slow motor units. This is an opposite behavior of that described in long term denervated or resting muscles. These effects of lifelong high level activity seems to act primarily on motor neurons, in particular on those always more active, i.e., on the slow motoneurons. The preferential reinnervation that follows along decades of increased activity maintains neuron and myofibers. All together the results open interesting perspectives for applications of FES and electroceuticals for rejuvenation of aged muscles to delay functional decline and loss of independence that are unavoidable burdens of advanced aging.Trial Registration: ClinicalTrials.gov: NCT01679977

Molecular analyses provide insight into mechanisms underlying sarcopenia and myofibre denervation in old skeletal muscles of mice

Molecular mechanisms that are associated with age-related denervation and loss of skeletal muscle mass and function (sarcopenia) are described for female C57Bl/6J mice aged 3, 15, 24, 27 and 29 months (m). Changes in mRNAs and proteins associated with myofibre denervation and protein metabolism in ageing muscles are reported, across the transition from healthy adult myofibres to sarcopenia that occurs between 15 and 24m. This onset of sarcopenia at 24m, corresponded with increased expression of genes associated with neuromuscular junction denervation including Chnrg, Chrnd, Ncam1, Runx1, Gadd45a and Myog. Sarcopenia in quadriceps muscles also coincided with increased protein levels for Igf1 receptor, Akt and ribosomal protein S6 (Rps6) with increased phosphorylation of Rps6 (Ser235/236) and elevated Murf1 mRNA and protein, but not Fbxo32: many of these changes are also linked to denervation. Global transcription profiling via microarray analysis confirmed these functional themes and highlighted additional themes that may be a consequence of pathology associated with sarcopenia, including changes in fatty acid metabolism, extracellular matrix structure and protein catabolism. Ageing was also associated with increased global gene expression variance, consistent with decreased control of gene regulation.

Long-Term High-Level Exercise Promotes Muscle Reinnervation With Age

The histologic features of aging muscle suggest that denervation contributes to atrophy, that immobility accelerates the process, and that routine exercise may protect against loss of motor units and muscle tissue. Here, we compared muscle biopsies from sedentary and physically active seniors and found that seniors with a long history of high-level recreational activity up to the time of muscle biopsy had 1) lower loss of muscle strength versus young men (32% loss in physically active vs 51% loss in sedentary seniors); 2) fewer small angulated (denervated) myofibers; 3) a higher percentage of fiber-type groups (reinnervated muscle fibers) that were almost exclusive of the slow type; and 4) sparse normal-size muscle fibers coexpressing fast and slow myosin heavy chains, which is not compatible with exercise-driven muscle-type transformation. The biopsies from the old physically active seniors varied from sparse fiber-type groupings to almost fully transformed muscle, suggesting that coexpressing fibers appear to fill gaps. Altogether, the data show that long-term physical activity promotes reinnervation of muscle fibers and suggest that decades of high-level exercise allow the body to adapt to age-related denervation by saving otherwise lost muscle fibers through selective recruitment to slow motor units. These effects on size and structure of myofibers may delay functional decline in late aging.

Reinnervation of Vastus lateralis is increased significantly in seniors (70-years old) with a lifelong history of high-level exercise

It has long been recognized that histological changes observed in aging muscle suggest that denervation contributes to muscle deterioration and that disuse accelerates the process while running activity, sustained for decades, protects against age-related loss of motor units. Here we show at the histological level that lifelong increased physical activity promotes reinnervation of muscle fibers. In muscle biopsies from 70-year old men with a lifelong history of high-level physical activity, we observed a considerable increase in fiber-type groupings (almost exclusively of the slow type) in comparison to sedentary seniors, revealing a large population of reinnervated muscle fibers in the sportsmen. Slow-type transformation by reinnervation in senior sportsmen seems to be a clinically relevant mechanism: the muscle biopsies fluctuate from those with scarce fiber-type transformation and groupings to almost fully transformed muscle, going through a process in which isolated fibers co-expressing fast and slow MHCs seems to fill the gaps. Taken together, our results suggest that, beyond the direct effects of aging on the muscle fibers, changes occurring in skeletal muscle tissue appear to be largely, although not solely, a result of sparse denervation. Our data suggest that lifelong exercise allows the body to adapt to the consequences of the age-related denervation and to preserve muscle structure and function by saving otherwise lost muscle fibers through recruitment to different, mainly slow, motor units. These beneficial effects on motoneurons and, subsequently on muscle fibers, serve to maintain size, structure and function of muscle fibers, delaying the functional decline and loss of independence that are commonly seen in late aging.

G.P.118 Targeting sarcopenia: Molecular basis of denervation of aged muscles and benefits of life-long exercise

Abstract With ageing, the progressive loss of muscle mass and function (sarcopenia) results in frailty, loss of independence and is a major cause of increased falls and fractures. The precise reasons for sarcopenia are unclear. We have previously described the time course of sarcopenia in female C57Bl/6J mice with significant loss of muscle mass at 24 and 28 months (m) of age (Shavlakadze et al., 2010: Biogerontology). Muscle function and maintenance of muscle mass require electrical stimulation by a nerve, yet denervation as a primary cause for sarcopenia has barely been considered. We have described altered morphology and striking denervation of neuromuscular junctions (NMJs) for muscles in geriatric (28 m) compared to young (3 m) female C57Bl/6J mice, using immunostaining of whole intact fast extensor digitorum longus (EDL) muscle (Chai et al., 2011: PlosONE). Myofibre type changes indicative of muscle denervation and re-innervation were observed in all muscles examined. However, there was no loss of alpha-motoneuron cell bodies in the lumbar spinal cord of geriatric (28 m) mice. These data suggest that changes in the muscles per se are a primary cause of the age-related denervation. Establishing these baseline data for geriatric mice is essential in order to take full advantage of the wealth of genetically modified mice available to study sarcopenia, and for testing therapeutic interventions. Our molecular analyses of muscles from mice aged 3, 15, 24 and 28 months aim to identify the molecular basis for this age-relate denervation. We have already demonstrated that life-long exercise significantly reduces sarcopenia (measured by loss of muscle mass) at 28 months of age (unpublished) and we are investigating the molecular basis for the benefits of this exercise on ageing muscles with a focus on NMJ innervation.

Advanced age-related denervation and fiber-type grouping in skeletal muscle of SOD1 knockout mice

In this study skeletal muscles from 1.5- and 10-month-old Cu/Zn superoxide dismutase (SOD1) homozygous knockout ( JLSod1 −/−) mice obtained from The Jackson Laboratory (C57Bl6/129SvEv background) were compared with muscles from age- and sex-matched heterozygous ( JLSod1 +/− ) littermates. The results of this study were compared with previously published data on two different strains of Sod1 −/− mice: one from Dr. Epstein's laboratory ( ELSod1 −/−; C57Bl6 background) and the other from Cephalon, Inc. ( CSod1 −/−; 129/CD-1 background). Grouping of succinate dehydrogenase-positive fibers characterized muscles of Sod1 −/− mice from all three strains. The 10-month-old Sod1 −/− C and JL mice displayed pronounced denervation of the gastrocnemius muscle, whereas the ELSod1 −/− mice displayed a small degree of denervation at this age, but developed accelerated age-related denervation later on. Denervation markers were up-regulated in skeletal muscle of 10-month-old JLSod1 −/− mice. This study is the first to show that metallothionein mRNA and protein expression was up-regulated in the skeletal muscle of 10-month-old JLSod1 −/− mice and was mostly localized to the small atrophic muscle fibers. In conclusion, all three strains of Sod1 −/− mice develop accelerated age-related muscle denervation, but the genetic background has significant influence on the progress of denervation.

Remodeling of the neuromuscular junction precedes sarcopenia related alterations in myofibers

Several mechanisms contributing to the etiology of sarcopenia (age-related loss of muscle size) have been postulated. One of these attributes the loss of muscle mass to a preceding age-related denervation of myofibers. The aim of this study was to determine if signs of denervation were apparent at the neuromuscular junction (NMJ) before fiber atrophy, or fiber type conversion could be documented, and to reveal if a muscle’s activity level impacts its sensitivity to age-related denervation. Plantaris and soleus muscles were obtained from young adult (10 months) and early aged (21 months) rats. Pre- and post-synaptic NMJ morphology was quantified with cytofluorescent staining of nerve terminal branches and endplate regions, respectively. Myofiber profiles (fiber size and fiber type composition) were assessed with histochemical procedures. Results show that in the lightly recruited plantaris, significant ( P < 0.05) signs of denervation were noted in aged rats, while the same muscles displayed no change in myofiber profile. In the heavily recruited soleus, however, there was little evidence of denervation, and again no alterations in myofiber profile. These results indicate that age-related denervation occurs before myofiber atrophy, and that high amounts of neuromuscular activity may delay the onset of age-related denervation and sarcopenia.

Motor neuron targeting of IGF-1 attenuates age-related external Ca 2+-dependent skeletal muscle contraction in senescent mice

A population of fast muscle fibers from aging mice is dependent on external Ca 2+ to maintain tetanic force during repeated contractions. We hypothesized that age-related denervation in muscle fibers plays a role in initiating this contractile deficit, and that prevention of denervation by IGF-1 overexpression would prevent external Ca 2+-dependent contraction in aging mice. IGF-1 overexpression in skeletal muscle prevents age-related denervation, and prevented external Ca 2+-dependent contraction in this work. To determine if the effects of IGF-1 overexpression are on muscle or nerve, aging mice were injected with a tetanus toxin fragment-C (TTC) fusion protein that targets IGF-1 to spinal cord motor neurons. This treatment prevented external Ca 2+-dependent contraction. We also show evidence that injections of the IGF-1-TTC fusion protein prevent age-related alterations to the nerve terminals at the neuromuscular junctions. We conclude that the slow age-related denervation of fast muscle fibers underlies dependence on external Ca 2+ to maintain tetanic force in a population of muscle fibers from senescent mice.

Effects of denervation on enzyme‐histochemical and morphometrical properties of the rat soleus muscle in relation to age

The soleus muscle of young adult (5 months), adult (10-11 months) and old (23 months) male Wistar rats was unilaterally denervated for a period of 3 weeks and studied with regard to enzyme-histochemical and morphometrical properties. Denervation caused a marked atrophy of all fibres, irrespective of age and enzyme-histochemical type. Fibres having myofibrillar ATPase staining characteristics intermediate to type I and type IIA fibres increased in number in all age groups and a reduction in the number/proportion of type I fibres was found in adult and old animals. These results indicate that a slow-to-fast shift in myofibrillar properties as a consequence of denervation, shown to occur in the soleus muscle of young animals, also takes place in old age. This supports the view that atrophic fibres with intermediate myofibrillar ATPase staining characteristics, and possibly also atrophic IIA fibres, seen in old soleus muscle could be type I fibres which have undergone a transformation in response to the age-related denervation process.

A neurogenic factor in faecal incontinence in the elderly.

Idiopathic faecal incontinence in middle-aged people has been found to be commonly associated with neurogenic damage to the anal sphincter musculature. Because neurogenic atrophy of skeletal muscle is a common feature of ageing, we have investigated the possibility that faecal incontinence in elderly people might be due to age-related denervation of the anal sphincter musculature. The technique of single fibre electromyography was used to measure the motor unit fibre density in the external and sphincter muscle in ten patients aged 78 to 99 years. Those patients whose continence was impaired had a markedly increased fibre density. Our findings suggest that neurogenic damage to the anal sphincter musculature may so reduce its functional reserve in elderly people that incontinence occurs when other factors, such as looseness of stool or depression of cerebral function, co-exist.