Sensitivity of subcellular components of neuromuscular junctions to decreased neuromuscular activity.

Muscle unloading imparts subtotal disuse on the neuromuscular system resulting in reduced performance capacity. This loss of function, at least in part, can be attributed to disruptions at the neuromuscular junction (NMJ). However, research has failed to document morphological remodeling of the NMJ with short term muscle unloading. Here, rather than quantifying cellular components of the NMJ, we examined subcellular active zone responses to 2 weeks of unloading in male Wistar rats. It was revealed that in the plantaris, but not the soleus muscles, unloading elicited significant (P ≤ 0.05) decrements in active zone staining as measured by Bassoon, and calcium channel expression. It was also discovered that unloading decreased the area of calcium channels staining relative to active zone areas of staining suggesting potential interference in the ability of calcium influx to trigger the release of vesicles docked at the active zone. Post-synaptic adaptations of the motor endplate were not evident. This presynaptic subcellular size reduction was not associated with atrophy of the underlying plantaris muscle fibers, although atrophy of the weight-bearing soleus fibers, where no subcellular remodeling was evident, was noted. These results suggest that the active zone is highly sensitive to alterations in neuromuscular activity, and that morphological adaptation of excitatory and contractile components of the NMJ can occur independently of each other.

Both aging and exercise training alter the rate of recovery of neuromuscular performance of male soleus muscles.

It is known that both exercise and aging influence neuromuscular performance; however their effects on post-exercise recovery are largely unknown. To examine how exercise training and aging might affect post-exercise recovery, the function of muscles taken from young, and aged male rats assigned to exercise, or control conditions was assessed with ex vivo procedures using indirect (nerve endings), and direct (sarcolemma) stimulation at different times (Initial, Final min of, and Recovery i.e. 1 min post, from 5 min of stimulation). Results revealed that initially, strength of indirectly stimulated young, male muscles was significantly (P = 0.05) greater than aged ones, but after continuous stimulation, aged and young muscles displayed similar strength, and controls showed more strength than trained muscles (P = 0.02). All groups except young controls exhibited significant recovery with 1 min of rest (P = 0.03). Compared to indirect stimulation, direct stimulation resulted in greater peak tension at each time point examined (P < 0.05); young muscles again were stronger than aged ones initially (P = 0.003), but not by the conclusion of stimulation (P = 0.20). One min following the direct stimulation protocol, no significant recovery was observed by any of the four treatment groups. These data indicate that motor neurons limit neuromuscular function, and that the effects of fatigue are more severe during stimulation of young, compared to aged muscle. Finally, results presented here indicate that age and training status do interact to influence post-exertional recovery, at least among male neuromuscular systems.

Neuromuscular adaptability of male and female rats to muscle unloading.

Previously, it has been shown that following muscle unloading, males and females experience different maladaptations in neuromuscular function. As a follow-up, the present investigation sought to determine if male and female neuromuscular systems demonstrated similar, or disparate morphological adaptations to muscle unloading. Twenty young adult male, and 20 young adult female rats were randomly assigned to one of two treatment protocols: muscle unloading, or control conditions. Following the 2-week intervention period, immunofluorescent procedures were used to quantify pre- and post-synaptic features of neuromuscular junctions (NMJs), and to assess myofiber profiles (size and fiber type composition) of the soleus, plantaris, and EDL muscles. A 2-way ANOVA with main effects for sex and treatment was then used to identify statistically significant (p ≤ .05) differences among structural parameters. Analysis of NMJs showed a consistent lack of differences between males and females. Overall, NMJs were also found to be resistant to the effects of unloading. When examining myofiber profiles, however, male myofibers were revealed to be significantly larger than female ones in each of the muscles examined. Unloading resulted in significant myofiber atrophy only in the primarily weight-bearing soleus muscle. Only the EDL showed unloading-induced differences in myofiber type distribution (Type II → I). These data indicate that different components of the neuromuscular system (NMJs, myofibers) respond uniquely to unloading, and that sex affects myofiber type profiles, but not NMJs. Moreover, it appears that only muscles that have their habitual activity patterns disturbed by unloading (i.e., the soleus, adapt to that intervention).

Neuromuscular junction degeneration in muscle wasting.

PURPOSE OF REVIEW: Denervation is a hallmark of age-related and other types of muscle wasting. This review focuses on recent insights and current viewpoints regarding the mechanisms and clinical relevance of maintaining the neuromuscular junction to counteract muscle wasting resulting from aging or neural disease/damage. RECENT FINDINGS: Activity-dependent regulation of autophagy, the agrin-muscle specific kinase-Lrp4 signaling axis, and sympathetic modulation are principal mechanisms involved in stabilizing the neuromuscular junction. These findings are derived from several animal models and were largely confirmed by human gene expression analysis as well as insights from rare neuromuscular diseases such as amyotrophic lateral sclerosis and congenital myasthenic syndromes. Based on these insights, agrin-derived fragments are currently being evaluated as biomarkers for age-related muscle wasting. Tuning of autophagy, of the agrin pathway, and of sympathetic input are being studied as clinical treatment of muscle wasting disorders. SUMMARY: Basic research has revealed that maintenance of neuromuscular junctions and a few signaling pathways are important in the context of age-dependent and other forms of muscle wasting. These findings have recently started to enter clinical practice, but further research needs to substantiate and refine our knowledge.

Gender-specific neuromuscular adaptations to unloading in isolated rat soleus muscles.

INTRODUCTION: The potential of gender to affect unloading-induced neuromuscular adaptations was investigated. METHODS: Twenty male and 20 female rats were assigned to control (CTL), or unloaded (UL) conditions. After 2 weeks of unloading, soleus muscles were removed, and neuromuscular function was assessed during a train of alternating indirect (neural) and direct (muscle) stimuli. RESULTS: In rested muscle, strength showed significant (P ≤ 0.05) main effects for gender (male > female) and treatment (CTL > UL). By the end of the testing protocol, when muscles showed fatigue, gender-related and treatment-related differences in strength had disappeared. Neuromuscular transmission efficiency and strength suffered a greater decline during the testing protocol in males than females. Unloaded male muscles displayed greater contractile velocity than female muscles both when rested and fatigued. CONCLUSIONS: Gender affected unloading-induced neuromuscular adaptations. The greater strength of rested male muscles was due to greater muscle mass and neuromuscular transmission efficiency. Muscle Nerve 54: 300-307, 2016.

Effect of resistance training on neuromuscular junctions of young and aged muscles featuring different recruitment patterns.

To examine the effects of aging on neuromuscular adaptations to resistance training (i.e., weight lifting), young (9 months of age) and aged (20 months of age) male rats either participated in a 7-week ladder climbing protocol with additional weight attached to their tails or served as controls (n = 10/group). At the conclusion, rats were euthanized and hindlimb muscles were quickly removed and frozen for later analysis. Longitudinal sections of the soleus and plantaris muscles were collected, and pre- and postsynaptic features of neuromuscular junctions (NMJs) were visualized with immunofluorescence staining procedures. Cross-sections of the same muscles were histochemically stained to determine myofiber profiles (fiber type and size). Statistical analysis was by two-way ANOVA (main effects of age and treatment) with significance set at P ≤ 0.05. Results revealed that training-induced remodeling of NMJs was evident only at the postsynaptic endplate region of soleus fast-twitch myofibers. In contrast, aging was associated with pre- and postsynaptic remodeling in fast- and slow-twitch myofibers of the plantaris. Although both the soleus and the plantaris muscles failed to display either training or aging-related alterations in myofiber size, aged plantaris muscles exhibited an increased expression of type I (slow-twitch) myofibers in conjunction with a reduced percentage of type II (fast-twitch) myofibers, suggesting early stages of sarcopenia. These data demonstrate the high degree of specificity of synaptic modifications made in response to exercise and aging and that the sparsely recruited plantaris is more vulnerable to the effects of aging than the more frequently recruited soleus muscle.

Aging obviates sex-specific physiological responses to exercise.

OBJECTIVES: Both sex and aging have been shown to affect physiological responses to exercise. The aim of the present investigation was to determine whether aging impacted the sex-specific nature of physiological responses to exercise commonly noted among young adults. METHODS: Ten aged men (69.0 ± 1.7 years; mean ± SE) and 10 aged women (71.6 ± 1.3 years) reporting similar levels of habitual physical activity performed a 30-min exercise session at 60-65% of their predetermined peak oxygen uptake. Cardiovascular, thermoregulatory, and metabolic variables were assessed before exercise, at the 15th and 30th min of exercise, and at 5 and 15 min into a passive postexercise recovery period. Variables of interest were statistically analyzed via two-way analysis of variance with repeated measures; significance was set at P < 0.05. RESULTS: Significant effects of time (i.e., exercise) for each physiological variable of interest were identified, but not once was a significant effect of group (i.e., sex) detected. CONCLUSIONS: Exercise-induced physiological responses to prolonged, moderate intensity exercise were similar among aged men and aged women. This evidence that the sexually dimorphic nature of physiological responses to exercise is obviated with age should be taken into account when prescribing health-related exercise training programs for older individuals.

Cellular and Subcellular Characteristics of Neuromuscular Junctions in Muscles with Disparate Duty Cycles and Myofiber Profiles.

The neuromuscular system accounts for a large portion (~40%) of whole body mass while enabling body movement, including physical work and exercise. At the core of this system is the neuromuscular junction (NMJ) which is the vital synapse transducing electrical impulses from the motor neurons to their post-synaptic myofibers. Recent findings suggest that subcellular features (active zones) of the NMJ are distinctly sensitive to changes in activity relative to cellular features (nerve terminal branches, vesicles, receptors) of the NMJ. In the present investigation, muscles with different recruitment patterns, functions, and myofiber type profiles (soleus, plantaris, extensor digitorum longus [EDL]) were studied to quantify both cellular and subcellular NMJ characteristics along with myofiber type profiles. Results indicated that, in general, dimensions of subcellular components of NMJs mirrored cellular NMJ features when examining inter-muscle NMJ architecture. Typically, it was noted that the NMJs of the soleus, with its most pronounced recruitment pattern, were larger (p < 0.05) than NMJs of less recruited muscles. Moreover, it was revealed that myofiber size did not dictate NMJ size as soleus muscles displayed the smallest fibers (p < 0.05) while the plantaris muscles exhibited the largest fibers. In total, these data show that activity determines the size of NMJs and that generally, size dimensions of cellular and subcellular components of the NMJ are matched, and that the size of NMJs and their underlying myofibers are uncoupled.

Mitochondria transplant therapy improves regeneration and restoration of injured skeletal muscle.

BACKGROUND: Injection of exogenous mitochondria has been shown to improve the ischaemia-damaged myocardium, but the effect of mitochondrial transplant therapy (MTT) to restore skeletal muscle mass and function has not been tested following neuromuscular injury. Therefore, we tested the hypothesis that MTT would improve the restoration of muscle function after injury. METHODS: BaCl2 was injected into the gastrocnemius muscle of one limb of 8-12-week-old C57BL/6 mice to induce damage without injury to the resident stem cells. The contralateral gastrocnemius muscle was injected with phosphate-buffered saline (PBS) and served as the non-injured intra-animal control. Mitochondria were isolated from donor mice. Donor mitochondria were suspended in PBS or PBS without mitochondria (sham treatment) and injected into the tail vein of BaCl2 injured mice 24 h after the initial injury. Muscle repair was examined 7, 14 and 21 days after injury. RESULTS: MTT did not increase systemic inflammation in mice. Muscle mass 7 days following injury was 21.9 ± 2.1% and 17.4 ± 1.9% lower (P < 0.05) in injured as compared with non-injured intra-animal control muscles in phosphate-buffered saline (PBS)- and MTT-treated animals, respectively. Maximal plantar flexor muscle force was significantly lower in injured as compared with uninjured muscles of PBS-treated (-43.4 ± 4.2%, P < 0.05) and MTT-treated mice (-47.7 ± 7.3%, P < 0.05), but the reduction in force was not different between the experimental groups. The percentage of collagen and other non-contractile tissue in histological muscle cross sections, was significantly greater in injured muscles of PBS-treated mice (33.2 ± 0.2%) compared with MTT-treated mice (26.5 ± 0.2%) 7 days after injury. Muscle wet weight and maximal muscle force from injured MTT-treated mice had recovered to control levels by 14 days after the injury. However, muscle mass and force had not improved in PBS-treated animals by 14 days after injury. The non-contractile composition of the gastrocnemius muscle tissue cross sections was not different between control, repaired PBS-treated and repaired MTT-treated mice 14 days after injury. By 21 days following injury, PBS-treated mice had fully restored gastrocnemius muscle mass of the injured muscle to that of the uninjured muscle, although maximal plantar flexion force was still 19.4 ± 3.7% (P < 0.05) lower in injured/repaired gastrocnemius as compared with uninjured intra-animal control muscles. CONCLUSIONS: Our results suggest that systemic mitochondria delivery can enhance the rate of muscle regeneration and restoration of muscle function following injury.

Factors relating to gender specificity of unloading-induced declines in strength.

INTRODUCTION: This investigation aimed to: (1) confirm whether women were more vulnerable to the negative neuromuscular adaptations elicited by muscle unloading and if so, (2) determine which physiological mechanism(s) explain those gender-related differences. METHODS: Healthy young men (20.7 ± 0.3 years) and women (20.3 ± 0.3 years)-(N = 12/group)-participated by completing neuromuscular functional tests before and after 7 days of unloading. RESULTS: During isokinetic testing of peak torque, work performed, and power, women displayed significantly (P ≤ 0.05) greater declines in performance than men at 1.05 and 2.09, but not 0.53 rads/s. During maximal isometric contractions, women experienced greater strength decrements. Similar gender-specific adaptations to unloading were found in EMG activity, but not muscle mass, neuromuscular transmission, or force relative to EMG. CONCLUSIONS: Women are more susceptible to the adaptations of muscle unloading, and disturbances in neural drive from the central nervous system are probably responsible.

Adaptive Remodeling of the Neuromuscular Junction with Aging.

Aging is associated with gradual degeneration, in mass and function, of the neuromuscular system. This process, referred to as "sarcopenia", is considered a disease by itself, and it has been linked to a number of other serious maladies such as type II diabetes, osteoporosis, arthritis, cardiovascular disease, and even dementia. While the molecular causes of sarcopenia remain to be fully elucidated, recent findings have implicated the neuromuscular junction (NMJ) as being an important locus in the development and progression of that malady. This synapse, which connects motor neurons to the muscle fibers that they innervate, has been found to degenerate with age, contributing both to senescent-related declines in muscle mass and function. The NMJ also shows plasticity in response to a number of neuromuscular diseases such as amyotrophic lateral sclerosis (ALS) and Lambert-Eaton myasthenic syndrome (LEMS). Here, the structural and functional degradation of the NMJ associated with aging and disease is described, along with the measures that might be taken to effectively mitigate, if not fully prevent, that degeneration.

Juvenile Neuromuscular Systems Show Amplified Disturbance to Muscle Unloading.

Muscle unloading results in severe disturbance in neuromuscular function. During juvenile stages of natural development, the neuromuscular system experiences a high degree of plasticity in function and structure. This study aimed to determine whether muscle unloading imposed during juvenile development would elicit more severe disruption in neuromuscular function than when imposed on fully developed, mature neuromuscular systems. Twenty juvenile (3 months old) and 20 mature (8 months old) rats were equally divided into unloaded and control groups yielding a total of four groups (N = 10/each). Following the 2 week intervention period, soleus muscles were surgically extracted and using an ex vivo muscle stimulation and recording system, were examined for neuromuscular function. The unloading protocol was found to have elicited significant (P ≤ 0.05) declines in whole muscle wet weight in both juvenile and mature muscles, but of a similar degree (P = 0.286). Results also showed that juvenile muscles displayed significantly greater decay in peak force due to unloading than mature muscles, such a finding was also made for specific tension or force/muscle mass. When examining neuromuscular efficiency, i.e., function of the neuromuscular junction, it again was noted that juvenile systems were more negatively affected by muscle unloading than mature systems. These results indicate that juvenile neuromuscular systems are more sensitive to the effects of unloading than mature ones, and that the primary locus of this developmental related difference is likely the neuromuscular junction as indicated by age-related differences in neuromuscular transmission efficiency.

Effects of exercise training on neuromuscular junctions and their active zones in young and aged muscles.

The neuromuscular junction (NMJ) connects the motor neuron with myofibers allowing muscle contraction. Both aging and increased activity result in NMJ remodeling. Here, the effects of exercise were examined in young and aged soleus muscles. Using immunofluorescent staining procedures, cellular and active zone components of the NMJ were quantified following a treadmill running program. Immunofluorescence was employed to determine myofiber profiles (size and type). Two-way analysis of variance procedures with main effects of age and treatment showed that when analyzing NMJs at the cellular level, significant (p ≤ 0.05) effects were identified for age, but not treatment. However, when examining subcellular active zones, effects for exercise, but not for age, were detected. Myofiber cross-sectional area showed that aging elicited atrophy and that among younger muscles endurance exercise training yielded decrements in myofiber size. Conversely, among aged muscles training elicited whole muscle and myofiber trends (p < 0.10) toward hypertrophy. Thus, different components of the neuromuscular system harbor unique sensitivities to various stimuli enabling proper adaptations to attain optimal function under differing conditions.

A comparison of physiological variables in aged and young women during and following submaximal exercise.

Previously, we have examined how aging affects the physiological responses of men to endurance exercise. In the present investigation, we aimed to extend our assessment of the influence of aging on exercise-induced responses by focusing on women. Ten young (20.3 +/- 0.3 years; mean +/- SE) and 10 aged (75.5 +/- 1.2 years) women performed 30 min of cycling at 60-65% of their predetermined peak oxygen uptake. Data for respiratory exchange ratio (RER), heart rate, blood pressure, rectal temperature, and plasma metabolites were collected before exercise, at the 15th and 30th min of exercise, and at 5 and 15 min postexercise. A two-way, repeated measures ANOVA with main effects of age and time was conducted on each variable. Our findings showed that age affected exercise-induced responses of each variable quantified. Although RER, heart rate, temperature, and lactate were significantly (P < 0.05) higher among young women, blood pressure and glucose values were greater among aged women. Moreover, unlike previous results noted among men where age-related differences primarily occurred during postexercise recovery, in women the effect of aging was detected during exercise itself. The data presented here indicate that aging impacts physiological responses of women to prolonged endurance exercise even when relative intensity (% of peak oxygen uptake) is held constant. Combined with our earlier study on men, these findings suggest that gender interacts with aging to determine whether age-related differences are manifested during exercise itself, or during postexercise recovery.

Gender influences neuromuscular adaptations to muscle unloading.

Muscle unloading results in decreased strength. This is partly attributed to reduced neural activation. This study examined whether men and women experienced different adaptations to muscle unloading. Ten men (21.4 +/- 0.8 years; mean +/- SE) and ten women (20.9 +/- 0.2 years) were subjected to 1 week of muscle unloading. Muscle function was quantified during knee extensions before, and following unloading. Electromyography (EMG) was assessed during maximal isometric contractions before, and after unloading. Results showed greater (P < 0.05) unloading-induced decrement in muscle performance in women than men. The loss of peak torque was significantly correlated (r = 0.69) with the decline in EMG consequent to unloading, and women displayed significantly larger reductions in EMG. These findings indicate that: (1) women are more affected by muscle unloading than men, and (2) gender-related differences in strength declines are associated with similar disparities in the nervous system's capacity to maximally stimulate muscle.

Adaptations to short-term muscle unloading in young and aged men.

INTRODUCTION: The purpose of this investigation was to determine whether young (21.7 yr) and aged (68.5 yr) men experienced similar responses to 7 d of muscle unloading (N = 10 per group). METHODS: Unilateral lower limb suspension (ULLS) was used to impose muscle unloading of the knee extensors. To compare the effects of unloading on aged and young men, a repeated-measures factorial ANOVA was used to assess those effects on isometric strength, as well as strength, total work, and average power during isokinetic contractions conducted at 0.53, 1.05, and 2.09 rad.s(-1). RESULTS: Data showed that at slower speeds of movement, only a main effect of unloading was identified with young and aged men displaying similar and significant (P < 0.05) ULLS-induced decrements in strength, work, and power. The decrease in isometric strength correlated well with loss of electromyographic activity of contracting muscles (r = 0.79, P = 0.0002). At higher speeds of isokinetic contractions, not only was a main effect of age detected (young > aged), but it was also revealed that aged men, but not young men, experienced significant unloading-induced declines in muscle performance. Moreover, unloading resulted in a significant increase in plasma cortisol, a potent catabolic hormone, only among aged men. In contrast to other variables assessed, muscle endurance, quantified during 30 repetitions completed at 3.14 rad.s(-1), did not differ between age groups, nor was it altered by unloading. CONCLUSION: These findings suggest that young and aged men respond differently to muscle unloading, but in assessing muscle performance, these differences are manifested only during faster contractile velocities.

Muscle fibers and their synapses differentially adapt to aging and endurance training.

BACKGROUND: This project aimed to determine the adaptability of the neuromuscular system to the stimuli of exercise training, and aging. METHODS: Young adult, and aged male rats were randomly assigned to either exercise training, or sedentary control groups. Exercise training featured an 8 week program of treadmill running. At the end of the intervention period, neuromuscular function was quantified with ex vivo stimulation procedures on isolated soleus muscles. Morphological adaptations were determined by quantifying myofiber profiles (fiber size and type) of soleus muscles. RESULTS: Ex vivo procedures confirmed that rested (fresh) young muscles were significantly (P < 0.05) stronger than aged ones. By the end of the 5 min stimulation protocol, however, young and aged muscles displayed similar levels of strength. Neuromuscular transmission efficacy as assessed by comparing force produced during indirect (neural) and direct (muscle) stimulation was unaffected by aging, or training, but under both conditions significantly declined over the stimulation protocol mimicking declines in strength. Myofiber size was unaffected by age, but training caused reductions in young, but not aged myofibers. Aged solei displayed a higher percentage of Type I fibers, along with a lower percentage of Type II fibers than young muscles. CONCLUSIONS: The greater strength of young muscles has a neural, rather than a muscular focal point. The loss of strength discerned over the 5 min stimulation protocol was linked to similar fatigue-related impairments in neuromuscular transmission. The two components of the neuromuscular system, i.e. nerves and muscles, do not respond in concert to the stimulus of either aging, or exercise training.

Chronic Resistance Training Does Not Ameliorate Unloading-Induced Decrements in Neuromuscular Function.

OBJECTIVE: The aim of this study was to assess the efficacy of long-term resistance training in preventing the detrimental effects of muscle unloading on neuromuscular function. DESIGN: Eleven untrained men and 11 men with extensive backgrounds in resistance training were tested for several parameters of neuromuscular function at various isokinetic contractile velocities before and after 7 days of muscle unloading. Measurements included muscle mass, strength, power, total work, electromyography, and neuromuscular transmission efficiency using superimposed electrical stimulation of maximally contracting muscles. RESULTS: Muscle performance was superior in resistance-trained subjects before and after unloading. In both groups of participants, unloading resulted in significantly (P < 0.05) diminished muscle performance, but only at isometric or slower isokinetic contractile velocities. Electromyography activity was significantly higher in trained than in untrained subjects before and after unloading. Thigh muscle mass was greater among trained subjects before and after unloading. Neither electromyography activity, thigh muscle mass, nor neuromuscular transmission efficiency was significantly altered by unloading in trained or untrained participants. CONCLUSIONS: Chronic resistance training was found to be ineffective in neutralizing the deleterious effects of unloading on neuromuscular function. It appears that positive adaptations associated with long-term resistance training provide no prophylactic effect when neuromuscular systems are subjected to unloading.

Aged men experience disturbances in recovery following submaximal exercise.

BACKGROUND: Physiological responses to exercise of moderate intensity and duration among aged compared to young adults have yet to be clearly defined. Further, the effects of aging on the rate and effectiveness of postexercise recovery are unknown. METHODS: Here, selected physiological responses during and following exercise of the same relative intensity were examined in untrained young and aged men. RESULTS: Generally, the two groups displayed similar responses during 30 minutes of exercise. During recovery, however, numerous age-related differences were manifested. Relative heart rate (% peak) was higher during recovery among the aged group. Postexercise lactate remained increased longer among aged men, and blood glucose regulation was impaired during recovery. This difference in circulating glucose was associated with insulin responses whereby young, but not aged men experienced a postexercise spike. Unlike that in young men, rectal temperature among aged men continued to increase through the entire recovery period. CONCLUSIONS: These data suggest that aged men encounter problems in recovering from submaximal exercise.