Gransee, Heather M.

This study found that inhibiting TrkB kinase activity decreases the extent of neuromuscular transmission failure during repetitive stimulation at low frequency while increasing failure with high-frequency stimulation. The frequency-dependent effect of 1NMPP1 treatment suggests that the role of TrkB signaling in maintaining neuromuscular transmission during repetitive stimulation may differ between motor unit types (slow-twitch, fatigue-resistant vs. fast-twitch, fatigable) and likely reflect effects at the presynaptic terminal without major effects on the muscle itself.

Introduction The nerve-derived growth factor neuregulin (NRG) plays a role in the regulation of skeletal muscle mass through Akt and mTOR signaling transduction pathways that regulate both protein synthesis and degradation. We previously reported that NRG increases muscle protein synthesis (~20%) in a PI3 kinase (PI3K)/Akt-dependent manner. However, the effects of NRG on protein degradation are still poorly understood and are needed to elucidate the role of NRG in the maintenance of skeletal muscle protein balance. Methods Neonatal diaphragm muscle ex vivo preparations were pharmacologically treated with NRG and pharmacological inhibitors of PI3K (LY294002, 50 μM), MEK (PD98059, 50 μM) or mTOR (rapamycin, 100 nM). Tyrosine release from muscle was used as a surrogate measure of protein degradation. Results We report that basal protein degradation in the neonatal rat diaphragm muscle is significantly reduced by NRG treatment (19%). Basal protein degradation was increased following treatment with inhibitors of PI3K, MEK or mTOR, with inhibition of each pathway sufficient to increase basal protein degradation greater than 30%. Importantly, NRG treatment in the presence of each of these inhibitors blunts the increase in protein degradation induced by inhibition of PI3K, MEK or mTOR. NRG effects were significantly blunted by rapamycin (p < 0.05 compared to NRG alone), but not by LY294002 or PD98059. Discussion We suggest that both the PI3K/Akt and MAP kinase pathways are important for NRG effects on protein degradation, but that mTOR may be a critical modulator of these effects and thus of protein balance in skeletal muscle.

ABSTRACT Motor neuron plasticity and survival depend on several trophic factors, with signaling via the tropomyosin receptor kinase B (TrkB) receptor being particularly important beyond development and across the lifespan. We hypothesized that differences in motor neuron properties may reflect differential trophic influences across motor units within a single motor unit pool. Based on extensive previous characterization of rat phrenic motor neurons (PhMN), somal surface area was used as an indicator of motor unit type (smaller neurons innervating slow‐twitch, fatigue‐resistant units versus larger neurons innervating fast‐twitch, fatigable units). To examine whether TrkB expression reflects these size‐related properties, TrkB mRNA transcripts were studied in individual PhMNs labeled retrogradely with Alexa Fluor 488‐conjugated cholera toxin subunit β from five adult male Sprague–Dawley rats. Longitudinal sections of the cervical C3–C5 spinal cord (10 μm thick) were processed using fluorescence in situ hybridization (RNAscope) and imaged using confocal microscopy to measure individual PhMN size and full‐length TrkB mRNA transcripts in nuclear and cytoplasmic compartments. Across 287 PhMNs, significant size variation existed both within and between animals. Across all animals, somal surface area averaged 4385 ± 1627 μm 2 with mean values per animal ranging from 3544 to 5461 μm 2 . PhMNs were classified into tertiles within each animal based on somal surface area. Individual PhMNs averaged 1070 ± 685 TrkB mRNA transcripts per motor neuron. Larger PhMNs in the upper tertile by size expressed a greater total number of TrkB mRNA transcripts per motor neuron, with values on average threefold higher compared to smaller motor neurons in the lower tertile ( p < 0.001). Cytoplasmic and nuclear TrkB mRNA density (transcripts/volume) displayed no significant effect of PhMN somal surface area tertile. Overall, the greater TrkB mRNA levels in larger motor units responsible for higher force, expulsive motor behaviors may underlie their functional adaptations and resilience to injury or disease. image

Abstract Muscle plasticity is defined as the ability of a given muscle to alter its structural and functional properties in accordance with the environmental conditions imposed on it. As such, respiratory muscle is in a constant state of remodeling, and the basis of muscle's plasticity is its ability to change protein expression and resultant protein balance in response to varying environmental conditions. Here, we will describe the changes of respiratory muscle imposed by extrinsic changes in mechanical load, activity, and innervation. Although there is a large body of literature on the structural and functional plasticity of respiratory muscles, we are only beginning to understand the molecular‐scale protein changes that contribute to protein balance. We will give an overview of key mechanisms regulating protein synthesis and protein degradation, as well as the complex interactions between them. We suggest future application of a systems biology approach that would develop a mathematical model of protein balance and greatly improve treatments in a variety of clinical settings related to maintaining both muscle mass and optimal contractile function of respiratory muscles. © 2012 American Physiological Society. Compr Physiol 2:1441‐1462, 2012.

Autophagy impairments have been implicated in various aging conditions. Previous studies in cervical motor neurons show an age-dependent increase in the key autophagy proteins LC3 and p62, reflecting autophagy impairment and autophagosome accumulation. Chloroquine is commonly used to inhibit autophagy by preventing autophagosome–lysosome fusion and may thus emulate the effects of aging on the neuromuscular system. Indeed, acute chloroquine administration in old mice decreases maximal transdiaphragmatic pressure generation, consistent with aging effects. We hypothesized that chloroquine alters diaphragm muscle neuromuscular junction (NMJ) morphology and increases denervation. Adult male and female C57BL/6 × 129J mice between 5 and 8 months of age were used to examine diaphragm muscle NMJ morphology and denervation following daily intraperitoneal injections of chloroquine (10 mg/kg/d) or vehicle for 7 days. The motor end-plates and pre-synaptic terminals were fluorescently labeled with α-bungarotoxin and anti-synaptophysin, respectively. Confocal microscopy was used to assess pre- and post-synaptic morphology and denervation. At diaphragm NMJs, chloroquine treatment decreased pre-synaptic volume by 12% compared to the vehicle (p < 0.05), with no change in post-synaptic volume. Chloroquine treatment increased the proportion of partially denervated NMJs by 2.7-fold compared to vehicle treatment (p < 0.05). The morphological changes observed were similar to those previously reported in the diaphragm muscles of 18-month-old mice. These findings highlight the importance of autophagy in the maintenance of the structural properties at adult NMJs in vivo.

Aging disrupts multiple homeostatic processes, including autophagy, a cellular process for the recycling and degradation of defective cytoplasmic structures. Acute treatment with the autophagy inhibitor chloroquine blunts the maximal forces generated by the diaphragm muscle, but the mechanisms underlying neuromuscular dysfunction in old age remain poorly understood. We hypothesized that chloroquine treatment increases the presynaptic retention of the styryl dye FM 4-64 following high-frequency nerve stimulation, consistent with the accumulation of unprocessed bulk endosomes. Diaphragm-phrenic nerve preparations from 24-month-old male and female C57BL/6 × 129 J mice were incubated with FM 4-64 (5 µM) and either chloroquine (50 µM) or vehicle during 80 Hz phrenic nerve stimulation. Acute chloroquine treatment significantly decreased FM 4-64 intensity at diaphragm neuromuscular junctions following 80 Hz phrenic nerve stimulation, consistent with disrupted synaptic vesicle recycling. A similar reduction was evident in regions with the greatest FM 4-64 fluorescence intensity, which most likely surround synaptic vesicle release sites. In the absence of nerve stimulation, chloroquine treatment significantly increased FM 4-64 intensity at diaphragm neuromuscular junctions. These findings highlight the importance of autophagy in regulating presynaptic vesicle retrieval (including vesicle recycling and endosomal processing) and support the role of autophagy impairments in age-related neuromuscular dysfunction.

Abstract Ageing‐related neuromuscular dysfunction is associated with reduced tropomyosin‐related kinase receptor subtype B (TrkB) signalling and accumulation of damaged cytoplasmic aggregates in motor neurons. Autophagy functions to remove these damaged aggregates, and we previously reported increased cervical motor neuron expression of LC3 and p62 in old age. We hypothesized that inhibition of TrkB kinase activity results in an increase in the relative expression of both LC3 and p62 in cervical motor neurons, consistent with impaired progression of autophagy. TrkB F616A mice, which possess a mutation that renders TrkB kinase activity susceptible to rapid inhibition by 1NMPP1, were treated at 6, 18 or 24 months of age with vehicle or 1NMPP1 for 7 days. Immunofluorescence intensity was measured to determine LC3 and p62 expression in choline acetyltransferase‐positive motor neurons in the cervical spinal cord. The effect of inhibiting TrkB kinase activity on progression of autophagy was age dependent. In 6‐month‐old mice, inhibiting TrkB kinase activity increased cervical motor neuron expression of LC3 by 11% ( P < 0.001) and p62 by 8% ( P = 0.019) compared with vehicle treatment. In 18‐ and 24‐month‐old mice, there was no effect of inhibiting TrkB kinase activity on motor neuron LC3 or p62 expression. We provide evidence that inhibition of TrkB signalling impairs progression of autophagy in motor neurons of young mice, similar to the response to ageing. Accordingly, a reduction of TrkB signalling in old age might contribute to neuromuscular dysfunction by impairing progression of autophagy in motor neurons.

Chloroquine is an antimalarial agent commonly used as an autophagy inhibitor by preventing autolysosomal fusion. Autophagy is an important cellular process through which defective cytoplasmic structures are recycled and degraded that has been implicated in aging neuromuscular effects. We recently showed that acute chloroquine treatment blunts the maximal forces generated by the diaphragm muscle, likely by impairing neuromuscular transmission generally consistent with aging effects in the diaphragm muscle. In addition, old mice show increased accumulation of autophagy proteins in phrenic motor neurons, consistent with impaired autophagy flux. In the present study, we hypothesized that chloroquine treatment results in accumulation of bulk endosomes pre-synaptically. Each hemidiaphragm with attached phrenic nerve was excised from 24-month-old male and female C57BL/6 mice to evaluate frequency dependent (0 vs 80 Hz stimulation) uptake of the styryl dye FM4-64, given that 80-Hz stimulation frequency induces bulk endocytosis after synaptic vesicle retrieval. Diaphragm-phrenic nerve preparations were incubated in 5 μM FM4-64 for 1 h prior to 6-min nerve stimulation (0.5 ms supramaximal pulses in 80 Hz, 200 ms trains delivered at 40% duty cycle) followed by a 5-min post-stimulation incubation. FM4-64 uptake at diaphragm neuromuscular junctions (NMJs) was measured using confocal microscopy. Preparations were randomly assigned to co-incubation with chloroquine (50 μM) or vehicle (Rees-Simpson solution). There was minimal FM4-64 uptake in the absence of phrenic nerve stimulation, consistent with the activity-dependent uptake of the styryl dye. With 80 Hz stimulation, high levels of FM4-64 uptake were evident in both the chloroquine and vehicle-treated groups without significant differences across groups. FM4-64 fluorescence was evident throughout the presynpatic terminal with few areas of dense accumulation, consistent with retention of endosomes. There was no evidence of greater endosome accumulation with chloroquine treatment. Of note, a slight increase in FM4-64 uptake was evident in the chloroquine treated groups compared to vehicle in the non-stimulated NMJs. These results are consistent with FM4-64 uptake reflecting bulk endocytosis that mainly occurs during high frequency stimulation. The impairment in autophagy flux following chloroquine treatment is expected to result in endosome accumulation but it is possible that the stimulation protocol was insuffcient to elucidate this possibility. Collectively, the findings of this study are consistent with impaired autophagy at diaphragm presynaptic terminals which may underlie neuromuscular deficits observed in old age. Supported by NIH grant R01 AG057052 and the Mayo Clinic. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chloroquine (CQ), an antimalarial agent, inhibits autophagy by preventing the autophagosome from fusing with the lysosome, emulating the effects of aging. Autophagy is a cellular digestion process that contributes to homeostasis through degradation of cytoplasmic structures in the cell. Aging effects include autophagy impairment, leading to the accumulation of altered proteins and organelles, thus contributing to subsequent cell damage and death. Previous studies in phrenic motor neurons show an age-dependent increase in key autophagy proteins LC3 and p62, reflecting autophagy impairment and autophagosome accumulation. In addition, acute chloroquine administration in old mice decreases maximal transdiaphragmatic pressure generation, suggesting a neuromuscular effect in aging. We hypothesized that treatment with the autophagy inhibitor chloroquine alters pre-synaptic morphology and increases the proportion of denervated diaphragm muscle fibers. Adult male and female 6-month C57BL6 mice were used to examine diaphragm muscle neuromuscular junction (NMJ) morphology and denervation. The autophagy inhibitor chloroquine (10 mg/kg/d) or vehicle was administered via daily intraperitoneal injections for 7 days. Confocal microscopy was used to compare pre- and post-synaptic morphology and denervation. There was evidence of a decrease in pre-synaptic volume without evidence of post-synaptic volume changes in chloroquine treated mice compared to vehicle. As expected, vehicle treated mice did not show evidence of substantial denervation. In contrast, chloroquine treated mice showed an increased proportion of denervated NMJs. The morphological changes observed were similar to those previously reported in the diaphragm muscles of 24-month-old mice. These findings reveal the effects at the neuromuscular junction of inhibiting autophagy and implicate impaired autophagy as a mechanism of aging effects. Supported by NIH R01 AG057052 and Mayo Clinic. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The diaphragm muscle is essential for breathing, and its dysfunctions can be fatal. Many disorders affect the diaphragm, including muscular dystrophies. Despite the clinical relevance of targeting the diaphragm, there have been few studies evaluating diaphragm function following a given experimental treatment, with most of these involving anti-inflammatory drugs or gene therapy. Cell-based therapeutic approaches have shown success promoting muscle regeneration in several mouse models of muscular dystrophy, but these have focused mainly on limb muscles. Here we show that transplantation of as few as 5000 satellite cells directly into the diaphragm results in consistent and robust myofiber engraftment in dystrophin- and fukutin-related protein-mutant dystrophic mice. Transplanted cells also seed the stem cell reservoir, as shown by the presence of donor-derived satellite cells. Force measurements showed enhanced diaphragm strength in engrafted muscles. These findings demonstrate the feasibility of cell transplantation to target the diseased diaphragm and improve its contractility.