Citations

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.

The neuromotor control of the diaphragm muscle (DIAm) in response to hypercapnia is not well understood. We show that both the number of motor units recruited and their discharge rates increase with hypercapnia, consistent with increased respiratory drive during hypercapnia. Potentially in response to this increased drive, the greatest effect of hypercapnia is on during the postinspiratory (descending) ramp of DIAm EMG activity, which shortens to facilitate higher respiratory rates.

The heterogeneity of diaphragm muscle (DIAm) activity in awake animals reflects real motor behavior diversity but makes assessments of eupneic breathing challenging. The present article uses an unsupervised machine learning model to identify eupneic breathing amidst a deluge of different DIAm electromyography (EMG) burst patterns in awake rats. This technique offers a scalable and reliable tool that improves efficiency of DIAm EMG analysis and minimizes potential sources of bias.

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 (DIAm) is the major inspiratory pump for breathing. Neural control of the DIAm during breathing involves the recruitment of fatigue resistant motor units comprising smaller phrenic motor neurons (PhMNs), which innervate type I and IIa fibers. More fatigable DIAm motor units comprise larger PhMNs that innervate type IIx/IIb fibers, which generate greater forces but are more fatigable. The fatigable motor units are only infrequently recruited for expulsive behaviors of the DIAm. Thus, there are marked differences in the activation history of DIAm motor units. Consistent with their higher activity levels, previous studies from our lab have shown that smaller PhMNs and type I and IIa DIAm fibers have greater mitochondrial volume density (MVD). PhMNs located in the cervical spinal cord (C3-C5) receive direct excitatory input from medullary premotor neurons, which is primarily ipsilateral. Thus, C2 spinal cord hemisection (C2SH) disrupts the neural pathway essential for breathing and significantly reduces DIAm activity on the same side. Therefore, we hypothesized that C2SH significantly reduces MVD of smaller PhMNs. In Sprague Dawley rats, PhMNs on both sides were retrogradely labeled by intrapleural injection of cholera toxin B (CTB) 3 days prior to C2SH (right side). In addition, DIAm EMG activity was recorded on both sides by inserting bipolar electrodes. Mitochondria were labeled by intraspinal injection of MitoTracker Red and imaged in identified PhMNs using 3D confocal microscopy. A series of optical slices (0.5 mm) of PhMNs were deconvolved to improve contrast, then thresholded for MitoTracker labeling followed by 3D reconstruction, providing detailed images of mitochondrial morphometry and distribution within PhMNs. In sham rats, the MVD of smaller (lower somal surface area) PhMNs was higher compared to larger PhMNs. C2SH reduced DIAm EMG activity on the ipsilateral side across a 14-day period, while activity on the contralateral side increased. After 14 days, C2SH reduced MVD by ~25% in smaller PhMNs on the ipsilateral side. We conclude that the reduced activity of smaller PhMNs induced by C2SH induced mitochondrial morphological changes, most likely due to reduced mitochondrial biogenesis. These results indicate that reduced DIAm activity associated with mechanical ventilation may impose mitochondrial remodeling that reduces mitochondrial oxidative capacity in PhMNs. These adaptations may disrupt mitochondrial function and impair weaning from mechanical ventilation. Research support by NIH grants HL166204, HL146114 and AG44615. 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 (DIAm) is the major pump muscle for breathing. Neural control of the DIAm involves recruitment of phrenic motor neurons (PhMNs) comprising motor units that vary in their mechanical and fatigue properties. Smaller PhMNs innervating fatigue resistant DIAm fibers are highly active being recruited during breathing, while larger PhMNs are infrequently recruited during more forceful expulsive behaviors. Previously, we found that to support their higher activity levels, smaller PhMNs have higher mitochondrial volume density and are less fragmented compared to larger PhMNs. With aging, larger PhMNs are more vulnerable and ~30% are lost, and those surviving display greater mitochondrial fragmentation. We also found that older rats exhibit elevated levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFa), which in other tissue has been linked to mitochondrial fragmentation via activation of the IRE1a S724-mediated endoplasmic reticulum (ER) stress pathway. Accordingly, we hypothesized that in 24-month-old rats, increased TNFa levels activate the pIRE1aS724-mediated ER stress pathway, leading to XBP1s splicing, which transcriptionally increases expression of cyclin-dependent kinases (CDK1 and CDK5) that mediate phosphorylation of DRP1 at Serine 616 (pDRP1S616), and ultimately mitochondrial fragmentation. We found that serum TNFa levels increased ~3.5 fold in 24-month rats compared to 6-month rats. Consistent with elevated TNFa, we found increased pIRE1aS724 and XBP1s splicing in the cervical ventral horn of 24-month rats. In retrogradely labeled PhMNs, we found XBP1s was localized to the nucleus, consistent with transcriptional activation of gene expression. Bioinformatic analysis and ChIP assay confirmed XBP1s binding to the CDK1 and CDK5 promoter regions in rat cervical ventral horn. Both CDK1 and CDK5 mRNA and protein were elevated in the cervical ventral of 24-month rats. In aged rats, pDRP1S616 phosphorylation increased with a corresponding increase in mitochondrial fragmentation. Our results support our hypothesis that chronic inflammation (elevated TNFa levels) in older rats activate the pIRE1aS724-mediated ER stress pathway, leading to XBP1s splicing, which transcriptionally targets CDK1 and CDK5 expression mediating pDRP1S616 phosphorylation and mitochondrial fragmentation. Research Supported by NIH R01 grant AG44615. 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.

Neural control of the diaphragm muscle (DIAm) is essential to sustain breathing. During breathing, fatigue resistant DIAm motor units are recruited comprising smaller phrenic motor neurons (PhMNs) and type I and IIa muscle fibers. Larger PhMNs comprise more fatigable DIAm motor units that are infrequently active during expulsive behaviors. The difference in activation history is reflected in the higher mitochondrial volume density in smaller PhMNs. In PhMNs, brain-derived neurotrophic factor (BDNF) signals through its high-affnity receptor, tropomyosin receptor kinase B (TrkB.FL). Previously, we found that intrathecal BDNF treatment induces CREB phosphorylation at Serine 133 (pCREBs133). We hypothesized that pCREBs133 binds to the PGC1a promoter, transcriptionally targeting PGC1a expression, mediating downstream increased expression of NRF1, NRF2, and TFAM and mitochondrial biogenesis. To test this hypothesis, we used a chemosensitive TrkBF616 rat model with a knock-in allele sensitive to 1NMPP1 in that TrkB kinase is inhibited following 1NMPP1, a small molecule derivative of the protein kinase inhibitor protein phosphatase 1 that plays a significant role inhibiting the TrkB phosphorylation and activation. Systemic treatment of 1NMPP1 in TrkBF616 rats inhibits TrkB kinase activity. A bioinformatic analysis was performed to identify a putative binding site for pCREBs133 in the PGC1a promoter sequence in rats. Using a chromatin immunoprecipitation (ChIP) assay we found that pCREBS133 binds to the PGC1a promoter in rat cervical ventral horn. We showed that intrathecal BDNF treatment increased pCREBs133 phosphorylation, which was blocked by 1NMPP1 treatment in TrkBF616 rats. PGC1a, NRF1/2, and TFAM protein was compared in cervical ventral horn lysates from BDNF- and BDNF+1NMPP1-treated TrkBF616 rats by qRT-PCR and Western Blot. Relative mtDNA copy number (normalized to nuclear DNA) was quantified by qPCR to assess the mitochondrial biogenesis. All results were analyzed by paired t-test. Following 1NMPP1-induced inhibition of TrkB kinase, PGC1a, NRF1/2, and TFAM mRNA and protein expression was reduced. Consistent with these findings, mtDNA copy number was decreased following TrkB kinase inhibition by 1NMPP1. We conclude that inhibition of TrkB kinase by 1NMPP1 reduces PGC1a-mediated mitochondrial biogenesis in rat cervical ventral horn. Supported by NIH grants AG44615 and HL146114. 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 (DIAm) is the primary inspiratory muscle in mammals. At baseline, expiration is passive; however, with increased CO2 expiration becomes active, involving the inhibition of postinspiratory DIAm activity. The neuromotor control of the DIAm involves both the recruitment and frequency coding of DIAm motor units (MU’s) and it is not clear which aspect plays a greater role with exposure to hypercapnia. The activity of DIAm MU’s can be split into three phases: recruitment, sustained activity, and derecruitment. The goal of the present study was to evaluate the effects of hypercapnia on the neuromotor control of the DIAm in awake Sprague-Dawley rats. We hypothesized that hypercapnia would lead to increased respiratory drive as estimated by the root-mean-square (RMS) EMG amplitude at the end of the recruitment phase. Accordingly, we implanted chronic fine-wire EMG electrodes into the mid-costal region of the right side of the DIAm in 10 (5 female, 5 male) anesthetized rats. Three days after recovery from surgery, we recorded DIAm EMG in lightly restrained awake rats during normocapnia and hypercapnia (7% CO2, 21% O2, balance N2). We estimated the duration of motor unit recruitment, sustained activity, and derecruitment by assessing the stationarity of DIAm EMG activity and used these durations to determine the RMS EMG amplitude at the completion of recruitment, at the peak, and immediately prior to derecruitment (normalized to the average peak RMS EMG amplitude during normocapnia within each animal). We found that the RMS EMG amplitude at the completion of recruitment was increased from ~75% to 85% with hypercapnia. Peak RMS EMG amplitude during hypercapnia increased from 100% to ~120%, while the amplitude at the onset of derecruitment increased from ~50% to 95%. These data suggest that ~70% of the total RMS EMG peak amplitude can be attributed primarily to motor unit recruitment, while the remaining ~30% can be attributed to frequency modulation during hypercapnia. Importantly the onset-to-offset amplitude difference was ~25% during normocapnia and ~15% during hypercapnia, suggesting that DIAm MU’s were derecruited at higher discharge rates with hypercapnia, likely due to inhibition. We also found that compared to normocapnia, respiratory rate increased from ~85 breaths/min to 125 breaths/min while the burst duration decreased from ~600 ms to ~400 ms during hypercapnia. The duty cycle was maintained around 80% for both, however the duty cycle of the sustained activity duration (i.e. the period during which MU’s are remaining activated) decreased from ~56% to ~28%. Taken together, our data suggest that hypercapnia in awake rats results in an increase in the number of motor units recruited and their discharge rates as well as changes in the phases of DIAm MU activity indicative of active expiration. Funded by NIH HL-146114. 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 (DIAm) is the major inspiratory muscle. Neural control of DIAm activation involves recruitment of phrenic motor neurons. Activation of the DIAm during breathing involes recruitment of fatigue resistant motor units comprising smaller PhMNs and type I and IIa muscle fibers. More force, but infrequent non-ventilatory behaviors of the DIAm involes the recruitment of fatigable motor units comprising larger PhMNs and type IIx/IIb fibers. This study investigated the role of brain derived neurotrophic factor (BDNF) signaling, through its high-affnity receptor, tropomyosin receptor kinase B (TrkB.FL), in the survival of PhMNs in young (6 months old) and old (24 months old) Sprague Dawley rats. Previous research in Fischer 344 rats revealed an age-related loss of larger PhMNs. We hypothesize that both BDNF/TrkB signaling and PhMN activity are crucial for PhMN survival, but that the influence of BDNF/TrkB decreases with age due to reduced TrkB.FL expression. PhMNs were retrogradely labeled and imaged using 3D confocal microscopy in both young and old rats. We found a similar reduction in larger PhMNs in Sprague Dawley rats as previously observed in Fischer 344 rats. In contrast to younger rats, intrathecal BDNF treatment did not induce phosphorylation of the transcription factor CREB at Serine133 in older rats, consistent with reduced TrkB.FL expression and signaling in older rats. In both old age and following TrkB kinase inhibition, smaller PhMNs survived perhaps due to the continued activation driving pCREB, while larger PhMNs may require BDNF/TrkB signaling to maintain pCREB and survival. Research Supported by NIH R01 grants AG44615, HL146114 and R56-HL166204. 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.