Chloroquine impairs maximal transdiaphragmatic pressure generation in old mice

Abstract

Aging results in increased neuromuscular transmission failure and denervation of the diaphragm muscle, as well as decreased force generation across a range of motor behaviors. Increased risk for respiratory complications in old age is a major health problem. Aging impairs autophagy, a tightly regulated multistep process responsible for clearing misfolded or aggregated proteins and damaged organelles. In motor neurons, aging-related autophagy impairment may contribute to deficits in neurotransmission, subsequent muscle atrophy, and loss of muscle force. Chloroquine is commonly used to inhibit autophagy. We hypothesized that chloroquine decreases transdiaphragmatic pressure (Pdi) in mice. Old mice (16–28 mo old; n = 26) were randomly allocated to receive intraperitoneal chloroquine (50 mg/kg) or vehicle 4 h before measuring Pdi during eupnea, hypoxia (10% O₂)-hypercapnia (5% CO₂) exposure, spontaneous deep breaths (“sighs”), and maximal activation elicited by bilateral phrenic nerve stimulation (Pdi_max). Pdi amplitude and ventilatory parameters across experimental groups and behaviors were evaluated using a mixed linear model. There were no differences in Pdi amplitude across treatments during eupnea (∼8 cm H₂O), hypoxia-hypercapnia (∼10 cm H₂O), or sigh (∼36 cm H₂O), consistent with prior studies documenting a lack of aging effects on ventilatory behaviors. In vehicle and chloroquine-treated mice, average Pdi_max was 61 and 46 cm H₂O, respectively. Chloroquine decreased Pdi_max by 24% compared to vehicle (P < 0.05). There were no sex or age effects on Pdi in older mice. The observed decrease in Pdi_max suggests aging-related susceptibility to impairments in autophagy, consistent with the effects of chloroquine on this important homeostatic process.

NEW & NOTEWORTHY Recent findings suggest that autophagy plays a role in the development of aging-related neuromuscular dysfunction; however, the contribution of autophagy impairment to the maintenance of diaphragm force generation in old age is unknown. This study shows that in old mice, chloroquine administration decreases maximal transdiaphragmatic pressure generation. These chloroquine effects suggest a susceptibility to impairments in autophagy in old age.

INTRODUCTION

Aging is characterized by gradual functional, structural, and biochemical changes in physiologic processes that lead to lower quality of life and death. Aging effects on respiratory muscles including the diaphragm muscle contribute to the decline in respiratory function, increased risk for infectious complications, and death (1–4). In previous studies, aging-related diaphragm neuromuscular dysfunction is evident by increased neuromuscular transmission failure (5, 6), reduced presynaptic terminal volume (7), increased neuromuscular junction fragmentation (8), loss of larger phrenic motor neurons (9), and diaphragm muscle sarcopenia (10). Measurements of transdiaphragmatic pressure (Pdi) are a surrogate for diaphragm muscle force across a range of motor behaviors (11, 12). In previous studies, we found that Pdi generated during maximal activation elicited by bilateral phrenic nerve stimulation (Pdi_max) significantly decreased in old age (∼25%), whereas Pdi during eupnea, hypoxia-hypercapnia exposure, and spontaneous deep breaths (“sighs”) remained unchanged (13, 14). This aging-related decrease in higher force nonventilatory behaviors that are required to clear the airway likely contributes to the higher incidence of respiratory complications in the elderly (15, 16).

The mechanisms that lead to aging-related changes in diaphragm muscle function are not fully elucidated. However, emerging evidence suggests that autophagy, a key regulator of neuron homeostasis and plasticity, may play an important role (17–20). Autophagy is a tightly regulated multistep process responsible for clearing misfolded or aggregated proteins and damaged organelles. Autophagy is impaired with age (17, 21–23), leading to aging-related cellular dysfunction and neurodegeneration (24–26). In a previous study evaluating autophagy protein expression in phrenic motor neurons, we found that p62 expression (marker of degradation phase of autophagy) was significantly increased in 18-mo-old mice compared to 6-mo-old mice (17). These findings suggest that autophagy impairment may precede sarcopenia (10) and may contribute to aging-related neurodegeneration evident in motor units.

Chloroquine is an antimalarial drug that inhibits the fusion of autophagosomes with lysosomes, an essential step of autophagy (27–30), and is commonly used to inhibit autophagy across various systems and model organisms. Chloroquine also has anticancer and immune modulation effects across a range of dose and treatment regimens (31–35). Previous studies in mice found that chloroquine decreased neuromuscular transmission and diaphragm contractility (36, 37); however, at such doses, effects distinct from autophagy inhibition are likely. The effects of chloroquine on the maintenance of diaphragm force generation in old age have not been directly studied, particularly at doses that inhibit autophagy. The present study aims to test the hypothesis that chloroquine decreases Pdi in 16- to 26-mo-old mice (75%–95% survival rate).

METHODS

Animals

In these experiments, we used adult male (n = 10) and female (n = 16) C57BL/6 mice aged between 16 and 26 mo old. Animals were caged by sex and maintained in a 12-h light cycle with free access to food and water at Mayo Clinic facilities. Institutional Animal Care and Use Committee approved all protocols in compliance with the National Institute of Health guidelines.

Experimental Treatment

Mice were assigned in randomized, blinded fashion to intraperitoneal injection of either 50 mg/kg of chloroquine (MilliporeSigma, Burlington, MA), or vehicle (0.9% saline) 4 h before data collection. The dose of chloroquine is equivalent with previous work documenting acute autophagy inhibition (29, 30, 38) and functional effects on neuromuscular transmission (36, 39, 40). The group allocation was revealed once we finished data analysis.

Transdiaphragmatic Pressure Measurements

Mice were anesthetized using fentanyl (0.3 mg/kg), diazepam (5 mg/kg), and droperidol (15 mg/kg) (41, 42). Adequate anesthetic depth was guaranteed by continuous monitoring of heart rate, respiratory rate, and periodic deep tissue stimulation (approximately every 15 min). Anesthetic agents were redosed (one-third of the initial dose) as needed. We monitored heart rate and O₂ saturation using a thigh pulse oximeter (MouseOx Plus, Starr Life Sciences Corp, Oakmont, PA). Oxygen saturation in all animals was maintained at >90% with supplemental oxygen as needed.

The procedure for Pdi measurements was performed as described previously (12–14, 42–45). Briefly, we cannulated the trachea (19 G) and placed two pressure transducers: one in the stomach and the second in the esophagus (Mikro-Tip catheter transducer, 3.5 F, No. 8405249, SPR-524; Millar Instruments, Houston, TX). The data were digitized using PowerLab 16/35 and visualized using LabChart 8 (ADInstruments, Colorado Springs, CO). The direction of signal deflection of the esophageal pressure (P_eso) and gastric pressure (P_gas) was assessed to confirm the accurate positioning of the catheters throughout the experiment (Fig. 1). This was followed by postmortem evaluation of catheter position. As in previous studies (11, 12, 14, 42, 44), the Pdi was calculated as the difference between P_gas and P_eso. The abdomen was bound with a piece of medical gauze to approximate near-isometric conditions for diaphragm activation. The Pdi signal was band-pass filtered between 0.3 Hz and 30 Hz. The data were exported for post hoc analyses using MATLAB (MathWorks, Natick, MA 01760).

Figure 1.

Representative transdiaphragmatic pressure (Pdi) tracings from Pdi_max. We monitored the direction of signal deflection of the esophageal pressure (P_eso) and gastric pressure (P_gas) to confirm the accurate positioning of the catheters throughout the experiment.

We obtained Pdi measurements during quiet breathing of room air (eupnea), exposure to a hypoxic (10% O₂) and hypercapnic (5% CO₂) gas mixture for 5 min, and Pdi_max elicited by bilateral phrenic nerve stimulation, as previously described (12–14, 42–49). We allowed mice to rest for at least 2 min between behaviors to allow Pdi amplitude to return to the eupneic baseline. Deep breaths (“sighs”) were defined as large and spontaneous inspiratory events of at least two times the average eupneic Pdi, followed by an apneic period with the elimination of at least one full breath. Subsequently, a microdissection of the ventral cervical region was performed to expose and isolate the phrenic nerves bilaterally. Straight parallel bipolar electrodes (FHC, Cat. No. 30211, Bowdoin, ME) connected to a stimulator (Grass S88, Grass Telefactor, Warwick, RI) were used to stimulate both phrenic nerves with 0.02 ms duration pulses at a frequency of 150 Hz in a 330 ms duration train repeated every second for three to five trains. Mineral oil bath was used in the neck surrounding the isolated phrenic nerve to avoid current spread. Stimulation current was then adjusted until a maximal Pdi response was elicited. During stimulation, deflections in P_eso and P_gas were verified to confirm the appropriate direction (positive for the gastric signal, negative for esophageal) and stimulus duration (∼330 ms/ train) (Fig. 1). The traces in the individual P_eso, P_gas, or the resulting Pdi signal were also examined for movement artifacts and excluded from further analysis, if present.

We previously reported an algorithm to automatically detect individual respiratory events and mark individual breath onsets and offsets across a range of motor behaviors (46). The terminal point of each breath was identified by return to baseline. Based on previous studies in anesthetized mice (42, 43, 49), Pdi during eupnea and hypoxia-hypercapnia is consistent such that we excluded individual breaths with a Pdi amplitude greater than 2 SD away from the mean, assuming technical issues, such as variances in electrode placement, anesthetic depth, and/or animal health, contributed to this variance. This approach was used to facilitate analysis of within-subject variability.

For each behavior, we measured Pdi amplitude and duration, duty cycle, and rate of rise. We analyzed individual respiratory events for 1 to 2 min of eupnea, the last 2 min of hypoxia-hypercapnia, and identified the largest sigh recorded during eupnea or hypoxia-hypercapnia exposure and the largest bilateral phrenic nerve stimulation event for Pdi_max (12, 46). The rate of rise of Pdi for each motor behavior was determined by measuring the Pdi amplitude at 75 ms after onset (Pdi₇₅), consistent with prior studies (50, 51).

Statistical Analyses

A standard software was used to perform all statistical analyses (JMP 14, SAS Institute Inc., Cary, NC). Agostino and Pearson omnibus test was used to assess normality. A mixed linear model was used to evaluate Pdi amplitude and ventilatory parameters across experimental groups and behaviors. The variables behavior, sex, treatment group, and their interaction were used as fixed effects and animal as a random effect. Based on previous reports of Pdi in mice (12, 14, 42), we estimated that nine animals per group would be sufficient to detect a 20% change in Pdi_max following treatment, with 80% power and α = 0.05. A post hoc analysis was performed using the Tukey–Kramer Honestly Significant Difference (HSD) test when appropriate. We established statistical significance at P < 0.05. Experimental data in the text of the manuscript are presented as means (SD), unless otherwise stated.

RESULTS

Animals

We treated all mice (16 females and 10 males) with chloroquine or vehicle (0.9% saline) 4 h before the data collection. Four animals died during the experiment. Animals in both treatment groups were of similar ages. The chloroquine group was an average of 20 mo old, from 17 to 23 mo; vehicle group was an average of 20 mo old, from 16 to 28 mo. Pdi_max values from four animals were excluded due to a priori defined criteria (see methods). There was no significant difference between male and female weight (male: 33 ± 4 g; female: 31 ± 4 g; F_1,21 = 2; P = 0.17).

Transdiaphragmatic Pressure Measurements

Pdi was measured during eupnea, hypoxia-hypercapnia, and Pdi_max in every animal in the chloroquine (n = 10) and vehicle (n = 12) groups. Equal anesthetic depth was maintained across animals by continuous monitoring of heart rate, respiratory rate, O₂ saturation, as well as assessment of deep pain response and corneal reflex.

The mixed linear model for Pdi amplitude did not show an effect of sex (F_1,19 < 1; P = 0.89), so all the presented data are a combination of male and female values. Figure 2 shows the representative Pdi tracings across all behaviors. Figure 3 shows the plot of the mean Pdi amplitudes across behaviors. There was a significant effect on the Pdi amplitude of behavior (F_3,56 = 131; P < 0.001), and the interaction between behavior and group (F_3,56 = 3; P = 0.02). There were no significant differences between the vehicle and chloroquine-treated groups in Pdi amplitude during eupnea, hypoxia-hypercapnia, or sighs. During eupnea, the mean Pdi amplitude for both the vehicle and chloroquine group was the same (8 ± 2 cm H₂O). During hypoxia-hypercapnia, the mean Pdi amplitude was 9 ± 2 cm H₂O for vehicle and 10 ± 2 cm H₂O for chloroquine group. During sigh, the Pdi amplitude was 37 ± 15 cm H₂O for the vehicle and 35 ± 8 cm H₂O for the chloroquine group. Pdi amplitude during sighs was ∼4 times higher than the amplitude during eupnea across treatment groups. During bilateral phrenic nerve stimulation, Pdi was 24% lower in the chloroquine group (46 ± 12 cm H₂O) compared to the vehicle group (61 ± 16 cm H₂O; P < 0.05).

Figure 2.

Representative transdiaphragmatic pressure (Pdi) tracings from a single, aged mouse from each treatment group during eupnea (breathing room air), hypoxia-hypercapnia exposure (10% O₂–5% CO₂), spontaneous deep breaths (sighs), and maximal Pdi generated by bilateral phrenic nerve stimulation at 150 Hz (Pdi_max). We treated mice with vehicle (0.9% saline) or 50 mg/kg of chloroquine 4 h before data collection.

Figure 3.

Summary results of transdiaphragmatic pressure (Pdi) generated during eupnea (breathing room air) and hypoxia-hypercapnia exposure (10% O₂–5% CO₂), spontaneous deep breaths (sighs), and bilateral phrenic nerve stimulation at 150 Hz (Pdi_max) in C57BL/6 mice treated intraperitoneally with vehicle (0.9% saline; n = 4 males, 8 females) or 50 mg/kg of chloroquine (n = 4 males, 6 females) 4 h before data collection. Data were analyzed using a mixed linear model with animal as a random effect (see methods). There was a significant effect on the Pdi amplitude of behavior (F_3,56 = 131; P < 0.001), and the interaction between behavior and group (F_3,56 = 3; P = 0.02). Boxplot shows the median, first and third quartiles (box), minimum and maximum values (whiskers). *Significantly different compared with vehicle-treated group (P < 0.05).

There was a significant effect on the Pdi amplitude at 75 ms after onset (Pdi₇₅) of behavior (F_3,5309 = 43; P < 0.001), but no effect of group (F_1,2 <1; P = 0.82) or the interaction between behavior and group (F_3,16 <1; P = 0.94). The mean Pdi₇₅ across groups was 3 ± 1 cm H₂O during eupnea, 5 ± 2 cm H₂O during hypoxia-hypercapnia, and 4 ± 2 cm H₂O during sigh. The mean Pdi₇₅ during bilateral phrenic nerve stimulation was significantly higher than the other behaviors (22 ± 13 cm H₂O; P < 0.05).

Ventilatory Parameters

Respiratory rate, inspiratory duration, and duty cycle were calculated from the Pdi signal during eupnea and hypoxia-hypercapnia (Table 1). There was no interaction between sex, behavior, or treatment in respiratory rate, inspiratory duration, or duty cycle. There was an overall significant effect on the respiratory rate of behavior (F_1,35 = 50; P < 0.001) and sex (F_1,36 = 16; P < 0.001), but no effect of treatment group (F_1,58 <1; P = 0.34). However, post hoc analysis revealed no differences in the respiratory rate between sexes for each behavior and group, so the data in Table 1 reflect males and females combined. The respiratory rate was significantly higher (∼50%) during hypoxia-hypercapnia than during eupnea. Overall, the respiratory rate increased to 147 ± 30 min⁻¹ during hypoxia-hypercapnia, compared to 95 ± 20 min⁻¹ during eupnea. There was an overall significant effect on inspiratory duration of behavior (F_1,35 = 21; P <0.001) and sex (F_1,36 = 14; P <0.001), but no effect of treatment group (F_1,35 < 1; P = 0.68). Post hoc analysis revealed that females had a lower hypoxia-hypercapnia inspiratory duration (229 ± 41 ms) compared to males in the chloroquine group (353 ± 56 ms; P < 0.05). Finally, there was an overall effect on the duty cycle of behavior (F_1,35 = 12; P < 0.001) and treatment group (F_1,35 = 11; P < 0.001), but no effect of sex (F_1,19 = 1; P = 0.22). Overall, the duty cycle increased to 63 ± 6% during hypoxia-hypercapnia compared with 57 ± 7% during eupnea.

Table 1.

Ventilatory parameters during eupnea and hypoxia-hypercapnia

	Vehicle	Chloroquine
Respiratory rate, min−1*
Eupnea	99 ± 23	92 ± 16
Hypoxia-hypercapnia	146 ± 32	148 ± 29
Inspiratory duration, ms*
Eupnea	378 ± 7	354 ± 4
Hypoxia-hypercapnia	270 ± 8	279 ± 8
Duty cycle, %*Ɨ
Eupnea	59 ± 7	54 ± 6
Hypoxia-hypercapnia	66 ± 6	60 ± 5

C57BL/6 mice were treated intraperitoneally with vehicle (0.9% saline; n = 4 males, 8 females) or 50 mg/kg of chloroquine (n = 4 males, 6 females) 4 h before data collection. Data analyzed using a mixed linear model with animal as a random effect and presented as means ± 95% confidence interval (CI). *Overall effect of behavior (P < 0.001; see results); ƗOverall effect of treatment group (P < 0.001; see results).

DISCUSSION

The present study shows that acute treatment with chloroquine decreases Pdi during bilateral phrenic nerve stimulation in aged mice, reflecting a decreased higher force generation capacity. Chloroquine did not affect Pdi during lower forces necessary for ventilatory behaviors. Chloroquine is commonly used as an autophagy inhibitor across various model organisms (27, 28, 34, 35). Given the importance of autophagy to aging-related changes in motor neurons (17–20), the purpose of this study was to evaluate the effect of a known autophagy inhibitor, chloroquine, on Pdi in aged mice. The effects of chloroquine on Pdi in old age are novel and serve as the basis for future studies to determine the possible role of autophagy in aging-related neuromuscular dysfunction (17, 21, 52, 53). Our results are also consistent with the aging-related reduction in Pdi_max in mice, rats, and humans (10, 13, 54, 55).

Mechanism of Chloroquine Effects in Old Age

Chloroquine is an antimalarial drug that inhibits the fusion of autophagosomes with lysosomes, an essential step of autophagy (28). Intraperitoneal administration of chloroquine in mice at similar doses as in the present study (50–60 mg/kg) increases LC3 II protein expression by 4 h in liver (29), heart (38), and brain (56), and 2 wk of chloroquine administration (50 mg/kg/day) increases LC3 immunofluorescence in motor neurons (57). These changes in autophagy marker LC3 expression are similar to those seen in phrenic motor neurons of aged mice (17), suggesting that chloroquine may exacerbate age-related impairments in autophagy and impose functional decline.

Although a major mode of action of chloroquine is on autophagy, it likely impacts multiple cell processes independent of autophagy. In nutrient-deficient endothelial cells, chloroquine (50 µM) increases intracellular reactive oxygen species generation (58). Chloroquine also has immune modulation effects (32), for example 100-µM chloroquine produces proinflammatory responses by human astroglial cells through activation of NF-κB (59). The beneficial anticancer effects of chloroquine include reducing tumor growth in mouse models of melanoma when given at 50–100 mg/kg/day for 20 days and was found to be independent of autophagy (31). Accordingly, it is possible that our observed effect of chloroquine is not due to autophagy inhibition. However, the doses used were considerably higher or repeated chronically than those in the present study. Based on the results of the present study, the mechanism by which chloroquine reduces maximal Pdi generation in aged mice must be investigated in future studies.

Potential Sites of Chloroquine Action in the Neuromuscular System

Several prior reports have examined chloroquine effects on force generation and neuromuscular transmission. Several studies indicate direct effects of chloroquine on muscle contractions that were dose- and treatment duration-dependent (60, 61). In vitro, decreased muscle contraction induced by acetylcholine or caffeine is evident at chloroquine concentrations >10 µM (61). In the diaphragm muscle, acute and chronic treatment with chloroquine reduced twitch and tetanic forces (60), likely reflecting impaired intracellular Ca²⁺ handling. Importantly, in humans, the administration of chloroquine did not affect muscle protein synthesis or turnover rates (62), thus clouding the interpretation of the mechanisms responsible for chloroquine-induced myopathy (63). These studies notwithstanding, the contribution of pre- and postsynaptic effects has not been elucidated. Chloroquine at concentrations ≥5 µM decreases quantal content and reduces synaptic vesicle release probability at mouse diaphragm neuromuscular junctions studied electrophysiologically, suggesting an effect on presynaptic vesicle release and recycling (36). Experiments in frog gastrocnemius-soleus nerve-muscle preparations showed that chloroquine at concentrations >17 µM caused a reduction in neuromuscular transmission after 12–35 min of chloroquine incubation (39). In agreement, chloroquine concentrations in the range of ∼0.16–20 µM decrease indirect muscle contraction elicited by supramaximal nerve stimulation in mouse diaphragm-phrenic nerve preparations (37) and in frog gastrocnemius and cat tibialis anterior muscles (40). Accordingly, the chloroquine dose in the present study is expected to elicit effects primarily at presynaptic sites of diaphragm neuromuscular junctions. In oocytes expressing adult muscle acetylcholine receptors, minimal effects of 0.2 µM of chloroquine on acetylcholine-evoked currents were reported (64). At concentrations >10 µM, chloroquine decreased motor neuron action potential amplitude (39), as well as presynaptic calcium channel currents and action potential firing rate in the brain (65). In aggregate, these studies support an effect of chloroquine on neuromuscular transmission that includes disruption of presynaptic vesicle release and recycling.

Autophagy and Neuromuscular Transmission

Autophagy and endocytosis are interconnected pathways (66) and share common molecules (67). Inhibiting autophagy could contribute to impaired neuromuscular transmission by disrupting synaptic vesicle cycling, especially during higher frequency stimulation (>100 Hz) when bulk endocytosis becomes necessary for replenishing synaptic vesicle pools (68) (Fig. 4). Bulk endocytosis requires early endosome machinery to process large sections of presynaptic membrane into new synaptic vesicles and replenish the vesicle pool (69–72). Late endosomes fuse with autophagosomes and lysosomes to generate autolysosomes, a step mediated by SNAP-29, Stx17, and VAMP8 (73). Chloroquine inhibits mobilization of SNAP-29 to autophagosomes and hence the formation of SNAP-29/Stx17 complexes (28, 73), leading to early endosome accumulation and preventing endocytic processing to replenish synaptic vesicles (74). Disruption of endolysosomal trafficking may also decrease synaptic vesicle fusion and release via inhibiting effects of accumulated proteins such as VAMP4 (75). Inhibiting Rab 7 decreases synaptic vesicle fusion in hippocampal neurons due to the accumulation of VAMP4, an essential cargo molecule for activity-dependent bulk endocytosis (75). VAMP4 accumulation decreases vesicle release probability during higher frequency (40 Hz) stimulation in primary hippocampal neurons (75). In addition, aberrant autophagy and endocytosis may lead to accumulation of proteins like Synaptotagmin 7 or n-Sect/Munc-18 that negatively regulate synaptic vesicle release (76, 77).

Figure 4.

Model for chloroquine effects at the neuromuscular junction. During lower stimulation frequencies, mechanisms like clathrin-mediated endocytosis predominate for vesicle retrieval (1). After endocytosis, retrieved vesicles are processed and prepared for release as part of the recycling or reserve pools of synaptic vesicles (2). Infrequently, retrieved vesicles fuse with the sorting endosome (3). During high-frequency stimulation, vesicles are retrieved by bulk endocytosis (4). Bulk endosomes fuse with sorting endosomes (3) to process synaptic vesicles into the reserve pool. Sorting endosomes also fuse with autophagosomes (5), which are processed by fusing with lysosomes to create autolysosomes (6). Both Stx17/SNAP-29 complex (present on autophagosomes) and VAMP 8 (on lysosomes) mediate these fusion events. Chloroquine inhibits autolysosome generation by inhibiting Stx17/SNAP-29 complex formation, impairing autophagy flux and vesicle/endosome trafficking. Furthermore, accumulation of proteins like VAMP4, Synaptotagmin 7, or n-Sect/Munc-18 may directly inhibit synaptic vesicle release (7). Accordingly, chloroquine administration is expected to impair neurotransmission particularly during behaviors that require higher stimulation frequencies and decrease maximal muscle force generation. Figure created with BioRender.com.

Autophagy and Aging

Autophagy is impaired as a result of aging in many cells, including motor neurons (17, 78–81). Indeed, in a previous study, we found that the autophagy protein p62 increases by 18 mo of age, and LC3 increases by 24 mo in mouse phrenic motor neurons (17), supporting aging-related autophagosome accumulation and impaired autophagy. Aging-related neuromuscular changes include increased neuromuscular transmission failure in phrenic nerve-diaphragm muscle preparations (5, 6), morphological changes at diaphragm neuromuscular junctions that are expected to impact synaptic vesicle cycling and release (decreased presynaptic terminal volume) (6, 7), as well as diaphragm muscle sarcopenia (10, 13, 49, 82–84). There is evidence that aging effects are selective, including loss of predominantly larger phrenic motor neurons (9) and reduced cross-sectional area of type IIx and/or IIb diaphragm muscle fibers (10, 84). Furthermore, there is increased susceptibility to neuromuscular transmission failure of type IIx and/or IIb diaphragm muscle fibers (5, 6) in older rats (24 mo old) compared to in young adult rats (6 mo old), with minimal effects on fatigue-resistant type I or IIa diaphragm muscle fibers. Overall, these aging-related changes at phrenic motor units show selectivity for fast-twitch, fatigable units, with a relative sparing of fatigue-resistant units. In a previous study in rat diaphragm neuromuscular junctions, lipid raft uptake was found to increase significantly at higher frequency stimulation (80 Hz) compared with lower frequency (20 Hz) and unstimulated (0 Hz) conditions (85). Fast-twitch fatigable motor units are physiologically activated at the higher frequencies (11, 86, 87). Greater lipid raft uptake reflects activity-dependent bulk endocytosis (88), and thus is likely susceptible to aging-related disruption in autophagy. Indeed, the susceptibility of more fatigable, fast-twitch motor unit effects may reflect the requirement for unimpaired autophagy in maintaining synaptic vesicle cycling and neuromuscular transmission at these neuromuscular junctions.

DATA AVAILABILITY

Data will be made available upon reasonable request.

GRANTS

This work was supported by NIH R01 AG057052.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

C.A.S., H.M.G., G.C.S., and C.B.M. conceived and designed research; C.A.S., M.H.A., and O.U.K. performed experiments; C.A.S., M.H.A., O.U.K., H.M.G., and C.B.M. analyzed data; C.A.S., M.H.A., H.M.G., and C.B.M. interpreted results of experiments; C.A.S., H.M.G., and C.B.M. prepared figures; C.A.S., H.M.G., and C.B.M. drafted manuscript; C.A.S., H.M.G., and C.B.M. edited and revised manuscript; C.A.S., M.H.A., O.U.K., H.M.G., G.C.S., and C.B.M. approved final version of manuscript.