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.

Keywords: aging; autophagy; diaphragm muscle; motor neuron.

Graphical abstract

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.

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).

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.