Time-dependent changes in autophagy, mitophagy and lysosomes in skeletal muscle during denervation-induced disuse.

Denervation is an experimental model of peripheral neuropathies as well as muscle disuse, and it helps us understand some aspects of the sarcopenia of aging. Muscle disuse is associated with reduced mitochondrial content and function, leading to metabolic impairments within the tissue. Although the processes that regulate mitochondrial biogenesis are understood, those that govern mitochondrial breakdown (i.e., mitophagy) are not well characterized in this context. Autophagy and mitophagy flux, measured up to the point of the lysosome (pre-lysosomal flux rates), were increased in the early stages of denervation, along with mitochondrial dysfunction, but were reduced at later time points when the degree of muscle atrophy was highest. Denervation led to progressive increases in lysosomal proteins to accommodate mitophagy flux, yet evidence for lysosomal impairment at later stages may limit the removal of dysfunctional mitochondria, stimulate reactive oxygen species signaling, and reduce muscle health as denervation time progresses.Deficits in skeletal muscle mitochondrial content and quality are observed following denervation-atrophy. This is due to alterations in the biogenesis of new mitochondria as well as their degradation via mitophagy. The regulation of autophagy and mitophagy over the course of denervation (Den) remains unknown. Further, the time-dependent changes in lysosome content, the end-stage organelle for mitophagy, remains unexplored. Here, we studied autophagic as well as mitophagic pre-lysosomal flux in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria from rat muscle subjected to Den for 1, 3, or 7 days. We also assessed flux at 1-day post-denervation in transgenic mt-keima mice. Markers of mitochondrial content were reduced at 7 days following Den, and Den further resulted in rapid decrements in mitochondrial respiration, along with increased ROS emission. Pre-lysosomal autophagy flux was upregulated at 1- and 3-days post-Den but was reduced compared to time-matched sham-operated controls at 7-days post-Den. Similarly, pre-lysosomal mitophagy flux was enhanced in SS mitochondria as early as 1- and 3-days of Den but decreased in both SS and IMF subfractions following 7 days of Den. Lysosome protein content and transcriptional regulators TFEB and TFE3 were progressively enhanced with Den, an adaptation designed to enhance autophagic capacity. However, evidence for lysosome dysfunction was apparent by 7 days, which may limit degradation capacity. This may contribute to an inability to clear dysfunctional mitochondria and increased ROS signaling, thereby accelerating muscle atrophy. Thus, therapeutic targeting of lysosome function may help to maintain autophagy and muscle health during conditions of muscle disuse or denervation. Abstract figure legend This study investigates the temporal regulation of the autophagy-lysosome system in rat skeletal muscle following neuromuscular denervation (Den) with a focus on mitochondrial decay through mitophagy. We show that mitochondrial dysfunction is time-dependant, with elevations at 3-days post-Den and further at 7 days, preceding decrements in mitochondrial protein content. Deficits in mitochondrial content may be explained by prior elevations in mitophagy as early as 1- and 3-days post-Den, but these elevations were bi-phasic, returning to lower values by 7-days post-Den. To meet the demands of increased autophagy, lysosome protein content was progressively upregulated with 3- and 7-day of Den, but evidence of lysosome dysfunction was evident, and this could impede the removal of poor-quality mitochondria. Overall, these changes in the autophagy-lysosome system following neuromuscular denervation and provide insight into the processes that contribute to Den-induced muscle atrophy. Representative graphs are Den/Sham, with the dotted line representing sham-operated control values. This article is protected by copyright. All rights reserved.