Physical exercise is widely recognized for its numerous health benefits, impacting multiple tissues and systems. While the benefits are well-established, the precise mechanisms, particularly the coordinated tissue responses and age-related differences, remain unclear. Most research has focused on skeletal muscle, identifying key signaling pathways like IGF1/PI3K/Akt, AMPK, mTOR, and PGC-1α. However, a comprehensive understanding requires investigating non-skeletal muscle tissues across the whole body. Furthermore, the impact of exercise on young versus aged individuals, and their respective responses to external stimuli such as infection, is poorly understood. Previous studies have explored exercise's role in restoring mitochondrial function and reactivating aged adult stem cells, but a systemic transcriptomic analysis comparing young and old animals is lacking. This study aimed to address these gaps by creating a comprehensive single-cell transcriptomic atlas of exercise effects across multiple tissues and organs in young and aged mice, considering the influence of acute infectious challenges.

The literature extensively documents the health benefits of exercise, emphasizing its positive effects on the musculoskeletal, cardiovascular, pulmonary, and nervous systems. Several studies have explored specific signaling pathways activated by exercise within skeletal muscle, highlighting the importance of pathways such as IGF1/PI3K/Akt, AMPK, mTOR, and PGC-1α. However, research on the systemic effects of exercise beyond skeletal muscle has been limited. Similarly, while exercise is known to combat age-related decline, the underlying mechanisms and specific effects on different tissues and cell types at a single-cell resolution remain largely unexplored. Previous studies on aging and exercise interventions have used techniques like caloric restriction and heterochronic parabiosis, yet comprehensive single-cell transcriptome analyses of exercise's systemic impact, especially in aged animals, were lacking before this study. The authors highlighted the need for a more systemic and integrative approach to understand how exercise benefits multiple systems and organs and how these benefits differ with age.

Young (2 months old) and old (16 months old) male C57BL/6J mice were subjected to either 12 months of voluntary exercise or standard housing. Real-time monitoring of voluntary exercise activity was conducted. After 12 months, physical function was assessed through various tests, including body weight measurement, treadmill and rotarod tests, grip strength assessment, and Y-maze performance for spatial learning and memory. Plasma concentrations of inflammatory cytokines (IL-1β) and liver damage indicators (AST/ALT ratio) were measured. Genome-wide RNA sequencing (RNA-seq) was performed on 13 tissue types (brain, spinal cord, skeletal muscle, heart, lung, aorta, kidney, liver, small intestine, testis, spleen, bone marrow, and peripheral blood) from all four groups (young exercise, young control, old exercise, old control). Single-cell RNA-seq (scRNA-seq) and single-nucleus RNA-seq (snRNA-seq) were performed on subsets of these tissues to achieve single-cell resolution. To investigate the protective effects of exercise against acute infection, a subset of mice received an intraperitoneal injection of lipopolysaccharide (LPS). Differential gene expression analysis was performed to identify exercise-related differentially expressed genes (DEGs) in young and old mice, and the protective effects of exercise on LPS-induced inflammation were also analyzed. Finally, the reversal of aging-related gene expression changes by exercise was investigated by identifying aging-related DEGs and their responses to exercise in aged mice. Multiple phenotypic validations, including histology and immunostaining, were performed to confirm the molecular findings.

Long-term voluntary exercise resulted in reduced body weight, improved physical performance (rotarod, treadmill, grip strength), and enhanced spatial learning and memory in aged mice. Plasma IL-1β levels were reduced in aged exercising mice, and the AST/ALT ratio, an indicator of liver damage, was also improved. RNA-seq and scRNA-seq/snRNA-seq analyses revealed extensive age-dependent and tissue-specific gene expression changes in response to exercise. In young mice, exercise protected against LPS-induced inflammation by suppressing the expression of pro-inflammatory genes and pathways. This protective effect was less pronounced in aged mice. In aged mice, exercise reversed many aging-related gene expression changes across multiple tissues, with the most significant effects observed in the nervous system and vascular endothelial cells. Many aging-related pathways were reversed by exercise, including those involved in inflammation, apoptosis, and intercellular communication. Furthermore, exercise restored the expression of circadian clock genes, particularly BMAL1, which was identified as a central mediator of the exercise-induced geroprotective effects. Experimental manipulation of BMAL1 in cardiac endothelial cells (CAECs) confirmed its role in delaying cellular senescence and protecting against LPS-induced damage. Histological analyses confirmed the reversal of aging-related phenotypes in several organs.

This study provides a comprehensive, single-cell resolution view of the systemic effects of exercise on both young and aged mice. The findings confirm exercise's multifaceted protective effects, highlighting its role in combating inflammation, protecting against acute infection (particularly in young animals), and reversing aging-related changes across numerous tissues. The prominent role of BMAL1 and the circadian clock in mediating the beneficial effects of exercise suggests that targeting this pathway might be a promising strategy for developing 'exercise mimetics.' The age-dependent differences in the response to both exercise and LPS challenge emphasize the importance of considering age-related factors when developing exercise interventions or therapies to combat age-related diseases.

This study comprehensively maps the effects of long-term exercise on gene expression across multiple tissues and cell types in young and aged mice. The research shows that exercise offers robust protection against acute inflammation in young mice and reverses aging-related changes in multiple tissues in older mice. The study highlights the importance of the circadian clock, particularly BMAL1, in mediating these effects, paving the way for potential therapeutic interventions targeting this pathway to mimic the benefits of exercise.

The study was conducted using male C57BL/6J mice, limiting the generalizability of the findings to other mouse strains or genders. The voluntary exercise model, while reflecting natural behavior, may not perfectly replicate structured exercise protocols used in human studies. Further studies are necessary to translate these findings to humans.