
Space Sciences
Skeletal muscle gene expression dysregulation in long-term spaceflights and aging is clock-dependent
D. Malhan, M. Yalçin, et al.
This groundbreaking research by Deeksha Malhan, Müge Yalçin, Britt Schoenrock, Dieter Blottner, and Angela Relógio uncovers how circadian clock disruptions during long-term spaceflights and aging affect skeletal muscle gene expression. The study reveals critical insights into combating musculoskeletal atrophy in astronauts by maintaining circadian function through factors like exercise and fasting.
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Introduction
Skeletal muscle, a highly dynamic organ, is significantly impacted by aging, resulting in sarcopenia (muscle wasting). This process involves a gradual loss of muscle function due to an imbalance between protein synthesis and degradation, influenced by nutritional status and physical activity. The circadian clock, a fundamental biological timekeeper, regulates gene expression in various tissues, including skeletal muscle. Over 2300 genes related to striated muscle display circadian patterns. The mammalian circadian clock, composed of a central pacemaker (suprachiasmatic nucleus, SCN) and peripheral oscillators in tissues like the liver, orchestrates physiological processes including metabolism, myogenesis, and immune function. The SCN synchronizes peripheral clocks via neuronal and hormonal signals. Zeitgebers, such as light, meal timing, and exercise, reset the clock, aligning it with the environment. At the molecular level, transcriptional-translational feedback loops (TTFLs) involving BMAL1, CLOCK, PERs, and CRYs generate circadian rhythms in gene expression. RORA/RORB/RORC and NR1D1/NR1D2 further fine-tune BMAL1 activity. Emerging evidence suggests a critical role for the circadian clock in skeletal muscle homeostasis; disruption of the clock (e.g., Bmal1 knockout) leads to sarcopenia. Aging disrupts circadian rhythms, causing reduced amplitude and phase shifts, and studies on immobility (bed rest) and spaceflight reveal significant muscle loss and circadian phase delays in astronauts. These circadian disruptions have broader health implications, contributing to reduced workforce health, disease development, and decreased lifespan. Prior transcriptomics studies have shown altered circadian signaling in various tissues due to spaceflight, but a detailed analysis of skeletal muscle circadian clock alterations during spaceflight is lacking. This research investigates whether exercise or fasting can act as preventative measures against spaceflight-induced muscle loss, given their influence on skeletal muscle gene expression. This study systematically analyzes the effects of both intrinsic (genetic manipulation, aging) and extrinsic factors (spaceflight, bed rest) on skeletal muscle circadian rhythms using data from 28 published omics studies to characterize the functional consequences of clock disruptions.
Literature Review
The literature extensively documents the impact of aging on skeletal muscle, leading to sarcopenia and reduced function. The circadian clock's role in regulating various physiological processes, including muscle function, has gained increasing attention. Studies on circadian gene expression atlases reveal the temporal organization of gene expression in different tissues, including the skeletal muscle. The molecular mechanisms underlying circadian rhythms, specifically the transcriptional-translational feedback loops (TTFLs) and the roles of BMAL1, CLOCK, PERs, CRYs, RORs, and NR1Ds are well-established. The importance of the circadian clock in skeletal muscle homeostasis is highlighted by studies demonstrating the link between clock gene disruptions and sarcopenia. Existing research also shows the impact of aging on circadian parameters, resulting in amplitude decrease and phase shifts. Studies on the effects of prolonged immobility and spaceflight on skeletal muscle highlight significant muscle loss and circadian rhythm disruptions, particularly concerning core body temperature shifts. The literature also indicates that disrupted circadian rhythms contribute to health issues such as cancer, metabolic disorders, and mental health problems. However, a comprehensive molecular characterization of spaceflight-induced circadian clock alterations in skeletal muscle has been lacking, making this research essential to further understanding the implications of altered circadian rhythms in this context.
Methodology
This study employed a systematic analysis of publicly available omics datasets (microarray, RNA-seq, LC-MS) from skeletal muscle tissue or cells. The data encompassed various conditions: healthy human and mouse skeletal muscle (to establish a baseline circadian phenotype), mouse models with core-clock gene alterations (Bmal1 knockout, Clock mutant, CLOCK knockdown), mice exposed to spaceflight (various durations), mice undergoing bed rest (analogue to spaceflight), mice subjected to hypergravity, and human and mouse data representing aging and the effects of exercise and fasting (as potential countermeasures). A total of 28 published studies provided the data. Data preprocessing involved quality control using FastQC and Trimmomatic, alignment to reference genomes (human, mouse, rat) using STAR aligner, transcript quantification with Salmon, and gene-level summarization with tximport and edgeR normalization. Similar methods were applied to microarray and LC-MS data. Rhythmicity analysis was performed using RAIN to identify 24-hour rhythmic genes, and Limorhyde was used for differential rhythmicity analysis to assess changes in acrophase and amplitude. Differential expression analysis was carried out using limma (and Proteus for LC-MS data) to identify significantly up- or down-regulated genes. Network analysis, using Cytoscape, STRING, and MCODE, was conducted to visualize interactions between differentially expressed genes in spaceflight, aging, exercise, and fasting datasets. Skeletal muscle and circadian clock-associated pathways were retrieved from KEGG and Reactome databases, supplemented with literature-curated genes. Pearson correlation analysis was performed to assess the relationships between core clock genes in spaceflight and aging datasets. The study analyzed both circadian rhythmicity and differential expression of pre-defined genes of interest, including core clock elements and skeletal muscle pathway genes, across all datasets.
Key Findings
The study revealed that mammalian skeletal muscle exhibits circadian rhythms in gene expression related to metabolism and myogenic capacity. Analysis of human and mouse data showed circadian expression of genes associated with muscle signaling pathways (SUN2, HMOX1, WWTR1, RORA) and core clock genes. Altering core-clock components (Bmal1 or Clock) in mice led to significant changes in circadian properties and gene expression in several skeletal muscle pathways, including those involving ATF4, FGF2, and NDUFA4. Analysis of spaceflight data showed that alterations in clock- and skeletal muscle-associated pathways correlated with spaceflight duration. Longer spaceflights led to more substantial changes in gene expression, including upregulation of genes like Arntl, Clock, Cry1, Npas2, Myod1, Nfat5, and Fos, and downregulation of Cry2, Nr1d2, Per2, Per3, and Dbp. Bed rest, as a spaceflight analogue, resulted in similar transcriptomic changes. Aging studies revealed differential expression of genes like MYH8 (upregulated), ACTN3, and ATP2A1 (downregulated), which play roles in muscle contraction and calcium regulation. A comparison of spaceflight and aging datasets showed that numerous genes were similarly upregulated (CASQ2, FGF1) or downregulated (NOS1, FABP3, FGF13) in both conditions. The effect of exercise and fasting on gene expression varied based on the type, intensity, and timing of exercise. Some genes showed similar changes in response to both exercise and fasting as those observed in spaceflight and aging datasets. Overall, long-term spaceflight and aging shared common molecular dysregulation in clock- and skeletal muscle-associated pathways. Network analysis revealed interactions among differentially expressed genes, providing further insight into the regulatory mechanisms influenced by both spaceflight and aging.
Discussion
The findings highlight the significant role of the circadian clock in maintaining skeletal muscle homeostasis. The observed similarities in gene expression changes between long-term spaceflight and aging on Earth point towards common underlying mechanisms. The dysregulation of specific pathways during spaceflight may accelerate an aging-like phenotype in astronauts. The results support the potential use of external Zeitgebers, such as exercise and fasting, as countermeasures to mitigate these effects. The ability of these interventions to reverse or prevent the dysregulation of specific genes, like ATF4, NDUFA4L2, FABP3, FGF2, and Wnt5a, suggests that timed exercise or fasting protocols could be beneficial for astronauts during long-duration space missions and potentially for combating age-related muscle loss on Earth. While the study primarily uses mouse models and human data, the findings suggest that monitoring and manipulation of the circadian rhythm could be important in future space travel.
Conclusion
This study provides compelling evidence of the interconnectedness between circadian rhythms and skeletal muscle health in the context of long-duration spaceflight and aging. The overlap in gene expression alterations between these conditions, coupled with the potential of exercise and fasting as countermeasures, suggests novel strategies for mitigating muscle loss in astronauts. Future research should focus on larger-scale studies and time-course experiments to validate the findings and optimize countermeasure protocols. The study's findings have implications not only for space medicine but also for developing interventions against age-related sarcopenia.
Limitations
The study relies on publicly available datasets, which may have inherent limitations in terms of sample size, experimental protocols, and data quality. The lack of comprehensive time-course data for spaceflight and aging conditions limited the scope of the circadian analysis. The diverse muscle types and species used in the analysis potentially introduces some heterogeneity. Further research is necessary to confirm these findings in larger, more controlled experiments and to define optimal timing and intensity of countermeasure interventions.
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