Muscle-Specific Upregulation of Timeless Mediates Exercise-Induced Amelioration of Age-Related Circadian Rhythm Disruption and Cardiac Dysfunction in Drosophila

Muscle-Specific Upregulation of Timeless Mediates Exercise-Induced Amelioration of Age-Related Circadian Rhythm Disruption and Cardiac Dysfunction in Drosophila | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Muscle-Specific Upregulation of Timeless Mediates Exercise-Induced Amelioration of Age-Related Circadian Rhythm Disruption and Cardiac Dysfunction in Drosophila Dengtai Wen, Shouzhi Lv, Jingyao Sun, Ying-qi Chen, Ying Lin, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9316587/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract The core genes of the circadian pathway, such as Timeless(Tim) , not only participate in the regulation of biological rhythms, but also play significant roles in DNA damage repair, chronic inflammation, and metabolism of cells. However, it remains unclear whether exercise can delay the age-related phenotypic degeneration by regulating muscle Tim gene. In here, we first carried out the expression regulation of muscle Tim gene in the Drosophila by constructing the Mhc-gal4/Tim-UAS system, and then subjected the flies to a 4-week endurance exercise intervention. The results showed that knockdown of the muscle Tim gene accelerated aging-related phenotypic deterioration in Drosophila , manifesting as increased nighttime activity, decreased climbing speed, elevated heart rate and reduced cardiac output, shortened time to hypoxic heart failure, and shortened lifespan. This is accompanied by reductions in muscle tissue levels of Clk gene, Sir2 gene, PGC-1α gene, Mhc gene, MRCC-I protein, and SOD protein, along with a significant increase in ROS. Conversely, overexpression of the muscle Tim gene delayed aging-related phenotypic changes in aged Drosophila. Exercise not only effectively counteracts the acceleration of aging-related phenotypic deterioration caused by muscle Tim gene knockdown but also further delayed aging-related phenotypic changes in aged Drosophila on the basis of muscle Tim gene overexpression. In summary, this study highlights the role of the muscle Tim gene in the aging of skeletal muscles and the heart, as well as the relationship between exercise and the muscle Tim gene, providing strategies for the prevention and treatment of age-related diseases. Aging Timeless circadian rhythm exercise mitochondrial oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction Regardless of China or the entire world, population aging is becoming increasingly severe. According to UN data, in 2023, the global population aged 65 and over was approximately 800 million. It is projected that the global elderly population will exceed 1 billion for the first time in the 2030s. Global aging is accelerating, with currently about 10% of the world's population being over 65[ 1 ]. In China, the population aged 60 and over is 310.31 million (approximately 310 million), accounting for 22.0% of the national population. China has already entered the stage of moderate aging and is progressing towards deep aging[ 2 ]. With aging, both animals and humans experience a decline in tissue and organ function, as well as the occurrence of age-related diseases such as sarcopenia, insomnia, and coronary heart disease[ 3 ]. These diseases not only severely reduce the quality of life of the elderly but also impose a heavy economic burden on families and society[ 4 ]. Therefore, it is particularly important for humanity to continuously delve into the molecular mechanisms of aging and then implement precise interventions to delay aging and reduce the incidence of age-related diseases. The Tim gene is a crucial regulatory gene in organisms, highly conserved from flies to humans. It plays a dual role in organisms, primarily acting as a regulator of the core circadian clock and a maintainer of DNA damage repair. In model organisms such as Drosophila , the core of the circadian clock consists of molecular feedback loops. The CLK/CYC heterodimer activates the transcription of Per and Tim genes, while the PER and TIM repressor proteins accumulate in the cytoplasm. Upon entering the nucleus, PER and TIM inhibit transcription by directly binding to the CLK/CYC heterodimer. This repression persists until the PER and TIM proteins are degraded, thereby allowing a new round of transcription. Light exposure causes rapid degradation of the TIM protein, resetting the circadian clock and enabling the organism to adapt to the day-night cycle[ 5 , 6 ]. In mammals (including humans), although the Tim homolog is also involved in circadian clock regulation, its core role has shifted during evolution, becoming more focused on the cell cycle and DNA repair, while its circadian function is partially undertaken by other genes (such as Cry genes). When encountering DNA damage or replication stress, TIM stabilizes the replication fork, preventing its collapse[ 7 , 8 ]. Furthermore, TIM interacts with proteins such as Tipin and participates in activating the S-phase checkpoint. This means that if DNA is damaged, TIM helps send signals to pause the cell cycle, providing the cell with sufficient time to repair the damaged DNA, thereby preventing the transmission of mutations to daughter cells[ 9 , 10 ]. Consequently, by regulating the circadian clock, TIM indirectly influences the expression of downstream metabolism-related genes, thereby affecting glucose metabolism and energy balance[ 11 ]. However, the role of the muscle Tim gene in aging and aging-related diseases remains unclear. Numerous studies have confirmed that aerobic exercise is an effective means delay aging and mitigate many aging-related diseases, including coronary heart disease, Alzheimer's disease, circadian rhythm disorders, and sarcopenia[ 12 – 15 ]. However, the interactive relationship between exercise and the muscle Tim gene in the context of aging and aging-related diseases remains unclear. This study leverages the advantages of Drosophila transgenic technology, their relatively short lifespan, and the conservation of exercise-related mechanisms. By constructing an Mhc/UAS-Tim system to achieve specific regulation (upregulation, normal expression, and downregulation) of Tim gene expression in Drosophila muscles, we will employ classic gain-of-function and loss-of-function approaches to investigate the effects of the muscle Tim gene on aging-related circadian rhythms, exercise capacity, cardiac function, and the time to heart failure under hypoxia. Furthermore, we will analyze the underlying molecular mechanisms from the perspectives of muscle mitochondria, oxidative stress, and contractile proteins. Subsequently, we will verify the effects of exercise intervention under different conditions of muscle Tim expression, aiming to provide new strategies for delaying aging and preventing aging-related diseases. 2 Materials and methods 2.1 Fly Stocks, Husbandry, Grouping, Diet, and Exercise Training Protocols The Tim -UAS-overexpression( Tim -UAS-OE) flies (stock ID: 80686; FlyBase Genotype: w*; P{UAS- Tim .Y}2 − 1/CyO), and the Mhc-gal4 (stock ID: 55133; FlyBase Genotype: w*; P{Mhc-GAL4.K}2/TM3, Sb1) flies were obtained from the Bloomington Stock Center. The Tim -UAS-RNAi flies (stock ID: v2885; FlyBase Genotype: w 1118 ; P{GD1267}v2885) were obtained from the Vienna Drosophila Resource Center. Female Mhc-gal4 flies were crossed with male Tim-UAS-OE and Tim -UAS-RNAi flies. The F1 generation male offspring from these crosses were collected, and age-matched male flies were divided into the following groups: Tim -UAS-OE > Mhc-gal4( OE ), Tim -UAS-OE > Mhc-gal4 + Exercise ( OE -E), Tim -UAS-RNAi > Mhc-gal4 ( RNAi) , and Tim -UAS-RNAi > Mhc-gal4 + Exercise ( RNAi-E) . Additionally, age-matched male Tim-UAS-OE(UAS) , Tim-UAS-OE +Exercise (UAS-E) , Tim-UAS-RNAi(UAS) , Tim-UAS-RNAi +Exercise ( UAS-E ) , and Mhc-gal4 (Gal4) flies were used as control groups to account for genetic background. All Drosophila were maintained and experimentally manipulated under controlled conditions (25°C, 50% humidity), with food replacement performed every other day. The food consisted of 2.0% yeast, 6.7% cornmeal, 0.7% agar, 1.6% soybean powder, 4.8% sucrose, 4.8% maltose, and 0.3% propionic acid [ 16 ]. Flies were exercised in vials with an 8-cm length. The vials were rotated at 60 rad/s. After each up-and-down turn, the vials were held stationary for 10 seconds to allow the flies to climb. Flies were exercised for 60 or 65 minutes per day (Table 1 ). The exercise regimen consisted of two days of training followed by one day off, and then two days of training followed by two days off. All exercise groups began training at 2 weeks of age and underwent a 4-week-long exercise program[ 17 ]. Table 1 Training protocols Day Exercise Rest Exercise Rest Exercise Rest Exercise Rest Monday 15min 5min 15min 5min 15min 5min 15min 5min Tuesday 20min 5min 15min 5min 15min 5min 15min 5min Wednesday Rest Thursday 15min 5min 15min 5min 15min 5min 15min 5min Friday 20min 5min 20min 5min 15min 5min 15min 5min Saturday Rest Sunday Rest 2.2 Circadian rhythm measurement The circadian rhythm of the flies was measured at the end of 1 week and 5 weeks of age. Ten flies from each group were placed in a culture tube and recorded for 24 hours using a digital camera (infrared camera mode at night, Sony, 56 MEGA PIXELS). The video was analyzed using QQ video software (version: 4.6.3.1104). Each fly in the video was labeled "1" if it changed position within a 1-minute interval and "0" if it remained stationary (Fig. 1 ). Using these labels, the following parameters were calculated: average activity rate per minute, average activity rate per hour (Average activity rate with 60 minutes), average activity rate during 12 hours of daytime, Average activity rate during 12 hours of night, and average activity rate over 24 hours, and the 24-hour average activity rate curve of flies. These metrics provided a comprehensive assessment of the flies' circadian activity patterns. 2.3 Climbing ability, cardiac function and heart failure, and lifespan assay The climbing speed of Drosophila was used to reflect their motor performance. Specifically, the measurement procedure was as follows: the test vials containing flies were placed on a Power Tower platform, and the Power Tower mechanically tapped the vials every 10 seconds to knock the flies to the bottom[ 18 ]. A video camera was then activated to record three consecutive climbing trials. The recordings were subsequently analyzed by AVS software, and screenshots were taken to capture the climbing height achieved within 3 seconds after each mechanical tap. The highest climbing height from each vial was recorded for analysis. Each group consisted of 20 flies, and the test was repeated five times per group[ 19 ]. Cardiac function assay: after the fruit flies were anesthetized in ether for 3 minutes, the head, ventral thorax, and ventral abdominal cuticle of anesthetized flies were removed, and the hearts were exposed. Oxygenated artificial hemolymph could maintain the normal function of the heart in Drosophila . High-speed cameras captured video of a fruit fly’s heart beating, and the video was took 30 seconds. The video of heart rate and stroke volume was analyzed using AVS software[ 20 ]. Stroke volume=(Square of dilation radius - Square of contraction radius) × π × 1. Heart failure assay: after the fruit flies completed the video analysis of their heart functions, the lymph fluid from the fruit flies was removed using a pipette, and 5 microliters of new lymph fluid was then re-introduced into the abdomen of the fruit flies. The time it took for the fruit fly's heart to go from normal beating to stopping beating was observed every 5 minutes. The time of fruit fly heart failure was analyzed by plotting the curve based on the duration of heart stoppage. Dead flies were recorded daily to monitor mortality. The lifespan of each fly was calculated as the number of days from eclosion (emergence as an adult) until death. Survival curves were generated to characterize the lifespan distribution for each group. The sample sizes ranged from 200 to 210 flies per group to ensure robust statistical analysis[ 21 ]. 2.4 Transmission electron microscopy of skeletal muscle For electron microscopic observation, muscle tissues were meticulously dissected in an ice-cold fixative solution containing 2.5% glutaraldehyde in 0.1 mol/L PIPES buffer (pH 7.4). After fixation at 4°C for 10 h, the samples were rinsed with 0.1 mol/L PIPES buffer. Post-fixation was then carried out with 1% OsO₄ for 30 min, followed by staining with 2% uranyl acetate for 1 h. Dehydration was performed through a graded ethanol series (50%, 70%, and 100%), after which the samples were embedded in epoxy resin. Ultrathin sections were prepared and examined using an HT-7700 transmission electron microscope, and images were acquired for subsequent analysis[ 22 ]. 2.5 ELISA assay The levels of MRCC-I, SOD, and ROS were quantified using commercial ELISA kits (Insect MRCC-I, SOD ELISA Kits, and ROS Kits, MLBIO, Shanghai, China). Muscles from 30 flies were homogenized in PBS (pH 7.2–7.4). The homogenates were snap-frozen in liquid nitrogen and stored at 2–8°C after thawing. Samples were further homogenized using mechanical grinders and centrifuged at 2000–3000 rpm for 20 min, after which the supernatant was collected. For the assay, absorbance was measured at 450 nm within 15 min after adding the Stop Solution, using the blank well as the zero reference[ 19 ]. 2.6 qRT-PCR At the end of the 5th week, skeletal muscle (thorax) samples from 30 Drosophila were collected per group and immersed in 1000 µL of Trizol reagent for subsequent analysis of relevant pathway gene expression (sample collection was repeated three times per group for experimental replicates). Each cDNA sample was prepared in triplicate for PCR amplification. Quantitative analysis was performed using the CT method[ 22 ]. The primer sequences of PGC-1α were as follows: F: 5’-TGTTGCTGCTACTGCTGCTT-3’; R: 5’-GCCTCTGCATCACCTACACA-3’. Primer sequences of Tim were as follows: F: 5’-TAACACGAAGCCACGGAATAAA-3’, R: 5'-CTCGATGGTGTTCTCGGTGA-3’. Primer sequences of Clk were as follows: R: 5’-GCTGTAACCCTTGAGGAGGAAAT-3’, F:5'-TCGGATTCAACGTCCATGTC − 3’. Primer sequences of Sir2 were as follows: F: 5’-GCAGT GCCAGCCC AATAA-3’, R: 5’-AGCCGATCACGATC AGTAGA-3’. Primer sequences of Rp49 were as follows: F: 5 -CTAAGCTGTCGCACAAATGG-3’, R: 5’- AACTTCTTGAATCCGGTGGG-3’. 2.7 Statistical analyses The differences in various indicators among the Gal4 group, UAS group, and RNAi group or OE group were compared using one-way ANOVA. The differences in various indicators between 1-week-old and 5-week-old Drosophila, as well as between the exercise and non-exercise groups, were analyzed using an independent samples t-test. The P-values for the survival curves and heart failure time curves were determined using the log-rank test. Experimental data are presented as mean ± standard deviation (SD), with statistical significance defined as α = 0.05 (or 0.01 where specified). 3 Results 3.1 The muscle Tim gene is involved in regulating age-related phenotypic changes in Drosophila From fruit flies to humans, aging-related phenotypic changes are highly conserved, manifested as decreased locomotor capacity, disrupted circadian rhythms, and weakened cardiac function[ 23 – 25 ]. Among these, aging-related lifespan is the most direct and ultimate phenotype[ 26 ]. Both genetic predisposition and environmental factors can lead to differential susceptibility to aging among individuals, resulting in healthy aging versus pathological aging, and even the emergence of premature aging phenotypes. For instance, mutations in the Sirt1 gene or long-term high-fat dietary intake can induce premature aging phenotypes[ 27 , 28 ]. Alterations in circadian rhythms are among the earliest emerging phenotypes of aging and are closely associated with abnormal changes in the neuronal circadian pathway[ 29 ]. However, it remains unclear whether the circadian pathway in muscle participates in the regulation of the aging process. In this study, we established a Tim-UAS-RNAi/Mhc-Gal4 system in F1-generation Drosophila through genetic crosses to achieve muscle-specific knockdown of Tim gene expression. By assessing aging-related phenotypes and investigating the underlying mechanisms of muscle aging, we aimed to elucidate the role of muscle Tim in systemic aging, as well as in skeletal and cardiac muscle aging (Fig. 2 -A). Our results showed that muscle-specific knockdown of Tim had no significant effects on 24-hour activity, 12-hour daytime activity, 12-hour nighttime activity, climbing speed, heart rate, stroke volume, or time to hypoxic cardiac failure in 1-week-old young flies (Fig. 2 -B to I, P > 0.05). In 5-week-old aged flies, however, although muscle Tim RNAi did not significantly affect 24-hour or daytime activity (Fig. 2 -J and K, P > 0.05), it led to a significant increase in nighttime activity (Fig. 2 -L and M, P < 0.05 or P < 0.01), a marked decline in climbing speed (Fig. 2 -N, P < 0.05 or P < 0.01), a significant elevation in heart rate (Fig. 2 -O, P < 0.05 or P < 0.01), and significant reductions in stroke volume (Fig. 2 -P, P < 0.05 or P < 0.01), time to hypoxic cardiac failure (Fig. 2 -Q, P < 0.05 or P < 0.01), and lifespan (Fig. 2 -R and S, P < 0.05 or P < 0.01). In addition, the results showed that in flies with different genetic backgrounds (UAS, Gal4, RNAi), there were no significant differences in 24-hour activity or 12-hour daytime activity between 5-week-old aged flies and 1-week-old young flies (Fig. 3 -A and B, P > 0.05). However, the 12-hour nighttime activity of 5-week-old aged flies was significantly increased compared to that of 1-week-old young flies (Fig. 3 -C to F, P < 0.05 or P < 0.01). Furthermore, compared to 1-week-old young flies, the 5-week-old aged flies exhibited significantly reduced climbing speed and heart rate (Fig. 3 -G and H, P < 0.05 or P < 0.01), a significantly increased stroke volume (Fig. 3 -I, P < 0.05 or P < 0.01), and a significantly shortened time to hypoxic cardiac failure (Fig. 3 -K to L, P < 0.05 or P < 0.01). These results suggest that muscle-specific Timeless gene RNAi accelerated the deterioration of aging-related behavioral phenotypes(Fig. 3 -M). To further confirm the role of the muscle-specific Tim gene in aging, we generated a Tim-UAS-Overexpression (OE)/Mhc-Gal4 system in F1-generation Drosophila through genetic crosses to achieve Tim overexpression(OE) specifically in muscle tissue. The results showed that muscle-specific overexpression of Tim had no significant effects on the 24-hour activity rate, 12-hour daytime activity rate, 12-hour nighttime activity rate, climbing speed, heart rate, stroke volume, or time to hypoxic cardiac failure in 1-week-old young flies (Fig. 4 -A to H, P > 0.05). In 5-week-old aged flies, however, although muscle-specific overexpression of Tim did not significantly affect their 24-hour or 12-hour daytime activity rates (Fig. 4 -I and J, P > 0.05), it significantly reduced their nighttime activity rate (Fig. 4 -K and L, P < 0.05 or P < 0.01), significantly increased their climbing speed (Fig. 4 -M, P < 0.05 or P < 0.01), significantly decreased their heart rate (Fig. 4 -N, P < 0.05 or P < 0.01), and significantly enhanced their stroke volume (Fig. 4 -O, P < 0.05 or P < 0.01), time to hypoxic cardiac failure (Fig. 4 -P, P < 0.05 or P < 0.01), and lifespan (Fig. 4 -Q and R, P < 0.05 or P < 0.01). In addition, the results showed that in flies with different genetic backgrounds (UAS, Gal4, OE), there were no significant differences in 24-hour activity or 12-hour daytime activity between 5-week-old aged flies and 1-week-old young flies (Fig. 5 -A and B, P > 0.05). However, the 12-hour nighttime activity of 5-week-old aged flies was significantly increased compared to that of 1-week-old young flies (Fig. 5 -C to F, P < 0.05 or P < 0.01). Furthermore, compared to 1-week-old young flies, the 5-week-old aged flies exhibited significantly reduced climbing speed and heart rate (Fig. 5 -G and H, P < 0.05 or P < 0.01), a significantly increased stroke volume (Fig. 5 -I, P < 0.05 or P < 0.01), and a significantly shortened time to hypoxic cardiac failure (Fig. 5 -J and K, P < 0.05 or P < 0.01). These results suggest that muscle-specific Timeless gene RNAi delayed the deterioration of aging-related behavioral phenotypes 3.2 Physiological mechanisms of the impact of muscular Tim gene on the aging of skeletal muscle and heart Skeletal muscle and cardiac aging are accompanied by a series of changes in intrinsic physiological mechanisms, such as increased oxidative stress, mitochondrial dysfunction, and loss of contractile proteins[ 30 – 32 ]. Although studies have shown that the Tim gene is closely associated with cellular rhythms and DNA damage repair[ 33 ], its impact on the physiological mechanisms of cellular aging requires further investigation. In this study, we first employed real-time quantitative PCR to detect the mRNA expression levels of the muscular Tim gene and others, in order to verify the successful construction of the Mhc-Gla4/Tim-UAS structure in the F1 generation of Drosophila. The results showed that the Mhc-Gla4/Tim-UAS-RNAi structure significantly reduced the relative expression of the muscular Tim gene (Fig. 6 -A, P < 0.05 or P < 0.01). Furthermore, knockdown of the muscular Tim gene significantly downregulated the expression of muscle Clk gene, Sir2 gene, and PGC-1α gene, as well as the protein level of MRCC-I and Mhc gene expression, and the activity level of SOD, while significantly increasing the level of muscle ROS (Fig. 6 -B to H, P < 0.05 or P < 0.01). Results from Mhc protein immunofluorescence histochemistry indicated that knockdown of the muscular Tim gene significantly reduced Mhc protein expression. Transmission electron microscopy images of skeletal muscle tissue suggested that knockdown of the muscular Tim gene significantly decreased mitochondrial number and myofibril integrity (Fig. 5 -I). In contrast, the results showed that the Mhc-Gla4/Tim-UAS-overexpression structure significantly up regulated the relative expression of the muscular Tim gene (Fig. 6 -J, P < 0.05 or P < 0.01). Furthermore, overexpression of the muscular Tim gene significantly up regulated the expression of muscle Clk gene, Sir2 gene, and PGC-1α gene, as well as the protein level of MRCC-I and Mhc gene expression, and the activity level of SOD, while significantly decreasing the level of muscle ROS (Fig. 6 -K to Q, P < 0.05 or P < 0.01). Results from Mhc protein immunofluorescence histochemistry indicated that overexpression of the muscular Tim gene significantly increased Mhc protein expression. Transmission electron microscopy images of skeletal muscle tissue suggested that overexpression of the muscular Tim gene significantly increased mitochondrial number and myofibril integrity (Fig. 5 -R). These results indicated that the Timeless gene in muscle played an important role in the aging of skeletal muscle, heart, and circadian rhythm, and its mechanism was closely related to its ability to regulate the activity status of the muscle Timeless/Clock pathway and the Timeless/Sir2/PGC-1α pathway. 3.3 Exercise attenuates aging in skeletal muscle and the heart via the upregulation of muscle Tim gene Accumulating evidence suggests that regular aerobic exercise helps delay aging-related phenotypic changes, including improved locomotor activity, enhanced circadian rhythms, and a reduced incidence of cardiovascular diseases in aged individuals[ 34 – 36 ]. These benefits are associated with the ability of exercise to ameliorate aging-related physiological changes, such as enhanced antioxidant capacity, reduced oxidative stress, and the preservation of mitochondrial function. However, it remains unclear whether these effects are mechanistically linked to the Tim gene in muscle. The results of this study showed that exercise significantly reduced the 24-hour activity rate and the 12-hour nocturnal activity rate in flies with muscle-specific Tim gene RNA interference (P < 0.05 or P 0.05) (Fig. 7 -A to E). Moreover, exercise significantly improved the climbing speed of flies with muscle-specific Tim gene RNAi, significantly reduced their heart rate, and markedly enhanced their stroke volume, and it also significantly prolonged the time to heart failure under hypoxia and extended their lifespan (P < 0.05 or P < 0.01) (Fig. 7 -F to M). Furthermore, exercise significantly increased the mRNA expression of Tim, Clk, Sir2, PGC-1α , and Mhc , elevated MRCC-I protein levels, and transmission electron microscopy images revealed a notable increase in mitochondrial number and a reduction in mitochondrial damage (P < 0.05 or P < 0.01) (Fig. 7 -N to T). Finally, exercise significantly increased SOD activity and markedly reduced ROS levels in the muscles of muscle-specific Tim RNAi flies (P < 0.05 or P < 0.01) (Fig. 7 -U to V). Similarly, muscle-specific Tim gene overexpression flies, the results of this study showed that exercise also significantly reduced the 24-hour activity rate and the 12-hour nocturnal activity rate in flies with muscle-specific Tim gene overexpression (P < 0.05 or P 0.05) (Fig. 8 -A to E). Moreover, exercise significantly improved the climbing speed of flies with muscle-specific Tim gene overexpression, significantly reduced their heart rate, and markedly enhanced their stroke volume, and it also significantly prolonged the time to heart failure under hypoxia and extended their lifespan (P < 0.05 or P < 0.01) (Fig. 8 -F to M). Furthermore, exercise significantly increased the mRNA expression of Tim, Clk, Sir2, PGC-1α , and Mhc , elevated MRCC-I protein levels, and transmission electron microscopy images revealed a notable increase in mitochondrial number and a reduction in mitochondrial damage (P < 0.05 or P < 0.01) (Fig. 8 -N to T). Finally, exercise significantly increased SOD activity and markedly reduced ROS levels in the muscles of muscle-specific Tim overexpression flies (P < 0.05 or P < 0.01) (Fig. 8 -U to V). These findings suggested that exercise acted as an upstream regulator of the muscle Timeless gene, delaying aging-related deterioration in skeletal muscle, heart, and circadian rhythms by activating the Timeless/Clock and Timeless/Sir2/PGC-1α pathways. 4 Discussion Aging is an intrinsic process that occurs in nearly all living organisms, characterized by the progressive decline or loss of function across cellular, tissue, and organ systems over time, ultimately leading to the end of life. In humans, the central nervous system is among the most vulnerable to aging, making disruptions in sleep–wake rhythms—such as phase advancement and sleep fragmentation—some of the earliest indicators of the aging process [ 37 ]. These alterations have also been observed in Drosophila [ 38 ], suggesting that age-related changes in circadian rhythms are evolutionarily conserved. In mammals, the molecular clock typically exhibits reduced activity during aging, as reflected by decreased amplitude in core clock genes such as Clock, Bmal1, and Per2, while the expression of Per1, Cry1, and Cry2 remains relatively stable. TIM protein directly interacts with the cell cycle checkpoint proteins ATR and CHK1 [ 39 ]. Consequently, the age-related decline in circadian TIM expression may compromise the function of the CHK1: ATR3 complex in DNA damage checkpoint responses [ 40 ]. In mammals, the Tim gene produces two alternatively spliced transcripts, enabling TIM to serve as a molecular link between the cell cycle checkpoint machinery and the circadian clock [ 41 ]. In Drosophila , aging suppresses Tim expression, which may contribute to circadian disruption[ 42 ]. Collectively, growing evidence implicates Tim , a core circadian regulator, in the aging process. However, its specific role in aging skeletal muscle and cardiac tissue remains to be elucidated. This study employed Drosophila genetic crosses to establish a UAS-Tim/Mhc-Gal4 system in F1 progeny, enabling muscle- specific knockdown and overexpression of Tim gene to investigate its role in circadian rhythm aging, skeletal muscle aging, and cardiac aging. The results showed that muscle-specific Tim knockdown or overexpression had no significant effect on the average daily locomotor activity in one-week-old young flies. However, in five-week-old aged flies, Tim knockdown led to a significant increase in average nighttime activity, whereas Tim overexpression significantly reduced nighttime activity. These findings suggest that muscle-specific Tim knockdown accelerates age-related nighttime sleep fragmentation in Drosophila, while Tim overexpression helps preserve healthy sleep patterns during aging. These findings are consistent with previously reported studies on circadian rhythms and aging, both in Drosophila and mammalian models. In both mammals and Drosophila , skeletal muscle and heart—as vital organs—undergo structural and functional decline with aging, accompanied by an increased incidence of age-related diseases such as sarcopenia and heart failure. Age-related sarcopenia is a major cause of reduced locomotor activity and diminished motor capacity in aged individuals, with underlying mechanisms involving loss of contractile proteins in skeletal muscle cells, decreased mitochondrial function, and elevated oxidative stress damage[ 43 – 45 ]. Similarly, age-associated heart failure is characterized by reduced cardiac contractility, decreased cardiac output and ejection fraction, and increased incidence of arrhythmias, with physiological mechanisms analogous to those observed in skeletal muscle aging[ 46 , 47 ]. During aging, the reduction in cardiomyocyte number due to apoptosis and necrosis leads to hypertrophy of the remaining cardiomyocytes in an attempt to maintain pump function. However, this hypertrophy increases cardiac stiffness and oxygen consumption[ 48 ]. The myocardium is one of the most oxygen-consuming tissues in the human body. Hypoxia impairs aerobic oxidation (the most efficient energy production pathway) in cardiomyocytes, forcing a shift towards inefficient anaerobic glycolysis. This results in insufficient energy supply in cardiomyocytes, further deteriorating their systolic and diastolic functions[ 49 ]. Additionally, hypoxia induces mitochondrial dysfunction, generating excessive reactive oxygen species (free radicals) and triggering oxidative stress responses. This damages DNA and proteins within cardiomyocytes while activating inflammatory pathways, thereby accelerating cardiomyocyte death and fibrosis[ 50 ]. Hypoxia can also alter the electrophysiological stability of cardiomyocytes, increasing the risk of arrhythmias such as atrial fibrillation, which in turn exacerbates heart failure[ 51 ]. Therefore, aging reduces the heart's tolerance to hypoxia and increases the incidence of hypoxic heart failure[ 52 ]. In this study, muscle-specific knockdown or overexpression of the Tim gene had no significant effect on climbing speed or cardiac function in one-week-old young flies. However, in five-week-old aged flies, Tim knockdown significantly impaired climbing speed and cardiac function, and shortened the time to heart failure under hypoxic conditions, while Tim overexpression produced the opposite effects. These findings suggest that the muscle Tim gene plays a critical role in skeletal muscle and cardiac aging. Sirt1/Sir2, as key NAD⁺-dependent deacetylases, play a core protective role in skeletal muscle and cardiac aging through multi-level regulation of mitochondrial function. For instance, in the heart and skeletal muscle, Sirt1/Sir2 activate PGC-1α via deacetylation, enhancing its transcriptional activity and promoting the expression of NRF1, TFAM, and other factors, thereby facilitating mitochondrial biogenesis and maintaining mitochondrial network stability[ 53 – 55 ]. Additionally, Sirt1/Sir2 activate the FOXO pathway, upregulating antioxidant enzymes such as SOD2 and catalase to scavenge excess ROS and mitigate oxidative damage in both cardiac and skeletal muscle[ 56 ]. Conversely, studies have shown that aging is accompanied by a decline in SIRT1/SIRT3 levels, leading to impaired mitochondrial fusion and respiratory function in cardiomyocytes, which subsequently results in defective cardiac contractility[ 57 ]. SIRT1 deficiency renders the hearts of young mice susceptible to an aging-like phenotype in response to ischemia/reperfusion injury[ 58 ]. Although Sirt1/Sir2 are core regulators of skeletal muscle and cardiac aging, the interactive relationship between the Tim gene and Sirt1/Sir2 in these tissues remains unclear. This study provides the first evidence in Drosophila that muscle Tim gene activates the Sir2/PGC-1α/MRCC-I pathway and the Tim-Clk pathway, enhances antioxidant capacity (SOD activity), and reduces oxidative stress (ROS), thereby protecting skeletal and cardiac myocytes from age-related decline and attenuating the loss of contractile proteins during aging. Accumulating evidence suggests that exercise plays an indispensable and beneficial role in delaying aging. For instance, regular exercise effectively ameliorates age-related sleep disturbances in the elderly, reduces sleep fragmentation, and enhances circadian rhythm stability in aged individuals[ 59 , 60 ]. Moreover, both resistance and aerobic exercise attenuate the age-related loss of skeletal muscle mass, thereby counteracting the decline in physical and locomotor capacity associated with aging[ 61 , 62 ]. Exercise also delays cardiac aging by improving cardiac function and reducing the incidence of age-related cardiac diseases and heart failure[ 63 ]. These beneficial effects of exercise on circadian rhythms, skeletal muscle function, and cardiac function are evolutionarily conserved across mammals, humans, and even Drosophila [ 64 , 65 ]. However, whether these anti-aging benefits of exercise are linked to the function of the muscle Tim gene remains unclear. The results of this study demonstrate that, regardless of whether the muscle Tim gene is knocked down, expressed at normal levels, or overexpressed, exercise improves circadian rhythms (as evidenced by reduced nighttime activity), enhances locomotor capacity and cardiac function, and prolongs the time to hypoxic heart failure in aged Drosophila . These findings suggest that exercise may act as an upstream regulator of the muscle Tim gene to modulate skeletal muscle and cardiac aging, although the underlying molecular mechanisms remain to be elucidated. The molecular mechanisms by which exercise delays skeletal muscle and cardiac aging also involve the regulation of Sirt1/Sir2 and its associated pathways. Studies have shown that exercise enhances Sirt1/Sir2 activity by upregulating NAD⁺ levels, thereby activating downstream targets such as PGC-1α and FOXO. In skeletal muscle, exercise-induced activation of the Sirt1/PGC-1α signaling axis promotes mitochondrial biogenesis, enhances oxidative metabolism, and upregulates antioxidant enzyme expression, thereby mitigating age-related oxidative damage and protein loss[ 66 – 68 ]. Similarly, in the heart, exercise improves mitochondrial function in cardiomyocytes, enhances contractility, and reduces the incidence of age-related cardiac lipotoxicity and heart failure through Sirt1-dependent mechanisms, including the activation of downstream targets such as PGC-1α and FOXO[ 67 , 69 , 70 ]. Notably, this study found that the muscle Tim gene activates the Sir2/PGC-1α/MRCC-I pathway, and exercise also regulates this same pathway, suggesting a potential interaction between Tim and Sirt1/Sir2 in mediating the anti-aging effects of exercise. To further elucidate the relationship between exercise and the muscle Tim gene, we examined the relative mRNA expression of Tim in the muscle tissue of aged Drosophila . The results showed that exercise significantly upregulated Tim mRNA expression in the muscle tissue of Tim-knockdown, normal-expression, and overexpression flies, accompanied by an increase in muscle Clk gene expression. These findings suggest that in aging muscle, exercise acts as an important upstream regulator of the Tim gene, positively modulating the muscle Tim/Clk pathway, the Sir2/PGC-1α/MRCC-I pathway, and antioxidant capacity, thereby reducing oxidative stress damage and contractile protein loss associated with skeletal muscle and cardiac aging, ultimately enhancing tissue function. 5 Conclusion Based on the current findings, the Timeless gene in muscle played an important role in the aging of skeletal muscle, heart, and circadian rhythm, and its mechanism was closely related to its ability to regulate the activity status of the muscle Timeless/Clock pathway and the Timeless/Sir2/PGC-1α pathway. Exercise acted as an upstream regulator of the muscle Timeless gene, delaying aging-related deterioration in skeletal muscle, heart, and circadian rhythms by activating the Timeless/Clock and Timeless/Sir2/ PGC-1α pathways. This study provides potential molecular targets for the prevention and treatment of age-related diseases affecting skeletal muscle and the heart. Declarations Additional Information Competing Interests: Authors have no conflicts of interest. Funding: This work is supported by the College Youth Innovation Team Program of Shandong Province (No. 2023RW057) and the National Natural Science Foundation of China (No. 32000832). Author Contributions Research idea and study design: D.t.W.; data acquisition: D.t.W. H.w.Q., Y.L, J.y.S; data analysis/interpretation: Y.q.C., G.b.S, T.s.Y.,D.y.S.; statistical analysis: D.t.W.; supervision: H.w.Q. Each author contributed during manuscript drafting or revision and approved the final version of the manuscript. Acknowledgements We thank the Core Facility of Drosophila Resource and Technology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences for providing fly stocks and reagengts. Associated Data Data Availability Statement. 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Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 12 May, 2026 Reviews received at journal 06 May, 2026 Reviewers agreed at journal 14 Apr, 2026 Reviewers agreed at journal 13 Apr, 2026 Reviewers invited by journal 13 Apr, 2026 Editor assigned by journal 07 Apr, 2026 Submission checks completed at journal 06 Apr, 2026 First submitted to journal 03 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9316587","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":623648248,"identity":"78a43a8f-62cc-46b3-9622-71d0e8b0ccc0","order_by":0,"name":"Dengtai Wen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYBACefnDBw58KGAGsQ2I02I4gy3x4QwDUrQw3OAxNuYhSQvj7AYzaRsD68QG9uZtEgw1dwhrYZc5kCadY5Ce2MBzrEyC4dgzImxpSDgG1HI4sUEix0yCseEwES47kNgmbQHSIv+GWC03kpmNGcC28BCpxbDnGOPDHoN04zaetGKLhGNEaJFn7/9w4EeFtWw/++GNNz7UEOMwGGADEQkkaBgFo2AUjIJRgAcAALs6NzxcEOgkAAAAAElFTkSuQmCC","orcid":"","institution":"Ludong University","correspondingAuthor":true,"prefix":"","firstName":"Dengtai","middleName":"","lastName":"Wen","suffix":""},{"id":623648249,"identity":"121b5e2b-792f-4087-847c-451d09ca5f42","order_by":1,"name":"Shouzhi Lv","email":"","orcid":"","institution":"Ludong University","correspondingAuthor":false,"prefix":"","firstName":"Shouzhi","middleName":"","lastName":"Lv","suffix":""},{"id":623648250,"identity":"0dfb3bdd-64b7-4bd8-a472-a19a8148534e","order_by":2,"name":"Jingyao Sun","email":"","orcid":"","institution":"Ludong University","correspondingAuthor":false,"prefix":"","firstName":"Jingyao","middleName":"","lastName":"Sun","suffix":""},{"id":623648251,"identity":"9a5e5901-6db3-425e-8fae-4b59a518aea6","order_by":3,"name":"Ying-qi Chen","email":"","orcid":"","institution":"Ludong University","correspondingAuthor":false,"prefix":"","firstName":"Ying-qi","middleName":"","lastName":"Chen","suffix":""},{"id":623648252,"identity":"7bbb5a84-6342-439d-841d-530ee7acda12","order_by":4,"name":"Ying Lin","email":"","orcid":"","institution":"Ludong University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Lin","suffix":""},{"id":623648256,"identity":"cdc58e25-9701-4d36-ad47-0dce79a3df95","order_by":5,"name":"Zhongrui Du","email":"","orcid":"","institution":"Ludong University","correspondingAuthor":false,"prefix":"","firstName":"Zhongrui","middleName":"","lastName":"Du","suffix":""},{"id":623648257,"identity":"5e0846dd-65f7-48d5-b0f8-319bebd5b7aa","order_by":6,"name":"Gangbin Sun","email":"","orcid":"","institution":"Anhui Institute of Information Technology","correspondingAuthor":false,"prefix":"","firstName":"Gangbin","middleName":"","lastName":"Sun","suffix":""},{"id":623648258,"identity":"80841069-7285-4dca-8f28-6eb5f90a53d3","order_by":7,"name":"Tian-shuo Yuan","email":"","orcid":"","institution":"Ludong University","correspondingAuthor":false,"prefix":"","firstName":"Tian-shuo","middleName":"","lastName":"Yuan","suffix":""},{"id":623648259,"identity":"51daec54-cc5d-49b3-8c42-5c0172177dd6","order_by":8,"name":"Deyu Shu","email":"","orcid":"","institution":"Ludong University","correspondingAuthor":false,"prefix":"","firstName":"Deyu","middleName":"","lastName":"Shu","suffix":""},{"id":623648260,"identity":"46a44021-ebd7-4ff4-bd03-60df83fcc053","order_by":9,"name":"Ji-ying Wang","email":"","orcid":"","institution":"Ludong University","correspondingAuthor":false,"prefix":"","firstName":"Ji-ying","middleName":"","lastName":"Wang","suffix":""},{"id":623648261,"identity":"0c6c3e9e-4fa3-45ac-a662-f941ce06cb12","order_by":10,"name":"Wen-qi Hou","email":"","orcid":"","institution":"Ludong University","correspondingAuthor":false,"prefix":"","firstName":"Wen-qi","middleName":"","lastName":"Hou","suffix":""}],"badges":[],"createdAt":"2026-04-04 00:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9316587/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9316587/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107323643,"identity":"8c40cdb0-7de8-4ea7-866c-c672a4a23886","added_by":"auto","created_at":"2026-04-20 11:01:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2169436,"visible":true,"origin":"","legend":"\u003cp\u003eMethod for analyzing locomotor activity rhythm of \u003cem\u003eDrosophila\u003c/em\u003e. A 24-hour video recording was conducted on fruit flies in culture tubes. During analysis, the positions of the fruit flies in the video were analyzed at one-minute intervals. If the position remained unchanged, it was scored as \"0\"; if the position changed, it was scored as \"1\". The average activity rate per tube was then calculated (number of fruit flies with position changes divided by total number of fruit flies). As shown in the figure, during the time period from 10:20 to 10:21, six fruit flies exhibited position changes, resulting in an average activity rate of 0.4.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9316587/v1/54923e4e7df15b53446016b3.png"},{"id":107323644,"identity":"5129e49e-2a8c-45e2-a7d3-9145a7668256","added_by":"auto","created_at":"2026-04-20 11:01:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10168446,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of muscle-specific \u003cem\u003eTim\u003c/em\u003eknockdown on aging-related phenotypes in \u003cem\u003eDrosophila\u003c/em\u003e. (A) Generation of Drosophila UAS/Mhc-GAL4 system through genetic cross. (B) Average 24-hour locomotor activity of 1-week-old flies. (C) Average 12-hour daytime locomotor activity of 1-week-old flies. (D) Average 12-hour nighttime locomotor activity of 1-week-old flies. (E) Locomotor activity profile over 24 hours of 1-week-old flies. (F) Climbing speed of 1-week-old flies. (G) Heart rate of 1-week-old flies. (H) Cardiac output of 1-week-old flies. (I) Time to anoxic heart failure in 1-week-old flies. (J) Average 24-hour locomotor activity of 5-week-old flies. (K) Average 12-hour daytime locomotor activity of 5-week-old flies. (L) Average 12-hour nighttime locomotor activity of 5-week-old flies. (M) Locomotor activity profile over 24 hours of 5-week-old flies. The locomotor activity of 5-week-old flies was significantly higher than that of UAS and GAL4 flies during the period from 20:01 to 04:00, suggesting that may cause severe fragmentation of nighttime sleep in aged flies. (N) Climbing speed of 5-week-old flies. (O) Heart rate of 5-week-old flies. (P) Cardiac output of 5-week-old flies. (Q) Time to anoxic heart failure in 5-week-old flies. (R) Survival curve of flies. (S) Average lifespan of flies. For comparisons among multiple groups (Gal4, UAS, and RNAi), one-way analysis of variance (ANOVA) followed by least significant difference (LSD) post hoc tests was conducted. The log-rank test was used to calculate P-values for lifespan curve and time to anoxic heart failure comparisons. Data are presented as means ± standard error of the mean (SEM). *P \u0026lt; 0.05; **P \u0026lt; 0.01; ns means no significant difference.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9316587/v1/d0e7dfe1470eb114c0ae8ee2.png"},{"id":107323645,"identity":"b2e8cc2a-546b-41f3-af98-ea287ddd7985","added_by":"auto","created_at":"2026-04-20 11:01:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2438908,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of aging on locomotor activity rhythms, climbing speed, and cardiac function in RNAi flies. (A) Comparison of average 24-hour locomotor activity between 1-week-old and 5-week-old RNAi flies. (B) Comparison of average 12-hour daytime locomotor activity between 1-week-old and 5-week-old RNAi flies. (C) Comparison of average 12-hour nighttime locomotor activity between 1-week-old and 5-week-old RNAi flies. (D) Comparative analysis of 24-hour average locomotor activity profiles between 1-week-old and 5-week-old GAL4 flies. (E) Comparative analysis of 24-hour average locomotor activity profiles between 1-week-old and 5-week-old UAS flies. (F) Comparative analysis of 24-hour average locomotor activity profiles between 1-week-old and 5-week-old RNAi flies. Regardless of whether it is GAL4 flies, UAS flies, or RNAi flies, aging significantly increases their nighttime activity rate, suggesting that aging may lead to increased fragmentation of nighttime sleep in Drosophila. However, RNAi may exacerbate this aging-related nighttime sleep fragmentation. (G) Comparison of climbing speed between 1-week-old and 5-week-old RNAi flies. (H) Comparison of heart rate between 1-week-old and 5-week-old RNAi flies. (I) Comparison of cardiac stroke volume between 1-week-old and 5-week-old RNAi flies. (J) Comparison of time to anoxic heart failure between 1-week-old and 5-week-old Gal4 flies. (K) Comparison of time to anoxic heart failure between 1-week-old and 5-week-old UAS flies. (L) Comparison of time to anoxic heart failure between 1-week-old and 5-week-old RNAi flies. (M) \u003cstrong\u003eMuscle-specific \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTimeless\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene RNAi accelerated the deterioration of aging-related behavioral phenotypes. \u003c/strong\u003eIndependent-sample t-tests were used to compare the one-week old fliesand five-week oldflies. The log-rank test was used to calculate P-values for time to anoxic heart failure comparisons. Data are presented as means ± standard error of the mean (SEM). A significance threshold sets at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, and ns means no significant difference.*P \u0026lt; 0.05; **P \u0026lt; 0.01; ns means no significant difference.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9316587/v1/fa5b514e379b41aa9c8854df.png"},{"id":107323646,"identity":"25888256-21e9-4287-bcfe-3c6aa27d079c","added_by":"auto","created_at":"2026-04-20 11:01:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7597882,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of muscle-specific \u003cem\u003eTim\u003c/em\u003eoverexpression(OE) on aging-related phenotypes in \u003cem\u003eDrosophila\u003c/em\u003e. (A) Average 24-hour locomotor activity of 1-week-old OE flies. (B) Average 12-hour daytime locomotor activity of 1-week-old OE flies. (C) Average 12-hour nighttime locomotor activity of 1-week-old OE flies. (D) Locomotor activity profile over 24 hours of 1-week-old OE flies. (E) Climbing speed of 1-week-old OE flies. (F) Heart rate of 1-week-old OE flies. (G) Cardiac output of 1-week-old OE flies. (H) Time to anoxic heart failure in 1-week-old OE flies. (I) Average 24-hour locomotor activity of 5-week-old OE flies. (J) Average 12-hour daytime locomotor activity of 5-week-old OE flies. (K) Average 12-hour nighttime locomotor activity of 5-week-old OE flies. (L) Locomotor activity profile over 24 hours of 5-week-old OE flies. The nighttime locomotor activity of 5-week-old OE flies was significantly lower than that of UAS and GAL4 flies, suggesting that OE may alleviate the fragmentation of nighttime sleep in aged flies. (M) Climbing speed of 5-week-old OE flies. (N) Heart rate of 5-week-old OE flies. (O) Cardiac output of 5-week-old OE flies. (P) Time to anoxic heart failure in 5-week-old OE flies. (Q) Survival curve of OE flies. (R) Average lifespan of OE flies. For comparisons among multiple groups (Gal4, UAS, and OE), one-way analysis of variance (ANOVA) followed by least significant difference (LSD) post hoc tests was conducted. The log-rank test was used to calculate P-values for lifespan curve and time to anoxic heart failurecomparisons. Data are presented as means ± standard error of the mean (SEM). *P \u0026lt; 0.05; **P \u0026lt; 0.01; ns means no significant difference.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9316587/v1/4f981284e8ce798c8b53776f.png"},{"id":107323647,"identity":"eb544743-725c-438a-8ce5-0bdbbaebd21a","added_by":"auto","created_at":"2026-04-20 11:01:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6848172,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of aging on locomotor activity rhythms, climbing speed, and cardiac function in OE flies. (A) Comparison of average 24-hour locomotor activity between 1-week-old and 5-week-old OE flies. (B) Comparison of average 12-hour daytime locomotor activity between 1-week-old and 5-week-old OE flies. (C) Comparison of average 12-hour nighttime locomotor activity between 1-week-old and 5-week-old OE flies. (D) Comparative analysis of 24-hour average locomotor activity profiles between 1-week-old and 5-week-old GAL4 flies. (E) Comparative analysis of 24-hour average locomotor activity profiles between 1-week-old and 5-week-old UAS flies. (F) Comparative analysis of 24-hour average locomotor activity profiles between 1-week-old and 5-week-old OE flies. Regardless of whether it is GAL4 flies, UAS flies, or RNAi flies, aging significantly increases their nighttime activity rate, suggesting that aging may lead to increased nighttime sleep fragmentation in Drosophila. However, this phenomenon was not observed in OEflies, indicating that OE may reduce nighttime sleep fragmentation in aged flies. (G) Comparison of climbing speed between 1-week-old and 5-week-old OE flies. (H) Comparison of heart rate between 1-week-old and 5-week-old OE flies. (I) Comparison of cardiac stroke volume between 1-week-old and 5-week-old RNAi flies. (J) Comparison of time to anoxic heart failure between 1-week-old and 5-week-old UAS flies. (K) Comparison of time to anoxic heart failure between 1-week-old and 5-week-old OE flies. Independent-sample t-tests were used to compare the one-week old flies and five-week old flies. The log-rank test was used to calculate P-values for time to anoxic heart failure comparisons. Data are presented as means ± standard error of the mean (SEM). A significance threshold sets at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, and ns means no significant difference. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ns means no significant difference.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9316587/v1/680361e10c1dd62190f4ef6d.png"},{"id":107323648,"identity":"fd7a9694-7b01-44b3-b874-55e6f2843fca","added_by":"auto","created_at":"2026-04-20 11:01:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8935092,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of muscle-specific Tim RNAi and overexpression(OE) on physiological and biochemical indicators related to Drosophila musculature. (A) Relative expression of Tim gene in muscles of RNAi flies. (B) Relative expression of Clk gene in muscles of RNAi flies. (C) Relative expression of Sir2 gene in muscles of RNAi flies. (D) Relative expression of PGC-1α gene in muscles of RNAi flies. (E) Mitochondrial HRCC-I protein levels in muscles of RNAi flies. (F) Relative expression of Mhc gene in muscles of RNAi flies. (G) SOD activity levels in muscles of RNAi flies. (H) ROS levels in muscles of RNAi flies. (I) Immunofluorescence of Mhc protein and transmission electron microscopy images of muscles in RNAi flies. (J) Relative expression of Tim gene in muscles of OE flies. (K) Relative expression of Clk gene in muscles of OE flies. (L) Relative expression of Sir2 gene in muscles of OE flies. (M) Relative expression of PGC-1α gene in muscles of OE flies. (N) Mitochondrial HRCC-I protein levels in muscles of OE flies. (O) Relative expression of Mhc gene in muscles of OE flies. (P) SOD activity levels in muscles of OE flies. (Q) ROS levels in muscles of OE flies. (R) Immunofluorescence of Mhc protein and transmission electron microscopy images of muscles in OE flies. For comparisons among multiple groups (Gal4, UAS, and RNAi or OE), one-way analysis of variance (ANOVA) followed by least significant difference (LSD) post hoc tests was conducted. Data are presented as means ± standard error of the mean (SEM). *P \u0026lt; 0.05; **P \u0026lt; 0.01; ns means no significant difference.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9316587/v1/90b2a5b350eca525b0c69d1b.png"},{"id":107484729,"identity":"bba0b17e-f8c5-4324-8653-3a70b6701bb5","added_by":"auto","created_at":"2026-04-22 02:32:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":11174156,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of exercise on muscle-specific \u003cem\u003eTim\u003c/em\u003e RNAi Drosophila. (A) Average 24-hour activity rate of flies. (B) Average 12-hour daytime activity rate of flies. (C) Average 12-hour nighttime activity rate of flies. (D) 24-hour average activity rate curve of UAS flies. (E) 24-hour average activity rate curve of RNAi flies. (F) Climbing speed of flies. (G) Heart rate of flies. (H) Cardiac output of flies. (I) Time to anoxic heart failure in UAS flies. (J) Time to anoxic heart failure in RNAi flies. (K) Survival curve of UAS flies. (L) Survival curve of RNAi flies. (M) Lifespan of flies. \u0026nbsp;(N) Relative expression of \u003cem\u003eTim\u003c/em\u003e gene in muscles. (O) Relative expression of Clk gene in muscles. (P) Relative expression of Sir2 gene in muscles. (Q) Relative expression of PGC-1α gene in muscles. (R) Mitochondrial HRCC-I protein levels in muscles. (S) Relative expression of Mhc gene in muscles of RNAi flies. (T) Immunofluorescence of Mhc protein and transmission electron microscopy images of muscles in RNAi flies. (U) SOD activity levels in muscles. (V) ROS levels in muscles.Data are presented as means ± standard error of the mean (SEM). A significance threshold sets at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, and ns means no significant difference. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ns means no significant difference.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9316587/v1/37bb675732dd0557a443db95.png"},{"id":107323650,"identity":"56d6c4f9-1019-4110-a16b-a69f74bb69e2","added_by":"auto","created_at":"2026-04-20 11:01:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":11915554,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of exercise on muscle-specific \u003cem\u003eTim\u003c/em\u003e overexpression Drosophila. (A) Average 24-hour activity rate of flies. (B) Average 12-hour daytime activity rate of flies. (C) Average 12-hour nighttime activity rate of flies. (D) 24-hour average activity rate curve of UAS flies. (E) 24-hour average activity rate curve of overexpression flies. (F) Climbing speed of flies. (G) Heart rate of flies. (H) Cardiac output of flies. (I) Time to anoxic heart failure in UAS flies. (J) Time to anoxic heart failure in overexpression flies. (K) Survival curve of UAS flies. (L) Survival curve of overexpression flies. (M) Lifespan of flies. \u0026nbsp;(N) Relative expression of \u003cem\u003eTim\u003c/em\u003egene in muscles. (O) Relative expression of Clk gene in muscles. (P) Relative expression of Sir2 gene in muscles. (Q) Relative expression of PGC-1α gene in muscles. (R) Mitochondrial HRCC-I protein levels in muscles. (S) Relative expression of Mhc gene in muscles. (T) Immunofluorescence of Mhc protein and transmission electron microscopy images of muscles in overexpression flies. (U) SOD activity levels in muscles. (V) ROS levels in muscles. Data are presented as means ± standard error of the mean (SEM). A significance threshold sets at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, and ns means no significant difference. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ns means no significant difference.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9316587/v1/0ef0ebfa1aacc9ed2e0b7846.png"},{"id":107487263,"identity":"414fae99-185a-4d99-9ae0-d5b6458c123c","added_by":"auto","created_at":"2026-04-22 02:40:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":61505562,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9316587/v1/1bebc816-fb2b-42f1-9db1-6379d337525c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Muscle-Specific Upregulation of Timeless Mediates Exercise-Induced Amelioration of Age-Related Circadian Rhythm Disruption and Cardiac Dysfunction in Drosophila","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eRegardless of China or the entire world, population aging is becoming increasingly severe. According to UN data, in 2023, the global population aged 65 and over was approximately 800\u0026nbsp;million. It is projected that the global elderly population will exceed 1\u0026nbsp;billion for the first time in the 2030s. Global aging is accelerating, with currently about 10% of the world's population being over 65[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In China, the population aged 60 and over is 310.31\u0026nbsp;million (approximately 310\u0026nbsp;million), accounting for 22.0% of the national population. China has already entered the stage of moderate aging and is progressing towards deep aging[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. With aging, both animals and humans experience a decline in tissue and organ function, as well as the occurrence of age-related diseases such as sarcopenia, insomnia, and coronary heart disease[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These diseases not only severely reduce the quality of life of the elderly but also impose a heavy economic burden on families and society[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, it is particularly important for humanity to continuously delve into the molecular mechanisms of aging and then implement precise interventions to delay aging and reduce the incidence of age-related diseases.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eTim\u003c/em\u003e gene is a crucial regulatory gene in organisms, highly conserved from flies to humans. It plays a dual role in organisms, primarily acting as a regulator of the core circadian clock and a maintainer of DNA damage repair. In model organisms such as \u003cem\u003eDrosophila\u003c/em\u003e, the core of the circadian clock consists of molecular feedback loops. The CLK/CYC heterodimer activates the transcription of \u003cem\u003ePer\u003c/em\u003e and \u003cem\u003eTim\u003c/em\u003e genes, while the PER and TIM repressor proteins accumulate in the cytoplasm. Upon entering the nucleus, PER and TIM inhibit transcription by directly binding to the CLK/CYC heterodimer. This repression persists until the PER and TIM proteins are degraded, thereby allowing a new round of transcription. Light exposure causes rapid degradation of the TIM protein, resetting the circadian clock and enabling the organism to adapt to the day-night cycle[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In mammals (including humans), although the \u003cem\u003eTim\u003c/em\u003e homolog is also involved in circadian clock regulation, its core role has shifted during evolution, becoming more focused on the cell cycle and DNA repair, while its circadian function is partially undertaken by other genes (such as \u003cem\u003eCry\u003c/em\u003e genes). When encountering DNA damage or replication stress, TIM stabilizes the replication fork, preventing its collapse[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, TIM interacts with proteins such as Tipin and participates in activating the S-phase checkpoint. This means that if DNA is damaged, TIM helps send signals to pause the cell cycle, providing the cell with sufficient time to repair the damaged DNA, thereby preventing the transmission of mutations to daughter cells[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Consequently, by regulating the circadian clock, TIM indirectly influences the expression of downstream metabolism-related genes, thereby affecting glucose metabolism and energy balance[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, the role of the muscle \u003cem\u003eTim\u003c/em\u003e gene in aging and aging-related diseases remains unclear.\u003c/p\u003e \u003cp\u003eNumerous studies have confirmed that aerobic exercise is an effective means delay aging and mitigate many aging-related diseases, including coronary heart disease, Alzheimer's disease, circadian rhythm disorders, and sarcopenia[\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, the interactive relationship between exercise and the muscle \u003cem\u003eTim\u003c/em\u003e gene in the context of aging and aging-related diseases remains unclear. This study leverages the advantages of \u003cem\u003eDrosophila\u003c/em\u003e transgenic technology, their relatively short lifespan, and the conservation of exercise-related mechanisms. By constructing an \u003cem\u003eMhc/UAS-Tim\u003c/em\u003e system to achieve specific regulation (upregulation, normal expression, and downregulation) of Tim gene expression in Drosophila muscles, we will employ classic gain-of-function and loss-of-function approaches to investigate the effects of the muscle \u003cem\u003eTim\u003c/em\u003e gene on aging-related circadian rhythms, exercise capacity, cardiac function, and the time to heart failure under hypoxia. Furthermore, we will analyze the underlying molecular mechanisms from the perspectives of muscle mitochondria, oxidative stress, and contractile proteins. Subsequently, we will verify the effects of exercise intervention under different conditions of muscle \u003cem\u003eTim\u003c/em\u003e expression, aiming to provide new strategies for delaying aging and preventing aging-related diseases.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Fly Stocks, Husbandry, Grouping, Diet, and Exercise Training Protocols\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eTim\u003c/em\u003e-UAS-overexpression(\u003cem\u003eTim\u003c/em\u003e-UAS-OE) flies (stock ID: 80686; FlyBase Genotype: w*; P{UAS-\u003cem\u003eTim\u003c/em\u003e.Y}2\u0026thinsp;\u0026minus;\u0026thinsp;1/CyO), and the Mhc-gal4 (stock ID: 55133; FlyBase Genotype: w*; P{Mhc-GAL4.K}2/TM3, Sb1) flies were obtained from the Bloomington Stock Center. The \u003cem\u003eTim\u003c/em\u003e-UAS-RNAi flies (stock ID: v2885; FlyBase Genotype: w\u003csup\u003e1118\u003c/sup\u003e; P{GD1267}v2885) were obtained from the Vienna Drosophila Resource Center. Female Mhc-gal4 flies were crossed with male \u003cem\u003eTim-UAS-OE\u003c/em\u003e and \u003cem\u003eTim\u003c/em\u003e-UAS-RNAi flies. The F1 generation male offspring from these crosses were collected, and age-matched male flies were divided into the following groups: \u003cem\u003eTim\u003c/em\u003e-UAS-OE\u0026thinsp;\u0026gt;\u0026thinsp;Mhc-gal4(\u003cem\u003eOE\u003c/em\u003e), \u003cem\u003eTim\u003c/em\u003e-UAS-OE\u0026thinsp;\u0026gt;\u0026thinsp;Mhc-gal4\u0026thinsp;+\u0026thinsp;Exercise (\u003cem\u003eOE\u003c/em\u003e-E), \u003cem\u003eTim\u003c/em\u003e-UAS-RNAi\u0026thinsp;\u0026gt;\u0026thinsp;Mhc-gal4 (\u003cem\u003eRNAi)\u003c/em\u003e, and \u003cem\u003eTim\u003c/em\u003e-UAS-RNAi\u0026thinsp;\u0026gt;\u0026thinsp;Mhc-gal4\u0026thinsp;+\u0026thinsp;Exercise (\u003cem\u003eRNAi-E)\u003c/em\u003e. Additionally, age-matched male \u003cem\u003eTim-UAS-OE(UAS)\u003c/em\u003e, \u003cem\u003eTim-UAS-OE\u003c/em\u003e+Exercise \u003cem\u003e(UAS-E)\u003c/em\u003e, \u003cem\u003eTim-UAS-RNAi(UAS)\u003c/em\u003e, \u003cem\u003eTim-UAS-RNAi\u003c/em\u003e+Exercise \u003cem\u003e(\u003c/em\u003eUAS-E\u003cem\u003e)\u003c/em\u003e, and \u003cem\u003eMhc-gal4 (Gal4)\u003c/em\u003e flies were used as control groups to account for genetic background.\u003c/p\u003e \u003cp\u003eAll \u003cem\u003eDrosophila\u003c/em\u003e were maintained and experimentally manipulated under controlled conditions (25\u0026deg;C, 50% humidity), with food replacement performed every other day. The food consisted of 2.0% yeast, 6.7% cornmeal, 0.7% agar, 1.6% soybean powder, 4.8% sucrose, 4.8% maltose, and 0.3% propionic acid [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFlies were exercised in vials with an 8-cm length. The vials were rotated at 60 rad/s. After each up-and-down turn, the vials were held stationary for 10 seconds to allow the flies to climb. Flies were exercised for 60 or 65 minutes per day (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The exercise regimen consisted of two days of training followed by one day off, and then two days of training followed by two days off. All exercise groups began training at 2 weeks of age and underwent a 4-week-long exercise program[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTraining protocols\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExercise\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRest\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExercise\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRest\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eExercise\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRest\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eExercise\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eRest\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMonday\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTuesday\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWednesday\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e \u003cp\u003eRest\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eThursday\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFriday\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSaturday\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e \u003cp\u003eRest\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSunday\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e \u003cp\u003eRest\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Circadian rhythm measurement\u003c/h2\u003e \u003cp\u003eThe circadian rhythm of the flies was measured at the end of 1 week and 5 weeks of age. Ten flies from each group were placed in a culture tube and recorded for 24 hours using a digital camera (infrared camera mode at night, Sony, 56 MEGA PIXELS). The video was analyzed using QQ video software (version: 4.6.3.1104). Each fly in the video was labeled \"1\" if it changed position within a 1-minute interval and \"0\" if it remained stationary (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Using these labels, the following parameters were calculated: average activity rate per minute, average activity rate per hour (Average activity rate with 60 minutes), average activity rate during 12 hours of daytime, Average activity rate during 12 hours of night, and average activity rate over 24 hours, and the 24-hour average activity rate curve of flies. These metrics provided a comprehensive assessment of the flies' circadian activity patterns.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Climbing ability, cardiac function and heart failure, and lifespan assay\u003c/h2\u003e \u003cp\u003eThe climbing speed of \u003cem\u003eDrosophila\u003c/em\u003e was used to reflect their motor performance. Specifically, the measurement procedure was as follows: the test vials containing flies were placed on a Power Tower platform, and the Power Tower mechanically tapped the vials every 10 seconds to knock the flies to the bottom[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. A video camera was then activated to record three consecutive climbing trials. The recordings were subsequently analyzed by AVS software, and screenshots were taken to capture the climbing height achieved within 3 seconds after each mechanical tap. The highest climbing height from each vial was recorded for analysis. Each group consisted of 20 flies, and the test was repeated five times per group[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCardiac function assay: after the fruit flies were anesthetized in ether for 3 minutes, the head, ventral thorax, and ventral abdominal cuticle of anesthetized flies were removed, and the hearts were exposed. Oxygenated artificial hemolymph could maintain the normal function of the heart in \u003cem\u003eDrosophila\u003c/em\u003e. High-speed cameras captured video of a fruit fly\u0026rsquo;s heart beating, and the video was took 30 seconds. The video of heart rate and stroke volume was analyzed using AVS software[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Stroke volume=(Square of dilation radius - Square of contraction radius) \u0026times; π\u0026thinsp;\u0026times;\u0026thinsp;1.\u003c/p\u003e \u003cp\u003eHeart failure assay: after the fruit flies completed the video analysis of their heart functions, the lymph fluid from the fruit flies was removed using a pipette, and 5 microliters of new lymph fluid was then re-introduced into the abdomen of the fruit flies. The time it took for the fruit fly's heart to go from normal beating to stopping beating was observed every 5 minutes. The time of fruit fly heart failure was analyzed by plotting the curve based on the duration of heart stoppage.\u003c/p\u003e \u003cp\u003eDead flies were recorded daily to monitor mortality. The lifespan of each fly was calculated as the number of days from eclosion (emergence as an adult) until death. Survival curves were generated to characterize the lifespan distribution for each group. The sample sizes ranged from 200 to 210 flies per group to ensure robust statistical analysis[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Transmission electron microscopy of skeletal muscle\u003c/h2\u003e \u003cp\u003eFor electron microscopic observation, muscle tissues were meticulously dissected in an ice-cold fixative solution containing 2.5% glutaraldehyde in 0.1 mol/L PIPES buffer (pH 7.4). After fixation at 4\u0026deg;C for 10 h, the samples were rinsed with 0.1 mol/L PIPES buffer. Post-fixation was then carried out with 1% OsO₄ for 30 min, followed by staining with 2% uranyl acetate for 1 h. Dehydration was performed through a graded ethanol series (50%, 70%, and 100%), after which the samples were embedded in epoxy resin. Ultrathin sections were prepared and examined using an HT-7700 transmission electron microscope, and images were acquired for subsequent analysis[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 ELISA assay\u003c/h2\u003e \u003cp\u003eThe levels of MRCC-I, SOD, and ROS were quantified using commercial ELISA kits (Insect MRCC-I, SOD ELISA Kits, and ROS Kits, MLBIO, Shanghai, China). Muscles from 30 flies were homogenized in PBS (pH 7.2\u0026ndash;7.4). The homogenates were snap-frozen in liquid nitrogen and stored at 2\u0026ndash;8\u0026deg;C after thawing. Samples were further homogenized using mechanical grinders and centrifuged at 2000\u0026ndash;3000 rpm for 20 min, after which the supernatant was collected. For the assay, absorbance was measured at 450 nm within 15 min after adding the Stop Solution, using the blank well as the zero reference[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 qRT-PCR\u003c/h2\u003e \u003cp\u003eAt the end of the 5th week, skeletal muscle (thorax) samples from 30 Drosophila were collected per group and immersed in 1000 \u0026micro;L of Trizol reagent for subsequent analysis of relevant pathway gene expression (sample collection was repeated three times per group for experimental replicates). Each cDNA sample was prepared in triplicate for PCR amplification. Quantitative analysis was performed using the CT method[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The primer sequences of \u003cem\u003ePGC-1α\u003c/em\u003e were as follows: F: 5\u0026rsquo;-TGTTGCTGCTACTGCTGCTT-3\u0026rsquo;; R: 5\u0026rsquo;-GCCTCTGCATCACCTACACA-3\u0026rsquo;. Primer sequences of \u003cem\u003eTim\u003c/em\u003e were as follows: F: 5\u0026rsquo;-TAACACGAAGCCACGGAATAAA-3\u0026rsquo;, R: 5'-CTCGATGGTGTTCTCGGTGA-3\u0026rsquo;. Primer sequences of \u003cem\u003eClk\u003c/em\u003e were as follows: R: 5\u0026rsquo;-GCTGTAACCCTTGAGGAGGAAAT-3\u0026rsquo;, F:5'-TCGGATTCAACGTCCATGTC\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026rsquo;. Primer sequences of \u003cem\u003eSir2\u003c/em\u003e were as follows: F: 5\u0026rsquo;-GCAGT GCCAGCCC AATAA-3\u0026rsquo;, R: 5\u0026rsquo;-AGCCGATCACGATC AGTAGA-3\u0026rsquo;. Primer sequences of \u003cem\u003eRp49\u003c/em\u003e were as follows: F: 5 -CTAAGCTGTCGCACAAATGG-3\u0026rsquo;, R: 5\u0026rsquo;- AACTTCTTGAATCCGGTGGG-3\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical analyses\u003c/h2\u003e \u003cp\u003eThe differences in various indicators among the Gal4 group, UAS group, and RNAi group or OE group were compared using one-way ANOVA. The differences in various indicators between 1-week-old and 5-week-old Drosophila, as well as between the exercise and non-exercise groups, were analyzed using an independent samples t-test. The P-values for the survival curves and heart failure time curves were determined using the log-rank test. Experimental data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), with statistical significance defined as α\u0026thinsp;=\u0026thinsp;0.05 (or 0.01 where specified).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The muscle \u003cem\u003eTim\u003c/em\u003e gene is involved in regulating age-related phenotypic changes in \u003cem\u003eDrosophila\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eFrom fruit flies to humans, aging-related phenotypic changes are highly conserved, manifested as decreased locomotor capacity, disrupted circadian rhythms, and weakened cardiac function[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Among these, aging-related lifespan is the most direct and ultimate phenotype[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Both genetic predisposition and environmental factors can lead to differential susceptibility to aging among individuals, resulting in healthy aging versus pathological aging, and even the emergence of premature aging phenotypes. For instance, mutations in the \u003cem\u003eSirt1\u003c/em\u003e gene or long-term high-fat dietary intake can induce premature aging phenotypes[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Alterations in circadian rhythms are among the earliest emerging phenotypes of aging and are closely associated with abnormal changes in the neuronal circadian pathway[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, it remains unclear whether the circadian pathway in muscle participates in the regulation of the aging process.\u003c/p\u003e \u003cp\u003eIn this study, we established a \u003cem\u003eTim-UAS-RNAi/Mhc-Gal4\u003c/em\u003e system in F1-generation \u003cem\u003eDrosophila\u003c/em\u003e through genetic crosses to achieve muscle-specific knockdown of \u003cem\u003eTim\u003c/em\u003e gene expression. By assessing aging-related phenotypes and investigating the underlying mechanisms of muscle aging, we aimed to elucidate the role of muscle \u003cem\u003eTim\u003c/em\u003e in systemic aging, as well as in skeletal and cardiac muscle aging (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-A). Our results showed that muscle-specific knockdown of \u003cem\u003eTim\u003c/em\u003e had no significant effects on 24-hour activity, 12-hour daytime activity, 12-hour nighttime activity, climbing speed, heart rate, stroke volume, or time to hypoxic cardiac failure in 1-week-old young flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-B to I, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In 5-week-old aged flies, however, although muscle \u003cem\u003eTim\u003c/em\u003e RNAi did not significantly affect 24-hour or daytime activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-J and K, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), it led to a significant increase in nighttime activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-L and M, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), a marked decline in climbing speed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-N, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), a significant elevation in heart rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-O, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and significant reductions in stroke volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-P, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), time to hypoxic cardiac failure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-Q, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and lifespan (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e-R and S, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the results showed that in flies with different genetic backgrounds (UAS, Gal4, RNAi), there were no significant differences in 24-hour activity or 12-hour daytime activity between 5-week-old aged flies and 1-week-old young flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-A and B, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, the 12-hour nighttime activity of 5-week-old aged flies was significantly increased compared to that of 1-week-old young flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-C to F, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, compared to 1-week-old young flies, the 5-week-old aged flies exhibited significantly reduced climbing speed and heart rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-G and H, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), a significantly increased stroke volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-I, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and a significantly shortened time to hypoxic cardiac failure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-K to L, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results suggest that muscle-specific \u003cem\u003eTimeless\u003c/em\u003e gene RNAi accelerated the deterioration of aging-related behavioral phenotypes(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-M).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further confirm the role of the muscle-specific \u003cem\u003eTim\u003c/em\u003e gene in aging, we generated a \u003cem\u003eTim-UAS-Overexpression (OE)/Mhc-Gal4\u003c/em\u003e system in F1-generation \u003cem\u003eDrosophila\u003c/em\u003e through genetic crosses to achieve \u003cem\u003eTim\u003c/em\u003e overexpression(OE) specifically in muscle tissue. The results showed that muscle-specific overexpression of \u003cem\u003eTim\u003c/em\u003e had no significant effects on the 24-hour activity rate, 12-hour daytime activity rate, 12-hour nighttime activity rate, climbing speed, heart rate, stroke volume, or time to hypoxic cardiac failure in 1-week-old young flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-A to H, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In 5-week-old aged flies, however, although muscle-specific overexpression of \u003cem\u003eTim\u003c/em\u003e did not significantly affect their 24-hour or 12-hour daytime activity rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-I and J, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), it significantly reduced their nighttime activity rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-K and L, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), significantly increased their climbing speed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-M, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), significantly decreased their heart rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-N, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and significantly enhanced their stroke volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-O, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), time to hypoxic cardiac failure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-P, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and lifespan (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-Q and R, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the results showed that in flies with different genetic backgrounds (UAS, Gal4, OE), there were no significant differences in 24-hour activity or 12-hour daytime activity between 5-week-old aged flies and 1-week-old young flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-A and B, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, the 12-hour nighttime activity of 5-week-old aged flies was significantly increased compared to that of 1-week-old young flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-C to F, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, compared to 1-week-old young flies, the 5-week-old aged flies exhibited significantly reduced climbing speed and heart rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-G and H, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), a significantly increased stroke volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-I, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and a significantly shortened time to hypoxic cardiac failure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-J and K, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results suggest that muscle-specific \u003cem\u003eTimeless\u003c/em\u003e gene RNAi delayed the deterioration of aging-related behavioral phenotypes\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 Physiological mechanisms of the impact of muscular\u003c/b\u003e \u003cb\u003eTim\u003c/b\u003e \u003cb\u003egene on the aging of skeletal muscle and heart\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSkeletal muscle and cardiac aging are accompanied by a series of changes in intrinsic physiological mechanisms, such as increased oxidative stress, mitochondrial dysfunction, and loss of contractile proteins[\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Although studies have shown that the \u003cem\u003eTim\u003c/em\u003e gene is closely associated with cellular rhythms and DNA damage repair[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], its impact on the physiological mechanisms of cellular aging requires further investigation.\u003c/p\u003e \u003cp\u003eIn this study, we first employed real-time quantitative PCR to detect the mRNA expression levels of the muscular \u003cem\u003eTim\u003c/em\u003e gene and others, in order to verify the successful construction of the \u003cem\u003eMhc-Gla4/Tim-UAS\u003c/em\u003e structure in the F1 generation of Drosophila. The results showed that the \u003cem\u003eMhc-Gla4/Tim-UAS-RNAi\u003c/em\u003e structure significantly reduced the relative expression of the muscular Tim gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-A, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, knockdown of the muscular \u003cem\u003eTim\u003c/em\u003e gene significantly downregulated the expression of muscle \u003cem\u003eClk\u003c/em\u003e gene, \u003cem\u003eSir2\u003c/em\u003e gene, and \u003cem\u003ePGC-1α\u003c/em\u003e gene, as well as the protein level of MRCC-I and \u003cem\u003eMhc\u003c/em\u003e gene expression, and the activity level of SOD, while significantly increasing the level of muscle ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-B to H, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Results from Mhc protein immunofluorescence histochemistry indicated that knockdown of the muscular \u003cem\u003eTim\u003c/em\u003e gene significantly reduced Mhc protein expression. Transmission electron microscopy images of skeletal muscle tissue suggested that knockdown of the muscular Tim gene significantly decreased mitochondrial number and myofibril integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, the results showed that the \u003cem\u003eMhc-Gla4/Tim-UAS-overexpression\u003c/em\u003e structure significantly up regulated the relative expression of the muscular \u003cem\u003eTim\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-J, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, overexpression of the muscular \u003cem\u003eTim\u003c/em\u003e gene significantly up regulated the expression of muscle \u003cem\u003eClk\u003c/em\u003e gene, \u003cem\u003eSir2\u003c/em\u003e gene, and \u003cem\u003ePGC-1α\u003c/em\u003e gene, as well as the protein level of MRCC-I and \u003cem\u003eMhc\u003c/em\u003e gene expression, and the activity level of SOD, while significantly decreasing the level of muscle ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e-K to Q, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Results from Mhc protein immunofluorescence histochemistry indicated that overexpression of the muscular \u003cem\u003eTim\u003c/em\u003e gene significantly increased Mhc protein expression. Transmission electron microscopy images of skeletal muscle tissue suggested that overexpression of the muscular \u003cem\u003eTim\u003c/em\u003e gene significantly increased mitochondrial number and myofibril integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-R).\u003c/p\u003e \u003cp\u003eThese results indicated that the \u003cem\u003eTimeless\u003c/em\u003e gene in muscle played an important role in the aging of skeletal muscle, heart, and circadian rhythm, and its mechanism was closely related to its ability to regulate the activity status of the muscle Timeless/Clock pathway and the Timeless/Sir2/PGC-1α pathway.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Exercise attenuates aging in skeletal muscle and the heart via the upregulation of muscle\u003c/b\u003e \u003cb\u003eTim\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAccumulating evidence suggests that regular aerobic exercise helps delay aging-related phenotypic changes, including improved locomotor activity, enhanced circadian rhythms, and a reduced incidence of cardiovascular diseases in aged individuals[\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These benefits are associated with the ability of exercise to ameliorate aging-related physiological changes, such as enhanced antioxidant capacity, reduced oxidative stress, and the preservation of mitochondrial function. However, it remains unclear whether these effects are mechanistically linked to the \u003cem\u003eTim\u003c/em\u003e gene in muscle.\u003c/p\u003e \u003cp\u003eThe results of this study showed that exercise significantly reduced the 24-hour activity rate and the 12-hour nocturnal activity rate in flies with muscle-specific \u003cem\u003eTim\u003c/em\u003e gene RNA interference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while no significant effect was observed on their diurnal activity rate (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-A to E). Moreover, exercise significantly improved the climbing speed of flies with muscle-specific \u003cem\u003eTim\u003c/em\u003e gene RNAi, significantly reduced their heart rate, and markedly enhanced their stroke volume, and it also significantly prolonged the time to heart failure under hypoxia and extended their lifespan (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-F to M). Furthermore, exercise significantly increased the mRNA expression of \u003cem\u003eTim, Clk, Sir2, PGC-1α\u003c/em\u003e, and \u003cem\u003eMhc\u003c/em\u003e, elevated MRCC-I protein levels, and transmission electron microscopy images revealed a notable increase in mitochondrial number and a reduction in mitochondrial damage (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-N to T). Finally, exercise significantly increased SOD activity and markedly reduced ROS levels in the muscles of muscle-specific \u003cem\u003eTim\u003c/em\u003e RNAi flies (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e-U to V).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, muscle-specific \u003cem\u003eTim\u003c/em\u003e gene overexpression flies, the results of this study showed that exercise also significantly reduced the 24-hour activity rate and the 12-hour nocturnal activity rate in flies with muscle-specific \u003cem\u003eTim\u003c/em\u003e gene overexpression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while no significant effect was observed on their diurnal activity rate (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e-A to E). Moreover, exercise significantly improved the climbing speed of flies with muscle-specific \u003cem\u003eTim\u003c/em\u003e gene overexpression, significantly reduced their heart rate, and markedly enhanced their stroke volume, and it also significantly prolonged the time to heart failure under hypoxia and extended their lifespan (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e-F to M). Furthermore, exercise significantly increased the mRNA expression of \u003cem\u003eTim, Clk, Sir2, PGC-1α\u003c/em\u003e, and \u003cem\u003eMhc\u003c/em\u003e, elevated MRCC-I protein levels, and transmission electron microscopy images revealed a notable increase in mitochondrial number and a reduction in mitochondrial damage (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e-N to T). Finally, exercise significantly increased SOD activity and markedly reduced ROS levels in the muscles of muscle-specific \u003cem\u003eTim\u003c/em\u003e overexpression flies (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e-U to V).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese findings suggested that exercise acted as an upstream regulator of the muscle \u003cem\u003eTimeless\u003c/em\u003e gene, delaying aging-related deterioration in skeletal muscle, heart, and circadian rhythms by activating the Timeless/Clock and Timeless/Sir2/PGC-1α pathways.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eAging is an intrinsic process that occurs in nearly all living organisms, characterized by the progressive decline or loss of function across cellular, tissue, and organ systems over time, ultimately leading to the end of life. In humans, the central nervous system is among the most vulnerable to aging, making disruptions in sleep\u0026ndash;wake rhythms\u0026mdash;such as phase advancement and sleep fragmentation\u0026mdash;some of the earliest indicators of the aging process [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These alterations have also been observed in \u003cem\u003eDrosophila\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], suggesting that age-related changes in circadian rhythms are evolutionarily conserved.\u003c/p\u003e \u003cp\u003eIn mammals, the molecular clock typically exhibits reduced activity during aging, as reflected by decreased amplitude in core clock genes such as Clock, Bmal1, and Per2, while the expression of Per1, Cry1, and Cry2 remains relatively stable. TIM protein directly interacts with the cell cycle checkpoint proteins ATR and CHK1 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Consequently, the age-related decline in circadian TIM expression may compromise the function of the CHK1: ATR3 complex in DNA damage checkpoint responses [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In mammals, the \u003cem\u003eTim\u003c/em\u003e gene produces two alternatively spliced transcripts, enabling TIM to serve as a molecular link between the cell cycle checkpoint machinery and the circadian clock [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In \u003cem\u003eDrosophila\u003c/em\u003e, aging suppresses \u003cem\u003eTim\u003c/em\u003e expression, which may contribute to circadian disruption[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Collectively, growing evidence implicates \u003cem\u003eTim\u003c/em\u003e, a core circadian regulator, in the aging process. However, its specific role in aging skeletal muscle and cardiac tissue remains to be elucidated.\u003c/p\u003e \u003cp\u003eThis study employed Drosophila genetic crosses to establish a \u003cem\u003eUAS-Tim/Mhc-Gal4\u003c/em\u003e system in F1 progeny, enabling muscle- specific knockdown and overexpression of \u003cem\u003eTim\u003c/em\u003e gene to investigate its role in circadian rhythm aging, skeletal muscle aging, and cardiac aging. The results showed that muscle-specific Tim knockdown or overexpression had no significant effect on the average daily locomotor activity in one-week-old young flies. However, in five-week-old aged flies, \u003cem\u003eTim\u003c/em\u003e knockdown led to a significant increase in average nighttime activity, whereas \u003cem\u003eTim\u003c/em\u003e overexpression significantly reduced nighttime activity. These findings suggest that muscle-specific \u003cem\u003eTim\u003c/em\u003e knockdown accelerates age-related nighttime sleep fragmentation in Drosophila, while \u003cem\u003eTim\u003c/em\u003e overexpression helps preserve healthy sleep patterns during aging. These findings are consistent with previously reported studies on circadian rhythms and aging, both in \u003cem\u003eDrosophila\u003c/em\u003e and mammalian models.\u003c/p\u003e \u003cp\u003eIn both mammals and \u003cem\u003eDrosophila\u003c/em\u003e, skeletal muscle and heart\u0026mdash;as vital organs\u0026mdash;undergo structural and functional decline with aging, accompanied by an increased incidence of age-related diseases such as sarcopenia and heart failure. Age-related sarcopenia is a major cause of reduced locomotor activity and diminished motor capacity in aged individuals, with underlying mechanisms involving loss of contractile proteins in skeletal muscle cells, decreased mitochondrial function, and elevated oxidative stress damage[\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Similarly, age-associated heart failure is characterized by reduced cardiac contractility, decreased cardiac output and ejection fraction, and increased incidence of arrhythmias, with physiological mechanisms analogous to those observed in skeletal muscle aging[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. During aging, the reduction in cardiomyocyte number due to apoptosis and necrosis leads to hypertrophy of the remaining cardiomyocytes in an attempt to maintain pump function. However, this hypertrophy increases cardiac stiffness and oxygen consumption[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The myocardium is one of the most oxygen-consuming tissues in the human body. Hypoxia impairs aerobic oxidation (the most efficient energy production pathway) in cardiomyocytes, forcing a shift towards inefficient anaerobic glycolysis. This results in insufficient energy supply in cardiomyocytes, further deteriorating their systolic and diastolic functions[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Additionally, hypoxia induces mitochondrial dysfunction, generating excessive reactive oxygen species (free radicals) and triggering oxidative stress responses. This damages DNA and proteins within cardiomyocytes while activating inflammatory pathways, thereby accelerating cardiomyocyte death and fibrosis[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Hypoxia can also alter the electrophysiological stability of cardiomyocytes, increasing the risk of arrhythmias such as atrial fibrillation, which in turn exacerbates heart failure[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Therefore, aging reduces the heart's tolerance to hypoxia and increases the incidence of hypoxic heart failure[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In this study, muscle-specific knockdown or overexpression of the \u003cem\u003eTim\u003c/em\u003e gene had no significant effect on climbing speed or cardiac function in one-week-old young flies. However, in five-week-old aged flies, \u003cem\u003eTim\u003c/em\u003e knockdown significantly impaired climbing speed and cardiac function, and shortened the time to heart failure under hypoxic conditions, while \u003cem\u003eTim\u003c/em\u003e overexpression produced the opposite effects. These findings suggest that the muscle \u003cem\u003eTim\u003c/em\u003e gene plays a critical role in skeletal muscle and cardiac aging.\u003c/p\u003e \u003cp\u003eSirt1/Sir2, as key NAD⁺-dependent deacetylases, play a core protective role in skeletal muscle and cardiac aging through multi-level regulation of mitochondrial function. For instance, in the heart and skeletal muscle, Sirt1/Sir2 activate PGC-1α via deacetylation, enhancing its transcriptional activity and promoting the expression of NRF1, TFAM, and other factors, thereby facilitating mitochondrial biogenesis and maintaining mitochondrial network stability[\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Additionally, Sirt1/Sir2 activate the FOXO pathway, upregulating antioxidant enzymes such as SOD2 and catalase to scavenge excess ROS and mitigate oxidative damage in both cardiac and skeletal muscle[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Conversely, studies have shown that aging is accompanied by a decline in SIRT1/SIRT3 levels, leading to impaired mitochondrial fusion and respiratory function in cardiomyocytes, which subsequently results in defective cardiac contractility[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. SIRT1 deficiency renders the hearts of young mice susceptible to an aging-like phenotype in response to ischemia/reperfusion injury[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Although Sirt1/Sir2 are core regulators of skeletal muscle and cardiac aging, the interactive relationship between the \u003cem\u003eTim\u003c/em\u003e gene and Sirt1/Sir2 in these tissues remains unclear. This study provides the first evidence in \u003cem\u003eDrosophila\u003c/em\u003e that muscle \u003cem\u003eTim\u003c/em\u003e gene activates the Sir2/PGC-1α/MRCC-I pathway and the \u003cem\u003eTim-Clk\u003c/em\u003e pathway, enhances antioxidant capacity (SOD activity), and reduces oxidative stress (ROS), thereby protecting skeletal and cardiac myocytes from age-related decline and attenuating the loss of contractile proteins during aging.\u003c/p\u003e \u003cp\u003eAccumulating evidence suggests that exercise plays an indispensable and beneficial role in delaying aging. For instance, regular exercise effectively ameliorates age-related sleep disturbances in the elderly, reduces sleep fragmentation, and enhances circadian rhythm stability in aged individuals[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Moreover, both resistance and aerobic exercise attenuate the age-related loss of skeletal muscle mass, thereby counteracting the decline in physical and locomotor capacity associated with aging[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Exercise also delays cardiac aging by improving cardiac function and reducing the incidence of age-related cardiac diseases and heart failure[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. These beneficial effects of exercise on circadian rhythms, skeletal muscle function, and cardiac function are evolutionarily conserved across mammals, humans, and even \u003cem\u003eDrosophila\u003c/em\u003e[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. However, whether these anti-aging benefits of exercise are linked to the function of the muscle \u003cem\u003eTim\u003c/em\u003e gene remains unclear. The results of this study demonstrate that, regardless of whether the muscle \u003cem\u003eTim\u003c/em\u003e gene is knocked down, expressed at normal levels, or overexpressed, exercise improves circadian rhythms (as evidenced by reduced nighttime activity), enhances locomotor capacity and cardiac function, and prolongs the time to hypoxic heart failure in aged \u003cem\u003eDrosophila\u003c/em\u003e. These findings suggest that exercise may act as an upstream regulator of the muscle \u003cem\u003eTim\u003c/em\u003e gene to modulate skeletal muscle and cardiac aging, although the underlying molecular mechanisms remain to be elucidated.\u003c/p\u003e \u003cp\u003eThe molecular mechanisms by which exercise delays skeletal muscle and cardiac aging also involve the regulation of Sirt1/Sir2 and its associated pathways. Studies have shown that exercise enhances Sirt1/Sir2 activity by upregulating NAD⁺ levels, thereby activating downstream targets such as PGC-1α and FOXO. In skeletal muscle, exercise-induced activation of the Sirt1/PGC-1α signaling axis promotes mitochondrial biogenesis, enhances oxidative metabolism, and upregulates antioxidant enzyme expression, thereby mitigating age-related oxidative damage and protein loss[\u003cspan additionalcitationids=\"CR67\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Similarly, in the heart, exercise improves mitochondrial function in cardiomyocytes, enhances contractility, and reduces the incidence of age-related cardiac lipotoxicity and heart failure through Sirt1-dependent mechanisms, including the activation of downstream targets such as PGC-1α and FOXO[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, this study found that the muscle \u003cem\u003eTim\u003c/em\u003e gene activates the Sir2/PGC-1α/MRCC-I pathway, and exercise also regulates this same pathway, suggesting a potential interaction between \u003cem\u003eTim\u003c/em\u003e and Sirt1/Sir2 in mediating the anti-aging effects of exercise. To further elucidate the relationship between exercise and the muscle \u003cem\u003eTim\u003c/em\u003e gene, we examined the relative mRNA expression of Tim in the muscle tissue of aged \u003cem\u003eDrosophila\u003c/em\u003e. The results showed that exercise significantly upregulated Tim mRNA expression in the muscle tissue of Tim-knockdown, normal-expression, and overexpression flies, accompanied by an increase in muscle Clk gene expression. These findings suggest that in aging muscle, exercise acts as an important upstream regulator of the \u003cem\u003eTim\u003c/em\u003e gene, positively modulating the muscle \u003cem\u003eTim/Clk\u003c/em\u003e pathway, the Sir2/PGC-1α/MRCC-I pathway, and antioxidant capacity, thereby reducing oxidative stress damage and contractile protein loss associated with skeletal muscle and cardiac aging, ultimately enhancing tissue function.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eBased on the current findings, the \u003cem\u003eTimeless\u003c/em\u003e gene in muscle played an important role in the aging of skeletal muscle, heart, and circadian rhythm, and its mechanism was closely related to its ability to regulate the activity status of the muscle Timeless/Clock pathway and the Timeless/Sir2/PGC-1α pathway. Exercise acted as an upstream regulator of the muscle \u003cem\u003eTimeless\u003c/em\u003e gene, delaying aging-related deterioration in skeletal muscle, heart, and circadian rhythms by activating the Timeless/Clock and Timeless/Sir2/ PGC-1α pathways. This study provides potential molecular targets for the prevention and treatment of age-related diseases affecting skeletal muscle and the heart.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAdditional Information \u003c/strong\u003e\u003cbr\u003e Competing Interests: Authors have no conflicts of interest.\u003cbr\u003e Funding: This work is supported by the College Youth Innovation Team Program of Shandong Province (No. 2023RW057) and the National Natural Science Foundation of China (No. 32000832). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003cbr\u003eResearch idea and study design: D.t.W.; data acquisition: D.t.W. H.w.Q., Y.L, J.y.S; data analysis/interpretation: Y.q.C., G.b.S, T.s.Y.,D.y.S.; statistical analysis: D.t.W.; supervision: H.w.Q. Each author contributed during manuscript drafting or revision and approved the final version of the manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003cbr\u003eWe thank the Core Facility of Drosophila Resource and Technology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences for providing fly stocks and reagengts.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAssociated Data\u003c/strong\u003e\u003cbr\u003e Data Availability Statement.\u003cbr\u003e All the generated data and the analysis developed in this study are included in this article.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMafra, A., et al., \u003cem\u003eThe global multiple myeloma incidence and mortality burden in 2022 and predictions for 2045\u003c/em\u003e. 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FASEB JOURNAL, 2023. 37.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Biology](https://bmcbiol.biomedcentral.com/)","snPcode":"12915","submissionUrl":"https://submission.springernature.com/new-submission/12915/3","title":"BMC Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Aging, Timeless, circadian rhythm, exercise, mitochondrial, oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-9316587/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9316587/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe core genes of the circadian pathway, such as \u003cem\u003eTimeless(Tim)\u003c/em\u003e, not only participate in the regulation of biological rhythms, but also play significant roles in DNA damage repair, chronic inflammation, and metabolism of cells. However, it remains unclear whether exercise can delay the age-related phenotypic degeneration by regulating muscle \u003cem\u003eTim\u003c/em\u003e gene. In here, we first carried out the expression regulation of muscle \u003cem\u003eTim\u003c/em\u003e gene in the \u003cem\u003eDrosophila\u003c/em\u003e by constructing the \u003cem\u003eMhc-gal4/Tim-UAS\u003c/em\u003e system, and then subjected the flies to a 4-week endurance exercise intervention. The results showed that knockdown of the muscle \u003cem\u003eTim\u003c/em\u003e gene accelerated aging-related phenotypic deterioration in \u003cem\u003eDrosophila\u003c/em\u003e, manifesting as increased nighttime activity, decreased climbing speed, elevated heart rate and reduced cardiac output, shortened time to hypoxic heart failure, and shortened lifespan. This is accompanied by reductions in muscle tissue levels of \u003cem\u003eClk\u003c/em\u003e gene, \u003cem\u003eSir2\u003c/em\u003e gene, \u003cem\u003ePGC-1α\u003c/em\u003egene, \u003cem\u003eMhc\u003c/em\u003e gene, MRCC-I protein, and SOD protein, along with a significant increase in ROS. Conversely, overexpression of the muscle \u003cem\u003eTim\u003c/em\u003e gene delayed aging-related phenotypic changes in aged Drosophila. Exercise not only effectively counteracts the acceleration of aging-related phenotypic deterioration caused by muscle \u003cem\u003eTim\u003c/em\u003e gene knockdown but also further delayed aging-related phenotypic changes in aged \u003cem\u003eDrosophila\u003c/em\u003e on the basis of muscle \u003cem\u003eTim\u003c/em\u003e gene overexpression. In summary, this study highlights the role of the muscle \u003cem\u003eTim\u003c/em\u003e gene in the aging of skeletal muscles and the heart, as well as the relationship between exercise and the muscle \u003cem\u003eTim\u003c/em\u003e gene, providing strategies for the prevention and treatment of age-related diseases.\u003c/p\u003e","manuscriptTitle":"Muscle-Specific Upregulation of Timeless Mediates Exercise-Induced Amelioration of Age-Related Circadian Rhythm Disruption and Cardiac Dysfunction in Drosophila","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-20 11:01:21","doi":"10.21203/rs.3.rs-9316587/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-13T03:08:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T16:54:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"96066893946774103868373332689946892970","date":"2026-04-14T14:15:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49489853909090726198989903143957316778","date":"2026-04-13T14:38:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-13T12:06:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-07T12:49:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-06T07:00:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Biology","date":"2026-04-04T00:02:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Biology](https://bmcbiol.biomedcentral.com/)","snPcode":"12915","submissionUrl":"https://submission.springernature.com/new-submission/12915/3","title":"BMC Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ccd022b2-85b0-43c9-97a6-a6d8ae77b13e","owner":[],"postedDate":"April 20th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-13T03:08:29+00:00","index":13,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T16:54:26+00:00","index":12,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T11:01:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-20 11:01:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9316587","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9316587","identity":"rs-9316587","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}