Inactivation of Myostatin Delays Senescence via TREX1-SASP in Bovine Skeletal Muscle Cells

The myostatin (MSTN) gene also regulates the developmental balance of skeletal muscle after birth, and has long been linked to age-related muscle wasting. Many rodent studies have shown a correlation between MSTN and age-related diseases. It is unclear how MSTN and age-associated muscle loss in other animals are related. In this study, we utilized MSTN gene-edited bovine skeletal muscle cells to investigate the mechanisms relating to MSTN and muscle cell senescence. The expression of MSTN was higher in older individuals than in younger individuals. We obtained consecutively passaged senescent cells and performed senescence index assays and transcriptome sequencing. We found that senescence hallmarks and the senescence-associated secretory phenotype (SASP) were decreased in long-term-cultured myostatin inactivated (MT-KO) bovine skeletal muscle cells (bSMCs). Using cell signaling profiling, MSTN was shown to regulate the SASP, predominantly through the cycle GMP-AMP synthase-stimulator of antiviral genes (cGAS-STING) pathway. An in-depth investigation by chromatin immunoprecipitation (ChIP) analysis revealed that MSTN influenced three prime repair exonuclease 1 (TREX1) expression through the SMAD2/3 complex. The downregulation of MSTN contributed to the activation of the MSTN-SMAD2/3-TREX1 signaling axis, influencing the secretion of SASP, and consequently delaying the senescence of bSMCs. This study provided valuable new insight into the role of MSTN in cell senescence in large animals.


Introduction
Myostatin (MSTN) is also known as GDF-8 [1].Natural mutations of MSTN are associated with an increase in muscle mass [2].Currently, multiple species of MSTN gene inactivation mutant animals have been obtained through gene editing technology [3][4][5], and all the MSTN mutant animals show "double-muscled" phenotypes.Inactivation of the MSTN gene can not only improve individual growth levels and exercise abilities but also increase muscle production in livestock and poultry production, generating more economic benefits [6].MSTN inhibits the development of skeletal muscle before birth and regulates muscle tissue homeostasis in adult animals.In addition, because of the similarity to human physiology, anatomy, genetics, and size, animals raised for food are also useful animal models for human diseases [7].
Skeletal muscle tissue is composed of muscle fibers, which are primarily divided into two types based on the proportion of myosin heavy chain (MHC) isoforms: Type I (slow oxidative fibers) and Type II (fast glycolytic fibers) [8].The extracellular matrix (ECM) connects cylindrical fibers.Each fiber is enveloped by an ECM layer, which includes the perimysium and the basal lamina.Collagen fibers interweave with extensive vascular and nerve networks to form the perimysium, which is the outermost membrane.The reticular layer, composed of collagen fibers, and the basal layer form the basal lamina, which is located between the perimysium and the plasma membrane [9].The plasma membrane contains many proteins, such as the dystrophin-glycoprotein complex that is involved in connecting fibers and the ECM [9].Skeletal muscle's ability to regenerate itself steadily decreases with age [10].The aging process is linked to a reduction in the number and function of satellite cells, which severely impairs or even eliminates the remodeling ability of skeletal muscle [11].Hyaluronan (HA) is ubiquitously expressed in the extracellular matrix (ECM) of mammals [12].Researchers have also explored the potential of hyaluronans (HAs) in promoting cell growth and proliferation, as well as rescuing cells under oxidative stress, through three different experimental setups, the study indicates that different hyaluronan-based gels have a protective effect in the recovery of stressed cells and muscle atrophy [13].According to previous studies, inhibiting myostatin is a potential way to alleviate age-related muscle atrophy [14,15].
The secretion of many inflammation factors, which is also known as SASP, has been identified in recent research as a characteristic of senescent cells [16].This can affect the function of neighboring cells and tissues, promoting tissue inflammation and deficiency, which could cause cellular senescence.Studies revealed that 1,25(OH)2D3 deficiency could cause cellular senescence and SASP in skeletal muscle cells to induce sarcopenia [17].The innate DNA sensing pathway is essential to regulating SASP and senescence [18].The SASP is triggered by the abnormal activation of the cytoplasmic DNA sensing mechanism, the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) [19].TREX1 is a 3 ′ DNA exonuclease.Reports have shown that the loss of TREX1 results in the inability of cells to undergo a normal cell cycle, resulting in defects in the G1/S phase transition in cells [20,21].At the same time, MSTN negatively regulates the process of the G1/S phase, thereby inhibiting the cell cycle and maintaining the quiescent state of satellite cells [22].This, in turn, affects the proliferation of skeletal muscle cells.MSTN can affect the expression of the cell cycle through the downstream transcription factor SMAD.The knockout of MSTN promotes cell proliferation, and the overexpression of MSTN inhibits cell proliferation and DNA synthesis [23].In pre-senescent cells, the nucleus effectively clears cytoplasmic DNA fragments through the action of TREX1 and DNase2.However, in senescent cells, the expression of DNase is decreased, resulting in the accumulation of nuclear DNA in the cytoplasm [19].Following TREX1 depletion, there is an increased content of ssDNA in the cytoplasm [24].Subsequently, this triggers intracellular DNA sensing mechanisms, which, in turn, activate signaling pathways for pro-inflammatory factors [25,26].
Although the respective roles of MSTN and SASP in skeletal muscle aging have been reported, the mechanism by which the MSTN gene influences SASP and delays the aging of skeletal muscle cells is still unclear.In this study, we used skeletal muscle cells from consecutive passages of MSTN gene-edited bovine to explore the role of the MSTN gene in skeletal muscle aging.This exploration is of significant importance for understanding the generation of animal muscle mass and overall organism health, as well as for the treatment of aging-induced related diseases.

MSTN Gene Inactivation Delayed the Senescence of Long-Term Cultured bSMCs
To evaluate the effect of MSTN inactivation on the senescence of the long-term cultured bSMCs, we used WT and MT-KO bovine skeletal muscle cells (bSMCs) from our laboratory [27], where the expression of MSTN in MT-KO cells decreased more than in the WT cells (Figure 1a).Through a continuous passage, we obtained cells of different generations and established aging models of WT and MT-KO bSMCs.After many passages, WT and MT-KO cells tended to be flat, larger than the low passage cells, and they lacked plasticity (Figure 1b,c).The survival of cells through the number of passages was statistically plotted for both the WT and MT-KO groups, with the highest number of passages in the WT group being around 30 generations, while three cell lines in the MT-KO group were passed to less than 40 generations (Figure 1d).
passages, WT and MT-KO cells tended to be flat, larger than the low passage cells, and they lacked plasticity (Figure 1b,c).The survival of cells through the number of passages was statistically plotted for both the WT and MT-KO groups, with the highest number of passages in the WT group being around 30 generations, while three cell lines in the MT-KO group were passed to less than 40 generations (Figure 1d).

Inactivation of the MSTN Alleviated Senescence Caused by the Long-Term Culture of bSMCs
Although some advancement has been made in knowing the properties and mechanisms of senescent cells, researchers proposed the multi-marker method to assess the efficiency of cellular senescence; this method involves assessing senescence-associated beta-galactosidase (SA-β-gal) activity, co-staining markers typically related to cell proliferation, and p16, p21 expression, and elements anticipated to change in specific senescence scenarios [28].We analyzed the senescence indicators in continuously passaged cells and compared the WT cells with the MT-KO cells.Telomere length decreased with the increase in passage number, and the telomere length in the WT cells was shorter than that of the MT-KO cells in LP cells (Figure 2a).We observed an increased percentage of cells positive for SA-β-gal staining (WT vs. MT-KO, EP 15.33% ± 2.52 vs. 8.33% ± 1.53; MP 32.67% ± 2.52 vs. 22% ± 2.65; LP 84% ± 3.61 vs. 74.33%± 3.06) (Figure 2b), a decreased percentage of ki67-positive cells (WT vs. MT-KO, EP 89.69% ± 4.47 vs. 97.96%± 0.37; MP 55.05% ± 0.44 vs. 84.96%± 2.14; LP 18.86% ± 4.88 vs. 41.54%± 2.22) (Figure 2c), a decrease in the G1/S-phase of the cell cycle (Figure 2d), and an upregulation of p16 protein expression (Figure 2e).These results showed that the downregulation of MSTN delayed cellular senescence in bovine skeletal muscle cells.

Inactivation of the MSTN Downregulated the SASP in Skeletal Muscle Senescence Cells
Western blot analysis was used to detect the expression of the MSTN protein in the EP, MP, and LP of both WT and MT-KO cells.The results revealed an increase in MSTN expression with generation, yet significantly lower levels were observed in MT-KO cells compared to WT cells (Figure 3a).Then, we selected different stages of WT and MT-KO cells for transcriptome sequencing.To evaluate the dynamic changes in the trends of WT and MT-KO cells after continuous subculture, an analysis using genome-wide RNA sequencing (RNA-seq) was carried out.STEM analyses of WT (EP, MP, LP) and MT-KO (EP, MP, LP) cells were clustered into eight profiles for each sample.Profile 6 (8324), Profile 1 (1457), and Profile 7 (700) of the WT cells and Profile 2 (3404) and Profile 6 (1581) of the MT-KO cells were significantly different from the other profiles (Figure 3b).The genes with significant differences were analyzed using the GO and KEGG pathways (Figure 3c,d).The KEGG pathway analysis showed that 29, 726, and 894 pathways were mapped in EP, MP, and LP cells, of which 96, 917 pathways were significantly different, respectively (q value < 0.05), including cellular senescence, cell cycle, DNA replication, and the NF-κB signaling pathway (Figure 3c).A heatmap of the enriched genes related to cellular senescence was created and revealed that the GO terms "aging" and "SASP" were primarily linked to genes that were expressed differently in WT and MT-KO cells (Figure 3d,e).The mRNA expression of increased genes and reduced genes in the heatmap of SASP factors were analyzed (Figure 3f).These results indicate that the inactivation of the MSTN gene downregulated the SASP in skeletal muscle senescence cells.The basis of MSTN expression in different generations of cells was analyzed, and MP cells were selected for subsequent experiments.

Inactivation of the MSTN Downregulated the SASP through the Upregulation of TREX1
According to reports, the innate DNA sensing system is essential to regulate the SASP and senescence [29].Compared with the WT long-term-cultured bSMCs, TREX1 expression was increased in the MT-KO cells, and with the increase in generation, TREX1 expression was lower than in the early passages of WT and MT-KO (Figure 4a).In addition, the downstream factors of the cGAS-STING pathway were observed to decrease in the MT-KO cells (Figure 4b-d).Through the above, in bSMCs, MT-KO will affect the expression of TREX1, but how MSTN affects TREX1 needs to be explored.As SMAD2/3 acts as a downstream complex transcription factor of MSTN, we investigated its binding ability to the TREX1 promoter region.Through prediction analysis using the JASPAR database, it was discovered that SMAD2/3 is linked to the TREX1 (NC_037349.1)promoter region between −1446 and −1437.The ChIP-qPCR findings indicated that there is binding of SMAD2/3 to the TREX1 promoter (Figure 4e).Using the luciferase reporter assay, we were able to detect transcriptional activity following SMAD3 overexpression to investigate the impact of SMAD2/3 on the transcriptional activity of TREX1.It was discovered that SMAD3 inhibits the TREX1 promoter's transcriptional activity, and, after interfering with SMAD3, the activity of the TREX1 promoter was significantly upregulated (Figure 4f).These results suggest that SMAD3 has a negative regulatory effect on the TREX1 promoter.

Aging Prematurely Due to TREX1 Absence in bSMCs
To investigate the effect of TREX1 deficiency in MT-KO_MP bSMCs, we transfected the TREX1 shRNA in MT-KO_MP bSMCs and evaluated the efficiency of interference.shTREX-1, with the highest interference efficiency, was selected for subsequent experiments (Figure 6a).The loss of TREX1 protein expression in TREX1-deficient MT-KO_MP bSMCs was verified by Western blotting (Figure 6b).There were higher proportions of SA-β-gal in the TREX1-deficient cells than in the MT-KO_MP cells (28.67% ± 1.15 vs. 6.67% ± 1.53) (Figure 6c), and a lower proportion of ki67-positive cells (13.95% ± 2.85 vs. 30.83%± 7.47) (Figure 6d).The protein expression of downstream genes, including cGAS, STING, p-TBK1, and p-IRF3, exhibited a significant increase (Figure 6e,f), and the IRF3, and TBK1 total protein expression levels showed no obvious difference (Figure 6f,g).In shTREX1 MT-KO_MP bSMCs, p65 protein expression was higher in the nucleus than in the cytoplasm (Figure 6h).The mRNA expression of the SASP-related genes IL-6 and IL-8 was higher in the TREX1-deficient cells than in the MT-KO_MP cells (Figure 6i).

Discussion
The skeletal muscle, as the largest organ in the human body, is essential for the survival of the organism and performs a variety of functions [30].Although it has great regenerating capacity, after damage, this capacity decreases with age.With age increase, interventions such as exercise will not be able to help one avoid muscle atrophy as well as decreased muscle mass due to other diseases [31].Baumann et al. discovered that the myostatin protein content increased with age from 1.5 to 27 months in the rat's gastrocnemius muscle [32].The levels of myostatin and SA-β-gal in mouse skeletal muscle increased with age [33].The knockdown of myostatin reveals significant muscle hypertrophy.Throughout the process of development, satellite cell-derived myoblasts multiply and differentiate continually before fusing with pre-existing muscle fibers to enlarge them [34].Compared to wild-type animals, adult Mstn −/− mice exhibit a notable rise in both the number of satellite cells per muscle fiber and the percentage of activated satellite cells.Introducing myostatin shRNAs via injection in rats led to a more than twofold augmentation in satellite cell numbers [22].
For the study of cellular senescence, the most commonly used method is to continuously pass the cells to obtain a replicative cellular senescence model [35].This experiment was based on this continuous passaging culture until the cells were difficult to collect.Compared to low-passage cells, high-passage cells from both the WT and MT-KO groups exhibited significant morphological changes.Studies have reported significant changes in cell phenotype, differentiation potential, and gene expression after passages [36,37].Therefore, we examined telomere length, SA-β-gal activity, ki67-positive cells, cell cycle dynamics, and the expression of p16 with increasing passage numbers.Both the WT and MT-KO groups exhibited noticeable changes in these aging-related indicators as the passage number increased.Through the detection of these aging indicators, we confirmed the continuously passaged senescent cells were suitable for subsequent experiments.Then, transcriptome sequencing analysis was conducted on the obtained continuously passaged senescent cells, and the result highlighted genes related to cellular senescence.We analyzed these genes and found that most of them were associated with the SASP.It is increasingly evident that the release of inflammatory mediators is one aspect of aging [38].Senescent cells demonstrate cell cycle arrest [39], tissue repair ability loss [40], and proinflammatory cytokine release [41].Several mechanisms have been demonstrated to be involved in regulating the activity of SASP factors, including transcription-related variables, positive regulators of SASP factor expression Nuclear Factor κB (NF-κB) [42], and the cGAS-STING signaling pathway.DNA damage leads to the accumulation of damaged DNA in the cytoplasm of cells containing cGAS, which is regarded as a cytoplasmic DNA sensor [43].It can identify and detect damage to DNA, and when cytoplasmic DNA (mtDNA, cDNA, and pieces of cytoplasmic chromatin) accumulates in senescent cells, it produces cGAMP, which sets off the SASP.In inflammatory conditions, aberrant DNA metabolism and the proinflammatory impact of cfDNA buildup may be exacerbated by decreased TREX1 expression, which could ultimately cause normal cells to progressively acquire the SASP [44].
Combining transcriptome data, we conducted an expression-level analysis of TREX1 at different passages and found that with increasing passages, both TREX1 mRNA and protein expression decreased.In the WT group, the decrease occurred after EP, while in the experimental group, it occurred after MP.TREX1-deficient mice induce autoimmune disorders by activating downstream signaling pathways including the cGAS [45].We simultaneously detected the mRNA and protein expression levels of cGAS and STING downstream of TREX1.As compared to the WT group, the MT-KO group's expression levels were lower, according to the data.This result is in line with earlier studies.However, the regulatory effect of MSTN on TREX1 and cGAS-STING signaling pathways remains unclear.Then, we explored the potential association between MSTN absence and TREX1.
The results indicate that MSTN regulates TREX1 through SMAD2/3.SASP-phenotyping senescent cells that express GATA4 and NF-κB factors produce IL-6 and IL-8, proteases, and matrix metalloproteinases [46].p65 is an important member of the NF-κB transcription factor family, playing crucial roles in both the cytoplasm and the nucleus, undergoing dynamic translocation under different environmental and conditional cues [47].In its inactive state, p65 exists in the cytoplasm, and when it becomes active, it moves to the nucleus and takes part in biological processes such as the immune response, inflammation, and cell proliferation [48].To further confirm this, TREX1 was overexpressed in the WT group, and TREX1 was interfered with in the MT-KO group.It was found that after MSTN inactivation, by modulating the activity of SMAD2/3 and TREX1, the downstream cGAS-STING signaling pathway was affected, influencing the secretion of SASP, and consequently delaying the senescence of bMSCs cells (Figure 7).Conversely, MSTN skeletal muscle cells are more susceptible to replicative senescence effects.
unclear.Then, we explored the potential association between MSTN absence and T The results indicate that MSTN regulates TREX1 through SMAD2/3.SASP-pheno senescent cells that express GATA4 and NF-κB factors produce IL-6 and IL-8, pro and matrix metalloproteinases [46].p65 is an important member of the NF-κB tran tion factor family, playing crucial roles in both the cytoplasm and the nucleus, unde dynamic translocation under different environmental and conditional cues [47].In active state, p65 exists in the cytoplasm, and when it becomes active, it moves to t cleus and takes part in biological processes such as the immune response, inflamm and cell proliferation [48].To further confirm this, TREX1 was overexpressed in t group, and TREX1 was interfered with in the MT-KO group.It was found that after inactivation, by modulating the activity of SMAD2/3 and TREX1, the downstream STING signaling pathway was affected, influencing the secretion of SASP, and quently delaying the senescence of bMSCs cells (Figure 7).Conversely, MSTN s muscle cells are more susceptible to replicative senescence effects.
In this study, we only discussed nuclear DNA.When DNA is in the cytoplas DNA sensing mechanism is activated, thereby activating the pro-inflammatory cy pathway [20,21,49].Mitochondrial DNA exists in the mitochondrial matrix and is a lar molecule of double-stranded dsDNA, adjacent to the electron transport chain, is the main source of reactive oxygen species.Therefore, it is particularly suscept oxidation, leading to mtDNA mutations, which may lead to disease and aging [50 application of mtDNA and cGAS-STING signals in skeletal muscle aging deserves f exploration.

Cell Culture and Isolation
Three 2-year-old MSTN +/− (MT-KO) and three wild-type (WT) heifer bovine s muscle cells from our laboratory were chosen [27].Wild-type and MSTN +/− bovine m tissues were obtained after they were slaughtered, and then the tissues were rinsed In this study, we only discussed nuclear DNA.When DNA is in the cytoplasm, the DNA sensing mechanism is activated, thereby activating the pro-inflammatory cytokine pathway [20,21,49].Mitochondrial DNA exists in the mitochondrial matrix and is a circular molecule of double-stranded dsDNA, adjacent to the electron transport chain, which is the main source of reactive oxygen species.Therefore, it is particularly susceptible to oxidation, leading to mtDNA mutations, which may lead to disease and aging [50].The application of mtDNA and cGAS-STING signals in skeletal muscle aging deserves further exploration.

Cell Culture and Isolation
Three 2-year-old MSTN +/− (MT-KO) and three wild-type (WT) heifer bovine skeletal muscle cells from our laboratory were chosen [27].Wild-type and MSTN +/− bovine muscle tissues were obtained after they were slaughtered, and then the tissues were rinsed in PBS with 2% penicillin-streptomycin (Gibco, Grand Island, NY, USA) three times, treated with 75% ethanol, and followed by treatment with PBS three times.The tissues were treated with 1 mg/mL collagenase IV (Thermo Fisher Scientific, Waltham, MA, USA) for 2-3 h to digest into cells and were cultured using DMEM medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 20% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA) and 10% horse serum (HS, Thermo Fisher Scientific, Waltham, MA, USA) at 38.5°C with 5% CO 2 .The obtained cells were cultured and passage culture was performed when the cell confluence reached 90%until the cells were difficult to collect and the passage was finished [27].Due to the difficulty in sampling cells after more than 30 generations, cells from the 30-generation period were chosen as the late passage (LP).Cells from the p10-generation period were taken as early passage (EP) samples, and cells from the p20generation period were taken as middle-passage (MP) samples for experimentation.

Telomere Length Assay
DNA of the different generation cells was extracted.PCR was performed to amplify specific segments of both telomere DNA and the single-copy gene.The threshold cycle (CT) values for telomere DNA and the single-copy gene were detected.∆CT was calculated by subtracting the CT value of the single-copy gene from that of telomere DNA.The formula T/S = 2 −∆CT was used to calculate the T/S ratio.

SA-β-Gal Staining
Cells of different generations were first fixed with fixative (0.2% glutaraldehyde and 2% formaldehyde mixture) for 5 min at 25 • C, and then stained using SA-β-gal staining solution (Beyotime, shanghai, China, C0602) overnight at 25 • C. Finally, the percentage of cells positive for SA-β-gal was counted.

Immunofluorescent Staining
Cells from various generations were fixed using 4% paraformaldehyde for 30 min at 25 • C. Subsequently, they were rinsed 3 times with PBS and then incubated overnight at 4 • C with anti-Ki67 antibodies (Abcam, Cambridge, MA, USA, ab15580, diluted 1:500).After another three washes with PBS solution, the cells were incubated with secondary antibodies for 15 min at 25 • C. Finally, nuclear staining was performed using DAPI.Images were captured using a confocal microscope (A1R, Nikon, Tokyo, Japan).

The use of Fluorescence-Activated Cell Sorting for Cell Cycle Assay (FACS)
Cells of different generations were collected into a centrifuge tube and resuspended using ice-cold PBS.They were fixed for 30 min at 4 • C using cold 70% ethanol, and then washed with PBS followed by propidium iodide (PI) staining, following the instructions in the assay kit (Beyotime, shanghai, China, C1052).Then, they were incubated at 37 • C for 30 min in the dark.The treated cells were detected by flow cytometry (ModFIT LT v3.1) for cell cycle analysis within 24 h.

Real-Time PCR
Using the RNAiso Plus kit, the total RNA of cells of different generations was extracted (Takara, Tokyo, Japan).Next, the RNA was reverse transcribed into cDNA.The expression of genes was detected by real-time qPCR with SYBR Green fluorescence dye (Takara, Tokyo, Japan) using LC480 (Rocho, Basel, Switzerland).Supplementary Table S1 contains the PCR primers that Sangon Biotech generated.The PCR amplification protocol was as below: 95 • C for 30 s, 95 • C for 5 s, and 60 • C for 34 s for 40 cycles.Using GAPDH as the reference gene, the relative expression of genes was calculated using the 2 −∆∆Ct method.

Chromatin Immunoprecipitation Assay
ChIP assays were carried out using kits (Thermo Fisher Scientific, USA).Briefly, cells were fixed using formaldehyde and quenched using glycine.MNase was used to lyse and digest DNA.Cells were subsequently sonicated to break the nuclear envelope.The resulting lysates were subsequently incubated overnight at 4 • C with SMAD2/3 antibodies (Abcam, USA, ab202445) and protein G beads.After that, DNA was restored with the use of a DNA purification kit.Quantitative PCR was used to determine the purity of the DNA.

Luciferase Reporter
The amplified TREX1 gene promoter region from bovine genomic DNA was integrated with the pGL3-Basic vector (Promega, Madison, WI, USA) and transfected to the seeded HEK293T.Then, 48 h later, luciferase activity was assessed by a dual-luciferase reporter assay system (Promega, USA).

Statistics Analysis
The mean ± SD of three different experiments was used to express all the data.The error bars in the graphs represent one standard deviation, whereas the bars in the graphs represent the means.For statistical analysis, Welch's t-test was employed to compare data from two groups with various standard deviations.A recurring one-way ANOVA was utilized when comparing two or more groups.* p <0.05 indicated statistically significant results.The phototypesetting was carried out in Adobe Photoshop CS3, and the histograms were created in Prism 8.0 (GraphPad, La Jolla, CA, USA).

Conclusions
In this study, through the continuous passaging of bovine skeletal muscle cells, a replicative senescence model of bovine skeletal muscle cells was established, and an analysis of the transcriptome data revealed that they were mainly genes associated with the SASP.MSTN regulates the SASP through the MSTN-SMAD2/3-TREX1 signaling axis.The overexpression of TREX1 in the WT group delayed the aging process in accelerated aging skeletal muscle cells.It interfered with the TREX1 MT-KO group, accelerating the aging of skeletal muscle cells.This study expands the current knowledge on MSTN and skeletal muscle cell senescence, demonstrating that the inactivation of MSTN is involved in delaying the aging of bovine skeletal muscle cells.

Figure 1 .
Figure 1.Characteristics of long-term cultured inactivation of the MSTN bSMCs.(a) Expression of MSTN mRNA and protein in bSMCs.(b) Muscle cells derived from bovine at different periods in the continuous subculture; scale bar, 1000 µm.(c) The difference in the area of passaged cells (µm 2 ).(d) Survival curves of different generations in WT and MT-KO groups.The mean ± SD is used to present the data (n = 3).* p < 0.05; *** p < 0.001; **** p < 0.0001 (t-test).

Figure 1 .
Figure 1.Characteristics of long-term cultured inactivation of the MSTN bSMCs.(a) Expression of MSTN mRNA and protein in bSMCs.(b) Muscle cells derived from bovine at different periods in the continuous subculture; scale bar, 1000 µm.(c) The difference in the area of passaged cells (µm 2 ).(d) Survival curves of different generations in WT and MT-KO groups.The mean ± SD is used to present the data (n = 3).* p < 0.05; *** p < 0.001; **** p < 0.0001 (t-test).

Figure 2 .
Figure 2. Hallmarks of the senescence phenotype in long-term-cultured bSMCs.(a) Changes in lomere length in skeletal muscle cells at different passage numbers.(b) SA-β-gal staining of WT a MT-KO cells and the percentages of SA-β-gal-positive cells of WT and MT-KO, the red arrows re to the stained senescent cells.Scale bar, 1000 µm.(c) ki67 staining and ki67-positive cells perce Scale bar, 50 µm.(d) Bar chart displaying the percentage of WT and MT-KO cells of the cell cyc (e) Protein expression of p16 at different passage numbers.EP, MP, and LP stand for early passag middle passage, and late passage, respectively.Early passage cells are around p10, middle passa cells are around p20, and late passage cells are around p30, same as below.The mean ± SD is us to present the data (n = 3).* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, p > 0.05 (t-test).

Figure 2 .
Figure 2. Hallmarks of the senescence phenotype in long-term-cultured bSMCs.(a) Changes in telomere length in skeletal muscle cells at different passage numbers.(b) SA-β-gal staining of WT and MT-KO cells and the percentages of SA-β-gal-positive cells of WT and MT-KO, the red arrows refer to the stained senescent cells.Scale bar, 1000 µm.(c) ki67 staining and ki67-positive cells percent.Scale bar, 50 µm.(d) Bar chart displaying the percentage of WT and MT-KO cells of the cell cycle.(e) Protein expression of p16 at different passage numbers.EP, MP, and LP stand for early passage, middle passage, and late passage, respectively.Early passage cells are around p10, middle passage cells are around p20, and late passage cells are around p30, same as below.The mean ± SD is used to present the data (n = 3).* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, p > 0.05 (t-test).

Figure 3 .Figure 3 .
Figure 3. Inactivation of the MSTN downregulated the SASP in senescent bSMCs.(a) The expressio of MSTN in cells with different passage numbers was examined using Western blotting.(b) STEM analysis: different colors represent significant differences, the number in the upper left corner indi cates the profile number, the number in the upper right corner indicates the gene number, and th number in the bottom left corner indicates the significance of the differences.(c,d) Enriched KEGG and GO pathways.(e) KEGG enrichment of cell senescence-associated genes.(f) The mRNA expres sion of increased genes and reduced genes in heatmap analysis.The mean ± SD is used to presen the data (n = 3).* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (t-test).

Figure 5 .
Figure 5. Overexpression of TREX1 can rejuvenate aged bSMCs.(a,b) TREX1 mRNA and protein expression of overexpression TREX1 WT_MP group and WT_MP group.(c) SA-β-gal staining and positive cell percent of overexpression of TREX1 WT_MP and WT_MP cells, the red arrows refer to the stained senescent cells.scale bar, 1000 µm.(d) ki67 staining and positive cell percent of overexpression of TREX1 WT_MP and WT_MP cells; scale bar, 50 µm.(e-g) Protein expression of cGAS,

18 Figure 6 .
Figure 6.TREX1 deficiency resulted in premature aging of bSMCs.(a) Transfected the TREX1 shRNA in MT-KO_MP bSMCs and evaluated the efficiency of interference.(b) TREX1 protein expression after deficiency of TREX1 in MT-KO_MP in bSMCs.(c) SA-β-gal staining and positive cells with percent of shTREX1_MP and MT-KO_MP cells, the red arrows refer to the stained senescent cells.scale bar, 1000 µm.(d) Ki67 staining and positive cell percent of shTREX1 and MT-KO cells; scale bar, 50 µm.(e-g) Protein expression of cGAS, STING, IRF3, p-IRF3, TBK1, and p-TBK1, of shTREX1_MP and MT-KO_MP cells.(h) Nuclear and cytoplasmic protein expression of p65 of

Figure 6 .
Figure 6.TREX1 deficiency resulted in premature aging of bSMCs.(a) Transfected the TREX1 shRNA in MT-KO_MP bSMCs and evaluated the efficiency of interference.(b) TREX1 protein expression after

Figure 7 .
Figure 7. Schematic of the mechanism of MT-KO in bSMCs senescence.In the MSTN inact model, MSTN formed a complex and ActRIIB then combined with the type I receptor.The ac MT-KO signal then activated phosphorylated SMAD2/3, where up on SMAD2/3 it was dec and combined with SMAD4.By combining with the TREX1 promoter inside the nucleus, th pound enhanced TREX1's activity.The increase in TREX1 reduced the damaged DNA in th plasm, decreased cGAS-STING signaling, decreased secretion of the SASP factors, and dela nescence.

Figure 7 .
Figure 7. Schematic of the mechanism of MT-KO in bSMCs senescence.In the MSTN inactivation model, MSTN a complex and ActRIIB then combined with the type I receptor.The activated MT-KO signal then activated phosphorylated SMAD2/3, where up on SMAD2/3 it was decreased and combined with SMAD4.By combining with the TREX1 promoter inside the nucleus, this compound enhanced TREX1's activity.The increase in TREX1 reduced the damaged DNA in the cytoplasm, decreased cGAS-STING signaling, decreased secretion of the SASP factors, and delayed senescence.