SESN1 is a FOXO3 effector that counteracts human skeletal muscle ageing

Abstract Sarcopenia, a skeletal muscle disorder in which loss of muscle mass and function progresses with age, is associated with increased overall frailty, risk of falling and mortality in the elders. Here, we reveal that SESN1 safeguards skeletal muscle from ageing downstream of the longevity gene FOXO3, which we recently reported is a geroprotector in primate skeletal muscle. Knockdown of SESN1 mimicked the human myotube ageing phenotypes observed in the FOXO3‐deficient human myotubes, whereas genetic activation of SESN1 alleviated human myotube senescence. Of note, SESN1 was identified as a protective secretory factor against muscle atrophy. Administration of recombinant SESN1 protein attenuated senescence of human myotubes in vitro and facilitated muscle regeneration in vivo. Altogether, we unveil a key role of SESN1 downstream of FOXO3 in protecting skeletal muscle from ageing, providing diagnostic biomarkers and intervention strategies for counteracting skeletal muscle ageing and related diseases.

phenotypes observed in the FOXO3-deficient human myotubes, whereas genetic activation of SESN1 alleviated human myotube senescence. Of note, SESN1 was identified as a protective secretory factor against muscle atrophy. Administration of recombinant SESN1 protein attenuated senescence of human myotubes in vitro and facilitated muscle regeneration in vivo. Altogether, we unveil a key role of SESN1 downstream of FOXO3 in protecting skeletal muscle from ageing, providing diagnostic biomarkers and intervention strategies for counteracting skeletal muscle ageing and related diseases.

| INTRODUCTION
Skeletal muscle, comprising 30%-40% of total body mass, controls movement and maintains posture by attaching to bones directly or indirectly. 1,2 By secreting myokines, skeletal muscle exerts systemic effects on the whole body and plays an important role in maintaining the homeostasis and health of the body. However, with ageing, decline in skeletal muscle mass and function, known as sarcopenia, reduces capacity for movement, maintaining balance and other physical functions, increases the risk of falls, disability and even death. 3,4 Therefore, understanding the mechanisms underlying maintenance of skeletal muscle homeostasis and how these are impacted by ageing are of great scientific and clinical significance.
To a limited extent, sarcopenia can be slowed down by physical therapy or nutritional approaches. [5][6][7] Regular training can improve muscle mass and strength by increasing protein synthesis, myofibrillar count and muscle fibre cross-sectional area. 8 Nutrition interventions, through ensuring adequate intake of protein, creatine, vitamin D, Omega-3 fatty acids and so on, primarily maintain muscle mass. 9,10 In general, the molecular basis of skeletal muscle ageing has been challenging to pin down, and as a result, progress towards the development of intervention therapies has been slow. Nevertheless, identifying and validating the regulatory mechanism of skeletal muscle ageing remains an important goal for the development of future effective and evidencebased intervention measures for skeletal muscle ageing.
Sestrins (Sesns) are a family of highly conserved and ubiquitous metabolic proteins that are induced in cells in response to environmental stresses, including oxidative stress, DNA damage, hypoxia and starvation. [11][12][13] Previous studies have shown that Sesns can play protective roles in various diseases such as metabolic disorders, lipid accumulation and insulin resistance. 14 Vertebrates express three different types of Sesns, namely SESN1, SESN2 and SESN3. 15 SESN1, the earliest known protein in the Sestrins family, also known as PA26, has been identified as a target of the tumour-suppressing protein p53. 16 Although SESN1 has been reported to have multiple effects on maintaining physiological homeostasis, to our knowledge, it has not previously been linked to primate skeletal muscle ageing.
In this study, we identified and experimentally validated that downregulation of the FOXO3-SESN1 axis is a mechanism that triggers skeletal muscle ageing in primates. An important finding with clinical implications is that genetic activation of endogenous SESN1 or pharmacological treatment with recombinant SESN1 protein counteracted the onset of human myotube senescence in vitro and enabled skeletal muscle regeneration in vivo. Our study strengthens the understanding about the mechanistic underpinning of primate skeletal muscle ageing and facilitates the development of novel therapeutical intervention strategies to combat age-associated muscle degenerative disorders.

| Experimental animals
C57BL/6J mice (16 months old) were purchased from SPF Biotechnology Co., Ltd and housed conventionally in a constant temperature (25 C) and humidity (50%-60%) animal room, with a 12 h light-dark cycle and free access to normal diet and water.

| Tissue sampling
Skeletal muscle tissues were harvested from human and mice as previously described. 17,20 The skeletal muscle tissues, removed of attached fat or fascia tissues, were fixed in 4% paraformaldehyde (PFA) at 4 C for 24 h and embedded in paraffin or Tissue-Tek optimum cutting temperature (O.C.T) compound (Sakura Finetek) for the following histological analysis. The remaining tissues were stored in liquid nitrogen for other RNA or protein analyses.

| Cardiotoxin injury-induced skeletal muscle damage in mice
Skeletal muscle injury was performed as described previously. 17 Briefly, C57BL/6J male mice (16 months old) were randomly divided into an uninjured group treated with phosphate buffered saline (PBS) (referred to Sham group), and the injured group treated with PBS (referred to post-injury-Vehicle group) or recombinant SESN1 protein (referred to post-injury-rSESN1 group). For the injured group, 10 μM of cardiotoxin (CTX, Latoxan) in 25 or 50 μL PBS was injected twice into tibialis anterior muscle or quadriceps muscle, respectively. In the following 7 days, recombinant SESN1 protein (0.5 μg/mL) in 30 or 60 μL PBS was injected into tibialis anterior muscle or quadriceps muscle, respectively.  21,22 Briefly, mice in Sham, post-injury-Vehicle and postinjury-rSESN1 groups were held by the tail and allowed to hold the grid of the apparatus. Mice were gently pulled away from the grid until they released the grid. The peak pull force was measured 10 times at 1-min interval for each test, and the mean of the values was recorded as the grip strength of each mouse.

| Rotarod test
Motor coordination was assessed in mice in Sham, post-injury-Vehicle and post-injury-rSESN1 groups with a rotarod test, performed on a rotating rod (Yiyan Tech, YLS-4C) that accelerated from 4 to 44 rpm/ min with an acceleration of 8 rpm/min approximately per minute. The time spent on the rod (before falling) per trial was recorded. Animals were trained three trials per day for 3 consecutive days before formal experiment, with measurements at 5-min intervals. For each mouse, the average time from start of each trial to dropping down was recorded.

| Treadmill performance test
Mice were trained on a treadmill (SANS Bio Instrument, SA101) at a 5 incline with an electrical stimulation (2 mA) over 3 days before formal experiments. Each trial sustained for 20 min with the speed accelerated from 5 to 20 m/min. On the test day, the mice ran on the treadmill with an acceleration of 2 rpm/min. The maximal speed, time and distance to exhaustion were recorded when the mice were unable to return to the treadmill and stayed on the electrode for more than 10 s.

| Immunofluorescence staining
Immunofluorescence staining was performed as previously described with slight modifications. 23 For tissues, the muscle tissue embedded in the O.C.T. were cut into 10 μm cryosections by the Leica CM3050S cryostat. The cryosections were air-dried for 15 min and washed three times in PBS before fixed with 4% PFA for another 20 min. Next, these sections were permeabilized with 0.4% Triton X-100 in PBS for 1 h, and again rinsed with PBS three times. Cells were fixed with 4% PFA for 20 min and rinsed with PBS twice, and permeabilized with 0.4% Triton X-100 (Sigma-Aldrich) for 1 h at room temperature (RT). After blocking for 1 h at RT, the sections or cells were incubated with primary antibodies overnight at 4 C. Subsequently, the samples were washed several times with PBS and incubated with fluorescently labelled secondary antibodies for 1 h at RT. The nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific), and washed three times in PBS and then mounted in VECTERSHIELD anti-fading mounting medium (Vector Laboratories). The image was acquired using a confocal laser scanning microscope (Leica TCS SP5 II). The antibodies used for immunofluorescence analysis are listed in Table S3.   Table S4. 2.11 | Activation of endogenous expression of SESN1 using CRISPR/dCas9 transcriptional activation system

| Lentivirus packaging
The CRISPR/dCas9-mediated gene activation system was performed as previously described. 18 Briefly, guide RNAs, designed to target the SESN1 locus at the transcription start site (TSS), and two non-targeting controls (NTCs) were constructed into lentiSAMv2 vector (Addgene). For the induction of endogenous expression of SESN1, FOXO3 À/À human myotube progenitor cells were co-transduced with 2.13 | Senescence-associated β-galactosidase staining Senescence-associated β-galactosidase (SA-β-gal) staining of hMyotube was conducted as previously described. 24 Briefly, cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde at RT for 5 min. Then, fixed cells were stained with fresh staining solution containing X-gal at 37 C overnight after washing twice with PBS. Fields of view were randomly selected in each well, and the percentages of SA-β-gal-positive cells was counted by ImageJ software.

| RNA isolation and real-time quantitative PCR
Total RNA was extracted using TRIzol Reagent (Life Technologies) according to the manufacturer's protocol. Then, the GoScript™ Reverse Transcription System (Promega) was used to reverse transcribe the cDNA. And samples were used for real-time quantitative PCR (RT-qPCR) assay with THUNDERBIRD SYBR qPCR Mix (Toyobo) on a CFX384 Real-Time PCR system (Bio-Rad), and statistical significances were assessed by an independent-sample t test. The primer pairs used in this study are listed in Table S4.

| Western blot analysis
Western blot analysis was performed as previously described. 25,26 Tissues or cells were lysed with SDS lysis buffer containing 2% SDS and 62.5 mM Tris-HCl (pH = 6.8) and incubated at 100 C for 10 min.
The protein concentration was quantified using a bicinchoninic acid (BCA) quantification kit. The protein lysates were subjected to SDS-PAGE and subsequently transferred to a PVDF (polyvinylidene fluoride) membrane (Millipore). After blocking in 5% skimmed milk powder (BBI Life Sciences) in 1Â TBST (Tris-buffered saline with 0.1% Tween 20), the membranes were incubated with the primary antibodies overnight at 4 C and with the HRP-conjugated secondary antibodies (ZSGB-BIO) for 1 h at RT, followed by visualization using the Chemi-Doc XRS system (Bio-Rad). The band intensity quantification was performed with ImageJ software. The antibodies used are listed in Table S3.

| Chromatin immunoprecipitation-qPCR
Chromatin immunoprecipitation (ChIP)-qPCR was performed following the previously published protocol with slight modification. 18 Briefly, hMyotubes were collected and washed in PBS, then cross-  Table S4. The antibodies used in this study are listed in Table S3.

| Luciferase reporter assay
Luciferase reporter assay was performed as previously described. 27 Partial SESN1 promoter was amplified by PCR and cloned into the  Table S4.

| Enzyme-linked immunosorbent assay
Protein levels of SESN1 in human serum and hMyotube-conditioned medium were measured by sandwich enzyme-linked immunosorbent assay (ELISA) as per the manufacturer's instructions (Fine Test). Protein levels of SESN1 in the hMyotube-conditioned medium were normalized to the number of nuclei of the hMyotubes. The absorbance of each well was scanned at 450 nm using Synergy H1 Hybrid Reader (Bio-Tek).

| Nuclei isolation and snRNA-seq on the 10Â Genomics platform
Isolation of nuclei was performed following a previously published protocol with some modifications. 28

| Bulk RNA-seq library construction and sequencing
RNA quality control, library construction and high-throughput sequencing were performed for each sample as previously described. 29  2.21 | Bulk RNA-seq data processing RNA-seq data were processed as previously described. 30 Briefly, to trim adapter sequence and remove low quality reads, the raw sequencing reads were first processed with Trim Galore (http://www.bioinformatics. babraham.ac.uk/projects/trim_galore/; version 0.6.6). Then, the cleaned reads were mapped against human reference hg19 downloaded from Ensembl 31 with HISAT2 (version 2.2.1), 32 and mapped reads counted with HTSeq (version 0.13.5). 33 Differentially expressed gene (DEG) analysis was conducted with DESeq2 (version 1.28.1) 34 in R, and DEGs were identified with the cutoff of jLog 2 FCj > 0.5 and adjusted p values < 0.05.

| Processing and quality control of snRNAseq data
Raw sequencing reads of mouse skeletal muscle were aligned to the pre-mRNA reference (Ensembl, mm10) and counted using Cell Ranger (version 4.0.0) with the default parameters. The raw count matrices were filtered using CellBender (version 0.2.0) software in order to eliminate the contamination of background mRNA. 35 Seurat (version 3.2.2) object of each sample was constructed from the decontaminated matrix and nuclei with genes fewer than 200 or mitochondrial ratio more than 5% were discarded. 36 Doublet removal was performed with DoubletFinder (version 2.0.3). 37 Afterwards, the clusters lacking specific marker genes and with relatively low gene content were also discarded. Marker genes for each cell type are shown in Table S2.  Table S1.

| Gene ontology enrichment analysis
Gene ontology (GO) enrichment analysis was performed with Metascape. 38 Representative terms were selected with the cutoff of p values < 0.01 and visualized with ggplot2 R package (version 3.3.2). 39

| Gene set score analysis
Regeneration-related gene set was acquired from Regeneration Roadmap, 40

| Analysis of FOXO3 target genes
A transcription factor regulatory network of ageing-associated DEGs in cynomolgus monkey skeletal muscle was established under SCENIC (Single-Cell Regulatory Network Inference and Clustering) standard workflow following previous work. 20,41 At first, the candidate target genes co-expressed with FOXO3 were identified using Genie3. The weight scores calculated by Genie3 represent the co-expression levels between FOXO3 and its candidate target genes. Next, each coexpression module was analysed using RcisTarget 41 to identify putative direct-binding target genes of FOXO3. Only target genes with highconfident motif enrichment were selected for the downstream analysis.

| Statistical analyses
All data were statistically analysed using the two-tailed t test or Wilcoxon Rank Sum test to compare differences between different groups, assuming equal variance with PRISM software (GraphPad 8 Software). p Values are presented in indicated figures.

| SESN1 acts as a major downstream gene of geroprotector FOXO3 in primate skeletal muscle
In a separate study, we recently established a single-nucleus transcriptome atlas of primate skeletal muscle ageing 20 ( Figure 1A). Our data indicate a higher susceptibility of myofibres to ageing, as evidenced by higher transcriptional noise, and more DEGs in aged myofibre cells than those in non-myofibre cells ( Figure 1B,C). Remarkably, downregulation of FOXO3, a well-known longevity gene, 20,42,43 was identified in aged myofibres and contributed to primate skeletal muscle dyshomeostasis and degeneration ( Figures 1D-F and S1A).
Next, we sought to investigate the molecular mechanism by which FOXO3 inactivation causes progressive muscle degeneration.
Through motif analysis of DEGs shared between aged muscle (Old vs. Young) and FOXO3-depleted human myotubes (hMyotubes) (FOXO3 À/À vs. FOXO3 +/+ ), we discovered two genes, SESN1 and SH3BGR, with potential FOXO3 binding sites 2 kb upstream of their transcription start sites (TSSs) ( Figure 1G). Particularly, SESN1 has a higher Genie3 weight ( Figure 1G), along with its higher relevance with FOXO3 via co-expression analysis, indicating its higher potential to be a target gene of FOXO3 ( Figure 1H). To evaluate whether FOXO3 is capable of binding to the predicted site, we performed ChIP-qPCR with an anti-FOXO3 antibody. Using FOXO3 À/À hMyotubes as a negative control, we observed specific binding between FOXO3 and the SESN1 promoter in hMyotubes ( Figure 1I). Subsequently, to query whether SESN1 is directly activated by FOXO3, we cloned the promoter of SESN1 upstream of the luciferase reporter and found that the promoter of SESN1 was indeed transcriptionally activated by FOXO3 ( Figure 1J). By contrast, we observed blunted SESN1 promoter activity upon FOXO3 depletion as assessed by luciferase reporter assay ( Figure 1J), and similarly, that a single nucleotide change (ACA to CCA) within the SESN1 promoter reduced FOXO3 binding and substantially . Right, the diameters of the hMyotubes were quantified as fold changes (FOXO3 À/À vs. FOXO3 +/+ ) and are presented as mean ± SEMs. n = 3 biological replicates. (F) Left, representative SA-β-gal staining images of FOXO3 +/+ and FOXO3 À/À hMyotubes. Scale bars, 100 and 50 μm (zoomed-in image). Right, the percentages of SA-β-gal-positive hMyotubes were quantified as fold changes (FOXO3 À/À vs. FOXO3 +/+ ) and are presented as mean ± SEMs. n = 3 biological replicates. (G) SESN1 and SH3BGR were identified through analysis of overlapping genes between FOXO3 target genes and genes shared by aging associated DEGs in monkey myofibre and DEGs (FOXO3 À/À vs. FOXO3 +/+ ) in hMyotubes. Table showing   repressed luciferase reporter activity ( Figure 1J). Consistently, SESN1 expression levels were markedly downregulated in FOXO3 À/À hMyotubes relative to wildtype (WT) ones ( Figure 1K,L). In contrast, we noticed elevated SESN1 expression levels of hMyotubes in which endogenous FOXO3 was activated, and in which a constitutively active version of FOXO3 was generated via gene editing-based alanine substitution on two of the three classical phosphorylation sites 42 ( Figure 1M).
Collectively, these observations support a role for FOXO3 in positively regulating SESN1 transcription.

| SESN1 mediates the geroprotective effects of FOXO3 on primate skeletal muscle
In line with the downregulation of FOXO3 in aged primate skeletal muscles, we also found a consistent downregulation of SESN1 mRNA and protein expression levels in almost all aged cynomolgus monkey myofibre cells and in skeletal muscles as an entity (Figure 2A-C).
More strikingly, we observed an age-dependent decline of SESN1 protein in human skeletal muscle, along with decreased expression in prolonged-cultured senescent hMyotubes ( Figure 2D,E). Altogether, these findings demonstrate that inactivation of the FOXO3-SESN1 axis is a pronounced molecular characteristic of primate skeletal muscle ageing.
We next asked whether SESN1 mediates the geroprotective effect of FOXO3 in primate skeletal muscle. Indeed, we found that  (Table S1), we found that shared upregulated genes were associated with catabolic proteolysis and oxidative stress, whereas shared downregulated genes were associated with muscle functions (i.e., muscle structure development) ( Figure 2J).
Given these overlaps, we wondered whether the ageing phenotypes in FOXO3-deficient hMyotubes could be rescued by overexpression of SESN1. To this end, we induced endogenous SESN1 expression using the CRISPR-dCas9 transcriptional activation system ( Figure 3A,B). As expected, we observed that ageing features in the  Table S1). Taken together, these data suggest that SESN1, as a major downstream effector of FOXO3, plays a pivotal role in safeguarding skeletal muscle against ageing.
3.3 | SESN1 serves as a protective secretory factor against human myotube senescence Intriguingly, SESN1 was recently recognized to be a secreted protein. 44 Given our data showing that its expression was downregulated in skeletal muscle (the largest organ in the human body) with age, we next asked whether its blood level in human individuals decreases with age. As expected, we found that SESN1 levels were significantly lower in serum of older human individuals than in younger individuals ( Figure 3E). Consistently, SESN1 levels were also reduced in conditioned medium of prolonged-cultured senescent hMyotubes ( Figure 3F).
Given these findings, we asked if exogenous supplementation of recombinant SESN1 protein could restore ageingassociated compromised skeletal muscle function. Impressively, in FOXO3 À/À hMyotubes, we found that treatment with recombinant SESN1 protein at a concentration of 0.5 μg/mL rescued the senescent phenotypes, as evidenced by increased myotube diameter and reduced SA-β-gal activity ( Figure 3G-I). These results indicate that SESN1 serves as a secreted factor that protects against skeletal muscle ageing, suggesting directions for exploring SESN1-based intervention strategies for delaying skeletal muscle ageing.

| Recombinant SESN1 protein boosts muscle regeneration in vivo
Since senescent cells accumulate with age and hamper muscle regeneration, declined regeneration capability associated with age-accumulated damage is a hallmark feature of the elderly. 45,46 Thus, we next asked whether recombinant SESN1 protein treatment harbours a beneficial effect on skeletal muscle regeneration in vivo. To this end, we treated aged mice (16 months old) with CTX to obtain a classic muscle injury and regeneration model, 47,48 and subsequently administrated daily recombinant SESN1 protein for 7 days (Figures 4A and S2A,B). Indeed, our data showed that administration of recombinant SESN1 protein, relative to Vehicle-treated control, improved grip strength and physical endurance, as reflected by prolonged grid-hanging time, as well as increased maximal running time and running distance ( Figure 4B-D), indicating that exogenous SESN1 protein supplementation substantially enhances athletic ability after muscle injury in mice.
As quiescent muscle stem cells (MuSCs) become activated and proliferate to generate myofibres in response to physiological or pathological stimuli (e.g., toxin drug-induced injury), 17 we next examined the changes of MuSCs and found an elevated proportion of Pax7-positive cells and Ki67-positive mitotic cells after SESN1 protein treatment ( Figure 4E). Furthermore, a shift towards larger embryonic MHC (eMHC)-positive fibres, a classic trait for skeletal muscle regeneration, 17,49 was also observed in the SESN1-supplemented muscle ( Figure S2C). Most importantly, when we measured the crosssectional area of skeletal muscle fibres, we noticed a marked increment in the diameter of myofibres in SESN1-treated mice ( Figure 4F,G). Overall, these findings suggest recombinant SESN1 protein treatment in mice effectively enhance generation ability after muscle injury.   Figure 5D-F). 50 Importantly, the scores of regeneration-related genes were higher in SESN1-treated skeletal muscle and particularly in Fast IIB myofibre and FAP relative to Vehicle-treated counterparts ( Figure 5G).
Taken together, these results suggest that recombinant SESN1 protein has therapeutic potential for promoting muscle regeneration and delaying muscle ageing.

| DISCUSSION
Age-related decrease in skeletal muscle mass and strength is a hallmark of sarcopenia. 51 As such, this decline impairs physical performance and increases metabolic disease risk in the elders. Here, we identified the FOXO3-SESN1 axis as a gatekeeper mechanism protecting against primate skeletal muscle ageing. Notably, SESN1 levels were reduced in the serum of elderly human individuals, implying that SESN1 could function as a potent circulating biomarker to predict progressive skeletal muscle atrophy. More importantly, we propose that recombinant SESN1 protein may have potential as a therapeutic agent capable of attenuating myofibre senescence and promoting muscle regeneration ( Figure 5H). These novel findings identify SESN1 as a robust mediator antagonizing myofibre senescence, paving the way for development of novel diagnostics and intervention therapies for human skeletal muscle ageing.
In this study, we identified SESN1 as a major effector downstream of FOXO3 that exerts a potent protective role against skeletal muscle ageing. In broadly related work, FOXO1, another member of the FOXO family, was reported to directly regulate expression levels of the SESN1 homologue SESN3 in human tumour cells, 52 supporting our observations and suggesting a conserved mechanism by which FOXO proteins transcriptionally regulate members of the Sestrin family.
To be noted, we detected diminished secretion of SESN1 in the senescent human myotube model and lower SESN1 protein level in serum from human elderly. Interestingly, a recent study reported decreased SESN1 protein levels in serum of the elderly with sarcopenia relative to the elderly not afflicted with sarcopenia. 44 These clinical observations, together with our data, suggest that reduced secretion of myokine SESN1 may represent a potential predictive biomarker of muscle ageing, and an advance warning sign of sarcopenia. Most strikingly, we show that exogenous supplementation of recombinant SESN1 protein alleviated human myotube senescence in vitro and boosted muscle repair and regeneration in vivo. 17 Although resistance exercise and nutritional supplementation are known to augment muscle mass accretion and improve muscle strength, these intervention strategies are less realistic in the elderly population, most of whom have massive impairments in physical performance and anorexia. 1,53,54 On the other hand, beneficial effects of hormone replacement therapy on muscle atrophy, such as pharmacological interventions with testosterone and growth hormone in patients with low-baseline levels, remain controversial 1,3 and are associated with cardiovascular risks and other safety issues, 3 presenting significant challenges for clinical translation. Therefore, there are no effective and safe targeted interventions to mitigate or reverse age-related muscle loss. Given the high unmet need, the data showing that exogenous supplementation of myokine SESN1 alleviating ageingrelated muscle degeneration alongside promoting the muscle regeneration and repair in preclinical models are encouraging. Since human recombinant protein can be produced in large quantities with high quality, this study paves the way for exploring a myokine-based therapeutic strategy with more attractive safety and convenience features.
In conclusion, based on our previously established single-nucleus transcriptomic landscape of primate skeletal muscle ageing, 20 we explored the potential of SESN1 as a novel biomarker of human skeletal muscle ageing. In functional models, we also demonstrate that treatment with recombinant SESN1 protein inhibits myofibre ageing and promotes its regeneration and repair. Our approach supports further explorations of additional diagnostic biomarkers of skeletal muscle ageing and development of novel therapeutic interventions to treat ageing-associated muscle diseases.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.