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. 2023 May;56(5):e13455.
doi: 10.1111/cpr.13455. Epub 2023 May 17.

SESN1 is a FOXO3 effector that counteracts human skeletal muscle ageing

Ying Jing  1   2   3   4 , Yuesheng Zuo  2   5   6 , Liang Sun  7   8   9 , Zheng-Rong Yu  10 , Shuai Ma  9   11   12   13 , Huifang Hu  11   12   13 , Qian Zhao  3   4 , Daoyuan Huang  3   4 , Weiqi Zhang  2   5   6   9   12   14   15 , Juan Carlos Izpisua Belmonte  16 , Yang Yu  17   18 , Jing Qu  1   2   9   12   13 , Guang-Hui Liu  2   3   9   11   12   13 , Si Wang  3   4   9   19
Affiliations

SESN1 is a FOXO3 effector that counteracts human skeletal muscle ageing

Ying Jing et al. Cell Prolif. 2023 May.

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIGURE 1
FIGURE 1
SESN1 is transactivated by FOXO3 in primate skeletal muscle. (A) Uniform manifold approximation and projection (UMAP) plot showing the 14 cell types of cynomolgus monkey skeletal muscle. Cells are annotated to the right. EC, endothelial cell; Fib/FAP, fibroblast/fibro‐adipogenic progenitor; Mac, macrophage; MuSC, muscle stem cell; PMF, postsynaptic muscle fibre; SMC, smooth muscle cell; Tendo, Tendon fibroblast; tSC, terminal Schwann cell. (B) Scatter plot showing the log2 ratio of transcriptional noise between old and young samples as calculated using sample averages and single cells on the X and Y axes (n = 16), respectively. (C) Pie charts showing the proportions of ageing‐related differentially expressed genes (DEGs) (adjusted p values < 0.05 and |LogFC| > 0.25) between old and young groups for each cell type. Left, upregulated DEGs; right, downregulated DEGs. (D) Violin plots showing FOXO3 mRNA expression levels across Fast IIA, Fast IIX and Slow I cell types in cynomolgus monkey skeletal muscles between old and young groups. (E) MyHC immunofluorescence staining in FOXO3 +/+ and FOXO3 −/− human myotubes (hMyotubes). Representative images are shown on the left. Scale bars, 50 and 25 μm (zoomed‐in image). Right, the diameters of the hMyotubes were quantified as fold changes (FOXO3 −/− vs. FOXO3 +/+ ) and are presented as mean ± SEMs. n = 3 biological replicates. (F) Left, representative SA‐β‐gal staining images of FOXO3 +/+ and FOXO3 −/− 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 −/− 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 −/− vs. FOXO3 +/+ ) in hMyotubes. Table showing the weight information of SESN1 and SH3BGR in FOXO3 regulated transcriptional network. (H) Scatter plot showing the relative expression levels of FOXO3 and SESN1. Every 20 nuclei of myofibre in young and old groups were consolidated as a bin. The gene expression levels of each bin were calculated as the average expression of nuclei inside the specific bin. Each point marked in grey or blue represents a cell bin of young or old group, respectively. (I) ChIP‐qPCR analysis of the occupancy of FOXO3 on the SESN1 promoter in myotubes using an anti‐FOXO3 antibody. The diagram above depicts the relative position of primers (arrows) used for ChIP‐qPCR. Data are presented as mean ± SEMs. n = 3 technical replicates for each group. (J) Dual luciferase reporter assay showing transcriptional activation of SESN1 by FOXO3. FOXO3 +/+ and FOXO3 −/− hMyotube progenitor cells were co‐transfected with plasmid expressing Renilla and vectors carrying wildtype (WT) or mutant (Mut) pGL3‐SESN1 promoter. (K) RT‐qPCR analysis showing the mRNA levels of SESN1 in FOXO3 +/+  and FOXO3−/− hMyotubes. Data are presented as mean ± SEMs. n = 3 biological replicates. (L) Western blot analysis showing the protein levels of SESN1 in FOXO3 +/+ and FOXO3 −/− hMyotubes. GAPDH was used as the loading control. Band intensities were quantified as fold changes (FOXO3 −/− vs. FOXO3 +/+ ) and is presented as mean ± SEMs. n = 3 independent experiments. (M) Western blot analysis showing the protein levels of SESN1 in FOXO3 +/+ and FOXO3 2SA/2SA hMyotubes. GAPDH was used as the loading control. Band intensities were quantified as fold changes (FOXO3 2SA/2SA vs. FOXO3 +/+ ) and is presented as mean ± SEMs. n = 3 independent experiments. ChIP, chromatin immunoprecipitation; RT‐qPCR, real‐time quantitative PCR.
FIGURE 2
FIGURE 2
SESN1 exerts a key protective role in counteracting primate skeletal muscle ageing. (A) Box plots showing SESN1 mRNA expression levels across Fast IIA, Fast IIX and Slow I cell types in monkey myofibres between old and young groups. (B) RT‐qPCR analysis showing the SESN1 mRNA expression changes in young and old monkey skeletal muscles. SESN1 mRNA levels were quantified as fold changes (old vs. young) and are presented as mean ± SEMs. n = 8 monkeys for each group. (C) Western blot analysis and band intensity quantification of SESN1 protein levels in young and old monkey skeletal muscle samples. Data were quantified as fold changes (old vs. young) and are presented as mean ± SEMs. n = 8 monkeys for each group. (D) Left, western blot analysis and band intensity quantification of SESN1 protein levels in human skeletal muscle samples. GAPDH was used as the loading control. Middle, bar plot showing the relative expression of SESN1 protein levels for each individual. Right, the negative correlation of relative SESN1 protein levels in skeletal muscle across different ages. The shadow indicates the 0.95 confidence interval around smooth. n = 7 donors. (E) Western blot analysis showing the protein levels of SESN1 in WT hMyotubes after a prolonged‐culture for 6, 10 and 12 days. GAPDH was used as the loading control. Band intensities were quantified as fold changes in hMyotubes at Day 10 or Day 12 versus at Day 6 and is presented as mean ± SEMs. n = 3 independent experiments. (F) Schematic showing the method used to generate SESN1‐knockdown hMyotubes. (G) Western blot analysis showing the protein levels of SESN1 in hMyotubes transfected with si‐NC or si‐SESN1. GAPDH was used as the loading control. Band intensities were quantified as fold changes (si‐SESN1 vs. si‐NC) and is presented as mean ± SEMs. n = 3 independent experiments. (H) MyHC immunofluorescence staining of the hMyotubes transfected with si‐NC or si‐SESN1. Representative images are shown on the left. Scale bars, 50 and 25 μm (zoomed‐in image). Right, the diameters of the hMyotubes were quantified as fold changes (si‐SESN1 vs. si‐NC) and are presented as mean ± SEMs. n = 3 biological replicates. (I) Representative images of SA‐β‐gal‐positive hMyotubes transfected with si‐NC or si‐SESN1. Representative images are shown on the left. Scale bars, 100 and 50 μm (zoomed‐in image). Data were quantified as fold changes (si‐SESN1 vs. si‐NC) and are presented as mean ± SEMs on the right. n = 3 biological replicates. (J) GO terms shared by DEGs between old and young monkey skeletal muscle (mkMuscle) and DEGs between si‐SESN1 and si‐NC hMyotubes. DEG, differentially expressed gene; GO, gene ontology; RT‐qPCR, real‐time quantitative PCR; WT, wildtype.
FIGURE 3
FIGURE 3
Activation of SESN1 gene expression and supplementation of recombinant SESN1 protein attenuate human myotube senescence. (A) Schematic showing the method used to activate the endogenous SESN1 in FOXO3 −/− hMyotubes by CRISPR/dCas9‐mediated transcriptional activation system. (B) Western blot analysis showing the protein levels of SESN1 in hMyotubes upon the CRISPR/dCas9‐mediated activation of SESN1 gene expression. GAPDH was used as the loading control. Band intensities were quantified as fold changes (CRISPRa‐sgSESN1 vs. CRISPRa‐sgNTC) and is presented as mean ± SEMs. n = 3 independent experiments. (C) MyHC immunofluorescence staining of the hMyotubes upon the CRISPR/dCas9‐mediated activation of SESN1 gene expression. Representative images are shown on the left. Scale bars, 50 and 25 μm (zoomed‐in image). Right, the diameters of the hMyotubes were quantified as fold changes (CRISPRa‐sgSESN1 vs. CRISPRa‐sgNTC) and are presented as mean ± SEMs. n = 3 biological replicates. (D) SA‐β‐gal‐positive cells of the hMyotubes upon the CRISPR/dCas9‐mediated activation of SESN1 gene expression. Representative images are shown on the left. Scale bars, 100 and 50 μm (zoomed‐in image). Data were quantified as fold changes (CRISPRa‐sgSESN1 vs. CRISPRa‐sgNTC) and are presented as mean ± SEMs on the right. n = 3 biological replicates. (E) ELISA analysis showing the SESN1 content in the sera from young and old individuals. Data are presented as mean ± SEMs. n = 40 donors per group. (F) ELISA analysis showing the SESN1 content in conditioned medium of WT hMyotubes after a prolonged‐culture for 6, 10 and 12 days. Data are presented as mean ± SEMs. n = 3 biological replicates. (G) Schematic showing the method of recombinant SESN1 (rSESN1) protein treatment in FOXO3 −/− hMyotubes. (H) MyHC immunofluorescence staining of the FOXO3 −/− hMyotubes treated with Vehicle or rSESN1 protein. Representative images are shown on the left. Scale bars, 50 and 25 μm (zoomed‐in image). Right, the diameters of the hMyotubes were quantified as fold changes (rSESN1 vs. Vehicle) and are presented as mean ± SEMs. n = 3 biological replicates. (I) Representative images of SA‐β‐gal‐positive cells of FOXO3 −/− hMyotubes treated with Vehicle or rSESN1 protein. Representative images are shown on the left. Scale bars, 100 and 50 μm (zoomed‐in image). Data were quantified as fold changes (rSESN1 vs. Vehicle) and are presented as mean ± SEMs on the right. n = 3 biological replicates. ELISA, enzyme‐linked immunosorbent assay; WT, wildtype.
FIGURE 4
FIGURE 4
Recombinant SESN1 protein facilitates skeletal muscle regeneration in vivo. (A) Schematic diagram showing the experimental designs and time course of the Sham, cardiotoxin (CTX)‐induced mouse skeletal muscle injury control (post‐injury‐Vehicle) or recombinant SESN1 protein treatment (post‐injury‐rSESN1). (B) Grip test strength evaluation in mice of Sham, post‐injury‐Vehicle and post‐injury‐SESN1 groups at Day 9. Data were quantified and are presented as mean ± SEMs on the right. n = 6 mice for each group. (C) Grid‐hanging capacity evaluation in Sham, post‐injury‐Vehicle and post‐injury‐rSESN1 groups at Day 9. Data were quantified and are presented as mean ± SEMs on the right. n = 6 mice for each group. (D) Distance and time to exhaustion and maximal speed analyses in mice of Sham, post‐injury‐Vehicle and post‐injury‐rSESN1 groups at Day 9. Data were quantified and are presented as mean ± SEMs. n = 6 mice for each group. (E) Top, the representative immunofluorescence staining images of Pax7 and Ki67 in skeletal muscles of Sham, post‐injury‐Vehicle and post‐injury‐rSESN1 groups at Day 10. Scale bars, 25 μm. Bottom, the relative Pax7‐ and Ki67‐positive cells were quantified as fold changes and are presented as mean ± SEMs. n = 6 mice for each group. (F) Muscle fibre cross‐sectional area in mice of Sham, post‐injury‐Vehicle and post‐injury‐rSESN1 groups at Day 10. Top, representative immunofluorescence staining images of cross‐sectional area of fibres, which were labelled by Laminin antibody in green. Scale bars, 100 μm and 50 μm (zoomed‐in image). Bottom, the cross‐sectional area of fibres was quantified and is presented as mean ± SEMs. n = 6 mice for each group. (G) Top, frequency distribution of muscle fibre cross‐section area in Sham, post‐injury‐Vehicle and post‐injury‐rSESN1 groups at Day 10. Bottom, the calculated lower quartile (Q1), the middle quartile (Q2; median of the data), the upper quartile (Q3) and the maximum value in the range (Q4) of the fibre size distribution. n = 6 mice for each group.
FIGURE 5
FIGURE 5
Single‐nucleus transcriptomic analysis of skeletal muscle from mice treated with recombinant SESN1 protein. (A) UMAP plot showing mouse skeletal muscle cell types by single‐nucleus RNA‐sequencing. Cells are coloured by types and annotated to the right. (B) UMAP plot showing the cell distribution in Sham, post‐injury‐Vehicle and post‐injury‐rSESN1 groups. (C) Dot plot showing the expression of representative marker genes for each cell type in mouse skeletal muscle. (D) Venn diagram showing the number and percentage of upregulated (top) and downregulated (bottom) DEGs induced by CTX treatment and rescued by rSESN1 protein administration. Rescued DEGs exhibiting the opposite changes upon CTX‐induced injury and rSESN1 protein treatment. (E) Heatmaps showing the distribution of DEGs across different cell types between post‐injury‐Vehicle and Sham groups, and between post‐injury‐rSESN1 and post‐injury‐Vehicle groups, respectively. Each row represents one gene, and each column represents one cell type. (F) GO term and pathway enrichment analysis for upregulated rescued DEGs. The colour key from white to red indicates ‐Log10 (p value) from low to high. (G) Left, ridge map showing the global distribution density of regeneration‐related gene set score for DEGs in post‐injury‐Vehicle and post‐injury‐rSESN1 groups treated mouse skeletal muscle. The corresponding dashed line represents the peak position of each group. Right, box plot showing the regeneration‐related gene set score across Fast IIB, PMF and FAP types of mouse muscle in post‐injury‐Vehicle and post‐injury‐rSESN1 groups. (H) A working model for FOXO3‐SESN1 axis in the homeostatic regulation during skeletal muscle aging. CTX, cardiotoxin; DEG, differentially expressed gene; GO, gene ontology; UMAP, uniform manifold approximation and projection.
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