1. Cell Biology
  2. Chromosomes and Gene Expression

LSD1 defines the fiber type-selective responsiveness to environmental stress in skeletal muscle

  1. Hirotaka Araki
  2. Shinjiro Hino  Is a corresponding author
  3. Kotaro Anan
  4. Kanji Kuribayashi
  5. Kan Etoh
  6. Daiki Seko
  7. Ryuta Takase
  8. Kensaku Kohrogi
  9. Yuko Hino
  10. Yusuke Ono
  11. Eiichi Araki
  12. Mitsuyoshi Nakao  Is a corresponding author
  1. Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Japan
  2. Department of Metabolic Medicine, Faculty of Life Sciences, Kumamoto University, Japan
  3. Department of Muscle Development and Regeneration, Institute of Molecular Embryology and Genetics, Kumamoto University, Japan
  4. Department of Molecular Bone Biology, Graduate School of Biomedical Sciences, Nagasaki University, Japan
https://doi.org/10.7554/eLife.84618
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Cite this article
  1. Hirotaka Araki
  2. Shinjiro Hino
  3. Kotaro Anan
  4. Kanji Kuribayashi
  5. Kan Etoh
  6. Daiki Seko
  7. Ryuta Takase
  8. Kensaku Kohrogi
  9. Yuko Hino
  10. Yusuke Ono
  11. Eiichi Araki
  12. Mitsuyoshi Nakao
(2023)
LSD1 defines the fiber type-selective responsiveness to environmental stress in skeletal muscle
eLife 12:e84618.
https://doi.org/10.7554/eLife.84618
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7 figures, 1 table and 6 additional files

Figures

Figure 1 with 7 supplements
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Muscle-specific lysine-specific demethylase-1 (LSD1)-knockout exacerbates dexamethasone (Dex)-induced fast muscle atrophy.

(A) Experimental design: LSD1 deletion was induced by tamoxifen injection for 5 days (days 0–4), and Dex was administered for 7 days (days 3–9) at 1 mg/kg. (B) Muscle weight normalized by the body …

Figure 1—figure supplement 1
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Generation of lysine-specific demethylase-1 (LSD1)-mKO mice.

(A) LSD1-mKO mice. Upon tamoxifen administration, LSD1 was depleted in differentiating and differentiated muscle cells, in which human ACTA1 promoter was activated. We performed the dexamethasone …

Figure 1—figure supplement 1—source data 1

Original blots for Figure 1—figure supplement 1C.

https://cdn.elifesciences.org/articles/84618/elife-84618-fig1-figsupp1-data1-v1.zip
Figure 1—figure supplement 2
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Effect of lysine-specific demethylase-1 (LSD1)-mKO at the postnatal developmental stage.

(A) Experimental design: postnatal day 11 male wild type (WT) or LSD1-mKO mice were injected with tamoxifen for 5 consecutive days. (B) The expression of differentiation markers of muscle fiber and …

Figure 1—figure supplement 3
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Effect of dexamethasone (Dex) dosage on atrophic phenotype.

(A) Experimental design: 8-week-old male wild type (WT) mice were injected with phosphate buffered saline (PBS) or Dex (1 or 5 mg/kg) for 7 days, consecutively. To arrange an experimental setting …

Figure 1—figure supplement 4
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Effects of dexamethasone (Dex) treatment in lysine-specific demethylase-1 (LSD1)-mKO mice.

(A) Changes in body weight during the Dex administration period (days 3–9) in wild type (WT) (n=40) and LSD1-mKO (n=44) mice. (B) Liver weight in WT (n=14) and LSD1-mKO (n=9) mice without Dex …

Figure 1—figure supplement 5
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Effects of lysine-specific demethylase-1 (LSD1)-mKO on muscle atrophy- and hypertrophy-associated signaling pathways.

(A) Western blot analysis of an autophagy protein LC3 in EDL muscles from dexamethasone (Dex)-treated wild type (WT) and LSD1-mKO mice. Autophagic signaling was evaluated based on the LC3-II/LC3-I …

Figure 1—figure supplement 5—source data 1

Original blots for Figure 1—figure supplement 5.

https://cdn.elifesciences.org/articles/84618/elife-84618-fig1-figsupp5-data1-v1.zip
Figure 1—figure supplement 6
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Increase in the number of slow fibers in dexamethasone (Dex)-treated lysine-specific demethylase-1 (LSD1)-mKO mice.

(A) Single color images of type I and IIA fibers shown in Figure 1D. Scale bars, 300 μm. (B) (Upper panel) The cross-sectional areas and numbers of each fiber type were determined by analyzing the …

Figure 1—figure supplement 7
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Fiber-type composition in dexamethasone (Dex)-treated lysine-specific demethylase-1 (LSD1)-mKO mice.

(A) Staining of individual fiber types in soleus (Sol) muscles from wild type (WT) and LSD1-mKO mice after tamoxifen administration (without Dex treatment). Scale bars, 100 μm. Representative images …

Figure 2 with 3 supplements
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Transcriptome analysis reveals the upregulation of muscle atrophy and slow fiber genes in fast-dominant muscles in dexamethasone (Dex)-treated lysine-specific demethylase-1 (LSD1)-mKO mice.

(A) Comparison of the transcriptome data from LSD1-mKO and wild type (WT) gastrocnemius (Gas) muscles. The red and blue dots indicate significantly up- and downregulated genes, respectively, in …

Figure 2—source data 1

Lists of genes described in Figure 2A.

https://cdn.elifesciences.org/articles/84618/elife-84618-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
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Effects of lysine-specific demethylase-1 (LSD1)-mKO on the expression of atrophy-, hypertrophy-, and fiber type-associated genes.

(A, B) Gene expression profiles in the muscles from dexamethasone (Dex)-treated wild type (WT) (n=8) and LSD1-mKO (n=11) mice. The expression of hypertrophy-associated genes in the gastrocnemius …

Figure 2—figure supplement 2
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Transcriptome analysis of the soleus (Sol) muscle in lysine-specific demethylase-1 (LSD1)-mKO mice after dexamethasone (Dex) treatment.

(A) Comparison of the transcriptome data of the muscles from the Dex-treated WT (n=3) and LSD1-mKO (n=3) mice. The magenta and cyan dots represent the genes that were significantly upregulated and …

Figure 2—figure supplement 2—source data 1

Lists of genes described in Figure 2—figure supplement 2A.

https://cdn.elifesciences.org/articles/84618/elife-84618-fig2-figsupp2-data1-v1.xlsx
Figure 2—figure supplement 3
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The role of lysine-specific demethylase-1 (LSD1) in streptozotocin (STZ)-induced muscle atrophy.

(A) Experimental design: 8-week-old male wild type (WT) or LSD1-mKO mice were administered tamoxifen intraperitoneally to WT mice for 5 consecutive days and the STZ was intraperitoneally injected at …

Figure 3 with 2 supplements
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Lysine-specific demethylase-1 (LSD1) regulates the expression of atrophy-associated genes through the modulation of Foxk1.

(A) Chromatin immunoprecipitation (ChIP)-qPCR analyses of H3K4me3 at Sln, Gadd45a, and Ddit4 gene promoters in LSD1-mKO gastrocnemius (Gas) muscles after dexamethasone (Dex) treatment. The data are …

Figure 3—source data 1

Original blots for Figure 3E.

https://cdn.elifesciences.org/articles/84618/elife-84618-fig3-data1-v1.zip
Figure 3—figure supplement 1
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Lysine-specific demethylase-1 (LSD1) interacts with Foxk1 and increases the nuclear retention.

(A) Enrichment of LSD1 at the atrophy gene loci in C2C12 myoblasts. Chromatin immunoprecipitation (ChIP)-seq data reported by Tosic M et al. (Nat. Commun. 9, No. 366, 2018) was analyzed by …

Figure 3—figure supplement 1—source data 1

Original blots for Figure 3—figure supplement 1B and C.

https://cdn.elifesciences.org/articles/84618/elife-84618-fig3-figsupp1-data1-v1.zip
Figure 3—figure supplement 2
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Foxk1 cooperates with lysine-specific demethylase-1 (LSD1) to control the expression of atrophy-associated genes.

(A) Foxk1 protein expression in Foxk1-KO C2C12 cells. C2C12 cells were transfected with control or Foxk1-KO Double Nickase Plasmids and subjected to puromycin selection. (B) The expression of the …

Figure 3—figure supplement 2—source data 1

Original blots for Figure 3—figure supplement 2A and D.

https://cdn.elifesciences.org/articles/84618/elife-84618-fig3-figsupp2-data1-v1.zip
Figure 4 with 1 supplement
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Lysine-specific demethylase-1 (LSD1)-mKO augments the effects of voluntary wheel running (VWR) by enhancing muscle endurance and increasing the number of oxidative fibers.

(A) Experimental design: after familiarization with a cage equipped with a running wheel, LSD1 deletion was induced by tamoxifen injection (days 0–4). Mice were subjected to VWR in the same cage for …

Figure 4—figure supplement 1
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Lysine-specific demethylase-1 (LSD1)-mKO increased the number of oxidative fibers in slow dominant muscle after voluntary wheel running (VWR) training.

(A) LSD1 mRNA expression in soleus (Sol) muscles from wild type (WT) (n=6) and LSD1-mKO (n=6) mice after VWR training. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) values …

Figure 5 with 1 supplement
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Lysine-specific demethylase-1 (LSD1)-mKO augments the effects of voluntary wheel running (VWR) by increasing the expression of ERRγ and its target genes.

(A) RNA-seq analysis of wild type (WT) and LSD1-mKO soleus (Sol) muscles after voluntary wheel running (VWR) training. K-means clustering identified a group of genes that were upregulated in mKO …

Figure 5—source data 1

Original blots for Figure 5E.

https://cdn.elifesciences.org/articles/84618/elife-84618-fig5-data1-v1.zip
Figure 5—figure supplement 1
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Expression of Esrrg and its target genes remain unaffected in lysine-specific demethylase-1 (LSD1)-mKO gastrocnemius (Gas) and tibialis anterior (TA) muscles after voluntary wheel running (VWR).

(A) The expression of Esrrg and functionally related genes in sedentary wild type (WT) (n=4) and LSD1-mKO (n=4) soleus (Sol) muscles. (B) The expression of Esrrg and its target genes in the Gas and …

Figure 5—figure supplement 1—source data 1

Original blots for Figure 5—figure supplement 1D.

https://cdn.elifesciences.org/articles/84618/elife-84618-fig5-figsupp1-data1-v1.zip
Figure 6
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Lysine-specific demethylase-1 (LSD1) expression is decreased in the aged muscles in mice and humans.

(A) Muscle weight in young (2.5 months of age, n=4) and old (28 months of age, n=3) male C57BL/6J mice. (B) The expression of fiber type-specific genes and atrophy-associated genes in the …

Figure 6—source data 1

Original blots for Figure 6C.

https://cdn.elifesciences.org/articles/84618/elife-84618-fig6-data1-v1.zip
Figure 7
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Lysine-specific demethylase-1 (LSD1) serves as an ‘epigenetic barrier’ that defines stress sensitivities in the skeletal muscle.

LSD1 attenuates glucocorticoid (GC)-induced atrophy in the fast fiber-dominant muscles, in collaboration with Foxk1, an anti-autophagic transcription factor. On the other hand, LSD1 attenuates …

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)Lsd1-floxed mice (B6.129-Kdm1atm1.1Sho/J)Jackson LaboratoriesStrain #:023969
RRID:IMSR_JAX:023969
Strain, strain background (Mus musculus)Tg(ACTA1-cre/Esr1*)2Kesr/JJackson LaboratoriesStrain #:025750
RRID:IMSR_JAX:025750
Strain, strain background (Mus musculus)Lsd1flox/flox; TgACTA1-CreERT2/+ conditional knockout mice (LSD1-mKO mice)This paperN/ASee ‘Animal studies’ in Methods
Strain, strain background (Mus musculus)C57BL6/J miceCharles River LaboratoriesN/A
Cell line (Mus musculus)C2C12RIKENRCB0987
Cell line (Ruttus norvegicus)H9c2ATCCCRL-1446
AntibodyRabbit polyclonal anti-LSD1abcamab17721WB (1:500), ChIP (5 μg), Co-IP (3 μg)
AntibodyRabbit polyclonal anti-Foxk1abcamab18196WB (1:1000), ChIP (5 μg), Co-IP (3 μg)
AntibodyRabbit monoclonal anti-ERR gammaabcamab128930WB (1:1000)
AntibodyMouse monoclonal anti-MHC type IDHSBBA-F8IHC (1:50)
AntibodyMouse monoclonal anti-MHC type IIADHSBSC-71IHC (1:50)
AntibodyRabbit polyclonal anti-LamininSigma-AldrichL9393IHC (1:500)
AntibodyRabbit polyclonal anti-AktCell Signaling Technology#9272WB (1:1000)
AntibodyRabbit polyclonal anti-Phospho-AktCell Signaling Technology#9271WB (1:1000)
AntibodyRabbit polyclonal anti-LC3MBLPM036WB (1:1000)
AntibodyMouse monoclonal anti- Phospho-4E-BP1 (Thr37/46)Cell Signaling Technology#2855WB (1:1000)
AntibodyRabbit polyclonal anti-4E-BP1Cell Signaling Technology9454WB (1:1000)
AntibodyMouse monoclonal anti- SQSTM1(p62)Santa Cruzsc-28359WB (1:1000)
AntibodyRabbit polyclonal anti-GAPDHSanta Cruzsc-25778WB (1:6000)
AntibodyRabbit polyclonal anti-trimethyl-Histone H3 (Lys4)Millipore07-473ChIP (3 μg)
AntibodyRabbit polyclonal anti-Histone H3abcamab1791WB (1:5000)
Recombinant DNA reagentControl Double Nickase PlasmidSanta Cruz Biotechnologysc-437281
Recombinant DNA reagentFOXK1 Double Nickase PlasmidSanta Cruz Biotechnologysc-437282-NIC
Recombinant DNA reagentpENTR4-H1RIKEN BRC DNA BANKRDB04395
Recombinant DNA reagentpCAG-HIVgpRIKEN BRC DNA BANKRDB04394
Recombinant DNA reagentpCMV-VSV-G-RSV-RevRIKEN BRC DNA BANKRDB04393
Recombinant DNA reagentCS-RfA-EVBsdRIKEN BRC DNA BANKRDB06090
Recombinant DNA reagentCS-RfA-EVBsd shFoxk1This paperN/ASee ‘Construction of lentiviral expression vectors and their transduction’ in Methods
Recombinant DNA reagentCS-RfA-EVBsd shGL3This paperN/ASee ‘Construction of lentiviral expression vectors and their transduction’ in Methods
Recombinant DNA reagentCSII-EF-3xFLAG-hLSD1-BsdThis paperN/AA lentiviral construct used for the forced expression of human LSD1
Sequence-based reagentPrimers for qRT-PCRThis paperN/ASee Supplementary file 3
Sequence-based reagentSee Table S2 for primers for ChIP-qPCRThis paperN/ASee Supplementary file 4
Commercial assay or kitNEBNext Ultra DNA Library Prep KitNew England BiolabsE7370S
Commercial assay or kitNEBNext Poly(A) mRNA Magnetic Isolation ModuleNew England BiolabsE7490S
Chemical compound, drugTamoxifenSigma-AldrichT5648
Chemical compound, drugDexamethasoneSigma-AldrichD4902
Chemical compound, drugInsulinnacalai tesque19251-24
Chemical compound, drugStreptozotocinWako197-15153
Chemical compound, drugT-3775440 hydrochlorideMedChemExpressHY-103085
Chemical compound, drugFuGENE 6 Transfection ReagentPromegaE2691
Chemical compound, drugPuromycin dihydrochlorideSigma-AldrichP8833
Chemical compound, drugBlasticidin SFUJIFILM Wako Chemicals029-18701
Chemical compound, drugCan Get Signal immunostain Solution ATOYOBONKB-501
Chemical compound, drugmalinolMuto Pure Chemicals2009-1
Chemical compound, drugCan Get Signal Solution 1TOYOBONKB-201
Chemical compound, drugCan Get Signal Solution 2TOYOBONKB-301
Chemical compound, drugTRIzol RNA Isolation ReagentThermo Fisher Scientific15596026
Chemical compound, drugReverTra Ace qPCR RT Master MixTOYOBOFSQ-201
Chemical compound, drugTHUNDERBIRD SYBR qPCR MixTOYOBOQPS-201
Software, algorithmBZ-X Analyzer softwareKEYENCEN/A
Software, algorithmHomer v4.9.1Heinz et al., 2010http://homer.ucsd.edu/homer/
Software, algorithmSTARDobin et al., 2013https://github.com/alexdobin/STAR; Dobin, 2023
Software, algorithmEdge RRobinson et al., 2010http://bioconductor.org/packages/release/bioc/html/edgeR.html
Software, algorithmHeatmapperBabicki et al., 2016http://www.heatmapper.ca/expression/
Software, algorithmDEseq2Love et al., 2014https://bioconductor.org/packages/release/bioc/html/DESeq2.html
Software, algorithmComplexHeatmapGu et al., 2016https://github.com/jokergoo/ComplexHeatmap; Gu, 2022
Software, algorithmMolecular Signature Database v7.4Subramanian et al., 2005https://www.gsea-msigdb.org/gsea/msigdb/
Software, algorithmHOMER V4.9.1Heinz et al., 2010http://homer.ucsd.edu/homer/
Software, algorithmJMP (version 10.0.2d1)SAS Institute IncN/A
Software, algorithmGraphPad Prism version 8.4.3GraphPad SoftwareN/A
Software, algorithmIntegrative Genomics Viewer (IGV)James T et al., 2011https://software.broadinstitute.org/software/igv/

Additional files

Supplementary file 1

Top 50 differentially regulated genes in dexamethasone (Dex)-treated lysine-specific demethylase-1 (LSD1)-mKO mice.

https://cdn.elifesciences.org/articles/84618/elife-84618-supp1-v1.xlsx
Supplementary file 2

Cluster 6 genes in Figure 5A.

https://cdn.elifesciences.org/articles/84618/elife-84618-supp2-v1.xlsx
Supplementary file 3

Primers used in this study.

https://cdn.elifesciences.org/articles/84618/elife-84618-supp3-v1.xlsx
Supplementary file 4

Chromatin immunoprecipitation (ChIP) primers used in this study.

https://cdn.elifesciences.org/articles/84618/elife-84618-supp4-v1.xlsx
Supplementary file 5

Full uncropped blots.

https://cdn.elifesciences.org/articles/84618/elife-84618-supp5-v1.pdf
MDAR checklist
https://cdn.elifesciences.org/articles/84618/elife-84618-mdarchecklist1-v1.pdf

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