Journal: bioRxiv

Article Title: Transient hypoxia followed by progressive reoxygenation is required for efficient skeletal muscle repair through Rev-ERBα modulation

doi: 10.1101/2024.05.02.592180

Figure Lengend Snippet: A. Experimental design. Acute muscle injury was induced by cardiotoxin (CTX) intramuscular (IM) injection in the tibialis anterior (TA) of 8-week-old wild-type mice. Mice were then housed in standard normoxic atmosphere (21%O 2 ) or in a normobaric hypoxic chamber (10%O 2 ) from 0 to 28 days post-injury (dpi). B. Representative images of LAMININ + myofiber (white) and nuclei (DAPI, blue) staining on cross-sections of CTX-injured TAs from mice exposed to normoxia or prolonged hypoxi, at 7, 14 and 28 dpi. Scale bar: 50μm. C. Quantification of myofiber diameters (μm) on cross-sections of CTX-injured TAs from mice exposed to normoxia or prolonged hypoxia, from 0 to 28 dpi. D. Quantification of CTX-injured TA cross-section areas of wild-type mice exposed to normoxia or prolonged hypoxia, at 14 and 28 dpi. E. Histograms showing muscle weights (g) of CTX-injured vs contralateral non-injured TAs from mice exposed normoxia or prolonged hypoxia, at 14 and 28 dpi. F. Quantification of the total number of myofibers in CTX-injured TAs from wild-type mice exposed to normoxia or prolonged hypoxia, at 14 and 28 dpi. G. Representative images of type-I (blue), type-IIA (red), type-IIB (green) myofiber and laminin (white) stainings performed on CTX-injured TA transversal muscle sections of wild-type mice exposed to normoxia or prolonged hypoxia, at 28 dpi. Scale bar 200μm. H. Quantification of myofiber type distribution (%) on CTX-injured TAs of wild-type mice exposed to normoxia or prolonged hypoxia, at 28 dpi. I. Quantification of the number of centronuclei per myofiber in CTX-injured TAs of wild-type mice exposed to normoxia (n = 6-7) or prolonged hypoxia (n = 6-8), at 14 and 28 dpi. Statistics : Results are expressed as means ± SEM. For C, D, E, F, H and I. Unpaired t-test. n=5-8 per time point.

Article Snippet: Then, mice have been placed in a normoxic environment at 21%O 2 or in a normobaric hypoxic chamber (Biospherix) at 10%O 2 inhaled to induce a systemic hypoxia during 5, 7, 10, 14 or 28 days post-CTX injury.

Techniques: Injection, Staining

Journal: bioRxiv

Article Title: Transient hypoxia followed by progressive reoxygenation is required for efficient skeletal muscle repair through Rev-ERBα modulation

doi: 10.1101/2024.05.02.592180

Figure Lengend Snippet: A. Experimental design. Acute muscle injury was induced by cardiotoxin (CTX) intramuscular (IM) injection in the tibialis anterior (TA) of 8-week-old wild-type mice. Intraperitoneal (IP) injection of pimonidazole hypoxic probe were performed at 0 and 5 days post-injury (dpi). B. Representative co-immunofluorescence staining of pimonidazole hypoxic probe (red), PAX7 + cells (green) and nuclei (DAPI, blue) on CTX-injured TA cross-sections. White arrows show PAX7 + cells. Scale bar: 10 μm. C. Experimental design. Acute muscle injury was induced by CTX injection in the TA of 8-week-old wild-type mice. Mice were then housed in standard normoxic atmosphere (21%O 2 ) or in a normobaric hypoxic chamber (10%O 2 ), from 0 to 28 days post-injury (dpi). D. Representative pictures of PAX7 + cells (red), LAMININ + myofibers (white) and nuclei (DAPI, blue) on CTX-injured TA cross-sections of wild-type mice exposed to normoxia or prolonged hypoxia, at 7, 14 and 28 dpi. Scale bars: 50 μm (overviews) and 12.5 μm (insets). E. Quantification of PAX7 + cells/mm on CTX-injured TA cross-sections of wild-type mice exposed to normoxia or prolonged hypoxia. F. Representative co-immunofluorescence staining of PAX7 (red), MYOD (red), KI67 (yellow) and nuclei (DAPI, blue) on CTX-injured TA cross-sections of wild-type mice exposed to normoxia or prolonged hypoxia, at 7 dpi. Scale bar: 50 μm. G. Percentage of quiescent (PAX7 + /MYOD - /KI67 - ), activated (PAX7 + /MYOD + /KI67 - ), proliferating (PAX7 + /MYOD + /KI67 + ) and differentiated (PAX7 - /MYOD + /KI67 - ) cell populations on CTX-injured TA cross-sections of wild-type mice exposed to normoxia or prolonged hypoxia, at 7 dpi. H. Experimental design. Single myofibers were isolated from Extensor Digitorum Longus (EDL) muscles of 8-week-old wild-type mice and cultured ex vivo under physioxia (8%O 2 ), prolonged hypoxia (1%O 2 ) or transient hypoxia flowed by progressive reoxygenation (1-8%O 2 ) during 72h. I. Representative pictures of PAX7 (green), MYOD (red) and nuclei (DAPI, blue) staining on isolated myofibers after 72h of culture under 8%, 1% or 1-8%O 2 . Scale bar: 20 μm. J. Quantification of the number of cells and clusters per myofiber and the number of cells per cluster on isolated myofibers after 72h of culture under 8%, 1% or 1-8%O 2 . K. Quantification of the percentage of quiescent PAX7 + /MYOD - , activated PAX7 + /MYOD + and differentiated PAX7 - /MYOD + cells on isolated myofibers cultivated under 8%, 1% or 1-8%O 2 for 72h. At 8%O 2 , 187 cells were counted on 25 myofibers; at 1%O 2 , 164 cells were counted on 32 myofibers; at 1-8%O 2 , 581 cells were counted on 32 myofibers. Statistics : Results are expressed as means ± SEM. For E. Unpaired t-test. n=5-8 per time point per group. For G. Unpaired t-test. n=5 per atmosphere exposure. For J. and K. One-way ANOVA followed by Tukey’s post-test. n=4 per O 2 level.

Article Snippet: Then, mice have been placed in a normoxic environment at 21%O 2 or in a normobaric hypoxic chamber (Biospherix) at 10%O 2 inhaled to induce a systemic hypoxia during 5, 7, 10, 14 or 28 days post-CTX injury.

Techniques: Injection, Immunofluorescence, Staining, Isolation, Muscles, Cell Culture, Ex Vivo

Journal: bioRxiv

Article Title: Transient hypoxia followed by progressive reoxygenation is required for efficient skeletal muscle repair through Rev-ERBα modulation

doi: 10.1101/2024.05.02.592180

Figure Lengend Snippet: A. Experimental design. Acute muscle injury was induced by cardiotoxin (CTX) intramuscular (IM) injection in tibialis anterior (TA) muscles from 8-week-old HIF CTRL (Pax7 CreERT /+ ; HIF-1α fl/fl without tamoxifen) and HIF cKO (Pax7 CreERT /+ ; HIF-1α fl/fl with Cre recombinase induction by tamoxifen) mice. Mice were then housed in standard normoxic atmosphere (21%O ) or in a normobaric hypoxic chamber (10%O ), from 0 to 28 days post-injury (dpi). B. Representative pictures of LAMININ (white) staining on CTX-injured TA cross-sections from HIF CTRL and HIF cKO mice exposed to normoxia or prolonged hypoxia, at 28 dpi. Scale bar: 50μm. C. Quantification of the myofiber diameter (μm) and the number of myofiber per mm of CTX-injured TA cross-sections from HIF CTRL and HIF cKO mice exposed to normoxia or prolonged hypoxia, at 28 dpi. D. Experimental design. Single myofibers were isolated from Extensor Digitorum Longus (EDL) muscles of 8-week-old HIF CTRL or HIF cKO mice and cultured ex vivo under physioxia (8%O 2 ), prolonged hypoxia (1%O 2 ) or transient hypoxia followed by progressive reoxygenation (1-8%O 2 ) during 72h. E. Representative staining of PAX7 (green), MYOD (red) and nuclei (DAPI, blue) on isolated myofibers from HIF CTRL or HIF cKO after 72h of culture under 8%, 1%O 2 or 1-8%O 2 . Scale bar: 20 μm. F. Percentage of quiescent (PAX7 + /MYOD - ), activated (PAX7 + /MYOD + ) and differentiated (PAX7 - /MYOD + ) cell populations obtained on isolated myofibers from EDL of HIF CTRL or HIF cKO mice, after 72h of culture under 8%O 2 , 1%O 2 or 1-8%O 2 . For HIF CTRL, n=3 with a counting of 654 cells on 38 myofibers at 8%O 2 , 337 cells on 43 myofibers at 1%O 2 and 1076 cells on 49 myofibers at 1-8%O 2 . For HIF cKO, n=4 with a counting of 643 cells on 47 myofibers at 8%O 2 , 292 cells on 57 myofibers at 1%O 2 and 788 cells on 62 myofibers at 1-8%O 2 . G. Experience design. FACS-sorted MuSCs were isolated from HIF CTRL or HIF cKO mice and plated with appropriate densities to ensure same confluence after 72h of culture under physioxia (8%O 2 ), prolonged hypoxia (1%O 2 ) and transient hypoxia followed by progressive reoxygenation (1-8%O 2 ). H. Representative pictures of MHC + (Myosin Heavy Chain) myotubes and nuclei (DAPI, blue) staining after 72h of culture under 8%, 1% or 1-8% O 2 . Scale bar: 50 μm. I. Evaluation of fusion index (%) and number of nuclei per myotube after 72h of culture under 8%, 1% or 1-8% O 2 . Statistics : Results are expressed as means ± SEM. For C, F and I. 2-way ANOVA followed by Sidak’s post-tests. n=4 per mice group per atmosphere exposure (C), n=3-4 per mice group per O 2 level (F) and n=5-6 per mice group per per O 2 level (I).

Article Snippet: Then, mice have been placed in a normoxic environment at 21%O 2 or in a normobaric hypoxic chamber (Biospherix) at 10%O 2 inhaled to induce a systemic hypoxia during 5, 7, 10, 14 or 28 days post-CTX injury.

Techniques: Injection, Muscles, Staining, Isolation, Cell Culture, Ex Vivo

Journal: bioRxiv

Article Title: Transient hypoxia followed by progressive reoxygenation is required for efficient skeletal muscle repair through Rev-ERBα modulation

doi: 10.1101/2024.05.02.592180

Figure Lengend Snippet: A. Experimental design. Acute muscle injury was induced by cardiotoxin (CTX) intramuscular (IM) injection in the tibialis anterior (TA) of 8-week-old Tg:Pax7nGFP mice. Mice were then housed in standard normoxic atmosphere (21%O 2 ) or in a normobaric hypoxic chamber (10%O 2 ), from 0 to 7 days post-injury (dpi). FACS-sorted PAX7+ (GFP+) cells were isolated from CTX-injured TAs at 7 dpi to perform a bulk RNAseq analysis. n=4 mice per atmosphere exposure. B. Differential Gene Expression (DEG) of PAX7+ cells isolated from injured TAs of mice exposed to hypoxic versus normoxic atmosphere. Volcano plot was obtained from p-values > 0.05. C. Violin plots representing myogenic quiescence ( Pax7 ), differentiation ( MyoD1, MyoG ) and fusion ( Mymk = myomaker) genes significatively modulated in PAX7+ cells exposed to hypoxia. D. Top 3 of signalling pathways obtained from KEGG enrichment analysis of all DEGs with FDR (False Discovery Rate) ≤ 0.05 and fold-change [0.5 ≤ ; ≥ 2]. E. Gene set enrichment analysis (GSEA) of circadian rhythm pathway and Heatmap showing the main 6 genes actors of circadian rhythm significatively (FDR ≤ 0.05 and fold-change [0.5 ≤ ; ≥ 2]) upregulated (red) or down-regulated (blue) in our RNAseq. F. Network representation of the protein-protein interactions of genes differentially regulated in PAX7+ cells exposed to hypoxia with FDR ≤ 0.05, obtained by using STRING.

Article Snippet: Then, mice have been placed in a normoxic environment at 21%O 2 or in a normobaric hypoxic chamber (Biospherix) at 10%O 2 inhaled to induce a systemic hypoxia during 5, 7, 10, 14 or 28 days post-CTX injury.

Techniques: Injection, Isolation, Expressing

Journal: bioRxiv

Article Title: Transient hypoxia followed by progressive reoxygenation is required for efficient skeletal muscle repair through Rev-ERBα modulation

doi: 10.1101/2024.05.02.592180

Figure Lengend Snippet: A. Experimental design. Acute muscle injury was induced by cardiotoxin (CTX) intramuscular (IM) injection in the tibialis anterior (TA) of 8-week-old Tg:Pax7nGFP mice. Mice were then housed in standard normoxic atmosphere (21%O 2 ) or in a normobaric hypoxic chamber (10%O 2 ), from 0 to 7 days post-injury (dpi). FACS-sorted PAX7+ (GFP+) cells were isolated from CTX-injured TAs at 7 dpi to perform a gene expression analysis. B. Histograms showing the relative expression of circadian clock effectors Nr1d1 ( nuclear receptor subfamily 1 group D member 1), Cry1 (cryptochrome circadian regulator 1) and Per2 (period circadian regulator 2) normalized to Tbp (TATA-box Binding Protein) on FACS-sorted PAX7+ (GFP+) cells isolated from CTX-injured TAs at 7 dpi. C. Experimental design. FACS-sorted PAX7+ (GFP+) cells were isolated from 8-week-old Tg:Pax7nGFP mice and plated with appropriate densities to ensure same confluence after 72h of culture under physioxia (8%O 2 ) or prolonged hypoxia (1%O 2 ), in the presence of the Rev-ERBα antagonist, SR8278 or its vehicle. D. Representative pictures of MHC + (Myosin Heavy Chain) myotubes and nuclei (DAPI, blue) staining after 72h of culture under 8% or 1%O 2 , with the Rev-ERBα antagonist SR8278 or its vehicle. Scale bar: 50 μm. E. Evaluation of fusion index (%) after 72h of culture under 8% or 1%O 2 , with the Rev-erbα antagonist SR8278 or its vehicle. F. Quantification of the number of small myotubes containing between 2 and 10 nuclei/mm , after 72h of myogenic myogenic cell culture under physioxia (8%O ) or hypoxia (1%O ), with the Rev-erbα antagonist SR8278 or its vehicle. G. Quantification of the number of large myotubes containing more than 10 nuclei/mm , after 72h of culture under 8% or 1%O , with the Rev-erbα antagonist SR8278 or its vehicle. H. Experimental design. FACS-sorted PAX7+ (GFP+) cells were isolated from 8-week-old Tg:Pax7nGFP mice and plated with appropriate densities to ensure same confluence after 72h of culture under physioxia (8%O 2 ) in the presence of the Rev-ERBα agonist, SR9009 or its vehicle. I. Representative pictures of MHC + (Myosin Heavy Chain) myotubes and nuclei (DAPI, blue) staining after 72h of culture under 8%O 2 , with the Rev-ERBα agonist SR9009 or its vehicle. Scale bar: 50 μm. J. Evaluation of fusion index (%) after 72h of culture under 8% with the Rev-erbα agonist SR9009 or its vehicle. F. Quantification of the number of small myotubes containing between 2 and 10 nuclei/mm , after 72h of myogenic myogenic cell culture under physioxia (8%O ) with the Rev-erbα agonist SR9009 or its vehicle. G. Quantification of the number of large myotubes containing more than 10 nuclei/mm , after 72h of culture under 8% with the Rev-erbα agonist SR9009 or its vehicle. Statistics : Results are expressed as means ± SEM. For E, F and G. 2-way ANOVA followed by Sidàk’s post-tests. n=4 per O 2 level and per condition. For J, K and L. Unpaired t-test. n=5 per condition.

Article Snippet: Then, mice have been placed in a normoxic environment at 21%O 2 or in a normobaric hypoxic chamber (Biospherix) at 10%O 2 inhaled to induce a systemic hypoxia during 5, 7, 10, 14 or 28 days post-CTX injury.

Techniques: Injection, Isolation, Expressing, Binding Assay, Staining, Cell Culture