BMAL1 drives muscle repair through control of hypoxic NAD+ regeneration in satellite cells

Abstract

The process of tissue regeneration occurs in a developmentally timed manner, yet the role of circadian timing is not understood. Here, we identify a role for the adult muscle stem cell (MuSC)-autonomous clock in the control of muscle regeneration following acute ischemic injury. We observed greater muscle repair capacity following injury during the active/wake period as compared with the inactive/rest period in mice, and loss of Bmal1 within MuSCs leads to impaired muscle regeneration. We demonstrate that Bmal1 loss in MuSCs leads to reduced activated MuSC number at day 3 postinjury, indicating a failure to properly expand the myogenic precursor pool. In cultured primary myoblasts, we observed that loss of Bmal1 impairs cell proliferation in hypoxia (a condition that occurs in the first 1-3 d following tissue injury in vivo), as well as subsequent myofiber differentiation. Loss of Bmal1 in both cultured myoblasts and in vivo activated MuSCs leads to reduced glycolysis and premature activation of prodifferentiation gene transcription and epigenetic remodeling. Finally, hypoxic cell proliferation and myofiber formation in Bmal1-deficient myoblasts are restored by increasing cytosolic NAD⁺ Together, we identify the MuSC clock as a pivotal regulator of oxygen-dependent myoblast cell fate and muscle repair through the control of the NAD⁺-driven response to injury.

Keywords: NAD+; circadian rhythm; hypoxia; muscle regeneration; muscle stem cell.

Figure 1.

Muscle regeneration following injury depends on time of day and the muscle stem cell-autonomous circadian clock. (A) Experimental design: WT mice were injured with cardiotoxin (CTX) injection into TA muscles at ZT4 or ZT16. Muscles were harvested at the opposite time point at 6.5 d postinjury (dpi). n = 5 mice per group. (B) Representative H&E- and laminin-stained sections of regenerating TA muscles at the site of injury. Scale bar, 200 µm. (C,D) Muscle fiber size (cross-sectional area [CSA]) distribution (C) and quantification of the mean CSA (D) of nascent myofibers of regenerating TA muscles from WT mice injured at ZT4 and ZT16. (**) P < 0.01 by two-sided unpaired Student's t-test. (E) Experimental design: Adult control and Bmal1^musc mice received five consecutive daily intraperitoneal injections of 100 mg/kg tamoxifen in corn oil. After 7 d, mice were injected with CTX in TA muscles and examined for muscle regeneration at 7 and 14 dpi. n = 6 mice per group. (F) Representative H&E-stained sections of regenerating TA muscles from control and Bmal1^musc mice at 7 and 14 dpi. Scale bar, 200 µm. (G,H) Fiber size (CSA) distribution (G) and quantification of the CSA (H) of nascent myofibers of regenerating TA muscles from control and Bmal1^musc mice at 7 and 14 dpi. Data are represented as mean ± SEM. (***) P < 0.001 by two-sided unpaired Student's t-test.

Figure 2.

The myoblast circadian clock controls cell proliferation and differentiation in hypoxia. (A) Cell number quantification by hemocytometer of WT and Bmal1^–/– (KO) primary mouse myoblasts following exposure to normoxia (N; 21% O₂) or hypoxia (H; 1% O₂) for 24 h. (*) P < 0.05, (**) P < 0.01 by two-way ANOVA with Tukey's multiple comparisons test. (n.s.) Nonsignificant. (B) Experimental design: RNA and ATAC sequencing of WT and Bmal1^–/– myoblasts following 6 h in normoxia or hypoxia. n = 3 per condition. (C) Gene ontology analysis using ShinyGO to determine pathway enrichment of differentially expressed genes (DEGs) between WT and Bmal1^–/– myoblasts in normoxia and hypoxia. A subset of genes with detectable expression (average normalized counts >0) from the myoblast RNA-seq data set (19,862 genes in total) was used as the background gene set. (D) Heat map of DEGs important for cell proliferation and cell differentiation that are enriched in WT and Bmal1^–/– myoblasts, respectively. (E) MRF DNA motif enrichment within promoters of the DEGs between Bmal1^–/– and WT myoblasts. A subset of genes with detectable expression (average normalized counts >0) from the myoblast RNA-seq data set (19,862 genes in total) was used as the background gene set. Dot sizes are proportional to the number of target sequences containing the indicated motif. Dot colors are proportional to adjusted P-value. (H) Hypoxia, (N) normoxia. (F) Experimental design: measurement of myogenic capacity in WT and Bmal1^–/– myoblasts following 24 h of preconditioning in normoxia versus hypoxia. (G,H) Myosin heavy chain (MHC, top) and DAPI (bottom) staining (G) and quantification of differentiation index presented as the percentage of MHC⁺ nuclei (H) following 72 h in differentiation medium. Scale bar, 1000 µm. Data are represented as mean ± SEM. (*) P < 0.05, (**) P < 0.01 by two-way ANOVA with Tukey's multiple comparisons test. (n.s.) Nonsignificant.

Figure 3.

The myoblast circadian clock controls glycolytic and oxidative fuel selection. (A) KEGG pathway k-means clustering and enrichment analysis using integrated differential expression and pathway analysis (iDEP) demonstrating enrichment in metabolic pathways in DEGs between Bmal1^–/– (KO) and WT myoblasts. (B) qPCR quantification of DEGs involved in metabolic pathways such as glycolysis and FAO. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001 by two-way ANOVA with Tukey's multiple comparisons test. (n.s.) Nonsignificant. (C, left graph) ECAR from WT and Bmal1^–/– myoblasts treated sequentially with glucose, oligomycin (complex V inhibitor), and 2-deoxyglucose (glycolysis inhibitor). (Right graph) OCR from WT and Bmal1^−/− myoblasts treated sequentially with oligomycin, FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; uncoupling agent), and antimycin A/rotenone (ETC inhibitors). n = 6 wells per condition. (***) P < 0.001 by two-way ANOVA with Tukey's multiple comparisons test. (D) OCR measurements from WT and Bmal1^–/– myoblasts treated without or with 125 µM CoCl2 for 6 h, followed by palmitate-BSA with either 4 µM etomoxir (FAO inhibitor) or vehicle control. OCR measurements were taken as described in C. n = 6 wells per condition. (**) P < 0.01, (***) P < 0.001 by one-way ANOVA with Tukey's test for multiple comparisons. (E) Relative ATP, AMP/ATP, and NAD⁺ contents in WT and Bmal1^–/– myoblasts cultured in normoxia and hypoxia for 6 h. n=3 wells per condition. (*) P < 0.05 by two-sided Student's t-test comparing only genotype effect within each oxygen condition. (n.s.) Nonsignificant. (F) Immunoblots of histone acetylation at H3K9 and H4K16 in WT versus Bmal1^–/– myoblasts following 0 and 6 h in hypoxia. Numbers below the gel images indicate average band intensity relative to total H3. (N) Normoxia, (H) hypoxia. (G) Distribution of read count frequency within TSS regions from ATAC sequencing of WT and Bmal1^–/– myoblasts. (H) Gene ontology biological process analysis using genes that display both increased mRNA expression in Bmal1^–/– myoblasts versus WT and nearby increases in chromatin accessibility. (I) IGV genome browser tracks showing RNA sequencing reads from known myogenic genes and ATAC sequencing reads in nearby enhancer regions. (J) CentriMo DNA motif analysis of ATAC sequencing data demonstrating higher frequency of MRF motifs near peak centers in Bmal1^–/– versus WT myoblasts. Data are represented as mean ± SEM.

Figure 4.

MuSC clock regulates in vivo ASC proliferation as well as glucose and protein metabolism following injury. (A) FACS quantification of quiescent (from uninjured TAs) and activated (from TAs at 3 dpi) stem cells isolated from Bmal1^musc and control mice. (*) P < 0.05 by unpaired Student's t-test comparing the difference between Bmal1^musc and control SCs under a given status. (B) Experimental design: Bmal1^musc and control mice received five consecutive daily intraperitoneal injections of 100 mg/kg tamoxifen in corn oil. After 7 d, mice were injected in the TA muscles with CTX, and ASCs were isolated for RNA sequencing at 3 dpi. n = 5 mice per genotype. (C) GSEA plot of enrichment of cell proliferation- and myogenesis-related genes among DEGs from Bmal1^musc and control ASCs. (D) Volcano plot showing 23 down-regulated and 89 up-regulated genes in Bmal1^musc ASCs compared with controls at 3 dpi. (E) GSEA plot showing enrichment and heat map of fold change of fatty acid metabolism-related genes in Bmal1^musc and control ASCs. (F) PCA plot demonstrating distinct metabolite profiles upon stem cell activation and loss of Bmal1. n = 5 control, n = 8 Bmal1^musc mice. (G) Quantification of significantly (P < 0.05) different metabolites between Bmal1^musc and control ASCs at 3 dpi. Data are represented as mean ± SEM.

Figure 5.

Pyruvate supplementation or NAD⁺ replenishment restores hypoxic growth and myogenesis in Bmal1^–/– myoblasts. (A) Experimental design: Primary myoblasts isolated from Bmal1^musc mice were infected with control (Adv-control) or Cre-expressing (Adv-Cre) adenovirus for 48 h to induce deletion of the Bmal1 gene. Myoblasts were cultured in “conditional” growth medium (GM) with the indicated nutrients and/or drugs for either 48 h in 1% O2 for cell growth measurements (top) or 6 h in 1% O₂ before changing to differentiation medium (DM) in normoxia to allow for myogenic differentiation (bottom). (B) Cell number quantification by hemocytometer of WT and Bmal1^–/– (KO) primary myoblasts after culturing in hypoxia for 48 h. (*) P < 0.05 by multiple unpaired Student's t-tests. (C,D) MHC and DAPI staining (C) and quantification of differentiation index (percentage of MHC⁺ nuclei) following hypoxic pretreatment under the indicated conditions (D; as described in A). (*) P < 0.05, (***) P < 0.001 by multiple unpaired Student's t-tests. Scale bar, 400 μm. (E) Cell number quantification by hemocytometer of WT and Bmal1^–/– (KO) primary myoblasts with or without overexpression of LbNOX after culturing in hypoxia for 48 h. (*) P < 0.05 by multiple unpaired Student's t-tests. (n.s.) Nonsignifciant. (F,G) MHC and DAPI staining (F) and quantification of differentiation index in WT and Bmal1^–/– primary mouse myoblasts with or without overexpression of LbNOX (G; as described in A). (*) P < 0.05, (**) P < 0.01 by two-way ANOVA with Tukey's multiple comparisons test. (EV) Empty plasmid vector. Data are represented as mean ± SEM.