Glucose Metabolism Drives Histone Acetylation Landscape Transitions that Dictate Muscle Stem Cell Function

Abstract

The impact of glucose metabolism on muscle regeneration remains unresolved. We identify glucose metabolism as a crucial driver of histone acetylation and myogenic cell fate. We use single-cell mass cytometry (CyTOF) and flow cytometry to characterize the histone acetylation and metabolic states of quiescent, activated, and differentiating muscle stem cells (MuSCs). We find glucose is dispensable for mitochondrial respiration in proliferating MuSCs, so that glucose becomes available for maintaining high histone acetylation via acetyl-CoA. Conversely, quiescent and differentiating MuSCs increase glucose utilization for respiration and have consequently reduced acetylation. Pyruvate dehydrogenase (PDH) activity serves as a rheostat for histone acetylation and must be controlled for muscle regeneration. Increased PDH activity in proliferation increases histone acetylation and chromatin accessibility at genes that must be silenced for differentiation to proceed, and thus promotes self-renewal. These results highlight metabolism as a determinant of MuSC histone acetylation, fate, and function during muscle regeneration.

Figure 1.. Histone Acetylation Dynamics during Regeneration Revealed by CyTOF

(A) Visualization of t-distributed stochastic neighbor embedding (viSNE) plot of uninjured and injured (D6 after notexin injection) muscle. Sca1/CD31/CD11b/ CD45⁻ (Lin⁻) α7-integrin⁺ cells were clustered based on PAX7, MYOD1, and MYOG (Myogenin). viSNE plots of combined biological replicates (n = 3) for each time point shown. Color scale shows CD34, PAX7, MYOD1, or MYOG normalized expression. Significance determined using one-way ANOVA with Tukey’s test for multiple comparisons across samples (injured and uninjured), and repeated-measures ANOVA was used for comparisons between Activated or Self-Renewing Stem and Differentiated subsets within biological samples. Subsets outlined are as follows: (a) quiescent PAX7⁺CD34⁺MYOD⁻ MuSCs (D0), gray; (b) self-renewing PAX7⁺CD34⁻MYOD⁻ MuSCs (D6), black; (c) activated PAX7⁺MYOD⁺ MuSCs (D6), blue; and (d) differentiating PAX7⁻MYOD⁺MYOG⁺ progenitors (D6), red. (B) Expression of PAX7, CD34, MYOD1, and MYOG in subsets in (A). (C) IdU incorporation subsets in (A). (D) Histone acetylation normalized for total histone H3 content in subsets outlined in (A). (E) Representative contour plot of CD34 versus MYOG at D0 and D6. Cells are Lin⁻α7-integrin⁺. Quiescent (CD34⁺MYOG⁻), Activated (CD34^LowMYOG⁻), and Differentiated (CD34^LowMYOG⁺) subsets are indicated. (F) H4K16 and H3K18 acetylation in Quiescent, Activated, and Differentiated subsets. Acetylation normalized for Quiescent MuSCs. n = 4–11 biological replicates. Significance determined using one-way ANOVA with Tukey’s test for multiple comparisons across samples (injured and uninjured), and Student’s paired t test was used for comparisons between within biological samples. (G) Immunostaining of H4K16ac on extensor digitorum muscle fibers. Freshly isolated fibers were compared to those cultured for 72 h. Quiescent MuSCs identified as PAX7⁺MYOD1⁻, activated MuSCs as PAX7⁺MYOD⁺, and early differentiated progenitors as PAX7⁻/MYOD⁺ cells. Scale bar (white): 10 μm. Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001). See Figures S1 and S2 and Table S1.

Figure 2.. Histone Acetylation Is Reduced in MYOG⁺ Progenitors, and upon Induction of Myogenic Differentiation by Serum Removal, but Spares the Myogenic Program

(A) Representative histogram (MYOG) and contour plot (H3K18ac versus MYOG) of confluent, heterogeneously differentiated myoblasts in GM (~15%–30% MYOG⁺). MYOG⁺ and MYOG⁻ gates are in red and black. (B) Normalized histone acetylation or total histone H3 intensity for MYOG⁻ versus MYOG⁺ myoblasts within same samples. n = 3–4 biological replicates are shown. Student’s paired t test was used for comparisons. (C) Histone acetylation in sub-confluent myoblasts in GM versus 24 h in DM. The percentage of MYOG+ cells in GM and DM was <5% and 40%–60%, respectively (Figures S3B and S3C). n = 3–4 biological replicates. Significance determined using one-way ANOVA with Tukey’s test for multiple comparisons across samples (GM and DM), and Student’s paired t test was used for comparisons between MYOG⁻ and MYOG⁺ cells within biological samples. (D) Histone acetylation in GM and 24-h DM myoblasts by LC-MS/MS. Lysine acetylation calculated as a fraction relative to each histone peptide (relative abundance). Histone peptides analyzed from the following, left to right: H3 9–17; H3 18–26; H4 4–17; and H2A1–11. n = 3 biological replicates were run on a mass spectrometer three independent times. For statistical comparisons, two-way ANOVA was used with matching for experimental runs. (E) Enrichment of H3K9∣14 acetylation across the genome for a 2-kbp window around transcriptional start sites (TSSs). ChIP-sequencing and gene expression data used from the Mouse ENCODE Consortium (GSE36023). (F) Histone acetylation ChIP-sequencing from C212 myoblasts and myotubes (D7 of differentiation) analyzed from Blum et al. (2012) and Asp et al. (2011) (H3K9, H4K12, H3K18, and H3K27ac, respectively). Genes associated with each peak were identified within a 2-kb window around the TSS. A total of 8,963 genes showed reduced histone acetylation on any one or more mark in myotubes (left) and 1,967 genes were identified with increased histone acetylation in myotubes (right). Shown are the number of genes associated with loss or gain of any one, two, three, or all four histone acetylation marks. Gene set enrichment for genes shown below. (G) HOMER Motif enrichment analysis of promoters of genes associated with loss (top) or increase (bottom) of histone acetylation on one or more marks. No significant motifs identified for genes with loss of acetylation. Promoters of genes with increased histone acetylation enriched for MADS, E-Box, Homeobox, and NR (nuclear receptor) consensus motifs. Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001). See also Figure S3.

Figure 3.. Myogenic Fate Corresponds to Distinct Glycolytic States

(A) Flux from glucose to histone acetylation. (B) Expression of glycolytic genes in freshly isolated and 2-day hydrogel cultured MuSCs and primary myoblasts grown on collagen-coated plastic. Color scale shows Log₂Fold compared to mean TPM (transcripts per million) of fresh MuSCs (left) or proliferating myoblasts (right). Genes in orange indicate ATP-generating phase/lactate-producing stage of glycolysis. Right, Log₂Fold change shown for MuSC-derived myoblasts versus day 2 versus day 5 of induced differentiation of published microarray data (GSE24811; Soleimani et al., 2012). For statistical comparisons of all replicates of RNA-seq data, see Table S2. (C–F) Bioenergetic profile of confluent primary myoblasts in growth media (GM) or after 24 h in differentiation media (DM). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XF96 extracellular flux analyzer using the Mitochondrial Stress or Glycolytic Stress kits. n = 5–8 biological replicates. One-way ANOVA with Tukey’s test for multiple comparisons used. (C) Oxygen consumption profile for GM (15% FBS) or 24-h DM (5% horse serum) myoblasts with or without 25 mM glucose. (D) Basal respiration, ATP production, maximal respiration, and spare capacity for GM and DM ± 25 mM glucose. (E) Glycolytic stress profile for GM versus 24-h DM. (F) Glycolytic rate, capacity, and reserve for GM and DM myoblasts. (G) Immunostaining for pPDH, PAX7, and MYOD1 on isolated single fibers from soleus fixed immediately after isolation, or after 48 h in culture in GM. (H) pPDH/PDH by flow cytometry for freshly isolated versus 48-h cultured MuSCs on hydrogels. n = 4 independent experiments. Student’s paired t test was used for statistical comparison. (I) Quantification of pPDH/PDH for primary myoblasts in GM versus 24-h DM. n = 3 independent experiments. Student’s paired t test was used for statistical comparisons. (J) pPDH/PDH of quiescent, self-renewing, and activated (MYOD⁻ and MYOD⁺) MuSCs, and MYOG⁺ differentiating progenitors CyTOF (as characterized in Figure 1). n = 3 biological replicates, one-way ANOVA with Tukey’s test for multiple comparisons used for comparisons against quiescent MuSCs, and repeated-measures ANOVA used to compare activated versus differentiated subsets within biological replicates. Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001). See also Figure S4 and Table S2.

Figure 4.. Glucose Flux toward Histone Acetylation Decreases upon Myogenic Differentiation

(A) Scheme of ¹³C glucose labeling. Sub-confluent primary myoblasts in GM were switched to GM or GM + DCA (10 mM) or DM for 6 h. In parallel, 24-h differentiated, confluent myoblasts were switched to labeled DM for 6 h. ¹³C glucose was added to a final concentration of 25 mM. n = 3 biological replicates used for each condition. (B) Quantification of ¹³C incorporation by LC-MS/MS as outlined in (A). Incorporation of labeled glucose quantified as fraction of labeled (+2-Da shift) over total acetylation (labeled + unlabeled) for each mark. Incorporation on singly acetylated lysines was analyzed: H3 9–17; H3 18–26; H4 4–17; and HH2A 4–11. n = 3 biological replicates were run on a mass spectrometer three independent times. For comparisons, two-way ANOVA was used with matching for each experimental run. (C) H3K9∣14, H3K18, H4K16, H2bK5, H3K27, and H3K56 acetylation of proliferating myoblasts after 24-h DCA, normalized for untreated controls. n = 3 biological replicates; statistics calculated by Student’s paired t test. (D) Effect of DCA on differentiation. Cells were untreated (−/−), treated with DCA only in DM (−/+), treated in GM but not DM (+/−), or treated with DCA in both GM and DM (+/+). Differentiation quantified as percentage positive for MYOG. n = 5–6 biological replicates. (E) Histogram of MYOG in myoblasts differentiated for 24 h following DCA treatment in GM, DM, or both (−/−, −/+, +/−, +/+). MYOG⁺ gate in gray. (F) Contour plot of MYOG versus H3K9∣14ac following 72-h treatment with scrambled siRNA or siAcly. MYOG+ gate outlined. (G) H3K9∣14 histone acetylation following siAcly or SiScramble. DCA or vehicle added after 24-h knockdown and treatment continued for 48 h, for a total of 72-h siRNA treatment. n = 3. (H) MYOG⁺ quantification following siAcly or SiScramble, +/− DCA as described in (G). n = 3. (I) H3K9∣14 acetylation following siCs or SiScramble, +/− DCA by flow cytometry. n = 3. (J) MYOG⁺ quantification following siCs or SiScramble, +/− DCA. n = 3. (K) H3K9∣14 histone acetylation following DMSO (vehicle) or 20 μM Mdivi-1 treatment of myoblasts in GM or DM for 24 h. (L) Histogram, MYOG⁺ after 24-h treatment with DMSO or 20 μM Mdivi-1 in GM or DM. (M) MYOG⁺ quantification of myoblasts in GM or DM treated with DMSO or Mdivi-1 for 24 h. n = 3. Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001). Unless otherwise indicated, significance was calculated by one-way ANOVA with Tukey’s test for multiple comparisons for n = 3 biological replicates. See also Figure S5.

Figure 5.. Genomic Characterization of Glucose-Metabolism Mediated Histone Acetylation

(A) ChIP-sequencing of H3K9∣14 acetylation of proliferating myoblasts in GM ± DCA for 24 h. Enrichment shown for a ±1-kb window around TSSs. (B) Genomic tracks for representative housekeeping (left) and myogenic genes (right). (C) ChIP-sequencing signal for peaks in control versus DCA treated myoblasts. The dotted line indicates slope of 1 (x = y). (D) Gene set enrichment analysis of genes associated with increased ChIP-sequencing peak signal in DCA versus control myoblasts. Overlap with genes with gain or loss of at least one histone acetylation mark in myotube versus myoblasts (see Figure 2F) shown. (E) Average change in expression in absolute transcripts per million (TPM) following DCA treatment. Change in expression of all genes was compared with those with increased ChIP-sequencing peaks following DCA treatment in proximal promoters (±2 kb) or distal enhancer (−100 kb). Statistical comparison was made versus average change in TPM over all genes (0). (F) Comparison of ChIP-sequencing and ATAC-seq. Chromatin accessibility by ATAC-seq was assayed in myoblasts in GM ± DCA. Change in ChIP-sequencing signal shown as a function of distance to nearest ATAC-seq peak. (G) Comparison of ChIP-sequencing and ATAC-seq. Chromatin accessibility by ATAC-seq was assayed in myoblasts in GM with or without DCA treatment. Change in ChIP-sequencing signal is shown as a function of number of associated ATAC-seq peaks. (H) Genomic track of H3K9∣14 ChIP-sequencing and ATAC-seq peaks for control and DCA-treated myoblasts. (I) ATAC-seq signal of peaks in control versus DCA treated myoblasts. The dotted line shows slope of 1 (x = y). (J) Change in expression in absolute transcripts per million (TPM) following DCA treatment. Expression of all genes was compared with those with increased ATAC-seq peaks following DCA treatment in proximal promoters (±2 kb) or distal enhancer (−100 kb). Statistical comparison made versus average change in TPM over all genes (0). (K) HOMER motif enrichment analysis of genes that show increased accessibility by ATAC-seq following DCA treatment. Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001). Unless otherwise indicated, significance was calculated by one-way ANOVA with Tukey’s test for multiple comparisons. See also Table S3.

Figure 6.. PDK dKO Mice Show Muscle Regeneration Defects and MuSC-Specific Histone Acetylation and Engraftment Capacity

(A) Real-time qPCR of Pdk(1-4), Pdhal, and Pax7 expression in cultured MuSCs isolated from WT or dKO animals. n = 3 biological replicates. (B) Immunohistochemistry for pPDH in WT and dKO animals at D0, or D6 and D12 after injury by notexin injection. (C) Quantification of Pax7⁺ cells per area (1 mm²) at D0, D6, and D12. n = 4–6 animals per time point. (D) Representative image of Pax7⁺ MuSCs in of WT and dKO muscle at D12. DAPI, Laminin co-staining. (E) H3K18 and H4K16 acetylation by flow cytometry for uninjured (D0) MuSCs (Lin⁻α7-integrin⁺CD34⁺MYOG⁻). Histone acetylation normalized for uninjured WT controls in each experiment. n = 4–10 animals. (F) H3K18 and H4K16 acetylation for injured (D6) MuSCs (Lin⁻α7-integrin⁺CD34⁺MYOG⁻). Histone acetylation normalized for injured WT controls in each experiment. n = 3–12 animals. (G) Histogram of MYOG⁺ cells within Lin⁻α7-integrin⁺ population at D6 after injury. (H) MYOG⁺ quantification of cells as described in (G). n = 3. (I) Histogram of fiber cross-sectional area in dKO and WT, D12 after injury. (J) Median fiber cross-sectional area of WT and dKO at D12 after injury. n = 3 animals. (K) Transplant assay. MuSCs were isolated from WT or dKO donors, and transduced with a Luciferase reporter virus. After 24 h, 500 cells were transplanted into irradiated NSG recipient mice. MuSCs from WT and PDK mice were transplanted into contralateral legs to minimize biological variability. (L) BLI signal for dKO and WT; images shown for same animal. (M) BLI signal of engrafted MuSCs 28 days after transplantation, calculated as radiance (photons/second). n = 5. Student’s unpaired t test was used for all statistical comparisons. Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001). See also Figure S6.