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. 2022;2(2):155-169.
doi: 10.1038/s43587-021-00164-x. Epub 2022 Jan 27.

Scinderin promotes fusion of electron transport chain dysfunctional muscle stem cells with myofibers

Xun Wang  1 , Spencer D Shelton  1 , Bogdan Bordieanu  1   2 , Anderson R Frank  3   4 , Yating Yi  5   6 , Siva Sai Krishna Venigalla  1 , Zhimin Gu  1 , Nicholas P Lenser  1   7 , Michael Glogauer  8 , Navdeep S Chandel  9   10 , Hu Zhao  5   11 , Zhiyu Zhao  1 , David G McFadden  3   4   12 , Prashant Mishra  1   12   13
Affiliations

Scinderin promotes fusion of electron transport chain dysfunctional muscle stem cells with myofibers

Xun Wang et al. Nat Aging. 2022.

Abstract

Muscle stem cells (MuSCs) experience age-associated declines in number and function, accompanied by mitochondrial electron transport chain (ETC) dysfunction and increased reactive oxygen species (ROS). The source of these changes, and how MuSCs respond to mitochondrial dysfunction, is unknown. We report here that in response to mitochondrial ROS, murine MuSCs directly fuse with neighboring myofibers; this phenomenon removes ETC-dysfunctional MuSCs from the stem cell compartment. MuSC-myofiber fusion is dependent on the induction of Scinderin, which promotes formation of actin-dependent protrusions required for membrane fusion. During aging, we find that the declining MuSC population accumulates mutations in the mitochondrial genome, but selects against dysfunctional variants. In the absence of clearance by Scinderin, the decline in MuSC numbers during aging is repressed; however, ETC-dysfunctional MuSCs are retained and can regenerate dysfunctional myofibers. We propose a model in which ETC-dysfunctional MuSCs are removed from the stem cell compartment by fusing with differentiated tissue.

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Figures

Extended Data Figure 1:
Extended Data Figure 1:. Cox10 deletion in MuSCs results in defective muscle regeneration.
a, Cox10 transcript and protein in MuSCs 5 days post 1st dose of tamoxifen. H2B: Histone2B. b, MuSC TMRE profiles in the indicated buffers. Each column represents MuSCs from an individual animal. pyr/mal, pyruvate/malate. rot, rotenone. c, Mitochondrial genome content (mtDNA/nDNA) and mass (Mitotracker (MT) DeepRed) in MuSCs at 5 days post 1st dose of tamoxifen. nDNA: nuclear DNA. d, Representative MuSC FACS profiles at 5 days post 1st tamoxifen dose. The CD34+ frequency is reported as a percentage of CD31−CD45−DAPI− cells. e, Histology (H&E) of TA muscle cross-sections subject to cryoinjury. dpi, days post injury. f, Immunofluorescence of Myog (red), Laminin (green), and DAPI (blue) from TA muscle cross-sections 2 days post-cryoinjury. g, Immunofluorescence of MYH3 (red), laminin (green), and DAPI (blue) from TA muscle cross-sections 5 days post-cryoinjury. h, Quantitation of Myog+ cells (2 days post cryoinjury) and MYH3+ fibers (5 days post cryoinjury), normalized to muscle area. i, CD34+ MuSC (as a percentage of CD31−CD45−DAPI− cells assessed at 5 days post 1st tamoxifen dose. j, Pax7+ MuSC numbers (normalized to muscle area) from TA muscle cross-sections at timepoints after a single tamoxifen dose. p-values reflect comparisons with 0hr data for each genotype. k, Histology (H&E) of TA muscle cross-sections at 5 days post cryo-injury. l, Histology (H&E) of TA muscle cross-sections at 5 days post-BaCl2 injury. m, mRNA transcripts at 5 days post 1st tamoxifen dose. Statistical significance was assessed using two-tailed Mann-Whitney (a), two-tailed t-test (c,h,i), or two-way ANOVA (j,m) tests, with adjustments for multiple comparisons. Box plots indicate median values and interquartile ranges; whiskers are plotted using the Tukey method. The number of biological replicates per group is indicated. All scale bars are 50μm. Experiments were repeated 3x (panel e), 6x (panel f), 8-10x (panel g), and 3x (panels k,l); all with similar results.
Extended Data Figure 2:
Extended Data Figure 2:. Cox10−/− MuSCs rapidly deplete following a single dose of tamoxifen.
a, Schematic of tamoxifen administration protocol used in this figure: A single, high-dose tamoxifen (TMX) administration to induce recombination and Cox10 deletion. b, Quantitation of Dendra2+ mononuclear cells at the indicated timepoints post-tamoxifen administration, in wild-type reporter mice (Pax7-CreERT2(KARDON); Df/f;). Mean and standard deviation (error bars) are indicated. c, PCR-based genotyping of unrecombined mito-Dendra2 allele (Dflox; expected size 265bp) and recombined mito-Dendra2 allele (Dex; expected size 645bp) in isolated MuSCs at the indicated timepoints post-tamoxifen administration. d, Cox10 levels in isolated MuSCs from Cox10−/− mice, at the indicated timepoints post tamoxifen administration. Histone 2B (H2B) are shown as a loading control. e, MuSC frequency at indicated timepoints post-tamoxifen administration in mice with Cox10f/f or Cox10−/− MuSCs. p-values reflect comparisons with t=0 hr data for each genotype. f, Pax7+ cell numbers (normalized to muscle area) at different times post-tamoxifen administration. p-values reflect comparisons with t=0 hr data for each genotype. g, Representative images of endogenous Pax7+ cells (red), laminin (green) and DAPI (blue) from tibialis anterior (TA) cross-sections of indicated mice at indicated timepoints post-tamoxifen administration. Scale bar, 50 μm. Statistical significance was assessed using two-way ANOVA (e,f) tests, with adjustments for multiple comparisons. Box plots indicate median values and interquartile ranges; whiskers are plotted using the Tukey method. The number of biological replicates per group is indicated in the figure. Experiments were repeated 4 times for panel g, with similar results.
Extended Data Figure 3:
Extended Data Figure 3:. Cox10−/− MuSCs do not display features of activation or premature differentiation.
a, Histology (H&E) of TA muscle cross-sections from animals of the indicated genotype, at different timepoints following a single dose of tamoxifen. b, Ki-67 (green) and DAPI (blue) staining of TA muscle cross-sections from animals, as described in (a). The positive control is from a wild-type animal at 2 days post cryoinjury. c, Same as b, but MYH3 (red) and DAPI (blue) staining. The positive control is from a wild-type animal at 5 days post cryoinjury. d, Same as b, but Myogenin (red) and DAPI (blue) staining. The positive control is from a wild-type animal at 2 days post cryoinjury. All scale bars are 50 μm; insets are magnified 3x. Experiments were repeated 3 times for panel a-d, with similar results.
Extended Data Figure 4:
Extended Data Figure 4:. Cox10−/− MuSCs do not display features of senescence or apoptosis.
a, MyoD (red) and DAPI (blue) immunofluorescent staining of TA muscle cross-sections from animals of the indicated genotype, at different timepoints following a single dose of tamoxifen. The positive control is from a wild-type animal at 2 days post cryoinjury. b, Same as a, but TUNEL (red) and DAPI (blue) staining. For the positive control, a wild-type TA muscle cross-section was treated with DNase. c, Same as a, but β-galactosidase staining. d, Same as a, but Cleaved Caspase-3 staining (red) and DAPI (blue) staining. e, Cleaved Caspase-3 levels in MuSCs of the indicated genotype at 48hr post-tamoxifen, assessed by western blot. Histone 2B (H2B) is shown as a loading control. The positive control is protein extract from Cox10f/f MuSCs treated with cytochrome c (0.25mg/mL for 1hr at room temperature) to induce Caspase-3 cleavage. All scale bars are 50 μm; insets are magnified 3x. Experiments were repeated 3 times for panel a-d, with similar results.
Extended Data Figure 5:
Extended Data Figure 5:. Cox10−/− MuSCs fuse into neighboring myofibers.
a, Representative immunofluorescence images from soleus muscles of Cox10−/− animals at 7 days post-the 1st dose of tamoxifen. mito-Dendra2+ fibers are labeled in green; type I (slow) fibers are labeled in red based on myosin heavy chain staining. Scale bar, 50 μm. b, Same as (a), but instead type II (fast) fibers are labeled in red based on myosin heavy chain staining. Scale bar, 50 μm. c, Quantitation of MuSC-myofiber fusion into type I and type II fibers from n=4 Cox10−/− animals. Each fiber was classified based on fiber type (type I vs. type II) and Dendra2 positivity, and the number of fibers in each category are indicated. The chi-squared statistic and p-value are indicated. d, Representative immunofluorescent images of isolated single myofibers from Cox10−/− animals. Mito-Dendra2 domains are colored green; total mitochondrial content is imaged based on Tomm20 staining (red). Scale bar, 50 μm. e, Representative images of myofiber segments containing mito-Dendra2 domains (green). Tomm20 staining for mitochondrial content is shown in red. Scale bar, 50 μm. f, Mean fluorescent profiles and standard error envelopes for aligned mito-Dendra2 domains (green), and mitochondrial content in these regions (based on Tomm20 staining, red), for n=15 fibers. AU, arbitrary units. Experiments were repeated 4 times for panel a,b, and 15 times for panels d,e; with similar results.
Extended Data Figure 6:
Extended Data Figure 6:. Myomaker is required for MuSC-myofiber fusion.
a, Dendra2+ fiber numbers (normalized to muscle area) in TA and soleus muscles of the indicated genotypes at different times post the 1st dose of tamoxifen. b, mito-Dendra2+ domain number (normalized to muscle area) in TA and soleus muscles of the indicate genotype at different post a single-high dose of tamoxifen, based on protocol in EDF 2a. c, Longitudinal imaging of tdTomato signal in TA and soleus muscles of the indicated genotype at 21 days post the 1st dose of tamoxifen administration. Scale bar, 200 μm. d, tdTomato domain number (normalized to muscle area) in myofibers of TA and soleus muscles at 21 days post the 1st dose of tamoxifen. e, Fusion indices in wt and Cox10−/− MuSCs subjected to in vitro differentiation. f, Cox10 and Myomaker (Mymk) transcript levels (normalized to β2-microglobulin) in isolated MuSCs of the indicated genotype at 5 days after the 1st dose of tamoxifen. g, Longitudinal images of mito-Dendra2 signal in TA and soleus muscles of mice of the indicated genotype, 21 days after the 1st dose of tamoxifen. Scale bar, 200 μm. h, Pax7+ cell numbers (normalized to muscle area) in TA muscle cross-sections of mice of the indicated genotype at 21 days after the 1st dose of tamoxifen. i, Relative mean TMRE fluorescence in isolated MuSCs of the indicated genotype, incubated with the indicated substrates and inhibitors. pyr/mal, pyruvate/malate. j, Relative mean superoxide levels in isolated MuSCs of the indicated genotype. Statistical significance was assessed using two-way ANOVA (b), Kruskal-Wallis (a,i), two-tailed t-test (d,e), or one-way ANOVA (f,h,j) tests, with adjustments for multiple comparisons. Box plots indicate median values and interquartile ranges; whiskers are plotted using the Tukey method. The number of biological replicates per group is indicated in the figure. Experiments were repeated 6 times for panels c,g, with similar results.
Extended Data Figure 7:
Extended Data Figure 7:. Reactive oxygen species regulate MuSC-myofiber fusion.
a, Ndufa9, Qpc and Tfam levels in MuSCs at 5 days post 1st tamoxifen dose. H2B: Histone2B. b, Mean MuSC TMRE levels in the indicated buffers. pyr/mal, pyruvate/malate. c, MuSC frequency (mito-Dendra2+ cells as a percentage of DAPI− mononuclear cells) at 5 days post 1st tamoxifen dose. p-values reflect comparisons with wt. d, Mean MuSC mitochondrial superoxide and mitochondrial total ROS (mitoROS) levels at 5 days post 1st tamoxifen dose. p-values reflect comparisons with wt. e, Mean MuSC superoxide levels from mice treated with vehicle (PBS) or N-acetylcysteine (NAC) at 5 days post 1st tamoxifen dose. f, Mean MuSC mitochondrial superoxide and mitoROS levels at 5 days post 1st tamoxifen dose, from mice treated with PBS or the indicated antioxidant. p-values reflect comparisons with wt. g, mito-Dendra2 domain numbers (normalized to muscle area) in muscle at 5 days post 1st tamoxifen dose from mice treated with the indicated antioxidants (or PBS). p-values reflect comparisons with ‘PBS’. h, Mean MuSC superoxide levels from wild-type mice treated with vehicle (PBS) or BSO for 21 days. i, MuSC frequency (Pax7+ cell number or CD34+ frequency) in wild-type mice treated with vehicle (PBS) or BSO for 21 days. j, Mean MuSC mitochondrial superoxide and mito ROS levels from sedentary (SED) or exercised (EXE) wild-type animals treated with PBS or the indicated antioxidant. p-values reflect comparisons with ‘SED’. k, mito-Dendra2 domain numbers (normalized to muscle area) in muscle of sedentary (SED) or exercised (EXE) mice treated with the indicated antioxidants (or PBS). p-values reflect comparisons with ‘SED’. Statistical significance was assessed using one-way ANOVA (c,d,f,j,k), two-way ANOVA (b,e,g), or two-tailed t-tests (h,i), with adjustments for multiple comparisons. Box plots indicate median values and interquartile ranges; whiskers are plotted using the Tukey method. The number of biological replicates per group is indicated in the figure.
Extended Data Figure 8:
Extended Data Figure 8:. Scinderin is required for MuSC-myofiber fusion.
a, Histology (H&E) from TA cross-sections at 5 days post BaCl2 injury. b, Immunofluorescence of TA cross-sections, 5 days post BaCl2 injury, stained for MYH3 (red), Laminin (green), and DAPI (blue). c, MYH3+ regenerative myofibers (normalized to muscle area) at 5 days post BaCl2 injury. d, Fusion indices in MuSCs subjected to in vitro differentiation. e, Cox10 and Scin transcript levels (normalized to β2-microglobulin) in MuSCs at 5 days post 1st tamoxifen dose. f, MuSC superoxide levels at 5 days post 1st tamoxifen dose. g, Mean MuSC TMRE fluorescence in the indicated conditions. pyr/mal, pyruvate/malate. h, mito-Dendra2 domain numbers (normalized to muscle area) in muscle of PBS or BSO-treated mice (for 21 days). i, mito-Dendra2 domain numbers (normalized to muscle area) in muscle of sedentary (SED) or exercised (EXE) mice. j, Representative traces of maximal twitch force from TA muscle at 21 days post-BaCl2 injury. k, Specific force for maximal twitch from TA muscles at 21 days post-BaCl2 injury. l, Representative traces of tetanic force generation from TA muscles (at 21 days post-BaCl2 injury) subjected to a 20Hz stimulus. m, Force-frequency relationship from TA muscles at 21 days post-BaCl2 injury. Statistical significance was assessed using two-tailed t-test (c,d,k), Kruskal-Wallis (h), one-way ANOVA (e,f), or two-way ANOVA (g,h,i,m) tests, with adjustments for multiple comparisons. Box plots indicate median values and interquartile ranges; whiskers are plotted using the Tukey method. The number of biological replicates per group is indicated. All scale bars are 50 μm. Experiments were repeated 3x (panel a), 6x (panel b); all with similar results.
Extended Data Figure 9:
Extended Data Figure 9:. Aging MuSCs accumulate mtDNA mutations.
a, Representative images for FACS isolation of murine MuSCs. Cells were first gated by forward scatter (FSC) and side scatter (SSC) to remove doublets. MuSCs were identified by negative gating for DAPI, CD31, CD45, Sca1, and positive gating for CD34. b, Read depth across the mitochondrial genome for all samples (gray traces), displayed using a log2 scale. Average read depth for all samples shown in orange. The red arc indicates the position of the common “7193-8950” deletion. c, Deletion frequency of the “7193-8950” deletion detected in MuSCs from mice of the indicated age. n.d, not detected. d, Histogram of allelic frequencies for all detected mtDNA mutations in MuSCs from 18 month old and 27 month old mice. The number of identified mutations is indicated. e, Mutational signature of mtDNA substitutions in MuSCs from 18 month old and 27 month old mice. f, The observed frequencies of non-synonymous substitutions in each mtDNA gene from isolated MuSCs of 27 month old mice. The expected frequency is indicated as the dashed red line. p-values are calculated from chi-squared test. Box plots indicate median values and interquartile ranges; whiskers are plotted using the Tukey method. The number of biological replicates per group is indicated in the figure.
Extended Data Figure 10:
Extended Data Figure 10:. MuSC-myofiber fusion regulates MuSC numbers during aging.
a, Quantitation of MuSC frequency (CD34+ %) in mice of the indicated genotypes and ages. b, Normalized TA muscle weight at 21 days post-BaCl2 injury in mice of the indicated genotypes and ages. Statistical significance was assessed using two-way ANOVA (a,b) tests, with adjustments for multiple comparisons. Box plots indicate median values and interquartile ranges; whiskers are plotted using the Tukey method. The number of biological replicates per group is indicated in the figure.
Fig. 1:
Fig. 1:. Complex IV dysfunction induces rapid MuSC depletion in vivo.
a, Schematic for tamoxifen (TMX) administration protocol to induce recombination in adult MuSCs. b, FACS-based assay for ETC function: Isolated MuSCs are permeabilized with perfringolysin O (PFO), followed by TMRE staining in the presence or absence of mitochondrial substrates and inhibitors. c, Mean TMRE fluorescence of wild-type (Cox10f/f) and Cox10−/− MuSCs, in response to indicated substrates and inhibitors. ns, no substrate; pyr/mal, pyruvate/malate; rot, rotenone. d, MuSC frequency at indicated timepoints after the 1st dose of tamoxifen in mice with Cox10f/f or Cox10−/− MuSCs. p-values reflect comparisons with t=0 day data for each genotype. e, Pax7+ cell numbers (normalized to muscle area) at different times post the 1st dose of tamoxifen. p-values reflect comparisons with t=0 day data for each genotype. f, Representative images of endogenous Pax7+ cells (red), Laminin (green) and DAPI (blue) from tibialis anterior (TA) cross-sections of indicated mice at different times post-tamoxifen administration. Scale bar, 50 μm. g, Histology (H&E staining) and immunofluorescence images of TA cross-sections at 5 days post-BaCl2 injury (dpi). Regenerative myofibers can be identified by their centrally localized nuclei and MYH3-positive staining at this timepoint. Scale bar, 50 μm. h, Quantitation of regenerative (central nuclei) myofibers (normalized to muscle area) in TA muscles at 5 days post-BaCl2 injury. i, TA weight (normalized to body weight) at various time points after BaCl2 injury. dpi, days post-injury. Statistical significance was assessed using two-way ANOVA (c,d,e,i), or two-tailed t-test (h), tests with adjustments for multiple comparisons. Box plots indicate median values and interquartile ranges; whiskers are plotted using the Tukey method. The number of biological replicates in each group and p-values are indicated in the figure. Experiments were repeated 5 times in panel f, and 6 times in panel g; both with similar results.
Fig. 2:
Fig. 2:. Complex IV dysfunction induces MuSC-myofiber fusion in vivo and in vitro.
a, Longitudinal imaging of mito-Dendra2 signal in various muscles at 21 days post the 1st dose of tamoxifen. Scale bar, 200 μm. b, mito-Dendra2 domain number (normalized to muscle area) in myofibers of TA and soleus muscles at different times post the 1st dose of tamoxifen. c, In vitro MuSC-myotube fusion assay: Dendra2+ (green) freshly-isolated MuSCs of the indicated genotype were overlaid on DsRed+ (red) C2C12 myotubes and assessed for fusion with Myosin+ (magenta) myotubes after 4 days. Cells were stained with Myosin (magenta) and DAPI (blue). Scale bar, 50 μm. d, Quantitated myotube fusion indices for MuSCs of the indicated genotype. e, mito-Dendra2 domain numbers in TA and soleus muscles at 21 days post the 1st dose of tamoxifen in mice of the indicated genotype. Mymk, Myomaker. f, MuSC (CD34+) frequency of the indicated genotypes at 21 days post the 1st dose of tamoxifen. Same color scheme as d,e. Statistical significance was assessed using two-way ANOVA (b), two-tailed t-test (d,e), Kruskal-Wallis (b), two-tailed Mann-Whitney (e), or one-way ANOVA (f) tests with adjustments for multiple comparisons. Box plots indicate median values and interquartile ranges; whiskers are plotted using the Tukey method. The number of biological replicates in each group and p-values are indicated in the figure. Experiments were repeated 3 times in panel a, and 5-6 times in panel c; both with similar results.
Fig. 3:
Fig. 3:. MuSC-myofiber fusion is regulated by reactive oxygen species.
a, Representative longitudinal images of mito-Dendra2 domains in TA and soleus muscles of mice of the indicated genotype (21 days post the 1st dose of tamoxifen). Scale bar, 200 μm. b, Quantitation of mito-Dendra2 domain numbers (normalized to muscle area) at 21 days post the 1st dose of tamoxifen. p-values reflect comparisons with the wt group. c, Relative superoxide levels in MuSCs of the indicated genotype at 5 days post the 1st dose of tamoxifen. Same color scheme as b. p-values reflect comparisons with the wt group. d, mito-Dendra2 domain numbers (normalized to muscle area) in mice of the indicated genotype pre-treated with vehicle (PBS) or N-acetylcysteine (NAC) at 5 days post the 1st dose of tamoxifen. e, Representative longitudinal images of mito-Dendra2 domains in TA and soleus muscles of mice treated with vehicle (PBS) or BSO for 21 days. Scale bar, 200 μm. f, mito-Dendra2 domain numbers (normalized to muscle area) in mice treated with PBS or BSO for 21 days. g, Representative longitudinal images of mito-Dendra2 domains in TA and soleus muscles of sedentary (SED) mice, exercised mice treated with PBS (EXER+PBS), or exercised mice treated with NAC (EXER+NAC). Scale bar, 200 μm. h, mito-Dendra2 domain numbers (normalized to muscle area) in mice of the indicated groups. Statistical significance was assessed using one-way ANOVA (b,c,h), two-way ANOVA (d), two-tailed t-test (f), and two-tailed Mann-Whitney (f) tests with adjustments for multiple comparisons. Box plots indicate median values and interquartile ranges; whiskers are plotted using the Tukey method. The number of biological replicates in each group and p-values are indicated in the figure. Experiments were repeated 5-6 times in panel a, 5 times in panel e, and 10-11 times in panel g; all with similar results.
Fig. 4:
Fig. 4:. Scinderin induces actin network changes and MuSC-myotube fusion.
a, Volcano plot of gene expression changes in Cox10−/− vs. wt MuSCs. Log2(Fold Change) is plotted against the log10(adjusted p-value) for each gene. b, Gene ontology analysis of upregulated genes in Cox10−/− MuSCs. c, Normalized Scinderin transcript levels in isolated MuSCs of the indicated genotype at 5 days post the 1st dose of tamoxifen. p-values reflect comparisons with the wt group. d, Scinderin protein levels in isolated MuSCs of the indicated genotype at 5 days after the 1st dose of tamoxifen. Histone 2B (H2B) levels are shown as a loading control. e, Scinderin protein levels in isolated MuSCs of the indicated genotype and treatment at 5 days post the 1st dose of tamoxifen. H2B levels are shown as a loading control. f, Scinderin protein levels in C2C12 cells infected with empty or Scinderin overexpressing virus. H2B levels are shown as a loading control. g, Violin plots of actin protrusion numbers (per cell) for control and Scinderin-overexpressing C2C12 cells. h, Representative images of actin networks (stained by Acti-stain555 phalloidin (red)) and nuclei (DAPI, blue) in control and Scinderin-overexpressing C2C12 cells. Scale bar, 10 μm. i, Scinderin (α-HA; green) and actin (phalloidin; red) localization in cells overexpressing Scinderin. Scale bar, 10 μm. j, Representative images from an in vitro MuSC-myotube fusion assay: 10,000 Dendra2+ (green) freshly-isolated control or Scin-overexpressing MuSCs were overlaid on DsRed+ (red) C2C12 myotubes and assessed for fusion with Myosin+ myotubes after 4 days. Cells were stained with Myosin (magenta) and DAPI (blue). Scale bar, 50 μm. k, Quantitated myotube fusion indices for MuSCs of the indicated genotype. Statistical significance was assessed using one-way ANOVA (c), two-tailed Mann-Whitney test (g) or two-tailed t-test (k). Box and violin plots indicate median values and interquartile ranges; whiskers are plotted using the Tukey method. The number of biological replicates per group and p-values are indicated in the figure. Experiments were repeated 3 times in panels h,i, and 4 times in panel j; all with similar results.
Fig. 5:
Fig. 5:. Scinderin is required for MuSC-myofiber fusion.
a, Representative longitudinal images of mito-Dendra2 domains in TA and soleus muscles of mice of the indicated genotype, 21 days post the 1st dose of tamoxifen. Scale bar, 200 μm. b, mito-Dendra2 domain numbers (normalized to muscle area) at 21 days post the 1st dose of tamoxifen. c, Representative images of endogenous Pax7+ cells (red), laminin (green) and DAPI (blue) from tibialis anterior (TA) cross-sections from mice of the indicated genotype at 21 days post the 1st dose of tamoxifen. Scale bar, 50 μm. d, MuSC (CD34+) and Pax7+ frequency in mice of the indicated genotype at 21 days post the 1st dose of tamoxifen. e, Representative images of histology (H&E), MYH3+ fibers (red) and complex IV activity (COX) in cross-sections of TA muscles from mice of the indicated genotypes. Muscles were harvested and assessed 5 days or 21 days post-BaCl2 injury. For immunofluorescent staining, sections were stained with α-laminin (green), α-MYH3 (red), and DAPI (blue). Scale bar: 50 μm. d.p.i, days post-injury. f, Quantitation of regenerative fiber numbers (normalized to muscle area) in TA muscles at 5 days and 21 days post-BaCl2 injury. g, Normalized COX activity from regenerative myofibers at 5 days and 21 days post-BaCl2 injury. The number of analyzed fibers per group is indicated. h, TA weight (normalized to body weight) at 21 days after BaCl2 injury. Statistical significance was assessed using one-way ANOVA (b,d,f,h) or two-tailed Mann-Whitney (g) tests, with adjustments for multiple comparisons. Box and violin plots indicate median values and interquartile ranges; whiskers are plotted using the Tukey method. The number of biological replicates in each group and p-values are indicated in the figure. Experiments were repeated 5 times for panel a, 6 times for panel c, and 5-8 times for panel e; all with similar results.
Fig. 6:
Fig. 6:. Muscle stem cells accumulate mtDNA mutations with age.
a, Quantitation of MuSC number (frequency of CD34+ cells) from wild-type mice of the indicated ages. b, Representative superoxide FACS profiles of MuSCs from mice of the indicated age. NC: unstained negative control. Gating for the SOXHI (SuperoxideHI) population is indicated. c, Quantitation of mean superoxide levels in MuSCs of mice of the indicated age. d, Quantitation of SOXHI population frequency from MuSCs of mice of the indicated age. e, Number of identified mtDNA substitutions from deep mtDNA sequencing of isolated MuSCs from mice of the indicated age. f, Number of identified mtDNA indels (insertions/deletions) from isolated MuSCs of mice of the indicated age. g, Mapped positions of identified somatic mtDNA mutations (indels, non-coding, synonymous and non-synonymous substitutions) in MuSCs from 27 month old mice. h, Abundance of mtDNA protein-coding indels (as a percentage of total indels) observed in isolated MuSCs from mice of the indicated age. The expected frequency is indicated by the red line. i, Abundance of mtDNA non-synonymous substitutions (as a percentage of total protein-coding substitutions) observed in isolated MuSCs from mice of the indicated age. The expected frequency is indicated by the red line. Statistical significance was assessed using one-way ANOVA (a,c), Kruskal-Wallis (d,e,f), and chi-squared (h,i) tests with adjustments for multiple comparisons. Box plots indicate median values and interquartile ranges; whiskers are plotted using the Tukey method. The number of biological replicates per group and p-values are indicated in the figure.
Fig. 7:
Fig. 7:. Scinderin is required for regenerating healthy tissue in aged animals.
a, Schematic of aging experiments in wild-type, Mymk−/− and Scin−/− mice. Recombination is induced in MuSCs of animals at 6 weeks of age via tamoxifen (TMX) administration, followed by aging and the indicated analysis at various timepoints (2, 12, 24 and 30 months of age). At each timepoint, MuSCs are analyzed in situ and by FACS; in addition, tibialis anterior (TA) muscles are injured by BaCl2 and assess for regeneration at 21 days post-injury (d.p.i.). b, Pax7+ cell numbers (normalized to muscle area) in animals of the indicated genotype and age. p-values represent comparison with wt for each age group. c, Representative FACS profiles of Superoxide (SOX) levels in MuSCs isolated from animals of the indicated genotype and age. Gating for SOXHI population is indicated. d, Quantitation of SOXHI population frequency from MuSCs of mice of the indicated age and genotype. p-values represent comparison with wt for each age group. e, Representative images of histology (H&E) in cross-sections of TA muscles from mice of the indicated genotypes and ages. Muscles were harvested and assessed 21 days post-BaCl2 injury. Scale bar: 50 μm. f, Quantitation of fiber size from regenerative fibers (21 days post-injury) in TA muscles of mice of the indicated genotypes and ages. g, Representative images of COX activity in cross-sections of TA muscles from mice of the indicated genotypes and ages, at 21 days post-BaCl2 injury. h, Quantitation of relative COX activity in regenerative fibers (21 days post-injury) from TA muscles of mice of the indicated genotypes and ages. i, Representative images of SDH activity in cross-sections of TA muscles from mice of the indicated genotypes and ages, at 21 days post-BaCl2 injury. j, Quantitation of relative SDH activity in regenerative fibers (21 days post-injury) from TA muscles of mice of the indicated genotype and ages. Statistical significance was assessed using two-way ANOVA (b,d) or two-tailed Mann-Whitney (f,h,j) tests, with adjustments for multiple comparisons. Box and violin plots indicate median values and interquartile ranges; whiskers are plotted using the Tukey method. The number of biological replicates in each group and p-values are indicated in the figure. Experiments were repeated 5-8 times in in panels e,g,I; all with similar results.
Fig. 8:
Fig. 8:. Proposed model: MuSC-myofiber fusion regulates removal of ETC-dysfunctional MuSCs during aging.
a, In young wild-type animals, healthy MuSCs predominate and are competent to regenerate healthy myofibers in response to injury. b, In aged, wild-type animals, ETC-dysfunctional MuSCs (depicted with red mitochondria) are sensitive to elevated ROS and Scinderin levels, and thereby induced to fuse into neighboring myofibers. In this manner, the remaining MuSC population contains healthy mitochondria, and generates healthy myofibers in response to injury. c, In the absence of Scinderin during aging, MuSC-myofiber fusion is not available to remove ETC-dysfunctional MuSCs. In this setting, injury can trigger the activation, proliferation and fusion of damaged MuSCs, resulting in de novo myofibers with mitochondrial dysfunction.
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