Proteome dynamics reveal Leiomodin 1 as a key regulator of myogenic differentiation

Figures

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  Figure 1.. Analysis of the proteome shows dynamic alterations during myogenic differentiation. (A) Experimental workflow for analyzing the 5-day differentiation time course using mass spectrometry (Supplementary file 1) and qRT-PCR. Primary myoblasts isolated from five individual mice were seeded and differentiated for up to 5 days. (B) Principal component analysis (PCA) of proteomics data. Ellipses represent a 95% confidence interval for each day of differentiation. (C) qRT-PCR analysis showing the relative mRNA expression of Pax7, MyoD, and Myogenin, normalized to Gapdh and day 0. (D) Mass-spectrometry-based quantification of PAX7, MYOD, and MYOGENIN protein levels normalized to day 0. (E) Pie chart for proteome dynamics. 6098 protein groups were subjected to k-means clustering analysis, 2259 were unaffected, while 3839 protein groups showed significant changes (Absolute AVG log2 ratio >0.58 and Q value<0.25) in expression levels in at least one-time point compared to day 0. Protein groups were classified into six clusters based on their expression dynamics using k-means clustering. (F) Clusters showing distinct protein abundance dynamics during myogenic differentiation and KEGG pathways…

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  Figure 2.. LMOD1 increases during early myogenic differentiation and is upregulated in aged MuSCs. (A) Protein quantification of LMOD1, LMOD2, and LMOD3 during myogenic differentiation, based on mass spectrometry data and normalized to the protein amount of day 0. Error bars indicate SD, and colored dots indicate the mean value of the biological replicates (n=5). (B) LMOD1, LMOD2, and LMOD3 protein abundance estimated from mass spectrometry data in different skeletal muscles (gastrocnemius, G; soleus, S; tibialis anterior, TA; extensor digitorum longus, EDL). Displayed values are averages of n=5 samples from individual mice. Mass spectrometry data from Schüler et al., 2021. (C) LMOD1 and LMOD2 protein quantity in MuSCs obtained from young (n=10), old (n=8), and geriatric (n=5) mice; nd = not detected. Mass spectrometry data from Schüler et al., 2021. (D) Representative immunofluorescence images of LMOD1 (purple), PAX7 (green), and Hoechst (blue) of TA sections from n=3 individual mice per age group: young (3-month-old), old (18-months-old), and geriatric (33-months-old) mice. Scale bar: 5 µm. (E) Ratio of PAX7 +and LMOD1 + cells normalized to the cells positive for PAX7+ (Figure 3—figure…

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  Figure 2—figure supplement 1.. LMOD1 in MuSCs and skeletal muscle. (A) LMOD1 protein coverage in proteomics data from MuSCs. Multiple unique (proteotypic) peptides were identified. Data from Schüler et al., 2021. (B) Barplot showing log2 fold changes of Lmod1 mRNA abundance in activated vs. quiescent MuSCs (in young and old) from previously published transcriptome data sets (GSE81096; Lukjanenko et al., 2016) and (GSE47177; Liu et al., 2013). *: adjusted p-value <0.05, ***: adjusted p-value <0.001; a - activated, q - quiescent, o - old, y - young. (C) Immunofluorescence images of TA sections from different mice (n=3) per age group of young (3 months old) and geriatric (33 months old) mice. PAX7 (green), LMOD1 (purple), and Hoechst (blue). (D) Number of PAX7 + cells per mm2 cells in muscle sections from young, old, and geriatric mice and (E) Quantification of PAX7 + cells showing cytoplasmic LMOD1 localization. For (D) and (E): One-way ANOVA; *: p-value ≤0.05, ***: p-value ≤0.001, ns: not significant. In all bar plots, each black dot represents a biological replicate, and the error bars indicate the SD. Related to Figure 2.

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  Figure 3.. Knockdown of Lmod1 impairs myogenic differentiation and reduces muscle regeneration, while overexpression enhances it. (A) Schematic of the Lmod1 knockdown experiment after 3 days of differentiation. siRNA directed against Lmod1 or scramble (siCtrl) control was used to transfect primary myoblasts isolated from individual mice. Differentiation was induced by a change to differentiation medium at 0 hr. (B) Overview of quantified different cell types based on their expression of myogenic markers; Myogenin (red), Myosin heavy chain (MHC) (green), and Hoechst (blue). Scale bar: 50 µm. (a) Non-proliferating myoblasts: Hoechst+/Myogenin-/MHC- and undifferentiated. (b) Myogenin+/MHC-: Myogenin positive, MHC negative, just started to differentiate. (c) Myogenin+/MHC+: co-express both in the process of differentiation. (d) Myogenin-/MHC+: non-elongated. (e) Fully differentiated myotubes: quantified by nuclei count to assess the fusion process. (C) Representative immunofluorescence images after three days of differentiation and siCtrl or siLmod1 transfection; MHC (green), nuclei (blue). Scale bar: 50 µm. (D–F). Quantification of the number of nuclei per myotube (D), Myogenin-/MHC + cells…

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  Figure 3—figure supplement 1.. Validation of knockdown efficiency and statistical analysis of siLmod1/Lmod2 knockdown and LMOD1 overexpression. (A) Schematic of the Lmod1 knockdown experiment under proliferating conditions. siRNA directed against Lmod1 or scramble (siCtrl) control was used to transfect primary myoblasts isolated from individual mice. (B) qRT-PCR analysis showing the relative expression of Lmod1 after siLmod1 knockdown compared to siCtrl transfected cells, normalized to Gapdh expression levels. (C) Immunoblot and quantification of LMOD1 after transfections with siLmod1 and siCtrl and two days of proliferation normalized to GAPDH. Paired t-test: *: p-value ≤ 0.05. (D) Immunofluorescence analysis of primary myoblasts stained for the proliferation marker Ki67 (red) and Hoechst (blue). Scale bar: 100 µm. (E) Quantification of cell populations identified by immunofluorescence staining: non-proliferating myoblasts being Hoechst+/Ki67- and cells being Hoechst+/Ki67+ in siCtrl and siLmod1 transfected conditions and the total counted cells per condition. (F) and (G) Validation of the siLmod1 knockdown compared to siCtrl transfected cells under differentiation conditions. qRT-PCR for Lmod1 normalized to…

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  Figure 4.. LMOD1 interacts with SIRT1. (A) BioID workflow. Lmod1 and Lmod2 were C-terminally fused to a promiscuous biotin ligase (BirA*) and expressed in HEK293T cells. BirA* alone served as a control (Ctrl) to assess non-specific biotinylation. Overexpression of fusion proteins was induced by addition of tetracycline. Exogenous biotin was introduced to label interaction partners in close proximity. Biotinylated proteins were captured using streptavidin enrichment, followed by mass spectrometry analysis to identify and quantify proximal interactors. (B) and (C) Immunofluorescence analyses of LMOD1-BirA*-FLAG and LMOD2-BirA*-FLAG in HEK293T cells 4 days after seeding without addition of any substance (-tet/-bio), with addition of only tetracycline for 4 days (+tet/-bio) or with addition of both tetracycline for 4 days and biotin for 1 day (+tet/+bio); FLAG (green), streptavidin (red), nuclei (blue). Scale bar: 20 µm. (D) Principal component analysis (PCA) of the BioID data. Ellipses represent 95% confidence intervals. (E) Volcano plot of proteins enriched by streptavidin pull-down and analyzed by mass spectrometry from LMOD1-BirA* and LMOD2-BirA*. n=4 biological replicates (Supplementary…

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  Figure 4—figure supplement 1.. BioID for LMOD1 and LMOD2. (A) Immunoblot of BirA* fusion proteins performed on lysates from HEK293T cells stably transfected with LMOD1-BirA*-FLAG, LMOD2-BirA*-FLAG or Ctrl-BirA*-FLAG following 24 hr incubation with (+tet) or without (−tet) tetracycline. Middle panel, streptavidin-HRP blot following induction of BirA* fusion proteins with tetracycline and supplementation of biotin for 24 hr. Ponceau stainings were used as loading control. HRP: horseradish peroxidase. (B) Volcano plot of proteins enriched by streptavidin pull-down and analyzed by mass spectrometry from LMOD1-BirA* and (C) LMOD2-BirA* compared to BirA*-Ctrl. Significantly enriched (AVG Log2 Ratio >0.58 and Q value <0.05), known interaction partners for LMOD1 and LMOD2 are highlighted in red, SIRT1 is highlighted in yellow (Figure 4 – figure supplement 1). (D) Quantification of LMOD1 and LMOD2 protein abundance after overexpressing the LMOD1-BirA* and LMOD2-BirA* fusion proteins in comparison to BirA*-Ctrl. (E) Quantification of selected known interaction partners of LMOD1-BirA* and LMOD2-BirA* in comparison to BirA*-Ctrl. For (D) and (E), each symbol represents a biological replicate, and error bars indicate the…

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  Figure 5.. Overexpression of LMOD1 influences the subcellular localization of SIRT1. (A) Representative immunofluorescence staining of LMOD1 and SIRT1 at different time points of myogenic differentiation; LMOD1 (purple), SIRT1 (yellow), nuclei (Hoechst, blue). Scale bar: 20 µm. (B and C) Cytoplasmic to nuclear SIRT1 (B) and LMOD1 (C) ratio at different days of differentiation, n=50 cells were analyzed per biological replicate per time point. (D–F) Correlation of the cytoplasmic to nuclear ratio of LMOD1 and SIRT1 at 2 days of proliferation (R2=0.47) (D), 1 day differentiation (R2=0.42) (E) and 2 days of differentiation (R2=0.08) (F) (G) SIRT1 protein abundance upon LMOD1 OE at different time points, 3 days proliferation (3dp), 1 day differentiation (1dd), 3 days differentiation (3dd), 5 days differentiation (5dd). Each symbol represents a biological replicate (n=4, primary myoblasts); error bars indicate SD. (H - K) Representative immunoblot of SIRT1 in the nuclear (H and I) and cytoplasmic (J and K) fraction, comparing GFP and LMOD1 OE at early time points of differentiation (0 hr: undifferentiated/proliferating, 6 hr, 12 hr, 24 hr of differentiated primary myoblasts). SIRT1 signal was…

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  Figure 5—figure supplement 1.. Subcellular localization of LMOD1 and SIRT1 during myogenic differentiation. (A and B) Total immunofluorescence intensity (nucleus and cytoplasm) of LMOD1 (A) and SIRT1 (B) during different time points of myogenic differentiation. (C) Representative immunoblot of the nuclear-cytoplasmic fractionation experiment in primary myoblasts at different time points: 2d proliferation, 1d differentiation, and 2d differentiation. SIRT1 and LMOD1 signal was analyzed in (n=3) immunoblots and normalized to the respective entire lane of the Ponceau staining. ALDOA signal was used as a cytoplasmic marker, and Histone 4 (H4) was used as a nuclear marker. Immunoblot analyses of LMOD1 and SIRT1 intensity for each cellular compartment are depicted in the bar plots. One-way ANOVA *: p-value ≤ 0.05, ns: not significant. (D) Immunoblot showing LMOD1 and SIRT1 protein abundance at different time points of myogenic differentiation (0 hr=proliferating conditions, 6 hr differentiation, 12 hr differentiation, 24 hr differentiation). Quantification of immunoblots for LMOD1 and SIRT1 shown in bar plots normalized to GAPDH. For all bar plots, each symbol represents a biological replicate, and the error bars…

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  Figure 6.. Reduced SIRT1 signaling can partially reverse siLmod1-induced impaired myogenic differentiation. (A) Illustration of the experimental setup. Primary myoblasts were co-transfected with Lmod1-specific siRNA (siLmod1) and siRNA against Sirt1 (siSirt1) (depicted in orange) or treated with SIRT1 inhibitor EX527 (depicted in pink), simultaneously to the induction of differentiation. (B/F) Representative immunofluorescence images after Lmod1 and Sirt1 knockdown with siRNA at day 3 of differentiation (B) or after Lmod1 knockdown and addition of 25 µM EX527 at day 3 of differentiation (F); MYOGENIN (red), devMHC (green), nuclei (Hoechst, blue). (C - E) Quantification of the number of nuclei per myotube (C), percentage of Myogenin-/MHC + cells (D), and measured length of differentiated myotubes (E) after siRNA transfection of SIRT1 and LMOD1. One-way ANOVA *: p-value ≤0.05, **: p-value ≤0.01. (G - I) Quantification of the number of nuclei per myotube (G), percentage of Myogenin-/MHC + cells (H), and measured length of differentiated myotubes (I) after siRNA knockdown of LMOD1 and EX527 treatment to inhibit SIRT1. One-way ANOVA *: p-value ≤0.05, **: p-value ≤0.01. (J). Illustration of the…

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  Figure 6—figure supplement 1.. Reduced SIRT1 signaling can partially reverse siLmod1-induced impaired myogenic differentiation. (A) Immunoblot validation of the LMOD1 and SIRT1 single knockdown and double knockdown (siLmod1/siSirt1) or a scrambled siRNA. Primary myoblasts were transfected at the initiation of differentiation and harvested after 3 days of differentiation. One-way ANOVA: *: p-value ≤0.05. **: p-value ≤0.01. (B) qRT-PCR analysis showing the relative expression of Sirt1 and Lmod1 in siLmod1 or siSirt1 single knockdown or double knockdown compared to siCtrl-transfected cells, normalized to Gapdh expression levels. Paired t-test *: p-value ≤0.05 **: p-value ≤0.01. (C) and (D) Percentage of cell populations found in immunofluorescence staining: non-proliferating myoblasts (Hoechst+/Myogenin-/MHC-) cells, Myogenin+/MHC- cells, Myogenin+/MHC+, fully differentiated myotubes, and total counted cells under differentiating conditions after siRNA-mediated knockdown of LMOD1 and SIRT1 (C.) related to Figure 6C-E or siRNA mediated knockdown of LMOD1 and EX527 treatment to inhibit SIRT1 (D.) related to Figure 6G-I (E) Normalized RNA-seq read counts of Lmod1 and Sirt1 after 1 day of differentiation after siCtrl…

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  Author response image 1.. Specificity of antibodies detecting LMOD1. Representative immunofluorescence images of LMOD1 in primary myoblast cultures following siLmod1 knockdown, LMOD1 overexpression, or controls transfected with a non-targeting siRNA (siCtrl) after one day of differentiation. LMOD1 (purple), SIRT1 (yellow), and nuclei (Hoechst, blue). Scale bar: 10 µm.