Analysis of the proteome shows dynamic alterations during myogenic differentiation

A. Experimental workflow for analyzing the five-day differentiation time course using mass spectrometry (Table S1) and qRT-PCR. Primary myoblasts isolated from five individual mice were seeded and differentiated for up to five 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 enriched (FDR <0.05) in each cluster (Table S1). G. Proteins from cluster 2 were chosen according to KEGG pathway annotation: striated muscle contraction and function, motor protein activity and cytoskeletal dynamics. The protein-protein interaction network was visualized with STRING (Szklarczyk et al. 2011), edge confidence: medium 0.4. For (C) and (D): In all bar plots, each symbol represents a biological replicate, and the error bars indicate the standard deviation. (SD). One-way, ANOVA. *q value ≤ 0.05, **: q value ≤ 0.01, ***: q value ≤ 0.001.

LMOD1 increases during early myogenic differentiation and is upregulated in aged MuSCs

A. Protein quantification of LMOD1 and LMOD2 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 and LMOD2 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 (orange), and Hoechst (blue) of TA sections from n = 3 individual mice per age group: young (3-month-old) and old (18-months-old), geriatric (33-months-old) mice. Scale bar: 5 µm. E. Ratio of PAX7+ and LMOD1+ cells normalized to the cells positive for PAX7+ (Figure S2C) and F. Quantification of the percentage of PAX7+ cells showing nuclear LMOD1 localization. Related to Figure S1. For (E) and (F): One-way, ANOVA. *: q value < 0.05, **: q value < 0.01, ***: q value < 0.001. In all bar plots, each black dot represents a biological replicate, and the error bars indicate the SD. G. Muscle regeneration time course: Immunoblot of LMOD1 levels in regenerating TA muscle after injury. Quantification of LMOD1 levels normalized to Ponceau and relative to the LMOD1 signal at day 0. Each symbol represents a biological replicate, and error bars represent SD. One-way ANOVA; **: p-value < 0.01, ***: p-value < 0.001. H. Representative immunofluorescence images of LMOD1 (purple), PAX7 (orange), LAMININ (grey), and Hoechst (blue) of TA sections at different days post-injury from young mice (3 months old). The white arrowhead shows PAX7+ cells at day 0, white arrows indicate newly formed LMOD1+ myofibers at day 5. Scale bar: 50 µm.

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. 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 (orange), LMOD1 (purple) and Hoechst (blue). C. Number of PAX7+ cells per mm2 cells in muscle sections from young, old and geriatric mice and D. Quantification of PAX7+ cells showing cytoplasmic LMOD1 localization. For (C) and (D): 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.

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 0h. B. Overview of quantified different cell types based on their expression of myogenic markers; Myogenin (red), Myosin heavy chain (MHC) (green), Hoechst (blue). Scale bar: 50 µm. (a) reserve cells: Hoechst+/Myogenin-/MHC- and undifferentiated. (b) Myogenin+/MHC-: Myogenin positive, MHC negative, just started to differentiate. (c) Myogenin+/MHC+: co-express bot 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: 100 µm. D - F. Quantification of the number of nuclei per myotube (D), Myogenin-/MHC+ cells (E) and length of differentiated myotubes (in µm) (F). Paired t-test; *: p-value < 0.05, **: p-value < 0.01. G. Schematic of the Lmod2 and Lmodl/Lmod2 double knockdown experiment after 3 days of differentiation. siRNA against Lmod1, Lmod2, both or scramble (siCtrl) was used to transfect primary myoblasts. Differentiation was induced by a change to differentiation medium at 0h. H. Representative immunofluorescence images of siLmod2 and siLmod1/Lmod2 double knockdown after three days of differentiation; devMHC (green), nuclei (blue). Scale bar: 50 µm. I - K. Quantification of the number of nuclei per myotube (I), Myogenin-/MHC+ cells (J), Length of differentiated myotubes (in µm) (K). One-way ANOVA; *: p-value < 0.05, ns: not significant. L. Experimental schematic for analysis of in vivo CTX-induced injury of TA muscles combined with injection of self-delivering siRNAs at 3 days post injury. n = 4 mice per group, 3 months old. M. Representative immunofluorescence images of MYOGENIN (green), LAMININ (grey), and Hoechst (blue) of TA-sections 5 days post-injury from young mice (3 months old). N. Quantification of the number of Myogenin+ cells normalized per area. Unpaired t-test; *: p-value < 0.05. O. Illustration of the experimental setup. Primary myoblasts isolated from individual mice stably overexpressing (OE) LMOD1 (purple) or GFP (green) were seeded and either collected during proliferation (3d proli), after 1 day (1d diff), 3 days (3d diff) or 5 days (5d diff) of differentiation. Differentiation was induced by a change to differentiation medium at 0h. Cells were harvested for immunofluorescence analysis (IF), qRT-PCR, immunoblot (WB) or mass spectrometry (MS). P. Representative immunofluorescence images of primary myoblasts after one day of differentiation, showing either stable expression of LMOD1 or GFP. devMHC (green), MYOGENIN (red) and nuclei (blue). Scale bar is 50 µm. Q and R. Quantification of nuclei per myotube of GFP OE or LMOD1 OE cells (Q) and cells expressing Myogenin+/MHC+ defined as just differentiated cells (R) after 1 day of differentiation. Paired t-test *: p-value < 0.05. Related to Figure S2. In all bar plots, each symbol represents a biological replicate, and the error bars indicate the SD.

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 siLmodl 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: reserve cells 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 Gapdh expression levels (F) and immunoblot analysis with quantification of LMOD1 signal normalized to GAPDH (G). Paired t-test *: p-value < 0.05, ***. H. Quantification of cell populations identified by immunofluorescence staining: reserve (Hoechst+/Myogenin-/MHC-) cells, Myogenin+/MHC-cells, Myogenin+/MHC+, fully differentiated myotubes and total counted cells under differentiating conditions. I. and J. Validation of the siLmod1 or siLmod2 (single) or siLmod1/Lmod2 (double) knockdown compared to siCtrl transfected cells under differentiation conditions. qRT-PCR for Lmod1 and Lmod2 normalized to Gapdh expression levels (I) and representative immunoblot with quantification of LMOD2 normalized to GAPDH (J). One-way ANOVA *: p-value < 0.05 or numbers are indicated. ns: not significant. K. Quantification of cell populations (in %) identified by immunofluorescence staining after siLmod1 (single), siLmod2 (single) and siLmod1/2 (double) knockdown under differentiating conditions: reserve (Hoechst+/Myogenin-/MHC-) cells, Myogenin+/MHC-cells, Myogenin+/MHC+, fully differentiated myotubes and total counted cells under differentiating conditions. Related to Figures 3I and 3J. L and M. Validation of LMOD1 OE compared to GFP OE cells after one day of differentiation. Relative Lmod1 expression assessed by qRT-PCR was compared to GFP OE cells and normalized to Gapdh expression levels (L) and immunoblot with quantification of LMOD1 normalized to GAPDH (M). Paired t-test: *: p-value < 0.05. N and O. Quantification of cell populations identified by immunofluorescence staining after LMOD1 OE compared to GFP OE: reserve (Hoechst+/Myogenin-/MHC-) cells, Myogenin+/MHC-cells, Myogenin+/MHC+, fully differentiated myotubes, total counted cells after and myotube length (in µm) (N). Paired t-test: numbers are indicated, ns: not significant. Related to Figures 3N and 3O. P. Gene set enrichment analysis (GSEA) was based on a gene set containing proteins that significantly increased at one day of differentiation compared to 0h/proliferating primary myoblasts (AVG Log2 Ratio > 0.58 and Q value < 0.05, 189 proteins) from the proteomic data generated in (Figure 1A). The GSEA was performed using this gene set on the comparison LMOD1 OE vs. GFP OE at 1 day of differentiation (Table S2). In all bar plots, each symbol or black dot represents a biological replicate, and the error bars indicate the SD.

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 (Table S3) F. Quantification of selected unique interaction partners of LMOD1-BirA* and LMOD2-BirA* in comparison to BirA*-Ctrl. Each symbol represents a biological replicate, error bars indicate the SD. One-way ANOVA, *: p-value < 0.05, **: p-value < 0.01. G. Validation of the SIRT1-LMOD1 interaction. Cells expressing either GFP or SIRT1-GFP were used for co-immunoprecipitation using GFP-trap. The eluates from the GFP-trap were analyzed by immunoblot using antibodies directed to BirA* or GFP. For quantification, BirA* was normalized to SIRT1-GFP intensity. H. Representative images of proximity ligation assay (PLA) (red) in primary myoblasts during proliferation and after one day of differentiation. Scale bar: 10 µm.

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 h 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 h. 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 (Table S3). D. Quantification of selected known interaction partners of LMOD1-BirA* and LMOD2-BirA* in comparison to BirA*-Ctrl. Each symbol represents a biological replicate, and error bars indicate the SD. One-way ANOVA, *: p-value < 0.05, **: p-value < 0.01.

Overexpression of LMOD1 influences the subcellular localization of SIRT1.

A. Representative immunofluorescence staining of LMOD1 and SIRT1 at different timepoints of myogenic differentiation; LMOD1 (purple), SIRT1 (yellow), nuclei (Hoechst, blue). Scale bar: 20 µm. B - 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 timepoints, 3 days proliferation (3dp), 1 day differentiation (1dd), 3 days differentiation (3dd), 5d 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 (0h: undifferentiated/proliferating, 6h, 12h, 24h of differentiated primary myoblasts). SIRT1 signal was analyzed in n = 3 immunoblots and was normalized to Ponceau. In bar plots, each symbol represents a biological replicate, and the error bars indicate the SD. Paired t-test *: p-value < 0.05, ns: not significant.

Sub-cellular localization of LMOD1 and SIRT1 during myogenic differentiation

A - B. Total immunofluorescence intensity (nucleus and cytoplasm) of LMOD1 (A) and SIRT1 (B) during different timepoints of myogenic differentiation. C. Representative immunoblot of the nuclear-cytoplasmic fractionation experiment in primary myoblasts at different timepoints: 2d proliferation, 1d differentiation and 2d differentiation. SIRT1 and LMOD1 signal was analyzed in (n = 3) immunoblots and was normalized to Ponceau. 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 barplots. One-way ANOVA *: p-value < 0.05, ns: not significant. D. Immunoblot showing LMOD1 and SIRT1 protein abundance at different timepoints of myogenic differentiation (0h = proliferating conditions, 6h differentiation, 12h differentiation, 24h differentiation). Quantification of immunoblots for LMOD1 and SIRT1 shown in barplots normalized to GAPDH. For all bar plots, each symbol represents a biological replicate, and the error bars indicate the SD.

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 experimental setup for the RNA-Seq experiment. Primary myoblasts were first seeded and then transfected with siRNA against LMOD1 or SIRT1 or siCtrl. After two days of incubation, the primary myoblasts were transfected a second time with siRNA and induced to differentiate simultaneously. After one day of differentiation, cells were harvested, and RNA was isolated for library preparation and RNAseq analysis. K. Heatmap indicating oppositely differentially expressed SIRT1 target genes from siSirt1 vs siCtrl an siLmod1 vs siCtrl with a log2 FC ratio > 0.58. SIRT1 target genes identified from ChipSeq experiment published in (Table S4) (Ryall et al. 2015). For all bar plots, each symbol represents a biological replicate, and the error bars indicate the SD.

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 three 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 treated 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: reserve (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 one day of differentiation after siCtrl and siLmod1 and siCtrl and siSirtl knockdown (Table S4). Paired t-test *: p-value < 0.05 ***: p-value < 0.001. For all bar plots, each symbol represents a biological replicate, and the error bars indicate the SD. F. Working model: During the proliferation stage, SIRT1 is found in the nucleus, repressing the expression of myogenic regulating factors. However, at the onset of myogenic differentiation, LMOD1 interacts with SIRT1 and affects its subcellular localization, leading to decreased levels of SIRT1 in the nucleus. This reduction in SIRT1 levels results in the de­repression of MRFs and the initiation of differentiation.

Primers for cloning

Primers for qRT-PCR

Antibody-List