Lamin A/C protects chromatin accessibility during mechanical loading in human skeletal muscle

Fig. 1. Characterisation of lamin A/C deficient myotubes. a Experimental timeline. In vitro muscle differentiation recapitulates myoblast fusion to form multinucleated cells named myotubes. Confluent human muscle progenitors were induced to differentiate into multinucleated myotubes, and treated with siRNA targeting LMNA at the onset of differentiation. Acute equibiaxial mechanical stretch (4 h), if any, was applied immediately before harvest at 72 h. b-d Validation of know-down efficiency by (b) western-blot analysis, (c) RT‑qPCR quantification (mean ± SD, p < 0.01; n ≥ 3), (d) immunostaining of Lamin A/C. e 72 h differentiated myotubes stained for myosin heavy chain (MF20, in green). Nuclei are stained with Hoechst (blue). Bar scale = 150 µm. f Quantification of fusion index was determined in 72 h differentiated present in a given field in scrambled (Ctl) and lamin A/C depleted (siLMNA) myotubes (mean ± SD, ** p < 0.01). Lamin A/C depletion significantly altered the fusion index. g Representative Hoechst staining images illustrating nuclear morphology in Ctl and siLMNA myotubes under static condition (scale bar = 5µm). h Lamin B1 immunostaining showing nuclear envelope in scrambled…

Fig. 2. ATAC-seq data in Ctl and siLMNA myotubes. a Volcanoplot illustrating differentially accessible regions (DARs) between Ctl and siLMNA myotubes in static condition. Criteria for significance: log twofold change > 0.5 and false discovery rate (FDR) < 0.05. b Over-representation analysis (ORA) of DARs in siLMNA vs Ctl myotubes. c Histogram depicting the distribution of significant up and down-regulated DARs relative to genomic features in siLMNA vs Ctl myotubes. d Chromosomal distributions of DARs adjusted for gene density and their annotations in Ctl and siLMNA myotubes under static condition

Fig. 3. RNA-seq analysis of differential gene expression in Ctl and siLMNA myotubes. a Volcanoplot illustrating differentially expressed genes (DEGs) between Ctls and LMNA-deficient myotubes (log2 fold change > 1, FDR < 0.05). Up-regulated genes are in orange, down-regulated genes are in blue and non-significant deregulated genes are in grey. b RT-qPCR validation of key DEGs, confirming the RNA-seq results. Expression levels are normalized to housekeeping genes and presented as mean ± SD. n = 6 per condition from 2 independent experiments, ** p < 0.01. c Over-representation analysis (ORA) of significant DEGs respecting threshold in siLMNA vs Ctl myotubes. d Integration of DEGs and DARs respecting FDR < 0.05 threshold in Ctl and siLMNA myotubes. e Subset of the concordant analysis to promoter regions in Ctl and siLMNA myotubes

Fig. 4. Histone marks in A-type lamin deficient (siLMNA) myotubes. a, b Representative images showing immunofluorescence staining of euchromatin marks H3K4me3 (a, red) and H3K4ac (b, green) in Ctl and siLMNA myotubes under static condition. Nuclei are stained with Hoechst (blue). c, d Violin plots quantifying the nuclear intensity of H3K4me3 and H3K4ac nuclear intensity in static condition (Ctl vs siLMNA). n > 150 per condition, **** p < 0.0001. e, f Representative images showing immunofluorescence staining of the heterochromatin marks H3K9me3 (green) and H3K27me3 (green) in myonuclei from Ctl and siLMNA myotubes under static condition. Nuclei are stained with Hoechst (blue). g-h Violin plots quantifying the nuclear intensity of H3K9me3 and H3K27me3 in static myotubes (Ctl and siLMNA). N > 150 per condition, **** p < 0.0001. The solid bars in each violin plots represent the median, the 25th and the 75th percentiles lines mark respectively

Fig. 5. Nuclear abnormalities in A-type lamin deficient (siLMNA) myotubes following mechanical stretch (S-Ctl and S–si-LMNA). a Representative images of nuclei in Ctl and siLMNA myotubes following mechanical stretch. Nuclei are stained with Hoechst. b Schematic illustrating nuclear shape (insert, width vs length of the nucleus), and violin plots quantifying the nuclear aspect ratio in Ctl and siLMNA myotubes under static condition (Ctl and siLMNA) and following mechanical stretch (S-Ctl and S-siLMNA). Data represent n > 50 nuclei from 3 independent experiments per group, *** p < 0.005; **** p < 0.001. c Violin plots quantifying the nuclear area (in µm2) in Ctl and siLMNA myotubes under static condition (Ctl and siLMNA) and after stretching (S-Ctl and S-siLMNA). Data represent n > 50 nuclei from 3 independent experiments per group, ** p < 0.01; *** p < 0.005; **** p < 0.001. d Representative images of YAP immunostaining in Ctl and siLMNA myotubes under static condition and following mechanical stretch. Bar scale = 10 µm. e Violin plots of YAP nucleo-cytoplasmic (N/C) ratio in static condition (Ctl and siLMNA) and after stretching (S-Ctl and S-siLMNA). Data represent n = 300 nuclei from 3…

Fig. 6. Chromatin accessibility and transcriptomic deregulation after mechanical stretching in Ctl and siLMNA myotubes. a Volcanoplot of differentially accessible regions (DARs) in Ctl myotubes following mechanical stretch (S-Ctl versus Ctl) (log twofold change > 0.5, FDR < 0.05). b Volcanoplot of DARs in siLMNA myotubes following mechanical stretch (S-siLMNA versus siLMNA) (log twofold change > 0.5, FDR < 0.05). c Over-representation analysis (ORA) of significant DARs identified in S-siLMNA vs siLMNA. d Volcanoplot of DEGs in Ctl myotubes following mechanical stretch (S-Ctl versus Ctl) (log twofold change > 0.5, FDR < 0.05). e Volcanoplot of DEGs in S-siLMNA versus siLMNA) (log twofold change > 0.5, FDR < 0.05). f ORA analysis of significant DEGs respecting threshold in S-siLMNA versus siLMNA

Fig. 7. Correlation chromatin accessibility and gene expression in Ctl and siLMNA myotubes after mechanical stretching. a Correlation analysis between DEGs and DARs (FDR < 0.05) in siLMNA myotubes under static and after stretch conditions. Each dot represents a gene significantly differentially expressed and associated with chromatin accessibility changes b Subset analysis focusing on DARs in promoter regions under static and stretched in siLMNA myotubes

Fig. 8. Stretch highly affects chromatin in LMNA deficient myotubes. a comparing stretched siLMNA and Ctl (S-siLMNA vs. S-Ctl). b Histogram showing the distribution of significantly up- and down-regulated DARs relative to genomic annotations in stretched Ctl and siLMNA. c Chromosomal peak distributions of DARs normalized by gene density and annotated by genomic region in stretched Ctl and siLMNA myotubes. d ORA analysis of DARs in S-siLMNA vs S-Ctl myotubes

Fig. 9. Histone modifications in A-type lamin deficient (siLMNA) myotubes following mechanical stretch a, b Representative images of immunofluorescence staining for H3K4me3 (a, red) and H3K4ac (b, green) in Ctl and siLMNA myotubes after stretching. Nuclei are stained with Hoechst (blue). c, d Violin plots showing nuclear H3K4me3 and H3K4ac nuclear intensity after stretching (S-Ctl and S–si-LMNA). N > 150 per condition, **** p < 0.0001. e, f Representative images of immunofluorescence staining for H3K9me3 (e, green) and H3K27me3 (f, green) in Ctl and siLMNA myotubes after stretching. Nuclei are stained with Hoechst (blue). g, h Violin plots showing nuclear intensity of H3K9me3 and H3K27me3 in S-Ctl and S-siLMNA). Data represent N > 150 per condition, **** p < 0.0001. The solid bars in each violin plots represent the median, the 25th and the 75th percentiles lines mark respectively