Recovery in the Myogenic Program of Congenital Myotonic Dystrophy Myoblasts after Excision of the Expanded (CTG)n Repeat

Figures

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  Figure 1. Myogenic fusion capacity of myoblasts with and without the expanded (CTG)2600 repeat. (A) Images of myotube formation after five days of differentiation in myoblast lineages with (4F9) and without (3B11) the (CTG)2600 repeat expansion. Myosin heavy chain (MHC) staining is shown in green, whilst DAPI is shown in magenta. Bar = 100 µm. (B) Fusion index: Each data point represents the mean of three independent differentiation experiments for one myoblast lineage (mean ± SEM). (C) Group-wise microscopic quantification of the mean grey values of MHC staining in immunofluorescent images of cells with and without a repeat. (D) Western blot visualization of MHC protein levels in proliferating myoblast cultures (day -2), at the start (day 0), and on day 5 of differentiation. (E) Quantification of MHC expression at day 5 by western blot analysis of extracts from two myoblast lineages with and two lineages without the (CTG)2600 repeat. Averaged MHC signal intensities (arbitrary units) determined from three independent differentiation experiments are shown (mean ± SEM). * p < 0.05, ** p < 0.01, *** p < 0.001 (two-way ANOVA). (F–H) Morphotyping of myotubes formed from myoblasts with and…

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  Figure 2. Gene expression of myotonic dystrophy type 1 (DM1) locus genes in proliferating myoblasts with and without the (CTG)2600 repeat. (A) Expression of the six genes flanking DMPK/DM1-AS (size and location on the scale depicted in blue arrows) was determined by RNA-seq analysis of poly(A)-containing RNA prepared from proliferating myoblasts with (red) and without (grey) the (CTG)2600 repeat. Based on single nucleotide polymorphism (SNP) sequence information, the contribution of the two alleles of each gene (on the DM1 chromosome or the unaffected chromosome 19) could be independently determined. It was assumed that methylation of the repeat-expanded allele decreased the expression of, e.g., DM1 protein kinase (DMPK) and SIX5 from this allele [34]. (None of the genes featured significant differential expression levels before and after excision of the (CTG•CAG)n repeat.) (B) DMPK expression levels, as determined via RT-qPCR. Each data point shows the expression (mean ± SEM) in four cell lines with and without the (CTG)2600 repeat. * p < 0.05, *** p< 0.001 (two-way ANOVA).

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  Figure 3. Excision of the (CTG)2600 repeat differentially alters the temporal expression of drivers and markers of myogenic differentiation. (A) The experimental set-up for the analysis of mRNA levels for myogenic transcription factors and structural proteins before and during myoblast–myotube differentiation in cells with and without the (CTG)2600 repeat. Time points for sampling and RT-qPCR analysis are indicated (top). Anticipated changes in the expression of muscle-specific factors are depicted (bottom). (B–E) RNA expression for myogenic transcription factors (“drivers”) MYOD, MYOG, MYF5, and PAX7 (in arbitrary units). (F) RNA expression for the MEF2D + β-exon factor isoform. (G–I) RNA expression for structural muscle proteins (myogenic progression markers) DMD, MHCp, and MHCe. Each data point shows the mean ± SEM of the four different cell lines either with or without the (CTG)2600 repeat expansion. *p < 0.05, **p < 0.01, ***p < 0.001 (two-way ANOVA).

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  Figure 4. Alternative splicing patterns for early markers in myotonic dystrophy type 1 (DM1) myoblasts change from embryonic-to-adult mode after (CTG)2600 repeat removal. Analysis of RNA-seq reads across alternatively used splice junctions in transcripts from eight mis-spliced DM1-related genes in myoblasts shows the reversal of aberrant splicing after (CTG)2600 repeat removal. Each data point represents one cell line with or without the (CTG)2600 repeat. ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA).

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  Figure 5. RNA-seq analysis shows few changes in RNA expression in cells with a (CTG)2600 repeat. Volcano plot showing global transcriptional changes in cells with a (CTG)2600 repeat. The log2-fold change in expression is represented on the x-axis. The y-axis shows the -log10 of the p-value. A p-value of 0.05 and a fold change of 1.5 are indicated by the black dashed lines. All 15,960 genes found in the eight cell lines are plotted. Each circle represents one gene. Twenty-four genes significantly and >1.5-fold downregulated in cell lines with the (CTG)2600 repeat are shown in blue, and 28 genes significantly and >1.5-fold upregulated in cell lines with the repeat are shown in red.

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  Figure 6. Proteomic analysis of myoblasts with and without the (CTG)2600 repeat. Volcano plot showing global changes in protein abundance in cells with a (CTG)2600 repeat. The log2 fold change is represented on the x-axis. The y-axis shows the -log10 of the p-value. A p-value of 0.05 and a fold change of 1.5 are indicated by the black dashed lines. All 5838 proteins detected in the eight cell lines are plotted. Each circle represents one protein. Proteins significantly and > 1.5-fold less abundant in cell lines with the (CTG)2600 repeat are shown in blue, and all proteins significantly and > 1.5-fold more abundant are shown in red.