Aberrant Myokine Signaling in Congenital Myotonic Dystrophy Masayuki Nakamori, Kohei Hamanaka, James D. Thomas, Eric T. Wang, Yukiko K. Hayashi, Masanori P. Takahashi, Maurice S. Swanson, Ichizo Nishino, Hideki Mochizuki  Cell Reports  Volume 21, Issue 5, Pages 1240-1252 (October 2017) DOI: 10.1016/j.celrep.2017.10.018 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 21, 1240-1252DOI: (10.1016/j.celrep.2017.10.018) Copyright © 2017 The Authors Terms and Conditions

Figure 1 Relationship between Muscle Immaturity and Splicing in CDM Muscles (A) Representative histological images from H&E-stained muscle sections from CDM patients. Left: the mild case shows slight variation in muscle fiber size. Center: the moderate case exhibits a moderate variety in fiber size and some fibers with centrally placed nuclei. Right: all muscle fibers are extremely small and have large nuclei in relation to the cytoplasm in the severe case. Scale bars, 20 μm. (B) The histological grade correlates with the percentage of type 2C fibers. Spearman’s r = 0.88, p = 0.00079. (C) Muscle immaturity and corrected age in CDM muscles. Corrected age in CDM muscles did not correlate with either histological grade (top, Spearman’s r = −0.040, p = 0.912) or the percentage of type 2C fibers (bottom, Pearson’s r = −0.097, p = 0.79). (D) Relationships between alternative splicing in biceps brachii muscles and the percentage of type 2C fibers (top) or histological grade (bottom) in CDM. For each splice event, the fractional inclusion of the indicated exon is shown. Two splicing events (NRAP, left; FXR1, right) strongly correlated with muscle immaturity in CDM. See also Table 1. Cell Reports 2017 21, 1240-1252DOI: (10.1016/j.celrep.2017.10.018) Copyright © 2017 The Authors Terms and Conditions

Figure 2 RNA-Seq Analysis (A) Heatmap of normalized expression values for genes identified as significantly upregulated (red) or downregulated (blue) in SMA (columns 1–3) compared with CDM (columns 4–6) skeletal muscle. (B) Bar graphs displaying the top GO categories showing significant enrichment for upregulated (top) and downregulated (bottom) genes in CDM skeletal muscle. (C) Bar graph displaying expression levels of representative up- and downregulated genes in CDM compared with SMA (the y axis shows log2FC). These genes were selected on the basis of their presence in the “inflammatory response” GO group (red) or the “myogenesis” GO group (blue). (D) Left: read coverage across an example inflammation-associated gene, SAA1, in CDM and controls. In SMA, few genes mapped to this locus, whereas a dramatic number of reads mapped in CDM. Right: box plots comparing the expression of SAA1 in SMA and CDM with adult control, DM1 proto-mutation, and DM1 skeletal muscle (tibialis anterior). Cell Reports 2017 21, 1240-1252DOI: (10.1016/j.celrep.2017.10.018) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Aberrant Activation of the IL-6 Signaling Pathway in CDM Muscles (A) Schematic of IL-6-induced signal transduction. The complex of IL-6 and its receptor (IL-6R) binds to the transmembrane protein IL6ST homodimer, which transmits the signal intracellularly. The major downstream signaling uses the STAT3 pathway. Activated STAT3 migrates to the nucleus and promotes transcription of target genes, such as SAA1 and MYC. (B) Relationship between expression of components in the IL-6 pathway and muscle immaturity in CDM. The histological grade (left) and percentage of type 2C fibers (right) correlated with the expression of IL6 (Spearman’s r = 0.77, p = 0.016; Pearson’s r = 0.59, p = 0.093, respectively), IL6R (r = 0.78, p = 0.014; r = 0.83, p = 0.0059, respectively), IL6ST (r = 0.95, p = 7.2E−5; r = 0.74, p = 0.022, respectively), STAT3 (r = 0.91, p = 0.00059; r = 0.83, p = 0.0022, respectively), SAA1 (r = 0.84, p = 0.0046; r = 0.95, p = 8.2E−5, respectively), and MYC (r = 0.75, p = 0.020; r = 0.93, p = 0.00025, respectively) normalized to 18S rRNA. (C) IL6 mRNA and protein levels were assayed in human myoblasts with or without ASO treatment by real-time RT-PCR (left) and ELISA (right), respectively. The expression levels of both IL6 mRNA and protein were higher in CDM myoblasts than in control myoblasts. ASO treatment reduced both IL6 mRNA and protein in CDM but not in control myoblasts. Data from ASO-treated and untreated myoblasts from the same patient are connected by a dotted line. (D) Myoblast differentiation was measured by fusion index. Compared with no treatment (no-Tx), ASO and anti-IL-6 antibody treatments improved the differentiation defects in CDM myoblasts. Data are presented as means ± SD. ∗p < 0.05, t test. Cell Reports 2017 21, 1240-1252DOI: (10.1016/j.celrep.2017.10.018) Copyright © 2017 The Authors Terms and Conditions

Figure 4 CpG Methylation Profiles around CTG Repeats in CDM Muscles (A) Positive correlation between repeat length and histological grade (left, r = 0.91, p = 0.00026) or percentage of type 2C fibers (right, r = 0.86, p = 0.0014) in CDM muscles. (B) Heatmap of methylation levels (black, 50% methylation; white, 0% methylation) at CpG sites in the DMPK 3′ UTR. Each row indicating CDM or SMA is from a different patient. CTCF binding sites are indicated by red boxes. (C) Cluster dendrogram of CpG methylation status (high, medium, and low) at the CTCF-I site in CDM muscles (left). Shown is the relationship between CpG methylation status and repeat size in CDM muscles (right, r = 0.62, p = 0.058). (D) Positive correlation between CpG methylation status and histological grade (r = 0.73, p = 0.016) in CDM muscles. (E) Relationship between CpG methylation status and sense transcription (left, r = 0.77, p = 0.0016) or antisense transcription (right, r = −0.68, p = 0.049) at the DMPK 3′ UTR, quantified by strand-specific real-time PCR. Cell Reports 2017 21, 1240-1252DOI: (10.1016/j.celrep.2017.10.018) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Short CAG Repeat RNA Production in CDM Muscles (A) Upregulation of short CAG repeat RNA expression in CDM muscles compared with SMA muscles (p = 0.034, Mann-Whitney U test). (B) Relationship between the expression level of short CAG repeat RNA normalized to RNU6B and CpG methylation status at the CTCF binding site (left, r = −0.87, p = 0.0025) or histological grade (right, r = −0.72, p = 0.028) in CDM muscles. (C) Negative correlation between expression levels of short CAG repeat RNA and CUG-containing transcripts, PAPSS2 (left, r = −0.74, p = 0.022) and CASK (right, r = −0.85, p = 0.0042), in CDM muscles. (D) Positive correlation between the expression level of short CAG repeat RNA and alternative splicing in CAPZB (left, r = 0.95, p = 8.7E−5, FDR q < 0.01) and CACNA1S (right, r = 0.87, p = 0.0021, FDR q < 0.05) in CDM muscles. Cell Reports 2017 21, 1240-1252DOI: (10.1016/j.celrep.2017.10.018) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Proposed Mechanism of Muscle Immaturity in CDM Large repeat expansion since the embryonic stage causes hypermethylation at the CTCF-I site and reduction in CTCF binding at the DMPK locus. The reduction in CTCF binding promotes alternative sense transcription and suppresses antisense transcription and production of short CAG repeat RNA that neutralizes CUGexp, resulting in enhanced RNA toxicity by CUGexp. Enhanced RNA toxicity causes activation of PKR and induction of ER stress and provokes NF-κB activation, leading to activation of the IL-6/STAT3 signaling pathway. The upregulation of IL-6 and its target genes contributes to muscle immaturity in CDM. Cell Reports 2017 21, 1240-1252DOI: (10.1016/j.celrep.2017.10.018) Copyright © 2017 The Authors Terms and Conditions