Article
Aberrant Myokine Signaling in Congenital Myotonic Dystrophy Graphical Abstract
Authors Masayuki Nakamori, Kohei Hamanaka, James D. Thomas, ..., Maurice S. Swanson, Ichizo Nishino, Hideki Mochizuki
Correspondence
[email protected]
In Brief Congenital myotonic dystrophy (CDM) manifests characteristic genetic (very large CTG repeat expansions), epigenetic (CpG hypermethylation upstream of the repeat), and phenotypic (muscle immaturity) features not seen in adult DM. Nakamori et al. find phenotype-genotype and epigenotype correlation in CDM muscle and reveal involvement of the IL-6 myokine signaling pathway in the disease process.
Highlights d
CTG repeat size and CpG methylation status correlate with disease severity in CDM
d
Aberrant methylation associates with dysregulated transcription around the repeat
d
Transcriptional dysregulation in both directions enhances CUGexp RNA toxicity
d
Enhanced RNA toxicity upregulates IL-6 myokine signaling pathway in CDM muscle
Nakamori et al., 2017, Cell Reports 21, 1240–1252 October 31, 2017 ª 2017 The Authors. https://doi.org/10.1016/j.celrep.2017.10.018
Data and Software Availability GSE97806
Cell Reports
Article Aberrant Myokine Signaling in Congenital Myotonic Dystrophy Masayuki Nakamori,1,6,* Kohei Hamanaka,2 James D. Thomas,3 Eric T. Wang,3 Yukiko K. Hayashi,4 Masanori P. Takahashi,1,5 Maurice S. Swanson,3 Ichizo Nishino,2 and Hideki Mochizuki1 1Department
of Neurology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan 3Department of Molecular Genetics and Microbiology, Center for NeuroGenetics and the Genetics Institute, University of Florida, College of Medicine, Gainesville, FL 32610, USA 4Department of Pathophysiology, Tokyo Medical University, Shinjuku, Tokyo 160-0022, Japan 5Department of Functional Diagnostic Science, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan 6Lead Contact *Correspondence:
[email protected] https://doi.org/10.1016/j.celrep.2017.10.018 2Department
SUMMARY
Myotonic dystrophy types 1 (DM1) and 2 (DM2) are dominantly inherited neuromuscular disorders caused by a toxic gain of function of expanded CUG and CCUG repeats, respectively. Although both disorders are clinically similar, congenital myotonic dystrophy (CDM), a severe DM form, is found only in DM1. CDM is also characterized by muscle fiber immaturity not observed in adult DM, suggesting specific pathological mechanisms. Here, we revealed upregulation of the interleukin-6 (IL-6) myokine signaling pathway in CDM muscles. We also found a correlation between muscle immaturity and not only IL-6 expression but also expanded CTG repeat length and CpG methylation status upstream of the repeats. Aberrant CpG methylation was associated with transcriptional dysregulation at the repeat locus, increasing the toxic RNA burden that upregulates IL-6. Because the IL-6 pathway is involved in myocyte maturation and muscle atrophy, our results indicate that enhanced RNA toxicity contributes to severe CDM phenotypes through aberrant IL-6 signaling. INTRODUCTION Myotonic dystrophy is one of the most common types of muscular dystrophy, with an estimated prevalence of 1:8,000 (Harper, 2001). This systemic disease presents with multiple symptoms, including myotonia, progressive muscle weakness, insulin resistance, and cardiac conduction defects. Myotonic dystrophy (DM) has two genetically distinct forms: DM type 1 (DM1) is caused by the expansion of a CTG repeat in the 30 UTR of DMPK, whereas DM type 2 (DM2) results from an expansion of CCTG repeats in the first intron of CNBP. Both mutations give rise to toxic RNAs that contain tandem repeats: an expanded CUG repeat in DM1 and a CCUG repeat in DM2 (Ranum and Cooper, 2006). The toxic RNA alters the
activity of RNA binding proteins involved in alternative splicing, such as muscleblind-like (MBNL) and CUG-binding protein 1 (CELF1), and perturbs the regulation of pre-mRNA splicing in affected tissues. So far, more than 100 misregulated splicing events have been reported in DM muscles, some of which correlate with muscle weakness in adult DM1 (Nakamori et al., 2013). Congenital DM1 (CDM) is an early severe form of DM1, presenting at birth with hypotonia and generalized muscle weakness, which results in difficulty swallowing and breathing (Harper, 2001). Some CDM patients present symptoms even in utero, such as reduced fetal movement and hydramnios later in pregnancy. Despite neonatal intensive support, mortality from respiratory failure remains high in affected infants. Although DM1 and DM2 share common features, such as clinical symptoms, toxic RNA gain of function, and splicing misregulation, CDM is only found in DM1. Furthermore, CDM might not merely be an early severe form of DM1 but, rather, it exhibits characteristic features not seen in either adult DM1 or DM2. CDM patients have extremely expanded CTG repeats at an early disease stage and highly methylated CpG sites around the repeat tract, especially in muscles (Barbe´ et al., 2017; Harley et al., 1993; Lo´pez Castel et al., 2011; Steinbach et al., 1998; Yanovsky-Dagan et al., 2015). Histology of CDM skeletal muscle exhibits severe muscle fiber immaturity (i.e., intense atrophic muscle fibers, less necrotic or regenerating fibers, and an increase in undifferentiated type 2C fibers), and CDM myoblasts show a delay in maturation (Farkas-Bargeton et al., 1988; Furling et al., 2001). These differences from adult DM1 and DM2 strongly suggest specific pathological mechanisms governing CDM features. Especially, there remain significant and unanswered questions: why do only expanded CTG repeats in the DMPK and not CCTG repeats in the CNBP gene result in CDM, how do highly methylated CpGs around the expanded repeats contribute to the disease process, and what causes severe muscle immaturity in CDM? To investigate the specific mechanisms responsible for CDM and address these questions, we performed a comprehensive study dissecting the histology, repeat size, CpG methylation, alternative splicing, and gene expression of CDM skeletal muscles and cell models.
1240 Cell Reports 21, 1240–1252, October 31, 2017 ª 2017 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
RESULTS Muscle Immaturity and Splicing Defects in CDM One of the most characteristic features in CDM is muscle immaturity, which is not observed in adult DM1 (Nonaka et al., 1996). To reveal the mechanism of muscle immaturity, we studied skeletal muscle tissues (biceps brachii) from ten CDM patients with a corrected age of less than 15 months. We used muscles from four age-matched patients with spinal muscular atrophy (SMA) as disease controls. SMA muscles show less degeneration and immune cell infiltration similar to CDM and also present muscle immaturity because of defective innervation (Tews and Goebel, 1997). Therefore, a comparison with SMA enables us to focus on a disease-specific mechanism of muscle immaturity in CDM rather than the secondary changes. First, we evaluated muscle immaturity in CDM muscles and classified them into three categories in accordance with histological grade: mild, moderate, and severe changes (Figure 1A). We also counted the percentage of undifferentiated type 2C fibers as another marker of immaturity. As expected, the histological grade correlated with the percentage of type 2C fibers in CDM muscles (Spearman’s r = 0.88, p = 0.00079; Figure 1B). The corrected age of patients at biopsy or autopsy did not correlate with either histological grade or percentage of type 2C fibers, indicating that both indices represent pathological, rather than physiological, immaturity (Figure 1C). Misregulated alternative splicing is a fundamental molecular feature of DM, affecting many genes involved in muscle homeostasis and function (Osborne and Thornton, 2006). Some misregulated splicing events displayed graded changes that correlated with muscle weakness in adult DM1 (Nakamori et al., 2013). To determine whether a similar relationship with muscle immaturity is observed in CDM, we studied splicing abnormalities of 38 splicing events that are well correlated with muscle weakness in adult DM1. Some of these mis-splicing events showed a tendency toward correlation with the histological grade or percentage of type 2C fibers in CDM (Table 1). Of these, NRAP and FXR1 splicing showed significant correlation with the percentage of type 2C fibers after correction of multiple testing (Pearson’s r = 0.95, p = 0.00012, false-discovery rate [FDR] q < 0.05; r = 0.90, p = 0.00093, FDR q < 0.05, respectively; Figure 1D). However, previous studies reported that misregulation of NRAP and FXR1 splicing was observed in other muscle diseases and damaged muscles, suggesting that the splicing change might be non-specific or reflect muscle damage (Bachinski et al., 2014; Orengo et al., 2011). Aberrant Activation of the Interleukin-6 Myokine Pathway in CDM To identify other factors related to muscle immaturity rather than splicing defects, we studied how gene expression is affected in severe CDM muscles by RNA sequencing (RNA-seq) analysis (Figure 2A). We compared gene expression with that in SMA, as we did in our previous study for identifying splicing defects (Thomas et al., 2017), by using gene ontology (GO) analysis to systematically characterize the biological processes affected in severe CDM (Figure 2B). As expected, genes showing decreased expression showed significant enrichment for the
terms ‘‘myogenesis’’ and ‘‘muscle structure development.’’ From the GO analysis, we also observed a significant upregulation of the biological processes associated with inflammation in CDM muscles. One of the largest changes observed was found in the serum amyloid A1 gene (SAA1) (Figure 2C). SAA1 was among the most dramatically increased genes in CDM muscle, but it was negligibly detected in any adult DM1 dataset (Figure 2D). Although it was not the largest log2FC, its p.adj value was incredibly high, representing a highly reproducible event. Because expression of SAA1 as well as SAA2 is regulated by interleukin-6 (IL-6), we focused on the IL-6 pathway, which reportedly contributes to satellite cell differentiation and muscle wasting and atrophy (Mun˜oz-Ca´noves et al., 2013). The components of the IL-6 pathway, such as IL6, IL6R, IL6ST, and STAT3, as well as target genes of IL-6/STAT3 signaling, such as SAA1 and MYC, were upregulated in CDM, and the expression levels significantly correlated with muscle immaturity (Figures 3A and 3B). Next, we evaluated IL6 expression in human primary myoblasts derived from different patients with CDM (n = 3) and control individuals (n = 3). Increased expression of IL6 was also observed in all lines of CDM myoblasts (Figure 3C). Because CDM muscles were too small to allow for protein analysis, we evaluated IL-6 activity by ELISA in CDM myoblasts and confirmed increased expression of IL-6 protein (Figure 3C). In contrast, the expression of IL6 in muscles from adult DM1, Duchenne muscular dystrophy, amyotrophic lateral sclerosis, or polymyositis patients was 104-fold lower than in CDM or less than the threshold of detection by real-time PCR (data not shown). Although IL-6 is a major cytokine derived from muscles and is known as a ‘‘myokine,’’ macrophages and T and B lymphocytes can also produce IL-6 (Mun˜oz-Ca´noves et al., 2013). To exclude the possibility that increased IL-6 in CDM was due to infiltration of inflammatory cells, we performed immunohistochemistry by using macrophage and T and B lymphocyte markers. Infiltration of these inflammatory cells in CDM muscles was not higher than that in disease controls (Figure S1; Table S2), strongly indicating that elevated IL-6 in CDM was produced in the muscles. In addition, we evaluated the mRNA expression of fibrosis and inflammatory cell makers (COL1A1, COL1A2, CD68, ITGAX, MPEG1, CD4, CD8A, CD19, and MS4A1) by real-time PCR and verified that these markers were not increased in CDM muscles (data not shown). To demonstrate an involvement of IL-6 in CDM pathogenesis, we subsequently performed treatment studies in CDM myoblasts by antisense oligonucleotides (ASOs) targeting expanded CUG repeats and anti-IL6 antibodies. ASO treatment reduced both IL6 mRNA and protein expression in all CDM myoblast lines (Figure 3C), and the anti-IL-6 antibody as well as the ASO improved the impaired muscle differentiation capacity in CDM myoblasts (Figure 3D). Phenotype-Genotype and Epigenotype Correlation in CDM The query that arises is why the IL-6 pathway is activated in CDM and not in adult DM1. To investigate the specific mechanism, we focused on characteristic genetic and epigenetic features in CDM. We studied repeat length in CDM muscles, which have extremely large CTG repeat expansions at an early disease stage Cell Reports 21, 1240–1252, October 31, 2017 1241
B
mild
moderate
% type 2C fibers
A
severe
80
r = 0.88
60 40 20 0
mild
moderate severe
Histological grade D
20 0
45
0
FXR1
r = -0.95 % ex15,16 inclusion
40
(% normal isoform)
90 % ex12 inclusion
(weeks)
Corrected age
NRAP
r = -0.04
60
0
40
100 (% normal isoform)
C
60
20
80
r = -0.90
% type 2C fibers
0
40
80
% type 2C fibers
Histological grade
20
% ex15,16 inclusion
40
(% normal isoform)
% ex12 inclusion
(weeks)
Corrected age
r = -0.79
90
45
0
0 0
20
40
60
80
SMA
% type 2C fibers
r = -0.87
100 (% normal isoform)
r = -0.097 60
60
20
SMA CDM
CDM
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 mm. (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.
(Ashizawa et al., 1993). Although expanded repeat length in blood cells inversely correlates with the age of onset in DM1 (Harley et al., 1993), our previous study showed that repeat size in adult DM1 muscles does not correlate with muscle weakness (Nakamori et al., 2013). However, in CDM muscles, repeat size significantly correlated with histological grade (Spearman’s r = 0.91, p = 0.00026) and percentage of type 2C fibers (Pearson’s r = 0.86, p = 0.0014) (Figure 4A; Figure S2A). Although muscle groups assessed in adult DM1 (tibialis anterior) and 1242 Cell Reports 21, 1240–1252, October 31, 2017
CDM (biceps brachii) were different, these results suggested that expanded repeat size at an early disease stage itself plays a significant role in muscle immaturity in CDM. Highly expanded CTG repeats in DM1 were suggested to associate with aberrant CpG methylation around the repeat tract (Brouwer et al., 2013; Santoro et al., 2015; Steinbach et al., 1998; Yanovsky-Dagan et al., 2015). Previous studies reported hypermethylated CpG sites upstream of the expanded repeats in CDM blood cells and tissues, with little such methylation in adult DM1
Table 1. Correlation Coefficient between Splicing Abnormalities and Muscle Immaturity Histological Grade
% Type 2C Fibers
Gene
Spearman’s R
p Value
Pearson’s R
p Value
NRAP
0.794
1.1E2
0.946
1.2E4a
FXR1
0.867
2.5E3a
0.900
9.3E4a
CAMK2B
0.767
1.6E2
0.825
6.2E3
BIN1 exon 11
0.767
1.6E2
0.806
8.8E3
DMD exon 78
0.913
5.9E4a
0.795
1.1E2
DMD exon 71
0.083
8.3E1
0.740
2.3E2
MYOM1
0.356
3.5E1
0.724
2.8E2
CACNA1S
0.694
3.8E2
0.706
3.3E2
NFIX
0.548
1.3E1
0.671
4.8E2
MLF1
0.621
7.4E2
0.669
4.9E2
KIF13A
0.402
2.8E1
0.667
5.0E2
MBNL2
0.575
1.1E1
0.616
7.7E2
PHKA1 exon 28
0.575
1.1E1
0.590
9.4E2
ANK2
0.429
2.5E1
0.590
9.5E2 1.0E1
VPS39
0.822
6.6E3
0.578
GFPT1
0.411
2.7E1
0.572
1.1E1
PDLIM3
0.329
3.9E1
0.556
1.2E1
CAPZB
0.621
7.4E2
0.538
1.4E1
NCOR2
0.329
3.9E1
0.509
1.6E1
SORBS1
0.393
3.0E1
0.506
1.6E1
IMPDH2
0.621
7.4E2
0.491
1.8E1
BIN1 exon 7
0.329
3.9E1
0.481
1.9E1
LDB3
0.356
3.5E1
0.410
2.7E1
PHKA1 exon 19
0.475
2.0E1
0.332
3.8E1
MBNL1
0.329
3.9E1
0.294
4.4E1
ATP2A1
0.183
6.4E1
0.145
7.1E1
ARFGAP2
0.356
3.5E1
0.073
8.5E1
SOS1
0.329
3.9E1
0.002
1.0E+0
PHKA2
0.073
8.5E1
0.079
8.4E1
TTN
0.041
9.2E1
0.107
7.9E1
COPZ2
0.256
5.1E1
0.114
7.7E1
INSR
0.137
7.3E1
0.242
5.3E1
CLCN1
0.064
8.7E1
0.274
4.8E1
ABLIM2
0.511
1.6E1
0.647
5.9E2
ALPK3
0.393
3.0E1
0.731
2.5E2
DTNA (DB1)
0.511
1.6E1
0.784
1.2E2
DTNA (DB2)
0.749
2.0E2
0.812
7.9E3
MYBPC1
0.420
2.6E1
0.822
6.6E3
FDR q < 0.05.
a
or unaffected individuals (Barbe´ et al., 2017; Steinbach et al., 1998). Lo´pez Castel et al. (2011) studied the methylation status in various CDM tissues; however, a specific methylation pattern has not been detected in CDM muscles. To study CpG methylation at the DMPK locus and its relation to disease severity in CDM muscles, we performed a detailed analysis by using next-generation sequencing following bisulfite modification and quantified the methylation status of each CpG site around
the repeat tract. As reported previously, the upstream region of the CTG repeat tract was highly methylated in CDM muscles whereas the downstream region was not (Figure 4B). In SMA muscles, only minimal methylation was observed around the repeat tract, which is similar in adult DM1 skeletal muscles, as reported in the former study (Lo´pez Castel et al., 2011). Previously, Filippova et al. (2001) identified two binding sites for CTCF, a multifunctional transcription regulator known to possess insulator activity (Phillips and Corces, 2009), in a flanking region of the CTG repeats in DMPK (CTCF-I, upstream of the repeat; CTCF-II, downstream of the repeat). They also revealed that CpG methylation of these sites prevents binding of CTCF, affecting chromatin dynamics (Cho et al., 2005; Filippova et al., 2001). Because the CpG methylation status in the CTCF binding site upstream of the repeat was quite variable among CDM patients in our study, CpGs at the CTCF-I site were highly methylated in CDM myoblasts (Figure S2B), and whether CTCF binds to the CTCF-II site is controversial (Yanovsky-Dagan et al., 2015), we focused on the CTCF-I region and performed a cluster analysis for hypermethylation status to determine whether CpG methylation relates to pathogenesis. We found a tendency for higher CpG methylation in the CTCF binding site with longer repeat size (Spearman’s r = 0.62, p = 0.058; Figure 4C) and a more severe histological grade (r = 0.73, p = 0.016; Figure 4D). Several previous studies reported a decreased level of DMPK and adjacent gene expression in CDM because of altered chromatin dynamics (Frisch et al., 2001; Klesert et al., 1997; Thornton et al., 1997). One might imagine that altered expression of these genes contributes to muscle immaturity in CDM. To test whether the suppression of total DMPK and neighboring genes relates to muscle immaturity, we assessed the expression of total DMPK, transcribed from its canonical promoter, and its two adjacent genes, SIX5 and DMWD. However, the expression levels were not correlated with either muscle immaturity in CDM muscles (Figure S3) or methylation status at the CTCF binding site (data not shown). Therefore, it is unlikely that suppression of total DMPK or adjacent genes contributes to muscle immaturity in CDM. Aberrant CpG Methylation and Transcriptional Dysregulation at the Repeat Locus CpG methylation at CTCF binding sites has been extensively studied in the ATXN7 locus (Sopher et al., 2011). Similar to DMPK, there are two CTCF binding sites flanking the CAG$CTG repeats in ATXN7, expansion of which causes spinocerebellar ataxia type 7. In addition, bidirectional transcription around the repeats occurs in both DMPK and ATXN7 loci. Hypermethylation of CpG at the CTCF binding sites in ATXN7 results in reduced binding with CTCF, activating an adjacent alternative sense promoter upstream of the repeats and suppressing an antisense promoter (Sopher et al., 2011). In line with such evidence, similar transcriptional regulation by CTCF could occur in the DMPK locus. To investigate this possibility, we first checked whether an adjacent alternative promoter exists upstream of the CTG repeats in the DMPK locus. Bioinformatics analysis using the PROSCAN database (Prestridge, 1995) predicted the existence of an alternative promoter upstream of the CTG repeat tract, and a previous study also suggested regulatory activity in this Cell Reports 21, 1240–1252, October 31, 2017 1243
B
control
CDM
Down-regulated
0
-1.6 -0.8 0.0 0.8 1.6 Z score
inflammatory response acute-phase response cellular response to zinc ion hypoxia chemical homeostasis carboxylic acid transport wound healing response to lipopolysaccharide
Up-regulated
A
4
8 12 -log10(P)
16
myogenesis muscle structure development heart contraction syndecan 1 pathway sarcoplasmic reticulum calcium ion transport 0
5
10 15 -log 10(P)
20
log2FC
14 12 10 8 6 4 2 0 -2 -4 -6
KR IL2 T16 MM 4 SE P1 HPRPIN B4 MM SA P1 0 SAA2 CC A2S SA L7 AA 4 A1
Inflammatory response
C
25
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).
TPM
M1 TP R1 RY N4A SC N3 1S P CA CNA 1 CA OM MY B3 LD H8 MY
muscles, we performed strand-specific real-time RT-PCR for sense and antisense transcripts. Although we could not evaluate allele-specific expression of sense Myogenesis transcripts at the DMPK 30 UTR because of the low frequency of disease-associD ated polymorphisms at the BpmI site 1000 in exon 10 (Krahe et al., 1995) (n = 3 of a total of 10 samples), CDM muscles with 800 SMA hypermethylated CpGs at the CTCF bind600 ing site tend to exhibit more sense transcripts and less antisense transcripts at 400 the repeat locus (Spearman’s r = 0.77, p = 0.0016, and r = 0.68, p = 0.049, CDM 200 respectively; Figure 4E), also suggesting a regulatory role of CTCF on bidirectional 0 SAA1 transcription at the DMPK locus. Notably, SMA CDM control proto DM1 compared with adult DM1, severe CDM muscles also showed these tendencies region (Yanovsky-Dagan et al., 2015). We performed luciferase for expression of more sense and fewer antisense transcripts reporter assays in human myoblasts and found promoter activity (Figure S2C). of a 1.3-kb region upstream of the repeat tract, although the activity was not as much as that of a canonical DMPK promoter Short CAG RNA Reduces the Toxicity of CUGexp (Figure S4A). Next, to study the effect of CTCF on convergent Our study indicates that aberrant CpG methylation at the CTCF transcription at the DMPK locus, we knocked down CTCF by binding site relates to transcriptional dysregulation at the region, small interfering RNA (siRNA) in human control myoblasts, where including expanded CTG repeats. The increased activity of an CpGs at the CTCF binding site are not methylated (Figure S2B). alternative sense promoter can produce more toxic RNA conReduction of CTCF resulted in mild upregulation of sense tran- taining expanded CUG repeats (CUGexp), increasing the toxic scripts and significant downregulation of antisense transcripts burden. Given this, how does antisense transcription influence (p = 0.010 and p = 8.4E5, respectively; Figure S4B), indicating RNA toxicity? Antisense transcription at the DMPK locus prothat the regulatory function of CTCF for transcription at the duces transcripts containing expanded CAG repeats in DM1. DMPK locus is similar in the ATXN7 locus (Figure S4C). To deter- Such transcripts with CAG expansion form hairpin structures mine whether the methylation status at the CTCF binding site that can be subjected to Dicer cleavage and processed to affects bidirectional transcription at the DMPK locus in CDM 21-nt short CAG repeat RNA (Krol et al., 2007). To study the 1244 Cell Reports 21, 1240–1252, October 31, 2017
A
IL6
Figure 3. Aberrant Activation of the IL-6 Signaling Pathway in CDM Muscles
10
r = 0.77
/18S
IL6ST
IL6R
IL6ST
10
B
IL6
10-1
10-1 r = 0.59
10-3
10-3
SMA 10
STAT3
IL6R
CDM
0
20
0
20
0
20
0
20
0
20
0
20
40
10
/18S
r = 0.78 5
5
r = 0.83
SMA
MYC
STAT3
0
0
SAA1
2.5
IL6ST
40
CDM 2.5
/18S
r = 0.95
C mRNA
3
1.5
1.5
protein
1500
r = 0.74 0.5
0.5
2 1
0 ASO
- +
- +
Control
CDM
1000 500
/18S
IL-6 (pg/ml)
IL6 /18S
SMA
0 ASO
2.5
STAT3
2.0
r = 0.91
2.5 2.0
1.5
1.5
1.0
1.0
0.5
0.5
- +
Control
CDM
SMA 106
r = 0.83
0
0
- +
40
CDM
SAA1
40
CDM 106
no Tx ASO IL-6 Ab
100
103
103
0
0
r = 0.95
80
SMA
*
*
60
*
40
*
*
16
MYC
16
8
8
20
r = 0.93 0
0
0 Line #1
40
CDM
r = 0.75
* /18S
Fusion index (%)
/18S
r = 0.84
D
SMA
#2
Control
#3
#1
#2
CDM
#3
40
CDM
Histological grade
expression of short CAG RNA in CDM, we extracted microRNAs from muscles and quantified the expression level by real-time PCR. We found significantly more 21-nt short CAG RNA in CDM muscle tissue than in SMA muscle without expanded CTG$CAG repeats (p = 0.034; Figure 5A). The expression levels of short CAG RNA correlated with the CpG methylation status at the CTCF binding site (Spearman’s r = 0.87, p = 0.0025; Figure 5B), which also suggested that the aberrant methylation affects antisense transcription. In addition, the expression of short CAG RNA negatively correlated with the histological grade (Spearman’s r = 0.72, p = 0.028; Figure 5B) (i.e., a lower expression level in severe cases), indicating a possible protective contribution to the observed muscle immaturity in CDM. Short CAG RNA generated from repeat hairpin structures can downregulate the expression of CTG-containing genes through a mechanism similar to that of siRNA (Krol et al., 2007). To determine whether such knockdown effects occur in CDM, we studied the expression levels of transcripts that contain CUG repeats. We found negative regulation of PAPSS2 and CASK, which have 8 and 16 CUG repeats, respectively, by the short CAG repeat RNA
(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 = 7.2E5; 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 = 8.2E5, 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 (noTx), 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.
% type 2C fibers
in CDM muscle (Pearson’s r = 0.74, p = 0.022; r = 0.85, p = 0.0042, respectively; Figure 5C). It is possible that short CAG repeat RNA also downregulates the toxic RNA with expanded CUG repeats transcribed from the DMPK locus and reduces CUGexp toxicity. To investigate this possibility, we established a C2C12 cell model (C2C12-800R-BD) for inducible bidirectional transcription of 800 CTG repeats flanked by the upstream CMV/ CBA promoter and the downstream Tet-responsive promoter. In the C2C12-800R-BD cells, sense transcripts with 800 CUG repeats were constitutively expressed, and antisense transcripts with 800 CAG repeats were driven in a doxycycline-inducible manner (Figure S5A). By introducing low-dose doxycycline in the model cells, we reproduced the ratio of sense/antisense transcription (sense/antisense = 1 ± 0.15:0.080 ± 0.026; Figure S5B), similar to that in DM1 muscle (Gudde et al., 2017). As expected, upon activation of antisense transcription in the cell model, the short CAG repeat RNA increased (p = 0.0016), whereas the level of sense transcription was not changed (Figure S5C). We also found an improvement in misregulation of Atp2a1 splicing and a reduction in CUG repeat RNA foci after the induction of antisense Cell Reports 21, 1240–1252, October 31, 2017 1245
Figure 4. CpG Methylation Profiles around CTG Repeats in CDM Muscles
A 3000
r = 0.91
Repeat length
Repeat length
3000
2000
1000
r = 0.86
2000
1000 0
20
60
80
% type 2C fibers
Histological grade CTCF
B
40
CTCF
DMPK 3′ UTR
DMPK 3′ UTR
(CTG)n
CTCF-I
CTCF-II
D1 D2
CDM
(CTG)n D10
SMA 50%
0% CpG methylation
D
C
CpG methylation 1000 at CTCF-I site
2000
r = 0.73
Methylation status
D9 D7 D2 D5 D1 D3 D6 D8 D10 D4
r = 0.62
3000
Repeat length E
Histological grade
Sense transcription r = 0.77
/18S
4
/18S
Antisense transcription 1.8
2
r = -0.68
0.9
0
0
SMA Methylation status
SMA CDM
Methylation status
1246 Cell Reports 21, 1240–1252, October 31, 2017
CDM
(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 30 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 30 UTR, quantified by strandspecific real-time PCR.
A
p = 0.034
B
r = -0.87 0.15
0.15 Short CAG/RNU6B (a.u.)
Short CAG/RNU6B (a.u.)
0.15 0.1 0.05 0
SMA
0.1
0.1
0.05
0.05
0
0
CDM Methylation status
PAPSS2
C
r = -0.74
1
Histological grade CASK
4 /18S (a.u.)
/18S (a.u.)
2
Figure 5. Short CAG Repeat RNA Production in CDM Muscles
r = -0.72
r = -0.85
3
(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 = 8.7E5, FDR q < 0.01) and CACNA1S (right, r = 0.87, p = 0.0021, FDR q < 0.05) in CDM muscles.
2 1
0
for conditional expression of expanded CUG repeats (Nakamori et al., 2015). In Short CAG/RNU6B Short CAG/RNU6B these cells, expression of (CUG)800 is trig(a.u.) (a.u.) gered by Cre excision of a transcription terminator cassette, exerting RNA D toxicity, such as ribonuclear focus formaCAPZB CACNA1S tion and splicing misregulation. A previ70 25 ous report suggested that expanded r = 0.95 r = 0.87 CUG repeat RNAs form hairpin structures 20 and activate the double-stranded RNA15 dependent protein kinase PKR by phos35 phorylation (Tian et al., 2000). In line with 10 this, our C2C12 model cells (C2C12 Cre5 800R) showed an increase in phosphory0 0 lated PKR after induction of CUGexp 0 0.05 0.1 0.15 0 0.05 0.1 0.15 (Figures S6A and S6B). Activated PKR is Short CAG/RNU6B Short CAG/RNU6B reported to phosphorylate IkBa at serine (a.u.) (a.u.) 32 and induce its degradation by the proteasome (Gil et al., 2000). The concomitant degradation of IkBa disentranscription, indicating ameliorated CUGexp toxicity by short CAG gages its binding partner, nuclear factor kB (NF-kB), allowing subrepeat RNA (p = 0.00073 and p = 0.013, respectively; Figures S5D sequent nuclear translocation and stimulation of expression of and S5E). In addition, the expression level of short CAG repeat NF-kB-responsive genes, such as IL6 (Pedersen and Febbraio, RNA in CDM muscles negatively correlated with abnormalities of 2008; Peterson et al., 2011). Indeed, in C2C12 cells expressing several splicing events (CAPZB, CACNA1S, PHKA1 exon 19, CUGexp, PKR activation resulted in phosphorylation and degradaPHKA1 exon 28, VPS39, LDB3, MLF1, and ATP2A1; Figure 5D; tion of IkBa and activated NF-kB by phosphorylation (Figure S6A). Table S1). Most of these events were previously reported to rely In addition, nuclear NF-kB and its activity increased, and IL6 on MBNL1 and/or CELF1, which are affected by toxic RNA (Kalso- expression was upregulated by expression of CUGexp, the tra et al., 2008; Kanadia et al., 2006; Nakamori et al., 2013; Tang effect of which was reversed by ASOs targeting CUGexp et al., 2012). This suggested that short CAG repeat RNA neutralizes (Figures S6B–S6D). To exclude the possibility that CUGexp affects CUGexp toxicity and exerts a protective role and that a reduction in mRNA stability because of repeat-induced changes in RNA binding proteins (RBPs), we assessed the decay of Il6 mRNA in the short CAG repeat RNA enhances RNA toxicity in severe CDM. C2C12 cells. The rate of Il6 mRNA decay was not changed by expression of CUGexp (Figure S6E). In addition, the expression Toxic CUGexp Induces IL-6 Upregulation through NF-kB Activation of RBPs such as ZC3H12A and RC3H1, which regulate the To reveal the consequence of enhanced toxicity by CUGexp, we stability of IL6 mRNA, was not altered in CDM muscles (data used another C2C12 cell model (C2C12 Cre-800R) engineered not shown). 0.05
0.1
0.15
0
0.05
0.1
0.15
% ex29 inclusion
% ex8 inclusion
0
Cell Reports 21, 1240–1252, October 31, 2017 1247
Large CTG repeat expansion since embryonic stage
Hyper-methylation at CTCF-I site & reduction of CTCF binding at DMPK locus
Promotion of alternative sense transcription
Suppression of antisense transcription
Increase in CUG repeat RNA
Reduced production of short CAG repeat RNA
Enhanced RNA toxicity by CUGexp Activation of PKR and induction of ER stress
NF-κB activation
Activation of IL-6/STAT3 signaling pathway
Muscle immaturity in CDM 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-kB 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.
NF-kB activation is also induced by endoplasmic reticulum (ER) stress (Kitamura, 2011), which is reportedly upregulated in adult DM1 (Ikezoe et al., 2007). We found upregulation of mouse ER stress markers, particularly Xbp1 splicing and Atf4 expression, in C2C12 cells by expression of CUGexp (Figure S7A). Furthermore, human ER stress markers, XBP1 splicing and ATF4 expression, were upregulated and significantly correlated with muscle immaturity in CDM (Figures S7B and S7C). These results suggested the involvement of PKR and/or ER stress in the induction of IL-6 via NF-kB activation in CDM.
DISCUSSION DM1 and DM2 are genetically distinct diseases but share a toxic gain-of-function mechanism via CUGexp in the DMPK gene and CCUGexp in the CNBP gene, respectively (Harper, 2001). Both types of toxic RNA affect splicing factors, such as MBNL and/ 1248 Cell Reports 21, 1240–1252, October 31, 2017
or CELF1 proteins, resulting in splicing defects overlapped in DM1 and DM2. In contrast, CDM, a severe congenital form only found in DM1, presents characteristic genetic (highly expanded CTG repeats), epigenetic (hypermethylation of CpGs), and phenotypic (muscle immaturity) features not seen in adult DM1 (Harley et al., 1993; Nonaka et al., 1996; Steinbach et al., 1998). However, the disease process of CDM has yet to be elucidated because of the difficulty of obtaining enough and appropriate muscle samples for analysis. In this study, despite the limited availability of samples, we used ten CDM muscles obtained from identical muscles in different patients of similar age to investigate the specific pathomechanism of CDM. We revealed the genotype-epigenotype-phenotype correlation in CDM muscles: muscle immaturity correlates with expanded repeat size and CpG methylation at the CTCF binding site upstream the repeat tract, the aberrant CpG methylation associates with transcriptional dysregulation of an alternative sense promoter and antisense promoter around the repeat locus, a decrease in antisense transcription suppresses the production of short CAG repeat RNA capable of neutralizing CUGexp, the reduction of short CAG repeat RNA and activation of the alternative sense promoter both enhance RNA toxicity of CUGexp, and the IL-6 signaling pathway is upregulated in severe CDM, possibly because of toxic CUGexp through the activation of NF-kB (Figure 6). IL-6 is a multifunctional cytokine, usually secreted from immune cells, such as macrophages, monocytes, and T and B lymphocytes, in response to infections and tissue injuries (Pedersen and Febbraio, 2008). IL-6 is also produced by skeletal muscle and recognized as a myokine that exerts paracrine, autocrine, or endocrine effects. In general, transient production and shortterm action of IL-6 contribute to positive regulation in skeletal muscle, such as energy metabolism and satellite cell proliferation (Mun˜oz-Ca´noves et al., 2013). On the contrary, consistent longlasting activation of IL-6 exerts negative effects on muscle homeostasis. Because activation of the IL-6/STAT3 signaling pathway promotes myocyte proliferation and prevents premature differentiation, tuned-down signaling is necessary for cessation of myoblast proliferation and commencement of differentiation (Serrano et al., 2008; Sun et al., 2007). A recent study demonstrated that treatment of C2C12 myogenic cells with recombinant IL-6 inhibits myoblast differentiation (Pelosi et al., 2014). In addition, a delay in differentiation was reported in CDM myoblasts, where we observed upregulation of IL6, but not in adult DM1 and DM2 myoblasts (Cardani et al., 2009; Furling et al., 2001; Loro et al., 2010). Such evidence supports our hypothesis that the activated IL-6 signaling pathway results in muscle immaturity in CDM. Furthermore, previous studies suggested that long-lasting, consistent activation of the IL-6 pathway induces muscle atrophy and wasting. A transgenic mouse model that overexpresses IL-6 displayed severe muscle atrophy, and blockade of IL-6 signaling by treatment with an IL-6R antibody reversed the muscle phenotype (Tsujinaka et al., 1995, 1996). Other studies also demonstrated that activation of the IL-6/STAT3 pathway causes a decrease in muscle mass or muscle wasting in different animal models (Bonetto et al., 2012; Janssen et al., 2005), and inhibition of the pathway stimulates muscle regeneration (Price et al., 2014; Tierney et al., 2014). In addition, the dystrophic
phenotype in mdx mice, a model for Duchenne muscular dystrophy (DMD), was ameliorated by inhibition of IL-6 activity by an IL-6R antibody and exacerbated via IL-6 overexpression (Pelosi et al., 2015a, 2015b). Our study also revealed upregulation of SAA1, a target gene of IL-6 signaling, in CDM muscles. Zhang et al. (2009) reported that IL6 and SAA1 synergy mediates muscle wasting via proteolysis. Simultaneous upregulation of IL6 and SAA1 was observed in muscles of patients with critical illness myopathy that presents severe muscle wasting (Langhans et al., 2014). Importantly, in our study, we demonstrated that treatment with an ASO targeting CUGexp reduced IL-6 levels and that the anti-IL-6 antibody as well as the ASO partially rescued differentiation defects in CDM myoblasts. Thus, our results, together with the previous studies, strongly suggest that upregulation of the IL-6/STAT3 signaling pathway contributes to the severe muscle phenotype in CDM. Upregulation of the IL-6 pathway in CDM patients could be due to infiltration of immune cells or fibrosis, or it could just reflect an increase in satellite cells because of muscle immaturity. However, immune cell infiltration was not increased in CDM muscles, and IL6 was not upregulated even in muscles from inflammatory myositis; elevated IL6 levels in CDM muscles were more than 104-fold higher than in DMD patients with severe fibrosis; IL6 levels were significant higher in CDM patients than in SMA patients, who also have muscle immaturity because of impaired innervation; fibrosis and inflammatory cell markers were not increased in CDM; and given the upregulation of IL6 observed in CDM myoblasts and in the C2C12 murine myoblast cell line conditionally expressing CUGexp, it is likely that IL-6 is produced in the myocytes of CDM muscles. Then what activates IL-6 production in CDM muscles? Transient IL-6 production from skeletal muscle is mainly triggered by muscle contraction. However, it is unlikely that upregulation of IL6 in CDM muscles is due to enhanced muscle contraction because all of our CDM patients presented with hypotonia and not myotonia (Table S2). IL-6 production in the muscle is also triggered by nitric oxide, p38 mitogen-activated protein kinase (MAPK), NF-kB, or calcineurin/nuclear factor of activated T cell (NFAT) signaling (Pedersen and Febbraio, 2008). In our study, we did not observe upregulation of neuronal nitric oxide (NO) synthase (nNOS), p38, calcineurin, or NFATs in CDM muscles (data not shown), but we observed activation of NF-kB in C2C12 cells that conditionally expressed CUGexp. We also detected the activation of PKR and induction of ER stress, both of which can trigger NF-kB activation, in these cells. PKR is phosphorylated and activated by double-stranded RNA, including CUGexp (Tian et al., 2000), but it does not affect the disease phenotype in HSALR mice, a model for adult DM1 (Mankodi et al., 2003). However, because IL-6 was not upregulated in muscles from either HSALR mice (data not shown) or adult DM1 patients, PKR activation triggered by enhanced RNA toxicity could be specific to CDM. Previously, we demonstrated an increase in ER stress in adult DM1 muscles (Ikezoe et al., 2007). In this study, we observed elevated ER stress markers in CDM muscles as well as in the C2C12 cell model. Because the degree of ER stress was correlated with muscle immaturity in CDM, elevated ER stress might also trigger NF-kB activation and upregulation of the IL-6 pathway.
Several previous studies reported hypermethylation of CpG sites upstream of the expanded CTG repeats in CDM (Barbe´ et al., 2017; Lo´pez Castel et al., 2011; Steinbach et al., 1998; Yanovsky-Dagan et al., 2015). However, the biological significance has yet to be determined. Our quantitative analysis for methylation status first demonstrated that aberrant methylation dysregulates transcription at the repeat locus. Even though we could not evaluate allele-specific expression of sense/antisense transcripts at the DMPK 30 UTR, the total expression (from normal and mutant alleles) of sense transcripts increased and that of antisense transcripts decreased, in accordance with the aberrant methylation status. We also revealed that the reduction of antisense transcription led to decreased production of short CAG repeat RNA, which could play a protective role by neutralizing toxic CUGexp. Thus, our results indicate that aberrant CpG methylation enhances RNA toxicity by increasing sense transcription for CUGexp and reducing antisense transcription and short CAG repeat RNA production in CDM. Although we found a tendency for the methylation status to correlate with repeat size, the detailed mechanism of how expanded repeats induce CpG methylation around the repeat tract, or vice versa, is still unknown. It has also been suggested that CpG methylation and CTCF binding regulate CTG$CAG repeat instability (Lo´pez Castel et al., 2011). Further study is warranted to elucidate the mechanism. Our study demonstrated that enhanced RNA toxicity by transcriptional dysregulation correlates with the IL-6 signaling pathway and might contribute to muscle immaturity in CDM. RNA toxicity is a common pathological mechanism in adult DM and causes multiple splicing defects. In this study, we found a significant correlation between muscle immaturity in CDM and the splicing abnormality in NRAP and FXR1. However, because both splicing defects are observed in other muscle diseases or damaged muscles (Bachinski et al., 2014; Orengo et al., 2011), they could reflect non-specific muscle damage. Nonetheless, we cannot exclude the possibility that enhanced RNA toxicity caused a critical splicing defect for the muscle phenotype in CDM. Recently, we reported enhanced splicing alteration in CDM muscle because of combined misregulation of MBNL proteins independent of IL-6 expression (Thomas et al., 2017). These results suggest that CUGexp exerts its toxicity through MBNL loss of function (splicing abnormalities) together with IL-6 upregulation, which is not explained by the MBNL-centric mechanism. In addition, other MBNL/CELF1-mediated processes, such as alternative polyadenylation, RNA localization, and mRNA stability (Wang et al., 2016), could be involved. It might be possible that differences between the developmental expression of the DMPK and CNBP genes and the stability of 30 UTR and intronic RNAs also contribute to the locus-specific RNA toxicity in CDM. NF-kB and IL-6 transduce their signals by upregulation or phosphorylation of downstream effectors (Pedersen and Febbraio, 2008; Peterson et al., 2011). Although we could not investigate phosphorylation of the components of the NF-kB or IL-6 pathway in CDM muscles because of the limited amount of samples, our study showed overall upregulation of the IL-6 myokine signaling and target genes, in concordance with muscle immaturity in CDM. Because the IL-6 pathway plays an important role in satellite cell maturation, muscle atrophy, and wasting, our results strongly indicate an involvement of the myokine pathway in CDM Cell Reports 21, 1240–1252, October 31, 2017 1249
pathogenesis. In addition, our study suggests the possibility of treatment for CDM through interventions in the NF-kB or IL-6 signaling pathways, which have been applied successfully for other diseases, such as rheumatoid arthritis (Schoels et al., 2013). EXPERIMENTAL PROCEDURES
AUTHOR CONTRIBUTIONS M.N. conceived and designed the study, performed the experiments, analyzed the data, and wrote the manuscript. K.H. and J.D.T. designed the study, performed the experiments, analyzed the data, and wrote the manuscript. E.T.W. performed the experiments and analyzed the data. I.N. and M.S.S. designed the study, analyzed the data, and wrote the manuscript. Y.K.H., M.P.T., and H.M. helped write the manuscript.
Patients and Muscle Tissue Samples Human skeletal muscle (biceps brachii) tissues were obtained from biopsies or autopsies of ten CDM patients and four patients with genetically confirmed SMA and a corrected age of less than 15 months (Table S2). The chronological age was corrected for the length of gestation, which is for the number of weeks different from the expected 40 weeks. Muscles from adult DM1, amyotrophic lateral sclerosis (ALS), and polymyositis patients were described previously (Nakamori et al., 2007). For RNA analysis, we omitted one autopsied sample because of low RNA quality (RNA integrity number < 4.5). Details of the cell models are described in the Supplemental Experimental Procedures. DNA and RNA were extracted from skeletal muscles and cells as reported previously (Nakamori et al., 2011, 2013) and analyzed as described in the Supplemental Experimental Procedures.
ACKNOWLEDGMENTS
Histological Analysis Frozen muscle sections (6 mm thick) were stained by standard histological and immunohistochemistry (IHC) methods. The percentage of type 2C fiber was calculated from more than 200 myofibers in each muscle stained by adenosine triphosphatase. Muscle immaturity was graded into three categories: mild, no or mild fiber size variation; moderate, moderate fiber size variation with a significant number of atrophic fibers; and severe, excessive fiber size variation with a predominance of atrophic fibers or all fibers are highly atrophic. IHC was performed with the Ventana immunohistochemistry detection system (Ventana Medical Systems). The sections were blocked with 2% BSA and 0.25% goat serum and incubated with the following primary antibodies: antiCD4 (1:100, sc-19641, Santa Cruz Biotechnology), anti-CD8 (1:100, DK25, Dako), anti-CD20 (1:50, M0755, Dako), and anti-CD68 (1:200, sc-9139, Santa Cruz Biotechnology). Afterward, the sections were incubated with secondary antibodies and enzymatically detected with the I-VIEW DAB universal kit (Roche).
Received: June 7, 2017 Revised: September 2, 2017 Accepted: October 4, 2017 Published: October 31, 2017
Statistics Associations of histological grade or methylation status were assessed with the Spearman’s rank-order correlation coefficient. Associations of splicing misregulation or percentage type 2C fibers were described with Pearson’s correlation coefficients. Correction for multiple testing was performed, and the FDR was controlled using the Benjamini-Hochberg approach. Cluster analysis was performed with the R software (http://www.r-project.org). Intergroup comparisons of human samples and C2C12 cell models were conducted with the Mann-Whitney U test and Student’s t test, respectively. Study Approval This research was approved by the institutional ethics committee, and informed consent was obtained. All of the procedures for animal experiments were approved by the institutional animal care and use committee of Osaka University.
The authors thank Kazu Iwasawa, Rika Manabe, and the Center for Medical Research and Education of Osaka University Graduate School of Medicine (Drs. Tatsuya Tanaka and Saki Ishino) for technical assistance and Dr. Genevieve Gourdon for advice on strand-specific RT-PCR. This work was supported by a Grant-in-Aid for Young Scientists (A), a Grant-in-Aid for Challenging Exploratory Research, and a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (KAKENHI grant numbers 25713034, 15K15339, and 16H05321 to M.N.), an Intramural Research Grant for Neurological and Psychiatric Disorders from the National Center of Neurology and Psychiatry (grant number 29-4 to I.N. and M.N.), and NIH grants NS058901 and NS098819 (to M.S.S.).
REFERENCES Ashizawa, T., Dubel, J.R., and Harati, Y. (1993). Somatic instability of CTG repeat in myotonic dystrophy. Neurology 43, 2674–2678. Bachinski, L.L., Baggerly, K.A., Neubauer, V.L., Nixon, T.J., Raheem, O., Sirito, M., Unruh, A.K., Zhang, J., Nagarajan, L., Timchenko, L.T., et al. (2014). Most expression and splicing changes in myotonic dystrophy type 1 and type 2 skeletal muscle are shared with other muscular dystrophies. Neuromuscul. Disord. 24, 227–240. Barbe´, L., Lanni, S., Lo´pez-Castel, A., Franck, S., Spits, C., Keymolen, K., Seneca, S., Tome´, S., Miron, I., Letourneau, J., et al. (2017). CpG Methylation, a Parent-of-Origin Effect for Maternal-Biased Transmission of Congenital Myotonic Dystrophy. Am. J. Hum. Genet. 100, 488–505. Bonetto, A., Aydogdu, T., Jin, X., Zhang, Z., Zhan, R., Puzis, L., Koniaris, L.G., and Zimmers, T.A. (2012). JAK/STAT3 pathway inhibition blocks skeletal muscle wasting downstream of IL-6 and in experimental cancer cachexia. Am. J. Physiol. Endocrinol. Metab. 303, E410–E421. Brouwer, J.R., Huguet, A., Nicole, A., Munnich, A., and Gourdon, G. (2013). Transcriptionally Repressive Chromatin Remodelling and CpG Methylation in the Presence of Expanded CTG-Repeats at the DM1 Locus. J. Nucleic Acids 2013, 567435. Cardani, R., Baldassa, S., Botta, A., Rinaldi, F., Novelli, G., Mancinelli, E., and Meola, G. (2009). Ribonuclear inclusions and MBNL1 nuclear sequestration do not affect myoblast differentiation but alter gene splicing in myotonic dystrophy type 2. Neuromuscul. Disord. 19, 335–343.
DATA AND SOFTWARE AVAILABILITY
Cho, D.H., Thienes, C.P., Mahoney, S.E., Analau, E., Filippova, G.N., and Tapscott, S.J. (2005). Antisense transcription and heterochromatin at the DM1 CTG repeats are constrained by CTCF. Mol. Cell 20, 483–489.
The accession number for the sequencing data reported in this paper is GEO: GSE97806.
Farkas-Bargeton, E., Barbet, J.P., Dancea, S., Wehrle, R., Checouri, A., and Dulac, O. (1988). Immaturity of muscle fibers in the congenital form of myotonic dystrophy: its consequences and its origin. J. Neurol. Sci. 83, 145–159.
SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, seven figures, and three tables and can be found with this article online at https://doi.org/10.1016/j.celrep.2017.10.018.
1250 Cell Reports 21, 1240–1252, October 31, 2017
Filippova, G.N., Thienes, C.P., Penn, B.H., Cho, D.H., Hu, Y.J., Moore, J.M., Klesert, T.R., Lobanenkov, V.V., and Tapscott, S.J. (2001). CTCF-binding sites flank CTG/CAG repeats and form a methylation-sensitive insulator at the DM1 locus. Nat. Genet. 28, 335–343. Frisch, R., Singleton, K.R., Moses, P.A., Gonzalez, I.L., Carango, P., Marks, H.G., and Funanage, V.L. (2001). Effect of triplet repeat expansion on
chromatin structure and expression of DMPK and neighboring genes, SIX5 and DMWD, in myotonic dystrophy. Mol. Genet. Metab. 74, 281–291. Furling, D., Coiffier, L., Mouly, V., Barbet, J.P., St Guily, J.L., Taneja, K., Gourdon, G., Junien, C., and Butler-Browne, G.S. (2001). Defective satellite cells in congenital myotonic dystrophy. Hum. Mol. Genet. 10, 2079–2087. Gil, J., Alcamı´, J., and Esteban, M. (2000). Activation of NF-kappa B by the dsRNA-dependent protein kinase, PKR involves the I kappa B kinase complex. Oncogene 19, 1369–1378. Gudde, A.E.E.G., van Heeringen, S.J., de Oude, A.I., van Kessel, I.D.G., Estabrook, J., Wang, E.T., Wieringa, B., and Wansink, D.G. (2017). Antisense transcription of the myotonic dystrophy locus yields low-abundant RNAs with and without (CAG)n repeat. RNA Biol., 1–15. Harley, H.G., Rundle, S.A., MacMillan, J.C., Myring, J., Brook, J.D., Crow, S., Reardon, W., Fenton, I., Shaw, D.J., and Harper, P.S. (1993). Size of the unstable CTG repeat sequence in relation to phenotype and parental transmission in myotonic dystrophy. Am. J. Hum. Genet. 52, 1164–1174. Harper, P.S. (2001). Myotonic Dystrophy (London: W.B. Saunders Company).
Nakamori, M., Kimura, T., Fujimura, H., Takahashi, M.P., and Sakoda, S. (2007). Altered mRNA splicing of dystrophin in type 1 myotonic dystrophy. Muscle Nerve 36, 251–257. Nakamori, M., Gourdon, G., and Thornton, C.A. (2011). Stabilization of expanded (CTG)d(CAG) repeats by antisense oligonucleotides. Mol. Ther. 19, 2222–2227. Nakamori, M., Sobczak, K., Puwanant, A., Welle, S., Eichinger, K., Pandya, S., Dekdebrun, J., Heatwole, C.R., McDermott, M.P., Chen, T., et al. (2013). Splicing biomarkers of disease severity in myotonic dystrophy. Ann. Neurol. 74, 862–872. Nakamori, M., Taylor, K., Mochizuki, H., Sobczak, K., and Takahashi, M.P. (2015). Oral administration of erythromycin decreases RNA toxicity in myotonic dystrophy. Ann. Clin. Transl. Neurol. 3, 42–54. Nonaka, I., Kobayashi, O., and Osari, S. (1996). Nondystrophinopathic muscular dystrophies including myotonic dystrophy. Semin. Pediatr. Neurol. 3, 110–121. Orengo, J.P., Ward, A.J., and Cooper, T.A. (2011). Alternative splicing dysregulation secondary to skeletal muscle regeneration. Ann. Neurol. 69, 681–690.
Ikezoe, K., Nakamori, M., Furuya, H., Arahata, H., Kanemoto, S., Kimura, T., Imaizumi, K., Takahashi, M.P., Sakoda, S., Fujii, N., and Kira, J. (2007). Endoplasmic reticulum stress in myotonic dystrophy type 1 muscle. Acta Neuropathol. 114, 527–535.
Osborne, R.J., and Thornton, C.A. (2006). RNA-dominant diseases. Hum. Mol. Genet. 15, R162–R169.
Janssen, S.P., Gayan-Ramirez, G., Van den Bergh, A., Herijgers, P., Maes, K., Verbeken, E., and Decramer, M. (2005). Interleukin-6 causes myocardial failure and skeletal muscle atrophy in rats. Circulation 111, 996–1005.
Pelosi, M., De Rossi, M., Barberi, L., and Musaro`, A. (2014). IL-6 impairs myogenic differentiation by downmodulation of p90RSK/eEF2 and mTOR/ p70S6K axes, without affecting AKT activity. BioMed Res. Int. 2014, 206026.
Kalsotra, A., Xiao, X., Ward, A.J., Castle, J.C., Johnson, J.M., Burge, C.B., and Cooper, T.A. (2008). A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart. Proc. Natl. Acad. Sci. USA 105, 20333–20338.
Pelosi, L., Berardinelli, M.G., De Pasquale, L., Nicoletti, C., D’Amico, A., Carvello, F., Moneta, G.M., Catizone, A., Bertini, E., De Benedetti, F., and Musaro`, A. (2015a). Functional and Morphological Improvement of Dystrophic Muscle by Interleukin 6 Receptor Blockade. EBioMedicine 2, 285–293.
Kanadia, R.N., Shin, J., Yuan, Y., Beattie, S.G., Wheeler, T.M., Thornton, C.A., and Swanson, M.S. (2006). Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy. Proc. Natl. Acad. Sci. USA 103, 11748–11753.
Pelosi, L., Berardinelli, M.G., Forcina, L., Spelta, E., Rizzuto, E., Nicoletti, C., Camilli, C., Testa, E., Catizone, A., De Benedetti, F., and Musaro`, A. (2015b). Increased levels of interleukin-6 exacerbate the dystrophic phenotype in mdx mice. Hum. Mol. Genet. 24, 6041–6053.
Kitamura, M. (2011). Control of NF-kB and inflammation by the unfolded protein response. Int. Rev. Immunol. 30, 4–15.
Peterson, J.M., Bakkar, N., and Guttridge, D.C. (2011). NF-kB signaling in skeletal muscle health and disease. Curr. Top. Dev. Biol. 96, 85–119.
Klesert, T.R., Otten, A.D., Bird, T.D., and Tapscott, S.J. (1997). Trinucleotide repeat expansion at the myotonic dystrophy locus reduces expression of DMAHP. Nat. Genet. 16, 402–406.
Phillips, J.E., and Corces, V.G. (2009). CTCF: master weaver of the genome. Cell 137, 1194–1211.
Krahe, R., Ashizawa, T., Abbruzzese, C., Roeder, E., Carango, P., Giacanelli, M., Funanage, V.L., and Siciliano, M.J. (1995). Effect of myotonic dystrophy trinucleotide repeat expansion on DMPK transcription and processing. Genomics 28, 1–14. Krol, J., Fiszer, A., Mykowska, A., Sobczak, K., de Mezer, M., and Krzyzosiak, W.J. (2007). Ribonuclease dicer cleaves triplet repeat hairpins into shorter repeats that silence specific targets. Mol. Cell 25, 575–586. Langhans, C., Weber-Carstens, S., Schmidt, F., Hamati, J., Kny, M., Zhu, X., Wollersheim, T., Koch, S., Krebs, M., Schulz, H., et al. (2014). Inflammationinduced acute phase response in skeletal muscle and critical illness myopathy. PLoS ONE 9, e92048. Lo´pez Castel, A., Nakamori, M., Tome´, S., Chitayat, D., Gourdon, G., Thornton, C.A., and Pearson, C.E. (2011). Expanded CTG repeat demarcates a boundary for abnormal CpG methylation in myotonic dystrophy patient tissues. Hum. Mol. Genet. 20, 1–15. Loro, E., Rinaldi, F., Malena, A., Masiero, E., Novelli, G., Angelini, C., Romeo, V., Sandri, M., Botta, A., and Vergani, L. (2010). Normal myogenesis and increased apoptosis in myotonic dystrophy type-1 muscle cells. Cell Death Differ. 17, 1315–1324. Mankodi, A., Teng-Umnuay, P., Krym, M., Henderson, D., Swanson, M., and Thornton, C.A. (2003). Ribonuclear inclusions in skeletal muscle in myotonic dystrophy types 1 and 2. Ann. Neurol. 54, 760–768. Mun˜oz-Ca´noves, P., Scheele, C., Pedersen, B.K., and Serrano, A.L. (2013). Interleukin-6 myokine signaling in skeletal muscle: a double-edged sword? FEBS J. 280, 4131–4148.
Pedersen, B.K., and Febbraio, M.A. (2008). Muscle as an endocrine organ: focus on muscle-derived interleukin-6. Physiol. Rev. 88, 1379–1406.
Prestridge, D.S. (1995). Predicting Pol II promoter sequences using transcription factor binding sites. J. Mol. Biol. 249, 923–932. Price, F.D., von Maltzahn, J., Bentzinger, C.F., Dumont, N.A., Yin, H., Chang, N.C., Wilson, D.H., Frenette, J., and Rudnicki, M.A. (2014). Inhibition of JAKSTAT signaling stimulates adult satellite cell function. Nat. Med. 20, 1174–1181. Ranum, L.P., and Cooper, T.A. (2006). RNA-mediated neuromuscular disorders. Annu. Rev. Neurosci. 29, 259–277. Santoro, M., Fontana, L., Masciullo, M., Bianchi, M.L., Rossi, S., Leoncini, E., Novelli, G., Botta, A., and Silvestri, G. (2015). Expansion size and presence of CCG/CTC/CGG sequence interruptions in the expanded CTG array are independently associated to hypermethylation at the DMPK locus in myotonic dystrophy type 1 (DM1). Biochim. Biophys. Acta 1852, 2645–2652. Schoels, M.M., van der Heijde, D., Breedveld, F.C., Burmester, G.R., Dougados, M., Emery, P., Ferraccioli, G., Gabay, C., Gibofsky, A., Gomez-Reino, J.J., et al. (2013). Blocking the effects of interleukin-6 in rheumatoid arthritis and other inflammatory rheumatic diseases: systematic literature review and meta-analysis informing a consensus statement. Ann. Rheum. Dis. 72, 583–589. Serrano, A.L., Baeza-Raja, B., Perdiguero, E., Jardı´, M., and Mun˜oz-Ca´noves, P. (2008). Interleukin-6 is an essential regulator of satellite cell-mediated skeletal muscle hypertrophy. Cell Metab. 7, 33–44. Sopher, B.L., Ladd, P.D., Pineda, V.V., Libby, R.T., Sunkin, S.M., Hurley, J.B., Thienes, C.P., Gaasterland, T., Filippova, G.N., and La Spada, A.R. (2011). CTCF regulates ataxin-7 expression through promotion of a convergently transcribed, antisense noncoding RNA. Neuron 70, 1071–1084. Steinbach, P., Gla¨ser, D., Vogel, W., Wolf, M., and Schwemmle, S. (1998). The DMPK gene of severely affected myotonic dystrophy patients is
Cell Reports 21, 1240–1252, October 31, 2017 1251
hypermethylated proximal to the largely expanded CTG repeat. Am. J. Hum. Genet. 62, 278–285. Sun, L., Ma, K., Wang, H., Xiao, F., Gao, Y., Zhang, W., Wang, K., Gao, X., Ip, N., and Wu, Z. (2007). JAK1-STAT1-STAT3, a key pathway promoting proliferation and preventing premature differentiation of myoblasts. J. Cell Biol. 179, 129–138. Tang, Z.Z., Yarotskyy, V., Wei, L., Sobczak, K., Nakamori, M., Eichinger, K., Moxley, R.T., Dirksen, R.T., and Thornton, C.A. (2012). Muscle weakness in myotonic dystrophy associated with misregulated splicing and altered gating of Ca(V)1.1 calcium channel. Hum. Mol. Genet. 21, 1312–1324. Tews, D.S., and Goebel, H.H. (1997). Apoptosis-related proteins in skeletal muscle fibers of spinal muscular atrophy. J. Neuropathol. Exp. Neurol. 56, 150–156. Thomas, J.D., Sznajder, L.J., Bardhi, O., Aslam, F.N., Anastasiadis, Z.P., Scotti, M.M., Nishino, I., Nakamori, M., Wang, E.T., and Swanson, M.S. (2017). Disrupted prenatal RNA processing and myogenesis in congenital myotonic dystrophy. Genes Dev. 31, 1122–1133.
Tierney, M.T., Aydogdu, T., Sala, D., Malecova, B., Gatto, S., Puri, P.L., Latella, L., and Sacco, A. (2014). STAT3 signaling controls satellite cell expansion and skeletal muscle repair. Nat. Med. 20, 1182–1186. Tsujinaka, T., Ebisui, C., Fujita, J., Kishibuchi, M., Morimoto, T., Ogawa, A., Katsume, A., Ohsugi, Y., Kominami, E., and Monden, M. (1995). Muscle undergoes atrophy in association with increase of lysosomal cathepsin activity in interleukin-6 transgenic mouse. Biochem. Biophys. Res. Commun. 207, 168–174. Tsujinaka, T., Fujita, J., Ebisui, C., Yano, M., Kominami, E., Suzuki, K., Tanaka, K., Katsume, A., Ohsugi, Y., Shiozaki, H., and Monden, M. (1996). Interleukin 6 receptor antibody inhibits muscle atrophy and modulates proteolytic systems in interleukin 6 transgenic mice. J. Clin. Invest. 97, 244–249. Wang, E.T., Taliaferro, J.M., Lee, J.A., Sudhakaran, I.P., Rossoll, W., Gross, C., Moss, K.R., and Bassell, G.J. (2016). Dysregulation of mRNA Localization and Translation in Genetic Disease. J. Neurosci. 36, 11418–11426.
Thornton, C.A., Wymer, J.P., Simmons, Z., McClain, C., and Moxley, R.T., 3rd. (1997). Expansion of the myotonic dystrophy CTG repeat reduces expression of the flanking DMAHP gene. Nat. Genet. 16, 407–409.
Yanovsky-Dagan, S., Avitzour, M., Altarescu, G., Renbaum, P., Eldar-Geva, T., Schonberger, O., Mitrani-Rosenbaum, S., Levy-Lahad, E., Birnbaum, R.Y., Gepstein, L., et al. (2015). Uncovering the Role of Hypermethylation by CTG Expansion in Myotonic Dystrophy Type 1 Using Mutant Human Embryonic Stem Cells. Stem Cell Reports 5, 221–231.
Tian, B., White, R.J., Xia, T., Welle, S., Turner, D.H., Mathews, M.B., and Thornton, C.A. (2000). Expanded CUG repeat RNAs form hairpins that activate the double-stranded RNA-dependent protein kinase PKR. RNA 6, 79–87.
Zhang, L., Du, J., Hu, Z., Han, G., Delafontaine, P., Garcia, G., and Mitch, W.E. (2009). IL-6 and serum amyloid A synergy mediates angiotensin II-induced muscle wasting. J. Am. Soc. Nephrol. 20, 604–612.
1252 Cell Reports 21, 1240–1252, October 31, 2017