Altered expression and splicing of Ca(2+) metabolism genes in myotonic dystrophies DM1 and DM2

Abstract

Aims: Myotonic dystrophy types 1 and 2 (DM1 and DM2) are multisystem disorders caused by similar repeat expansion mutations, with similar yet distinct clinical features. Aberrant splicing of multiple effector genes, as well as dysregulation of transcription and translation, has been suggested to underlie different aspects of the complex phenotypes in DM1 and DM2. Ca(2+) plays a central role in both muscle contraction and control of gene expression, and recent expression profiling studies have indicated major perturbations of the Ca(2+) signalling pathways in DM. Here we have further investigated the expression of genes and proteins involved in Ca(2+) metabolism in DM patients, including Ca(2+) channels and Ca(2+) binding proteins.

Methods: We used patient muscle biopsies to analyse mRNA expression and splicing of genes by microarray expression profiling and RT-PCR. We studied protein expression by immunohistochemistry and immunoblotting.

Results: Most of the genes studied showed mRNA up-regulation in expression profiling. When analysed by immunohistochemistry the Ca(2+) release channel ryanodine receptor was reduced in DM1 and DM2, as was calsequestrin 2, a sarcoplasmic reticulum lumen Ca(2+) storage protein. Abnormal splicing of ATP2A1 was more pronounced in DM2 than DM1.

Conclusions: We observed abnormal mRNA and protein expression in DM affecting several proteins involved in Ca(2+) metabolism, with some differences between DM1 and DM2. Our protein expression studies are suggestive of a post-transcriptional defect(s) in the myotonic dystrophies.

Figure 1

Alternative isoforms of ATP2A1 and RYR1 in muscle biopsies from DM1, DM2 and non-DM dystrophy (NMD) patients compared to muscle of normal individuals. Panels a and c show design for RT-PCR assays. Panels b and d show results of quantitative fluorescent RT-PCR. Percent inclusion of the aberrantly spliced exon is shown on the Y axis. Error bars represent the standard deviation for each group. DM1 was not significantly different from normal for RYR1 (p-value = 0.251), while DM2 was (p-value = 0.0239). For ATP2A1 both DM1 and DM2 were significantly different from normal (p-value = 0.0211 and p-value = 2.68E-11, respectively)

Figure 2

Quantification of proteins after western blotting showing differential expression in DM muscle biopsies with significant p-values (p < 0.05). The average of healthy controls (N, normal) in each group was set to 1.00. The rows are labeled as A immunoblotted protein band; B coomassie brilliant blue -stained MyHC band as loading control. Each band is labeled with a patient identifier (X = outlier omitted from analysis). Standard deviation is indicated by error bars. a, SERCA2 (observed band 115 kD) was decreased in DM2 vs. N (p-value = 0.0074). b, TRDN (100 kD) showed higher expression in DM2 vs. DM1 (p-value = 0.0012). c, JPH1 (80 kD) was reduced in DMs as a group (p-value = 0.0256), however, only DM2 showed significant decrease alone vs. N (p-value = 0.0147). d, Calcineurin (60 kD) showed higher expression in DM2 and lower expression in DM1 compared to N, however, only comparing DM1 vs. DM2 showed significant difference (p-value = 0.0423). e, NFATC3 (115 kD) appeared increased in DM2, but the p-value DM2 vs. N was not quite significant (p-value = 0.0590); comparing DM1 vs. DM2 showed that NFATC3 was more expressed in DM2 than DM1 (p-value = 0.0359).

Figure 3

Representative immunohistochemical stainings of Ca²⁺ handling proteins in DM. In DAB immunohistochemistry (a–k, m, n, p, q), the specific signal is dark brown, and the background staining (H&E) is blue. In the upper panel, in control both RYR1 (b) and DHPR (c) are slightly more intensely expressed in fast fibres, when compared to MyHC-II (a) (detecting fast fibres). Reduction of RYR1 immunoreactivity in DM2 (e), and reduction of both RYR1 and DHPR in DM1 (h, i) is evident. In the lower panel, normal SERCA1 staining, similar to control (j) is seen in both DM2 (m) and DM1 (p). Reduction of JPH1 is seen in DM1 (q), but not in DM2 (n). The last panel with immunofluorescent staining shows drastic reduction of CASQ2 (green) in DM2 (o), and a less extensive reduction in DM1 (r) compared to healthy control (l). (a–c, j, k) N_??_VL_03; (L) N_M40_G_004; (d–f, m–o) DM2_M38_VL_001; (g, h, p) DM1_M48_TA_004; (i) DM1_M55_TA_010; (q) DM1_M48_VL_005; (r) DM1_M46_TA_001