In the present study, an inducible and heart-specific DM1 mouse model expressing expanded CUG RNA in the context of DMPK
3′ UTR exhibited the functional, pathological, electrophysiological, and molecular features of DM1. These features include prolonged PR intervals and QRS duration, decreased diastolic and systolic velocities, dilated cardiomyopathy, hypertrophy of cardiomyocytes, and proliferation of mitochondria. Importantly, the EpA960/MCM model reproduced a feature of DM1 that has not to our knowledge been previously reproduced in heart tissue of a DM1 mouse model: increased steady-state levels of CUGBP1 protein. Combined in situ hybridization and immunofluorescent staining for CUGBP1 and its paralog, CUGBP2, demonstrated elevated levels of both proteins specifically in nuclei containing RNA foci. A time course of molecular events following induction of EpA960(R) mRNA expression demonstrated that colocalization of MBNL1 with RNA foci and increased CUGBP1 expression occurred early and coincided with reversion to an embryonic splicing pattern for Tnnt2
. CUGBP1 and CUGBP2 are the only CELF proteins expressed in heart, and both are normally downregulated by 3 weeks postnatally (ref. 24
and our unpublished observations). Our analysis in EpA960/MCM mice demonstrated that a rapid increase in CELF protein expression, recapitulating the embryonic expression patterns, was an early response to the mutant RNA.
Altered activities of MBNL and CELF proteins mediate the disrupted alternative splicing observed in DM1 (2
). In addition, altered CUGBP1 cytoplasmic function is proposed to alter regulated translation of key myogenic proteins in skeletal muscle contributing to muscle degeneration (32
). MBNL1 is sequestered by CUG repeat RNA nuclear foci, resulting in a loss of nuclear activity, while steady-state levels of CUGBP1 increase in DM1 heart and skeletal muscle (6
). Here we demonstrate that RNA foci formed and MBNL1 colocalized with foci within 6 hours — even as soon as 3 hours — following tamoxifen administration. Elevated CUGBP1 protein expression was detected by immunofluorescence within 6 hours of tamoxifen administration. The time between RNA accumulation and changes in MBNL and CELF expression must be substantially less than 6 hours, because time is required for tamoxifen uptake, Cre-mediated recombination, and accumulation of EpA960(R) RNA from the recombined allele. The timing of CUGBP1 nuclear accumulation and MBNL colocalization directly correlates with Tnnt2
splicing changes, suggesting that both events could contribute to the disease.
Increased CUGBP1 was previously demonstrated to be pathogenic in CUGBP1-overexpressing mice in which increased levels of CUGBP1 in heart and skeletal muscle or in skeletal muscle alone resulted in neonatal lethality (32
). A pathogenic effect of CUGBP1 was also demonstrated in a fly model for DM1 (35
). While loss of MBNL activity can be explained by sequestration on nuclear CUG repeat RNA, the mechanism of increased CUGBP1 expression in cells containing RNA foci is less straightforward. Our present results confirm results from other labs indicating that neither CUGBP1 nor CUGBP2 is sequestered by RNA foci (21
); however, this does not preclude a transient interaction between CUGBP1 and RNA foci or transient or even stable interactions of CUGBP1 with a form of CUG repeat RNA not associated with foci. Our recent results from EpA960/MCM mice and DM1 cells indicate that steady-state levels of CUGBP1 protein increase as a result of hyperphosphorylation and increased half-life. Furthermore, CUG repeat RNA mediates this effect via activation of PKC, which was found to be activated in heart tissue from EpA960/MCM mice treated with tamoxifen as well as in heart tissue and cultured cells from individuals with DM1 (37
A comparison of our results with results from individuals with DM1 or DM2 and with other mouse models strongly suggests that the context of the pathogenic repeats determines whether CUGBP1 protein levels are affected. CUGBP1 levels are not elevated by repeat-containing RNAs that lack the DMPK
3′ UTR, that is, in the HSALR
mouse model in which CUG repeats are expressed in the context of the human skeletal α-actin mRNA or in DM2 skeletal muscle in which a CCUG repeat within ZNF9
intron 1 causes disease. In both cases, MBNL1 colocalizes with RNA foci and splicing is altered, but CUGBP1 levels are not affected (5
). In contrast, CUGBP1 protein is elevated in heart and skeletal muscle tissues and in cultured myoblasts and fibroblasts from DM1 patients (7
). Recently, high expression of RNA in mice containing the DMPK
3′ UTR with only 5 CTG repeats, GFP, and a segment of DMPK
intron 1 exhibited histological changes, myotonia, and splicing changes in skeletal muscle that were characteristic of DM1 (38
). These mice also exhibited arrhythmias. The absence of RNA foci and lack of obvious MBNL sequestration suggested that elevated CUGBP1 promotes the splicing changes (39
); however, an alternative explanation is that MBNL is effectively inactivated by binding to RNA containing 5 CUGs without forming foci. Mice expressing repeat RNA exhibited elevated CUGBP1 in skeletal muscle but not in heart, correlating with higher levels of RNA expression in muscle than in heart (38
). The absence of overt cardiomyopathy and the limitation of the cardiac phenotype to the conduction system suggest that the effects of the repeat RNA is limited to cells involved in conduction. It is possible that elevated CUGBP1 expression was not sufficiently widespread to be detected by Western blot analysis. We find that low-level induction of CUGBP1 protein expression, particularly when limited to specific cell types, is detectable by immunofluorescence but not Western blot analysis (our unpublished observations). It would be of interest to use immunofluorescence to examine heart tissue for cell-specific elevated CUGBP1 expression.
Expression of EpA960(R) mRNA in the EpA960/MCM 1323 line was lethal within 2 weeks of tamoxifen induction, while an identical mRNA lacking repeats had no noticeable phenotype even when more than 5-fold the amount of RNA was expressed. These results are consistent with results from the mouse C2C12 myoblast cell line, in which only CTG repeats in the context of the DMPK
3′ UTR inhibited skeletal muscle differentiation, while either CTG or DMPK
alone had no effect (40
). Similarly, expression of RNA containing the DMPK
3′ UTR with 960 CUG repeats induced hyperphosphorylation of CUGBP1 and increased protein stability, while comparable expression of the 3′ UTR alone had no effect (our unpublished observations). These results indicate that one or more cis-acting elements within DMPK
exon 15 act in combination with the CUG repeat RNA to induce alterations of CUGBP1 expression.
Expression of expanded CUG RNA in the heart affected both electric activity and contractility in EpA960/MCM mice. The prominent arrhythmias observed in DM1 (increased PR interval and heart block) are consistent with the observed degeneration of the conduction system (11
) and suggest a mechanism in which cell loss is caused by toxicity of CUG repeat RNA. This does not rule out the possibility that arrhythmias result from misregulated alternative splicing of genes critical for proper electrophysiological properties of the heart. Several genes have been shown to be misspliced in DM1 cardiac tissues, such as TNNT2
, and ALP
) as well as FXR1h
, identified in the present study. Mutations within a large number of ion channels have been shown to cause familial forms of arrhythmias, and these genes often express multiple isoforms by alternative splicing (43
). The effect of EpA960(R) RNA expression on cardiac function is independent of the arrhythmias and indicates that CUG repeat RNA induces a functional defect within the myocardium. It remains to be determined whether this reflects an effect on alternative splicing or other functions of MBNL, CELF, or other proteins.