Myotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy and the second most common form of muscular dystrophy overall (1
). DM is dominantly inherited and affects multiple organs, including skeletal muscle, heart, brain, and the endocrine system (2
). In the more common form of DM, DM type 1 (DM1), cardiac involvement occurs in 80% of the patients (3
). The cardiac manifestations of DM1 are heterogeneous and include conduction defects, arrhythmia, and dilated cardiomyopathy (5
). Due to the complexity of the cardiac disease, treatment strategies are limited. In addition, the molecular events involved in DM1 heart pathogenesis are unknown.
The genetic basis of DM1 is the expansion of CTG repeats in the 3′ untranslated region of the dystrophica myotonia protein kinase (DMPK
) gene (2
). Nuclear accumulation of the DMPK
RNA with expanded CUG repeats triggers events that lead to disruption of developmentally regulated alternative splicing (6
), which result in some of the disease symptoms such as myotonia and insulin resistance (7
). At least 2 families of RNA–binding proteins are implicated in DM1 pathogenesis: CUGBP and ETR3-like proteins (CELF) and muscleblind like (MBNL). Loss of MBNL function and increased levels of the CELF protein, CUG-binding protein 1 (CUGBP1), correlate with at least some of the splicing changes and disease symptoms observed in DM1 patients (9
Expanded CUG repeats bind and sequester MBNL proteins, resulting in their loss of function (12
). In support of a role for MBNL1 in DM1 pathogenesis, deletion of MBNL1 isoforms that bind to expanded CUG repeats in mice leads to cataracts, myotonia, development-specific splicing changes, and histological changes in skeletal muscle (10
). Furthermore, restoration of MBNL1 expression by adeno-associated viral gene delivery in skeletal muscle of mice expressing RNA containing 250 CUG repeats reverses splicing abnormalities and myotonia (16
). While the role of MBNL1 in DM1 skeletal muscle pathology is clear, the involvement in DM1 heart pathogenesis remains to be characterized.
In addition to MBNL1 sequestration, expanded CUG repeats activate the PKC signaling pathway, leading to CUGBP1 protein hyperphosphorylation and stabilization (17
), consistent with elevated steady-state levels of CUGBP1 in DM1 heart and skeletal muscle tissues (9
). Overexpression of CUGBP1 in mouse heart and skeletal muscle leads to DM1 splicing changes and results in embryonic lethality (19
), strongly suggesting pathogenic effects in striated muscle. However, the role of CUGBP1 in DM1 cardiac pathogenesis has not yet been investigated.
We previously established an inducible DM1 mouse model, in which a transgene containing the last exon of DMPK with 960 CTG repeats (EpA960) is induced to express CUG repeat–containing RNA [EpA960(R)], after recombination by Cre-mediated removal of concatamerized polyadenylation sites (21
). Tamoxifen-inducible and heart-specific EpA960(R) RNA expression was obtained from bitransgenic progeny of EpA960 animals mated to MerCreMer (MCM) animals, which express a tamoxifen-inducible form of Cre in a heart-specific manner (22
). Within 3 weeks after induction of EpA960(R) RNA, these mice exhibited high mortality, conduction abnormalities, and systolic and diastolic dysfunction as well as molecular changes seen in DM1 patients, such as colocalization of MBNL1 with RNA foci and reversion of splicing to embryonic patterns (21
). Importantly, activated PKCα/βII and increased CUGBP1 levels were evident within 6 hours after induction of expanded CUG RNA expression (17
), strongly suggesting that these are primary responses to expression of the toxic CUG repeat–containing RNA that contribute to DM1 pathogenesis.
To determine whether PKC activation is required to elicit the pathogenicity of EpA960(R) RNA, we used the specific PKC inhibitor, Ro-31-8220, to prevent PKCα/βII activation following induction of EpA960(R) RNA. This inhibitor was previously well characterized in mouse heart tissue by its ability to inhibit PKC isozymes and improve cardiac contractility (23
). Here, we show that administration of Ro-31-8220 to the heart-specific DM1 mouse model prevents activation of PKCα/βII as well as hyperphosphorylation and upregulation of CUGBP1. Importantly, Ro-31-8220 administration significantly improved mortality and improved cardiac conduction/contractile dysfunction. Consistent with the hypothesis that EpA960(R) RNA–induced pathogenesis is mediated at least in part via upregulation of CUGBP1, aberrant splicing of CUGBP1-regulated pre-mRNA targets was prevented by Ro-31-8220 administration, while MBNL-regulated splicing events were not. These results suggest that MBNL1 and CUGBP1 have distinct roles in DM1 heart pathogenesis. Importantly, administration of Ro-31-8220 did not improve the mortality in a second mouse model, in which CUGBP1 is inducibly overexpressed in the heart, using tetracycline-inducible transgene. These results indicate that PKC inhibition has no effect on cardiopathology caused by PKC-independent elevation of CUGBP1. Our results suggest that activation of the PKC pathway is a primary pathogenic event and that early abrogation of PKC activity ameliorates pathogenic cardiac features of DM1.