RNA toxicity in myotonic muscular dystrophy induces NKX2-5 expression

DM1, an autosomal dominant disorder mapping to chromosome 19q, is the most common inherited neuromuscular disorder in adults, with an incidence of 1:8,000 globally. Common features of this multi-systemic disease in adults include myotonia, progressive skeletal muscle loss, cardiac conduction abnormalities, smooth muscle dysfunction, hypersomnolence, cataracts, testicular atrophy, frontal balding and insulin resistance1. DM1 is caused by an expansion of a (CTG)n triplet repeat in the 3′ UTR of the gene encoding DM protein kinase (DMPK) from a normal range (n = 5 to ~30) to greater than several thousand repeats (n = 50 to >2,000)2. Individuals with DM1 have a proclivity for cardiac conduction abnormalities, with about 60%–70% developing substantial problems that often result in sudden death3. These characteristic conduction disturbances consist primarily of sinoatrial and atrioventricular blocks of varying degrees of severity, resulting in slow and irregular heartbeats, and they are often accompanied by fibrosis, fatty infiltration and atrophy of the conduction system and the myocardium. As the mutation responsible for DM1 lies in a noncoding region of DMPK, several pathogenic mechanisms have been proposed (reviewed in ref. 4), but accumulating data suggest that mutant DMPK mRNAs have a role in many DM1 phenotypes5–9. The DM1 mutation results in an mRNA that is sequestered in the nucleus10. DM2, another almost identical clinical disorder, is caused by an expansion of a (CCTG)n sequence in the first intron of ZNF9 and also results in ribonuclear inclusions11. This discovery gave strong credence to the RNA toxicity hypothesis of myotonic dystrophy pathogenesis, wherein the mutant transcripts alter the function of RNA splicing factors, primarily members of the muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) family of RNA-binding proteins, either by sequestering them as part of the ribonuclear inclusions (as for MBNL1) or by increasing their expression (as for CUG-BP1). These proteins are RNA splicing factors, and altering their functional levels in adult tissues results in aberrant splicing and reversion to embryonic splicing patterns for various target mRNAs that are normally regulated under a mutual antagonism of MBNL1 and CUG-BP1 (ref. 12). The mechanisms underlying the cardiac conduction defects in DM1 are unknown. Thorough molecular analysis of tissues from individuals with myotonic dystrophy has not been performed owing to the inherent difficulties associated with obtaining cardiac tissues from living donors. The analysis of the few available autopsy samples is also complicated by the fact that the individuals usually have had a long history of disease and associated comorbidities that could result in secondary effects. Based on mouse models, haploinsufficiency of DMPK had been posited as a potential pathogenic mechanism13,14. However, with the discovery of the mutation causing DM2 and the postulation of a common mechanism, attention has been placed on the RNA toxicity pathogenesis. Several aberrant splicing events resulting in a reversion to embryonic splicing patterns have been identified in hearts from individuals with DM1, but their relationship to DM1 pathology is unclear15. We recently developed an inducible mouse model of RNA toxicity in DM1 in which we have clearly demonstrated the reversible nature of the DMPK 3′ UTR mRNA toxicity16. These strains (named 5-313 and 5-336) express a green fluorescent protein (GFP) gene fused with the DMPK 3′ UTR (denoted GFP-DMPK 3′ UTR) under the control of a tetracycline-inducible human DMPK promoter. Notably, upon induction, the mice overexpress a normal DMPK 3′ UTR mRNA with only (CUG)5 and have no apparent ribonuclear inclusions or evidence of reported RNA splicing defects in the heart16. They rapidly develop myotonia (the cardinal feature of myotonic dystrophy) and progressive heart block (Fig. 1a), a phenotypic combination seen only in individuals with myotonic dystrophy. This model represents the first clear demonstration of RNA toxicity resulting in cardiac conduction abnormalities. Here we explore the molecular basis of this phenomenon and report how this led to the unanticipated finding of induced expression of NKX2-5 in cardiac and skeletal muscle and describe the identification of NKX2-5 as a genetic modifier of the cardiotoxicity caused by the DMPK 3′ UTR mRNA. Figure 1 Cardiac conduction abnormalities. (a) ECG abnormalities in induced transgenic mice. P (atrial) and R (ventricular) waves are indicated. * denotes missing R waves in second-degree block. Note the lack of P waves in the complete heart block example. (b ...