Abstract

The genetic basis of myotonic dystrophy type I (DM1) is the expansion of a CTG tract located in the 3′ untranslated region of DMPK. Expression of mutant RNAs encoding expanded CUG repeats plays a central role in the development of cardiac disease in DM1. Expanded CUG tracts form both nuclear and cytoplasmic aggregates, yet the relative significance of such aggregates in eliciting DM1 pathology is unclear. To test the pathophysiology of CUG repeat encoding RNAs, we developed and analyzed mice with cardiac-specific expression of a beta-galactosidase cassette in which a (CTG)400 repeat tract was positioned 3′ of the termination codon and 5′ of the bovine growth hormone polyadenylation signal. In these animals CUG aggregates form exclusively in the cytoplasm of cardiac cells. A key pathological consequence of expanded CUG repeat RNA expression in DM1 is aberrant RNA splicing. Abnormal splicing results from the functional inactivation of MBNL1, which is hypothesized to occur due to MBNL1 sequestration in CUG foci or from elevated levels of CUG-BP1. We therefore tested the ability of cytoplasmic CUG foci to elicit these changes. Aggregation of CUG RNAs within the cytoplasm results both in Mbnl1 sequestration and in approximately a two fold increase in both nuclear and cytoplasmic Cug-bp1 levels. Significantly, despite these changes RNA splice defects were not observed and functional analysis revealed only subtle cardiac dysfunction, characterized by conduction defects that primarily manifest under anesthesia. Using a human myoblast culture system we show that this transgene, when expressed at similar levels to a second transgene, which encodes expanded CTG tracts and facilitates both nuclear focus formation and aberrant splicing, does not elicit aberrant splicing. Thus the lack of toxicity of cytoplasmic CUG foci does not appear to be a consequence of low expression levels. Our results therefore demonstrate that the cellular location of CUG RNA aggregates is an important variable that influences toxicity and support the hypothesis that small molecules that increase the rate of transport of the mutant DMPK RNA from the nucleus into the cytoplasm may significantly improve DM1 pathology.

Highlights

  • Myotonic dystrophy 1 (DM1) is a multi-system disorder characterized by skeletal myopathy and cardiac disease [1]

  • Construction and analyses of a-myosin heavy chain (a-MHC)-LacZ-(CTG)400 mice To characterize the pathological effects intrinsic to the expression of expanded CTG repeat tracts in cardiac muscle, we built a transgenic cassette encoding the gene for b-galactosidase (LacZ) followed by a tract of,400 uninterrupted CTG repeats, which was cloned in a linker sequence and inserted between the LacZ termination codon and the bovine growth hormone polyadenylation (BGH-PolyA) sequence

  • We examined the pattern of alternative splicing of cardiac troponin T (Tnnt2), Z-band alternatively spliced PDZ-motif protein (Zasp), alpha-actinine-2 associated LIM protein (Alp) and M-line Titin [33,34,35] RNAs

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Summary

Introduction

Myotonic dystrophy 1 (DM1) is a multi-system disorder characterized by skeletal myopathy and cardiac disease [1]. Cardiac symptoms include variable conduction disorders and wall motion abnormalities [2,3,4,5,6]. First degree atrioventricular (AV) block and intraventricular conduction disorders are seen in ,75% of DM1 patients [2]. Progressive deterioration of the conduction system resulting in complete AV block or ventricular arrhythmias are primarily responsible for sudden cardiac death [3,4]. Conduction disorders predominate in DM1, decreased ventricular systolic and diastolic functions, and hypertrophic and dilated cardiomyopathy, have been reported in severely affected patients [5,6,7,8]. Histological abnormalities include myofibrillar loss, fibrosis and fatty infiltration of both the working myocardium and the specialized conduction system. Electron microscopic examination shows aberrant Z lines and mitochondrial abnormalities in DM1 hearts [9]

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