Genetic and pharmacologic elimination of myotonia from myotonic dystrophy type 1

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Project Summary: Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy that features prominent muscular symptoms, including myotonia, weakness, and wasting, as well as a diverse array of extra-muscular manifestations, including ocular cataracts, cardiac conduction abnormalities, and insulin resistance among others. DM1 is an autosomal dominant disorder that has been linked to the CTG trinucleotide repeat (TNR) expansion in the 3' UTR of the Dystrophia Myotonica Protein Kinase (DMPK) gene. DM1 patients possess greater than 50 (CTG) repeats and transcribe toxic RNA products capable of sequestering RNA-binding proteins, such as the Muscleblind-like (MBNL) family of splicing factors. This alters the available pool of splicing factors leading to changes in the alternative splicing for more than 100 developmentally regulated transcripts. Overall, these changes in splicing display a reversion back to embryonic patterns, with one example being the splicing of transcripts for ClC-1, a voltage-gated chloride channel expressed in skeletal muscle. In DM1, there is increased inclusion of exon 7a in Clcn1 transcripts, which causes a frameshift, premature termination codon, and a non-functional ion channel. This yields decreased inhibitory chloride conductance that has been directly linked to muscle hyperexcitability causing myotonia. In patients with DM1, the distribution of myotonia tends to correlate with other muscular symptoms—including weakness and wasting—with the highest severity of these symptoms localized to muscles of the distal extremities and oropharynx. This combined with evidence that approximately one-fourth of the expression level changes of transcripts in myofibers could be due to myotonia and not toxic-RNA has led to the hypothesis that myotonia plays a central role in driving the other skeletal muscle pathology in DM1. To investigate this, we developed a novel mouse line (ClC-1∆E7a/∆E7a) resistant to myotonia caused by the aberrant inclusion of E7a to eliminate myotonia from models of DM1. We will cross this novel line with the Mbnl1-/- and Mbnl1-/-/Mbnl2-/+ mouse models of DM1 to eliminate myotonia from these models and compare the changes in muscle physiology both in vitro (e.g., force contraction) and in vivo (e.g., grip strength), histopathology (e.g., central nucleation, fiber-type distribution), and genetic regulation (RNAseq) between myotonic and non-myotonic progeny. Thus far, we have successfully eliminated myotonia in Mbnl1-/-/ClC-1∆E7a mice and have observed significant histological and splicing differences compared to myotonic Mbnl1-/-/ClC-1+/+ mice, with the non-myotonic mice featuring phenotypes closer to wild-type. Then, we will replicate these studies with long-term anti-myotonic treatment of DM1 model mice with ranolazine with the goal that this could represent a myo-protective approach to aid DM1 patients.