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Mice Lacking MBNL1 and MBNL2 Exhibit Sudden Cardiac Death and Molecular Signatures Recapitulating Myotonic Dystrophy

ABSTRACT: Myotonic dystrophy (dystrophia myotonica, DM) is caused by expansions of CTG (type 1; DM1) or CCTG (type 2; DM2) repeats in the non-coding regions of the DMPK and CNBP genes, and patients with DM1 or DM2 often suffer from sudden cardiac death due to lethal arrhythmia. Specific molecular changes that underlie DM cardiac pathology have been linked to repeat-associated depletion of Muscleblind-like (MBNL) 1 and 2 proteins and upregulation of CUGBP Elav-like family member 1. Aims: We aim to create an Mbnl KO mouse model recapitulating DM cardiogenic death not under anesthesia, since none of the DM mouse models have reached this goal. Besides, it is unclear whether dysfunction of cardiomyocytes, among other cell types in the heart, can solely drive phenotypes. Methods and Results: We generated Myh6-Cre DKO (Mbnl1-/-; Mbnl2cond/cond; Myh6-Cre+/-) mice that completely eliminate Mbnl1/2 expression in cardiomyocytes. In this model, we found increased myocardial fibrosis, dilated cardiomyopathy and various arrhythmias. Importantly, we observed spontaneous lethal arrhythmic events in the context of shortened lifespan. RNA sequencing (RNA-seq) revealed mis-splicing events involving sarcomeric structure, mitochondrial dynamics and vesicular trafficking that mimics DM hearts. Gene expression changes were also noted by RNA-seq, and confirmed by qPCR. Notably, immunoblotting revealed a nearly 6-fold increase of Calsequestrin 1 and 50% reduction of epidermal growth factor proteins. Conclusion: These results showed that complete elimination of MBNL1/2 in mouse cardiomyocytes is sufficient to elicit a full spectrum of DM cardiac phenotypes including cardiac-related sudden death, and therefore these mice could serve as a useful model for studying DM heart pathogenesis and related translational research.

ORGANISM(S): Mus musculus

PROVIDER: GSE184574 | GEO | 2022/01/01

REPOSITORIES: GEO

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Project description:For some neurological disorders, disease is primarily RNA-mediated due to expression of non-coding microsatellite expansion RNAs (RNAexp). Toxicity is thought to result from enhanced binding of proteins to these expansions and depletion from their normal cellular targets. However, experimental evidence for this sequestration model is lacking. Here, we use HITS-CLIP and pre-mRNA processing analysis of human control versus myotonic dystrophy (DM) brains to provide compelling evidence for this RNA toxicity model. MBNL2 binds directly to DM repeat expansions in the brain resulting in depletion from its normal RNA targets with downstream effects on alternative splicing and polyadenylation. Similar RNA processing defects were detected in Mbnl compound knockout mice, highlighted by dysregulation of Mapt splicing and fetal tau isoform expression in adults. These results demonstrate that MBNL proteins are directly sequestered by RNAexp in the DM brain and introduce a powerful experimental tool to evaluate RNA-mediated toxicity in other expansion diseases. HITS-CLIP analysis was performed to identify RNA binding sites of MBNL2 in control, DM type 1 (DM1), and DM type 2 (DM2) autopsy-derived brain (n=3). Two regions of the brain selected for the study included the frontal cortex and hippocampus. Libraries were sequenced and wiggle files were generated for each biological replicate as well as three pooled biological replicates (3BRs) per group (control, DM1, and DM2). In addition, differential CLIP analysis (dCLIP) was performed to normalize binding data between groups and identify statistically significant changes in binding. The dCLIP analysis generated bedgraph files representing normalized binding profiles of MBNL2 in each group (control, DM1, and DM2) for visualization and comparative analysis. PolyA-seq was performed on control, DM1, and DM2 autopsy-derived brain samples (hippocampus and frontal cortex, n=3) as well as wild-type (WT) and Mbnl1; Mbnl2 conditional double knockout (Mbnl1-/-; Mbnl2c/c; Nestin-Cre+/- or DKO) brain (n=3). Libraries were sequenced and the resultant files were processed and aligned to the reference genomes (hg19 and mm10). Further computational processing was performed to remove internal oligo(dT) mis-priming events, identify valid polyA sites, and trim to the exact polyA sites. BedGraph files were generated for each group in human (control, DM1, and DM2) and mouse (WT and Mbnl DKO) for comparative visualization.

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Mice Lacking MBNL1 and MBNL2 Exhibit Sudden Cardiac Death and Molecular Signatures Recapitulating Myotonic Dystrophy

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