Distinct pathological signatures in human cellular models of myotonic dystrophy subtypes
Ellis Y. Kim, … , Hao F. Zhang, Elizabeth M. McNally
Ellis Y. Kim, … , Hao F. Zhang, Elizabeth M. McNally
Published February 7, 2019
Citation Information: JCI Insight. 2019;4(6):e122686. https://doi.org/10.1172/jci.insight.122686.
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Research Article Cardiology Stem cells

Distinct pathological signatures in human cellular models of myotonic dystrophy subtypes

Abstract

Myotonic dystrophy (DM) is the most common autosomal dominant muscular dystrophy and encompasses both skeletal muscle and cardiac complications. DM is nucleotide repeat expansion disorder in which type 1 (DM1) is due to a trinucleotide repeat expansion on chromosome 19 and type 2 (DM2) arises from a tetranucleotide repeat expansion on chromosome 3. Developing representative models of DM in animals has been challenging due to instability of nucleotide repeat expansions, especially for DM2, which is characterized by nucleotide repeat expansions often greater than 5,000 copies. To investigate mechanisms of human DM, we generated cellular models of DM1 and DM2. We used regulated MyoD expression to reprogram urine-derived cells into myotubes. In this myogenic cell model, we found impaired dystrophin expression, in the presence of muscleblind-like 1 (MBNL1) foci, and aberrant splicing in DM1 but not in DM2 cells. We generated induced pluripotent stem cells (iPSC) from healthy controls and DM1 and DM2 subjects, and we differentiated these into cardiomyocytes. DM1 and DM2 cells displayed an increase in RNA foci concomitant with cellular differentiation. iPSC-derived cardiomyocytes from DM1 but not DM2 had aberrant splicing of known target genes and MBNL sequestration. High-resolution imaging revealed tight association between MBNL clusters and RNA foci in DM1. Ca2+ transients differed between DM1- and DM2 iPSC–derived cardiomyocytes, and each differed from healthy control cells. RNA-sequencing from DM1- and DM2 iPSC–derived cardiomyocytes revealed distinct misregulation of gene expression, as well as differential aberrant splicing patterns. Together, these data support that DM1 and DM2, despite some shared clinical and molecular features, have distinct pathological signatures.

Authors

Ellis Y. Kim, David Y. Barefield, Andy H. Vo, Anthony M. Gacita, Emma J. Schuster, Eugene J. Wyatt, Janel L. Davis, Biqin Dong, Cheng Sun, Patrick Page, Lisa Dellefave-Castillo, Alexis Demonbreun, Hao F. Zhang, Elizabeth M. McNally

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Figure 6

Fluorescence in situ hybridization (FISH) detected an increase in RNA foci after cardiomyocyte differentiation.

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Fluorescence in situ hybridization (FISH) detected an increase in RNA fo...
FISH was used to detect RNA-encoded nucleotide repeat expansions using probes specific to the repeat expansions in DM1 or DM2. RNA probes for FISH included either (CAG)10 to detect DM1 or (CAGG)5 to detect the DM2 repeat expansion. Probes were labeled with Cy3 (red), FISH was conducted, and the number of foci was quantified and compared in undifferentiated iPSCs and iPSCs that had been differentiated to cardiomyocytes (iPSC-CM). (A) Example images of RNA foci (red) visualized using Cy3-labeled probes specific for the myotonic disease subtype. Nuclei were labeled with Hoechst (blue). Magnified images are shown in the insets. Scale bar: 20 μm; 10 μm (inset). (B) DM1 iPSCs and iPSC-CMs had an increased number of RNA foci compared with healthy control cells (*P = 0.04, **P = 0.0008, respectively). DM2 iPSC-CMs had an increased number of RNA foci compared with healthy control cells (****P = 0.03), while iPSC cells trended toward significance when compared with control cells (P < 0.07). For both DM1 and DM2, differentiation of iPSCs into iPSC-CMs resulted in an increase number of RNA repeat foci in iPSC-CMs (***P = 0.008, *P = 0.04, respectively). The number of RNA foci did not change with differentiation in control cells (2-way ANOVA).