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 March 25, 2019; First 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 2

Myotonic dystrophy type 1 (DM1) myogenic cells have reduced dystrophin expression compared with DM2 and control cells.

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Myotonic dystrophy type 1 (DM1) myogenic cells have reduced dystrophin e...
Direct reprogramming of urine cells was used to generate myotubes in culture from healthy control, DM1, and DM2 subjects. The clinical features of these human subjects are indicated in Table 1. (A) Myotubes were immunostained with α-actinin (red) and dystrophin (green) to assess myotube formation, sarcomere, and membrane protein content using markers of the Z disc (α-actinin) and the membrane-associated marker dystrophin. Nuclei were labeled with Hoechst (blue). The left column shows elongated myotubes and the merged α-actinin and dystrophin staining. Higher-magnification images of the white dotted box in column 3 are shown in the right column. Scale bar: 100 μm (images in columns 1, 2, and 3). Scale bar: 25 μm (right column). Reduced dystrophin staining was evident in DM1 myotubes. (B) DM1 myotubes had reduced dystrophin fluorescence (arbitrary units, AU) compared with control and DM2 myotubes. The reduced expression of dystrophin is viewed as a sign of impaired differentiation of DM1 myotubes, consistent with previous reports of cultured myoblasts from muscle (33, 34), and it indicates that reprogrammed urine cells can be used to model myotonic dystrophy in culture.