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 1

Generating human myotonic dystrophy (DM) myogenic cell lines using direct reprogramming of urine-derived cells with the transcription factor MyoD.

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Generating human myotonic dystrophy (DM) myogenic cell lines using direc...
(A) Obtaining muscle biopsies requires an invasive muscle biopsy. In contrast, cells from urine can be obtained noninvasively, and following culturing, urine cells can be reprogrammed into cell types of interest (31). Urine cells were cultured from healthy controls and individuals with myotonic dystrophy type 1 (DM1) and DM2. Once cell cultures were established, cells were transduced with lentivirus expressing an inducible form of MyoD (iMyoD) in order to induce myogenic reprogramming. This lentivirus produces MyoD in the presence of tamoxifen. Tamoxifen was used to induce MyoD expression, which, in turn, stimulated multinucleated myotube formation in culture. Directly reprogrammed multinucleated myotubes were studied after 28 days in culture. (B) MyoD (red) protein expression was readily detected in the nucleus of tamoxifen-treated cells. Nuclei were labeled with Hoechst 33342 (blue). Scale bar: 100 μm. (C) With Hoechst costaining, the transduction efficiency of each transduction (example in B) could be estimated. Transduction efficiency with the iMyoD construct was similar among control, DM1, and DM2 cell lines and, in each case, averaged greater than 70%. Efficiencies are represented as percentages of MyoD positive nuclei relative to total number of nuclei.