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 3

Muscleblind-like splicing regulator 1 (MBNL1) form intranuclear foci in Myotonic dystrophy type 1 (DM1) myotubes.

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Muscleblind-like splicing regulator 1 (MBNL1) form intranuclear foci in ...
A hallmark of DM1 is the sequestration of the splicing factor MBNL1 into intranuclear protein aggregates (12). In DM1, MBNL1 foci associate with CUG repeat expansions expressed in RNA (12-15). To determine if urine-derived, reprogrammed myotubes reflected this same pattern, myotubes were immunostained with α-MBNL1 antibody (green), and nuclei were labeled with Hoechst (blue). In myogenic cells generated from healthy controls, the normal pattern of intranuclear MBNL1 protein was seen with its distribution throughout the nucleus in a diffuse pattern (control row). In DM1 myotubes, this diffuse MBNL pattern was lost and, instead, readily detectable MBNL1-positive intranuclear foci were apparent (DM1 row, green dots). In DM2 myogenic cells, the MBNL1 pattern resembled control cells with diffuse MBNL1 distribution throughout the nucleus, indicating that MBNL1 sequestration is more associated with DM1 compared with DM2. Scale bar: 2 μm.