Cardiomyocytes derived from human induced Pluripotent Stem Cells effectively model genetic disease

Background: Genetic diseases arising from repeat expansions are difficult to model in mice because of genomic instability and loss of repeat expansions. Therefore, we used induced pluripotent stem cells (iPSCs) to model myotonic dystrophy, a repeat expansion disorder that affects multiple systems including heart and muscle. Type 1 myotonic dystrophy (DM1) is characterized genetically by a CTG repeat expansion in the DMPK gene with repeat number >70 considered pathogenic. Type 2 myotonic dystrophy (DM2) arises from expansion of a tetranucleotide repeat (CCTG) to >5000 copies. In DM1, RNA transcription of these expanded repeats is associated with the formation of intranuclear foci that bind splicing factors such as muscleblind-like (MBNL) family of RNA-binding proteins. This results in a functional depletion in MBNL and leads to missplicing of many genes. The heart is frequently affected in DM, and cardiac arrhythmias are common ranging from atrial fibrillation to ventricular arrhythmias. The molecular mechanisms underlying cardiac dysfunction in DM are not clear, and the differences between DM1 and DM2 are not well delineated. Methods/Results: We established iPSCs from both DM1 and DM2 patients and differentiated these into cardiomyocytes in order to better understand the basis of arrhythmias in DM. Using RNA fluorescent in situ hybridization (FISH), we identified an increase in intranuclear repeat expansion in both undifferentiated iPSCs and iPSC-derived cardiomyocytes compared to normal control cells. With cardiomyocyte differentiation, DM1 and DM2 cells showed a trend toward an increase in number of foci per nuclei from their undifferentiated iPSC counterparts, suggesting that differentiation may increase repeat transcription or may actually increase the repeat expansion itself. Using indo-1 ratiometric dye, we examined the Ca2+-handling properties of DM1 and DM2 cardiomyocytes. Cells were paced at four different frequencies (0.25, 0.5, 0.75, and 1 Hz) and 10 to 15 transient measurements were averaged at each frequency. Compared to non-DM cardiomyocytes, both DM1 and DM2 cardiomyocytes showed significantly slower calcium release and uptake at each frequency. Interestingly, DM1 cardiomyocytes showed intranuclear formation of MBNL clusters by immunofluorescence, whereas DM2 cardiomyocytes showed only non-specific nuclear staining for MBNL. Conclusions: These findings indicate that DM1 and DM2 may share the mechanisms related to the formation of intranuclear foci especially during differentiation and changes in Ca2+ handling. However, DM1 and DM2 may have distinct pathogenesis regarding MBNL. These cells provide a format in which to better understand disease mechanisms and test therapies directed at correcting toxic RNA activities.