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Hum Mol Genet. 2017 May 1;26(9):1670-1677. doi: 10.1093/hmg/ddx073.

Allele-specific ablation rescues electrophysiological abnormalities in a human iPS cell model of long-QT syndrome with a CALM2 mutation.

Author information

1
Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan.
2
Department of Cardiovascular and Respiratory Medicine, Shiga University of Medical Science, Seta-Tsukinowa, Otsu 520-2192, Japan.
3
Department of Molecular Physiology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan.
4
Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.
5
Department of Pediatrics, Nagasaki University Hospital, Nagasaki 852-8501, Japan.

Abstract

Calmodulin is a ubiquitous Ca2+ sensor molecule encoded by three distinct calmodulin genes, CALM1-3. Recently, mutations in CALM1-3 have been reported to be associated with severe early-onset long-QT syndrome (LQTS). However, the underlying mechanism through which heterozygous calmodulin mutations lead to severe LQTS remains unknown, particularly in human cardiomyocytes. We aimed to establish an LQTS disease model associated with a CALM2 mutation (LQT15) using human induced pluripotent stem cells (hiPSCs) and to assess mutant allele-specific ablation by genome editing for the treatment of LQT15. We generated LQT15-hiPSCs from a 12-year-old boy with LQTS carrying a CALM2-N98S mutation and differentiated these hiPSCs into cardiomyocytes (LQT15-hiPSC-CMs). Action potentials (APs) and L-type Ca2+ channel (LTCC) currents in hiPSC-CMs were analyzed by the patch-clamp technique and compared with those of healthy controls. Furthermore, we performed mutant allele-specific knockout using a CRISPR-Cas9 system and analyzed electrophysiological properties. Electrophysiological analyses revealed that LQT15-hiPSC-CMs exhibited significantly lower beating rates, prolonged AP durations, and impaired inactivation of LTCC currents compared with control cells, consistent with clinical phenotypes. Notably, ablation of the mutant allele rescued the electrophysiological abnormalities of LQT15-hiPSC-CMs, indicating that the mutant allele caused dominant-negative suppression of LTCC inactivation, resulting in prolonged AP duration. We successfully recapitulated the disease phenotypes of LQT15 and revealed that inactivation of LTCC currents was impaired in CALM2-N98S hiPSC model. Additionally, allele-specific ablation using the latest genome-editing technology provided important insights into a promising therapeutic approach for inherited cardiac diseases.

PMID:
28335032
DOI:
10.1093/hmg/ddx073
[Indexed for MEDLINE]

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