Calmodulinopathy−Analysis of Human iPS Cell Model of Long-QT Syndrome with a CALM2 Mutation and Therapeutic Approach Using Genome Editing Technology−

Abstract

Calmodulin is a ubiquitous Ca2+ sensor molecule encoded by the three unique genes, CALM1–3. Recently, mutations in CALM1–3 have been reported to be associated with severe arrhythmias including 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）. In addition, we performed mutant allele-specific knockout using a CRISPR-Cas9 system. Electrophysiological properties of hiPSC-CMs were analyzed by the patch-clamp technique. Electrophysiological analyses revealed that LQT15-hiPSC-CMs exhibited significantly lower beating rates, prolonged action potential durations（APD）, and impaired inactivation of LTCC currents compared with control. Ablation of the mutant allele rescued the electrophysiological abnormalities of LQT15-hiPSC-CMs. These results indicate that the mutant allele caused dominant-negative suppression of LTCC inactivation, resulting in prolonged APD. We successfully recapitulated the disease phenotypes of LQT15 and revealed the pathophysiological mechanism 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.