Elsevier

Heart Rhythm

Volume 20, Issue 1, January 2023, Pages 89-99
Heart Rhythm

Experimental
Disrupted CaV1.2 selectivity causes overlapping long QT and Brugada syndrome phenotypes in the CACNA1C-E1115K iPS cell model

https://doi.org/10.1016/j.hrthm.2022.08.021Get rights and content

Background

A missense mutation in the α1c subunit of voltage-gated L-type Ca2+ channel–coding CACNA1C-E1115K, located in the Ca2+ selectivity site, causes a variety of arrhythmogenic phenotypes.

Objective

We aimed to investigate the electrophysiological features and pathophysiological mechanisms of CACNA1C-E1115K in patient-specific induced pluripotent stem cell (iPSC)–derived cardiomyocytes (CMs).

Methods

We generated iPSCs from a patient carrying heterozygous CACNA1C-E1115K with overlapping phenotypes of long QT syndrome, Brugada syndrome, and mild cardiac dysfunction. Electrophysiological properties were investigated using iPSC-CMs. We used iPSCs from a healthy individual and an isogenic iPSC line corrected using CRISPR-Cas9–mediated gene editing as controls. A mathematical E1115K-CM model was developed using a human ventricular cell model.

Results

Patch-clamp analysis revealed that E1115K-iPSC-CMs exhibited reduced peak Ca2+ current density and impaired Ca2+ selectivity with an increased permeability to monovalent cations. Consequently, E1115K-iPSC-CMs showed decreased action potential plateau amplitude, longer action potential duration (APD), and a higher frequency of early afterdepolarization compared with controls. In optical recordings examining the antiarrhythmic drug effect, late Na+ channel current (INaL) inhibitors (mexiletine and GS-458967) shortened APDs specifically in E1115K-iPSC-CMs. The AP-clamp using a voltage command obtained from E1115K-iPSC-CMs with lower action potential plateau amplitude and longer APD confirmed the upregulation of INaL. An in silico study recapitulated the in vitro electrophysiological properties.

Conclusion

Our iPSC-based analysis in CACNA1C-E1115K with disrupted CaV1.2 selectivity demonstrated that the aberrant currents through the mutant channels carried by monovalent cations resulted in specific action potential changes, which increased endogenous INaL, thereby synergistically contributing to the arrhythmogenic phenotype.

Introduction

CACNA1C encodes the pore-forming α1 subunit (CaV1.2) of the L-type Ca2+ channel (LTCC).1 In CaV1.2, Ca2+ selective filters are composed of 4 negatively charged glutamate (E) residues in equivalent positions in domains I–IV to form the “EEEE ring” (Figure 1A) in contrast to the “DEKA” ring in the NaV1 channels (Online Supplemental Figure S1), and EIII (E1115 in CaV1.2) exhibits the strongest influence on Ca2+ selectivity.2 A single amino acid substitution in this position drastically changes its selectivity from Ca to Na ions.2,3

A missense mutation at this critical site (CACNA1C-E1115K) (Figure 1A) was reported in a family with Brugada syndrome4 and a patient with long QT syndrome (LQTS),5 independently. In the latter report, functional analyses using heterologous expression in tsA201 cells confirmed that Ca2+ selectivity in the LTCC is ablated by this mutation. However, the heterologous overexpression system has some limitations, such as the lack of other ionic currents or the inability to assess their summation as an action potential (AP). Recently, human-induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) offer a platform to reproduce actual human CMs, allowing us to model the disease-causing mechanism associated with specific genetic backgrounds.

In this study, we established an iPSC model of CACNA1C-E1115K from an individual with overlapping phenotypes of LQTS, Brugada syndrome, and mild cardiac systolic dysfunction and revealed impaired ion selectivity of the encoded LTCC, thereby recapitulating the clinical phenotypes in the index patient. Further, we demonstrated the importance of late Na+ channel current (INaL) enhancement associated with the abnormal mutant LTCC and the efficacy of INaL inhibitors, potentially facilitating the design for novel therapeutics.

Section snippets

Methods

A detailed description of all methods is provided in the Online Supplement. All protocols were approved by the Committee on Human Research, Kyoto University (Kyoto, Japan) and conformed to the principles of the Declaration of Helsinki.

Clinical profile of the proband and family members

A 12-year-old Japanese boy presented with bradycardia, QT prolongation (corrected QT [QTc] interval 473 ms), and coved-type ST-segment elevation in lead V2 after a pilsicainide challenge test (Figures 1B and 1C). There was no history of syncope or arrhythmia. The patient was also diagnosed with autism at the age of 6. Echocardiography showed mildly reduced left ventricular (LV) systolic function (LV ejection fraction 54%; Online Supplemental Figure S4A). Genetic testing identified heterozygous

Discussion

Our iPSC model of CACNA1C-E1115K demonstrated the impaired ion selectivity of the LTCC characterized by APD prolongation and decreased APA during the plateau phase. Further, our data showed that endogenous INaL secondarily increased and contributed to APD prolongation.

Conclusion

Our iPSC model demonstrated the aberrant ion currents through the LTCC with the impaired selectivity filter and the specific AP morphology caused by CACNA1C-E1115K, which underlined the mixed LQTS and Brugada syndrome phenotypes. In addition, the enhancement of INaL has been shown to contribute to the arrhythmogenicity and might represent a pharmacological treatment target. Our multimodal analysis provides a powerful platform for facilitating the characterization of pathophysiological features

Acknowledgments

We thank the proband and his family for their participation in this study. We are also grateful for Kyoko Yoshida, MAgr, for her technical assistance.

References (23)

  • S.H. Heinemann et al.

    Calcium channel characteristics conferred on the sodium channel by single mutations

    Nature

    (1992)
  • Funding Sources: This work was supported by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science (JSPS KAKENHI) (Grant Number JP16K09499 and JP19K08538 to Dr Makiyama and JP19K17595 to Dr Wuriyanghai), the Japan Agency for Medical Research and Development (AMED) (Grant Number JP20bm0804022 and JP21bm0804022 to Dr Yoshida, and JP20bm0104001 to Dr Woltjen), the Suzuken Memorial Foundation (to Drs Makiyama and Kimura), and the Fujiwara Memorial Foundation (to Dr Kashiwa).

    Disclosures: Dr Yoshida owns stocks in iPS Portal. The other authors have reported no conflict of interest with the contents of this article.

    1

    These three authors equally contributed to the work as the co-corresponding authors.

    2

    Present addresses: Dr Thomas L.Maurissen, Roche Pharma Research and Early Development, Immunology, Infectious Diseases and Ophthalmology, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland; Dr Yuta Yamamoto, Division of Cardiovascular Medicine, Department of Medicine, Stanford University, Stanford, CA, United States; Dr Naomasa Makita, Department of Cardiology, Sapporo Teishinkai Hospital, Sapporo, Japan.

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