Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes.

Gaspare Mostacciuolo, Antonio Zaza, Alessandra Moretti, Massimiliano Gnecchi, Peter J Schwartz, Alfred L George, Gaetano M De Ferrari, Alberto Porta, Stefano Severi, Marcella Rocchetti, Eleonora Torre, Claudia Altomare, Luna Simona Pane, Manuela Mura, Daniel Sinnecker, Lia Crotti, Lisa Dreizehnter, Luca Sala

Journal: Cardiovascular research 2017;113(5):531-541

PMID: 28158429

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Abstract

AIMS

Calmodulin (CaM) is a small protein, encoded by three genes (CALM1-3), exerting multiple Ca2+-dependent modulatory roles. A mutation (F142L) affecting only one of the six CALM alleles is associated with long QT syndrome (LQTS) characterized by recurrent cardiac arrests. This phenotypic severity is unexpected from the predicted allelic balance. In this work, the effects of heterozygous CALM1-F142L have been investigated in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from a LQTS patient carrying the F142L mutation, i.e. in the context of native allelic ratio and potential gene modifiers.

METHODS AND RESULTS

Skin fibroblasts of the mutation carrier and two unrelated healthy subjects (controls) were reprogrammed to hiPSC and differentiated into hiPSC-CMs. Scanty IK1 expression, an hiPSC-CMs feature potentially biasing repolarization, was corrected by addition of simulated IK1 (Dynamic-Clamp). Abnormalities in repolarization rate-dependency (in single cells and cell aggregates), membrane currents and intracellular Ca2+ dynamics were evaluated as putative arrhythmogenic factors. CALM1-F142L prolonged repolarization, altered its rate-dependency and its response to isoproterenol. This was associated with severe impairment of Ca2+-dependent inactivation (CDI) of ICaL, resulting in augmented inward current during the plateau phase. As a result, the repolarization of mutant cells failed to adapt to high pacing rates, a finding well reproduced by using a recent hiPSC-CM action potential model. The mutation failed to affect IKs and INaL and changed If only marginally. Intracellular Ca2+ dynamics and Ca2+ store stability were not significantly modified. Mutation-induced repolarization abnormalities were reversed by verapamil.

CONCLUSION

The main functional derangement in CALM1-F142L was prolonged repolarization with altered rate-dependency and sensitivity to β-adrenergic stimulation. Impaired CDI of ICaL underlined the electrical abnormality, which was sensitive to ICaL blockade. High mutation penetrance was confirmed in the presence of the native genotype, implying strong dominance of effects.

Address: Department of Biotechnology and Bioscience, University of Milano-Bicocca, Milan, Italy.; Center for Cardiac Arrhythmias of Genetic Origin and Laboratory of Cardiovascular Genetics, IRCCS Istituto Auxologico Italiano, Milan, Italy.; I. Medical Department - Cardiology, Klinikum Rechts der Isar- Technische Universität München, Munich, Germany.; Department of Molecular Medicine - Unit of Cardiology, University of Pavia, Pavia, Italy.; Department of Cardiothoracic and Vascular Sciences - Coronary Care Unit and Laboratory of Clinical and Experimental Cardiology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.; Biomedical Engineering Laboratory D.E.I, University of Bologna, Cesena, Italy.; Department of Biomedical Sciences for Health, University of Milan, Milan, Italy.; Department of Cardiothoracic, Vascular Anesthesia and Intensive Care, IRCCS Policlinico San Donato, Milan, Italy.; Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.; Department of Medicine, University of Cape Town, Cape Town, South Africa.; DZHK (German Centre for Cardiovascular Research) - Partner Site Munich Heart Alliance, Munich, Germany.

DOI: 10.1093/cvr/cvx006