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

M. Rocchetti, L. Sala, L. Dreizehnter, L. Crotti, D. Sinnecker, M. Mura, L.S. Pane, C. Altomare, E. Torre, G. Mostacciuolo, S. Severi, A. Porta, G.M. De Ferrari, A.L. George, P.J. Schwartz, M. Gnecchi, A. Moretti, A. Zaza

Research output: Contribution to journalArticlepeer-review

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. © 2017 The Author.
Original languageEnglish
Pages (from-to)531-541
Number of pages11
JournalCardiovascular Research
Volume113
Issue number5
DOIs
Publication statusPublished - 2017

Keywords

  • Calmodulin
  • hiPSC-CMs
  • LQTS
  • Mutations
  • Sudden death
  • calcium binding protein
  • calcium ion
  • calm 1 protein
  • calm 2 protein
  • calm 3 protein
  • unclassified drug
  • beta adrenergic receptor stimulating agent
  • calcium calmodulin dependent protein kinase II
  • calcium channel blocking agent
  • calmodulin
  • calmodulin 1, human
  • genetic marker
  • action potential
  • adult
  • allele
  • arrhythmogenesis
  • Article
  • calcium cell level
  • cardiac muscle cell
  • cell differentiation
  • cell mutant
  • controlled study
  • electric activity
  • female
  • heart arrest
  • human
  • human cell
  • induced pluripotent stem cell
  • long QT syndrome
  • male
  • membrane current
  • modifier gene
  • phenotype
  • priority journal
  • repolarization
  • sarcoplasmic reticulum
  • skin fibroblast
  • whole cell patch clamp
  • calcium signaling
  • case control study
  • cell culture
  • cell reprogramming technique
  • cytology
  • drug effects
  • fibroblast
  • genetic predisposition
  • genetic transfection
  • genetics
  • heart pacing
  • heart rate
  • heterozygote
  • kinetics
  • membrane potential
  • metabolism
  • mutation
  • nuclear reprogramming
  • pathophysiology
  • skin
  • Adrenergic beta-Agonists
  • Calcium Channel Blockers
  • Calcium Signaling
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Cardiac Pacing, Artificial
  • Case-Control Studies
  • Cells, Cultured
  • Cellular Reprogramming
  • Cellular Reprogramming Techniques
  • Fibroblasts
  • Genetic Markers
  • Genetic Predisposition to Disease
  • Heart Rate
  • Heterozygote
  • Humans
  • Induced Pluripotent Stem Cells
  • Kinetics
  • Long QT Syndrome
  • Membrane Potentials
  • Mutation
  • Phenotype
  • Skin
  • Transfection

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