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Modelling CPVT in a dish: Characterization of two novel ryanodine receptor mutations using human induced pluripotent stem cell-derived cardiomyocytes
Topic 25 - Rhythmology, stimulation—C tion applied to intact sinoatrial tissues did not show significant effects under basal conditions, but induced a significant reduction of the spontaneous frequency (PA/min) under -adrenergic stimulation. Conclusion We here demonstrated that mitochondrial Ca2+ regulates intracellular Ca2+ dynamics, and that mitochondrial Ca2+ is needed to support cAMP production, by stimulating ATP synthesis during a -adrenergic stimulation. We propose that this mechanism is required to maintain a high spontaneous frequency, especially under physiological stress conditions. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2019.02.180 529
Mapping of the catecholaminergic automatic activity from its foci within the pulmonary veins to the left atria in rat C. Pasqualin 1,2,∗ , C. Guilloteau 2 , F. Gannier 1,2 , N. Peineau 2 , P. Bredeloux 1,2 , V. Maupoil 1,2 1 Groupe physiologie des cellules cardiaques et vasculaires, laboratoire STIM - EA 7349 2 Groupe physiologie des cellules cardiaques et vasculaires, université de Tours, Tours, France ∗ Corresponding author. E-mail address: [email protected] (C. Pasqualin) Introduction Paroxysmal atrial fibrillation (pAF) is triggered by ectopic foci of electrical activities located in pulmonary vein (PV) myocardial sleeves. Previously, we showed in rat that norepinephrine (NE) superfusion induced a catecholaminergic automatic activity (CAA) occurring in repetitive bursts of action potential (AP) in isolated PV strips but not in left atria (LA). Objective This study was realised to characterize the calcium activity induced during the CAA as well as its propagation through PV and LA. Results Ectopic foci induced by NE were located in both proximal and distal parts of PV. Depending on the preparation, one isolated or several ectopic foci were identified, within the same or in two different PV of the preparation. This could explain the complex aspect of some bursts composed of electrical activities from these different foci. CAA triggered by these foci spread to the LA or remained confined to the PV where it originated. In this case, microelectrode recordings showed that CAA induced DAD-like electrotonic membrane potential oscillations in the LA, far from the PV foci. However, these electrical waves were not associated with calcium oscillations. Moreover, during bursting activity, re-entrant circuits sometimes occured in PV. Conclusion Ectopic foci triggered by NE in rat PV present similarities with those observed during pAF in human. The conduction of CAA from PV to atria is variable, yet always causing electric oscillations of membrane potential which could be arrhythmogenic.
267 Disclosure of interest peting interest.
The authors declare that they have no com-
https://doi.org/10.1016/j.acvdsp.2019.02.181 328
Modelling CPVT in a dish: Characterization of two novel ryanodine receptor mutations using human induced pluripotent stem cell-derived cardiomyocytes S. De Waard 1,2,∗ , J. Montnach 1 , C. Cortinovis 1 , V. Forest 1 , A. Girardeau 1 , M. Ronjat 1,2 , M. De Waard 1,2 , P. Lemarchand 1 , N. Gaborit 1 1 Inserm, 44000, l’institut du thorax, CNRS, université de Nantes, Nantes 2 LabEx Ion Channels Science and Therapeutics, Valbone, France ∗ Corresponding author. E-mail address: [email protected] (S. De Waard) Background Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disease exposing afflicted patients to sudden cardiac death. Most CPVT cases have been linked to mutations of the gene encoding ryanodine receptor type 2 (RYR2). RyR2 is a calcium channel located in the membrane of the sarcoplasmic reticulum (SR). It mediates release of calcium stocks from the SR to the cytosol, playing a major role in excitation-contraction coupling. Although several studies have investigated mechanisms underlying CPVT in mice or in vitro models, little has been done concerning functional assessment of CPVT physiopathology in human cardiomyocytes. Objective We aimed at characterizing, at cellular level, CPVT phenotype of patients carrying two new RyR2 mutations. Method Human induced pluripotent stem cells (hiPSCs) were derived from three CPVT patients. Two patients belonging to the same family carry the R4959Q RyR2 pore mutation. The third patient harbors the Y2476D mutation located in the cytosolic domain of the protein. hiPSCs were differentiated into cardiomyocytes for functional studies. Action potentials were recorded using patch-clamp technique. Calcium handling and contractility were investigated using Ionoptix and CardioExcyte technologies. Results hiPS-derived cardiomyocytes (hiPS-CMs) from CPVT patients displayed impaired beating properties. Calcium handling properties were also disrupted with changes in calcium transients, accompanied with increased diastolic calcium leak. At last, CPVT hiPS-CMs showed characteristics changes in action potential and arrhythmic events. Coherently with the patients’ phenotype, arrhythmic events were more frequent under beta-adrenergic stress. Conclusion This work enabled us to characterize two novel CPVT mutations, providing more insight into functional mechanisms involved in this pathology, and further validating the use of human pluripotent stem cells for investigating complex cardiac rhythm disorders. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2019.02.182
Mapping of the catecholaminergic automatic activity from its foci within the pulmonary veins to the left atria in rat C. Pasqualin 1,2,∗ , C. Guilloteau 2 , F. Gannier 1,2 , N. Peineau 2 , P. Bredeloux 1,2 , V. Maupoil 1,2 1 Groupe physiologie des cellules cardiaques et vasculaires, laboratoire STIM - EA 7349 2 Groupe physiologie des cellules cardiaques et vasculaires, université de Tours, Tours, France ∗ Corresponding author. E-mail address: [email protected] (C. Pasqualin) Introduction Paroxysmal atrial fibrillation (pAF) is triggered by ectopic foci of electrical activities located in pulmonary vein (PV) myocardial sleeves. Previously, we showed in rat that norepinephrine (NE) superfusion induced a catecholaminergic automatic activity (CAA) occurring in repetitive bursts of action potential (AP) in isolated PV strips but not in left atria (LA). Objective This study was realised to characterize the calcium activity induced during the CAA as well as its propagation through PV and LA. Results Ectopic foci induced by NE were located in both proximal and distal parts of PV. Depending on the preparation, one isolated or several ectopic foci were identified, within the same or in two different PV of the preparation. This could explain the complex aspect of some bursts composed of electrical activities from these different foci. CAA triggered by these foci spread to the LA or remained confined to the PV where it originated. In this case, microelectrode recordings showed that CAA induced DAD-like electrotonic membrane potential oscillations in the LA, far from the PV foci. However, these electrical waves were not associated with calcium oscillations. Moreover, during bursting activity, re-entrant circuits sometimes occured in PV. Conclusion Ectopic foci triggered by NE in rat PV present similarities with those observed during pAF in human. The conduction of CAA from PV to atria is variable, yet always causing electric oscillations of membrane potential which could be arrhythmogenic.
267 Disclosure of interest peting interest.
The authors declare that they have no com-
https://doi.org/10.1016/j.acvdsp.2019.02.181 328
Modelling CPVT in a dish: Characterization of two novel ryanodine receptor mutations using human induced pluripotent stem cell-derived cardiomyocytes S. De Waard 1,2,∗ , J. Montnach 1 , C. Cortinovis 1 , V. Forest 1 , A. Girardeau 1 , M. Ronjat 1,2 , M. De Waard 1,2 , P. Lemarchand 1 , N. Gaborit 1 1 Inserm, 44000, l’institut du thorax, CNRS, université de Nantes, Nantes 2 LabEx Ion Channels Science and Therapeutics, Valbone, France ∗ Corresponding author. E-mail address: [email protected] (S. De Waard) Background Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disease exposing afflicted patients to sudden cardiac death. Most CPVT cases have been linked to mutations of the gene encoding ryanodine receptor type 2 (RYR2). RyR2 is a calcium channel located in the membrane of the sarcoplasmic reticulum (SR). It mediates release of calcium stocks from the SR to the cytosol, playing a major role in excitation-contraction coupling. Although several studies have investigated mechanisms underlying CPVT in mice or in vitro models, little has been done concerning functional assessment of CPVT physiopathology in human cardiomyocytes. Objective We aimed at characterizing, at cellular level, CPVT phenotype of patients carrying two new RyR2 mutations. Method Human induced pluripotent stem cells (hiPSCs) were derived from three CPVT patients. Two patients belonging to the same family carry the R4959Q RyR2 pore mutation. The third patient harbors the Y2476D mutation located in the cytosolic domain of the protein. hiPSCs were differentiated into cardiomyocytes for functional studies. Action potentials were recorded using patch-clamp technique. Calcium handling and contractility were investigated using Ionoptix and CardioExcyte technologies. Results hiPS-derived cardiomyocytes (hiPS-CMs) from CPVT patients displayed impaired beating properties. Calcium handling properties were also disrupted with changes in calcium transients, accompanied with increased diastolic calcium leak. At last, CPVT hiPS-CMs showed characteristics changes in action potential and arrhythmic events. Coherently with the patients’ phenotype, arrhythmic events were more frequent under beta-adrenergic stress. Conclusion This work enabled us to characterize two novel CPVT mutations, providing more insight into functional mechanisms involved in this pathology, and further validating the use of human pluripotent stem cells for investigating complex cardiac rhythm disorders. Disclosure of interest The authors declare that they have no competing interest. https://doi.org/10.1016/j.acvdsp.2019.02.182