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Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia.

Jung CB, Moretti A, Mederos y Schnitzler M, Iop L, Storch U, Bellin M, Dorn T, Ruppenthal S, Pfeiffer S, Goedel A, Dirschinger RJ, Seyfarth M, Lam JT, Sinnecker D, Gudermann T, Lipp P, Laugwitz KL - EMBO Mol Med (2012)

Bottom Line: In patient iPSC-derived cardiomyocytes, catecholaminergic stress led to elevated diastolic Ca(2+) concentrations, a reduced SR Ca(2+) content and an increased susceptibility to DADs and arrhythmia as compared to control myocytes.Dantrolene, a drug effective on malignant hyperthermia, restored normal Ca(2+) spark properties and rescued the arrhythmogenic phenotype.This suggests defective inter-domain interactions within the RYR2 channel as the pathomechanism of the S406L mutation.

View Article: PubMed Central - PubMed

Affiliation: Klinikum rechts der Isar, Technische Universität München, I. Medizinische Klinik, Kardiologie, München, Germany.

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Intracellular Ca2+ signalling in control and CPVT-iPSC-derived cardiomyocytesA. Images of Fura-2 Ca2+ recordings depicting normal (R) and aberrant (AR1, AR2 and AR3) Ca2+ cycling in electrically stimulated iPSC-derived myocytes (top, from CPVT cells) and their percentage occurrence during pacing at either 0.5, 1.0 or 1.5 Hz (bottom). Red lines indicate electric stimulation and n the number of cells analysed.B. Bar graphs comparing the average resting intracellular Ca2+ ([Ca2+]i) before electrical stimulation started in control (black, n = 191) and CPVT (red, n = 211) myocytes from three different iPCS lines per group. Data are means ± SEM from four independent differentiation experiments.C,D. Average of diastolic and systolic [Ca2+]i in control (black) and CPVT (red) rhythmic myocytes during sequential pacing at 0.5, 1.0 and 1.5 Hz in absence (circles) and in presence (squares) of 10 µM isoproterenol. Between 4 and 42 cells were analysed per group; no rhythmic cells were observed with isoproterenol at 1.5 Hz. Data are means ± SEM. ***p < 0.001 versus CPVT and Control + Iso, ###p = 0.001 versus Control in C; *p = 0.04, ***p < 0.001 versus same group without isoproterenol in D; two-tailed t-test.E. Average (±SEM) of maximum caffeine-induced [Ca2+]i as measurement of SR Ca2+ content, in control (black) and CPVT (red) myocytes in absence (basal, n = 33 vs. n = 8 cells) and in presence of isoproterenol (n = 24 vs. n = 17 cells); *p = 0.03 versus control basal and CPVT + Iso, two-tailed t-test.
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fig03: Intracellular Ca2+ signalling in control and CPVT-iPSC-derived cardiomyocytesA. Images of Fura-2 Ca2+ recordings depicting normal (R) and aberrant (AR1, AR2 and AR3) Ca2+ cycling in electrically stimulated iPSC-derived myocytes (top, from CPVT cells) and their percentage occurrence during pacing at either 0.5, 1.0 or 1.5 Hz (bottom). Red lines indicate electric stimulation and n the number of cells analysed.B. Bar graphs comparing the average resting intracellular Ca2+ ([Ca2+]i) before electrical stimulation started in control (black, n = 191) and CPVT (red, n = 211) myocytes from three different iPCS lines per group. Data are means ± SEM from four independent differentiation experiments.C,D. Average of diastolic and systolic [Ca2+]i in control (black) and CPVT (red) rhythmic myocytes during sequential pacing at 0.5, 1.0 and 1.5 Hz in absence (circles) and in presence (squares) of 10 µM isoproterenol. Between 4 and 42 cells were analysed per group; no rhythmic cells were observed with isoproterenol at 1.5 Hz. Data are means ± SEM. ***p < 0.001 versus CPVT and Control + Iso, ###p = 0.001 versus Control in C; *p = 0.04, ***p < 0.001 versus same group without isoproterenol in D; two-tailed t-test.E. Average (±SEM) of maximum caffeine-induced [Ca2+]i as measurement of SR Ca2+ content, in control (black) and CPVT (red) myocytes in absence (basal, n = 33 vs. n = 8 cells) and in presence of isoproterenol (n = 24 vs. n = 17 cells); *p = 0.03 versus control basal and CPVT + Iso, two-tailed t-test.

Mentions: To assess whether CPVT-iPSC-derived cardiomyocytes recapitulate the disease phenotype, we analysed Ca2+ handling properties in single cells at 3–4 months maturation. We first examined whether CPVT myocytes display altered control of Ca2+ release during excitation–contraction (EC) by measuring electrically evoked Ca2+ transients at different pacing rates in absence and in presence of isoproterenol to mimic catecholaminergic stress (Fig 3 and Fig S2 of Supporting information). Increasing stimulation frequencies from 0.5 to 1.5 Hz correlated with a higher percentage of cells with abnormal Ca2+ handling in both control and CPVT myocytes (Fig 3A). However, this effect was significantly more pronounced in the diseased cells and was comparable among different CPVT-iPSC lines (Fig 3A and Fig S3 of Supporting information). We could observe three types of stress-induced Ca2+ cycling abnormalities, which associated with different severities of arrhythmogenicity: Ca2+ alternans, in which Ca2+ transients alternate between large and small on successive beats (AR1); Ca2+ transient fusion, characterized by absence of triggered Ca2+ transients at every second stimulation (AR2); and very irregular Ca2+ oscillations (AR3). Thus, frequency-induced stress appears to be one major arrhythmic trigger in CPVT-iPSC-derived myocytes. Deeper analysis of Ca2+ cycling properties in rhythmic cells revealed that, under basal conditions, control and CPVT myocytes presented comparable resting Ca2+ levels, similar systolic and diastolic Ca2+ concentration during electrical stimulation at different rates and equal SR Ca2+ content, determined by caffeine application (Fig 3B–E and Fig S4 of Supporting information). However, in presence of isoproterenol diastolic Ca2+ was significantly elevated in CPVT compared to control cells, while systolic Ca2+ levels remained similar (Fig 3C and D). Moreover, in contrast to control myocytes, SR Ca2+ load was not increased by isoproterenol treatment in CPVT cells (Fig 3E). These data suggest that in situations of catecholamine-induced elevated luminal Ca2+ the S406L-mutation in the RYR2 channels results in diastolic Ca2+ leak from the SR. This effect may be attributable to an increased S406L-RYR2 Ca2+ sensitivity, which lowers the release threshold to produce spontaneous activity during the diastolic period (Eisner et al, 2009; Priori & Chen, 2011). To investigate whether CPVT-iPSC-derived myocytes indeed possess an enhanced spontaneous Ca2+ release during adrenergic stimulation, we measured Ca2+ sparks in single cells during rest (Fig 4 and Movies S1-S4 of Supporting information). Ca2+ sparks are the elementary release events in cardiac EC coupling and derive from the local activity of RYR2 channel clusters (Cheng et al, 1993). Under basal conditions, Ca2+ spark frequency did not differ between control and CPVT myocytes, although Ca2+ spark amplitude, full width at 50% peak amplitude and decay time were significantly higher in diseased cells (Fig 4A–C). Moreover, only in CPVT myocytes, abnormal Ca2+ sparks with a prolonged plateau phase were observed (Fig 4B, ii). Under catecholaminergic stress, Ca2+ spark frequency considerably increased in CPVT compared to control cells, and associated with a greater decay time constant and even longer abnormal sparks (Fig 4B and C and Movies S3 and S4 of Supporting information). These results indicate that elevated diastolic Ca2+ and reduced SR Ca2+ load during catecholaminergic challenge in CPVT-iPSC-derived myocytes are caused by hyperactivity of individual Ca2+ release units.


Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia.

Jung CB, Moretti A, Mederos y Schnitzler M, Iop L, Storch U, Bellin M, Dorn T, Ruppenthal S, Pfeiffer S, Goedel A, Dirschinger RJ, Seyfarth M, Lam JT, Sinnecker D, Gudermann T, Lipp P, Laugwitz KL - EMBO Mol Med (2012)

Intracellular Ca2+ signalling in control and CPVT-iPSC-derived cardiomyocytesA. Images of Fura-2 Ca2+ recordings depicting normal (R) and aberrant (AR1, AR2 and AR3) Ca2+ cycling in electrically stimulated iPSC-derived myocytes (top, from CPVT cells) and their percentage occurrence during pacing at either 0.5, 1.0 or 1.5 Hz (bottom). Red lines indicate electric stimulation and n the number of cells analysed.B. Bar graphs comparing the average resting intracellular Ca2+ ([Ca2+]i) before electrical stimulation started in control (black, n = 191) and CPVT (red, n = 211) myocytes from three different iPCS lines per group. Data are means ± SEM from four independent differentiation experiments.C,D. Average of diastolic and systolic [Ca2+]i in control (black) and CPVT (red) rhythmic myocytes during sequential pacing at 0.5, 1.0 and 1.5 Hz in absence (circles) and in presence (squares) of 10 µM isoproterenol. Between 4 and 42 cells were analysed per group; no rhythmic cells were observed with isoproterenol at 1.5 Hz. Data are means ± SEM. ***p < 0.001 versus CPVT and Control + Iso, ###p = 0.001 versus Control in C; *p = 0.04, ***p < 0.001 versus same group without isoproterenol in D; two-tailed t-test.E. Average (±SEM) of maximum caffeine-induced [Ca2+]i as measurement of SR Ca2+ content, in control (black) and CPVT (red) myocytes in absence (basal, n = 33 vs. n = 8 cells) and in presence of isoproterenol (n = 24 vs. n = 17 cells); *p = 0.03 versus control basal and CPVT + Iso, two-tailed t-test.
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Related In: Results  -  Collection

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fig03: Intracellular Ca2+ signalling in control and CPVT-iPSC-derived cardiomyocytesA. Images of Fura-2 Ca2+ recordings depicting normal (R) and aberrant (AR1, AR2 and AR3) Ca2+ cycling in electrically stimulated iPSC-derived myocytes (top, from CPVT cells) and their percentage occurrence during pacing at either 0.5, 1.0 or 1.5 Hz (bottom). Red lines indicate electric stimulation and n the number of cells analysed.B. Bar graphs comparing the average resting intracellular Ca2+ ([Ca2+]i) before electrical stimulation started in control (black, n = 191) and CPVT (red, n = 211) myocytes from three different iPCS lines per group. Data are means ± SEM from four independent differentiation experiments.C,D. Average of diastolic and systolic [Ca2+]i in control (black) and CPVT (red) rhythmic myocytes during sequential pacing at 0.5, 1.0 and 1.5 Hz in absence (circles) and in presence (squares) of 10 µM isoproterenol. Between 4 and 42 cells were analysed per group; no rhythmic cells were observed with isoproterenol at 1.5 Hz. Data are means ± SEM. ***p < 0.001 versus CPVT and Control + Iso, ###p = 0.001 versus Control in C; *p = 0.04, ***p < 0.001 versus same group without isoproterenol in D; two-tailed t-test.E. Average (±SEM) of maximum caffeine-induced [Ca2+]i as measurement of SR Ca2+ content, in control (black) and CPVT (red) myocytes in absence (basal, n = 33 vs. n = 8 cells) and in presence of isoproterenol (n = 24 vs. n = 17 cells); *p = 0.03 versus control basal and CPVT + Iso, two-tailed t-test.
Mentions: To assess whether CPVT-iPSC-derived cardiomyocytes recapitulate the disease phenotype, we analysed Ca2+ handling properties in single cells at 3–4 months maturation. We first examined whether CPVT myocytes display altered control of Ca2+ release during excitation–contraction (EC) by measuring electrically evoked Ca2+ transients at different pacing rates in absence and in presence of isoproterenol to mimic catecholaminergic stress (Fig 3 and Fig S2 of Supporting information). Increasing stimulation frequencies from 0.5 to 1.5 Hz correlated with a higher percentage of cells with abnormal Ca2+ handling in both control and CPVT myocytes (Fig 3A). However, this effect was significantly more pronounced in the diseased cells and was comparable among different CPVT-iPSC lines (Fig 3A and Fig S3 of Supporting information). We could observe three types of stress-induced Ca2+ cycling abnormalities, which associated with different severities of arrhythmogenicity: Ca2+ alternans, in which Ca2+ transients alternate between large and small on successive beats (AR1); Ca2+ transient fusion, characterized by absence of triggered Ca2+ transients at every second stimulation (AR2); and very irregular Ca2+ oscillations (AR3). Thus, frequency-induced stress appears to be one major arrhythmic trigger in CPVT-iPSC-derived myocytes. Deeper analysis of Ca2+ cycling properties in rhythmic cells revealed that, under basal conditions, control and CPVT myocytes presented comparable resting Ca2+ levels, similar systolic and diastolic Ca2+ concentration during electrical stimulation at different rates and equal SR Ca2+ content, determined by caffeine application (Fig 3B–E and Fig S4 of Supporting information). However, in presence of isoproterenol diastolic Ca2+ was significantly elevated in CPVT compared to control cells, while systolic Ca2+ levels remained similar (Fig 3C and D). Moreover, in contrast to control myocytes, SR Ca2+ load was not increased by isoproterenol treatment in CPVT cells (Fig 3E). These data suggest that in situations of catecholamine-induced elevated luminal Ca2+ the S406L-mutation in the RYR2 channels results in diastolic Ca2+ leak from the SR. This effect may be attributable to an increased S406L-RYR2 Ca2+ sensitivity, which lowers the release threshold to produce spontaneous activity during the diastolic period (Eisner et al, 2009; Priori & Chen, 2011). To investigate whether CPVT-iPSC-derived myocytes indeed possess an enhanced spontaneous Ca2+ release during adrenergic stimulation, we measured Ca2+ sparks in single cells during rest (Fig 4 and Movies S1-S4 of Supporting information). Ca2+ sparks are the elementary release events in cardiac EC coupling and derive from the local activity of RYR2 channel clusters (Cheng et al, 1993). Under basal conditions, Ca2+ spark frequency did not differ between control and CPVT myocytes, although Ca2+ spark amplitude, full width at 50% peak amplitude and decay time were significantly higher in diseased cells (Fig 4A–C). Moreover, only in CPVT myocytes, abnormal Ca2+ sparks with a prolonged plateau phase were observed (Fig 4B, ii). Under catecholaminergic stress, Ca2+ spark frequency considerably increased in CPVT compared to control cells, and associated with a greater decay time constant and even longer abnormal sparks (Fig 4B and C and Movies S3 and S4 of Supporting information). These results indicate that elevated diastolic Ca2+ and reduced SR Ca2+ load during catecholaminergic challenge in CPVT-iPSC-derived myocytes are caused by hyperactivity of individual Ca2+ release units.

Bottom Line: In patient iPSC-derived cardiomyocytes, catecholaminergic stress led to elevated diastolic Ca(2+) concentrations, a reduced SR Ca(2+) content and an increased susceptibility to DADs and arrhythmia as compared to control myocytes.Dantrolene, a drug effective on malignant hyperthermia, restored normal Ca(2+) spark properties and rescued the arrhythmogenic phenotype.This suggests defective inter-domain interactions within the RYR2 channel as the pathomechanism of the S406L mutation.

View Article: PubMed Central - PubMed

Affiliation: Klinikum rechts der Isar, Technische Universität München, I. Medizinische Klinik, Kardiologie, München, Germany.

Show MeSH
Related in: MedlinePlus