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Functional abnormalities in iPSC-derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations.

Novak A, Barad L, Lorber A, Gherghiceanu M, Reiter I, Eisen B, Eldor L, Itskovitz-Eldor J, Eldar M, Arad M, Binah O - J. Cell. Mol. Med. (2015)

Bottom Line: To test the hypothesis we generated induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CM) from CPVT1 and CPVT2 patients carrying the RyR2(R420Q) and CASQ2(D307H) mutations, respectively, and investigated in CPVT1 and CPVT2 iPSC-CM (compared to control): (i) The ultrastructural features; (ii) the effects of isoproterenol, caffeine and ryanodine on the [Ca(2+) ]i transient characteristics.Importantly, positive inotropic and lusitropic effects were not induced in mutated cardiomyocytes. (iii) The effects of caffeine and ryanodine in mutated cardiomyocytes differed from control cardiomyocytes.Our results show that iPSC-CM are useful for investigating the similarities/differences in the pathophysiological consequences of RyR2 versus CASQ2 mutations underlying CPVT1 and CPVT2 syndromes.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Technion, Haifa, Israel.

No MeSH data available.


Related in: MedlinePlus

The effect of caffeine on the intracellular Ca2+ cycling of control, CPVT1 and CPVT2 iPSC-CM. (A–C) [Ca2+]i transients from control (clone KTN3 day 68), CPVT1 (clone 15.1 day 53) and CPVT2 (clone 20S3 day 60) iPSC-CM, respectively, demonstrating the effect of caffeine. (D) The per cent change in caffeine-induced Ca2+ signal amplitude, compared to the pre-caffeine [Ca2+]i transient amplitude. (E) The per cent change in area of the caffeine-induced Ca2+ signal, compared to the pre-caffeine [Ca2+]i transient area. Control iPSC-CM (n = 7), CPVT1 iPSC-CM (n = 8), CPVT2 iPSC-CM (n = 8), *P < 0.05, **P < 0.001. Asterisks above columns represent statistically significant effect of caffeine, asterisk above bars connecting columns represent significant difference between groups. (F) A schematic model describing the effects of caffeine in the three groups. In control caffeine depletes SR free Ca2+ stores. Because in CPVT2 there is more (due to the mutated CASQ2) luminal free Ca2+ than in CPVT1 (due to ‘leaky’ RyR2), the response to caffeine is much more pronounced in CPVT2 than in control and CPVT1 iPSC-CM.
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fig08: The effect of caffeine on the intracellular Ca2+ cycling of control, CPVT1 and CPVT2 iPSC-CM. (A–C) [Ca2+]i transients from control (clone KTN3 day 68), CPVT1 (clone 15.1 day 53) and CPVT2 (clone 20S3 day 60) iPSC-CM, respectively, demonstrating the effect of caffeine. (D) The per cent change in caffeine-induced Ca2+ signal amplitude, compared to the pre-caffeine [Ca2+]i transient amplitude. (E) The per cent change in area of the caffeine-induced Ca2+ signal, compared to the pre-caffeine [Ca2+]i transient area. Control iPSC-CM (n = 7), CPVT1 iPSC-CM (n = 8), CPVT2 iPSC-CM (n = 8), *P < 0.05, **P < 0.001. Asterisks above columns represent statistically significant effect of caffeine, asterisk above bars connecting columns represent significant difference between groups. (F) A schematic model describing the effects of caffeine in the three groups. In control caffeine depletes SR free Ca2+ stores. Because in CPVT2 there is more (due to the mutated CASQ2) luminal free Ca2+ than in CPVT1 (due to ‘leaky’ RyR2), the response to caffeine is much more pronounced in CPVT2 than in control and CPVT1 iPSC-CM.

Mentions: In these experiments, we investigated the Ca2+ storage/release capacities of the mutated versus control cardiomyocytes, by rapid application of 10 mM caffeine (an opener of the RyR2 receptor) to paced cardiomyocytes. As depicted in Figure8A, in control cardiomyocytes clone KTN3 caffeine caused an abrupt increase in intracellular Ca2+ along with a sharp decline in the [Ca2+]i transient amplitude. Within 20 sec. after intracellular Ca2+ peaked, it declined and the [Ca2+]i transients attained a steady state level close to the their pre-caffeine amplitude. Similar results are shown for clone 24.5 in Figure S2A. In CPVT1 cardiomyocytes clone 15.1 (Fig.8B) the response to caffeine was smaller and shorter, and the resumption of pre-caffeine intracellular Ca2+ level and [Ca2+]i transient amplitude was much faster than in control cardiomyocytes. Similar results are shown for clone 15.4 in Figure S2B. In sharp contrast, in CPVT2 cardiomyocytes clone 20S3 (Fig.8C) the response to caffeine was markedly augmented, and the recovery time for both intracellular Ca2+ level and [Ca2+]i transient amplitude was much longer, in the order of 50–60 sec. Similar results are shown for clone 19S1 in Figure S2C. To quantify the response to caffeine we calculated two parameters: (i) The per cent change in caffeine-induced Ca2+ signal amplitude, compared to the pre-caffeine [Ca2+]i transient amplitude. (ii) The per cent change in the area of the caffeine-induced Ca2+ signal, compared to the pre-caffeine [Ca2+]i transient area. Hence, compared to control cardiomyocytes, both measures of the caffeine response were smaller (P < 0.05) in CPVT1 cardiomyocytes and larger (P < 0.05) CPVT2 cardiomyocytes (Fig.8D–E).


Functional abnormalities in iPSC-derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations.

Novak A, Barad L, Lorber A, Gherghiceanu M, Reiter I, Eisen B, Eldor L, Itskovitz-Eldor J, Eldar M, Arad M, Binah O - J. Cell. Mol. Med. (2015)

The effect of caffeine on the intracellular Ca2+ cycling of control, CPVT1 and CPVT2 iPSC-CM. (A–C) [Ca2+]i transients from control (clone KTN3 day 68), CPVT1 (clone 15.1 day 53) and CPVT2 (clone 20S3 day 60) iPSC-CM, respectively, demonstrating the effect of caffeine. (D) The per cent change in caffeine-induced Ca2+ signal amplitude, compared to the pre-caffeine [Ca2+]i transient amplitude. (E) The per cent change in area of the caffeine-induced Ca2+ signal, compared to the pre-caffeine [Ca2+]i transient area. Control iPSC-CM (n = 7), CPVT1 iPSC-CM (n = 8), CPVT2 iPSC-CM (n = 8), *P < 0.05, **P < 0.001. Asterisks above columns represent statistically significant effect of caffeine, asterisk above bars connecting columns represent significant difference between groups. (F) A schematic model describing the effects of caffeine in the three groups. In control caffeine depletes SR free Ca2+ stores. Because in CPVT2 there is more (due to the mutated CASQ2) luminal free Ca2+ than in CPVT1 (due to ‘leaky’ RyR2), the response to caffeine is much more pronounced in CPVT2 than in control and CPVT1 iPSC-CM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig08: The effect of caffeine on the intracellular Ca2+ cycling of control, CPVT1 and CPVT2 iPSC-CM. (A–C) [Ca2+]i transients from control (clone KTN3 day 68), CPVT1 (clone 15.1 day 53) and CPVT2 (clone 20S3 day 60) iPSC-CM, respectively, demonstrating the effect of caffeine. (D) The per cent change in caffeine-induced Ca2+ signal amplitude, compared to the pre-caffeine [Ca2+]i transient amplitude. (E) The per cent change in area of the caffeine-induced Ca2+ signal, compared to the pre-caffeine [Ca2+]i transient area. Control iPSC-CM (n = 7), CPVT1 iPSC-CM (n = 8), CPVT2 iPSC-CM (n = 8), *P < 0.05, **P < 0.001. Asterisks above columns represent statistically significant effect of caffeine, asterisk above bars connecting columns represent significant difference between groups. (F) A schematic model describing the effects of caffeine in the three groups. In control caffeine depletes SR free Ca2+ stores. Because in CPVT2 there is more (due to the mutated CASQ2) luminal free Ca2+ than in CPVT1 (due to ‘leaky’ RyR2), the response to caffeine is much more pronounced in CPVT2 than in control and CPVT1 iPSC-CM.
Mentions: In these experiments, we investigated the Ca2+ storage/release capacities of the mutated versus control cardiomyocytes, by rapid application of 10 mM caffeine (an opener of the RyR2 receptor) to paced cardiomyocytes. As depicted in Figure8A, in control cardiomyocytes clone KTN3 caffeine caused an abrupt increase in intracellular Ca2+ along with a sharp decline in the [Ca2+]i transient amplitude. Within 20 sec. after intracellular Ca2+ peaked, it declined and the [Ca2+]i transients attained a steady state level close to the their pre-caffeine amplitude. Similar results are shown for clone 24.5 in Figure S2A. In CPVT1 cardiomyocytes clone 15.1 (Fig.8B) the response to caffeine was smaller and shorter, and the resumption of pre-caffeine intracellular Ca2+ level and [Ca2+]i transient amplitude was much faster than in control cardiomyocytes. Similar results are shown for clone 15.4 in Figure S2B. In sharp contrast, in CPVT2 cardiomyocytes clone 20S3 (Fig.8C) the response to caffeine was markedly augmented, and the recovery time for both intracellular Ca2+ level and [Ca2+]i transient amplitude was much longer, in the order of 50–60 sec. Similar results are shown for clone 19S1 in Figure S2C. To quantify the response to caffeine we calculated two parameters: (i) The per cent change in caffeine-induced Ca2+ signal amplitude, compared to the pre-caffeine [Ca2+]i transient amplitude. (ii) The per cent change in the area of the caffeine-induced Ca2+ signal, compared to the pre-caffeine [Ca2+]i transient area. Hence, compared to control cardiomyocytes, both measures of the caffeine response were smaller (P < 0.05) in CPVT1 cardiomyocytes and larger (P < 0.05) CPVT2 cardiomyocytes (Fig.8D–E).

Bottom Line: To test the hypothesis we generated induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CM) from CPVT1 and CPVT2 patients carrying the RyR2(R420Q) and CASQ2(D307H) mutations, respectively, and investigated in CPVT1 and CPVT2 iPSC-CM (compared to control): (i) The ultrastructural features; (ii) the effects of isoproterenol, caffeine and ryanodine on the [Ca(2+) ]i transient characteristics.Importantly, positive inotropic and lusitropic effects were not induced in mutated cardiomyocytes. (iii) The effects of caffeine and ryanodine in mutated cardiomyocytes differed from control cardiomyocytes.Our results show that iPSC-CM are useful for investigating the similarities/differences in the pathophysiological consequences of RyR2 versus CASQ2 mutations underlying CPVT1 and CPVT2 syndromes.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Technion, Haifa, Israel.

No MeSH data available.


Related in: MedlinePlus