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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: CPVT is caused by abnormal intracellular Ca(2+) handling resulting from mutations in the RyR2 or CASQ2 genes.Our major findings were: (i) Ultrastructurally, CASQ2 and RyR2 mutated cardiomyocytes were less developed than control cardiomyocytes. (ii) While in control iPSC-CM isoproterenol caused positive inotropic and lusitropic effects, in the mutated cardiomyocytes isoproterenol was either ineffective, caused arrhythmias, or markedly increased diastolic [Ca(2+) ]i .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.

View Article: PubMed Central - PubMed

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

No MeSH data available.


Related in: MedlinePlus

Pluripotency of iPSC derived from CPVT1 RyR2R420Q and CPVT2 CASQ2D307H patients and healthy control. (A–C) Immunostaining of typical pluripotent markers shown for iPSCs derived from (A) healthy control clone HDF24.2, (B) CPVT1 patient, clone HDF15.4 and (C) CPVT2 patient, clone 20.1. Nuclei were stained with DAPI (blue), scale bar 50 μm. (D–F) Karyotype analysis of iPSC derived from (D) healthy control clone HDF24.2, (E) CPVT1 patient clone HDF15.4 and (F) CPVT2 patient clone 20.1. (G–I) Histological analysis of a representative teratoma obtained from in vivo differentiated cells of (G) control iPSC-24S9, (H) CPVT1 iPSC-15.4 and (I) CPVT2 iPSC-20.1. The formed teratomas contained derivatives of all three germ layers (ectoderm, mesoderm and endoderm). Mesoderm: (m) muscle, (c) cartilage, (b) blood cells. Endoderm: (e) epithelium, (g) endocrine glands. Ectoderm: (n) neural rosette. All images were obtained from formalin-fixed and paraffin-embedded teratoma sections stained with haematoxylin and eosin, scale bar 50 μm.
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fig01: Pluripotency of iPSC derived from CPVT1 RyR2R420Q and CPVT2 CASQ2D307H patients and healthy control. (A–C) Immunostaining of typical pluripotent markers shown for iPSCs derived from (A) healthy control clone HDF24.2, (B) CPVT1 patient, clone HDF15.4 and (C) CPVT2 patient, clone 20.1. Nuclei were stained with DAPI (blue), scale bar 50 μm. (D–F) Karyotype analysis of iPSC derived from (D) healthy control clone HDF24.2, (E) CPVT1 patient clone HDF15.4 and (F) CPVT2 patient clone 20.1. (G–I) Histological analysis of a representative teratoma obtained from in vivo differentiated cells of (G) control iPSC-24S9, (H) CPVT1 iPSC-15.4 and (I) CPVT2 iPSC-20.1. The formed teratomas contained derivatives of all three germ layers (ectoderm, mesoderm and endoderm). Mesoderm: (m) muscle, (c) cartilage, (b) blood cells. Endoderm: (e) epithelium, (g) endocrine glands. Ectoderm: (n) neural rosette. All images were obtained from formalin-fixed and paraffin-embedded teratoma sections stained with haematoxylin and eosin, scale bar 50 μm.

Mentions: All the clones we used expressed the pluripotent markers Oct4, Nanog, SSEA4 and TRA1-60 (Fig.1A–C), had normal karyotype (Fig.1D–F), and demonstrated pluripotency as illustrated by their ability to differentiate into endoderm, mesoderm and ectoderm (Fig.1G–I). Furthermore, using sequencing of the genomic PCR product we confirmed the switch of G to A at nucleotide 1378 in the RyR2 gene (Fig.2A) 10, and the switch of G to C at nucleotide 1183 in the CASQ2 gene (Fig.2B) 7,11. Next, control, CPVT1 and CPVT2 iPSC were spontaneously differentiated into functional cardiomyocytes 7,9. Immunofluorescence staining of micro-dissected contracting areas from EBs (Fig.3A) demonstrated that the cardiomyocytes co-express the typical cardiac markers, cardiac troponin C and α-sarcomeric actinin. Importantly, the cardiomyocytes exhibited areas of cross-striations, indicating organization towards myofibrilar structures. Finally, the spontaneous firing rates of CPVT1 and CPVT2 iPSC-CM were significantly (P < 0.01) lower than control iPSC-CM (Fig.3).


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)

Pluripotency of iPSC derived from CPVT1 RyR2R420Q and CPVT2 CASQ2D307H patients and healthy control. (A–C) Immunostaining of typical pluripotent markers shown for iPSCs derived from (A) healthy control clone HDF24.2, (B) CPVT1 patient, clone HDF15.4 and (C) CPVT2 patient, clone 20.1. Nuclei were stained with DAPI (blue), scale bar 50 μm. (D–F) Karyotype analysis of iPSC derived from (D) healthy control clone HDF24.2, (E) CPVT1 patient clone HDF15.4 and (F) CPVT2 patient clone 20.1. (G–I) Histological analysis of a representative teratoma obtained from in vivo differentiated cells of (G) control iPSC-24S9, (H) CPVT1 iPSC-15.4 and (I) CPVT2 iPSC-20.1. The formed teratomas contained derivatives of all three germ layers (ectoderm, mesoderm and endoderm). Mesoderm: (m) muscle, (c) cartilage, (b) blood cells. Endoderm: (e) epithelium, (g) endocrine glands. Ectoderm: (n) neural rosette. All images were obtained from formalin-fixed and paraffin-embedded teratoma sections stained with haematoxylin and eosin, scale bar 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4549051&req=5

fig01: Pluripotency of iPSC derived from CPVT1 RyR2R420Q and CPVT2 CASQ2D307H patients and healthy control. (A–C) Immunostaining of typical pluripotent markers shown for iPSCs derived from (A) healthy control clone HDF24.2, (B) CPVT1 patient, clone HDF15.4 and (C) CPVT2 patient, clone 20.1. Nuclei were stained with DAPI (blue), scale bar 50 μm. (D–F) Karyotype analysis of iPSC derived from (D) healthy control clone HDF24.2, (E) CPVT1 patient clone HDF15.4 and (F) CPVT2 patient clone 20.1. (G–I) Histological analysis of a representative teratoma obtained from in vivo differentiated cells of (G) control iPSC-24S9, (H) CPVT1 iPSC-15.4 and (I) CPVT2 iPSC-20.1. The formed teratomas contained derivatives of all three germ layers (ectoderm, mesoderm and endoderm). Mesoderm: (m) muscle, (c) cartilage, (b) blood cells. Endoderm: (e) epithelium, (g) endocrine glands. Ectoderm: (n) neural rosette. All images were obtained from formalin-fixed and paraffin-embedded teratoma sections stained with haematoxylin and eosin, scale bar 50 μm.
Mentions: All the clones we used expressed the pluripotent markers Oct4, Nanog, SSEA4 and TRA1-60 (Fig.1A–C), had normal karyotype (Fig.1D–F), and demonstrated pluripotency as illustrated by their ability to differentiate into endoderm, mesoderm and ectoderm (Fig.1G–I). Furthermore, using sequencing of the genomic PCR product we confirmed the switch of G to A at nucleotide 1378 in the RyR2 gene (Fig.2A) 10, and the switch of G to C at nucleotide 1183 in the CASQ2 gene (Fig.2B) 7,11. Next, control, CPVT1 and CPVT2 iPSC were spontaneously differentiated into functional cardiomyocytes 7,9. Immunofluorescence staining of micro-dissected contracting areas from EBs (Fig.3A) demonstrated that the cardiomyocytes co-express the typical cardiac markers, cardiac troponin C and α-sarcomeric actinin. Importantly, the cardiomyocytes exhibited areas of cross-striations, indicating organization towards myofibrilar structures. Finally, the spontaneous firing rates of CPVT1 and CPVT2 iPSC-CM were significantly (P < 0.01) lower than control iPSC-CM (Fig.3).

Bottom Line: CPVT is caused by abnormal intracellular Ca(2+) handling resulting from mutations in the RyR2 or CASQ2 genes.Our major findings were: (i) Ultrastructurally, CASQ2 and RyR2 mutated cardiomyocytes were less developed than control cardiomyocytes. (ii) While in control iPSC-CM isoproterenol caused positive inotropic and lusitropic effects, in the mutated cardiomyocytes isoproterenol was either ineffective, caused arrhythmias, or markedly increased diastolic [Ca(2+) ]i .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.

View Article: PubMed Central - PubMed

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

No MeSH data available.


Related in: MedlinePlus