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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

Genotyping the CPVT1 RyR2 R420Q mutation and CPVT2 CASQ2 D307H mutation. (A) Sequence chromatograms from the CPVT1 iPSC-HF15.1 and 15.4 clones (WT/M), and from a healthy non-carrier iPSC-KTN3 clone (WT/WT). The analysis shows a G to A substitution at nucleotide 1378 in exon 13, converting an arginine amino acid to glutamine at position 420 of the protein. (B) Sequence chromatograms from the CPVT2 iPSC-HDF19.1, 20.1 clones (M/M) and a healthy non-carrier iPSC-KTN3 clone (WT/WT). There is a G to C substitution at nucleotide 1183 in exon 9, converting aspartic acid to histidine at codon 307.
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fig02: Genotyping the CPVT1 RyR2 R420Q mutation and CPVT2 CASQ2 D307H mutation. (A) Sequence chromatograms from the CPVT1 iPSC-HF15.1 and 15.4 clones (WT/M), and from a healthy non-carrier iPSC-KTN3 clone (WT/WT). The analysis shows a G to A substitution at nucleotide 1378 in exon 13, converting an arginine amino acid to glutamine at position 420 of the protein. (B) Sequence chromatograms from the CPVT2 iPSC-HDF19.1, 20.1 clones (M/M) and a healthy non-carrier iPSC-KTN3 clone (WT/WT). There is a G to C substitution at nucleotide 1183 in exon 9, converting aspartic acid to histidine at codon 307.

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)

Genotyping the CPVT1 RyR2 R420Q mutation and CPVT2 CASQ2 D307H mutation. (A) Sequence chromatograms from the CPVT1 iPSC-HF15.1 and 15.4 clones (WT/M), and from a healthy non-carrier iPSC-KTN3 clone (WT/WT). The analysis shows a G to A substitution at nucleotide 1378 in exon 13, converting an arginine amino acid to glutamine at position 420 of the protein. (B) Sequence chromatograms from the CPVT2 iPSC-HDF19.1, 20.1 clones (M/M) and a healthy non-carrier iPSC-KTN3 clone (WT/WT). There is a G to C substitution at nucleotide 1183 in exon 9, converting aspartic acid to histidine at codon 307.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549051&req=5

fig02: Genotyping the CPVT1 RyR2 R420Q mutation and CPVT2 CASQ2 D307H mutation. (A) Sequence chromatograms from the CPVT1 iPSC-HF15.1 and 15.4 clones (WT/M), and from a healthy non-carrier iPSC-KTN3 clone (WT/WT). The analysis shows a G to A substitution at nucleotide 1378 in exon 13, converting an arginine amino acid to glutamine at position 420 of the protein. (B) Sequence chromatograms from the CPVT2 iPSC-HDF19.1, 20.1 clones (M/M) and a healthy non-carrier iPSC-KTN3 clone (WT/WT). There is a G to C substitution at nucleotide 1183 in exon 9, converting aspartic acid to histidine at codon 307.
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: 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