Limits...
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

Cardiac differentiation of iPSC-derived from CPVT1 RyR2R420Q and CPVT2 CASQ2D307H patients and healthy control. (A) Immunofluorescence expression of typical myofilament in cardiomyocytes (62–64 days old) of control (clone KTN3), CPVT1 (clone 15.4) and CPVT2 (clone 19.1) stained for the typical myofilament proteins; α-actinin and cardiac troponin, scale bar 10 μm. (B) Spontaneous beating rate of control-CM (n = 9), CPVT1-CM (n = 10) and CPVT2-CM (n = 10), bpm: beats per minute. Statistical analysis of one-way anova followed by Dunn’s test, *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4549051&req=5

fig03: Cardiac differentiation of iPSC-derived from CPVT1 RyR2R420Q and CPVT2 CASQ2D307H patients and healthy control. (A) Immunofluorescence expression of typical myofilament in cardiomyocytes (62–64 days old) of control (clone KTN3), CPVT1 (clone 15.4) and CPVT2 (clone 19.1) stained for the typical myofilament proteins; α-actinin and cardiac troponin, scale bar 10 μm. (B) Spontaneous beating rate of control-CM (n = 9), CPVT1-CM (n = 10) and CPVT2-CM (n = 10), bpm: beats per minute. Statistical analysis of one-way anova followed by Dunn’s test, *P < 0.05.

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)

Cardiac differentiation of iPSC-derived from CPVT1 RyR2R420Q and CPVT2 CASQ2D307H patients and healthy control. (A) Immunofluorescence expression of typical myofilament in cardiomyocytes (62–64 days old) of control (clone KTN3), CPVT1 (clone 15.4) and CPVT2 (clone 19.1) stained for the typical myofilament proteins; α-actinin and cardiac troponin, scale bar 10 μm. (B) Spontaneous beating rate of control-CM (n = 9), CPVT1-CM (n = 10) and CPVT2-CM (n = 10), bpm: beats per minute. Statistical analysis of one-way anova followed by Dunn’s test, *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Cardiac differentiation of iPSC-derived from CPVT1 RyR2R420Q and CPVT2 CASQ2D307H patients and healthy control. (A) Immunofluorescence expression of typical myofilament in cardiomyocytes (62–64 days old) of control (clone KTN3), CPVT1 (clone 15.4) and CPVT2 (clone 19.1) stained for the typical myofilament proteins; α-actinin and cardiac troponin, scale bar 10 μm. (B) Spontaneous beating rate of control-CM (n = 9), CPVT1-CM (n = 10) and CPVT2-CM (n = 10), bpm: beats per minute. Statistical analysis of one-way anova followed by Dunn’s test, *P < 0.05.
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