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Cardiomyocytes generated from CPVTD307H patients are arrhythmogenic in response to β-adrenergic stimulation.

Novak A, Barad L, Zeevi-Levin N, Shick R, Shtrichman R, Lorber A, Itskovitz-Eldor J, Binah O - J. Cell. Mol. Med. (2012)

Bottom Line: Our study focuses on the autosomal recessive form of the disease caused by the missense mutation D307H in the cardiac calsequestrin gene, CASQ2.Next, iPSCs were differentiated to cardiomyocytes (iPSCs-CMs), which expressed the mutant CASQ2 protein.Electron microscopy analysis revealed that compared with control iPSCs-CMs, CPVT iPSCs-CMs displayed a more immature phenotype with less organized myofibrils, enlarged sarcoplasmic reticulum cisternae and reduced number of caveolae.

View Article: PubMed Central - PubMed

Affiliation: The Sohnis Family Stem Cells Center, Haifa, Israel.

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Related in: MedlinePlus

Characterization of CPVT iPSCs-CMs. (A) Micro-dissected contracting areas from the control and CPVT iPSCs-CMs were stained for typical myofilament proteins. The cardiomyocytes were co-labelled with anti-cardiac troponin I (green) and anti-sarcomeric α-actinin (red). (B) Representative PCR analysis of the cardiac gene troponin-T, and the calcium associated genes CASQ2, calreticulin, RyR2, junctin, triadin, NCX1, SERCA2 and the housekeeping gene GAPDH (n = 2). (C) The spontaneous beating rate of control iPSCs-CMs (n = 10) and CPVT iPSCs-CMs (n = 20). bpm: beats per minute.**P < 0.01. Scale bar: 10 μm.
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fig03: Characterization of CPVT iPSCs-CMs. (A) Micro-dissected contracting areas from the control and CPVT iPSCs-CMs were stained for typical myofilament proteins. The cardiomyocytes were co-labelled with anti-cardiac troponin I (green) and anti-sarcomeric α-actinin (red). (B) Representative PCR analysis of the cardiac gene troponin-T, and the calcium associated genes CASQ2, calreticulin, RyR2, junctin, triadin, NCX1, SERCA2 and the housekeeping gene GAPDH (n = 2). (C) The spontaneous beating rate of control iPSCs-CMs (n = 10) and CPVT iPSCs-CMs (n = 20). bpm: beats per minute.**P < 0.01. Scale bar: 10 μm.

Mentions: Next, the control and CPVT iPSCs were spontaneously differentiated into functional cardiomyocytes as previously described [16, 17]. Immunofluorescence staining of micro-dissected contracting areas (Fig. 3A) demonstrated that the cardiomyocytes co-express the typical cardiac markers, cardiac troponin I and α-sarcomeric actinin. Importantly, the cardiomyocytes exhibited areas of cross-striations, indicating organization toward myofibrilar structures. Next, PCR analysis was performed in order to determine whether key genes involved in the Ca2+ handling process or interact with CASQ2, are expressed in CPVT cardiomyocytes. The PCR analysis (Fig. 3B) shows the expression of CASQ2, calreticulin, junctin, triadin, NCX1, SERCA2 and RyR2 in control and CPVT iPSCs-EBs, and in EBs from clone H9.2 of hESC. These findings were repeated in three experiments.


Cardiomyocytes generated from CPVTD307H patients are arrhythmogenic in response to β-adrenergic stimulation.

Novak A, Barad L, Zeevi-Levin N, Shick R, Shtrichman R, Lorber A, Itskovitz-Eldor J, Binah O - J. Cell. Mol. Med. (2012)

Characterization of CPVT iPSCs-CMs. (A) Micro-dissected contracting areas from the control and CPVT iPSCs-CMs were stained for typical myofilament proteins. The cardiomyocytes were co-labelled with anti-cardiac troponin I (green) and anti-sarcomeric α-actinin (red). (B) Representative PCR analysis of the cardiac gene troponin-T, and the calcium associated genes CASQ2, calreticulin, RyR2, junctin, triadin, NCX1, SERCA2 and the housekeeping gene GAPDH (n = 2). (C) The spontaneous beating rate of control iPSCs-CMs (n = 10) and CPVT iPSCs-CMs (n = 20). bpm: beats per minute.**P < 0.01. Scale bar: 10 μm.
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Related In: Results  -  Collection

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fig03: Characterization of CPVT iPSCs-CMs. (A) Micro-dissected contracting areas from the control and CPVT iPSCs-CMs were stained for typical myofilament proteins. The cardiomyocytes were co-labelled with anti-cardiac troponin I (green) and anti-sarcomeric α-actinin (red). (B) Representative PCR analysis of the cardiac gene troponin-T, and the calcium associated genes CASQ2, calreticulin, RyR2, junctin, triadin, NCX1, SERCA2 and the housekeeping gene GAPDH (n = 2). (C) The spontaneous beating rate of control iPSCs-CMs (n = 10) and CPVT iPSCs-CMs (n = 20). bpm: beats per minute.**P < 0.01. Scale bar: 10 μm.
Mentions: Next, the control and CPVT iPSCs were spontaneously differentiated into functional cardiomyocytes as previously described [16, 17]. Immunofluorescence staining of micro-dissected contracting areas (Fig. 3A) demonstrated that the cardiomyocytes co-express the typical cardiac markers, cardiac troponin I and α-sarcomeric actinin. Importantly, the cardiomyocytes exhibited areas of cross-striations, indicating organization toward myofibrilar structures. Next, PCR analysis was performed in order to determine whether key genes involved in the Ca2+ handling process or interact with CASQ2, are expressed in CPVT cardiomyocytes. The PCR analysis (Fig. 3B) shows the expression of CASQ2, calreticulin, junctin, triadin, NCX1, SERCA2 and RyR2 in control and CPVT iPSCs-EBs, and in EBs from clone H9.2 of hESC. These findings were repeated in three experiments.

Bottom Line: Our study focuses on the autosomal recessive form of the disease caused by the missense mutation D307H in the cardiac calsequestrin gene, CASQ2.Next, iPSCs were differentiated to cardiomyocytes (iPSCs-CMs), which expressed the mutant CASQ2 protein.Electron microscopy analysis revealed that compared with control iPSCs-CMs, CPVT iPSCs-CMs displayed a more immature phenotype with less organized myofibrils, enlarged sarcoplasmic reticulum cisternae and reduced number of caveolae.

View Article: PubMed Central - PubMed

Affiliation: The Sohnis Family Stem Cells Center, Haifa, Israel.

Show MeSH
Related in: MedlinePlus