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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

Electron microscopy of peripheral sarcoplasmic reticulum (pSR) in 60–62 days old control, clones KTN3 and HDF24.2 (A), CPVT1, clone 15.4 (B) and CPVT2, clones 20.1 and 19S1 (C) iPSC-CM. Ryanodine receptors (short arrows) are visible between pSR and sarcolemma in control (A) and CPVT1 (B) cardiomyocytes. Enlarged cisterna of pSR is seen in CPVT2 cardiomyocyte (B). Electron-dense linear structure formed by CASQ2 (thick white arrow) is visible within the SR lumen of control cardiomyocytes (A).
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fig05: Electron microscopy of peripheral sarcoplasmic reticulum (pSR) in 60–62 days old control, clones KTN3 and HDF24.2 (A), CPVT1, clone 15.4 (B) and CPVT2, clones 20.1 and 19S1 (C) iPSC-CM. Ryanodine receptors (short arrows) are visible between pSR and sarcolemma in control (A) and CPVT1 (B) cardiomyocytes. Enlarged cisterna of pSR is seen in CPVT2 cardiomyocyte (B). Electron-dense linear structure formed by CASQ2 (thick white arrow) is visible within the SR lumen of control cardiomyocytes (A).

Mentions: The ultrastructural analysis performed on 60–62 days old cardiomyocytes from control (clones KTN3 and HDF24.2), CPVT1 (clone 15.4) and CPVT2 iPSC-CM (clones 20.1 and 19S1, not included in our previous CPVT2 study) demonstrated that control (Fig.4A and D), CPVT1 (Fig.4B and E) and CPVT2 iPSC-CM (Fig.4C and F) differentiated into cardiomyocytes with immature features: myofibrils with variable degrees of organization in a sarcomeric pattern, high mass of glycogen and large amounts of mitochondria. The analysis further showed that control iPSC-CM myofibrils exhibit typical sarcomeric pattern (Fig.4) with parallel Z-bands lined up (Table1). Clusters of mitochondria, glycogen and rough endoplasmic reticulum were also noted. Sarcoplasmic reticulum cisternae were present between the parallel myofibrils and in the periphery of cardiomyocytes. Peripheral SR (pSR) showed uniform wide cisternae (Table1), and RyR complexes were visible as dense nanostructures (‘feet’) connecting the pSR and sarcolemma (Fig.5, Table1). Furthermore, small clumps of CASQ2 were visible in the lumen of few pSR cisternae in control iPSC-CM (Fig.5A). In contrast, CPVT1 iPSC-CM showed a more immature phenotype: large clusters of mitochondria, glycogen and lysosomes compared to control iPSC-CM (Fig.4). Compared to control (Table1) CPVT1 iPSC-CM had smaller number of sarcomeres/cell (P < 0.05) and Z-bands were slenderer (P < 0.05) and more disarrayed. Furthermore, the CPVT1 iPSC-CM pSR SR showed narrower cisternae (Fig.5B, Table1, P < 0.05), a much smaller number of ‘feet’ (Table1, P < 0.05), and no visible dense chain-like nanostructures were found within the pSR lumen. CPVT2 iPSC-CM exhibited the most immature phenotype with large masses of glycogen, clusters of mitochondria, lysosomes and disarrayed myofibrils (Fig.4). Specifically, CPVT2 iPSC-CM had smaller number of sarcomeres/cell than control (P < 0.05, Table1), and sarcomeres were narrower (P < 0.05). pSR often showed enlarged (P < 0.05) and empty cisternae (Fig.5C). In addition, uniform narrow cisternae containing fewer ‘feet’ (RyR) in contact with plasmalemma (Table1) were visible in CPVT2 iPSC-CM. Electron-dense chain-like nanostructures were undetected within the pSR lumen of CPVT2 iPSC-CM.


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)

Electron microscopy of peripheral sarcoplasmic reticulum (pSR) in 60–62 days old control, clones KTN3 and HDF24.2 (A), CPVT1, clone 15.4 (B) and CPVT2, clones 20.1 and 19S1 (C) iPSC-CM. Ryanodine receptors (short arrows) are visible between pSR and sarcolemma in control (A) and CPVT1 (B) cardiomyocytes. Enlarged cisterna of pSR is seen in CPVT2 cardiomyocyte (B). Electron-dense linear structure formed by CASQ2 (thick white arrow) is visible within the SR lumen of control cardiomyocytes (A).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: Electron microscopy of peripheral sarcoplasmic reticulum (pSR) in 60–62 days old control, clones KTN3 and HDF24.2 (A), CPVT1, clone 15.4 (B) and CPVT2, clones 20.1 and 19S1 (C) iPSC-CM. Ryanodine receptors (short arrows) are visible between pSR and sarcolemma in control (A) and CPVT1 (B) cardiomyocytes. Enlarged cisterna of pSR is seen in CPVT2 cardiomyocyte (B). Electron-dense linear structure formed by CASQ2 (thick white arrow) is visible within the SR lumen of control cardiomyocytes (A).
Mentions: The ultrastructural analysis performed on 60–62 days old cardiomyocytes from control (clones KTN3 and HDF24.2), CPVT1 (clone 15.4) and CPVT2 iPSC-CM (clones 20.1 and 19S1, not included in our previous CPVT2 study) demonstrated that control (Fig.4A and D), CPVT1 (Fig.4B and E) and CPVT2 iPSC-CM (Fig.4C and F) differentiated into cardiomyocytes with immature features: myofibrils with variable degrees of organization in a sarcomeric pattern, high mass of glycogen and large amounts of mitochondria. The analysis further showed that control iPSC-CM myofibrils exhibit typical sarcomeric pattern (Fig.4) with parallel Z-bands lined up (Table1). Clusters of mitochondria, glycogen and rough endoplasmic reticulum were also noted. Sarcoplasmic reticulum cisternae were present between the parallel myofibrils and in the periphery of cardiomyocytes. Peripheral SR (pSR) showed uniform wide cisternae (Table1), and RyR complexes were visible as dense nanostructures (‘feet’) connecting the pSR and sarcolemma (Fig.5, Table1). Furthermore, small clumps of CASQ2 were visible in the lumen of few pSR cisternae in control iPSC-CM (Fig.5A). In contrast, CPVT1 iPSC-CM showed a more immature phenotype: large clusters of mitochondria, glycogen and lysosomes compared to control iPSC-CM (Fig.4). Compared to control (Table1) CPVT1 iPSC-CM had smaller number of sarcomeres/cell (P < 0.05) and Z-bands were slenderer (P < 0.05) and more disarrayed. Furthermore, the CPVT1 iPSC-CM pSR SR showed narrower cisternae (Fig.5B, Table1, P < 0.05), a much smaller number of ‘feet’ (Table1, P < 0.05), and no visible dense chain-like nanostructures were found within the pSR lumen. CPVT2 iPSC-CM exhibited the most immature phenotype with large masses of glycogen, clusters of mitochondria, lysosomes and disarrayed myofibrils (Fig.4). Specifically, CPVT2 iPSC-CM had smaller number of sarcomeres/cell than control (P < 0.05, Table1), and sarcomeres were narrower (P < 0.05). pSR often showed enlarged (P < 0.05) and empty cisternae (Fig.5C). In addition, uniform narrow cisternae containing fewer ‘feet’ (RyR) in contact with plasmalemma (Table1) were visible in CPVT2 iPSC-CM. Electron-dense chain-like nanostructures were undetected within the pSR lumen of CPVT2 iPSC-CM.

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