Table 1.
Primer sequences for RT-PCR.
Figure 1.
Characterization of CPVT-iPSCs.
A, Mutation analysis confirming the RyR2-P2328S mutation with altered DNA cleavage (arrow). B, Morphology of an iPSC colony. Scale bar 200 µm. C, Normal karyotype. D, Expression of pluripotency markers at passage 4 shown by RT-PCR, β-actin serving as a housekeeping gene. All studied endogenous pluripotency genes are turned on. None of the exogenous genes are expressed at passage 4. E, Immunocytochemical staining showing expression of pluripotency markers. Scale bars 1000 µm. F, Teratomas made from a CPVT-iPSC line further confirms pluripotency. G, EBs express markers from all the three embryonic germ layers.
Figure 2.
Characterization of iPSC-derived CPVT CMs.
A, Immunocytochemical staining of cardiac markers, blue represents DAPI-staining for nuclei. Scale bars 100 µm. B, The expression of Ca2+ cycling genes in differentiated CMs shown by RT-PCR. GAPDH is used as a housekeeping gene.
Figure 3.
Intracellular Ca2+ cycling and analysis of rhythm.
A, representative traces of the four different rhythm categories. Regular rhythm of calcium release with stable amplitude R, regular rhythm with varying amplitude Ra, irregular rhythm with stable amplitude I, irregular rhythm with varying amplitude Ia. B, doughnut charts indicating the percentage of CPVT and control CMs under each rhythm category.
Figure 4.
Intracellular Ca2+ cycling and SR Ca2+ stores.
A, representative traces from a single CPVT cell demonstrating the experimental protocol. Bl; spontaneous baseline beating, Adr; adrenaline perfusion. Caffeine was added following 10 spontaneous or paced beats. B, diastolic level of intracellular Ca2+. C, amplitude of caffeine-induced Ca2+ transients. D, amplitude of Ca2+ transients divided by amplitude of caffeine-induced Ca2+ transient, indicating fractional SR Ca2+ release. Units in A and B are Fura-2 ratio units, in C ΔF/F0. Numbers of control vs CPVT CMs analyzed: Bl n = 54 vs n = 90, Bl+paced n = 25 vs n = 50, Adr n = 27 vs 47, Adr+paced n = 19 vs n = 35, respectively. Error bars, SEM. *P<0.05, **P<0.01, with student's t-test.
Figure 5.
CPVT-P2328S CMs display DADs and EADs. A
, Time course of APD50 (empty squares) and APD90 (filled squares) Adr indicates perfusion with adrenaline. B, bars 1–3 are 9 sec time courses enlarged from A. (1) baseline with DADs, (2) DADs in the presence of adrenaline, (3) DADs continue after adrenaline perfusion. MDP −70 mV. Arrows indicate DADs. C, A CPVT-P2328S CM showing an EAD (grey arrow) and a DAD (black arrow) followed by a spontaneous burst episode (MDP −50 mV, maximum upstroke amplitude 45 mV). D, Current clamp recording of a CPVT-P2328S CM showing occasional EADs (arrows). MDP −70 mV. Dashed lines indicate the zero reference potential.
Table 2.
Characteristics of spontaneous ventricular-like APs in control and CVPT-CMs during regular beating.
Figure 6.
CPVT patients show afterdepolarizations and corresponding ECG changes. A
, MAP recording from CPVT-P2328S patient showing DADs (arrow). B, Example MAP recording from a CPVT-P2328S patient showing an extrasystole and the following beat with EAD (arrow). C, Example 24-h ECG recording from a CPVT-P2328S patient showing the simultaneous occurrence of T2- (black arrow) and U-waves (grey arrow) representing clinical counterparts to EADs [11], [28] and DADs [8], respectively.