Fig 1.
RyR2 protein, mutations studied in the present study and mutation clusters 1–4.
Mutations in this study (arrows) are located in different parts of the RyR2 protein and mutation clusters. Clusters are represented as black lines numbered from 1 to 4. Cluster 1 comprises of amino acids (AA) 44–466, cluster 2 AA 2246–2534 and cluster 3 AA 3778–4201 and these three clusters are located in the N-terminal and central regions of the protein and form the cytoplasmic domain. Cluster 4 comprises of AA 4497–4959 and forms the transmembrane domain, which is located in the C-terminal region. The figure is modified from [11].
Table 1.
Electrocardiographic parameters.
Fig 2.
Number of PVCs in exercise stress test before and after administration of intravenous dantrolene and 24 hours after dantrolene wash out.
Fig 3.
ECG examples of a 38-year-old patient carrying the RyR2 P2328S mutation.
(A) Resting ECG showing sinus rhythm and normal QRS morphology. (B) Exercise ECG at the highest work load of 105 W in the baseline study before dantrolene. PVCs include couplets and polymorphic NSVTs. (C) Disappearance of ventricular arrhythmias after administration of dantrolene (work load 105 W). (D) Exercise test on day two after 20-hours wash-out of dantrolene showing return of PVCs (work load 105 W).
Fig 4.
Characterization of CPVT1 iPSCs.
(A) Expression of pluripotency markers shown by RT-PCR, β-actin or GAPDH serving as a housekeeping gene. (B) None of the exogenous genes are expressed in CPVT1 cell lines. (C) EBs express markers from all the three embryonic germ layers. (D) Immunocytochemical stainings and expression of pluripotency markers. Scale bar 200 μm. (E) Sequencing analysis confirmed the RyR2 mutation in each cell line. (F) All the cell lines had normal karyotype, example picture from Q4201R cell line. (G) Teratomas made from a CPVT-iPSC line further confirms pluripotency, example pictures from L4115F cell line.
Fig 5.
Characterization of CPVT-iPSCs derived CMs.
(A) Immunocytochemical stainings of cardiac markers where red represents troponin T, green connexin-43 and blue DAPI-staining for nuclei. Scale bars 200 μm. (B) Representative traces of a control CM showing normal regular Ca2+ transients and CPVT1 CMs showing abnormalities like multiple peaks, low peaks, irregular phases and oscillations in Ca2+ handling. (C) Quantification of percentage of CPVT1 and control iPSC CMs exhibiting abnormal Ca2+ transients at baseline (bl) and during adrenaline perfusion (adr). (D) Frequency and (E) Diastolic level of intracellular Ca2+ of all CPVT1 and control CMs. Numbers of cells analyzed in C, D, and E, exon 3 del n = 48, P2328S n = 72, T2538R n = 52, L4115F n = 110, Q4201R n = 63, V4653F n = 29, Controls (WT) n = 28. As an exception, number of WT cells analyzed in D, and E, in bl n = 54 and adr n = 27 and number of P2328S cells in bl n = 90 and adr n = 47. Grey bars indicate cells at baseline and black bars during adrenaline perfusion. Error bars, SEM. *P<0.05 CPVT1 versus control, with Student’s t-test. Significance’s of mutation specific differences, see S2 Table.
Fig 6.
iPSC derived CMs reproduced the clinical responses of dantrolene.
(A) In vivo and in vitro effects of dantrolene correspond within each RyR2 mutation. In vitro drug effects were categorized into three groups (responders, semi-responders and non-responders) depending on how dantrolene affected to the amount of Ca2+ abnormalities when compared to adrenaline response. In vivo responder group show the percentage of the abolished PVCs when compared to the baseline. Numbers of cells analyzed in exon 3 del n = 16, P2328S n = 32, T2538R n = 17, L4115F n = 36, Q4201R n = 22, V4653F n = 13. (B) Representative traces of dantrolene responder in vitro in an L4115F mutated CM. Adrenaline causes Ca2+ cycling abnormalities and dantrolene abolishes all the abnormalities. (C) Representative traces of semi-responder in vitro in a P2328S mutated CM. The cell has abnormal Ca2+ cycling at baseline and during adrenaline perfusion and dantrolene reduces the abnormalities by abolishing the oscillation but leaving some low peak Ca2+ spiking.