Author + information
- Received October 20, 2011
- Revision received January 25, 2012
- Accepted February 15, 2012
- Published online September 11, 2012.
- Ilanit Itzhaki, PhD⁎,
- Leonid Maizels, BSc⁎,
- Irit Huber, PhD⁎,
- Amira Gepstein, PhD⁎,
- Gil Arbel, MSc⁎,
- Oren Caspi, MD, PhD⁎,
- Liron Miller, PhD†,
- Bernard Belhassen, MD‡,
- Eyal Nof, MD†,
- Michael Glikson, MD† and
- Lior Gepstein, MD, PhD⁎,⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Lior Gepstein, Rappaport Institute, Technion–Israel Institute of Technology, POB 9649, Haifa 31096, Israel
Objectives The goal of this study was to establish a patient-specific human-induced pluripotent stem cells (hiPSCs) model of catecholaminergic polymorphic ventricular tachycardia (CPVT).
Background CPVT is a familial arrhythmogenic syndrome characterized by abnormal calcium (Ca2+) handling, ventricular arrhythmias, and sudden cardiac death.
Methods Dermal fibroblasts were obtained from a CPVT patient due to the M4109R heterozygous point RYR2 mutation and reprogrammed to generate the CPVT-hiPSCs. The patient-specific hiPSCs were coaxed to differentiate into the cardiac lineage and compared with healthy control hiPSCs-derived cardiomyocytes (hiPSCs-CMs).
Results Intracellular electrophysiological recordings demonstrated the development of delayed afterdepolarizations in 69% of the CPVT-hiPSCs-CMs compared with 11% in healthy control cardiomyocytes. Adrenergic stimulation by isoproterenol (1 μM) or forskolin (5 μM) increased the frequency and magnitude of afterdepolarizations and also led to development of triggered activity in the CPVT-hiPSCs-CMs. In contrast, flecainide (10 μM) and thapsigargin (10 μM) eliminated all afterdepolarizations in these cells. The latter finding suggests an important role for internal Ca2+ stores in the pathogenesis of delayed afterdepolarizations. Laser-confocal Ca2+ imaging revealed significant whole-cell [Ca2+] transient irregularities (frequent local and large-storage Ca2+-release events, broad and double-humped transients, and triggered activity) in the CPVT cardiomyocytes that worsened with adrenergic stimulation and Ca2+ overload and improved with beta-blockers. Store-overload–induced Ca2+ release was also identified in the hiPSCs-CMs and the threshold for such events was significantly reduced in the CPVT cells.
Conclusions This study highlights the potential of hiPSCs for studying inherited arrhythmogenic syndromes, in general, and CPVT specifically. As such, it represents a promising paradigm to study disease mechanisms, optimize patient care, and aid in the development of new therapies.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder caused by unstable sarcoplasmic reticulum (SR) calcium (Ca2+) storage leading to exercise- or emotion-induced ventricular tachyarrhythmias and sudden cardiac death (1,2). The majority of CPVT cases are associated with dominant mutations in the cardiac ryanodine receptor gene (RyR2) with variable penetrance (CPVT1) (3,4), whereas the minority of cases result from recessive mutations in the cardiac calsequestrin isoform 2 (CASQ2) gene (5). Investigation of the mutations giving rise to CPVT provided important insights into the mechanisms underlying this disease and to the processes governing cardiomyocyte Ca2+ handling in general (6). Nevertheless, among the hurdles in studying this disease process has been the lack of appropriate human cardiac tissue models and the inability to study patient-specific disease variations.
The development of the groundbreaking human- induced pluripotent stem cells (hiPSCs) technology (7,8) may provide a possible solution to the aforementioned challenges. The iPSCs approach, pioneered by Takahashi and Yamanaka, allows the reprogramming of adult somatic cells into pluripotent stem cells by ectopic expression of a set of transcription factors (9). Subsequent studies showed the robust ability to generate hiPSCs and to coax their differentiation into cardiomyocytes (10,11). More recent proof-of-concept studies demonstrated the ability to establish patient- and disease-specific hiPSCs-derived cardiomyocytes (hiPSCs-CMs) that could recapitulate the abnormal cardiac phenotypes of Leopard and long-QT syndromes (12–14). In the current report, our goal was to establish an hiPSCs model of CPVT. We hypothesized that the generated CPVT-hiPSCs-CMs will recapitulate the disease phenotype in vitro, providing insights into disease mechanisms and offering a unique platform to evaluate patient-specific therapies.
hiPSCs generation and cardiomyocyte differentiation
The patient-specific hiPSCs clones were generated by retroviral reprogramming of dermal fibroblasts with Oct4, Sox-2, and Klf-4 followed by valproic acid treatment as previously described (13,15). Cardiomyocyte differentiation was induced using the embryoid body (EB) differentiating system. Briefly, undifferentiated hiPSCs were removed from the mouse embryonic fibroblast feeder-layer, dispersed into cell-clumps using collagenase type IV (300 U/ml [Life Technologies Corporation, Grand Island, New York]), cultivated in suspension for 10 days as EBs, and plated on gelatin-coated culture dishes (11).
Specimens were fixed with 4% paraformaldehyde, permeabilized with 1% Triton, blocked with 5% horse serum, and incubated with primary antibodies targeting: Oct4, Tra1-60 (Santa-Cruz Biotechnology, Santa Cruz, California), Nanog (Peprotech, Rehovot, Israel), SSEA-4 (R&D Systems, Minneapolis, Minnesota), troponin I, ryanodine receptor (Chemicon, Merck Millipore, Billerica, Massachusetts), and sarcomeric alpha actinin (Sigma-Aldrich, St. Louis, Missouri). The preparations were incubated with secondary antibodies and examined using a laser-scanning confocal microscope (Zeiss LSM-510-PASCAL). Alkaline phosphatase staining of hiPSCs colonies was performed using a Sigma detection kit.
Undifferentiated hiPSCs were dissociated with collagenase-IV and injected subcutaneously into SCID/beige mice. Tumor samples were collected at 6 to 8 weeks, fixed in 4%-formaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.
Genomic DNA was isolated by using the high-pure polymerase chain reaction (PCR) template preparation kit (Roche, Basel, Switzerland). The relevant DNA fragment of the RYR2 gene was amplified by PCR using 100-ng genomic DNA. Primers are detailed in Online Table 1. PCR products were then sequenced.
Gene expression analysis
RNA was isolated by using the RNeasy Plus Mini Kit (Qiagen Inc., Valencia, California). Reverse transcription into cDNA was conducted with the high-capacity cDNA reverse-transcription kit (Applied Biosystems). Real-time polymerase chain reaction (RT-PCR) studies were performed by using the DreamTaq Master Mix (Ferments Molecular Biology Tools, Thermo Fisher Scientific, Waltham, Massachusetts). Primers are listed in Online Table 1. Each RT-PCR included 2 min at 93°C followed by 27 to 35 cycles of 30 s at 93°C, 60 s at 60°C, and 40 s at 72°C.
RT-PCR studies were conducted in triplicate by using the Fast SYBR Green Master Mix (Applied Biosystems, Life Technologies Corporation). Primers are detailed in Online Table 2. Samples were cycled by using the Fast ABI-7500 sequence detector. Conditions were as follows: 20 s at 95°C followed by 40 cycles of 3 s at 95°C and 30 s at 60°C. Cycle threshold was calculated by using default settings for the real-time sequence detection software (Applied Biosystems).
Whole-cell patch-clamp recordings
Contracting EBs were enzymatically dispersed (1 mg/ml collagenase B [Roche]) and attached to fibronectin-coated glass coverslips. Action potentials (APs) were recorded from spontaneously contracting and quiescent hiPSCs-CMs at 32°C under spontaneous and paced (1 Hz) rhythms. The tyrode solution included (in mmol/l: NaCl 140; KCl 4; CaCl2 2; MgCl2 1; HEPES 10; and glucose 5 (pH 7.4; NaOH). The pipette solution contained (in mmol/l): K-aspartate 120; KCl 20; MgCl2 1; Na2ATP 4; GTP 0.1; HEPES 10; and glucose 10 (pH 7.2; KOH). APs were recorded in the current-clamp mode with Axopatch 200B, Digidata 1322A, and pClamp 9 (Axon Instruments, Sunnyvale, California).
Microelectrodearray recordings (Multichannels Systems, Reutlingen, Germany) were performed as previously described (11,16). Local activation times were determined at each electrode and used for generation of color-coded activation maps.
Cells were loaded with 5μM fluo-4 fluorescent Ca2+ indicator (Molecular Probes, Life Technologies Corporation) to allow recordings of whole-cell [Ca2+]i transients as previously reported (15). Experiments were conducted during continuous tyrode perfusion that included (in mmol/l) NaCl 140; KCl 5.4; CaCl2 1.8; MgCl2 1; HEPES 10; and glucose 10. [Ca2+]i imaging was performed by using the Zeiss LSM-700 confocal system. To evaluate for store-overload–induced Ca2+ release (SOICR), we used a modification of the method described by Jiang et al. (17,18). In brief, repeated Ca2+ imaging from the hiPSCs-CMCs was performed at increasing extracellular [Ca2+]o concentrations (0.1 to 4 mmol/l) in the presence of tetrodotoxin (10 μM). Tetrodotoxin was added to eliminate AP-dependent Ca2+ releases, thereby unmasking only SOICR events.
Continuous variables are reported as mean ± SEM. Categorical variables are expressed as frequencies. Categorical differences between groups were evaluated by using the chi-square test. Differences between group means were compared by using the unpaired Student t test. These statistical tests were applied to comparisons between the cells obtained from the 2 individuals studied and cannot necessarily be generalized to differences between patient populations. p < 0.05 was considered statistically significant.
Derivation of the patient-specific CPVT-hiPSCs
Dermal fibroblasts were obtained from a 30-year-old woman diagnosed with CPVT1. The patient comes from a family with a significant history of sudden cardiac death (including the patient's father, brother, sister, and nephews) (19) and was implanted prophylactically with an implantable cardioverter-defibrillator. An episode of nonsustained ventricular tachycardia was documented during device follow-up and after an epinephrine stress test (Fig. 1A). Genetic workup revealed a heterozygous missense mutation (M4109R) in the RyR2 gene in all affected family members (19).
Several CPVT-hiPSCs clones were generated during reprogramming of the patient's fibroblasts, 2 of which were propagated and used for cardiomyocyte differentiation and characterization (Fig. 1B). Similar hiPSCs previously created from fibroblasts of a healthy individual (15) served as controls. Importantly, the M4109R mutation was identified in the CPVT-hiPSCs but not in the healthy control cells (Fig. 1C).
The CPVT-hiPSCs colonies exhibited characteristic human embryonic stem cell morphology; stained positively for the pluripotency markers NANOG, OCT4, SSEA-4, and TRA1-60 (Fig. 1B); displayed alkaline phosphatase activity (Fig. 1B [right panel]); and maintained a normal karyotype (data not shown). Pluripotency of the CPVT-hiPSCs was confirmed by the presence of cell-derivatives of all 3 germ-layers in differentiating EBs (Fig. 2A). Note the positive staining for nestin (ectodermal marker), alpha-fetoprotein (endoderm), and desmin (mesoderm) (Fig. 2A). Similarly, injection of undifferentiated CPVT-hiPSCs into SCID/beige mice led to the formation of teratomas, containing advanced tissue derivatives of all 3 germ layers (Fig. 2B). Finally, all clones showed silencing of the 3 retroviral transgenes (Fig. 2C) and reactivation of endogenous pluripotency genes (OCT4, SOX2, NANOG, FOXD3, and REX1) (Fig. 2D), indicating successful reprogramming.
We next used the EB differentiation system to coax the differentiation of the hiPSCs into the cardiac lineage. Both the healthy control and CPVT hiPSCs lines showed similar cardiomyocyte differentiation potential as judged by the number of contracting EBs generated during differentiation (Fig. 3A) and by comparable quantitative expression of cardiac-specific genes by the differentiating cells (Fig. 3B).
Gene expression analysis (Fig. 3C) revealed the expression of cardiac-specific transcription factors (NKX2.5) and structural genes (MLC-2V, MYH-6, MYH-7 and cTnI) by the CPVT-hiPSCs-CMCs. Similarly, immunostaining studies targeting the sarcomeric proteins troponin I and alpha-actinin confirmed the cardiomyocyte phenotype of these cells (Fig. 3D). These experiments also verified the presence of the major SR Ca2+ release channels (RyR2) in the CPVT-hiPSCs-CMs (Fig. 3E). No differences were noted in RyR2 expression between healthy and diseased hiPSCs-CMs at the mRNA level (Fig. 3F) and also in protein expression and subcellular localization as judged by immunostaining analysis (Fig. 3E).
Intracellular (patch-clamp) and extracellular (multielectrode) electrophysiological recordings established the presence of cardiac-specific APs by the CPVT-hiPSCs-CMs at the cellular level (Fig. 3G) and the development of a functional syncytium at the tissue level (Fig. 3H). Similar to previous reports (10,13), 3 types of AP morphologies were recorded from the CPVT-hiPSCs-CMs (Fig. 3G). The most dominant (57% of cells) was the ventricular-like morphology, with atrial-like (29%), and nodal-like (14%) types being less prevalent.
CPVT-hiPSCs-CMs display afterdepolarizations
Detailed analysis of AP properties (amplitude, upstroke-velocity, durations, and resting membrane potential) revealed no significant differences between the healthy control and CVPT hiPSCs-CMs (Online Table 3). In contrast, marked differences were noted in the cardiomyocytes' arrhythmogenic potential. Hence, clear delayed afterdepolarizations (DADs) developed in most CPVT-hiPSCs-CMs when paced at 0.5 to 1 Hz (Fig. 4A). These DADs were observed in 69% (22 of 32) of the CPVT-hiPSCs-CMs studied (Fig. 4C), with a mean amplitude of 13.0 ± 8.2 mV. In contrast, DADs were only seen in 11% (3 of 28; p < 0.01) of the healthy control hiPSCs-CMs (Figs. 4B and 4C) and their amplitude was significantly smaller (5.7 ± 3.9 mV; p < 0.01).
In the majority of CPVT-hiPSCs-CMs (17 of 22), afterdepolarizations were initiated during phase 4 of the AP (Fig. 4A [left]). In the remaining 5 cells, afterdepolarizations were also noted during late phase 3 (Fig. 4A [middle/right panels]). The severity of the afterdepolarizations in the CPVT-hiPSCs-CMs seemed to correlate with beating frequency (Fig. 4D), and they could be identified both during the stimulation train (Fig. 5A [top]) and immediately after termination of pacing (Fig. 5A [bottom]). Finally, similar percentages of cells displaying afterdepolarizations (62% and 73%) were found in 2 different independently derived CPVT-hiPSCs clones, thus assuring that the aforementioned findings were not related to clonal variations.
Because arrhythmias in CPVT are related to exercise or emotional stress, we next evaluated the impact of activation of the adrenergic signaling pathway. Application of forskolin (5 μM), a direct activator of adenylate cyclase, significantly increased the frequency and amplitude of DADs in 83% (5 of 6) of the paced CPVT-hiPSCs-CMs studied and even led to the formation of triggered activity beats in 2 cells (Fig. 5B [top]). We next studied quiescent CPVT-hiPSCs-CMs. Under baseline conditions, with cessation of pacing (Fig. 5A [bottom]), low-amplitude DADs were generated at relatively long coupling intervals. Application of forskolin led to development of multiple DADs in these CPVT-hiPSCs-CMs (exhibiting higher amplitudes and shorter coupling intervals) and even to DAD-mediated triggered activity (Fig. 5B [bottom]).
Application of the beta-agonist isoproterenol also had a proarrhythmic effect in the CPVT-hiPSCs-CMs. Thus, compared with baseline recordings (Fig. 5C), isoproterenol (1 μM) administration resulted in the development of afterdepolarizations in 4 of the 6 cells studied, both during the stimulation train (1 Hz; Fig. 5D), as well as in quiescent cells, after termination of pacing (Fig. 5E). In the latter case, triggered activity was also noted.
Effects of flecainide
A unique advantage of the hiPSCs technology is the potential to screen the effects of therapeutic interventions in a patient-specific manner. To test the feasibility of this concept, we evaluated the effects of flecainide, an antiarrhythmic agent suggested to display antiarrhythmic properties in CPVT (20). As shown in Figure 5F, flecainide 10 μM completely eliminated or significantly decreased afterdepolarizations in all of the patient-specific CPVT-hiPSCs-CMs studied (n = 6).
Role of intracellular Ca2+ stores
To verify the role of internal Ca2+ stores in the pathogenesis of the observed DADs, we treated the CPVT-hiPSCs-CMs with thapsigargin (10 μM), a specific inhibitor of the sarcoplasmic reticulum calcium ATPase pump. Blocking of Ca2+ reuptake into the SR gradually depletes intracellular Ca2+ stores. We next evaluated the ability of forskolin-treated cells to develop DADs before and after thapsigargin treatment. As shown in Figure 5G, thapsigargin significantly depressed postpacing afterdepolarizations, indicating the important role of internal Ca2+ stores in their formation.
To further evaluate calcium handling, we performed laser-confocal Ca2+-imaging of fluo4-loaded hiPSCs-CMs. As depicted in Figures 6A to 6C, the CPVT-hiPSCs-CMs displayed significant whole-cell [Ca2+]i transient abnormalities when compared with healthy control cells (Figs. 6D to 6E). These abnormalities were noted in 77% (23 of 30) of the CPVT-hiPSCs-CMs and were manifested by ≥1 of the following: 1) frequent local Ca2+ release events interspersed between the regular beats (Fig. 6A); 2) development of large-storage release events (Fig. 6B); and 3) broad double-humped transients (Fig. 6C). In contrast, only 42% (14 of 41) of control cells displayed local Ca2+ release events (Fig. 6E), and their amplitudes and durations were lower than those noted in the CPVT-cardiomyocytes. Importantly, none of the control cells developed the more severe whole-cell [Ca2+]i transient irregularities.
In a similar manner to the electrophysiological recordings, adrenergic stimulation also worsened the degree of Ca2+ transient abnormalities in the CPVT-hiPSCs-CMCs. This finding was manifested by the development of new triggered activity events and/or broad double-humped transients in 60% (6 of 10) of the CPVT-hiPSCs-CMs after isoproterenol (1 μM) application (Fig. 6F) and in 75% (6 of 8) of the cells after forskolin (5μM) administration. In contrast, none of the healthy control cells (n = 18 and 13, respectively) displayed such abnormalities following similar interventions (Fig. 6G). Because beta-blockers represent the mainstay treatment for CPVT, we next evaluated their potential effects on the CPVT-hiPSCs-CMs. Application of propranolol (5 μM) reversed the observed abnormalities induced by isoproterenol in 78% (7 of 9) of the cells studied (Fig. 6F).
We next evaluated the effect of Ca2+ overload on the degree of Ca2+ transient abnormalities in the hiPSCs-CMCs. To this end, the cells were perfused with increasing extracellular Ca2+ concentrations ([Ca2+]o). As noted in Figure 6H, this resulted in significant worsening of the Ca2+ transient abnormalities (with development of large, wide, and double-humped transients) with elevated [Ca2+]o in 75% (6 of 8) of the CPVT-hiPSCs-CMs evaluated.
Store-overload-induced Ca2+ release (SOICR)
It has long been appreciated that SR Ca2+ release can also occur in the absence of membrane depolarization through a mechanism referred to as SOICR (6,18). It was also suggested that some RyR2 mutations may markedly increase the occurrence of SOICR (6,18). To evaluate whether SOICR can be modeled by hiPSCs-CMs, we performed repeated Ca2+ imaging in tetrodotoxin-treated hiPSCs-CMs at increasing extracellular [Ca2+]o concentrations (0.1 to 4 mM). As shown in Figure 7A, SOICR events could be identified in both the CPVT and healthy control hiPSC-CMs, with the number of Ca2+-oscillating cells increasing as function of [Ca2+]o (Fig. 7B). The threshold for generation of SOICR, however, was significantly altered by the M4109R RyR2 mutation. Consequently, the CPVT-hiPSCs-CMs began to develop SOICR at much lower [Ca2+]o concentrations when compared with healthy control cells (Figs. 7A and 7B).
In the current study, we demonstrated the unique potential of hiPSCs for modeling inherited arrhythmogenic syndromes by establishing a patient-specific model of CPVT. By studying cardiomyocytes differentiated from the CPVT-hiPSCs, we were able to: 1) recapitulate the disease phenotype of the CPVT patient by showing the development of DADs in the patient-derived hiPSCs-CMs; 2) identify the role of internal Ca2+ stores in the pathogenesis of these afterdepolarizations; 3) reveal the contribution of the beta-adrenergic signaling pathway to these arrhythmias; 4) show the ability of the hiPSCs-CMs to develop SOICR with increasing Ca2+ concentrations and the significance of the M4109R RyR2 mutation in altering the threshold for such release events; and 5) demonstrate the potential of patient-specific CPVT-hiPSC-CMs for evaluating candidate drugs such as beta-blockers and flecainide.
Since the realization, more than a decade ago, that mutations in the cardiac ryanodine-receptor gene are responsible for CPVT1 (3,4), much has been learned about the disease pathogenesis. Experimental findings from heterologous expression systems, lipid bilayers, and knock-in and knockout mice models suggest that the basic mechanism underlying development of arrhythmias in this disorder involves a diastolic Ca+2 leak from the mutant RyR2 (6). This may lead to development of DADs through activation of the membrane Na+/Ca2+ exchanger, which can eventually result in triggered activity; the pivotal arrhythmogenic mechanism in CPVT.
One of the challenges in confirming such a mechanism also clinically was the inability to directly study human cardiomyocytes from patients with CPVT. The advent of the hiPSCs technology may solve this cell-sourcing problem. This unique “disease-in-a-dish” concept is based on the capacity to derive isogenic hiPSCs from an affected individual and to coax their differentiation into the desired cell lineage (cardiomyocytes), thus portraying the patient-specific disease phenotype.
We have previously shown that hiPSCs-CMs express the necessary functional components associated with Ca2+ handling (15). This was demonstrated by development of spontaneous whole-cell [Ca2+]i transients in the hiPSCs-CMs, which were dependent on both sarcolemmal Ca2+ entry via L-type Ca2+ channels and intracellular store Ca2+ release (15).
Here, using hiPSCs-CMs derived from a CPVT patient, we provide proof-of-concept evidence that the mechanism thought to underlie arrhythmogenicity in CPVT is also valid in the patient-specific cardiomyocytes. This was shown by the development of DADs in most CPVT-hiPSCs-CMs that became even more prominent after activation of the beta-adrenergic signaling pathway and could eventually give rise to triggered activity.
We next evaluated the role of intracellular Ca2+ stores in the formation of these DADs. By depleting internal Ca2+ stores with thapsigargin, we were able to suppress afterdepolarizations in the CPVT-hiPSCs-CMs, thus confirming the crucial role of these stores in DADs' pathogenesis. Moreover, in a similar manner to the electrophysiological recordings, laser-confocal Ca2+ imaging demonstrated significant Ca2+-handling irregularities in the CPVT-hiPSCs-CMs. These abnormalities, ranging from an increase in the incidence and magnitude of local Ca2+ release events to more severe global whole-cell Ca2+ irregularities and arrhythmogenesis, worsened with beta-adrenergic stimulation and Ca2+ overload.
It is believed that the diastolic Ca2+ wave that underlies the generation of DADs occurs when the SR Ca2+ content exceeds a threshold level. The mechanism for such a depolarization-independent SR Ca2+ release was termed SOICR (6,18), and this process was implicated in the arrhythmogenesis associated with intracellular Ca2+ overload, such as during digitalis intoxication. It was also suggested, in both heterologous expression systems (18,21) and in cardiomyocytes derived from a mouse model of CPVT (22), that several RyR2 mutations may markedly reduce the SR Ca2+ threshold required for SOICR. In the current study, we demonstrated the ability of the hiPSCs-CMs to recapitulate the SOICR phenomenon with the number of Ca2+-oscillating cells increasing with elevated Ca2+ concentrations. We also found that the M4109R RyR2 mutation decreases the threshold for such events, with SOICR occurring in the CPVT-hiPSCs-CMs at much lower Ca2+ concentrations then in healthy control cells.
Finally, feasibility studies were performed to demonstrate the potential of the CPVT-hiPSCs-CMs model for drug screening. These studies revealed that beta-blockers, which have been the mainstay treatment for CPVT, may also be beneficial in the patient-specific hiPSCs-CMs by reversing the Ca2+-handling irregularities observed after adrenergic stimulation. In addition to beta-blockers, potential new antiarrhythmic strategies for CPVT include agents targeting the abnormal diastolic Ca2+ leak (e.g., RyR2 stabilizers) (23) or those targeting the ionic mechanisms responsible for the generation of triggered activity (6). Flecainide was recently shown to possess antiarrhythmic properties in CPVT (20) through either a stabilizing effect on RyR2 (decreasing its opening probability) (20,24) or by increasing the threshold for triggered activity (through its Na+-channel blocking activity) (25). Although the current study was not designed to shed additional light on the specific antiarrhythmic mechanism of flecainide, it did reveal its ability to eliminate afterdepolarizations and triggered activity in the patient-derived hiPSCs-CMs. In addition, because this work involved a single CPVT patient, future studies will have to expand the results of this study to larger cohorts of patients.
Taken together, our data demonstrate the unique potential of hiPSCs-CMs for modeling CPVT. Using this approach, the disease phenotype presented by the patient at bedside could be readily recognized in the patient-derived cells in vitro as an anomalous electrophysiological signature. Importantly, this study also provided insights into arrhythmia mechanisms in this syndrome and highlighted the potential of the hiPSCs strategy for screening the effects of potential disease aggravators (adrenergic stimulation) and customized treatment options (beta-blockers and flecainide).
The authors thank Dr. Doron Aronson for his statistical advice and Dr. Edith Suss-Toby for her help in the imaging studies.
For supplementary tables, please see the online version of this article.
This study was funded by the European Research Council Ideas program (ERC-2010-StG-260830-Cardio-iPS) and by the Nancy and Stephen Grand philanthropic fund. The authors have reported that they have no relationships relevant to the contents of this paper to disclose. The first two authors contributed equally to this work.
- Abbreviations and Acronyms
- action potential
- cardiac calsequestrin isoform 2
- catecholaminergic polymorphic ventricular tachycardia
- delayed afterdepolarizations
- embryoid body
- human-induced pluripotent stem cell
- human-induced pluripotent stem cells–derived cardiomyocytes
- polymerase chain reaction
- real-time polymerase chain reaction
- ryanodine receptor isoform 2
- store-overload–induced Ca2+ release
- sarcoplasmic reticulum
- Received October 20, 2011.
- Revision received January 25, 2012.
- Accepted February 15, 2012.
- American College of Cardiology Foundation
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