Electrical dysfunctions in human-induced pluripotent stem cell-derived cardiomyocytes from a patient with an arrhythmogenic right ventricular cardiomyopathy

Abstract

Aims

Our aim is to investigate the arrhythmogenic mechanism in arrhythmogenic right ventricular cardiomyopathy (ARVC)-patients by using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).

Methods and results

Human-induced pluripotent stem cell-derived cardiomyocytes were generated from human skin fibroblasts of two healthy donors and an ARVC-patient with a desmoglein-2 (DSG2) mutation. Patch clamp, quantitative polymerase chain reaction, and calcium imaging techniques were employed for the study. The amplitude and maximal upstroke velocity (V_max) of action potential (AP) in ARVC-cells were smaller than that in healthy donor cells, whereas the resting potential and AP duration (APD) was not changed. The reduced V_max resulted from decreased peak sodium current. The reason for undetected changes in APD may be the counter-action of reduced transient outward, small conductance Ca²⁺-activated, adenosine triphosphate-sensitive, Na/Ca exchanger (I_NCX) currents, and enhanced rapidly delayed rectifier currents. Isoprenaline (Iso) reduced I_NCX and shortened APD in both donor and ARVC-hiPSC-CMs. However, the effects of Iso in ARVC-cells are significantly larger than that in donor cells. In addition, ARVC-hiPSC-CMs showed more frequently than donor cells arrhythmogenic events induced by adrenergic stimulation.

Conclusion

Cardiomyocytes derived from the ARVC patient with a DSG2 mutation displayed multiple ion channel dysfunctions and abnormal cellular electrophysiology as well as enhanced sensitivity to adrenergic stimulation. These may underlie the arrhythmogenesis in ARVC patients.

Arrhythmogenic right ventricular cardiomyopathy , Arrhythmia , Ion channel , Human-induced pluripotent stem cell-derived cardiomyocytes

What’s new?

• Cardiomyocytes derived from the arrhythmogenic right ventricular cardiomyopathy (ARVC)-patients with a desmoglein-2 mutation display multiple ion channel dysfunctions.

• Arrhythmogenic right ventricular cardiomyopathy-cardiomyocytes exhibit abnormal action potential characteristics.

• Arrhythmogenic right ventricular cardiomyopathy-cardiomyocytes, reprogrammed for human-induced pluripotent stem cells, present enhanced sensitivity of ion channel to adrenergic stimulation.

Introduction

Arrhythmogenic right ventricular cardiomyopathy (ARVC), which is usually also referred to as arrhythmogenic right ventricular dysplasia (ARVD), is a rare inheritable heart disease characterized by ventricular tachyarrhythmias, progressive loss of cardiomyocytes with fibrofatty replacement, and even sudden cardiac death (SCD).¹ The prevalence of ARVC is about 1:2000–1:5000, and more common in males (2–3:1).¹ ARVC usually manifests during adolescence, although can also appear in the elderly, and is a leading cause of SCD due to ventricular tachyarrhythmias in young athletes ≤35 years of age.² In the most typical form of ARVC, the right ventricle is primarily affected. As the disease progresses, the left ventricle may also be involved. So three subtypes of ARVC have been described: (i) the classical right dominant subtype; (ii) biventricular subtype with early biventricular involvement; and (iii) left dominant subtype with predominant left ventricular involvement.

Most cases of ARVC are associated to desmosomal gene mutations. However, the exact pathogenic mechanisms by which desmosomal mutations cause cardiomyocyte (CM) loss, fibrofatty infiltration and life-threatening arrhythmia remain unclear. Although some animal models have been established and provided some useful insights into the pathogenesis of ARVC,³ the significant differences between animal and human hearts impose limits on the interpretation and applicability of such data. In addition, pathogenic processes of ARVC are difficult to study in human cardiomyocytes because (i) obtaining cardiac samples from early stages of human ARVC hearts is not possible due to ARVC being commonly diagnosed at advance diseased stages or post-mortem, and (ii) primary cardiac tissues are difficult to biopsy safely from symptomatic ARVC patients due to the risk of cardiac perforation.^4,5 These limiting factors have markedly hindered the study of this disease. Therefore the human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide an alternative platform for the mechanistic and therapeutic studies on ARVC. In recent years, several groups have successfully produced in vitro cellular models of ARVC using patient-specific hiPSC-CMs, which could recapitulate some key features of the disease.⁵ It has been shown that hiPSC-CMs from patients with ARVC with plakophilin-2 (PKP2) mutations revealed reduced densities of PKP2, the associated desmosomal protein plakoglobin, and the gap-junction protein connexin-43, widened and distorted desmosomes and lipid accumulation as well as prolonged field potential rise time.^6,7 In addition, it has been demonstrated that the coactivation of peroxisome proliferator-activated receptor-alpha/gamma (PPARα/PPARγ), reactive oxygen species production, and fatty acid oxidation are important for the pathogenesis of ARVC.⁶ Regarding the connection between desmosomal mutations and arrhythmias, several studies demonstrated that some mutations in PKP2 and desmoglein-2 (DSG2) can cause sodium channel dysfunctions.^3,5 However, data about changes of many other important ion channels in cardiomyocytes are still lacking. In current study, we tested our hypothesis that a desmosomal mutation may influence different ion channel expression or functions in cardiomyocytes. We report here that hiPSC-CMs from a patient with ARVC carrying a gene mutation of DSG2, an integral component of desmosomes, displayed multiple ion channel dysfunction and arrhythmia events.

Methods

Ethics statement

The skin biopsies from two healthy donors and one ARVC patient were obtained with written informed consent. The study was approved by the Ethics Committee of the Medical Faculty Mannheim, University of Heidelberg (approval number: 2009-350N-MA) and by the Ethical Committee of University Medical Center Göttingen (approval number: 10/9/15). The study was carried out in accordance with the approved guidelines and conducted in accordance with the Helsinki Declaration of 1975, as revised in 1983.

Generation of human-induced pluripotent stem cells

Human iPS cells (hiPSCs) were generated from primary human fibroblasts derived from skin biopsies. Human-induced pluripotent stem cell line D1 (here abbreviated as D1) was generated using lentiviral particles carrying the transactivator rtTA and an inducible polycistronic cassette containing the reprogramming factors OCT4, SOX2, KLF4, and c-MYC as described previously.^8–11 Human-induced pluripotent stem cell lines ipWT1.1 (GOEi014-B.1), ipWT1.3 (GOEi014-B.3, here abbreviated as D2), and ipWT1.6 (GOEi014-B.6) were generated in feeder free culture conditions using the integration-free episomal 4-in-1 CoMiP reprogramming plasmid (Addgene, #63726) with the reprogramming factors OCT4, KLF4, SOX2, c-MYC and short hairpin RNA against p53, as described previously with modifications.¹² Human-induced pluripotent stem cell lines isARVCb1.2 (GOEi092-A.2), isARVCb1.3 (GOEi092-A.3), and isARVCb1.4 (GOEi092-A.4) were generated from fibroblasts in feeder free culture conditions using the integration-free CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, #A16517) with the reprogramming factors OCT4, KLF4, SOX2, c-MYC according to manufacturer’s instructions with modifications. The generated hiPSCs were characterized for their pluripotency and their in vitro differentiation potential (Supplementary material online, Figure S1). See Supplementary material online for further details.

Generation of human-induced pluripotent stem cell-derived cardiomyocytes

Frozen aliquots of hiPSCs were thawed and cultured without feeder cells and differentiated into hiPSC-CMs as described with some modifications.^10,13 At 35 to 50 days of culture with basic culture medium, cardiomyocytes were dissociated from 24 well plates and plated on Matrigel-coated 3.5 cm petri dishes for patch clamp measurements.

Polymerase chain reaction assays

To quantify the steady-state mRNA expression of the hiPSC-CMs, RNA was reverse transcribed and quantitative polymerase chain reaction (qPCR) was performed as described.¹⁰ Gene symbols, RefSeq No. and Cat. No. of the primers used for qPCR analyses in hiPSC-CMs characterization were listed in Supplementary material online, Table S1. For evaluation of the characteristics of the used hiPSC lines, reverse transcriptase-polymerase chain reaction (RT-PCR) was performed as follows: Total RNA was isolated using the SV Total RNA Isolation System (Promega, #Z3105) according to manufacturer’s instructions. 100 ng RNA was used for the first-strand cDNA synthesis by using MULV Reverse Transcriptase (Thermo Fisher Scientific, #N8080018) and Oligo d(T)16 (Thermo Fisher Scientific, #N8080128). One-tenth of cDNA was used as PCR template and amplified using the GoTaq G2 DNA polymerase (Promega, #M7845) according to manufacturer’s instructions. Primer sequences, annealing temperatures and cycles used for RT-PCR analyses of the hiPSC lines are listed in Supplementary material online, Table S1.

Immunofluorescence staining

Immunofluorescence staining was performed using appropriate primary antibodies and AlexaFluor conjugated secondary antibodies (ThermoFisher) according to the manufacturer’s instructions. All the other antibodies used for characterization of hiPSC lines are listed in Supplementary material online, Tables S2 and S3.

Patch clamp

Standard patch clamp recording techniques were used to measure the rapidly delayed rectifier potassium (I_Kr), slowly delayed rectifier potassium (I_Ks), transient outward potassium (I_to), small conductance calcium-activated potassium (I_SK), adenosine triphosphate (ATP)-sensitive potassium (I_KATP), Na/Ca exchanger (I_NCX), sodium (I_Na), and L-type calcium (I_CaL) currents as well as action potential (AP) in the whole-cell configuration at room temperature.

Measurement of intracellular calcium concentration

To measure the intracellular Ca²⁺ concentration ([Ca²⁺]_i), cells were loaded with the fluorescent Ca²⁺-indicator Fluo-3 AM. The fluorescence of the cells was measured by using Cairn Optoscan calcium imaging system (Cairn Research, UK). Fluorescence is excited by 488 nm and emitted at 520 nm. Changes in [Ca²⁺]_i were described by

$${[{\text{Ca}}^{2+}]}_i = {\mathit{k}}_{\mathrm{d}}\left(\frac{\mathit{F}}{{\mathit{F}}_{\max }-\mathit{F}}\right),$$

where k_d is the dissociation constant of Fluo-3 (400 nmol/L), F is the Fluo-3 fluorescence, F_max is the Ca²⁺-saturated fluorescence obtained at the end of each experiment.¹⁴

Statistical analysis

If not otherwise indicated data are shown as mean ± SEM and were analysed using InStat© (GraphPad, San Diego, CA, USA) and SigmaPlot 11.0 (Systat GmbH, Germany). By analysing the data with the Kolmogorov Smirnov test it was decided whether parametric or non-parametric tests were used for analysis. The Student’s t-test and the Mann–Whitney U test were used to compare continuous variables with normal and non-normal distributions, respectively. To compare categorical variables, the Fisher’s test was used for four groups and the χ² test for independence was used for more than four groups. For parametric data one-way analysis of variance with Bonferroni post-test for multiple comparisons was performed. For non-parametric data the Kruskal–Wallis test with Dunn’s multiple comparisons post-test was used. Paired t-test was used for comparisons of data before and after application of a drug. P < 0.05 (two-tailed) was considered significant.

Results

Clinical data

A 50-year-old male patient with ARVC due to identified missense mutation (G to A substitution at nucleotide p.Gly638Arg) in the DSG2 gene was recruited for this study. The ARVC was diagnosed 2 years ago. His daughter was admitted to the hospital at 13-year-old with frequent ventricular extrasystoles and unconsciousness within sport. An electrophysiological study showed increased arrhythmia events and ventricular extrasystoles after giving of epinephrine and using ventricular pacing. Myocardial biopsies showed fibrofatty replacement contributing to ARVC. Next-generation-sequencing (NGS) confirmed a missense mutation in DSG2. Family screening showed that her father (the patient for this study), aunt and her cousin are affected having the same mutation (Figure 1B). The electrocardiogram of the patient (Figure 1A) presented typical ARVC changes in precordial leads V1–3. Cardiac magnetic resonance imaging and echocardiography presented a normal biventricular function. Because his daughter rejected biopsy, we recruited the patient to investigate whether the mutation in DSG2 may predispose the patient to arrhythmic risk using hiPSC-CM platform.

Figure 1

Characterizations of the ARVC-patient and patient-specific iPS cell lines. (A) Electrocardiogram (ECG) from the patient showing inverted T-waves in precordial leads V1–3. (B) The family pedigree of patient. The arrow indicates the patient for this study. (C) The iPSC line isARVCb1.2 (a representative clone) generated from skin fibroblasts of the donors (upper panel) display a typical morphology for human pluripotent stem cells (lower panel). (D) Flow cytometry analysis of pluripotency markers OCT4 and TRA-1-60 reveals a homogeneous population of pluripotent cells in isARVCb1.2. (E) In comparison to patient’s fibroblasts, generated iPSC line isARVCb1.2 show expression of endogenous pluripotency markers SOX2, OCT4, NANOG, LIN28, FOXD3, and GDF3 at mRNA level proven by RT-PCR. Human embryonic stem cells (hESCs) were used as positive control, mouse embryonic fibroblasts (MEFs) were used as negative control. (F) Generated iPSC line isARVCb1.2 express pluripotency markers OCT4, SOX2, NANOG, LIN28, SSEA4, and TRA-1-60 as shown by immunofluorescence staining. Nuclei are co-stained with DAPI. (G) Spontaneous differentiation potential of generated iPSC lines was analysed by embryoid body formation and germ-layer specific marker expression. Immunocytochemical staining of spontaneously differentiated iPSC line shows expression of endodermal marker AFP, mesodermal-specific α-SMA, and ectodermal βIII-tubulin. Nuclei are co-stained with DAPI. Scale bars: 100 µm. AFP, alpha fetoprotein; ARVC, arrhythmogenic right ventricular cardiomyopathy; ECG, electrocardiogram; GAPDH, housekeeping gene; iPSC, induced pluripotent stem cell; RT-PCR, reverse transcriptase-polymerase chain reaction; SMA, smooth muscle actin.

Characterization of patient-specific human-induced pluripotent stem cells

Cardiomyocytes were generated from hiPS-cells (hiPSCs) derived from skin fibroblasts of two healthy donors and the ARVC-patient. The characterization of hiPSCs and hiPSC-CMS from the donors has been performed in this lab.^10,11 To confirm the successful generation of hiPSCs and hiPSC-CMs from the ARVC-patient, three clones of cells were characterized at the beginning (Day 0) and at different time points by immunostaining and qPCR analysis. The pluripotency of hiPSCs was proved by different pluripotent markers (Figure 1C–G and Supplementary material online, Figure S1). The cardiac differentiation was confirmed by the expression of Troponin T type 2 (TNNT2), alpha-actinin, myosin regulatory light chain 2 (MYL2) and myosin regulatory light chain 4 (MYL4) in hiPSC-CMs (Figure 2A, Supplementary material online, Figure S2B and C). The electrophysiological properties of the hiPSC-CMs from 35 to 50 days after onset of differentiation were also characterized. The cells displayed typical cardiac APs and functional I_Na, I_Ca-L, I_to, I_Kr, I_Ks, I_SK, I_KATP and I_NCX that are planned to be investigated in this study.

Figure 2

Quantitative polymerase chain reaction analysis of hiPSC-CMs from healthy donors and the patient. Relative mRNA levels (normalized with housekeeping gene (GAPDH)) of genes were analysed by qPCR analysis. (A) Mean values of relative mRNA expression of pluripotent and cardiac markers from Day 0 to Day 35. (B) Mean values of relative mRNA expression of desmoglein-2 and ion channels at Day 35. Results shown are mean ± SD. ARVC, arrhythmogenic right ventricular cardiomyopathy; hiPSC-CMs, human-induced pluripotent stem cell-derived cardiomyocytes; qPCR, quantitative polymerase chain reaction.

Abnormal action potentials in arrhythmogenic right ventricular cardiomyopathy-patients by using human-induced pluripotent stem cell-derived cardiomyocytes

Three selected iPSC lines from each individual were selected for differentiation into spontaneous beating embryoid bodies and for subsequent functional analyses. Beating cardiomyocytes were observed 8–12 days after starting the differentiation. The beating cardiomyocytes from the healthy volunteers and from the ARVC patient 35–50 days after differentiation were used for patch clamp measurements.

In AP-measurements, cardiomyocytes from the ARVC-patient exhibited a reduced action potential amplitude (APA) and maximal depolarization velocity (V_max) (Figure 3C and D). The resting potential (RP), action potential durations (APD 50 and APD90) were similar in control (D1 and D2) and ARVC-hiPSC-CMs (Figure 3A, E, and F). Of note, the AP-parameters in cells from both donors are similar.

Figure 3

Abnormal action potentials in ARVC-hiPSC-CMs. (A) Representative action potential recordings in hiPSC-CMs from two healthy donors (D1 and D2) and the patient (ARVC). (B) Mean values of RP. (C) Mean values of AP amplitude (APA). (D) Mean values of the maximal rate of upstroke of AP (V_max). (E) Mean values of action potential duration at 50% repolarization (APD 50). (F) Mean values of action potential duration at 90% repolarization (APD 90). Values given are mean ± SEM; ns, P > 0.05. AP, action potential; APA, action potential amplitude; APD, action potential duration; ARVC, arrhythmogenic right ventricular cardiomyopathy; hiPSC-CMs, human-induced pluripotent stem cell-derived cardiomyocytes; n, number of cells; RP, resting potential.

Changes in ion channel currents in arrhythmogenic right ventricular cardiomyopathy-patients by using human-induced pluripotent stem cell-derived cardiomyocytes

To uncover the contributed ion channels for the AP changes, voltage clamp recordings were performed for different current measurements. The measured currents in ARVC-hiPSC-CMs were compared with that measured in hiPSC-CMs from two healthy donors. The peak I_Na, I_NCX, I_to, I_SK, and I_KATP were significantly decreased in ARVC-cardiomyocytes compared with control cardiomyocytes from either donor one (D1) or donor two (D2) (Figures 4 and 5). On the contrary, I_Kr was enhanced in ARVC-hiPSC-CMs (Figure 5A–C). The late I_Na, I_CaL, and I_Ks were not significantly changed in ARVC-hiPSC-CMs compared with healthy donor cells (Figures 4 and 5). Polymerase chain reaction analysis detected significant decreases in SCN5A, SK3, and KCNJ11 gene supporting the reduction of I_Na, I_SK and I_KATP (Figure 2B).

Figure 4

I_Na, I_NCX, and I_to were reduced in ARVC-hiPSC-CMs (A) Representative traces of peak I_Na in donor cells (D1and D2) and ARVC cells. (B) I–V curves of peak I_Na in D1and D2 and ARVC cells. (C) Mean values of peak sodium currents I_Na at −40 mV in hiPSC-CMs form the two donors (D1 and D2) and the patient (ARVC). (D) Mean values of late sodium currents (I_Na) at −40 mV in hiPSC-CMs form the two donors (D1 and D2) and the patient (ARVC). (E–G) Representative traces (E), I–V curves (F), and the current density at 0 mV (G) of peak L-type calcium currents (I_Ca-L) in donor cells (D1 and D2) and ARVC cells. (H–J) Representative traces (H), averaged curves (I), and the current density at 50 mV and −85 mV (J) of Na/Ca exchanger currents (I_NCX) in donor cells (D1 and D2) and ARVC cells. The currents were recorded by a ramp pulse of 1600 ms from 60 mV to −100 mV with a holding potential of −40 mV. (K–M) Representative traces (K), I–V curves (L), and the current density at 70 mV (M) of transient outward K⁺ currents (I_to) in donor cells (D1 and D2) and ARVC cells. Values given are mean ± SEM; ns, P > 0.05. The dotted lines indicate the zero level of currents. ARVC, arrhythmogenic right ventricular cardiomyopathy; hiPSC-CMs, human-induced pluripotent stem cell-derived cardiomyocytes; n, number of cells.

Figure 5

I_SK and I_KATP were reduced but I_Kr was enhanced in ARVC-hiPSC-CMs. (A– C) Representative traces (A) and I–V curves (B) and the current density at 40 mV (C) of I_Kr in donor cells (D1 and D2) and ARVC cells. (D–F) Representative traces (D), I–V curves (E), and the current density at 40 mV (F) of I_Ks in donor cells (D1 and D2) and ARVC cells. (G–I) Representative traces (G), I–V curves (H), and the current density at 70 mV (I) of I_SK in donor cells (D1 and D2) and ARVC cells. (J–L) Representative traces (J), I–V curves (K), and the current density at −120 mV (L) of I_KATP in donor cells (D1 and D2) and ARVC cells. *P < 0.05 vs. D1. The dotted lines indicate the zero level of currents. ARVC, arrhythmogenic right ventricular cardiomyopathy; hiPSC-CMs, human-induced pluripotent stem cell-derived cardiomyocytes; n, number of cells.

Changes in the sensitivity of arrhythmogenic right ventricular cardiomyopathy-patients by using human-induced pluripotent stem cell-derived cardiomyocytes to adrenergic stimulation

The phenomenon that arrhythmias happen frequently during exercise in ARVC-patients led us to check whether the sensitivity of ARVC-hiPSC-CMs to adrenergic stimulation is different from that of healthy hiPSC-CMs hiPSC-CMs either from donors or from the patient were challenged by isoprenaline (Iso) of 0.1, 1.0 and 10 µM and APs as well as currents were measured before and after the Iso challenging. Isoprenaline shortened APD50 and APD90 in a concentration-dependent manner in both donor and ARVC-hiPSC-CMs. However, the APD-shortening effects of Iso in ARVC-cells are significantly larger than that in donor-cells (Figure 6A and B). In current recordings, we observed that 1 µM Iso reduced I_NCX more strongly in ARVC-hiPSC-CMs than in donor cells (Figure 6D). The enhanced effect of Iso on other currents was not detected (Figure 6).

Figure 6

Effects of isoprenaline (Iso) on ion channel currents and action potentials in donor- and ARVC-hiPSC-CMs. Isoprenaline (Iso, 1 µM) was applied to cells for testing its effects on APs and currents. Percentage changes (%) were calculated as: (V_Iso − V_Ctr)/V_Ctr × 100, where V_Iso is the value in presence of Iso, V_Ctr is the value in absence of Iso. (A) Exemplary traces of APs in absence (Ctr) and presence of Iso and percent changes of APD50 induced by Iso. (B) Percent changes of APD90 induced by Iso. (C) Exemplary traces of I_Na in absence (Ctr) and presence of Iso and percent changes of peak I_Na at −35 mV induced by Iso. (D) Exemplary traces of I_NCX in absence (Ctr) and presence of Iso and percent changes of I_NCX induced by Iso. (E) Exemplary traces of I_to in absence (Ctr) and presence of Iso and percent changes of peak I_to at 70 mV induced by Iso. (F) Exemplary traces of I_KATP in absence (Ctr) and presence of Iso and percent changes of I_KATP at −120 mV induced by Iso. Values given are mean ± SEM; ns, P > 0.05. APD, action potential duration; ARVC, arrhythmogenic right ventricular cardiomyopathy; hiPSC-CMs, human-induced pluripotent stem cell-derived cardiomyocytes; Iso, isoprenaline; n, number of cells.

Arrhythmia events were increased in arrhythmogenic right ventricular cardiomyopathy-patients by using human-induced pluripotent stem cell-derived cardiomyocytes

To check whether the calcium handling is normal in ARVC-hiPSC-CMs, we examined the intracellular calcium concentration. All the beating cells displayed spontaneous Ca²⁺-transients and no significant differences in diastolic and systolic intracellular Ca²⁺-level were detected among donor- and ARVC-cells (Figure 7A–C). But the ARVC-hiPSC-CMs showed arrhythmogenic [early after depolarization (EAD)-like or delayed after depolarization (DAD)-like] events more frequently than donor cells. Furthermore, when epinephrine (10 µM) was applied to regularly beating cells, more ARVC-cells displayed the epinephrine-induced arrhythmogenic events (Figure 7D and E).

Figure 7

Abnormal calcium transients in ARVC-hiPSC-CMs. The calcium transients in spontaneously beating cells were measured by Flo-3 calcium imaging. Epinephrine (10 µM) was applied to cells for mimicking adrenergic stimulation. (A) Representative traces of calcium transients in a donor (D1 and D2) and ARVC cell. (B) Mean values of diastolic Ca²⁺ concentration. (C) Mean values of systolic Ca²⁺ concentration. (D) Representative Ca²⁺ transients in donor- and ARVC-cells in presence of 10 µM epinephrine. The arrows indicate arrhythmogenic (DAD-like or EAD-like) events. (E) Percentages of cells showing epinephrine-induced arrhythmogenic events. APD, action potential duration; ARVC, arrhythmogenic right ventricular cardiomyopathy; hiPSC-CMs, human-induced pluripotent stem cell-derived cardiomyocytes.

Discussion

We have for the first time generated patient-specific induced pluripotent stem cell-derived cardiomyocytes from a patient with ARVC carrying a mutation in the DSG2 gene. This has allowed revealing electrophysiological abnormalities of the cardiomyocytes from the patient. We found (i) abnormal APs characterized by reduced V_max; (ii) multiple ion channel dysfunctions; (iii) enhanced sensitivity of ion channel to adrenergic stimulation in ARVC-hiPSC-CMs.

Arrhythmogenic right ventricular cardiomyopathy is inherited as an autosomal dominant trait. Till now, 13 genes have been linked to ARVC patients. Among those the desmosomal genes account for more than 50% of all cases, including plakoglobin (JUP), PKP2, desmoplakin (DSP), desmoglein-2 (DSG2), and desmocollin-2 (DSC2).¹⁵ In addition, some non-desmosomal genes such as transmembrane protein 43, desmin, titin, phospholamban, and ryanodine receptor 2 are also implicated in ARVC. Among desmosome genes, mutations in the PKP2 gene are most frequently revealed in patients with ARVC. Desmosomal gene mutations can cause dysfunction of intercellular connection and so the heart muscle cannot be kept together properly. The muscle cells become detached and fatty deposits build up in an attempt to repair the damage, leading to loss of cardiomyocytes and lipid accumulation. Furthermore, desmosomal gene mutations can lead to dysfunction of intercellular gap junction, which is important for electrical conduction between cells. Therefore ARVC can cause arrhythmias probably because the normal electrical impulses can be disrupted as they pass through affected areas. However, some human and animal studies showed that electrophysiological changes precede the structural changes.^3,16–18 This suggests extra mechanisms contributing to the occurrence of arrhythmias in ARVC.

Although our understanding about the pathology of ARVC especially the structure and metabolism changes in the disease has been progressed, the changes in the cellular electrophysiology remain still to be clarified. Due to the obstacle getting human cardiac myocytes, it is difficult to study the cellular electrophysiological in native human cardiomyocytes from ARVC patients. The hiPSC-CMs provide an opportunity to investigate the electrical properties on single cell level.

Several hiPSC-CM cell lines derived from ARVC patients carrying mutations in the PKP2 gene have been established for modelling the disease. In hiPSC-CMs carrying pAla325Cysfsn11 and pThr50Serfsn61, the protein level of desmosomal protein plakoglobin and gap-junction protein connexin-43 was reduced; the rising time of field potentials was prolonged; desmosomes were widened and distorted.⁷ Similarly, ARVC hiPSC-CMs from a patient carrying a pLeu614Pro missense mutation in the PKP2 gene displayed reduced level of PKP2 and plakoglobin proteins. Morphological study showed that the cells from the patient were larger and contained lipid droplets.⁶ These data demonstrate that hiPSC-CMs carrying mutations in PKP2 gene recapitulated an ARVC-phenotype characterized by structure and metabolism dysfunctions. Moreover, hiPSC-CMs from ARVC patients also displayed reduced sodium channel currents and abnormal calcium handling.^5,19 Therefore we used the hiPSC-CM platform to investigate the cellular electrophysiology in ARVC-cells and detected abnormal ion channel expression and currents as well as abnormal APs in hiPSC-CMs carrying a p.Gly638Arg mutation in DSG2 gene. The protein level of DSG2 was not reduced in our patient (Supplementary material online, Figure S2), suggesting that the mutation probably leads to a dysfunction instead of reduced expression of DSG2 proteins. Whether this mutation also leads to structural or metabolic dysfunctions similar to that induced by PKP2 gene mutations is planned to be investigated in a second study.

We observed abnormal APs with reduced APA and V_max in cells from the ARVC patient, consistent with previous studies showing reduced sodium channel currents. Action potential amplitude and V_max, especially V_max, are important for the conduction of excitation in and between cells. Therefore reduced V_max could be one of the reasons for occurrence of tachyarrhythmias in ARVC patients. The frequently discussed mechanism underlying the arrhythmias in ARVC is the conduct interruption between cells caused by cell-detaching or intercellular fat-deposit. Our data together with the previously reported data suggest that in ARVC, the cellular electrical dysfunction can also impair the conduction of excitation and contribute to the occurrence of arrhythmias in the disease.

Next, we assessed the changes of some ion channels that may influence APs. The observed changes in ion channels are reduced I_Na, I_to, I_SK, I_KATP, I_NCX, and enhanced I_Kr. The reduced I_Na, which is consistent with the previous observation in transgenic mice with a DSG2 mutation (DSG2-N271S),³ can be responsible for the reduced V_max and APA. Reduced I_to, I_SK, I_KATP may prolong APD. Reduced I_NCX and enhanced I_Kr should shorten APD. These data may explain why the APD was not significantly changed in ARVC cells compared with healthy donor cells. Due to changes in multiple ion channels in ARVC cells, it is possible that APD could be different in different ARVC patients carrying different gene mutations because of different changes in some ion channels.

Here an important question is how DSG2, a desmosomal protein, influences the expression and currents of different ion channels in cardiomyocytes. It was demonstrated that PKP2, another important desmosomal protein, colocalize with SCN5A, connexin-43, and AnkG (cytoskeletal adaptor protein Ankyrin-G) in a functional complex.^20,–22 Loss of expression of PKP2 led to decreased gap junction-mediated coupling and altered the amplitude and kinetics of sodium current in cardiac myocytes,²¹ and hence slowed the conduction velocity in cardiomyocytes.²⁰ These data indicate that molecular interaction exists between PKP2 and ion channels. It might be possible that other desmosomal proteins like DSG2 can influence ion channel expression or function in a similar way, although different mechanisms for different ion channel dysfunction cannot be excluded.

Arrhythmias happen frequently during exercise in ARVC-patients, suggesting involvement of adrenergic stimulation. This phenomenon has been explained by stretch of the heart caused by strong heart contractions during sports. The stretch may enhance the impairment of the intercellular conduction of excitation in diseased cells and trigger arrhythmias. We hypothesized that the sensitivity of the diseased cardiomyocytes to adrenergic stimulation may be changed in ARVC patients. Indeed, we found that Iso-induced inhibition of I_NCX and APD-shortening as well as epinephrine-induced arrhythmic (EAD- or DAD-like) events were enhanced in ARVC-cells. The Iso-induced APD-shortening can be explained by the reduction of the inward current I_NCX by Iso. It is well known that either prolongation or shortening of APD can be substrate of arrhythmias. Although the basal APD was not significantly different in our hiPSC-CMs, the Iso-induced shortening was stronger in ARVC cells than in donor cells, which is in agreement with previous data showing that APD was prolonged by coactivation of PPARα and PPARγ but the basal APD was similar. These data imply that not only ion channel dysfunctions but also some co-factors may be important for the occurrence of arrhythmias of ARVC. Thus in AVRC patients, especially in catecholamine stress, the enhanced APD-shortening by adrenergic stimulation may increase the susceptibility to arrhythmias. This suggests that ARVC patients should avoid catecholamine stress like sports or emotional stresses, which may trigger occurrence of arrhythmias due to the increased sensitivity of cardiac ion channels to catecholamine. In addition, ARVC patients may also profit from beta-blockers and some antiarrhythmic drugs for preventing or treating arrhythmias due to the enhanced adrenergic effects and ion channel dysfunctions.

Study limitations

Some limitations should be considered in extrapolating the data from the current study. All the currents and APs were recorded at room temperature using pipettes with low resistance (1–2 MΩ). Influences of temperature and recording errors cannot be ruled out. Human-induced pluripotent stem cell-derived cells from only two healthy donors and one ARVC-patient were used for this study. Therefore, we cannot rule out the differences among individuals. Because different mutations may lead to different changes in cardiomyocytes, and it is difficult have patients with the same nutation in the same gene, the study in hiPSC-CMs from a single patient may also provide some important information for rare inherited diseases or personized medicine.

Conclusion

In summary, this study demonstrated that the multiple ion channel dysfunctions and enhanced sensitivity to adrenergic stimulation may contribute to occurrence of arrhythmias in ARVC patients. The hiPSC-CMs recapitulated the electrophysiological phenotype (arrhythmia) of ARVC and may provide a good opportunity for further mechanistic and therapeutic studies.

Supplementary material

Supplementary material is available at Europace online.

Acknowledgements

We gratefully thank C. Liebetrau, M. Grohe, L. Rogge, and Y. Wiegräfe for excellent technical assistance. We acknowledge the financial support of the Deutsche Forschungsgemeinschaft and Ruprecht-Karls-Universität Heidelberg within the funding program Open Access Publishing. We thank the China Scholarship Council (CSC) for the financial support for Zhihan Zhao and Xin Li.

Conflict of interest: none declared.

Funding

DZHK (German Center for Cardiovascular Research) and the BMBF (German Ministry of Education and Research) (Grants to M.B., J.U., T.W., and W.Z.).