DOI: 10.5061/dryad.2rbnzs7rp
title: Patient-specific induced pluripotent stem cell properties implicate Ca2+-homeostasis in clinical arrhythmia associated with combined heterozygous RYR2 and SCN10A variants

description: We illustrate the use of induced pluripotent stem cells (iPSCs) as
 platforms for investigating cardiomyocyte phenotypes in a human family
 pedigree exemplified by novel heterozygous RYR2-A1855D and SCN10A-Q1362H
 variants occurring alone and in combination. The proband, a four-month-old
 boy, presented with polymorphic ventricular tachycardia (PVT). Genetic
 tests revealed double novel heterozygous RYR2-A1855D and SCN10A-Q1362H
 variants inherited from his father (F) and mother (M) respectively. His
 father showed ventricular premature beats (VPB); his mother was
 asymptomatic. Molecular biological characterisations demonstrated greater
 TNNT2 mRNA expression in the iPSCs-induced cardiomyocytes (iPS-CMs) than
 in the iPSCs. cTNTs became progressively organised, but cytoplasmic RYR2
 and SCN10A aggregations occurred in the iPS-CMs. Proband-specific iPS-CMs
 showed decreased RYR2 and SCN10A mRNA expression. The RYR2-A1855D variant
 resulted in premature spontaneous sarcoplasmic reticular (SR) Ca2+
 transients (PCTs), Ca2+ oscillations (COs), and increased action potential
 durations (APDs). SCN10A-Q1362H did not confer any specific phenotype.
 However, the combined heterozygous RYR2-A1855D and SCN10A-Q1362H variants
 in the proband iPS-CMs resulted in accentuated Ca2+ homeostasis disorders,
 AP prolongation and susceptibility to early afterdepolarisations (EADs) at
 high stimulus frequencies. These findings attribute the clinical phenotype
 in the proband to effects of the heterozygous RYR2 variant exacerbated by
 heterozygous SCN10A modification.
description: <strong>1. </strong><strong>Family pedigree </strong> The investigation was conducted following principles defined by the Helsinki Declaration and approved by the Ethics Committee of Xi’an Children’s Hospital (No. 2019-599), Affiliated Children’s Hospital of Xi’an Jiaotong University. The family pedigree (Fig. 1A) of a four-month-old boy with CPVT was obtained from the Department of Cardiology, Xi’an Children’s Hospital in 2019. Clinical phenotypes of the pedigree were deduced from the clinical history and physical, electrocardiographic (ECG) and ultrasoundcardiographic (UCG) examination. Written informed consent was obtained from the parents. We term the proband father as proband-F and the proband mother as proband-M <strong>2. Genetic analysis </strong> Genomic DNA was extracted and clinical whole exons were tested using next-generation sequencing (NGS) target capture (SinoPath Company, China). The pathological characteristics of suspicious variants were predicted using multiple bioinformatics software, including Polyphen-2, Provean, SIFT, and Variant Taster. The clinical significance of detected variants was assessed following American Society of Medical Genetics and Genomics guidelines. <strong>3. Generation of iPSCs and iPS-CM differentiation </strong> IPSCs were generated from primary human peripheral blood mononuclear cells. The cells were generated in feeder-free culture conditions using the integration-free CytoTune-iPS 2.0 Sendai Reprogramming Kit (Cat. #A16517, Thermo Fisher Scientific, USA) with the reprogramming factors OCT4, KLF4, SOX2, c-MYC, according to manufacturer’s instructions with modifications [23] (see Supplementary file: Figure 1). Cardiomyocytes (iPS-CMs) derived from iPSCs were induced by the widely used GiWi-protocol originally proposed by Liu et al. [24]. The iPS-CMs usually beat from 10-12 days after differentiation. <strong>4. qPCR </strong> Total RNA was extracted from cells using TRIzol Reagent (Invitrogen, Waltham, MA, USA). cDNA was synthesised using the 5 × PrimeScript RT Master Mix (Cat. #RR036A-1, Takara, Japan) as follows: 37˚C for 15 min; 50˚C for 5 min; and 98˚C for 5 min. RT-qPCR was performed on the platform of the CFX Connect Real-Time System<sup>TM</sup> (BIO-RAD, USA) using 2×TB Green Faster qPCR Mix (Cat. #RR430, TaKaRa, Japan) as follows: 95˚C for 10 sec; 52˚C for 10 sec; and 72˚C for 10 sec for 40 cycles. All primers are listed in Supplementary file: Table 2. The expression levels of mRNA were normalised to those of<em> GAPDH </em>and calculated using the 2<sup>-ΔΔCt</sup> method. <strong>5. Immunofluorescence staining</strong> Immunofluorescence staining was performed using appropriate primary antibodies and Alexa Fluor conjugated secondary antibodies. Cells were washed with pre-cooled DPBS for 3 times and fixed with 4% PFA at room temperature (RT) for 10 min. Then cells were permeabilised with 0.5 % Triton X-100 (Cat. #A600198-0500, BBI, China) for 20 min. Cells were blocked with 2.5% bovine serum albumin (BSA; Sigma Aldrich, Cat. #F7524) in PBS at RT for 1.5 hours. Primary antibodies to RYR2 (Cat. #19766-1-AP, Proteintech, China) and SCN10A (Cat. #33722A18, Invitrogen, USA) were applied in 1 % BSA overnight at 4°C. After being washed with DPBS for 10 min, cells were stained with diluted secondary antibody Alexa FluorTM 488 F(ab’)2 fragment of goat anti-Rabbit lgG (H+L) (Cat. #A11070, Invitrogen, USA) and Alexa FluorTM 555 F(ab’)2 fragment of goat anti-mouse lgG (H+L) (Cat. #A21425, Invitrogen, USA) (1:500 in DPBS), incubated for one hour avoiding light at RT followed by counterstaining with DAPI (Cat. #D1306, ThermoFisher, USA) for 10 min. Fluorescence images were captured with the Olympus Fluoview system (FV3000, Olympus, Japan) to assess in vitro expression. <strong>6.  </strong><strong>Measurements of cellular Ca<sup>2+</sup> homeostasis </strong> IPS-CMs were seeded on Matrigel-coated 14 mm glass-bottomed dishes for 3–5 days and Ca<sup>2+</sup> transients were measured when cells resumed beating. IPS-CMs were loaded with the cell-permeable fluorescent Ca<sup>2+</sup> indicator in media containing 2 μM of Fluo-4 AM (Cat. #F14201, Thermo Fisher Scientific) and 0.02 % F-127 (Cat. #P3000MP, Thermo Fisher Scientific) for 10 min. During imaging, the dishes were kept in a heated 37°C stage bottomed environmental chamber and Ca<sup>2+</sup> transients from single beating iPS-CMs were measured using Zeiss LSM 980 in line-scan mode. After finishing baseline recordings, appropriate amounts of 100 nM isoproterenol (ISO) (Cat#. I2760, Merck, Germany) were added into the recording dish dropwise. Fluorescent signals were normalised to the baseline cellular fluorescence (<em>F</em><sub>0</sub>). <strong>7. Electrophysiological studies</strong> Briefly, at 40 days after iPS-CM differentiation, the iPS-CMs were dissociated into single-cell suspension and transferred to a temperature-controlled chamber (MappingLab, USA). APs were measured at 37 °C using a modified Tyrode’s solution containing (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl<sub>2</sub>, 1.0 MgCl<sub>2</sub>, 5.5 glucose, 5 HEPES, pH 7.4 (NaOH). Pipettes were filled with (in mM) 125 K-gluconate, 20 KCl, 5 NaCl, 0.44 amphotericin-B, 10 HEPES, pH 7.2 (adjusted using KOH), and osmolality 290 ± 3 mOsm. Electrodes were fabricated from borosilicate glass (World Precision Instruments, Sarasota, FL, USA) with tip resistances of 4.5–6.5 MΩ when filled with internal solution containing (in mM): 110 K-gluconate, 20 KCl, 1CaCl<sub>2</sub>, 1 MgCl<sub>2</sub>, 10 HEPES, 5 EGTA-KOH, 5 ATP-Mg<sup>2+</sup>, and 5 Na-phosphocreatine. The pH was adjusted to 7.2 by KOH, and the osmolality to 290 ± 3 mOsm. An Axon 700B amplifer (Axon Instruments, USA) was used for recordings, and the signals were digitised by a Digidata 1550B A/D converter (Axon Instruments, USA) under software control (pClamp10.4, USA). <strong>8. Statistical analysis</strong> Data are presented as mean ± SEM. Statistical significance of differences for normally distributed data was tested by unpaired Student’s t-test to compare two groups and ANOVA with Tukey’s post-hoc test to compare multiple groups. Data were analysed by GraphPad Prism 8 and differences were deemed as statistically significant if <em>P</em> &lt; 0.05.
description: Prism, clampit, Origin8