Patient-specific induced pluripotent stem cell properties implicate Ca2+-homeostasis in clinical arrhythmia associated with combined heterozygous RYR2 and SCN10A variants
Zhang, Yanmin et al. (2023), Patient-specific induced pluripotent stem cell properties implicate Ca2+-homeostasis in clinical arrhythmia associated with combined heterozygous RYR2 and SCN10A variants, Dryad, Dataset, https://doi.org/10.5061/dryad.2rbnzs7rp
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.
1. Family pedigree
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
2. Genetic analysis
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.
3. Generation of iPSCs and iPS-CM differentiation
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  (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. . The iPS-CMs usually beat from 10-12 days after differentiation.
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 SystemTM (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 GAPDH and calculated using the 2-ΔΔCt method.
5. Immunofluorescence staining
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.
6. Measurements of cellular Ca2+ homeostasis
IPS-CMs were seeded on Matrigel-coated 14 mm glass-bottomed dishes for 3–5 days and Ca2+ transients were measured when cells resumed beating. IPS-CMs were loaded with the cell-permeable fluorescent Ca2+ 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 Ca2+ 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 (F0).
7. Electrophysiological studies
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 CaCl2, 1.0 MgCl2, 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, 1CaCl2, 1 MgCl2, 10 HEPES, 5 EGTA-KOH, 5 ATP-Mg2+, 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).
8. Statistical analysis
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 P < 0.05.
Prism, clampit, Origin8
The National Natural Science Foundation of China, Award: 81974014
The National Natural Science Foundation of China, Award: 81470452
Shaanxi Province Natural Science Foundation, Award: 2022JQ-968
Shaanxi Province Natural Science Foundation, Award: 2021JQ-024
Shaanxi Province Natural Science Foundation, Award: 2022JQ-877
Shaanxi Province Natural Science Foundation, Award: 2021JQ-929
Xi’an Talent Program, Award: XAYC200023
Xi'an Health Commission, Award: 2022qn10
Xi'an Health Commission, Award: 2022ms08
Xi'an Health Commission, Award: 2022ms09
Xi'an Children's Hospital, Award: 2021H02
Xi'an Children's Hospital, Award: 2021A01
Xi'an Children's Hospital, Award: 2021B02