Post-Translational Modifications and Diastolic Calcium Leak Associated to the Novel RyR2-D3638A Mutation Lead to CPVT in Patient-Specific hiPSC-Derived Cardiomyocytes

Background: Sarcoplasmic reticulum Ca2+ leak and post-translational modifications under stress have been implicated in catecholaminergic polymorphic ventricular tachycardia (CPVT), a highly lethal inherited arrhythmogenic disorder. Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. Objective: The aims were to obtain functional hiPSC-derived cardiomyocytes from a CPVT patient harboring a novel ryanodine receptor (RyR2) mutation and model the syndrome, drug responses and investigate the molecular mechanisms associated to the CPVT syndrome. Methods: Patient-specific cardiomyocytes were generated from a young athletic female diagnosed with CPVT. The contractile, intracellular Ca2+ handling and electrophysiological properties as well as the RyR2 macromolecular remodeling were studied. Results: Exercise stress electrocardiography revealed polymorphic ventricular tachycardia when treated with metoprolol and marked improvement with flecainide alone. We found abnormal stress-induced contractile and electrophysiological properties associated with sarcoplasmic reticulum Ca2+ leak in CPVT hiPSC-derived cardiomyocytes. We found inadequate response to metoprolol and a potent response of flecainide. Stabilizing RyR2 with a Rycal compound prevents those abnormalities specifically in CPVT hiPSC-derived cardiomyocytes. The RyR2-D3638A mutation is located in the conformational change inducing-central core domain and leads to RyR2 macromolecular remodeling including depletion of PP2A and Calstabin2. Conclusion: We identified a novel RyR2-D3638A mutation causing 3D conformational defects and aberrant biophysical properties associated to RyR2 macromolecular complex post-translational remodeling. The molecular remodeling is for the first time revealed using patient-specific hiPSC-derived cardiomyocytes which may explain the CPVT proband’s resistance. Our study promotes hiPSC-derived cardiomyocytes as a suitable model for disease modeling, testing new therapeutic compounds, personalized medicine and deciphering underlying molecular mechanisms.


Tables:
AP duration at 90% of repolarization corrected using the Bazett's formula. Stars indicate differences between HC and CPVT without ISO. # indicate differences between CPVT with and without ISO. No differences were found between HC with and without ISO.

Protein sequence analysis and molecular modelling of the RyR2-D3638A mutant
RyR2 protein sequence alignment between the position 3584 and 3643 was performed using Uniprot (www.uniprot.org/align). A total of 5 different species were compared. In order to understand how D3638A substitution may change the structure of human RyR2, we modelled this mutant based on the known structure of the pig RyR2 (PDB code 5GO9)[1] by using Homology and Discovery modules of InsightII program [2].

CPVT-hiPSC generation and maintenance of pluripotent stem cells
All subjects gave their informed consent for inclusion before they participated in the study.
The study was conducted in accordance with the Declaration of Helsinki, and the protocol (10- Generated CPVT-hiPSC clones as well as pluripotent stem cell lines that were used as healthy controls in this study, UEFhfiPS1.4 [3] and CCTL12 previously published [4,5] were maintained as colonies on mitotically inactivated MEF feeder in HES medium with 10 ng/ml human FGF2.
Cells were manually dissected and passaged every 4-6 days.

Karyotype analysis
For cytogenetic analysis hiPSC metaphase arrest was induced using 0.1 µg/ml Colcemid

Alkaline phosphatase activity
Alkaline phosphatase activity was determined in hiPSCs that were fixed with 4% paraformaldehyde (PFA) for 4 min at room temperature, and after washing with 1x phosphate-buffered saline (PBS), using Alkaline Phosphatase Blue Microwell Substrate kit (Sigma-Aldrich).

RT-PCR and qRT-PCR
One-month-old beating EBs and undifferentiated hiPSC colonies were used for RNA  Table S5.

Embryoid body dissociation and immunocytochemistry
Prior to the staining of cardiac markers, beating (contracting) EBs were dissociated in order to get isolated CMs by collecting and washing EBs two times in Ca 2+ -free solution (120 mM  Table S5.

Atomic force microscopy (AFM)
Homogenous EBs in size and shape of 30 to 60 days-old were plated on the gelatin-coated 40  Table   S3.

Measurement of cytosolic Ca 2+ variation
After dissociation of 30 to 60 day-old contracting EBs, hiPSC-CMs were loaded with 3 µM  Table S6.
All immunoblots were developed using the Odyssey system (LI-COR) with IR labeled secondary antibodies for 1 h at room temperature.

Statistical analysis
Normality was tested using the Shapiro-Wilk test. An unpaired t test was used to compare 2 independent groups with parametric distribution. A Mann-Whitney test was performed for comparing 2 independent groups non-parametric distribution. A 2-way-analysis of variance (ANOVA) was used to compare more than 2 groups ± treatment with normal distribution followed by post-hoc test. We performed a Kruskal-Wallis test to compare more than 2 groups ± treatment with non-parametric distribution. All data are expressed as mean ± SEM. A value of p < 0.05 was considered significant. *, p < 0.05, **, p < 0.01 otherwise specified. Data analysis and statistics were done with Prism.       Expression levels were normalized to GAPDH expression.