Molecular characterization of the calcium release channel deficiency syndrome

We identified a potentially novel homozygous duplication involving the promoter region and exons 1–4 of the gene encoding type 2 cardiac ryanodine receptor (RYR2) that is responsible for highly penetrant, exertion-related sudden deaths/cardiac arrests in the Amish community without an overt phenotype to suggest RYR2-mediated catecholaminergic polymorphic ventricular tachycardia (CPVT). Homozygous RYR2 duplication (RYR2-DUP) induced pluripotent stem cell cardiomyocytes (iPSC-CMs) were generated from 2 unrelated patients. There was no difference in baseline Ca2+ handling measurements between WT-iPSC-CM and RYR2-DUP-iPSC-CM lines. However, compared with WT-iPSC-CMs, both patient lines demonstrated a dramatic reduction in caffeine-stimulated and isoproterenol-stimulated (ISO-stimulated) Ca2+ transient amplitude, suggesting RyR2 loss of function. There was a greater than 50% reduction in RYR2 transcript/RyR2 protein expression in both patient iPSC-CMs compared with WT. Delayed afterdepolarization was observed in the RYR2-DUP-iPSC-CMs but not in the WT-iPSC-CMs. Compared with WT-iPSC-CMs, there was significantly elevated arrhythmic activity in the RYR2-DUP-iPSC-CMs in response to ISO. Nadolol, propranolol, and flecainide reduced erratic activity by 8.5-fold, 6.8-fold, and 2.4-fold, respectively, from ISO challenge. Unlike the gain-of-function mechanism observed in RYR2-mediated CPVT, the homozygous multiexon duplication precipitated a dramatic reduction in RYR2 transcription and RyR2 protein translation, a loss of function in calcium handling, and a calcium-induced calcium release apparatus that is insensitive to catecholamines and caffeine.

reference samples with known duplication status (i.e. homozygous wild-type, heterozygous RYR2 duplication, and homozygous RYR2 duplication) were included in each assay run.
Following amplification, the data was first analyzed using QuantStudio TM Real-Time PCR system Software v1.3 (Applied Biosystems) set to a manual CT threshold of 0.2 and auto baseline set to on and then transferred to CopyCaller TM software (Applied Biosystems) for automated CNV calling.

Cardiomyocyte Dissociation
Cardiomyocyte aggregate cultures were maintained in 2% FBS/DMEM media in 24-well plates.
At differentiation day 30, iPSC-CMs were subjected to enzymatic dissociation using cardiomyocyte dissociation media (STEMCELL Technologies, 05025) to obtain single cell suspensions of CMs. The iPSC-CMs were first washed with 1 mL of PBS for 20-30 seconds.
Following aspiration of the PBS, 250 µL of cardiomyocyte dissociation media was added to each well. After 2 minutes of incubation, the cardiomyocyte dissociation media was aspirated and the cells were incubated for 4-8 minutes in a 5% CO 2 incubator at 37 o C. Following incubation, 500 µL of DMEM with 20% FBS was added to each well. The solution was titrated 3-5 times with a 1000 µL pipet tip and cells were transferred to a Geltrex-coated 35 mm glass bottom dish (MatTek, P35G-1.5-10-C) and cultured at 37 o C, 5% CO 2 for 24 hours. The media was then changed to DMEM with 2% FBS and stored in a 5% CO 2 incubator at 37 o C until use.

Immunocytochemistry of iPSC-CM Germ Layers
To test for the formation of separate germ layers, cells were harvested from wells containing 10-15 day old differentiated iPSC-CMs using the STEMCELL Technologies protocol previously described and plated into glass bottom 24-well plates. Cells were allowed to grow for 5 days, then subjected to fixation with 4% paraformaldehyde for 10 minutes at room temperature (RT) followed by being washed 3 times with PBS. The iPSC-CMs were then permeabilized/blocked with 0.1% Triton X-100/ PBS (PBST)/5% goat serum for 45 minutes at RT, and subsequently incubated in primary antibody solution made of PBST/5% goat serum containing a 1:250 dilution of neuron (ectoderm)-specific TUJ-1/β-III-tubulin (Biotechne, MAB1195), sarcomere (mesoderm)-specific cTNT (Abcam, ab45932) and/or -actinin (Sigma, A7811-.5ML), and endoderm-specific SOX17 (ThermoFisher, PA5-23352) primary antibodies at RT overnight. The next day, cells were washed 3 times with PBST/5% goat serum at RT before being incubated in PBST/5% goat serum with a 1:250 dilution of Alexa Fluor® 488 goat-anti-rabbit (Invitrogen, A11008) and Alexa Fluor 594 goat-anti-mouse (Invitrogen, A11005) secondary antibodies at RT for 60 minutes; DAPI was added to each secondary antibody solution at a concentration of 1µg/mL. After secondary antibody incubation, cells were washed 3 times with PBST and finally covered in 500 µL of PBS for imaging. Images were acquired on a Zeiss LSM 780 confocal microscope in the Mayo Clinic Microscopy and Flow Cytometry Cell Analysis Core Facility (Supplemental Figure 2). For iPSC-CM cell size analysis, acquired images of single, cardiac marker-positive cells were analyzed using ImageJ Software to assess surface area as an

Real-Time Quantitative Polymerase Chain Reaction
Following physical enrichment of iPSC-CMs from beating clusters, total RNA was isolated from multiple biological replicates from all four homozygous RYR2 duplication mutant hiPSC-CM clones and control iPSC-CMs using the mirVana™ miRNA isolation kit (Invitrogen, PLUS chemiluminescent ECL substrate (ThermoFisher, 34577) for 5 minutes and then exposed to HyBlot CL autoradiography film (Denville Scientific Inc., E3012). Film was scanned and saved as JPEG file; the relative density of RyR2 to that of β-Actin was analyzed and quantified with ImageJ (National Institutes of Health, Bethesda, MD). There was no significant difference in the protein expression among the lines (data not shown).

Immunocytochemical Analysis of iPSC-CMs
To demonstrate that the CMs express the typical cardiac markers, immunofluorescence staining for cardiac troponin (cTnT), cardiac ryanodine receptor-2 (RyR2), and DAPI was performed.

Quantitative Immunofluorescence in Human Heart Tissue
Frozen sections of human heart were processed by the Mayo Clinic Histology Core Laboratory.
Frozen section was fixed with a cold (-20 o C) acetone (Sigma, 179124) and methanol (Fisher, A454-4) (50:50) mixture for 5 minutes at -20 o C. The sections were washed immediately at room temperature with PBS to re-hydrate tissue. Blocking and primary/secondary antibody staining was followed as described above. Imaged data was analyzed by ZEN lite software (Zeiss) to quantify the average intensity that was collected and was analyzed by the statistic software, GraphPad Prism (GraphPad Software).

Fluo-4-Measured Ca 2+ Imaging to Assess Calcium Handling
Cardiomyocyte sarcoplasmic reticulum (SR) Ca 2+ load and RyR2-mediated leakage were The raw data was exported to Excel software (Microsoft, Redmond, WA) and then analyzed with an in-lab developed Excel-based program.

Electrophysiological Patch-Clamp Action Potential Measurements
Cardiomyocyte aggregate cultures were maintained in DMEM media. At differentiation days of 30-60, the enriched iPSC-CMs were subjected to enzymatic dissociation to obtain single cell suspensions of cardiomyocytes. These cells were added to 0.1% gelatin coated glass coverslips maintained in DMEM media and stored in a 5% CO 2 incubator at 37 o C before use.

Assessment of Field Potential and Arrhythmic Activity
Arrhythmic activity was assessed using an xCELLigence ® RTCA CardioECR instrument (ACEA Biosciences) at baseline (DMEM/2%FBS media) and during ISO challenge (100 nM).
Patient and unrelated control iPSC-CMs were dissociated from 24-well plates and then plated and cultured on fibronectin-coated (Gibco, 33016-15) 48-well electronic microtiter plates (ACEA Biosciences, 300600940) at 37 o C, 5% CO 2 . Field potentials (FPs) were measured two times for 20 seconds (block duration) every 10 minutes as a routine baseline measurement.
Each 20 seconds recording counted as independent experiment. We counted the erratic electrical activity showing electrodes and divided this by total number of recording electrodes per experiment. For example, if no electrode showed erratic activity then we assigned a value of Zero and if 1 electrode out of 4 showed erratic activity, then we assigned a value of 0.25. Overall, this normalization give how many beatings show erratic activity in given time.

Drug Rescue of Arrhythmic Activity
Various pharmacotherapies were used to test their efficacy on the treatment of arrhythmic events occurring in the mutant iPSC-CMs as a proof-of-principle, treatment-in-a-dish pilot study.
Propranolol (1µM), nadolol (10µM) or flecainide (6µM) were administered in combination with ISO (100 nM) immediately before recording. For the drug treatment test, a 1 hour setting was used with a 5 minute interval and 20 second block duration. The percentages of wells presenting arrhythmic activity were recorded per treatment. Isoproterenol, propranolol, nadolol, and flecainide were purchased through Sigma/Millipore.

Statistical Analysis
Data points are shown as the mean value and bars represent the standard deviation unless specifically mentioned. A Student's t-test was performed to determine statistical significance between two groups and a one-way ANOVA was performed for comparisons among three groups. For multiple post-hoc ANOVA analyses, both Tukey's and Bonferroni tests were used.
A p<0.05 was considered to be significant.

Supplemental Figure 4. Erratic Beating Frequency in siRNA RYR2 Knockdown iPSC-CMs
Shown in A are representative field potential (FP) recordings from scramble (sc) RNA, siRNA #1, and siRNA #2 treated normal iPSC-CMs following ISO (100nM) treatment. Panel B depicts a bar graph summary showing the erratic beating frequency (i.e. arrhythmic events) present at baseline and following ISO treatment in scRNA, siRNA #1, and siRNA #2 treated iPSC-CMs. The number of responding electrodes for recording for each sample and treatment ranged from n=21 to n=141. Four different date/batches of independent experiments were performed. Shown in panel C is the RT-qPCR of RYR2 mRNA transcript normalized by cardiac troponin T (cTnT). Three independent technical replicates were included (N=6) for each RT-qPCR experiment. A Student's t-test was performed to determine statistical significance between two groups. A p<0.05 was considered to be significant. duplication from patient 2; clone 2; MDP = maximum diastolic potential, APD = action potential duration, * = p<0.05