Fig 2: Allele-specific RT-PCR based on rs684923 performed on RNA extracted from RYR2-hiPSC-CMs treated with allele-specific shRNAs and a scrambled shRNA. (a) Allelic expression was normalized to total RYR2 expression and is shown relative to the expression in the hiPSC-CMs treated with the scrambled shRNA. shRNA_11 led to a significant reduction in expression of the mutant allele (T allele) compared to treatment with the scrambled shRNA (* p < 0.05, student’s t-test). There was no significant difference (NS) between expression of the wild-type (C allele) in the hiPSC-CMs treated with shRNA_11 compared to those treated with the scrambled shRNA. n = 3 independent experiments. (b) The wild-type to mutant RYR2 allelic ratio in hiPSC-CMs treated with allele-specific shRNAs, shown relative to scrambled. shRNA_11 results in the largest increase in wild-type:mutant ratio (* p < 0.05, student’s t-test). n = 3 independent experiments

Fig 3: Clinical information regarding the proband and family in which the p.(Arg4790Ter) RYR2 variant was identified. (a) Resting ECG traces from the proband. (i) resting 12 lead ECG shows no evidence of QT prolongation. (ii) ECG performed in the brugada position with extra precordial leads V3 = right parasternal 3rd intercostal space, A1 = Left parasternal 3rd intercostal space, A2 = Right parasternal 2nd intercostal space, A3 = Left parasternal 2nd intercostal space. The ECG shows no evidence of a Brugada pattern (ST elevation in the precordial leads). (b) Pedigree showing the family in which the p.(Arg4790Ter) RYR2 variant was identified. Males are represented with squares and females with circles. The proband (III:4) is denoted with an arrow and individuals who were found to carry the p.(Arg4790Ter) variant are indicated with a black dot. Symptomatic individuals are shaded in red

Fig 4: Effect of extracellular calcium concentration on calcium handling in RYR2‐ and control hiPSC‐CMs. (a) Percentage of RYR2‐hiPSC‐CMs (black) and healthy‐control hiPSC‐CMs (white) displaying spontaneous calcium transients and small calcium releases when bathed in solutions with varying calcium concentrations. n = 29 and n = 36 for RYR2 and control hiPSC‐CMs respectively, ** p < 0.001 for all bath calcium concentrations, chi‐squared test. (b) Representative whole cell calcium traces from RYR2‐ and control hiPSC‐CMs at 0.1 mM and 4.0 mM

Fig 5: Calcium handling abnormalities observed in RYR2- and control hiPSC-CMs following incubation with isoproterenol. (a) Significantly more RYR2-hiPSC-CMs which displayed normal transients at baseline developed calcium handling abnormalities following incubation with isoproterenol (10 µM) compared to control hiPSC-CMs, 52.6% versus 19.0%, n = 19 and n = 21 for RYR2 and control cells respectively, * p < 0.05, chi-squared test. (b) Whole cell calcium transients in a RYR2-hiPSC-CM at baseline (i) and after treatment with isoproterenol (ii). Note the development of double and triple humped transients after treatment with isoproterenol