Characterization of the mechanism by which a nonsense variant in RYR2 leads to disordered calcium handling

FIGURE 1. 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

FIGURE 2. Calcium handling abnormalities in patient‐specific RYR2‐hiPSC‐CMs at baseline. (a) Line scan image showing changes in intracellular calcium in single RYR2‐hiPSC‐CMs at baseline illustrating the differences between double and triple peaked and broader multiple peaked transients. (a(i)) note the double and triple peaked transients (red arrows). (a(ii)) Note the broad multiple peaked transients (red arrows). (b) Line scan images showing changes in intracellular calcium in control hiPSC‐CMs at baseline. The abnormalities recorded in control hiPSC‐CMs tended to be less complex and less severe than those seen in RYR2‐hiPSC‐CMs. (c(i)) hiPSC‐CMs generated from both RYR2 clones (RYR2‐1 and RYR2‐3) displayed significantly more calcium handling abnormalities compared to healthy‐control hiPSC‐CMs (control 28.9% n = 121, RYR2‐1 70.9% n = 103, RYR2‐3 64.9% n = 259), ** p < 0.001, chi‐squared test. (c(ii)) In cells displaying abnormalities, a higher percentage of calcium transients displaying abnormalities was observed in the RYR2‐hiPSc‐CMs compared to control hiPSC‐CMs (RYR2 55.74% n = 20, control 33.69% n = 12, ** p < 0.001, chi‐squared test). (c(iii)) In cells displaying comparable…

FIGURE 3. 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

FIGURE 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

FIGURE 5. Effect of different beta‐blockers on the proportion of RYR2‐hiPSC‐CMs displaying calcium handling abnormalities. Treatment with 2 µM labetalol, 1 µM carvedilol and 1 µM nebivolol led to significant reductions in the proportion of cells displaying calcium transient abnormalities (* p < 0.05, ** p < 0.001 respectively, chi‐squared test). Treatment with the other beta‐blockers tested resulted in no significant difference. Baseline n = 362, vehicle control n = 27, bisoprolol n = 23, metoprolol n = 28, labetalol n = 29, carvedilol n = 14, S‐propranolol n = 27, nebivolol n = 22, nadolol n = 31

FIGURE 6. RYR2 expression in hiPSC‐CMs harbouring the nonsense variant. (a) Sequencing of DNA extracted from healthy‐control hiPSCs showed that the line is homozygous for the rs684923 SNP in RYR2 ((i)) whilst the RYR2 line is heterozygous for the SNP ((ii)). (b) Allele‐specific RT‐PCR confirmed the expression of both RYR2 alleles in hiPSC‐CMs generated from both clones of the RYR2 line (RYR2‐1 and RYR2‐3). Only the C allele was detected in control hiPSC‐CMs confirming the specificity of the primers. (c) RT‐PCR for total RYR2 expression performed on RNA extracted from hiPSC‐CMs derived from two different control lines (UN1‐22 and Ctrl) and the RYR2‐hiPSC‐CMs (n = 4, 2 and 5 respectively). In RYR2‐hiPSC‐CMs, total RYR2 expression was significantly reduced compared to expression in the control lines,* p < 0.05, student’s t‐test. There was no significant difference in RYR2 expression between the two healthy‐control lines

FIGURE 7. Western blot to assess RYR2 levels. (a) Representative western blots showing expression of N‐terminus and C‐terminus in protein extracted from RYR2‐hiPSC‐CMs and control hiPSC‐CMs. (b) Quantification of expression of N‐terminus (total) of RYR2 in RYR2 and control hiPSC‐CMs. Data normalized to total protein. Total RYR2 expression was significantly reduced in the RYR2‐hiPSC‐CMs compared to the control hiPSC‐CMs, *** p < 0.0005, student’s t‐test. (c) Quantification of expression of C‐terminus of RYR2 in RYR2‐ and control hiPSC‐CMs. Data normalized to total protein. Expression of the C‐terminus in RYR2 hiPSC‐CMs was significantly reduced compared to expression in control hiPSC‐CMs, ** p < 0.001, student’s t‐test. (d) Ratio of expression of C‐terminus to N‐terminus in RYR2‐hiPSC‐CMs shown relative to expression in control hiPSC‐CMs. No significant difference was seen between the RYR2‐ and control hiPSC‐CMs. n = 5 (RYR2) and n = 3 (control)

FIGURE 8. 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

FIGURE 9. The effect of treatment with shRNA_11 on calcium handling and RYR2 protein levels in RYR2‐hiPSC‐CMs. (a) Treatment with shRNA_11 led to a significant reduction in the proportion of cells displaying abnormalities of the calcium transient compared to those treated with the scrambled shRNA. 68.2% of cells treated with scrambled (n = 22) and 40.5% of cells treated with shRNA_11 (n = 27) displayed calcium transient abnormalities (* p < 0.05, chi‐squared test). (b) (i) Line scan image showing changes in intracellular calcium in a single RYR2‐hiPSC‐CM treated with the scrambled shRNA. (ii) Line scan image showing changes in intracellular calcium in a single RYR2‐hiPSC‐CM treated with shRNA_11. (c) The effect of treatment with shRNA_11 on total RYR2 protein levels. (i) Representative western blot showing total RYR2 expression in RYR2‐hiPSC‐CMs transduced with shRNA_11 and a scrambled shRNA and also untransduced RYR2‐hiPSC‐CMs. (ii) Treatment with shRNA_11 resulted in an increase in total RYR2 protein levels compared to those treated with the scrambled shRNA and the untransduced cells however this was not statistically significant. n = 3 each group