Fig 2: Co-knockdown of IKr and INa by hERG transcript-specific shRNA.(A) Effects of hERG1a or hERG1b silencing on channel mRNA expression levels detected by RT-qPCR (mean ±95% CI) in iPSC-CMs. A non-targeting shRNA (scrambled shRNA) is used as a control. (B) Effects of specific hERG1a or hERG1b silencing on ion channel mRNAs expressed alone in HEK293 cells. (C) Representative family of traces show IKr in presence of control (upper) or hERG1b shRNA (lower). (D) Summary of steady-state current density vs. test potential shows effect of hERG1b shRNA (mean ±SE). (E) Effects of 1b shRNA on peak tail current vs. pre-pulse potential (mean ±SE). (F) Representative family of traces recorded from iPSC-CMs showing effects of hERG1b-specific shRNA compared to control shRNA on peak INa. (G) Summary current-voltage plot of peak INa vs. test potential (mean ±SE). (H) Summary conductance (G)-voltage plot based on data from G (mean ±SE). (I) Late sodium current representative trace in control and 1b shRNA-transfected cells, measured by applying a single pulse protocol of 800 ms. (J) Summary statistics of peak INa showed a decrease upon transfection with hERG1b shRNA (mean ±SE). (K) Late INa measured as the integral from 50 to 800 ms from the beginning of the pulse showed a decrease upon transfection with hERG1b shRNA (mean ±SE).10.7554/eLife.52654.036Figure 7—source data 1.Co-knockdown of transcripts by qPCR raw data for Figure 7A.10.7554/eLife.52654.037Figure 7—source data 2.Specificity of shRNA raw data for Figure 7B.10.7554/eLife.52654.038Figure 7—source data 3.IKr is reduced upon hERG silencing raw data for Figure 7D–E.10.7554/eLife.52654.039Figure 7—source data 4.Reduction of peak INa after hERG silencing raw data for Figure 7G–H.10.7554/eLife.52654.040Figure 7—source data 5.Decrease of INa,late current upon hERG silencing raw data for Figure 7J–K.

Fig 3: Correlation analysis of hERG and sodium currents.(A) Voltage step protocol used to measure both sodium (INa) and hERG (IKr) currents in the same cell, using cesium as the charge carrier for hERG channels. The region from where IKr was measured is highlighted in the protocol (yellow square). Representative traces before (Control) and after perfusing E-4031 (10 µM) to block hERG channels, validating that the recorded current is IKr. (B) Current density of hERG channels were plotted in function of Nav1.5 current density and correlation coefficients are shown on top of the graph. One cell (red dot) was excluded from the correlation analysis as it meets the outlier criteria. n = 20 cells of 2 independent experiments (N = 2).

Fig 4: Impact of chaperone treatment on protein interactions in trafficking-deficient KV11.1 variants.A, heatmap and (B) violin plots showing comparison of log2 fold changes over WT of 572 enriched protein interactions in trafficking-deficient variants G601S and G965∗, with or without 24-h E-4031 treatment. Statistical significance was calculated by paired two-tailed t test, and p values are depicted by ∗∗∗∗ < 0.0001. C and D, correlation of proteasomal degradation protein interactions for (C) G601S and (D) G965∗ in absence (x-axis) or presence (y-axis) of E-4031 (all log2 fold changes normalized to WT with DMSO). The black dotted line indicates the normal line with a slope of 1 and intercept at the origin. The orange error bars from each individual protein represent the SEM for vehicle (0.1% DMSO, horizontal) and E-4031 (10 μM, vertical). E, heatmap showing the top five interactions from ten manually annotated GO terms sorted by largest difference in log2 fold change/WT in absence or presence of E-4031 for 24 h. Only three proteins responsible for autophagy were enriched for KV11.1 interaction in our dataset. Cells were treated with either vehicle (0.1% DMSO) or E-4031 (10 μM) for 24 h before experiments. DMSO, dimethyl sulfoxide; GO, gene ontology.

Fig 5: Cluster Analysis of trafficking-deficient KV11.1 interactomes.A, hierarchical clustering of 572 protein interactors arranged by intensity similarity. Abundances for G601S and G965∗ variants in relation to WT KV11.1 were transformed into an Euclidian distance matrix and clustered using Ward’s minimum variance method. Each row represents an interactor with color indicating log2 fold change normalized to WT. B, swarm plots for all clusters depicting the log2 fold change normalized to WT for each variant and cluster. Statistical significance was calculated by ANOVA with post hoc Tukey’s multiple comparisons test and p values are depicted by ∗∗∗∗ < 0.0001. Bars represent mean ± SD. C–H, bar charts showing fold change compared to WT for individual genes within the top enriched protein GO term for each cluster. All samples were treated with vehicle (0.1%) for 24 h before sample collection. GO, gene ontology.