A bifunctional actuator to correct loss-of-function deficits in cardiac voltage-gated sodium channels

Figures

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  Fig. 1.. A bifunctional actuator to restore NaV1.5 channel function in cardiac disease. (A) Increased late Na current (INaL) contributes to long QT syndrome (LQTS) symptoms, while reduced peak/transient Na current (INaT) can contribute to Brugada syndrome (BrS). Human variants that lead to increased INaL and decreased INaT are linked to mixed or overlap syndrome phenotype. FixR and ELIXIR are peptides that selectively inhibit INaL. Few strategies are available to enhance INaT. (B) Mixed syndrome E1784K variant shows increased ubiquitination. Immunoblots of Venus-tagged NaV1.5 WT or E1784K variant immunoprecipitated using GFP-trap and probed using anti-GFP (left) and anti-ubiquitin (right) antibodies. Cells were treated with MG132 to prevent proteasomal degradation. (C) Bar graph shows quantification of ubiquitin signal normalized to GFP signal intensity corresponding to total channel expression (n = 6, from three independent transfections). *P = 0.0411 by paired t test. (D) Design of a bifunctional actuator that increases INaT and decreases INaL. A high-affinity nanobody (Nb82) targeting NaV1.5 is functionalized by fusing FixR, a potent INaL blocker, and the catalytic domain of a DUB to…

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  Fig. 2.. Functional modulation of NaV1.5 by the bifunctional modulator FixR-UpR. (A) Whole-cell peak current density (Jpeak) for WT NaV1.5. Each dot represents means ± SEM from n = 10 cells (two transfections). Inset: exemplar current recordings. (B to D) Coexpression of FixR-Nb-DUB with WT NaV1.5 differentially tunes JPeak. FixR-Nb-Usp21 (B; n = 11 cells from two transfections); FixR-Nb-Otud1 (C, n = 12 cells from two transfections); FixR-Nb-Otud4 (D, n = 10 cells from two transfections). *P < 0.05, **P < 0.01, and ***P < 0.001 by multiple unpaired t test. (E) The NaV1.5 E1784K variant evokes a strong reduction in JPeak. Each dot represents means ± SEM from n = 9 cells. (F to H) Coexpression of NaV1.5 E1784K with FixR-Nb-Usp21 (F, n = 8 cells from two transfections), FixR-Nb-Otud1 (G, n = 8 cells from two transfections), and FixR-Nb-Otud4 (H, n = 8 cells from two transfections)] all increased JPeak. FixR-Nb-Otud1 is henceforth termed FixR-UpR. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by multiple unpaired t tests. (I) Steady-state inactivation of WT Nav1.5 (top) and E1784K variant (bottom) without (black) and with FixR-UpR (red). Each dot represents means ± SEM from n = 7…

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  Fig. 3.. Mechanism of FixR-UpR in up-regulating NaV1.5 currents. (A) Schematic shows the topology of NaV1.5 channel, with mixed syndrome (purple), and LQTS3 (red) variants highlighted along with known ubiquitinated residues for WT NaV1.5 (gray). (B) Immunoblots show overall ubiquitination of Venus-tagged NaV1.5 WT or E1784K variant in the presence of either FixR-UpR or catalytically inactive FixR-UpR C/S mutant. (C) Bar graph shows normalized ubiquitin signal for NaV1.5 WT or E1784K variant in the presence of FixR-UpR or catalytically inactive FixR-UpR C/S mutant (n = 6 from three independent transfections). **P = 0.0041 and ****P < 0.0001 by repeated measures one-way ANOVA with Tukey’s multiple comparisons’s test. (D and E) Whole-cell peak current densities of WT NaV1.5 (D) and E1784K (E) variant in the absence (black) and presence (orange) of a catalytically inactive FixR-UpR C320S mutant. Each dot represents means ± SEM from n = 7–11 cells from two independent transfections. (F) FixR-UpR fails to up-regulate JPeak of NaV1.5 triple mutant (K442R, K443R, and K496R) that lacks key ubiquitinated residues in the DI-DII linker. Each dot represents means ± SEM from n = 6 cells from two…

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  Fig. 4.. 2-hit mice with diastolic dysfunction show reduced NaV1.5 currents. (A) Increase in body weight in mice following 5 weeks of regular (control, black) or high-fat diet combined with L-NAME supplementation (2-hit, red). Each bar represents means ± SEM from 10 (control) and 19 animals (2-hit). ****P < 0.0001 by unpaired t test. (B) Representative left ventricular M-mode echocardiographic tracings from control (top) and 2-hit (bottom) mice. Images are representative of 8 independent control or 2-hit mice. (C to H) Bar graphs quantify changes in diastolic diameter (C), diastolic volume of left ventricle (D), relative wall thickness (E), percentage of wall thickening (F), ejection fraction (G), and fractional shortening (H). 2-hit mice show signs of diastolic dysfunction with no apparent change in systolic function. Each bar represents means ± SEM from 8 animals each. ***P < 0.001 and *P < 0.05 by unpaired t test. (I) Confocal images of ventricular cardiomyocytes isolated from control (top) or 2-hit (bottom) mice show colocalization of immunolabeled NaV1.5 (cyan) and a plasma membrane marker (WGA; magenta). (J) Colocalization of NaV1.5 with the plasma membrane marker quantified as…

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  Fig. 5.. In vivo expression of FixR-UpR expression reverses NaV1.5 dysfunction in 2-hit mice. (A) Increase in body weight in mice following 5 weeks of regular (control) or high-fat diet with L-NAME (2-hit), injected retro-orbitally with AAV9 encoding GFP or FixR-UpR, 3 weeks before experimentation. Each bar represents means ± SEM from 13 (control with GFP), 14 (2-hit with GFP) and 13 animals (2-hit with FixR-UpR). ****P < 0.0001, ***P = 0.004, and **P = 0.0011 by one-way ANOVA followed by Dunnett’s test. (B) Representative left ventricular M-mode echocardiographic tracings from control animals injected with GFP (top) or 2-hit mice injected with GFP (middle) or FixR-UpR (bottom). (C and D) Bar graphs show changes in RWT (C) and ejection fraction (D). Each bar represents means ± SEM from 8 animals. **P = 0.009 by one-way ANOVA followed by Dunnett’s test. (E) Confocal images of CMs from control mice with GFP (top) and 2-hit mice with GFP (middle) and FixR-UpR (bottom) show colocalization of NaV1.5 (cyan) with PM (WGA; magenta). Scale bar, 20 μm. (F) Bar graph shows colocalization of NaV1.5 with the PM marker assessed by Pearson’s correlation coefficient. Each bar represents means ± SEM from…

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  Fig. 6.. FixR-UpR tunes NaV1.5 function in BrS/mixed syndrome hiPSC-CMs. (A) Schematic shows adenoviral delivery of FixR-UpR or GFP control to BrS/Mixed Syndrome hiPSC-CM. (B) Whole-cell peak Na current densities from the E1784K+/− hiPSC-CM transduced with GFP (green) or with FixR-UpR (blue). FixR-UpR evokes a strong increase in Jpeak of INa in these cells. Each dot represents means ± SEM from n = 8 cells each. ***P < 0.01 by multiple unpaired t tests. (C) Confocal images of E1784K+/− hiPSC-CMs transduced with GFP (top), or FixR-UpR (bottom) show co-localization of immunolabeled NaV1.5 (cyan) with plasma membrane (WGA; magenta). GFP indicates positive transduction of eGFP or FixR-UpR. Scale bars, 20 μm. (D) Bargraph shows increased co-localization of NaV1.5 with plasma membrane marker in the presence of FixR-UpR. Each bar represents means ± SEM from n = 49 (GFP) and n = 52 cells (FixR-UpR) respectively. (E) Fluorescence AP recordings of E1784K+/− hiPSC-derived cardiomyocyte monolayers. (F and G) Bar graphs show changes in the upstroke velocity (F) and APD80 (G) upon expression of FixR-UpR compared to GFP control. Each bar represents means ± SEM from n = 8 monolayers. **P < 0.01 and ****P…