Fig. 4

Fig. 4 shRNA silencing of sodium channel interactors, Epn2 and Gsn, increase sodium current density in mouse ventricular cardiomyocytes.

a, Voltage clamp protocol (top) and representative sodium current (I_Na) traces measured from adult cardiomyocytes expressing only GFP (GFP; bottom left) or GFP as well as shRNA for Epn2 (Epn2 knockdown (KD); bottom right). b, Current (I) to voltage (V_m) relationship of I_Na obtained from cardiomyocytes that were not injected (‘control’; solid circles), expressing only GFP (GFP; open squares) or from expressing GFP and shRNA for Epn2 (Epn2 KD; open diamonds). The data show increased peak sodium current density in Epn2 KD (*P = 0.014, linear mixed-effects analysis followed by Bonferroni post hoc analysis for multiple comparison testing). c, Sodium current activation measured for Epn2 KD, GFP or uninjected controls. d, Representative I_Na traces in GFP-expressing cardiomyocytes (bottom left) and in Gsn2 KD cardiomyocytes (bottom right). e, I to V_m relationship of I_Na for Gsn KD cardiomyocytes compared to that of controls (maximum sodium current trending to be increased for Gsn KD, P = 0.181, linear mixed-effects analysis followed by post hoc Bonferroni correction). f, I_Na activation curves. The activation curve is negatively shifted in Gsn KD cardiomyocytes compared to that of myocytes expressing only GFP (V_{1/2,Gsn KD} = −57.4 ± 0.63 mV; V_1/2,GFP = −53.4 ± 0.95 mV, *P = 0.037 linear mixed-effects analysis, followed by Bonferroni post hoc analysis for multiple comparison tests, control: n = 10 cells obtained from 3 mice; GFP: n = 9 cells obtained from 3 mice; Epn2 KD: n = 9 cells obtained from 4 mice; Gsn KD: n = 8 cells obtained from 4 mice; data are presented as mean ± standard error of the mean). g, Two-color STORM images for Scn5a (green) and Gsn (red) show 30% of Gsn clusters localizing within 20 nm of Scn5a clusters, 15 cells obtained from 3 mice in independent experiments. ‘Control’ are cardiomyocytes isolated from WT animals. ‘GFP’ are cardiomyocytes isolated from animals injected with an empty AAV vector. ‘Epn2 KD’ are cardiomyocytes isolated from mice with Epn2 silencing and ‘Gsn KD’ from mice with Gsn silencing. Multiple animals per group were necessary due to the limited number of datapoints that can be obtained from a single animal.

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