Fig. 4: Tmem65 KD leads to losses of NaV1.5 channels at ICDs.

a An immunoblot showing reduced NaV1.5 and β1 protein levels in Tmem65 KD mouse hearts when compared to control samples (n = 4 hearts). **P < 0.01. Statistical analysis was performed by one-way ANOVA with Tukey’s post-hoc test. Data were expressed as mean ± standard error of means. b Immunofluorescence of ICD regions in scrambled and Tmem65 KD hearts, showing the loss of ICD-bound NaV1.5 in Tmem65 KD hearts. β1 staining showed scattered puncta (white arrows) or aggregates (yellow arrows) also in Tmem65 KD hearts. c Immunoprecipitation assays using NCAD antibody and mouse cardiac lysates showed a reduced NaV1.5/NCAD interaction in Tmem65 KD mouse hearts. d Representative images of sodium current (I_Na⁺) in control vs. Tmem65 KD cardiomyocytes. e Current density-voltage (I-V) plot shows that I_Na⁺ density did not significantly differ between scrambled (black) and Tmem65 KD (red) cardiomyocytes. f Quantification of peak I_Na⁺ density in scrambled and Tmem65 KD cardiomyocytes. No statistical difference was found. g Voltage-dependence of I_Na⁺ activation and steady-state inactivation in cardiomyocytes with the least-square fits to the Boltzmann function. A statistical significance was found between Tmem65 KD and control cardiomyocytes in activation state, but not in inactivation state. All measurements are summarized in Table 4. N = 16 control cells and 25 Tmem65 KD cells for in I_Na⁺. All cells were isolated from male mouse hearts. For patch-clamp experiments, data were presented as mean ± standard deviation. The difference of current density was compared using unpaired student t-test.