Extended Data Fig. 3: The effect of dextromethorphan on cardiac calcium and hERG channels in Timothy syndrome iPSC-derived cardiomyocytes.
From: Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome
(a-b) Representative traces of time-course calcium imaging in spontaneously contracting isogenic control (Ctrl, black) and Timothy syndrome (TS, red) cardiomyocytes. (c-d) Calcium transient frequency (c) and duration (d) analysis in TS cardiomyocytes during the 2-hr imaging (n = 32). (e) Current-voltage relationship of Ba2+ recordings in Dxm-treated (n = 12) and non-treated TS cardiomyocytes (n = 13). (f) Representative traces of Ba2+ currents in isogenic control cardiomyocytes without treatment (Ctrl) or treated with dextromethorphan (+Dxm). (g) Voltage-dependent calcium channel inactivation was not altered by Dxm (n = 10) in the isogenic control cardiomyocytes compared to non-treated cells (n = 13). (h) Representative epi-fluorescent and phase-contrast images of TS cardiomyocytes without and with Dxm (+Dxm) from proximity ligation assay (PLA, SIGMAR1-CaV1.2, red, DAPI, blue). Scale bar, 10μm. (i) SIGMAR1-CaV1.2 PLA quantification in Dxm-treated (n = 33) and non-treated TS cardiomyocytes (TS, n = 32). (j) IKr current steady-state amplitudes were significantly reduced in TS cardiomyocytes (n = 10) compared to isogenic control (n = 10) at -10, 0 and 10 mV steps from -40mV hold. (k) IKr tail currents were significantly reduced in TS cardiomyocytes (n = 10) compared to isogenic control (n = 10) at -10, 0, 10 and 20 mV steps. (l) Representative traces of IKr currents (E-4031-sensitive) in isogenic Ctrl cardiomyocytes (black) and TS cardiomyocytes treated with Dxm (purple), Dxm & NE-100 (green) or without treatment (red, TS). The IKr traces are shown in Fig. 2h. (m) There was no significant difference in IKr current (tail) between Dxm-treated (n = 9) and non-treated TS cardiomyocytes (n = 10) while Dxm & NE-100 (n = 10) significantly reduced tail currents compared to Dxm at 10, 20, 30, 40 and 50 mV steps and also to non-treated TS at 20 mV step from -40mV hold. All data are mean ± s.d. The treatment of Dxm or NE-100 for all experiments was 5 μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for c, d between the time points to before and used for m at each voltage step. Unpaired two-tailed Student’s t-test were used for g, i between the groups and used for e, j, k at each voltage step. * P < 0.05, n.s., no significant. The samples were from at least two independent differentiations.
