Extended Data Fig. 1: The effect of SIGMAR1 agonists on CDK5, contraction and calcium channel in Timothy syndrome iPSC-derived cardiomyocytes.

(a) Docking models of SIGMAR1-Fluvo, -Dxm and -PRE. (b) PRE-084 (5 μM, 2 hr) reduced CDK5 kinase activity in Timothy syndrome (TS) cardiomyocytes. PHA-793887 (PHA, 5 μM), a CDK5 inhibitor, was used as positive control for the assay (n = 8/group). (c-e) The effects of PRE (5 μM, 2 hr) on CDK5R1/p35 protein (c, d, n = 12 for baseline and n = 9 for 2 hr after treatment) and CDK5 protein (c, d, n = 5/group) and mRNA (e, n = 4/group). (f) Representative epi-fluorescent and phase-contrast images of TS cardiomyocytes without treatment, with PRE-084 (+PRE, 5 μM, 2 hr) or PRE-084 and NE-100 treatment (+PRE & NE-100, both 5 μM, 2 hr) from proximity ligation assay (PLA, SIGMAR1-CDK5, red, DAPI, blue). Scale bar, 10μm. (g) SIGMAR1-CDK5 PLA quantification in PRE-treated (n = 16), PRE&NE-100-treated (n = 38) and non-treated TS cardiomyocytes (n = 14). (h) Representative traces of relative motion analysis of TS cardiomyocyte contractions before (black) and after 2-hr PRE-084 treatment (blue). The representative traces were obtained from Supplementary Movies 1 and 2. (i-j) Relative changes of beating rate (i) and irregularity (j) (n = 11) of TS cardiomyocytes after PRE-084 treatment. (k) Representative traces of Ba²⁺ currents in TS cardiomyocytes without treatment or treated with PRE-084 (+PRE), or fluvoxamine (+Fluvo) (each, 5 μM, 2 hr) and in isogenic control cardiomyocytes (Ctrl). +Dxm representative trace is shown in Fig. 2f. (l) Voltage-dependent calcium channel inactivation was significantly enhanced by PRE (n = 10), Fluvo (n = 10) and Dxm (n = 16) treatment in TS cardiomyocytes compared to non-treated cells (n = 25). n.s., no significant differences between +PRE, + Fluvo, +Dxm and isogenic Ctrl groups (n = 13). (m) Representative traces of Ba²⁺ currents in TS cardiomyocytes before treatment and acutely treated with PRE (5 μM, ~5-10 mins). (n) Voltage-dependent calcium channel inactivation was not significantly changed by acute PRE treatment in TS cardiomyocytes (n = 10). All data are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons was used for b, g and One-way ANOVA with Sidak’s multiple comparisons was used for l. Paired two-tailed Student’s t-test was used for i, j, n, and unpaired two-tailed Student’s t-test was used for d, e. *P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant. Cell samples from at least two independent differentiations were used.

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