Extended Data Fig. 2: The effect of SIGMAR1 agonists on action potential and SIGMAR1 and ATF4 expression profiling in Timothy syndrome iPSC-derived cardiomyocytes.
From: Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome
(a-c) Action potential parameters, APD50 (a), resting potential (b) and peak amplitude (c) in Timothy syndrome (TS) cardiomyocytes without treatment (n = 15) or treated for 2 hrs with 5 μM SIGMAR1 agonists, PRE-084 (+PRE, n = 13), fluvoxamine (+Fluvo, n = 10) or dextromethorphan (+Dxm, n = 11). APD90 is shown in Fig. 1d. (d-f) Action potential parameters, APD50 (d), resting potential (e) and peak amplitude (f) in isogenic control (Ctrl) cardiomyocytes without treatment (n = 10) and treated for 2 hrs with 5 μM SIGMAR1 agonists, Fluvo (n = 10) or Dxm (n = 11). (g) Quantification of human SIGMAR1 transcripts (normalized to GAPDH) in Timothy syndrome (n = 9 from two independent lines) and control cardiomyocytes (n = 12 from four independent lines). (h) Representative immunoblots of human SIGMAR1 and GAPDH protein using lysates from TS and control iPSC-derived cardiomyocytes. (i-j) Quantification of SIGMAR1 25 kDa protein band (i) and 35 kDa protein expression (j, normalized to GAPDH) in TS iPSC-derived cardiomyocytes compared to the isogenic Ctrl (n = 6/group). The molecular weight of SIGMAR1 is ~25 kDa while the 35 kDa band (#) has been reported previously and might be a dimer of a full-length SIGMAR1 with a SIGMAR1 splice variant59. (k) Representative immunoblots of human ATF4 using the same lysates from TS and isogenic Ctrl iPSC-derived cardiomyocytes shown in Extended Data Fig. 2h. (l-n) Quantification of ATF4 38 kDa protein band (l, non-modified), 50 kDa (m, phosphorylated) and 70 kDa protein expression (n, ubiquitinated, normalized to GAPDH) in TS iPSC-derived cardiomyocytes compared to the isogenic Ctrl (n = 6/group). (o-r) ATF4 overexpression (o) significantly increased SIGMAR1 transcription (p) and protein expression (q,r) in normal human cardiomyocytes transfected with ATF4 plasmid (+ ATF4, o&p, n = 7, r, n = 5). The cardiomyocytes were harvested 24 hr after the lipofection. The empty vector was used as a negative control (Mock, o&p, n = 5, r, n = 4). (q) Representative immunoblots of human SIGMAR1 and GAPDH using the lysate from the transfected cardiomyocytes. All data are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons was used for a-f and unpaired two-tailed Student’s t-test was used for g, i, j, l, m, n, o, p, r. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n.s., not significant. Cell samples from at least two independent differentiations were used.
