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Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome

Nature Cardiovascular Research volume 1, pages 142–156 (2022)Cite this article

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Abstract

Some missense gain-of-function mutations in the CACNA1C gene, encoding calcium channel CaV1.2, cause a life-threatening form of long QT syndrome (LQTS) named Timothy syndrome with currently no clinically effective therapeutics. Here we report that pharmacological targeting of sigma non-opioid intracellular receptor 1 (SIGMAR1) can restore electrophysiological function in induced pluripotent stem cell (iPSC)-derived cardiomyocytes generated from patients with Timothy syndrome and two common forms of LQTS, type 1 (LQTS1) and type 2 (LQTS2), caused by missense trafficking mutations in potassium channels. Electrophysiological recordings demonstrate that a Food and Drug Administration (FDA)-approved cough suppressant, dextromethorphan, can be used as an agonist of SIGMAR1 to shorten the prolonged action potential in cardiomyocytes from patients with Timothy syndrome and human cellular models of LQTS1 and LQTS2. When tested in vivo, dextromethorphan also normalized the prolonged QT intervals in a mouse model of Timothy syndrome. Overall, our study demonstrates that SIGMAR1 is a potential therapeutic target for Timothy syndrome and possibly other inherited arrhythmias such as LQTS1 and LQTS2.

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Fig. 1: The effect of SIGMAR1 agonists on cardiac action potential phenotypes in cardiomyocytes derived from iPSCs of patients with TS.
Fig. 2: The effect of dextromethorphan on cardiac Ca2+ handling and ion channels in a human iPSC model of TS.
Fig. 3: The effect of dextromethorphan on phenotypes in LQTS1.
Fig. 4: The effect of dextromethorphan on phenotypes in LQTS2.
Fig. 5: The effect of dextromethorphan on the mouse model of TS.

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Acknowledgements

We thank J.C. Wu (Stanford University) for sharing iPSC lines from patients with LQTS1 or LQTS2; G. Pitt (Weill Cornell Medicine), R. Katz, A. Rinderspacher, S. Deng, B. Corneo, R. Robinson, H. Colecraft, S. Marx, R. Kass, E. Passagué and J. Stein (Columbia University) and R. Kaye (SSI Strategy) for helpful discussions; and S. Asano (Pfizer) for helpful input about image analysis. This work was supported by NIH K99/R00HL111345, NIH R01HL138486, the Columbia University Core Usage Funding Program and a Helmsley Stem Cell Seed Grant (to M.Y.), NIH R01HL136758 (to J.P.M.), NIH F31HL142239 (to J.R.Q.), a JSPS postdoctoral fellowship (to K.M.) and the Columbia TRANSFORM TL1 training program (to R.B. and D.A.).

Author information

Author notes

  1. These authors contributed equally: LouJin Song, Ramsey Bekdash, Kumi Morikawa, Jose R. Quejada.

Authors and Affiliations

  1. Columbia Stem Cell Initiative, Department of Rehabilitation and Regenerative Medicine, Columbia University, New York, NY, USA

    LouJin Song, Ramsey Bekdash, Kumi Morikawa, Jose R. Quejada, Alison D. Klein, Danielle Aina-Badejo, Kazushige Yoshida, Hannah E. Yamamoto, Amy Chalan, Risako Yang, Achchhe Patel, Dario Sirabella, Teresa M. Lee, Fuun Kawano & Masayuki Yazawa

  2. Department of Pharmacology, Columbia University, New York, NY, USA

    LouJin Song, Ramsey Bekdash, Jose R. Quejada & Masayuki Yazawa

  3. Barnard College, New York, NY, USA

    Hannah E. Yamamoto

  4. Colgate University, Hamilton, NY, USA

    Risako Yang

  5. Department of Genetics and Development, Columbia University, New York, NY, USA

    Achchhe Patel & Dario Sirabella

  6. Department of Pediatrics, Columbia University, New York, NY, USA

    Teresa M. Lee

  7. Department of Medicine, Columbia University, New York, NY, USA

    Leroy C. Joseph & John P. Morrow

  8. Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, UT, USA

    Junco S. Warren

  9. Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT, USA

    Junco S. Warren

  10. Division of Developmental Genetics, Institute of Resource Developmental and Analysis, Kumamoto University, Kumamoto, Japan

    Junco S. Warren

  11. Proteomics and Macromolecular Crystallography Shared Resource, Columbia University, New York, NY, USA

    Rajesh K. Soni

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Contributions

L.S. and M.Y. conceived of and designed this project. L.S., R.B., K.M., J.R.Q. and M.Y. designed experiments. L.S., R.B., K.M., J.R.Q., A.D.K., D.A., K.Y., H.E.Y., A.C., R.Y., A.P., D.S., L.C.J., T.M.L., F.K., R.K.S., J.P.M. and M.Y. performed experiments and analyzed data: M.Y. conducted whole-cell patch clamping; R.K.S. conducted proteomics; F.K. conducted computational modeling. L.S., R.B., K.M., J.R.Q., J.S.W. and M.Y. interpreted data. M.Y. wrote the manuscript. L.S., R.B., K.M., J.R.Q., D.A., T.M.L. and J.P.M. proofread and edited the manuscript. All authors approved the manuscript.

Corresponding author

Correspondence to Masayuki Yazawa.

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Competing interests

L.S., R.B., K.M., J.R.Q. and M.Y. (inventors) filed a patent (attorney docket no. 01001/005273-US1; status, filed, 4 October 2019) related to this manuscript. This patent is for using SIGMAR1 agonists to treat LQTS (types 1, 2 and 8) and related cardiac channelopathy. The remaining authors declare no competing interests.

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Extended data

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.

Source data

Extended Data Fig. 2 The effect of SIGMAR1 agonists on action potential and SIGMAR1 and ATF4 expression profiling in Timothy syndrome iPSC-derived cardiomyocytes.

(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.

Source data

Extended Data Fig. 3 The effect of dextromethorphan on cardiac calcium and hERG channels in Timothy syndrome iPSC-derived cardiomyocytes.

(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.

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Extended Data Fig. 4 The effect of dextromethorphan on potassium channels in human cardiomyocytes derived from isogenic control iPSCs.

(a) Representative traces of IKr current (E-4031-sensitive) in isogenic control (Ctrl) cardiomyocytes treated with Dxm (5 μM, 2 hr) or without treatment. (b-c) IKr current amplitude (b) and tail IKr current (c) were not significantly changed by Dxm in the isogenic Ctrl cardiomyocytes (n = 10/group). (d) Schematic representation of confocal imaging for proximity ligation assay (PLA, SIGMAR1-hERG, red) in human iPSC-derived cardiomyocytes. (e) Representative confocal fluorescent images of TS cardiomyocytes used for PLA (SIGMAR1-hERG, red) and DAPI (blue) staining. Dxm treatment (+Dxm, 5 μM, 2 hr) was conducted to examine the effect of Dxm on PLA signals in TS cardiomyocytes. #3 images are used in Fig. 2j. Scale bar, 10μm. (f) Representative epi-fluorescent and phase-contrast images of isogenic Ctrl cardiomyocytes used for PLA (red, SIGMAR1-hERG) and DAPI (blue) staining. Dxm treatment (+Dxm, 5 μM, 2 hr) was conducted to examine the effect of Dxm on PLA signals in the cardiomyocytes. Scale bar, 10μm. (g) Quantification of PLA signal number of SIGMAR1-hERG in Dxm-treated (n = 21) and non-treated (n = 21) isogenic Ctrl cardiomyocytes. (h) Representative traces of IKs current (Chromanol 293B-sensitive) in isogenic control (Ctrl) cardiomyocytes treated with Dxm or without treatment. (i) IKs current amplitude analysis in the isogenic Ctrl cardiomyocytes with Dxm (5 μM, 2 hr, n = 9) or without treatment (n = 10). (j) Representative epi-fluorescent and phase-contrast images of isogenic Ctrl cardiomyocytes used for PLA (red, SIGMAR1-KV7.1) and DAPI (blue) staining. Dxm treatment (+Dxm, 5 μM, 2 hr) was conducted to examine the effect of Dxm on PLA signals in the cardiomyocytes. Scale bar, 10μm. (k) Quantification of PLA signal number of SIGMAR1-KV7.1 in Dxm-treated (n = 40) and non-treated (n = 44) isogenic Ctrl cardiomyocytes. All data are mean ± s.d. Unpaired two-tailed Student t-test was used for g,k between the groups, and for b,c,i at each voltage step. * P < 0.05. n.s., not significant. The cell samples were from at least two independent differentiations.

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Extended Data Fig. 5 The effect of dextromethorphan on potassium channels in Timothy syndrome iPSC-derived cardiomyocytes.

(a) Representative confocal fluorescent images of Timothy syndrome cardiomyocytes without treatment (left, TS) and with dextromethorphan (+Dxm). Scale bar, 5μm. (b) Quantification of fraction of green (hERG) over red (WGA) fluorescence signals in TS cardiomyocytes without treatment (n = 38) and with Dxm (n = 31). (c) Quantification of KCNH2 transcript in TS cardiomyocytes with and without Dxm (n = 4/group). (d) Representative immunoblots of human hERG and GAPDH protein using lysates from non-treated and Dxm-treated TS cardiomyocytes. (e) Quantification of hERG protein expressions (normalized to GAPDH) in non-treated and Dxm-treated TS cardiomyocytes (n = 3/group). The trend towards a decreased hERG protein expression in the treated group might be driven by one sample (#) with a reduced loading amount compared with others. (f) IKs current steady-state amplitudes were significantly reduced in TS cardiomyocytes (n = 10) compared to isogenic control (n = 10) at 20 and 50 mV steps from -40mV hold. (g) Representative traces of IKs currents (Chromanol 293B-sensitive) in isogenic Ctrl cardiomyocytes (black) and TS cardiomyocytes treated with Dxm (purple), Dxm and NE-100 (green) or without treatment (red, TS). The IKs traces are shown in Fig. 2l. (h) Representative confocal fluorescent images of Timothy syndrome cardiomyocyte without treatment (left, TS) and treated with dextromethorphan (+Dxm). Scale bar, 5μm. (i) Quantification of fraction of green (KV7.1) over red (WGA) fluorescence signals in TS cardiomyocytes without treatment (n = 21) and treated with Dxm (n = 31). (j) Quantification of KCNQ1 transcripts (isoform 1 and 2) in TS cardiomyocytes without treatment and treated with Dxm (n = 4/group). (k) Representative immunoblots of human KV7.1 and GAPDH protein using lysates from non-treated and Dxm-treated TS cardiomyocytes. (l-m) Quantification of KV7.1 isoform 1 (l) and isoform 2 protein expressions (m, normalized to GAPDH) in non-treated and Dxm-treated TS cardiomyocytes (n = 3/group). All data are mean ± s.d. The treatment of Dxm or NE-100 for all experiments was 5 μM, 2hrs. Unpaired two-tailed Student’s t-test was used for b, c, e, i, j, l, m between the groups and used for f at each voltage step. * P < 0.05. n.s., not significant. The cell samples were from at least two independent differentiations.

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Extended Data Fig. 6 Generation of isogenic controls from long QT syndrome type 1 and 2 iPSCs.

(a-b) Sequencing results confirmed the successful generation of isogenic control (Ctrl) iPSC lines from long QT syndrome type 1 (a) and type 2 (b). (c) Characterization of the isogenic Ctrl iPSC lines (top, phase contrast image; middle, teratoma formation assay). Scale bar, 200μm. The isogenic Ctrl iPSC demonstrated normal karyotype (bottom). (d) Representative images of TRA-1-60 (red)/TRA-1-81 (green)/Hoechst (nuclei, blue) stain in isogenic Ctrl iPSC lines. Scale bar, 20μm. (e) Representative traces of 0.5Hz-paced action potentials in LQTS1 patient-specific isogenic Ctrl iPSC-derived cardiomyocytes without treatment or treated with Dxm. (f-i) Analysis of action potential parameters, APD90 (f), APD50 (g), resting potential (h) and peak amplitude (i), in isogenic Ctrl cardiomyocytes without treatment (n = 11) or treated with Dxm (n = 10). (j) Representative traces of IKs currents (Chromanol-293B-sensitive) in long QT syndrome type 1 (LQTS1) patient-specific cardiomyocytes and the isogenic Ctrl cardiomyocytes. (k) IKs current steady-state amplitudes were significantly reduced in LQTS1 cardiomyocytes (n = 10) compared to the isogenic control (n = 11) at all voltage steps from -40mV hold. (l) Representative traces of 0.5Hz-paced action potentials in long QT syndrome type 2 (LQTS2) patient-specific isogenic Ctrl iPSC-derived cardiomyocytes without treatment or treated with Dxm. (m-p) analysis of action potential parameters, APD90 (m), APD50 (n), resting potential (o) and peak amplitude (p), in the isogenic Ctrl cardiomyocytes without treatment (n = 13) or treated with Dxm (n = 15). (q) Representative traces of IKr currents (E4031-sensitive) in LQTS2 patient-specific cardiomyocytes and the isogenic Ctrl cardiomyocytes. (r) IKr current steady-state amplitudes were significantly reduced in LQTS2 cardiomyocytes (n = 10) compared to the isogenic control (n = 12) at 10 and 20 mV steps from -40mV hold. (s) IKr tail currents were significantly reduced in LQTS2 cardiomyocytes compared to the isogenic control at 10, 20, 30, 40 and 50 mV steps from -40mV hold. All data are mean ± s.d. The treatment of Dxm for the experiments was 5 μM, 2hrs. Unpaired two-tailed Student’s t-test were used for f, g, h, i, m, n, o, p between the groups and for k, r, s at each voltage step. * P < 0.05, ** P < 0.01, n.s., not significant. The cardiomyocytes were from at least two independent differentiations.

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Extended Data Fig. 7 The effect of dextromethorphan on the phenotypes in long QT syndrome type 1 and type 2.

(a-f) Analysis of APD50 (50% from peak, a, b), peak amplitude (c, d) and resting potential (e, f) in LQTS1 cardiomyocytes without treatment (n = 11) or treated with fluvoxamine (Fluvo, n = 10) or dextromethorphan (Dxm, n = 10) and in LQTS2 cardiomyocytes without treatment (n = 11) or treated with Fluvo (n = 10) or Dxm (n = 11). (g,h) IKr current (tail) was significantly increased by Dxm in LQTS1 cardiomyocytes at 40 mV step (g, LQTS1, n = 10; +Dxm, n = 9) and in LQTS2 cardiomyocytes at 10, 30 and 40 mV steps (h, LQTS2, n = 10; +Dxm, n = 9). (i,j) Representative traces of Ba2+ currents in LQTS1 (i) or LQTS2 cardiomyocytes(j) without treatment or treated with Dxm. (k,l) Voltage-dependent calcium channel inactivation was not significantly changed by Dxm in LQTS1(k) or LQTS2 cardiomyocytes (l) compared to non-treated cells (n = 10/group). (m,n) Current-voltage relationship of Ba2+ recordings in Dxm-treated (n = 10) and non-treated LQTS1 cardiomyocytes (n = 7)(m) and in Dxm-treated (n = 7) and non-treated LQTS2 cardiomyocytes (n = 8)(n). (o,p) Representative immunoblots of human SIGMAR1, ATF4, CDK5, CDK5R1/p35 and GAPDH protein using lysates from control (Ctrl) cardiomyocytes, LQTS1 cardiomyocytes (o) and LQTS2 cardiomyocytes (p). ^, p35 antibody used for Extended Data Fig. 1c was discontinued (Santa Cruz, sc-820). Therefore, another antibody (Cell Signaling Technology, C64B10) was used for this blotting series while its signal-to-noise ratio was not as high as sc-820. (q-s) Quantification of SIGMAR1 25 kDa protein band (q, r) and #35 kDa protein band (s, possibly a dimer of a full-length SIGMAR1 with a SIGMAR1 splice variant59) in LQTS1 or LQTS2 cardiomyocytes compared to Ctrl (n = 4/group). (t,u) Quantification of CDK5 (left) and CDK5R1/p35 protein expressions (right) in LQTS1 or LQTS2 cardiomyocytes compared to control cardiomyocytes (n = 4/group). (v,w) Quantification of ATF4 38 kDa protein band (left, non-modified), 50 kDa (center, phosphorylated) and 70 kDa protein expression (right, ubiquitinated) in LQTS1 or LQTS2 cardiomyocytes compared to control cardiomyocytes (n = 4/group). All data are mean ± s.d. The treatment of Dxm or Fluvo for the experiments was 5 μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for a,b,c,d,e,f. Unpaired two-tailed Student’s t-test was used for k,l,q,r,s,t,u,v,w between the groups and for g,h,m,n at each voltage step. * P < 0.05, ** P < 0.01, n.s., not significant. The cardiomyocytes were from at least two independent differentiations.

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Extended Data Fig. 8 The effect of dextromethorphan on Timothy syndrome mouse model.

(a) Plasma dextromethorphan (Dxm) concentrations in control mice at day 4 and day 11 after continuous dosing of Dxm in drinking water (n = 13, mean ± s.e.m.). Dxm was provided in water since day 1 at 8am. * 8 p.m. time point at day 4 had an outlier defined by statistics and the outlier was excluded from the final analysis. (b) Representative analysis procedures for ECG traces in Timothy syndrome (TS) mice without treatment or treated with Dxm at day 4 (also used in Fig. 5c,d). (c) QT intervals were significantly shortened by Dxm treatment in TS mice at day 11 (n = 8 for ctrl, n = 4 for TS, n = 7 for TS + Dxm, female, 12-week old). Three mouse heart samples from each group were used for the global proteomics analysis. All data of Extended Data Fig. 8 are mean ± s.d. except Extended Data Fig. 8a. One-way ANOVA with Tukey’s multiple comparisons were used for statistics. ** P < 0.01, n.s., not significant.

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Extended Data Fig. 9 The effect of dextromethorphan on the protein expression in mouse hearts.

Altered protein expressions between control hearts (Ctrl), non-treated (TS) and Dxm-treated TS hearts (TS + Dxm) (n = 3/group). A comprehensive heat map and summary of proteomics results are shown in Fig. 5e. All data are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons was used. * P < 0.05, ** P < 0.01, n.s., not significant.

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Extended Data Fig. 10 The effect of dextromethorphan on KV7.1 protein expression in mouse hearts.

(a) Representative immunoblots of KV7.1, β-tubulin and Gapdh protein using mouse heart lysates from TS (non-treated and Dxm-treated) mice and the control littermates (Ctrl) at day 11 (each, n = 3, using the same samples with the global proteomics shown in Fig. 5e). #, bands indicate putative degradation of KV7.1 in TS mouse hearts. (b-c) Quantification of β-tubulin (b) and Kcnq1/KV7.1 protein expression (c, normalized to Gapdh) in TS (non-treated and Dxm-treated) and Ctrl mouse hearts (n = 3/group). Altered β-tubulin expression has been reported previously in animal model of myocardial infarction and Gapdh was recommended as a more reliable loading control for Western blot analyses60. (d) Kcnq1/KV7.1 mRNA expression (normalized to Gapdh) in TS (non-treated and Dxm-treated) and Ctrl mouse hearts (n = 3/group). All data are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons was used. ** P < 0.01, n.s., not significant.

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Supplementary information

Supplementary Information

Supplementary Fig. 1

Reporting Summary

Supplementary Video 1

Representative time-lapse phase-contrast images of cardiomyocyte clusters derived from iPSCs of patients with TS before treatment with 5 μM PRE-084. Analyses are shown in Extended Data Fig. 1h–j. Red (bottom left), a marker used for this cluster area.

Supplementary Video 2

Representative time-lapse phase-contrast images of cardiomyocyte clusters derived from iPSCs of patients with TS after 2 h of treatment with 5 μM PRE-084. Analyses are shown in Extended Data Fig. 1h–j. Red (bottom left), a marker used for this cluster area.

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Data including the global proteomic dataset (Fig. 5e).

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Song, L., Bekdash, R., Morikawa, K. et al. Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome. Nat Cardiovasc Res 1, 142–156 (2022). https://doi.org/10.1038/s44161-021-00016-2

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