Simultaneous optical recording of action potentials and calcium transients in cardiac single cells differentiated from type 1 CPVT-iPS cells
Numerous reports investigating channelopathies, including Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), have successfully reproduced using cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs). However, the relationship between action potentials (AP)...
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Frontiers Media S.A.
2025-06-01
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| Series: | Frontiers in Physiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fphys.2025.1579815/full |
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| author | Tadashi Takaki Tadashi Takaki Tadashi Takaki Norihisa Tamura Norihisa Tamura Kenichi Imahashi Kenichi Imahashi Tomoyuki Nishimoto Tomoyuki Nishimoto Yoshinori Yoshida Yoshinori Yoshida |
| author_facet | Tadashi Takaki Tadashi Takaki Tadashi Takaki Norihisa Tamura Norihisa Tamura Kenichi Imahashi Kenichi Imahashi Tomoyuki Nishimoto Tomoyuki Nishimoto Yoshinori Yoshida Yoshinori Yoshida |
| author_sort | Tadashi Takaki |
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| description | Numerous reports investigating channelopathies, including Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), have successfully reproduced using cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs). However, the relationship between action potentials (AP) and calcium transient waveforms—especially after drug treatment—remains unclear. In this study, we simultaneously loaded a membrane potential dye FluoVolt and the new calcium indicator CalbryteTM 590 AM and optimized stimulation and detection of both dyes to successfully obtain a higher signal-to-noise (S/N) ratio than the conventional membrane potential dye-red fluorescence Ca2+ dye combination, thus enabling the simultaneous recording of both AP and calcium transient waveforms in single hiPSC-CMs, which continued even after gradual increases in drug concentration. In drug-loading experiments on CPVT1 (RyR2-I4587V) hiPSC-derived ventricular-like CMs, carvedilol and flecainide demonstrated some effectiveness, while JTV519 at 3 µM exhibited both efficacy and alterations in AP waveforms. The Ca2+/calmodulin-dependent serine-threonine protein kinase II (CaMKII) inhibitor KN-93 at 1 µM was highly effective (93%) at reducing Ca2+ transient abnormalities without altering AP waveforms. |
| format | Article |
| id | doaj-art-8e49054ff7a045dcab58d1da286879f6 |
| institution | DOAJ |
| issn | 1664-042X |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Physiology |
| spelling | doaj-art-8e49054ff7a045dcab58d1da286879f62025-08-20T03:07:41ZengFrontiers Media S.A.Frontiers in Physiology1664-042X2025-06-011610.3389/fphys.2025.15798151579815Simultaneous optical recording of action potentials and calcium transients in cardiac single cells differentiated from type 1 CPVT-iPS cellsTadashi Takaki0Tadashi Takaki1Tadashi Takaki2Norihisa Tamura3Norihisa Tamura4Kenichi Imahashi5Kenichi Imahashi6Tomoyuki Nishimoto7Tomoyuki Nishimoto8Yoshinori Yoshida9Yoshinori Yoshida10Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, JapanCardiomyopathy Project, Takeda-CiRA Joint Program (T-CiRA), Fujisawa, Kanagawa, JapanDepartment of Pancreatic Islet Cell Transplantation, National Institute of Global Health and Medicine, Japan Institute for Health Security, Tokyo, JapanCardiomyopathy Project, Takeda-CiRA Joint Program (T-CiRA), Fujisawa, Kanagawa, JapanT-CiRA Discovery, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, JapanCardiomyopathy Project, Takeda-CiRA Joint Program (T-CiRA), Fujisawa, Kanagawa, JapanT-CiRA Discovery, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, JapanCardiomyopathy Project, Takeda-CiRA Joint Program (T-CiRA), Fujisawa, Kanagawa, JapanT-CiRA Discovery, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, JapanDepartment of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, JapanCardiomyopathy Project, Takeda-CiRA Joint Program (T-CiRA), Fujisawa, Kanagawa, JapanNumerous reports investigating channelopathies, including Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), have successfully reproduced using cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs). However, the relationship between action potentials (AP) and calcium transient waveforms—especially after drug treatment—remains unclear. In this study, we simultaneously loaded a membrane potential dye FluoVolt and the new calcium indicator CalbryteTM 590 AM and optimized stimulation and detection of both dyes to successfully obtain a higher signal-to-noise (S/N) ratio than the conventional membrane potential dye-red fluorescence Ca2+ dye combination, thus enabling the simultaneous recording of both AP and calcium transient waveforms in single hiPSC-CMs, which continued even after gradual increases in drug concentration. In drug-loading experiments on CPVT1 (RyR2-I4587V) hiPSC-derived ventricular-like CMs, carvedilol and flecainide demonstrated some effectiveness, while JTV519 at 3 µM exhibited both efficacy and alterations in AP waveforms. The Ca2+/calmodulin-dependent serine-threonine protein kinase II (CaMKII) inhibitor KN-93 at 1 µM was highly effective (93%) at reducing Ca2+ transient abnormalities without altering AP waveforms.https://www.frontiersin.org/articles/10.3389/fphys.2025.1579815/fullFluoVoltmembrane potential dyeCalbryte 590calcium transientcatecholaminergic polymorphic ventricular tachycardiainduced pluripotent stem cells |
| spellingShingle | Tadashi Takaki Tadashi Takaki Tadashi Takaki Norihisa Tamura Norihisa Tamura Kenichi Imahashi Kenichi Imahashi Tomoyuki Nishimoto Tomoyuki Nishimoto Yoshinori Yoshida Yoshinori Yoshida Simultaneous optical recording of action potentials and calcium transients in cardiac single cells differentiated from type 1 CPVT-iPS cells Frontiers in Physiology FluoVolt membrane potential dye Calbryte 590 calcium transient catecholaminergic polymorphic ventricular tachycardia induced pluripotent stem cells |
| title | Simultaneous optical recording of action potentials and calcium transients in cardiac single cells differentiated from type 1 CPVT-iPS cells |
| title_full | Simultaneous optical recording of action potentials and calcium transients in cardiac single cells differentiated from type 1 CPVT-iPS cells |
| title_fullStr | Simultaneous optical recording of action potentials and calcium transients in cardiac single cells differentiated from type 1 CPVT-iPS cells |
| title_full_unstemmed | Simultaneous optical recording of action potentials and calcium transients in cardiac single cells differentiated from type 1 CPVT-iPS cells |
| title_short | Simultaneous optical recording of action potentials and calcium transients in cardiac single cells differentiated from type 1 CPVT-iPS cells |
| title_sort | simultaneous optical recording of action potentials and calcium transients in cardiac single cells differentiated from type 1 cpvt ips cells |
| topic | FluoVolt membrane potential dye Calbryte 590 calcium transient catecholaminergic polymorphic ventricular tachycardia induced pluripotent stem cells |
| url | https://www.frontiersin.org/articles/10.3389/fphys.2025.1579815/full |
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