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Eag Domains Regulate LQT Mutant hERG Channels in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Liu QN, Trudeau MC - PLoS ONE (2015)

Bottom Line: In previous studies, we showed that isolated eag (i-eag) domains rescued the dysfunction of long QT type-2 associated mutant hERG R56Q channels, by substituting for defective eag domains, when the channels were expressed in Xenopus oocytes or HEK 293 cells.Here, our goal was to determine whether the rescue of hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes.We expressed hERG R56Q channels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and measured electrical properties of the cells with whole-cell patch-clamp recordings.We found that, like in non-myocyte cells, hERG R56Q had defective, fast closing (deactivation) kinetics when expressed in hiPSC-CMs. We report here that i-eag domains slowed the deactivation kinetics of hERG R56Q channels in hiPSC-CMs. hERG R56Q channels prolonged the AP of hiPSCs, and the AP was shortened by co-expression of i-eag domains and hERG R56Q channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Human Ether á go-go Related Gene potassium channels form the rapid component of the delayed-rectifier (IKr) current in the heart. The N-terminal 'eag' domain, which is composed of a Per-Arnt-Sim (PAS) domain and a short PAS-cap region, is a critical regulator of hERG channel function. In previous studies, we showed that isolated eag (i-eag) domains rescued the dysfunction of long QT type-2 associated mutant hERG R56Q channels, by substituting for defective eag domains, when the channels were expressed in Xenopus oocytes or HEK 293 cells.Here, our goal was to determine whether the rescue of hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes. We expressed hERG R56Q channels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and measured electrical properties of the cells with whole-cell patch-clamp recordings. We found that, like in non-myocyte cells, hERG R56Q had defective, fast closing (deactivation) kinetics when expressed in hiPSC-CMs. We report here that i-eag domains slowed the deactivation kinetics of hERG R56Q channels in hiPSC-CMs. hERG R56Q channels prolonged the AP of hiPSCs, and the AP was shortened by co-expression of i-eag domains and hERG R56Q channels. We measured robust Förster Resonance Energy Transfer (FRET) between i-eag domains tagged with Cyan fluorescent protein (CFP) and hERG R56Q channels tagged with Citrine fluorescent proteins (Citrine), indicating their close proximity at the cell membrane in live iPSC-CMs. Together, functional regulation and FRET spectroscopy measurements indicated that i-eag domains interacted directly with hERG R56Q channels in hiPSC-CMs. These results mean that the regulatory role of i-eag domains is conserved in the cellular environment of human cardiomyocytes, indicating that i-eag domains may be useful as a biological therapeutic.

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Adenoviral i-eag domains rescue the gating-deficiencies of hERG1a(R56Q) channels expressed in HEK293 cells.A, Schematic depicting hERG channel subunit: a, WT hERG1a; b, hERG1a(R56Q); c, hERG1a(R56Q) + i-eag. B, C, D, Representative current recordings from HEK293 cells expressing: WT hERG1a.Ad, hERG1a(R56Q).Ad, hERG1a(R56Q).Ad + i-eag.Ad. Inset represents the voltage protocol used. E, IV relationships of WT hERG1a, hERG1a(R56Q), and hERG1a(R56Q).Ad + i-eag.Ad. The current at the end of the depolarizing step was normalized to the maximum tail current and plotted versus voltage. F, Representative steady-state inactivation current recording from WT hERG1a (top) using a three-pulse protocol (bottom). G, Steady-state activation and inactivation curves. Steady-state activation curves were generated by normalizing tail currents at -50 mV to the maximum tail current and plotted versus voltage. Steady-state inactivation curves were generated by normalizing the peak current and plotted versus voltage. Both steady-state activation and inactivation curves were fit with a Boltzmann function. All values were plotted as mean ± S.E.M; n = 5–13 cells.
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pone.0123951.g001: Adenoviral i-eag domains rescue the gating-deficiencies of hERG1a(R56Q) channels expressed in HEK293 cells.A, Schematic depicting hERG channel subunit: a, WT hERG1a; b, hERG1a(R56Q); c, hERG1a(R56Q) + i-eag. B, C, D, Representative current recordings from HEK293 cells expressing: WT hERG1a.Ad, hERG1a(R56Q).Ad, hERG1a(R56Q).Ad + i-eag.Ad. Inset represents the voltage protocol used. E, IV relationships of WT hERG1a, hERG1a(R56Q), and hERG1a(R56Q).Ad + i-eag.Ad. The current at the end of the depolarizing step was normalized to the maximum tail current and plotted versus voltage. F, Representative steady-state inactivation current recording from WT hERG1a (top) using a three-pulse protocol (bottom). G, Steady-state activation and inactivation curves. Steady-state activation curves were generated by normalizing tail currents at -50 mV to the maximum tail current and plotted versus voltage. Steady-state inactivation curves were generated by normalizing the peak current and plotted versus voltage. Both steady-state activation and inactivation curves were fit with a Boltzmann function. All values were plotted as mean ± S.E.M; n = 5–13 cells.

Mentions: In this study, our goal was to test whether the genetically encoded, isolated eag (i-eag) domains could rescue the function of a hERG LQTS mutant channel in the environment of cardiac myocytes. We chose to use hiPSC-CMs because they are human cells, can be cultured for several weeks (allowing for the expression of recombinant genes) and have some properties of cardiomyocytes, such as APs. We found that transfection of hERG channels in mammalian expression vectors (e.g. pcDNA3) was not effective for hiPSC-CMs, so to carry out these experiments, we first performed a positive control by testing the expression of adenovirally delivered wild-type hERG, hERG(R56Q) and hERG(R56Q) + i-eag domains (Fig 1A) in HEK293 cells.


Eag Domains Regulate LQT Mutant hERG Channels in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Liu QN, Trudeau MC - PLoS ONE (2015)

Adenoviral i-eag domains rescue the gating-deficiencies of hERG1a(R56Q) channels expressed in HEK293 cells.A, Schematic depicting hERG channel subunit: a, WT hERG1a; b, hERG1a(R56Q); c, hERG1a(R56Q) + i-eag. B, C, D, Representative current recordings from HEK293 cells expressing: WT hERG1a.Ad, hERG1a(R56Q).Ad, hERG1a(R56Q).Ad + i-eag.Ad. Inset represents the voltage protocol used. E, IV relationships of WT hERG1a, hERG1a(R56Q), and hERG1a(R56Q).Ad + i-eag.Ad. The current at the end of the depolarizing step was normalized to the maximum tail current and plotted versus voltage. F, Representative steady-state inactivation current recording from WT hERG1a (top) using a three-pulse protocol (bottom). G, Steady-state activation and inactivation curves. Steady-state activation curves were generated by normalizing tail currents at -50 mV to the maximum tail current and plotted versus voltage. Steady-state inactivation curves were generated by normalizing the peak current and plotted versus voltage. Both steady-state activation and inactivation curves were fit with a Boltzmann function. All values were plotted as mean ± S.E.M; n = 5–13 cells.
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Related In: Results  -  Collection

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pone.0123951.g001: Adenoviral i-eag domains rescue the gating-deficiencies of hERG1a(R56Q) channels expressed in HEK293 cells.A, Schematic depicting hERG channel subunit: a, WT hERG1a; b, hERG1a(R56Q); c, hERG1a(R56Q) + i-eag. B, C, D, Representative current recordings from HEK293 cells expressing: WT hERG1a.Ad, hERG1a(R56Q).Ad, hERG1a(R56Q).Ad + i-eag.Ad. Inset represents the voltage protocol used. E, IV relationships of WT hERG1a, hERG1a(R56Q), and hERG1a(R56Q).Ad + i-eag.Ad. The current at the end of the depolarizing step was normalized to the maximum tail current and plotted versus voltage. F, Representative steady-state inactivation current recording from WT hERG1a (top) using a three-pulse protocol (bottom). G, Steady-state activation and inactivation curves. Steady-state activation curves were generated by normalizing tail currents at -50 mV to the maximum tail current and plotted versus voltage. Steady-state inactivation curves were generated by normalizing the peak current and plotted versus voltage. Both steady-state activation and inactivation curves were fit with a Boltzmann function. All values were plotted as mean ± S.E.M; n = 5–13 cells.
Mentions: In this study, our goal was to test whether the genetically encoded, isolated eag (i-eag) domains could rescue the function of a hERG LQTS mutant channel in the environment of cardiac myocytes. We chose to use hiPSC-CMs because they are human cells, can be cultured for several weeks (allowing for the expression of recombinant genes) and have some properties of cardiomyocytes, such as APs. We found that transfection of hERG channels in mammalian expression vectors (e.g. pcDNA3) was not effective for hiPSC-CMs, so to carry out these experiments, we first performed a positive control by testing the expression of adenovirally delivered wild-type hERG, hERG(R56Q) and hERG(R56Q) + i-eag domains (Fig 1A) in HEK293 cells.

Bottom Line: In previous studies, we showed that isolated eag (i-eag) domains rescued the dysfunction of long QT type-2 associated mutant hERG R56Q channels, by substituting for defective eag domains, when the channels were expressed in Xenopus oocytes or HEK 293 cells.Here, our goal was to determine whether the rescue of hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes.We expressed hERG R56Q channels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and measured electrical properties of the cells with whole-cell patch-clamp recordings.We found that, like in non-myocyte cells, hERG R56Q had defective, fast closing (deactivation) kinetics when expressed in hiPSC-CMs. We report here that i-eag domains slowed the deactivation kinetics of hERG R56Q channels in hiPSC-CMs. hERG R56Q channels prolonged the AP of hiPSCs, and the AP was shortened by co-expression of i-eag domains and hERG R56Q channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Human Ether á go-go Related Gene potassium channels form the rapid component of the delayed-rectifier (IKr) current in the heart. The N-terminal 'eag' domain, which is composed of a Per-Arnt-Sim (PAS) domain and a short PAS-cap region, is a critical regulator of hERG channel function. In previous studies, we showed that isolated eag (i-eag) domains rescued the dysfunction of long QT type-2 associated mutant hERG R56Q channels, by substituting for defective eag domains, when the channels were expressed in Xenopus oocytes or HEK 293 cells.Here, our goal was to determine whether the rescue of hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes. We expressed hERG R56Q channels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and measured electrical properties of the cells with whole-cell patch-clamp recordings. We found that, like in non-myocyte cells, hERG R56Q had defective, fast closing (deactivation) kinetics when expressed in hiPSC-CMs. We report here that i-eag domains slowed the deactivation kinetics of hERG R56Q channels in hiPSC-CMs. hERG R56Q channels prolonged the AP of hiPSCs, and the AP was shortened by co-expression of i-eag domains and hERG R56Q channels. We measured robust Förster Resonance Energy Transfer (FRET) between i-eag domains tagged with Cyan fluorescent protein (CFP) and hERG R56Q channels tagged with Citrine fluorescent proteins (Citrine), indicating their close proximity at the cell membrane in live iPSC-CMs. Together, functional regulation and FRET spectroscopy measurements indicated that i-eag domains interacted directly with hERG R56Q channels in hiPSC-CMs. These results mean that the regulatory role of i-eag domains is conserved in the cellular environment of human cardiomyocytes, indicating that i-eag domains may be useful as a biological therapeutic.

Show MeSH