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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 restored slow deactivation in hiPSC-CMs.Representative tail currents recorded from hiPSC-CMs infected by: A, WT hERG1a.Ad; B, hERG1a(R56Q).Ad; C, hERG1a(R56Q).Ad + i-eag.Ad. Inset represents the voltage protocol used. D, Tails were fit with a double exponential function, and mean τfast (top) and τslow (bottom) values were plotted against voltage on a logarithmic scale. All values were plotted as mean ± S.E.M.; n> = 15 cells.
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pone.0123951.g005: Adenoviral i-eag domains restored slow deactivation in hiPSC-CMs.Representative tail currents recorded from hiPSC-CMs infected by: A, WT hERG1a.Ad; B, hERG1a(R56Q).Ad; C, hERG1a(R56Q).Ad + i-eag.Ad. Inset represents the voltage protocol used. D, Tails were fit with a double exponential function, and mean τfast (top) and τslow (bottom) values were plotted against voltage on a logarithmic scale. All values were plotted as mean ± S.E.M.; n> = 15 cells.

Mentions: We characterized the altered deactivation of hERG R56Q channels and examined the rescue of adenoviral i-eag domains in more detail, using a voltage command protocol to elicit a series of deactivating traces. Compared with that of hERG.Ad, hERG(R56Q).Ad channels exhibited accelerated deactivation kinetics (Fig 5A, 5B, 5D and 5E). Co-expression of adenoviral i-eag restored the slow deactivation in hERG R56Q channels over a range of voltages (Fig 5C, 5D and 5E; Table 2). These data suggested that hERG R56Q defective channel gating (faster deactivation) was maintained in hiPSC-CMs, as in HEK293 cells and oocytes, and the adenoviral i-eag domains rescued the aberrant deactivation kinetics.


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 restored slow deactivation in hiPSC-CMs.Representative tail currents recorded from hiPSC-CMs infected by: A, WT hERG1a.Ad; B, hERG1a(R56Q).Ad; C, hERG1a(R56Q).Ad + i-eag.Ad. Inset represents the voltage protocol used. D, Tails were fit with a double exponential function, and mean τfast (top) and τslow (bottom) values were plotted against voltage on a logarithmic scale. All values were plotted as mean ± S.E.M.; n> = 15 cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414485&req=5

pone.0123951.g005: Adenoviral i-eag domains restored slow deactivation in hiPSC-CMs.Representative tail currents recorded from hiPSC-CMs infected by: A, WT hERG1a.Ad; B, hERG1a(R56Q).Ad; C, hERG1a(R56Q).Ad + i-eag.Ad. Inset represents the voltage protocol used. D, Tails were fit with a double exponential function, and mean τfast (top) and τslow (bottom) values were plotted against voltage on a logarithmic scale. All values were plotted as mean ± S.E.M.; n> = 15 cells.
Mentions: We characterized the altered deactivation of hERG R56Q channels and examined the rescue of adenoviral i-eag domains in more detail, using a voltage command protocol to elicit a series of deactivating traces. Compared with that of hERG.Ad, hERG(R56Q).Ad channels exhibited accelerated deactivation kinetics (Fig 5A, 5B, 5D and 5E). Co-expression of adenoviral i-eag restored the slow deactivation in hERG R56Q channels over a range of voltages (Fig 5C, 5D and 5E; Table 2). These data suggested that hERG R56Q defective channel gating (faster deactivation) was maintained in hiPSC-CMs, as in HEK293 cells and oocytes, and the adenoviral i-eag domains rescued the aberrant deactivation kinetics.

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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