Pentamidine-Induced Long QT Syndrome and Block of hERG Trafficking

Kuryshev, Yuri A.; Ficker, Eckhard; Wang, Lu; Hawryluk, Peter; Dennis, Adrienne T.; Wible, Barbara A.; Brown, Arthur M.; Kang, Jiesheng; Chen, Xiao Liang; Sawamura, Kaoru; Reynolds, William; Rampe, David

doi:10.1124/jpet.104.073692

Cited by 251 publications

(188 citation statements)

References 29 publications

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“…Pentamidin decreases the Ikr channel expression on the cell membrane due to the inhibition of the Ikr channel trafficking. 15) Pentamidin decreased the beat counts after eighteen or more hours of treatment in the present study. These results demonstrated that the beating EBs had the structural and functional properties of myocardium.…”

Section: Identification Of Beating Ebs As Myocardiumsupporting

confidence: 51%

A Simple Protocol for the Myocardial Differentiation of Human iPS Cells

Aikawa

Suzuki

Takaba

2015

Biological & Pharmaceutical Bulletin

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We have developed a simple protocol for inducing the myocardial differentiation of human induced pluripotent stem (iPS) cells. Human iPS cell-derived embryonic bodies (EBs) were treated with a combination of activin-A, bone morphogenetic protein-4 and wnt-3a for one day in serum-free suspension culture, and were subsequently treated with noggin for three days. Thereafter, the EBs were subjected to adherent culture in media with 5% serum. All EBs were differentiated into spontaneously beating EBs, which were identified by the presence of striated muscles in transmission electron microscopy and the expression of the specific cardiomyocyte markers, NKX2-5 and TNNT2. The beating rate of the beating EBs was decreased by treatment with a rapidly activating delayed rectifier potassium current (Ikr) channel blocker, E-4031, an Ikr trafficking inhibitor, pentamidin, and a slowly activating delayed rectifier potassium current (Iks) channel blocker, chromanol 293B, and was increased by treatment with a beta-receptor agonist, isoproterenol. At a low concentration, verapamil, a calcium channel blocker, increased the beating rate of the beating EBs, while a high concentration decreased this rate. These findings suggest that the spontaneously beating EBs were myocardial cell clusters. This simple protocol for myocardial differentiation would be useful in providing a sufficient number of the beating myocardial cell clusters for studies requiring human myocardium. Key words human induced pluripotent stem cell; myocardial differentiation; embryonic bodyThe cardiotoxicity of drug candidates is one of the crucial findings that limits drug development. A large number of pharmaceutical companies hope to evaluate the risk of cardiotoxicity of drug candidates using human cardiomyocytes in the early stage of development. We have been trying to examine the embryotoxicity of drug candidates in vitro using human induced pluripotent stem (iPS) cells. A simple and highly efficient protocol for inducing the myocardial differentiation of human iPS cells using biological substances has been strongly desired, especially a system using the embryonic body (EB, spheroids of iPS cells) method.Primary human myocardium is difficult to obtain commercially because the cells do not proliferate in culture. Embryonic stem (ES) cells and iPS cells from humans are able to differentiate into myocardium, but the mechanisms are different from those required for ES and iPS cells of mice, and the induction has generally been inefficient. The myocardium is differentiated from the mesoderm, one of the three germ layers generated during the differentiation of pluripotent stem cells. Bone morphogenetic protein-4 (BMP-4), activin-A and wnt-3a are mesoderm inducers, and play a crucial role in its development.

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Section: Identification Of Beating Ebs As Myocardiumsupporting

confidence: 51%

A Simple Protocol for the Myocardial Differentiation of Human iPS Cells

Aikawa

Suzuki

Takaba

2015

Biological & Pharmaceutical Bulletin

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“…It is possible that in addition to Y652 and F656, other amino-acid residues may be molecular determinants of drug binding. Several drugs, including pentamidine [21] , fluoxetine [25] , celastrol [26] , escitalopram [27] , citalopram [27] , and desipramine [28] , are known to inhibit hERG indirectly. This inhibition, which occurs by the disruption of hERG channel protein trafficking to the plasma membrane, reduces the cell surface hERG channel density.…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies reported that some drugs such as fluconazole [15] , cardiac glycosides [20] , and pentamidine [21] are able to reduce hERG surface expression by interrupting protein trafficking to the cell membrane. Therefore, we studied the effect of ATV on hERG channel protein trafficking.…”

Section: Disruption Of Herg Protein Traffickingmentioning

confidence: 99%

The protease inhibitor atazanavir blocks hERG K+ channels expressed in HEK293 cells and obstructs hERG protein transport to cell membrane

et al. 2015

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Aim: Atazanavir (ATV) is a HIV-1 protease inhibitor for the treatment of AIDS patients, which is recently reported to provoke excessive prolongation of the QT interval and torsades de pointes (TdP). In order to elucidate its arrhythmogenic mechanisms, we investigated the effects of ATV on the hERG K + channels expressed in HEK293 cells. Methods: hERG K + currents were detected using whole-cell patch clamp recording in HEK293 cells transfected with EGFP-hERG plasmids. The expression of hERG protein was measured with Western blotting. Two mutants (Y652A and F656C) were constructed in the S6 domain within the inner helices of hERG K + channels that were responsible for binding of various drugs. The trafficking of hERG protein was studied with confocal microscopy. Results: Application of ATV (0.01-30 μmol/L) concentration-dependently decreased hERG K + currents with an IC 50 of 5.7±1.8 μmol/L. ATV (10 μmol/L) did not affect the activation and steady-state inactivation of hERG K + currents. Compared with the wild type hERG K + channels, both Y652A and F656C mutants significantly reduced the inhibition of ATV on hERG K + currents. Overnight treatment with ATV (0.1-30 μmol/L) concentration-dependently reduced the amount of fully glycosylated 155 kDa hERG protein without significantly affecting the core-glycosylated 135 kDa hERG protein in the cells expressing the WT-hERG protein. Confocal microscopy studies confirmed that overnight treatment with ATV obstructed the trafficking of hERG protein to the cell membrane. Conclusion: ATV directly blocks hERG K + channels via binding to the residues Y652 and F656 in the S6 domain, and indirectly obstructs the transport of the hERG protein to the cell membrane.

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“…What is new in the work by Cordes et al (2005) and Kuryshev et al (2005) is the recognition that some drugs causing LQTS can selectively reduce I Kr by disrupting protein trafficking and channel maturation, rather than by direct block of surface membrane channels. This represents a new and novel indirect mechanism for causing drug-induced LQTS, and it raises several interesting questions.…”

mentioning

confidence: 99%

Protein trafficking abnormalities: a new mechanism in drug‐induced long QT syndrome

Eckhardt

Rajamani

January

2005

British J Pharmacology

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Drug induced long QT syndrome (LQTS) can lead to cardiac arrhythmias and sudden death, and has emerged as a worldwide problem. Most drugs that cause this are thought to directly block a specific cardiac ion channel (KCNH2 or hERG) that carries the rapidly activating delayed rectifier potassium current, I Kr . In this issue of the British Journal of Pharmacology, evidence is presented to support a new mechanism for causing drug induced LQTS. The drug pentamidine, at near therapeutic concentrations that do not cause direct KCNH2 channel block, disrupts normal KCNH2 channel protein processing and maturation to reduce its surface membrane expression. This indirect mechanism for reducing I Kr is novel, and whether other drugs may cause similar protein trafficking abnormalities is largely unknown. (Roden, 2004), which is encoded by the human ether-a-go-go-related gene (hERG or KCNH2).Owing to its unique structure and drug-binding domain, the KCNH2 potassium channel pore is promiscuous in its sensitivity to block by a wide variety of drugs, and in the last decade block of KCNH2 channels has emerged as a major problem facing both pharmaceutical and regulatory agencies. Pentamidine, an antiprotozoal agent, has been known since at least 1987 to cause QT interval prolongation. The mechanism by which this drug alters repolarization has not been previously elucidated. In this issue of British Journal of Pharmacology, Cordes et al. (2005) describe the mechanism by which pentamidine reduces I Kr . These authors show that pentamidine, like many drugs, blocks KCNH2 channels stably expressed in a human embryonic kidney cell (HEK293) line, and does so in a concentration-and voltage-dependent manner. The authors suggest that block is independent of the state of the channel, which is somewhat unusual as most drugs interact preferentially with the open or inactivated states. The IC 50 for KCNH2 channel block was 252 mM, a value nearly 500 times higher than the therapeutic freepentamidine concentration.This drug concentration paradox was investigated further by incubating KCNH2 channel expressing HEK293 cells in low ('therapeutic') concentrations of pentamidine. A 2-day incubation in pentamidine (1-10 mM), compared to control cells, resulted in altered growth in some cells and a decrease in KCNH2 current density of 36-85% measured by whole-cell patch clamp. The authors also performed Western blot analysis and confocal immunofluorescence imaging of control and pentamidine incubated cells, and showed in drug treated cells disruption of normal protein trafficking of KCNH2 channels with a concomitant reduction in mature KCNH2 protein. The authors conclude that the chronic administration of pentamidine causes a protein trafficking abnormality of KCNH2 channels. This work amplifies the recent report by Rampe and co-workers (Kuryshev et al., 2005), who also showed that low concentrations of pentamidine selectively disrupted protein trafficking and maturation of KCNH2 channels but not of hKv1.5, KvLQT1/minK and Kv4.3 channels, and that in is...

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Pentamidine-Induced Long QT Syndrome and Block of hERG Trafficking

Cited by 251 publications

References 29 publications

A Simple Protocol for the Myocardial Differentiation of Human iPS Cells

A Simple Protocol for the Myocardial Differentiation of Human iPS Cells

The protease inhibitor atazanavir blocks hERG K+ channels expressed in HEK293 cells and obstructs hERG protein transport to cell membrane

Protein trafficking abnormalities: a new mechanism in drug‐induced long QT syndrome

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