Biomedical subjects

Arthur M Brown

Publications and source records attributed to Arthur M Brown.

15 recordsLinked to original sources

PubMed2005

HERG block, QT liability and sudden cardiac death.

Non-cardiac drugs may prolong action potential duration (APD) and QT leading to Torsade de Pointes (TdP) and sudden cardiac death. TdP is rare and QT is used as a surrogate marker in the clinic. For non-cardiac drugs, APD/QT liability is always associated with a reduction in hERG current produced by either direct channel block or inhibition of trafficking. hERG and APD liabilities correlate better when APDs are measured in rabbit versus canine Purkinje fibres. hERG and APD/QT liabilities may be dissociated when hERG block is offset by block of calcium or sodium currents. hERG liability may be placed in context by calculating a safety margin (SM) from the IC50 for inhibition of hERG current measured by patch clamp divided by the effective therapeutic plasma concentration of the drug. The SM is uncertain because literature values for IC50 may vary by 50-fold and small differences in plasma protein binding have large effects. With quality control, the IC50 95% confidence limits vary less than twofold. Ideally, hERG liability should be determined during lead optimization. Patch damp has insufficient throughput for this purpose. A novel high-throughput screen has been developed to detect drugs that block hERG directly and/or inhibit hERG trafficking.

Action Potentials↗

PubMed2005

HERG channel trafficking.

Mutations in the cardiac potassium channel hERG/IKr cause inherited long QT syndrome with increased susceptibility to ventricular arrhythmias. Several mutations in hERG produce trafficking-deficient channels that are retained in the endoplasmic reticulum (ER). Surface expression of certain mutations (i.e. hERG G601S) can be restored by specific channel blockers. Although hERG currents have been studied extensively, little is known about proteins in the processing pathway. Using biochemical and electrophysiological assays we show that the cytosolic chaperones Hsp70 and Hsp90 interact transiently with wild-type hERG. Inhibition of Hsp90 prevents maturation and reduces hERG/IKr currents. Trafficking-deficient mutants remain tightly associated with chaperones in the ER until trafficking is restored, e.g. by channel blockers. hERG/chaperone complexes represent novel targets for therapeutic compounds with cardiac liability such as arsenic, which is used in the treatment of leukaemias. Arsenic interferes with the formation of hERG/chaperone complexes and inhibits hERG maturation causing ECG abnormalities. We conclude that Hsp9O and Hsp70 are crucial for productive folding of wild-type hERG. Therapeutic compounds that inhibit chaperone function produce a novel form of acquired long QT syndrome not by direct channel block but by reduced surface expression due to an acquired trafficking defect of hERG.

Action Potentials↗

PubMed2004

Pentamidine-induced long QT syndrome and block of hERG trafficking.

The diamidine pentamidine is used to treat leishmaniasis, trypanosomiasis, and Pneumocystis carinii pneumonia. Treatment may be accompanied by prolongation of the QT interval of the electrocardiogram and torsades de pointes tachycardias. Up to now, it has been thought that therapeutic compounds causing QT prolongation are associated with direct block of the cardiac potassium channel human ether a-go-go-related gene (hERG), which encodes the alpha subunit of cardiac I(Kr) currents. We show that pentamidine has no acute effects on currents produced by hERG, KvLQT1/mink, Kv4.3, or SCNA5. Cardiac calcium currents and the guinea pig cardiac action potential were also not affected. After overnight exposure, however, pentamidine reduced hERG currents and inhibited trafficking and maturation of hERG with IC(50) values of 5 to 8 microM similar to therapeutic concentrations. Surface expression determined in a chemiluminescence assay was reduced on exposure to 10, 30, and 100 microM pentamidine by about 30, 40, and 70%, respectively. These effects were specific for hERG since expression of hKv1.5, KvLQT1/minK, and Kv4.3 was not altered. In isolated guinea pig ventricular myocytes, 10 microM pentamidine prolonged action potential duration APD(90) from 374.3 +/- 57.1 to 893.9 +/- 86.2 ms on overnight incubation. I(Kr) tail current density was reduced from 0.61 +/- 0.09 to 0.39 +/- 0.04 pA/pF. We conclude that pentamidine prolongs the cardiac action potential by block of hERG trafficking and reduction of the number of functional hERG channels at the cell surface. We propose that pentamidine, like arsenic trioxide, produces QT prolongation and torsades de pointes in patients by inhibition of hERG trafficking.

Action Potentials↗

PubMed2004

Mechanisms of arsenic-induced prolongation of cardiac repolarization.

Arsenic trioxide (As(2)O(3)) produces dramatic remissions in patients with relapsed or refractory acute promyelocytic leukemia. Its clinical use is burdened by QT prolongation, torsade de pointes, and sudden cardiac death. In the present study, we analyzed the molecular mechanisms leading to As(2)O(3)-induced abnormalities of cardiac electrophysiology. Using biochemical and electrophysiological methods, we show that long-term exposure to As(2)O(3) increases cardiac calcium currents and reduces surface expression of the cardiac potassium channel human ether-a-go-go-related gene (HERG) at clinically relevant concentrations of 0.1 to 1.5 microM. In ventricular myocytes, As(2)O(3) increases action potential duration measured at 30 and 90% of repolarization. As(2)O(3) interferes with hERG trafficking by inhibition of hERG-chaperone complexes and increases calcium currents by a faster cellular process. We propose that an increase in cardiac calcium current and reduced trafficking of hERG channels to the cell surface cause QT prolongation and torsade de pointes in patients treated with As(2)O(3). Our results suggest that calcium-channel antagonists will be useful in normalizing QT prolongation during As(2)O(3) therapy. As(2)O(3) is the first example of a drug that produces hERG liability by inhibition of ion-channel trafficking. Other drugs that interfere with proteins in the processing pathway of cardiac ion channels may be proarrhythmic for similar reasons.

Animals↗

PubMed2003

Role of the cytosolic chaperones Hsp70 and Hsp90 in maturation of the cardiac potassium channel HERG.

The human ether-a-gogo-related gene (hERG) encodes the alpha subunit of the cardiac potassium current IKr. Several mutations in hERG produce trafficking-deficient channels that may cause hereditary long-QT syndrome and sudden cardiac death. Although hERG currents have been studied extensively, little is known about the proteins involved in maturation and trafficking of hERG. Using immunoprecipitations, we show that the cytosolic chaperones heat shock protein (Hsp) 70 and Hsp90, but not Grp94, interact with hERG wild type (WT) during maturation. The specific Hsp90 inhibitor geldanamycin prevents maturation and increases proteasomal degradation of hERG WT, while reducing hERG currents in heterologous expression systems. In ventricular myocytes, inhibition of Hsp90 also decreases IKr, whereas geldanamycin had no effect on IKs or heterologously expressed Kv2.1 and Kv1.5 currents. Both Hsp90 and Hsp70 interact directly with the core-glycosylated form of hERG WT present in the endoplasmic reticulum but not the fully glycosylated, cell-surface form. For the trafficking-deficient LQT2 mutants, hERG R752W and hERG G601S, interactions with Hsp90 and Hsp70 are increased as both mutants remained tightly associated with Hsp90 and Hsp70 in the endoplasmic reticulum. Incubation at lower temperature for R752W or with the hERG blocker astemizole for G601S dissociates channel-chaperone complexes and restores trafficking. In contrast, nonfunctional but trafficking-competent hERG G628S is released from chaperone complexes during maturation comparable to WT. We conclude that Hsp90 and Hsp70 are crucial for the maturation of hERG WT as well as the retention of trafficking-deficient LQT2 mutants. The full text of this article is available online at http://www.circresaha.org.

Analysis of Variance↗

PubMed2003

Optical mapping reveals conduction slowing and impulse block in iron-overload cardiomyopathy.

Cardiac disease with arrhythmia or heart failure is the leading cause of death in patients with thalassemia major and a major complication of other forms of iron overload. Current antiarrhythmic treatment does not appear to alter the clinical course. Using a gerbil model of iron-overload cardiomyopathy, we previously observed a reduction in the fast inward sodium current in isolated cardiomyocytes. Electrocardiograms (ECGs) in the same gerbil model indicate PR-interval prolongation, QRS-interval widening, and arrhythmias. We hypothesize that such changes in the ECG in this model are the result of abnormal action-potential conduction at the level of the whole heart. To test this hypothesis, we took ECGs and recorded action potentials using high-resolution optical mapping from the anterior surface of 9 iron-overloaded and 9 age-matched control ventricular-paced, Langendorff-perfused gerbil hearts. The iron-overloaded gerbils received weekly iron-dextran injections of 800 mg/kg for 14 to 18 weeks. ECGs showed QRS- and PR-interval prolongation in iron-treated gerbils compared with that in controls. In addition, atrioventricular block was observed in 2 of 6 iron-treated gerbils but not in controls. Conduction velocity was significantly slower in iron-treated gerbils than in controls. At normal pacing rates, abnormal activation patterns caused by stable regions of conduction block were observed in iron-overloaded gerbils (33%) but not in controls. Such abnormal impulse conduction may be a mechanism of increased arrhythmia vulnerability in iron-overload cardiomyopathy.

Action Potentials↗

PubMed2003

Deferoxamine prevents cardiac hypertrophy and failure in the gerbil model of iron-induced cardiomyopathy.

To evaluate the effects of the iron chelator deferoxamine on the functional and structural manifestations of iron-induced cardiac dysfunction, we measured cardiac power, left ventricular systolic, and diastolic function as (dP/dt)max and (dP/dt)min, respectively, and left ventricular and septal wall thickness in isolated heart preparations derived from the Mongolian gerbil model of iron overload. We induced iron overload with weekly subcutaneous injections of iron dextran (800 mg/kg/wk); deferoxamine (DFO; 100 mg/kg) was administered twice daily by subcutaneous injection, 5 of 7 days each week; and control animals received weekly subcutaneous injections of dextran alone. Animals administered iron alone initially exhibited, at 5 weeks, increased cardiac power but by 12 to 20 weeks, cardiac power was severely diminished, with impairment of both systolic and diastolic function of the left ventricle and marked cardiac hypertrophy (P<.001 for all vs control animals). Administration of DFO with iron did not interfere with the initial augmentation of cardiac power at 5 weeks but prevented the subsequent deterioration in cardiac performance. After 12 to 20 weeks, gerbils given DFO with iron had mean values of cardiac power indistinguishable from those of control animals; both systolic and diastolic function were significantly enhanced not only in comparison with those of animals treated with iron alone but also with respect to controls. In addition, DFO prevented cardiac hypertrophy; mean ventricular and septal wall thickness in gerbils given DFO and iron were not significantly different from those in controls. In the gerbil model of iron overload, concurrent administration of DFO with iron prevents both the development of cardiac hypertrophy and the progressive deterioration in cardiac performance that are produced by chronic iron accumulation.

Animals↗

PubMed2003

Deferoxamine promotes survival and prevents electrocardiographic abnormalities in the gerbil model of iron-overload cardiomyopathy.

We investigated the time course of electrocardiographic (ECG) changes in the Mongolian gerbil model of iron overload and the effects of the iron chelator deferoxamine (DFO) on these changes. Iron overload was produced with weekly subcutaneous injections of low doses (200 mg/kg/wk) or high doses (800 mg/kg/wk) of iron-dextran. DFO was administered subcutaneously at a dose of 200 mg/kg/day to high-dose animals. Our results show that (1) survival of iron-overloaded gerbils is dose-dependent, with median survival times of 68 and 14 weeks for low- and high-dose animals, respectively; (2) both low and high doses produce prolongation of the PR interval and bradycardia in early stages and prolongation of the QT interval, premature ventricular contractions, variable degrees of atrioventricular block, changes in the ST segment, and T-wave inversion at later stages coinciding with the development of heart failure; (3) DFO prevented death during 20 weeks of high-dose iron-dextran; (4) DFO prevented ECG changes, although delayed prolongation of PR intervals and QRS complexes occurred; and (5) despite marked prolongation of survival and prevention of ECG changes, DFO had modest effects on total cardiac iron content. We speculate that DFO chelates a small iron pool located within the cytoplasm of iron-overloaded cardiomyocytes.

Animals↗

PubMed2002

Renal tubular epithelial cell apoptosis is associated with caspase cleavage of the NHE1 Na+/H+ exchanger.

Renal tubular epithelial cell (RTC) apoptosis causes tubular atrophy, a hallmark of renal disease progression. Apoptosis is generally characterized by reduced cell volume and cytosolic pH, but epithelial cells are relatively resistant to shrinkage due to regulatory volume increase, which is mediated by Na(+)/H(+) exchanger (NHE) 1. We investigated whether RTC apoptosis requires caspase cleavage of NHE1. Staurosporine- and hypertonic NaCl-induced RTC apoptosis was associated with cell shrinkage and diminished cytosolic pH, and apoptosis was potentiated by amiloride analogs, suggesting NHE1 activity opposes apoptosis. NHE1-deficient fibroblasts demonstrated increased susceptibility to apoptosis, which was reversed by NHE1 reconstitution. NHE1 expression was markedly decreased in apoptotic RTC due to degradation, and preincubation with peptide caspase antagonists restored NHE1 expression, indicating that NHE1 is degraded by caspases. Recombinant caspase-3 cleaved the in vitro-translated NHE1 cytoplasmic domain into five distinct peptides, identical in molecular weight to NHE1 degradation products derived from staurosporine-stimulated RTC lysates. In vivo, NHE1 loss-of-function C57BL/6.SJL-swe/swe mice with adriamycin-induced nephropathy demonstrated increased RTC apoptosis compared with adriamycin-treated wild-type controls, thereby implicating NHE1 inactivation as a potential mechanism of tubular atrophy. We conclude that NHE1 activity is critical for RTC survival after injury and that caspase cleavage of RTC NHE1 may promote apoptosis and tubular atrophy by preventing compensatory intracellular volume and pH regulation.

Animals↗

PubMed2002

Increased K+ efflux and apoptosis induced by the potassium channel modulatory protein KChAP/PIAS3beta in prostate cancer cells.

K(+) channel-associated protein/protein inhibitor of activated STAT (KChAP/PIAS3beta) is a potassium (K(+)) channel modulatory protein that boosts protein expression of a subset of K(+) channels and increases currents without affecting gating. Since increased K(+) efflux is an early event in apoptosis, we speculated that KChAP might induce apoptosis through its up-regulation of K(+) channel expression. KChAP belongs to the protein inhibitor of activated STAT family, members of which also interact with a variety of transcription factors including the proapoptotic protein, p53. Here we report that KChAP induces apoptosis in the prostate cancer cell line, LNCaP, which expresses both K(+) currents and wild-type p53. Infection with a recombinant adenovirus encoding KChAP (Ad/KChAP) increases K(+) efflux and reduces cell size as expected for an apoptotic volume decrease. The apoptosis inducer, staurosporine, increases endogenous KChAP levels, and LNCaP cells, 2 days after Ad/KChAP infection, show increased sensitivity to staurosporine. KChAP increases p53 levels and stimulates phosphorylation of p53 residue serine 15. Consistent with activation of p53 as a transcription factor, p21 levels are increased in infected cells. Wild-type p53 is not essential for induction of apoptosis by KChAP, however, since KChAP also induces apoptosis in DU145 cells, a prostate cancer cell line with mutant p53. Consistent with its proapoptotic properties, KChAP prevents growth of DU145 and LNCaP tumor xenografts in nude mice, indicating that infection with Ad/KChAP might represent a novel method of cancer treatment.

Adenoviridae↗

PubMed2002

Bimodal cardiac dysfunction in an animal model of iron overload.

Iron-overload cardiomyopathy is the most common cause of death in patients with thalassemia major, yet the associated changes in cardiac function have not been quantified. We studied the effects of iron overload on cardiac function in Mongolian gerbils, a species that responds to iron overload in the same manner as human beings. We injected iron-dextran or dextran alone at low subcutaneous doses (200 mg/kg/wk) for 20 to 60 weeks and at high doses (800 mg/kg/wk) for 6 to 20 weeks. At shorter durations for either dose, the mean values of cardiac work, coronary flow, left ventricular (dP/dt)(max) and left ventricular (dP/dt)(min) in isolated perfused hearts were significantly greater than control values; at longer durations, these values were significantly less than control values. Echocardiography in intact animals showed eccentric cardiac hypertrophy, increased cardiac output, and normal exercise tolerance at shorter durations of dosage. At longer durations, concentric cardiac hypertrophy developed, and cardiac output and exercise capacity were impaired. The response to iron overload in Mongolian gerbils progresses from an initial state of high cardiac output to a subsequent state of low-output failure similar to the course of cardiomyopathy that has been inferred in patients with transfusional iron overload.

Animals↗

PubMed2002

Variable expression of long QT syndrome among gene carriers from families with five different HERG mutations.

OBJECTIVES: This study assessed the phenotypic variability of LQTS in carriers with the same and with different mutations in the LQT2 gene. BACKGROUND: Mutations of ion-channel genes are known to cause the long QT syndrome (LQTS), a disorder associated with distinctive genotypic-specific electrocardiographic patterns and variable clinical expression. METHODS: Clinical and electrocardiographic characteristics were assessed in five large LQTS families, each with a different mutation of the HERG gene (LQT2; n = 469, 69% genotyped, 102 carriers). One mutation was located on the N-terminus and the other four on the C-terminus of the HERG channel protein. RESULTS: The QTc duration and the frequency of cardiac events (syncope and LQTS-related cardiac arrest/death) were similar among carriers with the five HERG mutations. QTc was as variable in carriers of the same mutation as it was among carriers with different HERG mutations (P = 0.19). Qualitative assessment of the electrocardiograms revealed extensive intra-and interfamilial variability in T-wave morphology. Among carriers with multiple electrocardiograms extending over 2 to 7 years, variation in QTc over time was minimal. A strong association was found between QTc and the occurrence of cardiac events in carriers of all five mutations. CONCLUSIONS: The clinical expression of LQTS was equally variable in carriers from families with the same or different HERG mutations. These findings highlight the complexity of the clinical phenotype in this Mendelian dominant disorder and suggest that one or more modifier genes contribute to the variable expression of this syndrome.

Adult↗

PubMed2001

The binding site for channel blockers that rescue misprocessed human long QT syndrome type 2 ether-a-gogo-related gene (HERG) mutations.

Mutations in the human ether-a-gogo-related gene (HERG) K(+) channel gene cause chromosome 7-linked long QT syndrome type 2 (LQT2), which is characterized by a prolonged QT interval in the electrocardiogram and an increased susceptibility to life-threatening cardiac arrhythmias. LQT2 mutations produce loss-of-function phenotypes and reduce I(Kr) currents either by the heteromeric assembly of non- or malfunctioning channel subunits with wild type subunits at the cell surface or by retention of misprocessed mutant HERG channels in the endoplasmic reticulum. Misprocessed mutations often encode for channel proteins that are functional upon incorporation into the plasma membrane. As a result the pharmacological correction of folding defects and restoration of protein function are of considerable interest. Here we report that the trafficking-deficient pore mutation HERG G601S was rescued by a series of HERG channel blockers that increased cell surface expression. Rescue by these pharmacological chaperones varied directly with their blocking potency. We used structure-activity relationships and site-directed mutagenesis to define the binding site of the pharmacological chaperones. We found that binding occurred in the inner cavity and correlated with hydrophobicity and cationic charge. Rescue was domain-restricted because the trafficking of two misprocessed mutations in the C terminus, HERG F805C and HERG R823W, was not restored by channel blockers. Our findings represent a first step toward the design of pharmacological chaperones that will rescue HERG K(+) channels without block.

Anti-Arrhythmia Agents↗

PubMedDate not supplied

Variability in the measurement of hERG potassium channel inhibition: effects of temperature and stimulus pattern.

INTRODUCTION: In vitro evaluation of drug effects on hERG K(+) channels is a valuable tool for identifying potential proarrhythmic side effects in drug safety testing. Patch-clamp recording of hERG K(+) current in mammalian cells can accurately evaluate drug effects, but the methodology has not been standardized, and results vary widely. Our objective was to evaluate two potential sources of variability: the temperature at which recordings are performed and the voltage pulse protocol used to activate hERG K(+) channels expressed in HEK293 cells. METHODS: A panel of 15 drugs that spanned a broad range of potency for hERG inhibition and pharmacological class was evaluated at both room and near-physiological temperatures using several patch-clamp voltage protocols. Concentration-response analysis was performed with three stimulus protocols: 0.5- and 2-s step pulses, or a step-ramp pattern. RESULTS: Block by 2 of the 15 drugs tested, d,l-sotalol (antiarrhythmic) and erythromycin (antibiotic), was markedly temperature sensitive. hERG inhibition measured using a 2-s step-pulse protocol underestimated erythromycin potency compared with results obtained with a step-ramp protocol. Using conservative acceptance criteria and the step-ramp protocol, the IC(50) values for hERG block differed by less than twofold for 15 drugs. DISCUSSION: Data obtained at near-physiological temperatures using a step-ramp pattern are highly repeatable and provide a conservative safety evaluation of hERG inhibition.

Cells, Cultured↗

PubMedDate not supplied

HERG-Lite: a novel comprehensive high-throughput screen for drug-induced hERG risk.

INTRODUCTION: Direct block of I(Kr) by non-antiarrhythmic drugs (NARDs) is a major cause of QT prolongation and torsades de pointes (TdP), and has made the hERG potassium channel a major target of drug safety programs in cardiotoxicity. Block of hERG currents is not the only way that drugs can adversely impact the repolarizing current I(Kr), however. We have shown recently that two drugs in clinical use do not block hERG but produce long QT syndrome (LQTS) and TdP by inhibiting trafficking of hERG to the cell surface. To address the need for an inexpensive, rapid, and comprehensive assay to predict both types of hERG risk early in the drug development process, we have developed a novel antibody-based chemiluminescent assay called HERG-Lite. METHODS: HERG-Lite monitors the expression of hERG at the cell surface in two different stable mammalian cell lines. One cell line acts as a biosensor for drugs that inhibit hERG trafficking, while the other predicts hERG blockers based on their ability to act as pharmacological chaperones. In this study, we have validated the HERG-Lite assay using a panel of 100 drugs: 50 hERG blockers and 50 nonblockers. RESULTS: HERG-Lite correctly predicted hERG risk for all 100 test compounds with no false positives or negatives. All 50 hERG blockers were detected as drugs with hERG risk in the HERG-Lite assay, and fell into two classes: B (for blocker) and C (for complex; block and trafficking inhibition). DISCUSSION: HERG-Lite is the most comprehensive assay available for predicting drug-induced hERG risk. It accurately predicts both channel blockers and trafficking inhibitors in a rapid, cost-effective manner and is a valuable non-clinical assay for drug safety testing.

Cell Line↗