RESUMO
Type 2 congenital long QT syndrome (LQT-2) is linked to mutations in the human ether a-go-go-related gene (HERG) and is characterized by rate-corrected QT interval (QTc) prolongation, ventricular arrhythmias, syncope, and sudden death. Recognized triggers of these cardiac events include emotional and acoustic stimuli. Here we investigated the repeated occurrence of fever-induced polymorphic ventricular tachycardia and ventricular fibrillation in 2 LQT-2 patients with A558P missense mutation in HERG. ECG analysis showed increased QTc with fever in both patients. WT, A558P, and WT+A558P HERG were expressed heterologously in HEK293 cells and were studied using biochemical and electrophysiological techniques. A558P proteins showed a trafficking-deficient phenotype. WT+A558P coexpression caused a dominant-negative effect, selectively accelerated the rate of channel inactivation, and reduced the temperature-dependent increase in the WT current. Thus, the WT+A558P current did not increase to the same extent as the WT current, leading to larger current density differences at higher temperatures. A similar temperature-dependent phenotype was seen for coexpression of the trafficking-deficient LQT-2 F640V mutation. We postulate that the weak increase in the HERG current density in WT-mutant coassembled channels contributes to the development of QTc prolongation and arrhythmias at febrile temperatures and suggest that fever is a potential trigger of life-threatening arrhythmias in LQT-2 patients.
Assuntos
Arritmias Cardíacas/fisiopatologia , Canais de Potássio Éter-A-Go-Go/genética , Febre/complicações , Síndrome do QT Longo/fisiopatologia , Adulto , Idoso , Arritmias Cardíacas/etiologia , Temperatura Corporal , Linhagem Celular , Canal de Potássio ERG1 , Eletrocardiografia , Eletrofisiologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Heterozigoto , Humanos , Ativação do Canal Iônico/fisiologia , Síndrome do QT Longo/etiologia , Síndrome do QT Longo/genética , Masculino , Mutação de Sentido Incorreto , Transporte Proteico , Síndrome de Romano-Ward/diagnóstico , Síndrome de Romano-Ward/fisiopatologia , Temperatura , TransfecçãoRESUMO
Evaluation of drug cardiotoxicity is essential to the safe development of novel pharmaceuticals. Assessing a compound's risk for prolongation of the surface electrocardiographic QT interval and hence risk for life-threatening arrhythmias is mandated before approval of nearly all new pharmaceuticals. QT prolongation has most commonly been associated with loss of current through hERG (human ether-a-go-go related gene) potassium ion channels due to direct block of the ion channel by drugs or occasionally by inhibition of the plasma membrane expression of the channel protein. To develop an efficient, reliable, and cost-effective hERG screening assay for detecting drug-mediated disruption of hERG membrane trafficking, the authors demonstrate the use of microfluidic-based systems to improve throughput and lower cost of current methods. They validate their microfluidics array platform in polystyrene (PS), cyclo-olefin polymer (COP), and polydimethylsiloxane (PDMS) microchannels for drug-induced disruption of hERG trafficking by culturing stably transfected HEK cells that overexpressed hERG (WT-hERG) and studying their morphology, proliferation rates, hERG protein expression, and response to drug treatment. Results show that WT-hERG cells readily proliferate in PS, COP, and PDMS microfluidic channels. The authors demonstrated that conventional Western blot analysis was possible using cell lysate extracted from a single microchannel. The Western blot analysis also provided important evidence that WT-hERG cells cultured in microchannels maintained regular (well plate-based) expression of hERG. The authors further show that experimental procedures can be streamlined by using direct in-channel immunofluorescence staining in conjunction with detection using an infrared scanner. Finally, treatment of WT-hERG cells with 5 different drugs suggests that PS (and COP) microchannels were more suitable than PDMS microchannels for drug screening applications, particularly for tests involving hydrophobic drug molecules.
Assuntos
Técnicas de Cultura de Células , Avaliação Pré-Clínica de Medicamentos/métodos , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Transporte de Íons/efeitos dos fármacos , Síndrome do QT Longo/induzido quimicamente , Microfluídica/métodos , Polímeros/química , Bloqueadores dos Canais de Potássio/efeitos adversos , Bloqueadores dos Canais de Potássio/farmacologiaRESUMO
The pro-arrhythmic Long QT syndrome (LQT) is linked to 10 different genes (LQT1-10). Approximately 40% of genotype-positive LQT patients have LQT2, which is characterized by mutations in the human ether-a-go-go related gene (hERG). hERG encodes the voltage-gated K(+) channel alpha-subunits that form the pore of the rapidly activating delayed rectifier K(+) current in the heart. The purpose of this study was to elucidate the mechanisms that regulate the intracellular transport or trafficking of hERG, because trafficking is impaired for about 90% of LQT2 missense mutations. Protein trafficking is regulated by small GTPases. To identify the small GTPases that are critical for hERG trafficking, we coexpressed hERG and dominant negative (DN) GTPase mutations in HEK293 cells. The GTPases Sar1 and ARF1 regulate the endoplasmic reticulum (ER) export of proteins in COPII and COPI vesicles, respectively. Expression of DN Sar1 inhibited the Golgi processing of hERG, decreased hERG current (I(hERG)) by 85% (n > or = 8 cells per group, *, p < 0.01), and reduced the plasmalemmal staining of hERG. The coexpression of DN ARF1 had relatively small effects on hERG trafficking. Surprisingly, the coexpression of DN Rab11B, which regulates the endosomal recycling, inhibited the Golgi processing of hERG, decreased I(hERG) by 79% (n > or = 8 cells per group; *, p < 0.01), and reduced the plasmalemmal staining of hERG. These data suggest that hERG undergoes ER export in COPII vesicles and endosomal recycling prior to being processed in the Golgi. We conclude that hERG trafficking involves a pathway between the ER and endosomal compartments that influences expression in the plasmalemma.