Life threatening cardiac rhythm disturbances due to cocaine abuse, and treatment with specific antimicrobial agents such as macrolide antibiotics and pentamidine are well known. However, their underlying molecular mechanisms are not. There is growing evidence that infection with HIV-1 can stimulate the formation of inflammatory cytokines such as TNF-alpha and IL1-beta. The long term goal of the proposed research is to investigate the cellular and molecular mechanisms responsible for the cardiac problems seen in AIDS patients that are associated with cocaine abuse and antimicrobial therapy. Preliminary evidence from the applicant's laboratory demonstrate that TNF-alpha can reduce the amplitude of human ether-a-go-go related gene (HERG) encoded K currents, an action that has been previously shown to result in prolongation of the QT interval of the electrocardiogram and development of an acquired long QT syndrome. This syndrome is associated with a high incidence of polymorphic ventricular arrhythmias, torsade de pointes, and even sudden death. Other preliminary data from the applicant's laboratory suggest that cocaine also can block HERG encoded K currents with an affinity that is higher than that which blocks the analogous current expressed in guinea pig heart. Several clinical reports strongly suggest that specific antimicrobial drugs used in the treatment of AIDS patients, including erythromycin and pentamidine, also may block HERG encoded K currents. The applicant proposes to test the following five hypotheses: 1) inflammatory cytokines modulate the amplitude and/or gating properties of human cardiac K, Ca and Na channels; 2) the effects of inflammatory cytokines on human cardiac ion channels are mediated by activation of protein kinase C; 3) cocaine and its major metabolites, as well as selected antimicrobial drugs block HERG-encoded K channels at clinically relevant concentrations; 4) inflammatory cytokines produce an additive or synergistic effect to reduce the amplitude of HERG currents when cells are simultaneously exposed to cytokines and either cocaine or antimicrobial drugs; 5) the effects of cocaine on HERG can be reduced by increasing extracellular pH and/or external [K+]. 6) Kv4.3 gene expression underlies the human atrial transient outward current. This last hypothesis stems from the fact that the human atrial transient outward current is known to be an important regulator of cardiac repolarization and the applicant suggests it is a putative target of cytokine induced cardiotoxicity. The applicant proposes seven specific aims to test these six hypotheses. They are: 1) To test the hypothesis that cocaine and its major metabolites block the K channel encoded by the human ether-a-go-go related gene (HERG), the K-channel blocking effects of cocaine, methylecgonine (the major pyrolysis product), cocaethylene, ecgonine- methylester, benzoylecgonine, and norcocaine will be defined in an HEK-293 cell line that stably expresses HERG. These studies should help clarify the mechanism responsible for cocaine's effect on the human QT interval. 2) The hypothesis that the effects of cocaine on HERG encoded K currents can be modulated by changes in extracellular pH and external [K+] will be tested. The administration of Na bicarbonate has been proposed as an effective treatment for cocaine cardiotoxicity, and modest increases in plasma [K+] have been reported to virtually eliminate QT prolongation produced by other drugs known to block HERG. These experiments should clarify whether alterations in plasma pH and/or [K+], which are known to occur during myocardial ischemia, cocaine-induced seizures, or treatment with Na bicarbonate, can modulate the K channel blocking actions of cocaine. 3) The hypothesis that pentamidine and the macrolide antibiotics (erythromycin, azithromycin and clarithromycine) can block HERG at clinically relevant concentrations will be tested. This hypothesis has direct relevance to the reported proarrhythmic effects of these drugs in the treatment of infections (in AIDS patients and patients with other inflammatory disorders). 4) The applicant will test hypotheses that (a) HIV-induced production of inflammatory cytokines (TNF-alpha- and IL-1beta) can reduce the amplitude of HERG encoded K currents, (b) that their effects are mediated by activation of protein kinase C, and (c) result in an additive effect to reduce HERG encoded K currents exposed to cocaine, pentamidine and erythromycin. These experiments may provide a molecular explanation for the acquired long QT syndrome reported in 30% of AIDS patients. 5) The hypotheses that human atrial ionic currents (Ito, IKur, ICa and INa) are modulated by inflammatory cytokines, and that these effects are mediated by activation of protein kinase C will be tested. These experiments may elucidate the ionic basis for the deleterious effects of inflammatory cytokines on conduction and excitability in the human heart. (You will now notice that with aims 6&7, there is a major shift in the emphasis of the proposal from that enunciated in the previous five tightly related aims) 6) The hypothesis that expression of the K channel gene Kv4.3 underlies the transient outward current (Ito) in human atrial myocytes will be tested by comparison of Ito amplitude and the levels of Kv4.3 gene expression in single myocytes lacking Ito (~30% of pediatric atrial myocytes) with those found in cells containing a small Ito (pediatric) vs. cells containing a large Ito (adult atrial myocytes).
This aim will be achieved though the use of expression profiling in which the RNA from single cells is amplified following measurements of current amplitude from the same cell. This technique will be utilized to define the atrial cell expression of the K channel mRNA's for Kvl.l, Kvl.4, Kvl.5, Kv2.l, minK, Kv4.3, HIRK, HERG, and the beta subunits of Kvl.2 and Kv2. 7) The hypothesis that Kv4.2 and/or Kv4.3 gene expression underlies the human atrial will be further tested by comparing the physiological and pharmacological properties of native Ito with those of K channels encoded by mammalian Kv4.2 and Kv4.3 genes that have been transfected into mouse LtK-cells. Variables to be compared will include the voltage dependence of channel activation and inactivation, characteristics of channel blockade by quinidine and 4-aminopyridine, sensitivity of channel gating and conductance to changes in extracellular potassium concentration, and modulation by activators of protein kinase C.
| Stanat, Scott J C; Carlton, Carol G; Crumb Jr, William J et al. (2003) Characterization of the inhibitory effects of erythromycin and clarithromycin on the HERG potassium channel. Mol Cell Biochem 254:1-7 |
| Ferreira, S; Crumb Jr, W J; Carlton, C G et al. (2001) Effects of cocaine and its major metabolites on the HERG-encoded potassium channel. J Pharmacol Exp Ther 299:220-6 |
