The Mechanism Underlying Supression Of Aconitine-induced Arrhythmias By Chloride Channel Blockers In Rat Heart

It has been postulated that Cl- channels may be of pathophysiological significance and play a role in the genesis of arrhythmias, since Cl- channels were identified in the heart. Indeed, Clemo et al. have observed that swelling-activated Cl- current is persistently activated in ventricular myocytes from dogs with tachycardia-induced congestive heart failure. Evidence suggests that Ca~2+-activated Cl- current contributes to delayed afterdepolarizations in single Purkinje and ventricular myocytes. Akar et al. reported that intracellular Cl~ was accumulated within atrial myocytes during atrial fibrillation in dog model, which may be a result of activation of Cl-channels. It is frequently reported that Cl~ channel blockers have profound effects on some other ion channels. Recently, we suggested that the effects ofCl- channel blockers and less permeant Cl- substitutes on cation channels may involve Cl- channels. However, whether such relationship between Cl-channels and cation channels plays a role in the arrhythmogenesis remains poorly understood.Voltage-gated Na~+ channel is primarily responsible for the generation and propagation of action potential (AP) in excitable tissues. In the hearts, disturbances of the activity of voltage-gated Na~+ channels markedly influence the excitation of cardiac myocytes, which may play a role in arrhythmogenesis. Therefore, the Na~+ channel is an important target for the action of antiarrhythmic drugs. Although some putative C\~ channel blockers have been reported to inhibit cardiac voltage-gated Na~+ current (I_Na), however, the mechanism underlying the action of Cl- channel blockers on I_Na remains poorly understood and whether Cl- channel blockers may protect against thearrhythmias induced by abnormal activity of Na+ channels is also unknown.Aconitine, a Na+ channel agonist that reduces the channel inactivation and prolongs the open state of sodium channels, is known to induce arrhythmias. Owing to this property, aconitine has long been widely used as a tool in the study of voltage-gated Na+ channels, in model of cardiac arrhythemias in various animals and in testing the effects of antiarrhythmic drugs. In the present study, we investigated the effect and mechanism of putative Cl~ channel blockers, NPPB and NFA, on aconitine-induced arrhythmias, and provide novel evidence that Cl" channel blockade may be a therapeutic strategy for control of ventricular tachyarrhythmias.Methods1. Perfusion of isolated hearts and electrocardiographic recordings: Sprague-dawley rats were killed by decapitation, and the hearts wereimmediately removed and cannulated via the arota. Retrograde perfusion were performed at 37℃ in a Langendorff apparatus under constant pressure. A water-filled balloon was inserted through the mitral valve and secured in the left ventricle. The balloon was connected to a pressure transducer and a physiological recorder to monitor LVP. ECG was recorded via electrodes placed on the apical region of the heart and the aorta. All hearts were initially equilibrated for 30 minutes before recording.2. Cell preparations : Ventricular myocytes were enzymatically isolated from adult Sprague-Dawley rats as reported previously. Briefly, the hearts were removed immediately after decapitation and retrogradely perfused at 37°C in turn with the following solutions: Tyrode's solution (5 min), Ca~2+-free Tyrode's solution (5 min), Ca~2+-free Tyrode's solution with 0.5 mg/ml collagenase Ⅱ and 1 mg/ml BSA (35 min), KB (high K+) solution (5 min). After dissociation collection, the cells were maintained in KB solution at room temperature (23~25 ℃) for electrophysiological recordings.3. Whole-cell patch-clamp experiments: Aliquots of cell suspension were transferred into a perfusion chamber on the stage of an inverted microscope (IMT-2, Olympus, Tokyo, Japan). Pipettes had tip resistances of 2-2.5 MΩ when filled with internal solution. Whole-cell recordings were performed at room temperature using a patch-clamp amplifier (Axopatch 200B, Axon Instruments, Foster, CA, USA). Liquid junction potentials were offset before the pipette touched the cell. Whole-cell series resistance was compensated to more than 80%. In the anion substitution experiments, an agar-salt bridge was used as the reference electrode. The current signals were low-pass filtered at 2 kHz and digitized with an analog-to-digital converter (Digidata 1322) and pCLAMP 8.1...