Antiarrhythmic Effects Of Dipfluzine And The Underlying Mechanisms

Heart diseases, including diverse arrhythmias, are the leading cause of death in clinical patients. There are plenty of antiarrhythmic agents; however, their usage is usually limited because of the adverse effects. Almost all of them can evoke new arrhythmias. It is valuable to research and develop novel antiarrhythmic agents.Dipfluzine, a novel Ca²⁺ channel blocker with similar structure to flunarizine, is synthesized by Pharmaceutical School of Hebei Medical University. Ca²⁺ channel blockers inhibited the accumulation of cytosolic Ca²⁺, thus possessed antiarrhythmic effects on the arrhythmias caused by Ca²⁺ overload. Our previous experiments revealed that dipfluzine decreased overshoot, action potential amplitude (APA), maximal rate of depolarization in phase 0 (Vmax), duration of plateau phase and durations of 50% and 90% repolarization of action potential (APD50 and APD90) in partially depolarized guinea-pig papillary muscles, and reduced APA, Vmax, velocity of diastolic (phase 4) depolarization, and rate of pacemaker firing in rabbit sinoatrial node pacemaker cells. Dipfluzine exerted preventive effects on delayed afterdepolarization induced by ouabain and early afterdepolarization induced by isoprenaline in guinea-pig papillary muscles. In addition, dipfluzine had inhibitory effect on delayed afterdepolarization and triggered activity induced by isoprenaline in human atrial fibers. These results indicated that dipfluzine might possess potential antiarrhythmic activities. The first part of this thesis was designed to investigate the antiarrhythmic effects of dipfluzine using the arrhythmic animal models.In these arrhythmic models used in the first part, defective cytosolic Ca²⁺ handling was recognized as a mediator of cardiac arrhythmias. Dipfluzine exerted antiarrhythmic effects in these models, which indicated that dipfluzine might possess modulation effect on the cytosolic Ca²⁺ homeostasis. To clarify the mechanisms of action, in the second part of the thesis, the effect of dipfluzine on intracellular free-calcium concentration was investigated in guinea-pig ventricular myocytes using Laser Scanning Confocal Microscopy. Previously study have found that dipfluzine had inhibitory effects on L-type calcium current (Ica-L) in guinea-pig ventricular myocytes, and preserved the intracellular calcium homeostasis, which could explain some electrophysiological effects of dipfluzine. But we couldn't elucidate the effects of dipfluzine on APA, overshoot and Vmax, which were more related to the rapid sodium channel. Therefore, the third part of the thesis was designed to investigate the effect of dipfluzine on Na⁺ current in isolated single guinea-pig ventricular myocytes by whole cell patch-clamp technique. The experimental projects and results were as follows.Part 1 Antiarrhythmic effects of dipfluzine on experimental arrhythmiasObject: To investigate whether dipfluzine possessed antiarrhythmic effects on experimental arrhythmias.Methods: Experimental arrhythmias were induced by drugs (ouabain or chloroform) or myocardial ischemia-reperfusion. Dipfluzine was administrated by gavage or duodenum. The surface lead II GCG was continuously recorded with three subcutaneous limb electrodes using an MP100 data acquisition system. The time at which various arrhythmias appeared or the incidences of arrhythmias were recorded.Results: 1. Effects of dipflzuine on the arrhythmias induced by ouabain in guinea-pig In vehicle group, the cumulative dosages of ouabain to induce VP, VT, VF and CA were 158±30, 190±47, 232±67 and 285±71μg·kg^-1, respectively. Pretreatment with dipfluzine 20 mg·kg^-1 and verapamil 20mg·kg^-1 significantly increased the dosage of ouabain required to induce arrhythmias. Pretreatment with dipfluzine 10 mg·kg^-1 could increase the dosage of ouabain required to induce VP, while pretreatment with flunarizine 20 mg·kg^-1 didn't show significant effects on ouabain-induced arrhythmias. 2. Effects of dipfluzine on arrhythmias induced by myocardial ischemia-reperfusion in rat In sham operation group, VP and VT occurred in one out of the 10 experimental animals during reperfusion. Ischemia-reperfusion-induced VP, VT, VF and CA occurred in all animals treated with vehicle. VP wasn't abolished by pretreatment with dipfluzine (5 mg·kg^-1, 10 mg·kg^-1 or 20 mg·kg^-1). Incidences of VF and CA were lowered by pretreatment with dipfluzine (10 mg·kg^-1 and 20 mg·kg^-1), and VT was inhibited in the group treated with dipfluzine 20 mg·kg^-1. Pretreatment with flunarinze 20 mg·kg-1 only reduced the incidence of CA, whereas verapamil 20 mg·kg-1 reduced the incidences of VP, VT, VF and CA.3. Effect of dipfluzine on arrhythmia induced by chloroform inhalation in mice In vehicle group, the incidence of VF was 90%. Pretreatment with dipfluzine 40 mg·kg^-1 and verapamil 40 mg·kg^-1 for 7 d significantly decreased the incidence of VF induced by chloroform, while dipfluzine (10 mg·kg^-1 or 20 mg·kg^-1) and flunarizine 40 mg·kg^-1 had no significant effect on the incidence of VF induced by chloroform.Conclusion: Dipfluzine inhibited ventricular premature contraction, ventricular tachycardia, ventricular fibrillation and cardiac arrest induced by ouabain,ventricular tachycardia, ventricular fibrillation and cardiac arrest induced by myocardial ischemia-reperfusion and ventricular fibrillation induced by chloroform. Antiarrhythmic effects of dipfluzine were more potent than that of flunarizine, the analogous compound.Part 2 Effect of dipfluzine on the intracellular free-calcium concentration in guinea-pig ventricular myocytesObject: To observe the effect of dipfluzine on intracellular free-calcium concentration ([Ca²⁺]_i) in isolated single guinea-pig ventricular myocytes.Methods: [Ca²⁺]_i was measured using the Laser Scanning Confocal Microscopy in isolated single guinea-pig ventricular myocytes which were prepared by enzymatic dissociation method and loaded with Fluo 3-Am. The change of [Ca²⁺]_i was represented as the change ratio of fluorescent intensity [(FI-FI0)/FI0].Results: 1. Effect of dipfluzine on [Ca²⁺]_i of guinea-pig ventricular myocytes in normal Tyrode's solution Dipfluzine reduced [Ca²⁺]_i of guinea-pig ventricular myocytes in normal Tyrode's solution in a concentration-dependent manner after a 5-min perfusion. Verapamil 1.0μmol·L^-1 also reduced [Ca²⁺]_i. The effect of verapamil 1.0μmol·L^-1 was similar to that of dipfluzine 1.0μmol·L^-1.2. Effect of dipfluzine on [Ca²⁺]_i of guinea-pig ventricular myocytes in Ca²⁺-free Tyrode's solution Dipfluzine (0.1 to 10.0μmol·L^-1) reduced [Ca²⁺]_i in Ca²⁺-free Tyrode's solution after a 5-min perfusion. Verapamil 1.0μmol·L^-1 also reduced [Ca²⁺]_i. The effect of verapamil 1.0μmol·L^-1 was similar to that of dipfluzine at the same concentration.3. Prophylactic effect of dipfluzine on Ca²⁺ overload caused by high extracellular Ca²⁺ levels When Ca²⁺ concentration in the extracellular medium was elevated from 1.0 to 10.0 mmol·L^-1, cytosolic [Ca²⁺]_i increased considerably and Ca²⁺ overload was evoked, some myocytes shrinked gradually. Pretreatment with dipfluzine or verapamil before elevation of extracellular Ca²⁺ in the medium, significantly restricted the increase of [Ca²⁺]_i and the morphological changes of myocytes, while the suppressive effect of verapamil was more remarkable than that of dipfluzine at the same concentration.4. Curative effect of dipfluzine on Ca²⁺ overload caused by high extracellular Ca²⁺ levels [Ca²⁺]_i was elevated when the myocytes were perfused with high Ca²⁺ Tyrode's solution. Dipfluzine (0.1, 1.0 or 10.0μmol·L^-1) decreased the levels of [Ca²⁺]_i in a concentration-dependent manner. The similar event occurred with application of verapamil, while the effect of verapamil was less than that of dipfluzine.Conclusion: Dipfluzine exerted suppressive effect on [Ca²⁺]_i by blocking calcium channel and by alleviating calcium release from sarcoplasmic reticulum in isolated single guinea-pig ventricular myocytes. In addition, dipfluzine possessed prophylactic and curative effects on intracellular Ca²⁺ overload in ventricular myocytes caused by high extracellular Ca²⁺ levels and the effects might be related to the improving effects on the calcium extrusion from the cytosol besides blocking calcium channel and alleviating calcium release from sarcoplasmic reticulum.Part 3 Effect of dipfluzine on the Na⁺ current in guinea-pig ventricular myocytesObject: To investigate the effect of dip on Na⁺ current (I_Na⁺) in isolated single guinea-pig ventricular myocytes.Methods: I_Na⁺ was measured by whole cell patch-clamp technique in single isolated guinea-pig ventricular myocytes which were prepared by enzymatic dissociation method.Results: 1. Cardiac I_Na⁺ was elicited by 50-ms pulses to +50 mV from the holding potential at–80 mV with a step of +10 mV, which could be blocked completely by TTX 10μmol·L^-1. The peak I_Na⁺ occurred at about–20 mV and the reversal potential for I_Na⁺ was about +30 mV.2. Dipfluzine inhibited cardiac I_Na⁺ in a concentration-dependent manner, with the half inhibitory concentration value of 43.5μmol·L^-1. The blocking effect of dipfluzine on I_Na⁺ was reversible.3. Dipfluzine suppressed the cardiac I_Na⁺, without modifying the maximum activation potential and the reversal potential. The peak of I_Na⁺ was decreased by about 46% at–20 mV and the shape of I-V curve was not altered by dipfluzine 40μmol·L^-1.4. Dipfluzine shifted the steady-state inactivation curve of I_Na⁺ towards more negative without change the slope factor and produced very little change in the steady-state activation curve towards more positive. The mean half activation voltage was -34.9±5.1 mV and slope factor was 6.0±4.8 mV under control condition and -33.7±3.6 mV (P>0.05) and 5.6±2.4 mV (P>0.05) following exposure to dipfluzine 40μmol·L^-1, and the half inactivation voltage was -73.0±4.6 mV and slope factor was 4.8±1.8 mV under control condition and -82.8±7.2 mV (P<0.01) and 4.8±1.8 mV (P>0.05) following dipfluzine 40μmol·L^-1 treatment.5. Dipfluzine delayed the recovery of cardiac I_Na⁺ from inactivation state. The time course of recovery was (36±11) ms in control group and (79±28) ms (P<0.05) in dipfluzine 40μmol·L^-1 group.6. Dipfluzine blocked cardiac I_Na⁺ in a use- and frequency-dependent manner.Conclusion: Dipfluzine inhibited cardiac I_Na⁺ in concentration- use- and frequency-dependent manners, and had higher affinity for the inactivated state than that for the resting state of Na⁺ channels.SUMMARY1. Dipfluzine inhibited ventricular premature contraction, ventricular tachycardia, ventricular fibrillation and cardiac arrest induced by ouabain,ventricular tachycardia, ventricular fibrillation and cardiac arrest induced by myocardial ischemia-reperfusion and ventricular fibrillation induced by chloroform. Antiarrhythmic effects of dipfluzine were more potent than that of flunarizine, the analogous compound.2. Dipfluzine exerted suppressive effect on [Ca²⁺]_i by blocking calcium channel and by alleviating calcium release from sarcoplasmic reticulum in isolated single guinea-pig ventricular myocytes. In addition, dipfluzine possessed prophylactic and curative effects on intracellular Ca²⁺ overload in ventricular myocytes caused by high extracellular Ca²⁺ levels and the effects might be related to the improving effects on the calcium extrusion from the cytosol besides blocking calcium channel and alleviating calcium release from sarcoplasmic reticulum.3. Dipfluzine inhibited cardiac I_Na⁺ in concentration- use- and frequency- dependent manners, and had higher affinity for the inactivated state than that for the resting state of Na⁺ channels.