| Background:Stress is an adaptive response of organism to environmental stimuli, When an organism exerts limited control over environmental stimuli, stress response may ultimately produce increased susceptibility to psychosomatic disorders such as cardiovascular disturbances. As a common and serious stress-induced diseases, arrhythmia has become an important risk factor to human health. In most cases, stress-induced arrhythmia is on the basis of original heart lesion. It has not been reported that stress model can induce cardiac arrhythmia in normal animal. Stress-induced disease is often a chronic process with a multi-stimuli, therefore, it is necessary to establish a chronic multi-stress model, which can induce arrhythmia in animals.The mechanism of arrhythmia is not yet clear. Several groups have investigated the mechanisms underlying electrical remodeling in various animal models of long-lasting rapid atrial pacing and in humans with chronic atrial fibrillation(AF). The electrical remodeling is characterized by a change in the electrical properties of the atrium, notably a shortening of the atrial effective refractory period (AERP). The remodeling of ion channel is the basis of the electrical remodeling. Studies have pointed to a marked reduction in the density of currents through L-type calcium channels (ICa,L) and potassium channels(Ito) as the major factor. The fewer expression of L-type calcium channel in AF myocytes was presumedly responsible for the reduction of ICa,L. However, some results that have been published conflict each other. Therefore, it is important to study the changes of ICa,L and Ito in the atrial arrhythmia. Expression of L-type calcium channel and potassium channel play a central role in atrial remodeling. Myocardial L-type calcium channel and potassium channel consist of several different sub-units, each plays a different role.In the present study, a chronic multi-stress model in rat was established. ECGs were recorded and arrhythmia occurrence was counted in every recording periods. Concentrations of intracellular Ca2+ in atrial myocardium were measured after the test. ICa,L and Ito were studyed. L-type calcium channel and potassium channel subunit expression were investigated. We used verapamil as calcium channel blocker in chronic multi-stress model.Methods1. Chronic multi-stress model in rats and stress-induced arrhythmia1) Multiple stressor consisted of five different means: noise combinated with footshock, force swimming and restraint combinated with light was used in establishment of the stress model and Verapamil was used as calcium channel blocker. Behavior changes of stressed animals were observed, blood pressure and food utilization were recorded. Concentrations of serum NE and Epi were detected by HPLC, concentrations of serum ACTH and Cor measured by RIA.2) Electrocardiogram was recorded and arrhythmia occurrence was counted. Concentrations of intracellular Ca2+ in atrial myocardium were measured with laser-scanning confocal microscopy.2. Electrical remodeling of atrial myocardium in chronic multi-stress rats was tested by whole-cell patch clamp technique.1) Rats were exposed to chronic multi-stress, atrial myocytes were isolated after stress.2) patch clamp microelectrodes were produced by two-step drawn.3) Whole-cell patch clamp ICa,L and Ito recordings were performed. Clampfit 9.2 software was used for data acquisition and analysis. I-V curves were obtained by Clampfit software. The activation and inactivation curves were produced by Boltzmann equation.3. The gene and protein expression changes of L-type calcium channel and potassium channel subunits of atrial myocardium in chronic multi-stress rats.1) The gene expression of L-type calcium channelα1 c,α2/δ1,β1,β2,β3 subunits and potassium channel Kv1.4, Kv4.2, Kv4.3 subunits were quantified by real time PCR .2) L-type calcium channel and potassium channel subunits protein expression were tested by Westernblot method.Results1. Evaluation of chronic multi-stress model1) The changes of behavior in stress rats: the stress animals showed excited and anxiety 1 w after exposur to stessors, the behaviors were displaced gradually by suppression and depression in the last week.2) The changes of rate and blood pressure in chronic multi-stress rats: the rate and blood pressure of stress group increased significantly(P <0.01), there was a similar change in Verapamil group.3). The weight growth and food utilization changes: in both stress and Verapamil groups, weight growth was slower than the control group, the food utilization was lower than the control group (P <0.05).4) The contents of serum ACTH and Cor increased in stress group.2. Chronic multi-stress induced arrhythmia in rats.1) Chronic multi-stress-induced arrhythmia and the effect of Verapamil: there was a more striking arrhythmia occurrence(including sinus tachycardia, sinus arrhythmia, atrial premature beats, ventricular premature beats and ventricular tachycardia) in stress group. A delayed arrhythmia occurrence was found in Verapamil group compared with stress group(P<0.05). A lower incidence of arrhythmias was observed in Verapamil group compared with stress group(P<0.01).2) Verapamil inhibited the increasing of serum catecholamines in chronic multiple stress rats: It appeared a lower elevations of plasma catecholamines (NE and Epi) in Verapamil group than stress group(P<0.01).3) Verapamil inhbited calcium overload in chronic multiple stress rats: it appeared a lower concentration of intracellular Ca2+ in Verapamil group compared with stress group at the end of the test(P <0.01).3. The changes of ICa,L and Ito of atrial myocytes in chronic multi-stress rats1) ICa,L peak current amplitude reduced significantly in chronic multi-stress rats, the peak pA/pF was lower than Verapamil group and control group (P <0.01, n = 6). V1/2 was significantly higher than that of Verapamil group and control group(P <0.01, n=6).2) Ito peak current magnitude and the peak current density was lower in stress group than Verapamil group and control group(P<0.01, n = 6).4. The expression changes of L-calcium channel and potassium channel of atrial myocardium in chronic multi-stress rats.1) Gene expression of L-type calcium channelα1c,α2/δ1,β1,β2,β3 subunits wese markedly lower in stress group and Verapamil group compared to control group(P <0.01). Levels ofβ1 andβ2 subunit mRNA were lower in stress group than Verapamil group ( P <0.05, P <0.01).2) The level of potassium channel Kv1.4, Kv4.2, Kv4.3 subunits mRNA were increased in Verapamil group compared with control group and stress group(P <0.01).3) Calcium channelα1c,α2/δ1,β1 subunits protein and potassium channels Kv1.4, Kv4.2, Kv4.3 subunits protein level were significantly lower in stess group compared with control group (P <0.01). In Verapamil group, calcium channelα1c,α2/δ1,β1 subunit and potassium channel Kv1.4, Kv4.2 subunit protein level were lower than that of control group (P <0.01), Potassium channel Kv4.3 subunit protein levels was lower in Verapamil group compared with the control group(P <0.05). In stress group, calcium channelα1c,β1 subunits and potassium channel Kv1.4 subunit protein expression was markedly lower compared with Verapamil group (P <0.01, P <0.05, P <0.05).Conclusion1. A chronic multi-stress model in rats was established. The stress animals showed a behaveor change. The HR and BP were increased in stressed rats. There was a higher elevations of plasma ACTH, Cor, NE and Epi in chronic multi-stress rats. Chronic multi-stress can induce arrhythmias occurrence.2. Chronic multi-stress-induced intracellular Ca2+ overloading of atrial myocardium may be one of the reasons of arrhythmias. Whole-cell patch clamp ICa,L and Ito recordings showed that atrial myocytes ICa,L and Ito were down regulated in stress-induced arrhythmias rats. L-type calcium channel activity was decreased. So ICa,L and Ito channels current decrease is one of the reasons of stress-incuced arrhythmias.3. The expression of atrial L-type calcium channel subunits and potassium channel subunits decreased in chronic multi-stress rats, which may be one of the reasons why atrial ICa,L and Ito were reduced in stress-incuced arrhythmias rats.4. Verapamil inhibited stress-induced arrhythmias and intracellular Ca2+ overloading of atrial myocardial. Verapamil inhibited stress-induced ICa,L and Ito downregulation. We indicated that Verapamil ameliorated expression downregulation of L-type calcium channel and potassium channel partly. We concluded that Verapamil plays a prevention and therapy role in stress-induced arrhythmias.
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