| Background:Chronic heart failure (CHF) is one of the major diseases that getting more and more attention, the prevalence is approximately 2.6% in the Western world. Approximately 50% of CHF patients die suddenly, with lethal ventricular arrhythmias (VA) being the predominant causes. VA pathogencsis of chronic heart failure was complicated, involving β-adrenergic receptor (β-AR) signaling pathway activated, ion channel remodeling, intracellular calcium homeostasis disorders, CX43 function and reduced myocardial fibrosis and other factors. At present the exact mechanisms for VA/sudden cardiac death (SCD) in CHF patients are unclear. Therefore, to explore the mechanism of VA/SCD in CHF and find more effective therapeutic targets to reduce the incidence of chronic heart failure, is the focus and direction of cardiovascular research workers.The sympathetic nervous system plays a vital role in the pathophysiology of CHF. Many studies have demonstrated that high cardiac sympathetic activity is correlated with risk of death from arrhythmias.β2 adrenergic receptor (β2-AR), a subtype of β adrenergic receptor (β-AR), is important in regulating cardiac structure and function. β2-AR genetic variants increase the risk of sudden cardiac death (SCD), and abnormal augmentation of cardiac responsiveness to β2-AR is also a risk factor for ventricular fibrillation (VF). Ikr is a major contributor to phase 3 repolarization of the action potential. Its reduction often increases action potential duration (APD) and leads to increase in the incidence of early afterdepolarizations, which can increase incidence of VA.β2-AR, a G protein-coupled receptor, regulates cardiac function upon binding to catecholamine, which results in activation of heterotrimeric G-proteins. β2-AR couples dually to Gs proteins and pertussis toxin (PTX)-sensitive Gi proteins. β2-AR couples to Gs protein, which leads to activation of AC and production of cAMP. Phosphodiesterases (PDE) regulate cAMP production through AC activation and cAMP breakdown to modulate PKA activity and myocardial contractility.β2-AR activation can increase the risk of VA and SCD in CHF. However, the possible mechanism of β2-AR activation in ventricular arrhythmia in CHF has not yet been determined. We hypothesized that β2-AR activation can induce abnormal repolarization and ventricular arrhythmia through Gs/cAMP/PKA and Gi/PDE.Objective:The study was aim to verify that β2-AR activation induce ventricular arrhythmia through Gs/cAMP/PKA and Gi/PDE in CHF. We will investigate these aspects:(1) To use β2-AR agonist and antagonist to perfuse isolated hearts which will be from aortic constriction induced-failing hearts of guinea pigs, and to observe electrophysiological changes in failing hearts; (2) To use β2-AR agonist and the antagonist of β2-AR signal pathway factor to treat ventricular myocytes, and to observe the changes of Ikr and APD in CHF and to further study its possible mechanism. This study will provide theoretical basis and experimental proofs for β2-AR as a new target for prevention and treatment of ventricular arrhythmia in CHF.Methods:1. The CHF model of guinea pigs was established by aortic constriction. The left ventricular diameter and left ventricular ejection fraction (LVEF) were measured by echocardiography; the heart rate, QT interval and corrected QT were detected by electrocardiogram. The whole cell patch clamp technique was used to measure Ikr and APD in myocytes from CHF guinea pigs. Effective refractoriness period (ERP) in the left ventricle from guinea pigs with CHF was examined by Lagendorff perfusion and programmed electrical stimulation (PES).2. Ex vivo ECG recordings and PES were used to observe the corrected QT (QTc) and incidence of VA after treatment with selective (32-AR agonist salbutamol(10 μmol) and antagonist ICI118551 (1 μmol).3. Rp-cAMP (100 μmol), KT5720 (2.5 μmol), PTX (400 ng) and amrinone (30 μmol/L) were used to pretreat the myocytes from Control and CHF groups, and then the change of Ikr and APD in cells perfused with salbutamol were detected by whole cell patch clamp technique.Results:1. Effects of CHF on Ikr, APD and ERPTwelve weeks after aortic constriction, the Ikr tail current density in the CHF group significantly decreased compared with that of the control group (0.88±0.65 vs. 1.33±0.071, n=6, p<0.01). The voltage dependence of activation showed that there was no statistical difference in half between the CHF group and the control group (0.75±1.84 mV vs.1.67±0.92 mV, p>0.05), and also no difference in slope factor between these two groups (15.54±1.74 vs.16.98±0.89, p>0.05).APD at 90% repolarization (APD90) were dramatically prolonged in the CHF group compared with the control group (179.0±8.89 ms vs.146.8±6.48 ms, p<0.01), but there was no significant difference in 50% repolarization (APD50) between the CHF and control groups (143.9±11.3 vs.126.7 ±7.83 ms, p>0.05).CHF dramatically prolonged LV ERP compared with the control group (82.50±4.79 ms vs.68.33±3.07 ms, p<0.05).2. Effects of β2-AR stimulation on QTc and VA from isolated failing heart:In the control hearts, the selective β2-AR agonist salbutamol slightly prolonged QTc compared with the control group (262.2±7.29 ms vs 240.8± 6.07 ms, p<0.05). However, salbutamol significantly broadened QTc of CHF hearts compared with CHF hearts perfused without salbutamol (293.5±6.60 ms vs.258.7±6.65 ms, p<0.01). The percentage increase in QTc was significantly greater in the CHF hearts than in the control hearts (13.52%±1.72%vs.8.87%±0.84%, p<0.05). ICI118551 significantly limited QTc prolongation induced by salbutamol in Control and CHF groups.In the CHF hearts, following salbutamol perfusion, PES significantly increased the incidence of VA compared with the CHF hearts and the control preparations perfused with salbutamol (66.7% vs.16.7%, p<0.05). ICI118551 significantly decreased inducibility of PES-evoked VA in the CHF hearts exposed to salbutamol (16.7% vs.66.7%, p<0.05).3. Gs/cAMP/PKA and Gi/PDE are involved in the induced effect of β2-AR stimulation on VA in CHF:Salbutamol significantly inhibited Ikr (p<0.05), broadened APD90 (p<0.05) and APD50 (p<0.05) in ventricular myocytes from failing hearts. Rp-cAMP reduced the inhibitory effect of salbutamol on Ikr in the control group from 22.78%±4.16% to 10.41%±2.97%(p<0.05), and significantly attenuated the inhibitory effect of salbutamol on Ikr in CHF group (from 36.97%± 3.56% to 11.44%± 1.75%, p<0.01). Salbutamol slightly broadened APD90 and APD50 in the control cells, but no significant difference was observed in these cells (p>0.05); in CHF cells, Rp-cAMP significantly reduced the percentages of prolongation of APD90 and APD50 (3.99%±0.80% vs.14.22%±1.18%, p<0.01; 3.64%±1.07% vs.11.84%±2.13%, p<0.05). Salbutamol-induced inhibition of Ikr was attenuated by KT5720 in control group (from 22.78%±4.16% to 12.73%±1.32%, p<0.05) and the CHF group (from 36.97%± 3.56% to 17.70%±1.88%, p<0.01). The prolongation effects of salbutamol on APD90 and APD50 were significantly prevented by KT5720 (4.74%±2.00% vs. 14.22%±1.18%, p<0.01; 3.75%±1.88% vs.11.84%±2.13%, p<0.05).The reduction of Ikr induced by salbutamol was significantly greater from the CHF group in the presence of PTX (46.63%±2.40% vs.36.97%±3.56%, p<0.05), and the percentages of prolongation of APD90 and APD50 induced by salbutamol were greater than no PTX was given (18.74%± 1.11% vs.14.22%± 1.18%, p<0.05; 18.3%± 1.85% vs.11.84%±2.13%, p<0.05). The effect of (32-AR activation on Ikr was augmented by amrinone in CHF cells (49.25%±1.05% vs.36.97%±3.56%, p<0.05), and the percentage prolongation of APD90 and APD50 was greater with salbutamol plus amrinone (19.95%±1.34% vs.14.22%1.18%, p<0.01, 18.79%±1.53% vs.11.84±2.13%, p<0.05).Conclusions:1. β2-AR activation increases the incidence of VA in CHF.2. β2-AR activation governs ventricular arrhythmogenesis in chronic heart failure via Gs/cAMP/PKA and Gi/PDE pathways; inhibition of Gs/cAMP/PKA pathway can reduce the incidence of ventricular arrhythmias induced by β2-AR activation in chronic heart failure and suppression of Gi/PDE pathway can increase incidence of ventricular arrhythmias. |
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