摘要
研究背景
心房颤动(atrial fibrillation AF)是临床上最常见的复杂心律失常之一,其发病率随年龄增长而显著增加,并成为一种独立的危险因素使患者残率、致死率增加,因而是当前心血管疾病的一个研究重点,也是心律失常领域中亟待解决的难题。目前房颤的治疗包括药物疗法和近十几年来开展的介入疗法和新的手术方法,但其效果有限,绝大多数患者的症状和预后得不到明显的改观。为此,加深对房颤机制的认识,针对病理基础寻求治疗手段是当前房颤研究的重点,其中对心房重构(包括心房电重构与心房解剖重构)的深入研究可能会使我们发现房颤的更深层次的机制,从而找到解决房颤的有效方法。
近年来人们观察到房颤具有自身延续性,慢性持续性房颤常由多次的阵发性房颤做为先导,房颤终止以后再次复发的可能性与房颤持续的时间密切相关。1995 年Wijffels等在山羊模型上发现,随着快速心房刺激时间的延长,有效不应期(AERP)进行性缩短,房颤的诱发率明显增高,持续时间逐渐延长;即房颤本身可诱发心房的电生理改变,使房颤容易发生和持续,人们将这种现象称为心房电重构(Atrial electrical remodeling AER),主要表现为有效不应期缩短和有效不应期频率适应不良。它在房颤的发生、持续及房颤引起的一系列心房功能改变中起着重要作用。作为持续性房颤产生机制的最新进展,心房电重构为房颤的防治提供了新的方向,成为目前的研究热点。
在对心房电重构不断深入的研究中,人们还发现, 房颤转复为窦性心律后,心房的电生理特性很快恢复到正常状态,而心房收缩功能却需几周甚至数月才能恢复正常。在快速起搏右心室产生心力衰竭后诱发房颤的动物模型中发现,心房有效不应期、房颤波长及其离散度没有发生明显改变,这说明除了心房电重构之外,还有其它因素参与了房颤的发生与维持。近来研究表明,心房肌细胞在房颤期间发生了非常明显的结构变化,即发生了结构重构(atrial anatomical remodeling AAR)。
多年来人们对心房电重构和结构重构进行了大量的研究和探索,取得了一些成绩,但有关房颤发生和维持的确切机制仍然不是很明确,临床治疗中药物的有效性和副作用等方面的很多问题仍没有得到很好的解决。因此有必要在房颤发生的电生理机制和药物干预方面做进一步的深入研究,为最终解决房颤问题提供实验依据。
Background: Atrial fibrillation (AF) is the most common arrhythmia in clinical practice. Its incidence increases with age and presents a major risk factor of causing disability and death. Therefore, it is the most intensively studied disease in cardiology while its still remaining a major challenge. There has been some progress in the management of AF, including chemotherapy, interventional therapy and surgery. However, a little improvement has been seen concerning the symptoms and prognosis of the AF patients. Further insight into the pathogenesis and mechanism-directed therapy would shed more light on the management of AF, especially that better understanding of the atrial remodeling (electrical remodeling as well as anatomic remodeling) is where the hope lies in.
Recently, AF was recognized to be autonomously continuous. Onset of chronic persistent AF is often led by paroxysmal atrial fibrillation; Risk of recurrence is closely related with the duration of the fibrillation episodes. Using a goat model in 1995, Wijffels et al found that as the stimulation prolonged, the effective refractory period (AERP) of atrial shortened progressively, the atrial fibrillation caused electrophysiological changes which in turn favored the occurrence and persistence of the AF. This phenomenon was termed as atrial electrical remodeling (AER). A major characteristic of the AER is the shortening and maladjustment of ERP. AER plays important roles in the onset, persistence and functional changes of AF, which provides a new direction to study the mechanism of AF.
As further study was made in the AER, people found that electrophysiology of the atrial turned normal as the AF was converted to sinus rhythm, while the atrial contraction wouldn’t be normal until weeks or months later. In animal model with AF after heart failure caused by right ventricular fast pacing, no significant changes have been found in the atrial ERP, fibrillation wave length and its vicariance. Taking together, it suggested that factors other than AER were involved in the occurrence and persistence of AF. Recent study suggested that atrial myotcytes underwent structural changes in AF (Atrial anatomical remodeling AAR).
Despite the great efforts in the study of AER and AAR, exact mechanism of AF remains
unclear. Clinical medication is still confronted with limited efficacy and various drug adverse effects. thereby, this study focuses on the electrophysiology as well as drug intervention of AF in hope of giving further insight into the pathogenesis of AF. Objectives: To study the mechanism of the involvement of Angiotensin II in atrial electrical remodeling and anatomical remodeling, investigate the effects of Angiotensin II antagonist on the atrial remodeling and provide the experimental basis for clinical use of Angiotensin II antagonist to treat AF. 1. Isolate and identify the atrial myocytes using 2-step collagenase digestion method; Culture tissue blocks in vitro to obtain atrial myocytes. 2. Study the effects of angiotensin II on ICa-L, Ito and IK1 of normal atrial myocyte and examine ICa-L, Ito and IK1 on AF atrial myocytes; Elucidate the role of Angitensin II in AER of AF; Observe the antagonistic effect of Temisartan (angiotensin II receptor antagonist). 3. Examine the calcium overload of the atrial myocytes in patients with or without AF; Verify the result in isolated atrial mypcytes treated with Angiotensin II; Study the effect of angiotensin II antagonist on calcium overload of the cells. 4. Observe the tissue structure, ultrastructure and gap junctions of the atrial myocytes from AF or non-AF patients with microscope and scanning electron microscope to understand the relationship between anatomical remodeling and AF. Experimental Methods 1. Acute isolation and identification of human atrial myocytes Tissue samples were taken from the right atrial of patients undergoing cardiac surgery before setting up extracorporeal circulation. 2 steps of digestions were performed using collagenase and protease; Single atrial myocytes were isolated under stirring and saturated oxygen. Recalcification was performed by concerntration gradient method. Cells were observed under invert microscope and pictures were taken; Whole cell patch clamp was used to record the IK1. Living cells were identified by trypan blue staining. Culture and identification of the human atrial myocytes: atrial tissue blocks underwent adherent culture; cells were passaged after digestion with trypsin; Photos were taken under microscope. Cell slides were prepared and stained with mouse anti-humanα-Actin antibody; Goat anti-human TnI, mouse anti-human α-Actin rabbit anti-goat IgG-FITC and goat
anti-mouse IgG-CY3 were used to perform immunofluorescenct examination; Electron microscope was used to observe the ultrastructure of the atrial myocytes. 2. Electrophysiology examination Whole cell patch clamp was used to record the membrane currents; Human atrial myocyte suspension in KB solution was added into the perfusion chamber of the inverted microscope. The cells were allowed to adhere. Different extracellular solutions were infused into the chamber to record different currents. Microelectrodes were drawn for patch clamp. High impedance seal was formed between the electrode tips and cell membrane. Whole cell recording mode was set up after the capacitance compensation. After recalcification, membrane ICa-L, Ito and IK1 current were recorded in the freshly isolated human atrial myocytes with whole-cell recording mode. Effects of angiotensin II and telmisartan on the individual current were observed. Atrial myocytes from patients with/without AF were acutely isolated. (Some of the AF patients were pretreated with telmisartan.) Membrane currents were recorded. 3. Laser scan cofocal microscopic measurement of Calcium concentrations Single atrial cells were loaded with Fluo-3/AM. Microfluorescence intensity was determined with cofocal microscope. Ca2+ distribution image was obtained through digital camera and software processing. Image analysis software was used to quantify the fluorescence intensity to get the time-dependent curve of the [Ca2+]i. Measurement of the [Ca2+]i was performed on the following cells: normal atrial cells, normal cells with extracellular angiotensin II or telmisartan perfusion, AF atrial cells and Non-AF atrial cells. 4. Right atrial tissue samples were taken from patients with or without AF, who underwent cardiac surgery. Tissue samples were fixed and sections prepared for microscopic examination. Electron microscope was used to study the ultrastructure of the cells. Gap junction protein CX40 was stained by immunohistochemistry and semi-quantified. Results 1. Acute isolation and culture of atrial myocyte Atrial cells isolated with modified 2-step digestion procedure took on the typical characteristics of atrial myocytes. Cells were rod-shaped, clear striae were seen in the cells. Cells had intact membrane. Contraction was observed in some of the isolated cells. IK
recorded by patch clamp was consistent with the normal atrial myocytes. 50-70% of the isolated cells were viable and could survive over 24 hr as determined by Trypan blue staining. Under in vitro culture, cells were seen spreading from the fringe of adherent tissue blocks after 4-5 days. Cells were fusiform in shape. Cells could reach 70-80% confluence after 10 days. Passage for every 4-5 days was needed. Cell could grow for 8 generations (~1.5 months). 90% of the cells were stained positive with non-specific α-Actin antibody and negative with smooth muscle specific α-Actin antibody. Cells were positively stained with TnI (cardiac cell antibody). Microfilaments/ myofilaments were observed with electron microscope; Cellular organelles were close to the nuclear, all of which were the characteristic features of the atrial myocytes. 2. Membrane currents recorded with whole cell patch clamp Angiotensin II decreased the peak current density of Ito. Telmisartan had no effects on Ito itself, but could reverse the effect of angiotensin II, weakening the decrease of Ito. With presence of antiotensin II, peak current density of ICa-L and IK1 was increased; Telmisartan antagonized the effect of angiotensin II, lessening the current increase. The peak current density was significantly lower for Ito and ICa-L in atrial myocytes with AF comparing with non-AF atrial cells, while peak density of IK1 increased in AF atrial cells. With presence of telmisartan, IK1 was decreased significantly with Ito increased. Slightly higher peak current density was observed in Non-AF atrial cells than those in AF cells but not statistically significant. 3. Cofocal microscopic examination Intracellular fluorescence intensity began to increase immediately after the addition of angiotensin II, which became significant higher after 15 min. Intracelluar Ca2+ fluorescence intensity and density were significantly lower in telmisartan-treated atrial myocytes (for 15 min) than that angiotensin II-treated controls. Intracellular [Ca2+]i was significantly higher in AF atrial cells. [Ca2+]i was markedly lower in atrial cells from patients pretreated with telmisartan. 4. Under microscopy, it was found that cell number in the AF atrial tissues decreased. Hyperplasia was obvious in connective tissue. Lysis of myocytes could be seen. At ultrastucture level, reduced contraction elements were seen in AF atrial cells. Mitochondia hyperplasia occurred in areas with myofilament lysis. Gap junction protein CX40 decreased in
AF atrial cells and disturbed arrangement of Gap junction was detected. Conclusion 1. The human atrial cells obtained from acute isolation and in vitro culture have the morphological and electrophysiological features of cardiac cells. 2. Angiotensin II has significant effects on the electrophysiology of the atrial cells by influencing the ICa-L Ito and IK1, and telmisartan has antagonistic effects on angiotensin II, which indicats that angiotensin II participates in the process of AF. 3. Angiotensin II increases the intracellular calcium concentration of the atrial cells, which can be reversed by telmisartan. Telmisartan can also decrease the intracellular calcium concentration in the AF atrial cells, suggesting that calcium overload in cardiac cells is involved in the process of AF. 4. AF atrial cells undergo structural and ultrastructural alterations as well as gap junction protein changes. These changes may play key roles in the onset and persistence of AF.
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