研究背景: 心房颤动(房颤)是临床上最常见的快速室上性心律失常,是导致心血管疾病发生与死亡的一个重要原因。有越来越多的证据表明,肥胖是促进房颤发生与维持的一个新的危险因素。然而,其参与介导细胞凋亡、肥厚、间质纤维化的机制,包括各种血液动力学、神经介质、代谢相关因素间的复杂相互作用、炎症以及氧化应激等,尚不十分明确。 目前已知,高热量的摄入与久坐不动的生活方式即导致肥胖高发生率的主要原因。长期大量高脂饮食(high-fat diet,HFD)可促进心血管疾病、卒中等疾病的发生与发展。尽管肥胖与房颤发生与维持之间存在明确相关性,但一些常见的肥胖伴随情况如高血压、睡眠呼吸暂停以及糖尿病等均可促进心房重构,进而诱发房颤发生发展。因此对于长期高脂饮食摄入的非肥胖人群是否存在同样的心房重构及致心律失常作用仍需进一步研究。 心房结构重构和缝隙连接(gap junction,GP)重构是心房重构的重要组成部分。结构重构包括两方面,即心房扩大与组织纤维化。已知转化生长因β1(transforming growth factor-beta1, TGF-β1)可促进心房纤维化,进而诱发房颤。既往多项研究证实, TGF-β1的表达与肥胖显著相关。经证实,p38MAPK、ERK等信号通路参与房颤相关TGF-β1通路激活。而长期高脂饮食能否诱导p38 MAPK、ERK通路活化,上调TGF-β1,促进纤维化,进而导致房颤发生、维持还需进一步验证。此外,基质金属蛋白酶2 (matrix metalloproteinases 2,MMP-2),在促进房颤纤维化中也发挥着重要的作用。研究表明, MMP-2表达在房颤患者及肥胖患者心房组织中均明显上调。以上多项研究结果提示, TGF-β1与MMP-2可能是参与肥胖促进房颤发生与维持过程中的重要纤维化调节因子。而TGF-β1与MMP-2及其通路在长期高脂饮食而非肥胖与房颤相关性中的变化尚不明确,因此需进一步研究证实。 目前证实,在房颤患者及动物的心房组织中均观察到缝隙连接蛋白(connexin, Cx)表达和/或分布异常。此外,许多动物试验已证实,肥胖动物模型的心室与心房肌细胞中Cx43表达和或分布存在明显改变。但是,肥胖相关的心房肌细胞中Cx40的表达及分布研究相对较少。而长期高脂饮食而非肥胖的心房肌细胞中Cx40及Cx43的表达及分布的相关研究则更是少之又少。 研究目的: 通过长期HFD喂养构建动物模型,观察造模前后各项代谢指标及心房结构变化,同时采用RT-PCR、western blot及免疫荧光等实验方法检测各纤维化相关信号通路及缝隙连接蛋白的表达及分布。此外采用现有房颤造模方法,观察对比不同条件大鼠房颤发生率及持续时间,探讨研究高脂饮食对房颤发生与维持的影响。并设立离体实验,进一步明确高脂环境对心肌的影响,探索可能的发生机制,为房颤的防治提供依据及支持。 研究方法: 1)动物模型建立 61只SD雌性大鼠随机分配为两组,对照组(CT,n=21),高脂饮食组(HFD, n=40),分别给予普通饮食和高脂饮食,连续喂养12w。12w后,根据体重增长水平,将HFD饮食大鼠分为HFD诱导肥胖组(HFD-OB)以及HFD喂养非肥胖组(HFD-NOB)。 HFD大鼠模型建立后,随机选取每组三分之一大鼠建立房颤模型。充分麻醉后,经尾静脉给予CaCl₂+ACh混合溶液,标记给药时间,全程心电图监测。 2)研究指标 在体实验:造模后观察三组大鼠体重、心房/体重比、血脂水平;H&E及Masson染色心房组织,观察心肌排列、胶原含量变化;采用RT-PCR、western blot检测心房p38MAPK、ERK1/2、TGF-β1、Smad2/3、MMP-2、TIMP-2、Cx40 及 Cx43 的 mRNA与蛋白表达水平;通过免疫荧光检测心房Cx40及Cx43分布情况。 离体实验:首先行3T3-L1前脂肪细胞成脂分化,并提取SD乳鼠心肌细胞,随后采用Transwell体系行两种细胞共培养,结束后采用western blot检测心肌细胞p38 MAPK、ERK1/2、TGF-β1、Smad2/3、MMP-2、TIMP-2 及 Cx43 的蛋白表达水平。 研究结果: 1)一般情况及代谢参数变化:12w喂养结束后,相较于CT组,HFD-OB组大鼠出现HFD诱导性肥胖,体重显著增加(P<0.01)。而HFD-NOB组大鼠体重与CT组相比无显著差异,但HFD-OB及HFD-NOB组大鼠均出现了脂质代谢紊乱(P<0.05),而两组间并无显著差异。 2)心房结构重构:HFD-OB组(10.08±3.37%)与 HFD-NOB 组(12.10±1.00%)大鼠心房体重比明显高于CT组(7.07±0.84%),其中HFD-NOB组更为显著(与CT组相比,HFD-OBP<0.05, HFD-N0BP<0.001 );心房H&E染色显示HFD-OB组和HFD-NOB组大鼠心房肌排列显著杂乱无序,肌纤维中断呈团状或条索状、无连续性,间隙增宽,组间无显著差异;Masson染色示HFD喂养组大鼠心房肌胶原纤维含量显著增加;RT-PCR及WB检测示HFD-OB及HFD-NOB大鼠心房肌中MAPK、ERK1/2 mRNA及蛋白表达增加(P<0.05),通路激活,TGF-β1、Smad2/3 mRNA及蛋白上调(P<0.05),此外,MMP-2 mRNA 及蛋白表达增加(P<0.05)、TIMP-2 mRNA 及蛋白表达降低(P<0.05),两者平衡紊乱,综上促进心房扩大及纤维化,即发生心房结构重构。 3)心房缝隙连接重构:RT-PCR及WB检测示HFD喂养两组大鼠心房中Cx40及Cx43 mRNA及蛋白表达水平均显著降低(P<0.05),两组间对比无显著差异。同时免疫荧光染色发现这两组心房组织中Cx40及Cx43不仅表达减少,分布也以侧-侧连接为主,而CT组分布则主要呈端-端连接。 4)房颤诱发:经尾静脉给药刺激后,HFD-OB组中共9只大鼠诱发出AF,诱发率90.0% (与CT组相比,P<0.01); HFD-NOB组5只,诱发率71.43% (与CT组相比,P<0.01); HFD-OB与HFD-NOB组间无显著区别。HFD-OB组室性心律失常发生率 30.0%,死亡率 20.0%(与 CT 组相比,P<0.01 ); HFD-NOB 组为别为 28.57%,14.28% (与CT组相比,P<0.01)。而CT组仅1例发生VT,后自行恢复窦律,室性心律失常发生率10.0%,无死亡发生。 5)离体实验结果:通过诱导分化3T3-L1细胞,提取乳鼠心肌细胞,并经Transwell共培养后,共培养心肌细胞较对照组普通培养心肌细胞,其p38 MAPK、ERK1/2、 TGF-β1、Smad2/3、MMP-2的蛋白表达水平显著上调(P<0.05),TIMP-2及Cx43表达显著减少(P<0.05),结果与在体实验一致。 结论: 1)长期高脂饮食摄入可导致大鼠机体脂质代谢紊乱; 2)高脂饮食可通过激活大鼠心房肌p38MAPK、ERK信号通路,促进TGF-β1表达,进一步作用于下游Smad2/3蛋白,诱发心房纤维化,导致心房结构重构; 3)高脂饮食可诱发大鼠心房组织MMPs/TIMPs失衡,从而促进胶原增生、结构紊乱,进一步加重心房纤维化; 4)长期高脂饮食摄入可导致大鼠心房肌组织Cx40与Cx43表达及分布异常,导致心房电重构; 5)长期高脂饮食可通过上述心房结构重构、Cx重构相关改变,最终导致大鼠心房颤动易感性增加。 关键词:房颤;肥胖;高脂饮食;结构重构;缝隙连接 论文类型:应用基础
Background: Atrial fibrillation (AF) is the most common supraventricular arrhythmia in ciinical practice and considered to be a growing cardiovascular epidemic . Emerging evidence demonstrates that aging alone does not account for the standing increase in AF prevalence . Obesity has been characterized as a new risk factor contributing to AF . Obesity is a global pandemic with more than two-thirds of adults being overweight or obese . It is well documented that high calories intake and sedentary lifestyles are the underlying causes of high prevalence of obesity . Exposure to chronic high-fat diet (HFD) not only link to diabetes, it also has strong association with cardiovascular disease, stroke and the like . although analysis of numerous studies over the last decades has displayed the effect of obesity on atrial structural and gap junctional (mainly focused on connexin43 expressed and distributed in ventricles) remodeling, the mechanisms remain incompletely elucidated. Despite the unequivocal linkage between obesity and AF, it remains currently unknown whether structural and gap junctional remodeling following a chronic HFD feeding and/or increased AF risks are prevalent in the absence of HFD-induced obesity. Objective: Our study fed rats with ND or HFD to build animal model, then analyzed the structural and gap junctional electrophysiological alterations in the atria, determined the impact of HFD on the susceptibility to atrial fibrillation,investigated whether this effect might be the same in both HFD-induced obesity (HFD-OB) and HFD fed non-obesity (HFD-NOB) rats, and aimed at identifying the underlying mechanisms. Method: 1.In vivo, 61 female rats were randomly fed a ND(n=21) or consistent HFD(n=40) for 12wk. After 12wk of HFD feeding, HFD fed rats were divided into HFD-induced obesity group (HFD-OB) and non-obesity group (HFD-NOB) according to the levels of weight increase. Then blood samples were collected to test the levels of total cholesterol (TC),high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and triglycerides (TG). In addition, we performed H&E and Masson stainings to observe fibrosis in atrial tissue of different groups. RT-PCR and western blot were performed to test the expressions of fibrosis prevelant moleculers, such as p38 MAPK, ERK1/2, TGF-β1, Smad1/2, MMP-2 and TIMP-2, and connexins, including Cx40 and Cx43. Further more, we used IF to observe the distribution of Cx40 and Cx43 in atria. 2.In vitro, we performed differetiation of 3T3-L1 pre-adipocytes and extracted primary rat cardiac cell, then co-cultured these two kinds of cells by using Transwell system. After the co-culture, we performed to test the expressions of fibrosis prevelant moleculers, such as p38 MAPK, ERK1/2, TGF-β1, Smad1/2, MMP-2 and TIMP-2, and connexin, i.e.Cx43. Results: 1.Female SD rats fed with HFD showed differences in weight increase: After 12wk of feeding, only approximate three fifths of HFD fed rats (HFD-OB, n=26) displayed significantly higher body weight increase than the rest of HFD fed rats (HFD-NOB, n=14) and those fed with ND (CT, n=20) . In comparison to the CT rats, no significant difference in weight increase was shown in NOB rats. 2.Both HFD-OB and HFD-NOB rats exhibited dyslipidemia: HFD-NOB rats displayed a marginal elevation in TG amounts, as well as significantly up-regulated levels of TC and LDL-C, as compared to the CT rats. Though TG, TC and LDL-C levels all markedly increased in HFD-OB rats. The HDL-C levels did not reveal any difference among these three groups. The results indicated a disturbed lipid homeostasis in HFD fed rats. 3.HFD fed rats exhibited more extensive atrial fibrosis: H&E staining of atrial paraffin sections revealed more broadened interstitial space among atrial myofibers as well as distinctly disordered layout of atrial myocytes in both HFD-OB and HFD-NOB rats instead of normal myocardial structure. H&E staining of atrial tissue of OB and NOB rats did not exhibit any difference. Significant fibrosis was shown in Masson staining of HFD-OB and HFD-NOB rats atrial sections. Plenty of fibrosis was especially visible around atrial myocytes and distributed disorderly. On the contrary, the tiny amounts of collagen fibers in CT rats atria were continuous and complete. Compared with HFD-OB rats, the collagen area of HFD-NOB rats atria was larger, while there was no significant difference between these two groups. 4.HFD feeding up-regulated expressions of mRNA and proteins related to atrial fibrosis: RT-PCR and WB analysis of p38, ERK1/2, TGF-β1,Smad2/3 and MMP-2 exhibited that the expressions of mRNA and proteins significantly up-regulated in HFD-OB and HFD-NOB rats atrial myocytes. While the expression of TIMP-2 mRNA and protein markedly down-regulated in HFD-OB and HFD-NOB rats atrial myocytes. No significant difference was observed between these two groups. 5.HFD feeding down-regulated expressions of gap junction mRNA and proteins: RT-PCR analysis displayed a significant down-regulation in the expression of Cx40 and Cx43 mRNA in HFD fed rats atrial tissue, meanwhile, such an marked decrease of the proteins expression of Cx40 and Cx43 was observed in western blotting assay as well. Neither the RT-PCR nor the western blotting analysis showed significant differences between HFD-OB and HFD-NOB groups. These altered expressions of gap junction connexins suggested that HFD could impact the electrical remodeling to a certain extent, which may further provide the substrate susceptible to AF. 6.HFD fed rats showed altered distribution of gap junction in atria: Cx40 and Cx43 were mainly located at the intercalated discs of atrial myocytes in CT rats. Cx40 proteins expressions displayed significant down-regulations in HFD rats atrial myocytes via western blotting, supporting this results are the data of immunofluorescence approach. In addition, Cx40 was showed to be laterally distributed along the longitudinal atrial myocytes membranes. Cx43 was markedly down-regulated and laterally distributed in HFD-OB rats atria as well. Nevertheless, in comparison to the CT rats, Cx43 signals seemingly did not show a significant decrease in HFD-NOB rats. Though lateralized distribution of Cx43 was still observed in HFD-NOB rats atrial myocytes. These alterations in expressions and distributions of gap junction revealed that it may be more susceptible to AF via the HFD-induced connexin remodeling. Besides, HFD-OB and HFD-NOB rats showed similar levels of on gap junctional remodeling induced by HFD. 7.HFD fed rats showed high AF inducing rate: After the AF inducing by drugs, AF inducing rate of HFD-OB rats was 90.0%(n=9), which was significantly higher than that of CT rats(P <0.01); the rate of HFD-NOB was 71.43%(n=5), which was also significantly higher than that of CT rats(P<0.01);No significant difference was observed between these two groups. Compared with CT rats, HFD-OB and HFD-NOB rats had higher rates of ventricular arrythmia and death(P<0.01). 8.In vitro, after differentiation of 3T3-L1 pre-adipocytes and extraction of primary rat cardiac cells, then co-culture of these two kinds of cells by using Transwell system was performed. Western blot revealed that the expressions of p38 MAPK、ERKl/2、TGF-β1、 Smad2/3 MMP-2 proteins were significantly up-regulated in both HFD-OB and HFD-NOB rats(P<0.05), while, the expressions of TIMP-2 and Cx43 proteins were significantly down-regulated in both HFD-OB and HFD-NOB rats(P<0.05).No significant difference was observed between these two groups. Conclusion: 1.Chronic exposure to high-fat diet may induce dyslipidemia. 2.HFD could activate p38 MAPK and ERK signalling pathways, up-regulate expression of TGF-β1, then effect Smad proteins, further increase atrial fibrosis, finally induce atrial remodeling. 3.HFD coule induce imbalance of MMPs/TIMPs, therefore increase collagen hyperplasia and disturb the structure of collagen, further increase atrial fibrosis. 4.Chronic HFD could induce abnormal expression and distribution of Cx40 and Cx43, lead to atrial electrical remodeling. 5.Chronic HFD could increase the risk of atrial fibrillation by promoting atrial structure and gap junction remodeling mentioned above. KEY WORDS: Atrial fibrillation; Obesity; High-fat diet; Structural remodeling; Gap juction TYPE OF DISSERTATION: Application Fundamentals