目的：心房颤动(atrial fibrillation)简称房颤，是临床常见的快速性心律失常。本研究通过对风湿性心脏病慢性房颤及窦性心律患者进行血浆蛋白质组学和代谢组学研究，从蛋白质组和代谢组水平揭示心房颤动时的细胞生物学改变，深入阐明房颤机理，为发现更安全有效的房颤防治方法奠定基础。

方法：选取2013年1月-2016年12月就诊于天津泰达国际心血管病医院并诊断为风湿性心脏病、进行心脏瓣膜置换手术的患者100例（慢性房颤患者70例，窦性心律患者30例）纳入研究。手术前一日采集患者外周静脉血2ml，离心后取上层血浆-80℃冻存备用。选取慢性房颤和窦性心律(sinus rhythm, SR)患者各12例进行蛋白质组学测定，采用同位素标记相对和绝对定量（isobaric tags for relative and absolute quantification，iTRAQ）技术标记样本后，利用质谱（mass spectrometry，MS）技术对样本中的肽段进行识别，将串联质谱数据与数据库中的理论质谱数据匹配分析后，得到蛋白质的鉴定结果。以蛋白质的差异变化倍数（fold change, FC）为1.2倍且p-value =< 0.05为标准筛选差异蛋白。对差异蛋白质进行生物信息学分析，包括GO富集分析、KEGG pathway分析和蛋白互作（PPI）分析。选取差异蛋白中的10种进行酶联免疫吸附测定（enzyme linked immunosorbent assay, ELISA）验证。利用气相色谱与质谱联用技术（gas chromatography-mass spectrometer，GC-MS）对30例慢性房颤患者和26例窦性心律患者的血浆进行代谢组学研究分析。采用多维分析和单维分析相结合的办法筛选组间差异代谢物，并对差异代谢物进行KEGG pathway分析。在血浆蛋白质组学和代谢组学研究的基础上，结合相关文献报道，利用生物信息学工具进行整合组学分析。

结果：iTRAQ测定共发现了27344个Unique 肽段，鉴别出447种非冗余蛋白质，以FC变化为1.2倍且p-value=<0.05为条件筛选出57种差异蛋白，其中33种蛋白质表达上调，24种蛋白质表达下调。KEGG pathway分析发现过氧化物酶体增殖剂激活受体α通路、肾素-血管紧张素-醛固酮系统及凝血和补体系统等多个通路在房颤的发生发展中发挥着重要的作用。选取载脂蛋白A1（apolipoprotein A1）、载脂蛋白A2（apolipoprotein A2）、LIM结构域蛋白7（LIM domain only protein 7）、凝血酶原片段1+2（prothrombin fragment 1+2）、玻连蛋白（vitronectin）、血管紧张素原（angiotensinogen）、ficolin-3、血清淀粉样蛋白P（serum amyloid P-component）、巨噬细胞集落刺激因子受体1（macrophage colony-stimulating factor 1 receptor）、嘌呤核苷磷酸化酶（purine nucleoside phosphorylase）等10种蛋白质进行ELISA检测，其中载脂蛋白A1（房颤组为499.5 ± 59.79ug/ml，窦性心律组为725.8 ± 83.75ug/ml）、LIM结构域蛋白7 (房颤组为22.31 ± 1.50pg/ml，窦性心律组为16.99 ± 1.88pg/ml)、巨噬细胞集落刺激因子受体1（房颤组为37.76 ± 2.65ng/ml，窦性心律组为66.30 ± 10.82ng/l）、玻连蛋白（房颤组为53.28 ± 7.20ug/ml，窦性心律组为84.89 ± 15.87ug/ml）、凝血酶原片段F1+2（房颤组为0.97±0.06nmol/ml，窦性心律组为0.76±0.05 nmol/ml）和血管紧张素原(房颤组为208.3 ± 18.29pg/ml，窦性心律组为294.1 ± 36.84pg/ml)在房颤组和窦性心律组差异表达。采用GC-MS代谢组学技术检测发现了322种代谢产物，以OPLS-DA模型VIP>1，且p值<0.05为标准筛选出差异代谢物55种，其中表达上调的差异代谢物有38种，表达下调的差异代谢物有17种。对差异代谢物进行KEGG pathway分析，发现半乳糖代谢、不饱和脂肪酸合成、亚油酸代谢、ABC转运子和淀粉与蔗糖代谢等代谢通路与慢性房颤有关。

结论：本研究发现风湿性心脏病慢性房颤患者与窦性心律患者的血浆中存在差异表达的蛋白质和代谢产物，这些差异蛋白和差异代谢物及其参与的过氧化物酶体增殖剂激活受体α通路、肾素-血管紧张素-醛固酮系统、凝血和补体系统、半乳糖代谢、不饱和脂肪酸合成、亚油酸代谢、ABC转运子和淀粉与蔗糖代谢等通路与房颤密切相关。另外，慢性房颤时心脏出现代谢重构，心肌对脂肪酸的利用减少，对葡萄糖的利用增加，而PPARα通路在心脏的代谢重构中发挥着重要作用。

Objectives: Atrial fibrillation(AF) is one of the most common arrhythmia encountered in the clinical practice. In this study, we aimed to identify the molecular biology changes and mechanisms responsible for chronic atrial fibrillation. This study will help to lay the foundation for discovering more safety and effective ways to prevent and treat AF in the future.

Methods: 100 patients with rheumatic heart diseases who came to TEDA-international cardiovascular hospital, Tianjin, China, for clinical treatment from January 2013 to December 2016 were enrolled in our study. 70 patients with chronic atrial fibrillation were assigned to the AF group and 30 patients with sinus rhythm were assigned to the SR group. Isobaric tags for relative and absolute quantification (iTRAQ) technique and gas chromatography-mass spectrometer (GC-MS) technique were performed on the two groups to identify proteins and metabolites. Bioinformatics analysis including Gene Oncology enrichment analysis, KEGG pathway analysis and protein-protein interaction analysis were carried out to identify novel biomarkers and pathways in atrial fibrillation. We then selected 10 proteins for proteomic validation to verify the results of iTRAQ analysis. KEGG pathway analysis were also performed in the GC-MS metabolomic study to help identify possible pathways involved in the atrial fibrillation process.

Results: iTRAQ combined with MS techniques identified 27344 peptides or proteins, of which 57 were found to be differentially expressed, i.e., the proteins fulfilled both the fold change(FC)>1.2 or FC<0.8 and p value=<0.05 criteria. Among the 57 differentially expressed proteins, 33 of which were elevated and the remaining 24 proteins were decreased in the AF group. KEGG pathway analysis discovered that peroxisome proliferators-activated receptors (PPAR) pathway, renin angiotensin aldosterone system (RAAS) and coagulation and complement systems were involved in the process of atrial fibrillation. We selected 10 proteins for ELISA validation, including apolipoprotein A-I, apolipoprotein A-II, LIM domain only protein 7, prothrombin fragment 1+2, vitronectin, angiotensinogen, ficolin-3, serum amyloid P-component, macrophage colony-stimulating factor 1 receptor and purine nucleoside phosphorylase. Apolipoprotein A-I (AF:499.5 ± 59.79ug/ml,SR:725.8 ± 83.75ug/ml), LIM domain only protein 7(AF: 22.31 ± 1.50pg/ml, SR: 16.99 ± 1.88pg/ml), angiotensinogen(AF:208.3 ± 18.29ng/ml, SR: 294.1 ± 36.84ng/ml), macrophage colony-stimulating factor 1 receptor (AF:37.76 ± 2.645ng/ml, SR: 66.30 ± 10.82ng/ml), prothrombin fragment 1+2(AF: 0.97±0.06nmol/ml, SR: 0.76±0.05 nmol/ml) and vitronectin(AF: 53.28 ± 7.20ug/ml, SR:84.89 ± 15.87ug/ml) were found differentially expressed among the two groups by ELISA test. GC-MS metabolomic analysis discovered 322 metabolites and 55 of them were differentially expressed between the AF and SR groups. 38 of the metabolites were elevated and 17 of them were decreased. KEGG pathway analysis of the differentially expressed metabolites revealed that galactose metabolism, biosynthesis of unsaturated fatty acids, linoleic acid metabolism, ABC transporters and starch and sucrose metabolism may be related with chronic atrial fibrillation.

Conclusions: We for the first time identified the differentially expressed plasma proteins and metabolites in chronic atrial fibrillation patients. Our study suggested that PPARα pathway, RASS, coagulation and complement systems, galactose metabolism, biosynthesis of unsaturated fatty acids, linoleic acid metabolism, ABC transporters and starch and sucrose metabolism were closely related with atrial fibrillation. Chronic atrial fibrillation may display metabolic remodeling, another form of remodeling other than electrical remodeling and structure remodeling. The utilization of lipids was decreased and the oxidation of glucose was increased in the cardiac tissue. PPARα pathway is an important cellular pathway modulating the process of energy metabolism and it is of vital importance in the atrial fibrillation metabolic remodeling process.