| Atrial fibrillation(AF)is a prevalent sustained cardiac arrhythmia that affects approximately 1%of the global population.AF often results in rapid and irregular heart rhythm,leading to decreased quality of life,increased healthcare burden,as well as significantly higher incidence of complications and mortality.Despite the identification of certain mechanisms underlying the occurrence and progression of AF,such as ion channel remodeling and fibrosis,the treatment of AF remains a significant challenge.This is primarily due to our incomplete understanding of the underlying mechanisms of atrial remodeling in AF.Hence,exploring novel therapeutic strategies targeting atrial remodeling may offer new insights and approaches for the prevention and treatment of AF.Structural remodeling of the atria plays a crucial role in the initiation and maintenance of AF,particularly through the process of fibrosis.Excessive accumulation of extracellular matrix proteins,such as collagen and fibronectin,during atrial fibrosis leads to the formation of fibrotic tissue,which occupies the space of normal atrial myocardial cells.This impairs the contraction and relaxation functions of the atria,disrupts electrical coupling between myocardial cells,and forms anatomical barriers to electrical conduction.The formation of these fibrotic anatomical barriers further slows down the conduction and spread of electrical signals,creating areas of irregular conduction,promoting reentry circuits,and facilitating ectopic electrical activities,thus acting as triggers and perpetuators of AF.Despite the pivotal role of atrial structural remodeling in the initiation and progression of AF,our current understanding of the underlying mechanisms is still limited due to the complexity of atrial tissue,impeding in-depth exploration of AF mechanisms.The advent of single-cell sequencing provides a unique opportunity to unravel the complexity of atrial tissue at the resolution of individual cells.Through single-cell sequencing,we can delve into the cellular composition of the atria and identify key cell types involved.With a focus on these key cell types,we further investigate the cellular characteristics and intercellular interactions during atrial structural remodeling in AF.These findings will provide us with a deeper understanding of the mechanisms underlying AF and offer novel insights for AF treatment.This study comprises the following three main components:Part Ⅰ Single-cell atlas of the adult atrium and key cells involved in atrial structural remodeling in atrial fibrillationBackground:Atrial fibrillation(AF)is the most common sustained clinical arrhythmia.Atrial structural remodeling is a key process that promotes the occurrence and maintenance of AF.However,the underlying mechanism of this process remains unclear.Therefore,constructing a single-cell atlas of the atrium from normal to AF and identifying key cell subsets would be of great significance in uncovering the mechanisms underlying atrial structural remodeling in AF.Objective:To construct a single-cell atlas of the adult atrium and identify key cell subsets involved in the process of atrial structural remodeling in AF.Methods and results:This study aimed to construct a single-cell atlas of adult atrium and identify key cells involved in the process of atrial structural remodeling in AF.All available datasets involving atrial samples from public databases were extracted for singlecell integration analysis,revealing that the atrium is primarily composed of eight cell types,including cardiomyocytes(CMs,31.0%),fibroblasts(FBs,26.1%),pericytes(PCs,13.0%),endothelial cells(ECs,11.3%),myeloid immune cells(6.2%),smooth muscle cells(4.0%),lymphoid immune cells(3.2%),and adipocytes(2.3%).Among these,ECs play a major regulatory role in normal and potentially remodeling atria,with the interaction with FBs being the most significant.To investigate the role of ECs in AF,we collected left atrial appendages from three patients undergoing surgical ablation of AF and corresponding parts of three donor hearts in sinus rhythm for single-cell sequencing.Pseudotime analysis revealed that atrial FBs were significantly activated in AF,leading to typical structural remodeling,such as fibrosis.This process was primarily regulated by ECs,with TGFB1 being the most critical cytokine acting on FBs.The spatial accessibility of this interaction was further verified by constructing a Colla2-Cre;R26RGFP tracer mouse for FBs.Additionally,during atrial structural remodeling in AF,ECs significantly upregulated mesenchymal markers,indicating their mesenchymal activation plasticity.Conclusion:The adult atrium is composed of multiple cell types,with ECs playing a crucial regulatory role and serving as the key cell subpopulation driving the structural remodeling of AF.Single-cell sequencing analysis has revealed that ECs activate FBs via the TGFB pathway to participate in the atrial structural remodeling in AF,and ECs exhibit significant mesenchymal activation and plasticity during this process.Part Ⅱ Lineage tracing verifies endothelial plasticity with mesenchymal activation during atrial structural remodeling in atrial fibrillationBackground:The single-cell atlas of atrial fibrillation(AF)has highlighted the plasticity of mesenchymal activation in endothelial cells(ECs),which plays an important role in the atrial structural remodeling.Nevertheless,there is still debate over the existence of endothelial-mesenchymal transition and the significance of its contribution to fibrosis.To gain more insight into the mesenchymal activation of ECs and its contribution to atrial structural remodeling in the context of AF,it is crucial to combine animal models of AF,lineage tracing technology,and single-cell sequencing.Objective:To identify an appropriate mouse model for AF-related structural remodeling,trace ECs in vivo,and clarify the mesenchymal activation characteristics of ECs and their contribution to fibrosis during atrial structural remodeling in AF.Methods and results:In this study,we constructed four mouse models,including an angiotensin II-induced atrial fibrosis model(AngⅡ),an aortic coarctation model(TAC),a myocardial infarction model(MI),and a guidewire injury valve model(Wire injury).Corresponding models were evaluated for atrial size,fibrosis,and electrophysiological phenotype at 4-,8-,8-,and 12-weeks post-operation.The size of the atrial chamber was assessed by ultrasound,and the anteroposterior diameter of the left atrium was measured.The activation characteristics of fibroblasts were assessed by Masson staining and immunofluorescence for fibrosis,and the induction rate of atrial fibrillation and the effective refractory period of the atrial chamber were measured by intracardiac catheterization for electrophysiological phenotype.The results demonstrated that,except for the Wire injury model,the AngⅡ,MI,and TAC models exhibited significantly enlarged atria,which were 1.3,1.2,and 1.4 times that of the sham operation group,respectively.Fibrosis was significantly increased,2.1,1.9,and 7.1 times that of the sham group,respectively.Among the four models,only the AngⅡ and TAC models significantly increased the induction rate of atrial fibrillation,which were 2.03 and 2.09 times that of the sham group,respectively.In all models,only subendocardial fibroblasts were activated after TAC,and the fibrosis distribution of the TAC model was the closest to that of human atrial tissue.TAC was selected as the subsequent modeling procedure for atrial fibrillation in ECs tracer mice(Cdh5-Cre;R26RRFP),and RFP cells were sorted before and after atrial intervention for single-cell sequencing.Pseudotime analysis and gene scoring revealed distinct differentiation trajectories of ECs during atrial remodeling,all of which exhibited features of mesenchymal activation.However,immunofluorescence showed that the number of ECs transformed into mesenchymal cells at the protein level was small,and there were differences in the mesenchymal activation of ECs at the transcriptional and protein levels.Conclusion:The TAC model has significant atrial fibrosis and a high induction rate of AF and is the most suitable mouse model for the study of atrial remodeling,especially structural remodeling in AF.On the basis of TAC,lineage tracing combined with singlecell sequencing clarified the plasticity of ECs mesenchymal activation during atrial structural remodeling in AF.However,there are differences in transcription and protein levels in this plasticity,which needs to be further explored and studied in the future.Part Ⅲ Mesenchymal-activated endothelial cells participate in atrial structural remodeling in atrial fibrillation by regulating fibroblast function through the TGFB1 pathwayBackground:Lineage tracing combined with single-cell sequencing has clarified the plasticity of ECs in mesenchymal activation during atrial structural remodeling in AF.However,this feature appears inconsistent at the transcriptional and protein levels.While mesenchymal activation is the most prominent feature in the differentiation trajectory of ECs at the transcriptional level,ECs that have completely transformed into mesenchymal cells are extremely rare at the protein level.This suggests that ECs do not participate in the process of atrial structural remodeling by transforming into mesenchymal cells.The significance of mesenchymal activation of ECs requires further exploration.Objective:To elucidate the significance of mesenchymal activation of ECs and the primary mechanism by which ECs contribute to atrial structural remodeling in AF.Methods and results:In this study,we employed immunofluorescence and highthroughput cell phenotype detection to confirm that mesenchymal activation of ECs seldom leads to their complete transformation into mesenchymal cells.Co-localization of ECs and COL 1A1,PDGFRα,DDR2,FAP,and LTBP2 with RFP was absent in ECs tracer mice(Cdh5-Cre;R26RRFP)after TAC,except for α-SMA.Only 0.1%of ECs tracer mice captured α-SMA+EC,which suggests that mesenchymal ECs were infrequent.Mesenchymal ECs were not observed in other models,including AngⅡ,MI,and Wire injury.Using high-throughput cell phenotype detection,we dynamically observed the mesenchymal activation process of ECs,which showed significant changes in cell area,length,and width.The roundness and width-to-length ratio decreased significantly,and the proportion of ECs transformed into mesenchymal cells was about 0.3%.The migration rate of ECs also significantly decreased,but the other parameters did not conform to the traditional phenomenon of endothelial-mesenchymal transition.Analysis of cell communication suggested that mesenchymal activation in ECs plays a role in regulating other cells,particularly FBs.To verify this hypothesis,we sorted human atrial primary ECs and FBs for cell interaction experiments,which demonstrated that mesenchymal ECs abnormally release TGFB1 to promote FB activation,proliferation,collagen deposition,and release.The TGFB1-specific receptor inhibitor SB431542 effectively inhibited this process.In vivo,overexpression of Tgfbl in ECs by the adeno-associated virus RGDLRVS-AAV9-Cdh5-Tgfb1 led to significant atrial enlargement,increased fibrosis,and prolonged duration of AF.Conclusion:In the process of atrial structural remodeling in AF,the main role of ECs in mesenchymal activation is not to transform themselves into mesenchymal cells to contribute to fibrosis,but rather to promote the activation,proliferation,collagen deposition,and release of FBs and other fibrotic phenotypes.This process is primarily mediated by the TGFB1 pathway. |
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