The Role Of NEXN In Ischemic Cardiomyopathy And Single-cell Analysis Of The Cellular Microenvironment Of Coronary Atherosclerosis

Coronary heart disease(CAD)is the most common cardiovascular disease.With the increase of population aging worldwide,despite the wide application of prophylactic drugs,the incidence of ischemic cardiomyopathy(ICM)caused by coronary atherosclerotic lesions is still increasing year by year,and the mortality of acute myocardial infarction is on the rise.Early recovery of blood perfusion is the most effective strategy for the treatment of myocardial ischemia.However,before the revascularization,how to save the viable myocardium and reduce cell hypoxia injury is the key direction for the treatment of ischemic cardiomyopathy.At the same time,the vascular stenosis caused by coronary atherosclerotic lesions is the culprit of ischemic cardiomyopathy.How to slow down the progress of coronary atherosclerotic lesions and reverse vascular stenosis has become a major problem to be solved in the cardiovascular field.Therefore,a systematic description of the composition and microenvironment of coronary atherosclerosis cells and a comprehensive description of the important cell types and functions involved in disease progression provide an important theoretical basis for clarifying the pathogenesis of atherosclerosis and exploring effective intervention targets.In the early stage of myocardial hypoxia,myocardial cell membrane structure damage is the first ultrastructural changes,the earliest can appear in 10-15 minutes after ischemic attack.The integrity of membrane structure and function is crucial to the fate of cardiomyocytes.Therefore,early intervention to maintain the integrity of the membrane structure has become a major direction of myocardial protection.As an important part of the membrane structure and electrophysiological activities of cardiomyocytes,Junctional Membrane Complexes(JMCs)are important structural units to maintain the function of cardiomyocytes.Therefore,the first part of this study explored the expression patterns of NEXN,one of the important components of JMCs,during myocardial ischemia and its application in maintaining cardiac systolic function.In addition,due to the high expression abundance of NEXN in myocardial tissue and its specific expression in muscle tissue,this study also preliminarily explored its application as a candidate marker for ischemic cardiomyopathy.Coronary atherosclerotic diseases involve multiple cells,and there are complex cellular transformations in different pathological periods.Previous studies only focused on the functional changes of some cells in the progression of the disease,lacking the whole-cell description of coronary atherosclerotic lesions,and failed to systematically clarify the key cell types involved in the lesions.With the emergence of single cell technology,our analysis of tissue cell subtypes has reached an unprecedented resolution.Therefore,in the second part of this study,we performed single-cell transcriptome analysis of human coronary atherosclerosis tissues to give full play to the advantages of different single-cell platforms,and further comprehensively described coronary atherosclerosis.In summary,this study focuses on the exploration of the mechanism of coronary heart disease and the application of single cell technology in cardiovascular diseases.The details of the two parts are summarized as follows:Part 1:The role of NEXN in maintaining cardiac systolic function and as a candidate protein biomarker for ischemic cardiomyopathyBackground:The normal function of cardiomyocytes depends on its highly refined subcellular structure,in which Junctional Membrane Complexes(JMCs)are the key structures for normal cardiac excitation-contraction coupling.The structure and function of JMCs depends on their constituent proteins such as JPH2,BIN1,CAV3 and NEXN,which play an important role in regulating the location and functional activity of JMCs.JMCs can show compensatory remodeling in the pathological process,but with the degradation of JMCs structure will lead to the damage of myocardial contractility and arrhythmia,which leads to the decompensation of cardiac function and the occurrence of heart failure.As a newly discovered JMCs constituent protein,the role of NEXN in myocardial ischemia remains to be further explored.Methods:The expression characteristics of NEXN in ischemic cardiomyopathy were explored through RNA-seq data of ischemic cardiomyopathy and single cell data of human heart.The in vivo and in vitro models were used to detect the expression changes of NEXN in myocardial ischemia,and then the effects of up-regulation of NEXN on cardiac systolic function were explored by different overexpression methods.RNA-seq and TMT quantitative proteomics were used to explore the changes in pathway function caused by overexpression of NEXN.To detect the expression pattern of NEXN in vitro and in vivo and explore its application prospect as a candidate protein biomarker of ischemic cardiomyopathy.Results:The transcriptome data of ischemic cardiomyopathy showed that NEXN was highly expressed in cardiac cardiomyocytes,and was down-regulated in the process of disease.In vivo and in vitro ischemia and hypoxia experiments confirmed that the expression of NEXN decreased gradually with the prolongation of ischemia time.By using the heart-specific expression vector of adeno-associated virus(AAV)for primary cardiomyocytes to construct the in vivo NEXN up-down-regulation model,we found that the down-regulation of NEXN led to the damage of cardiac systolic function in adult rats under physiological conditions,and the confocal experiment found that the down-regulation of NEXN led to the structural disorder of cardiomyocytes.Furthermore,it was found in the study of cardiac ischemia model that pre-intervention of NEXN(up-regulation)could reduce ischemic injury to a certain extent and maintain cardiac systolic function.Furthermore,we found that NEXN was up-regulated by transcriptome combined with proteome sequencing.Differential gene enrichment analysis showed that NEXN was related to calcium channel,myocardial contraction,sarcolemma stability and transcriptional activity regulation.Bioinformatics analysis showed that NEXN up-regulation could lead to the expression of key proteins of Rho/Rock pathway(Rho family,Rhoa,Rhoc)in its protein regulatory network and inhibit Rho/ROCK pathway to reduce cardiomyocyte apoptosis and inflammatory response.Further study on the release pattern of NEXN in vivo and in vitro under hypoxic injury showed that the release of NEXN during cardiac injury was higher than the baseline level,and it was one of the ideal candidate protein biomarkers for ischemic cardiomyopathy.Conclusion:The expression of NEXN decreased with myocardial ischemia,and gradually released with the prolongation of ischemia time,and could be stably detected in peripheral blood.In vivo and in vitro functional experiments showed that up-regulation NEXN expression could lead to maintain the structure and function of cardiomyocytes.In addition,the RNA-seq combined with the TMT quantitative proteomics found that up-regulation of NEXN contributed to myocardial contraction,calcium channel function and membrane structure stability,and may reduce myocardial cell apoptosis and inflammatory response by inhibiting Rho/ROCK pathway,thereby playing a myocardial protection role.Part 2:Single-Cell Transcriptome Analysis of the Landscape of Human coronary atherosclerotic plaque Background:The progression of atherosclerosis involves the participation of multiple cells.The proportion and transformation process of cells in different pathological periods are very complex,and the key cell types and functions are still unclear.Although the cell heterogeneity of atherosclerosis was studied in mice and other human arterial tissues,the cell composition of specific human cardiac coronary artery is still unclear.In this study,single cell transcriptome sequencing combined with coronary heart disease GWAS data was used to describe the distribution of human coronary atherosclerotic cell subsets in detail,and the key cell types affecting disease progression were analyzed.Methods:Single cell suspension was obtained by enzymatic digestion of coronary artery tissue from recipients with heart transplantation.Two sequencing platforms were used for single cell transcriptome sequencing.The function and type of cell subsets were analyzed by histopathology,immunofluorescence and flow cytometry combined with bioinformatics.Results:Single-cell sequencing was performed on 72554 cells in 69 coronary-tissues from 25 heart transplant recipients.By bioinformatics analysis,we identified 34 subgroups representing 8 major coronary artery cell types:vascular smooth muscle cells(6 subgroups),fibroblasts(6 subgroups),macrophages(5 subgroups),T cells(4 subgroups),endothelial cells(5 subgroups),NK cells/Mast cell(3 subgroups),myofibroblasts(3 subgroups),and B/plasma cells(2 subgroups).Interstitial cells(vascular smooth muscle cells,fibroblasts,and myofibroblasts)constitute the main components of coronary artery,followed by macrophages,T cells,endothelial cells,and NK cells.By comparing the cell composition changes in different pathological stages,some immune cell clusters and interstitial cell clusters were found to change with the disease.Interstitial cells have a wide range of intercellular transformation processes with the progression of diseases,and there may be special T cell cluster in immune cells that affect the repair process of plaque healing.Intercellular communication analysis showed that the communication between endothelial cells and macrophages was predominant in progressing condition.The enhanced effect of HGF-MET in specific cell subsets may be related to the repair of plaque tissue.Through the combined analysis of the susceptible genes related to coronary heart disease found in GWAS study,it was found that the susceptible genes were mainly enriched in macrophages and fibroblasts.Combined with the data of coronary atherosclerosis proteomic study,it was found that the up-regulated proteins in the early stage were enriched in myofibroblast cells,well the up-regulated proteins in advanced stage were significantly enriched in macrophages.In-depth understanding of the functional changes of key cell types in the progression of diseases is essential for effective intervention in the progression of atherosclerosis.Conclusion:In this study,the single cell panorama of human coronary artery tissue was drawn,and the cell composition at different stages of disease progression was characterized.Our results can be used as a high-quality single cell database for the process of human coronary atherosclerosis,and help to explore the therapeutic targets for such diseases.