Single-cell Sequencing Reveals Cardiac Cells Heterogeneity And Mechanism Of Myocardial Injury In Hypercholesterolemic Mice

BACKGROUND:Cardiovascular disease（CVD）is the leading cause of deaths globally,with high mortality and morbidity rates.According to statistics,the annual prevalence of cardiovascular disease in the world is extremely high.An estimated 26million adults in the United States have been diagnosed with CVD,and the number of patients with CVD in China is estimated to be as high as 330 million,including 11.39million with coronary heart disease.In addition,CVD mortality accounts for approximately 31%of all deaths worldwide,posing a serious threat to human health.Therefore,early prevention and treatment of CVD and intervention of risk factors are essential.Common cardiovascular diseases mainly include coronary heart disease,cardiomyopathy,myocardial damage complicated by metabolic diseases,arrhythmia and heart failure as the end-stage outcome of heart disease.As one of the primary risk factors for CVD,hypercholesterolemia primarily manifests as excessive lipid deposition in the blood vessel walls,which leads to vessel wall damage and plaque formation,leading to circulatory disorders in organs and tissues eventually.Recent studies have suggested that,in addition to vascular injury,cholesterol metabolism disorders can directly cause myocardial damage,but little is known about the specific mechanisms.Single-cell sequencing is a significant achievement of high-throughput sequencing technology in recent years.It enables the analysis of molecular and functional differences between the same sample or different samples,and between individual cells,and then reveal molecular and metabolic changes in healthy or diseased states,which is a crucial tool to explore the specific mechanisms of disease progression.Therefore,we used single-cell sequencing technology to specifically detect all cells in the heart tissue of mice in the early and late stages of hypercholesterolemia,to explore the cellular subtypes and genetic changes in heart tissue during the disease process,and to reveal the potential molecular mechanisms related to hypercholesterolemia myocardial injury.METHODS:Here,8-week-old APOE^-/-mice were randomly divided into the 8-week normal control group,the 8w high cholesterol diet（HCD）treatment group and the 16w HCD treatment group.The cardiac tissue of the mice was extracted at the corresponding time points for single-cell sequencing.All cells and genes in the heart tissue were specifically detected by sequencing analysis.Blood samples were collected and centrifuged for determination of serum lipid related indicators.At the same time,heart tissue,aorta and liver tissue were collected for immunohistochemistry,HE staining and oil-red O staining to verify the stability of the model.Cardiac function was analyzed using mouse cardiac echocardiography at the corresponding treatment time of HCD.According to the results of single-cell sequencing,immunohistochemistry,immunofluorescence,western blot（WB）,enzyme-linked immunosorbent assay（ELISA）,Masson and Sirius red staining were used to verify and explore the molecular and cellular changes in the disease.RESULTS:In this study,we successfully established a hypercholesterolemia model in APOE^-/-mice.Twelve major cell types were identified from heart tissue by single-cell sequencing analysis,and a comprehensive landscape of cardiomyocytes,macrophages,fibroblasts,T cells,and other cells in the hearts of the mice was mapped.Further cellular and molecular functional enrichment analysis revealed significant heterogeneity in cardiomyocytes,fibroblasts,T cells and macrophages.In the KEEG and GO analysis of cardiomyocytes,the diabetic cardiomyopathy related gene Pln was significantly up-regulated in the cardiomyocyte cluster,which was also verified by immunofluorescence co-location analysis of Pln and Myl7.Sequencing results showed that the number of inflammatory cells and fibroblasts accounted for a large proportion,and increased with the progression of the disease.Among the inflammatory cells,changes were most pronounced in T cells and macrophages.Genes associated with inflammatory mediators,such as pro-inflammatory factor Interleukin（IL）-17,were also significantly up-regulated.Immunofluorescence localization of IL-17 indicated that it was closely related to fibroblasts.Enrichment analysis of fibroblasts showed that IL-17-related signaling pathways were significantly enriched.In addition,the pathways related to collagen deposition were significantly enriched in fibroblasts,and the Wnt5a pathway associated with chronic fibrosis in fibroblasts was up-regulated.The results of collagenⅢimmunofluorescence,Wnt5a WB and Masson staining further confirmed that myocardial fibrosis was obvious.Enrichment analysis of macrophages revealed significant interactions among macrophages,T cells and fibroblasts.CONCLUSIONS:The enrichment of the Pln gene in the cardiomyocytes,the interaction and activation of macrophages,T cells and fibroblasts,highlights the importance of fibrosis and inflammatory activation in hypercholesterolemia related myocardial injury,partially sheds light on the specific mechanisms of hypercholesterolemia-related myocardial injury and motivates the development of clinical treatment strategies.