A Comparative Study Of Transcriptome Differences Between Cardiac Cells And Cardiac Nuclei

Background:As an emerging technology in recent years,single-cell sequencing technology is very helpful for us to deeply understand the characteristics and changes of the heart in development,homeostasis and disease.However,three major problems have hindered the application of this technology in the field of cardiac research:the difficulty of dissociating cardiomyocytes and the difficulty in obtaining single-cell suspensions of cardiomyocytes with high viability,the activation of stress-related genes brought about by dissociation,and the importantly,cardiomyocytes are too large for single-cell capture with high-throughput capture platforms.To solve these problems,single-cell nuclear sequencing technology came into being.However,whether single-cell nuclear sequencing can replace single-cell sequencing for widespread use in the cardiac field has not been systematically elucidated.No studies have systematically evaluated the sequencing quality,bias,resolution,and comparison with single-cell sequencing and single-cell nuclear sequencing of cardiac tissue.Here,we compared the nuclear and whole-cell transcriptome differences of cardiac cells and their subsequent application values at bulk and single-cell levels,respectively.Methods and results:1.The Langendorff method to dissociate adult mouse cardiomyocytes is optimized In order to solve the problem that the Langendorff instrument is too complicated and the cell dissociation operation is difficult to master,which affects the viability and purity of the obtained cells,cannot meet the sequencing requirements,and has poor repeatability and large batch differences,we propose a detailed optimization protocol,including improved the Langendorff apparatus and simplified experimental procedures and using gravity as perfusion pressure for dissociated adult mouse hearts.This method is easy to learn and operate,and improves the viability and purity of target cell populations isolated from adult mouse hearts.2.Optimization of the nuclei fraction method from adult mouse cardiomyocyte In order to better compare the transcriptome differences between cardiomyocyte nuclei and whole cardiomyocytes,we optimized the process and experimental method for the extraction of cardiomyocyte nuclei.We first isolated purified adult mouse cardiomyocytes by Langendorff,and then divided the cardiomyocytes into two parts,one for the whole cardiomyocyte group and the other for the extraction of cardiomyocyte nuclei.The advantage of this is that the two groups of samples go through the same sample preparation process as much as possible,so as to effectively avoid the influence of different sample acquisition processes on the experimental results,and to more realistically show the difference between cardiomyocyte nuclei and whole cardiomyocytes.3.Bulk RNA-seq compares transcriptome differences between adult mouse cardiomyocytes and cardiomyocyte nucleiUsing an optimized cardiomyocyte isolation method and cardiomyocyte nuclear extraction method,we extracted and purified adult mouse cardiomyocytes and nuclear RNA,and performed second-generation strand-specific bulk RNA-seq.We investigated differences in relative transcript abundance between cardiomyocyte nuclei and whole cells and focused on showing genes and gene classes that were more enriched in nuclei or whole cells.Our findings also provide important insights into the subcellular localization of nuclear-encoded mitochondrial genes in cardiomyocytes.4.Comparison of transcriptome differences between cardiomyocytes and cardiomyocyte nuclei at the single-cell levelWe developed an unbiased tissue processing and single-cell nuclear transcriptomesequencing pipeline to unravel cardiac cell-specific characterizations and applied them to human cardiac tissue.Our method proved feasible in cardiac tissue from multiple species and obtained sequencing quality superior to previous literature.Based on this,we used this method to perform single-nucleus sequencing in adult heart tissue and compared it with single-cell sequencing,revealing the similarities and differences between the two,and providing new insights into the differences in the cognition of cardiovascular diseases by technology platform selection.Conclusion:Through the above experiments,we obtained the following conclusions:1)It provides an optimized Langendorff perfusion system and a stable operation method.2)Established an innovative method to extract cardiomyocyte nuclei from suspended cardiomyocytes.3)We first provide the subcellular localization landscape of RNA transcripts in adult mouse cardiomyocytes.Significant differences in mRNA and lncRNA expression between whole cells and nuclei have been shown.We also provide new knowledge about the NEMP transcripts subcellular localization in cardiomyocyte.Our results suggest that the use of nuclear RNA in cardiac disease samples as a surrogate for whole cells may bias the results of analysis at the transcriptional level.4)snRNA-seq and scRNA-seq are consistent in cell grouping and subgroup analysis;snRNA-seq can effectively reduce the interference of stress genes compared with scRNA-seq;snRNA-seq can unbiased characterization of human cardiac cells.