Mechanisms And Characteristics Of Ischemic Cardiomyopathy Based On Cardiac Cells Profile

Part 1.A modified method for isolation of human cardiomyocytes to model cardiac diseasesBackground Cardiomyocytes derived from animal and induced pluripotent stem cells(i PSs)are two main cell models to study cardiovascular disease,however both cells may not precisely mimic mature cardiomyocytes response to disease or stress conditions.Therefore,human primary cardiomyocytes would be a perfect cell model.Previous established protocols for human primary cardiomyocyte isolation,nevertheless,cell yield/status or practicality impose great limitations for application.Objective To establish and optimize a practical method allowing the robust isolation human viable cardiomyocytes.Methods and Results Cardiomyocyte isolation procedure includes : tissue harvest,specimen transport,tissue excision,de-calcium and enzyme digestion.We optimized conditions in each step and introduced microtomes slicing tissue to significantly improve efficacy of enzyme digestion.Cardiomyocytes yield and status were similar to Langendorff isolated cells.Isolated cardiomyocyte showed retained contractility,ion flux,calcium handling,intact morphology,responses to neurohormonal stimulation.Moreover,we also assessed metabolism of cardiomyocytes derived from different conditions and further validated potential functional applications of isolated cardiomyocytes.Conclusions We present a novel,simplified method,demonstrating isolation of viable human cardiomyocytes without tissue restrictionPart 2.Human cardiomyocytes proteomics profiling reveals G protein subunit beta 2(GNB2)regulates apoptosis process in ischemic cardiomyopathyBackground Despite the improvement of reperfusion treatments in ameliorating the mortality of myocardial infarction,many survivors will eventually develop heart failure owing to the loss of cardiomyocytes.In this regard,new strategy to maintain myocyte number is an important determinant in the prevention of heart dysfunction.Proteomic analysis based on heart tissue revealed some pathological characteristics in the ischemic cardiomyopathy(ICM),this approach has been limited by the fact that the signals are derived from various cells of tissue sample.Objective Using human primary cardiomyocytes resource towards investigating therapeutic target for preventing chronic heart failure after ischemic attack.Methods and Results Our study applies quantitative proteomics based on the cardiomyocytes derived from infarcted and non-infarcted area of explanted hearts to study the regulatory networks of ICM.Significant differences in death related protein abundance between ICM patients and the normal control were found in our data,and major alterations in BCL2-antagonist of cell death protein(BAD),Fas associated factor family member2(FAF2)and Autophagy-related protein 9A(ATG9A).Besides those proteins mentioned above,we found that G protein subunit beta 2(GNB2)is activated in cardiomyocytes from ICM patients,knockdown GNB2 decreased cardiomyocytes apoptosis in response to ischemic stimuli.Conclusions This is the first proteomic study based on human primary cardiomyocytes.Our data suggests an increase in apoptotic protein abundance in ICM and knockdown GNB2 promotes cardiomyocytes survival after ischemic insult.Part 3.Resolving human ischemic heart failure microenvironment at single cell levelBackground Heart failure is a complex disease with sustained inflammation driven by multifaceted factors of the circulating immune cells and resident cells.However,the limited clinical benefit of current therapies makes it critical to reveal detailed property of cardiac microenvironment.Objective Precisely dissect the cellular basis pinpointing to direct relevant pathogenesis,is essential for therapeutic investigation.Methods and Results We performed single cell RNA sequencing in over 86435 human single cells,unravelling unprecedently diverse immune cell and non-immune cell subsets in failing hearts.We confirmed that CCR2~+ tissue-resident macrophages and blood monocyte-derived inflammatory macrophages were the major source of IL-1?.Meanwhile,we uncovered tissue-resident memory CD4~+ and CD8~+ T cells expanded in failing heart,combining with T cell receptor sequencing,we showed that CD8~+ T cells were expanded via both in situ clonal expansion and blood recruitment.We also defined a pro-inflammatory ACKR1~+ venous endothelial cell subset expanded in failing hearts involved in leukocyte recruitment.Moreover,multi-lineage interaction revealed that in severely fibrotic and inflamed niche,ACTA2~+ myofibroblasts secreted IL34 interacted with CCR2~+ tissue-resident macrophages via CSF1 R,which ultimately led to increased level of CXCL8 in CCR2~+ tissue-resident macrophages.Upon CXCL8 stimulation,ACKR1~+ endothelial cells upregulated genes involved in cell adhesion and leukocyte recruitment,further promoting inflammation.Conclusions Our work provides a comprehensive survey of cell type specific molecular changes in failing hearts at single cell level,hopefully our results would shed light on investigation of effective therapeutic targets in heart failure.