Cell Fusion Between Bone Marrow Stem Cells And Cardiomyocytes Rescues Cardiomyocyte From Undergoing Apoptosis

The coronary atherosclerotic heart disease patients, stenosis or obstruction happens in their coronary artery, because of atherosclerosis, leading to myocardial ischemia and hypoxia. Whether myocardial ischemia and hypoxia in coronary heart disease, or reperfusion injury in modern reperfusion therapy, they can cause myocardial apoptosis and necrosis. The oxidative stress caused by free radicals play an important role.Bone marrow stem cells are one big group of adult stem cells. They include bone marrow-derived mesenchymal stem cells and bone marrow hematopoietic stem cells. They all have the characteristics of pluripotent differentiation, and tissue repair abilities. Bone marrow-derived mesenchymal stem cells can differentiate into cartilage, bone cells, fat cells and smooth muscle cells at least. Studies have also found that bone stem cells can differentiate into cardiomyocyte-like cells. And that, Bone marrow hematopoietic cells can differentiate into a variety of blood cells and non-blood cells. In recent years, heart transplantation of bone marrow stem cells has been shown to improve the damaged heart function. The mechanism may be related to the following aspects: to promote angiogenesis and blood circulation of the heart; secretion of cytokines improving the cardiac micro-environment; stem cell regeneration of myocardial cells complementing the number of myocardial cells.Recent studies have found that bone marrow stem cells can fuse with liver cells, nerve cells and heat shocked human small airway epithelial cells. The fusion between bone marrow stem cells and cardiomyocytes is still needed to be studied. So we assume that: cell fusion between bone marrow stem cells and cardiomyocytes rescues cardiomyocyte from undergoing apoptosis. In order to prove the hypothesis, we designed in vitro and in vivo study. In vitro study, the neonatal cardiomyocytes suffered by hydrogen peroxide, so that the cardiomyocytes were undergoing apoptosis. Then they were co-cultured with bone marrow stem cells in certain poportion. Finally we detected the evidences of cell fusion and apoptosis; In vivo study, the RFP positive expressing mice were suffered the left anterior descending artery ligation, immediately the GFP-positive expressing bone marrow-derived mesenchymal stem cells were injected directly into the edge of necrotic area, a week after that, we detected the evidences of cell fusion.【Methods】1. The derivation, culture, labelling and oxidative stress treatment of the primary rat neonatal cardiomyocytes.2. The derivation, cultue and defination of rat bone marrow mesenchymal stem cells and hematopoietic stem cells.3. The TUNEL assay.4. The Annexin V assay. 5. Mouse model of acute myocardial infarction, cell transplantation and heart digestion.【Results】1. The oxidative stess can cause cardiomyocytes undergoing apoptosis. (Annexin V and TUNEL assay):1)There were more apoptotic cardiomyocytes after H2O2 treatment than the control group (without treatment), P<0.05;2)25mmol/L与50mmol/L H2O2 affected cardiomyocytes rather, P>0.05; 3)Cardiomyocytes were detected after H2O2 treatment, results had no significant differences at 48, 72, 96 hours' check points after treatment., P>0.05.2. BMSCs can fuse with cardiomyocytes undergoing apoptosis. (Flow cytometry and fluorescent microscopy)1) Fluorescent microscopy:a. Fusion cells can be found through cell fixation and fluorescent staining.b. Live cells were observed under fluorescent microscopy: cardiomyocytes self fusion; BMSCs self fusion and cell fusion between bone marrow mesenchymal stem cells and apoptotic cardiomyocytes. 2) Flow cytometry:a. BMSCs only fused with injured cardiomyocytes, not fused with cardimyocytes without H2O2 treatment (control group), P<0.05;b. The fusion rate has a H2O2 dose response. P<0.05;c. The fusion rate has a co-culture duration time response. P<0.05.3. Cell fusion between BMSCs and cardiomyocytes can rescue cardiomyocytes from undergoing apoptosis. (TUNEL and Annexin V assay)1) TUNEL assay:The apoptotic rate of co-culture group (19%) was obviously lower than the control group (non co-culture)(8.4%), at the decending of 55.8%, P<0.05;2) Annexin V assay: The apoptotic rate of co-culture group for culturing 48, 72 and 96 hours: 9.3%, 7.6%, 5.6%. They were much lower than the control group (non co- culture)(11.9%), at descending of 21.8%, 36.1%and 52.9%, P<0.05; We found that the fusion rate was highly related to the apoptotic rate, the correlation coefficient rate R2=0.9901. The apoptosis decreased with the increasing fusion.4. The HSCs can fuse with cardiomyocytes undering apoptosis. (Flow cytometry and Fluorescent microscopy)1) The defination of hematopoietic stem cells: HSCs were got through the immune magnetic cell sorting, and 98% of them were CD90.1 positive.2) Fluorescent microscopy: Fusion cells can be found through cell fixation and fluorescent staining.3) Flow cytometry:a. HSCs only fused with injured cardiomyocytes, not fused with cardiomyocytes without H2O2 treatment. P<0.05;b. The fusion rate of HSCs with injured cardiomyocytes (10.7%) was obviously higher than the fusion rate of mesenchymal stem cells with injured cardiomyocytes (7.9%), P<0.05.5. Cell fusion between BMSCs and cardiomyocytes can rescue cardiomyocytes from undergoing apoptosis. ( Annexin V assay)1) The apoptotic rate of co-culture group was 6.5%, which was obviously lower than the control group (non co-culture)(10.4%), at descending of 37.5%, P<0.05;2) HSCs rescue injured cardiomyocytes as much as mesenchymal stem cells rather. P>0.05.6. Only cardic myocardial working cells fused with bone marrow mesenchymal stem cells. (Annexin V) Injured cardiac smooth muscle cells and fibroblast did not fuse with mesenchymal stem cells, the fusion rate was zero. 7. SCF, IL-11 and TNF-αaffected the fusion between BMSCs and injured cardiomyocytes.1) TNF-αwas loaded into the co-culture medium, the fusion rate was 5.7%, which was obviously lower than the control group (7.9%), P<0.05;2) IL-11 was loaded into the co-culture medium, the fusion rate was 10.2%, which was obviously higher than the control group (7.9%), P<0.05;3) SCF was loaded into the co-culture medium, the fusion rate was 11.2%, which was obviously higher than the control group (7.9%), P>0.05;8. In vivo study confirmed that injured cardiomyocytes fused with bone marrow mesenchymal stem cells.1) The mice were LAD ligated. BMSCs were directly injected into the infarct border zone. The fusion rate reached 8.8%;2) The mice were not LAD ligated. BMSCs were directly injected into the infarct border zone. The fusion rate reached 4.7%;3) The mice were LAD ligated. Medium was directly injected into infarct border zone. The fusion rate was 0.03%. All groups had significant statistic difference to each other. P<0.05.【Conclusion】1. Oxidative stress can cause cardiomyocytes undergoing apoptosis.2. Bone marrow stem cells, not only mesenchymal stem cells but also hematopoietic stem cells, fuse with injured cardiomyocytes (mainly myocardial working cells), rescue cardiomyocytes from undergoing apoptosis.3. Cell fusion maybe is a way of self protection that injured tissue against damage. Cell fusion provides a good explanation for stem cells'differentiation and healing ability.4. The mechanism of fusion is still unknown, still needs investigation. But based on our study, we can infer some related factors, such as: 1) Direct cell-cell connection2)Cell injury, like oxidative stress 3) Cell factors in micro circumstance 4) Stem cells' multi-differentiation ability and high activity.5. In vivo study confirms the results of fusion of in vitro study, and provides a good explanation for injured heart function improving after mesenchymal stem cells transplantation.6. Electrophysiology and myocardial protein expression in fusion cells still need farther investigation.