MicroRNA-155 Promotes Survival Of Cardiomyocyte Progenitor Cells In Vitro And In Vivo

BackgroundMyocardial infarction is a life-threatening disease, characterized by massive cardiomyocytes loss and consequently deterioration of cardiac function. Enormous effort has been put to investigate alternatives to restore better cardiac function after myocardial infarction. Until recently, the paradigm that heart is a post-mitotic organ is challenged by the accumulating evidence that the heart habors cardiac progenitor cells. The existence of cardiac progenitor cells provides an opportunity to regenerate cardiomyocytes, compensating the loss of cells after damage. Different research group identified different progenitor cell populations with different characteristics, respectively, using various isolation methods. Hierlihy and colleagues demonstrated for the first time that a putative cardiac progenitor cell population exists in the adult heart, the side population (SP), which have the ability to efflux Hoescht or Rhodamine dyes through ATP-binding transporters and to differentiate into cardiac lineage. Further, Beltrami et al. reported the existence of c-kit+CPCs, which possess cardiac potential and express neither cardiac lineage transcription factors such as Nkx2.5, GATA-4 nor membrane and cytoplasimc proteins[2]. Mouse Sca-1+CPCs [3,4] and Isl-1+cells [5]were identified by different groups, showing the capability for cardiomyocytes regeneration.CPCs are potentially an ideal candidate due to their capability of differentiating into all cell types required for cardiac repair; functional cardiomyocytes, endothelial cells and smooth muscle cells, all required for cardiac repair. Previously, we reported the isolation of cardiomyocyte progenitor cells (CMPCs) from the human heart that are able to proliferate and efficiently differentiate into functional cardiomyocytes without requiring co-culture with neonatal myocytes[6,7]. Three months after transplantation of CMPCs in a mouse model of myocardial infarction, we observed less outward remodeling and improved cardiac function as compared to control injections[8].Although high numbers of cells were injected in several studies, few implanted cells survived, limiting their potential contribution for myocardial repair. Most of the engrafted cells died in the first 48h after transplantation, partially due to the hostile microenvironment of the ischemic myocardium[9-11]. Therefore there is a lot to gain if we can find a strategy to improve cell survival after implantation.Since the last decade, microRNAs have been identified and considered as important gene-regulaitng molecules. They are 20-22 nucleotide length, endogenously expressed, non-coding small RNAs. MicroRNAs mostly downregulate genes expression by binding to the 3'UTR of the targets, thereby blocking the translation of target proteins or directly degrading target mRNAs. They are involved in organ development, cell proliferation, differentiation etc. Accumulating evidence has suggested that miRNAs also play an important role in cell survival. MicroRNA-155 (miR-155) is demonstrated to be involved in cell death in several cell lines, e.g. marcrophages and pancreatic cells. Recently, we observed that miR-155 is expressed in growing CMPCs, and demonstrated that miR-155 is not involved in cellular proliferation. In this study, we to investigate:1)whether mir-155 could improve cardiomyocyte progenitor cells survival after oxidative stress and the underlined mechanism.2)whether mir-155 could enhance cell survival after transplantation in a MI mouse model, using bioluminescence imaging (BLI).Part I MicroRNA-155 blocks necrosis induced by oxidative stress in cardiomyocyte progenitor cells (CMPCs) via targeting RIP1Objective:To investigate whether a candidate microRNA, microRNA-155 could improve cell survival after oxidative stress and the possible mechanism.Methods:1. cardiomyocyte progenitor cells isolation from human fetal heart using mouse anti-Sca-1 antibody MACS2. Taqman microRNA PCR to detect microRNA-155 expression level upon oxidative stress3. microRNA tranfection:pre-miR155/anti-miR155/scr-miR to overexpress miR-155 in CMPCs.3'UTR luciferase activity assay confirms the effectiveness of small miR molecules. 4. flow cytometry analysis:AnnexinV/7AAD staining to detect viable, apoptotic and necrotic cells5. Homogeneous caspases activity assay detects the endogenous active caspases activity upon PI staining to confirm the necrotic cells6. PCR and western blot detect mRNA and protein expression of RIP1 in miR-155 transfected CMPCs 7. RIP1 inhibitor Nec-1 or small RNA interference (siRIP1) mediated knockdown CMPCs8. PCR of 84 Akt-prosurvival pathway related genes, apoptosis related genes PCR arrayResults:1. Using quantitative PCR, we observed a four-fold increase of miR-155 when CMPCs were exposed to hydrogen-peroxide stimulation2. Flow cytometric analysis of cell viability, apoptosis, and necrosis showed that necrosis is the main cause of cell death. Overexpressing miR-155 in CMPCs attenuated necrotic cell death by 40±2.3% via targeting RIP1, receptor interacting protein 1.3. inhibiting RIP1, either by pre-incubating the cells with a RIP1 specific inhibitor, necrostatin-1 (Nec-1) or siRNA mediated knockdown, reduced necrosis by 38±2.5% and 33±1.9%, respectively.4. analyzing gene expression using a PCR-array showed that increased miR-155 levels did not change cell survival and apoptotic related gene expression.Conclusion:By targeting RIP1, miR-155 repressed necrotic cell death of CMPCs, independent of activation of Akt pro-survival pathway. MiR-155 provides the opportunity to block necrosis, a conventionally thought non-regulated process, and might be a potential novel approach to improve cell engraftment for cell therapy.PartⅡMicroRNA-155 improves CMPCs survival after transplantation in a murine myocardial infarction modelObjective:To investigate whether microRNA-155 could promote CMPCs survival after implantation using bioluminescence imaging.Methods:1. transduce fetal CMPCs with pLV-CMV-Luc2-GFP vector2. FACS analysis confirms and detect the effective transduction by detecting GFP positive cells 48h after transduction3. culture transduced CMPCs and compare transduced cells with untransduced cells in phenotype, proliferation speed aspects.4. transfect luc-GFP-CMPCs with miR-155 and stimulate with hydrogen perioxide, followed by cell counting for viable cells and dead cells5. miR-155 transfected luc-GFP-CMPC transplantation in MI SCID-NOD mice6. detect oxyluciferin signal by bioluminescence imaging 2days and 4days post transplantation.Results: 1. Transduced CMPCs stably express luciferase and GFP2. miR-155 protects luc-GFP-CMPCs from death induced by oxidative stress3. Overexpression miR-155 in CMPCs shows a strong trend to improve cell survival after transplantationConclusions:1. miR-155 inhibits cell death induced by H2O2 in luc-GFP-CMPCs in vitro.2. miR-155 might offer a molecular strategy to improve cell survival after transplantation, thereby promoting the effect of cell-based therapy for ischemic heart disease.