Trop2 Is A Key Player In Promoting Proliferation And Anti-apoptosis Of Mouse Cardiac C-kit~+ Cells

ObejiectiveThe evidence that Cardiac Progenitor Cells (CPCs) resided within adult myocardium are capable of differentiating into multiple cells types---including myocyte, smooth muscle cells and endothelial cells---has updated cardiac biology and shifted the paradigm that heart is a postmitotic organ. However, CPCs only have a limited ability of the preservation of heart function. In addition to their rare number, the mechanisms underlying insufficient efficient of CPCs repairing damaged myocardium appear to mediate by their low survival in rigor environmental cue of myocardium infarction. Several subpopulations of CPC have been identified in heart, and c-kit+ cells have been proposed as the main province for rejuvenating cardiac performance in myocardium infaction. Trop2 is a cell-surface glycoprotein most often expressed by cells with characters of rapid growth, and activation of Trop2 could enhance proliferation and anti-apoptosis ability in many kinds of cells. Here, we investigated whether Trop2 can be expressed on cardiac c-kit+ cells, and if yes, the functional roles and relative molecular mechanism underlying these effects response to activated Trop2.MethodsObserving the rate change of Trop2+c-kit+ cells after myocardium infactionTo learn the dynamic change of Trop2+c-kit+ cells in acute phase of myocardium infaction, the experimental mouse myocardium infaction model was inducted, and immunochemistry and flow cytometric analysis were performed to quantify the number of Trop2+c-kit+ cells.Isolation of cardiac c-kit+ cell subpopulationsCardiac c-kit+Trop2+ and c-kit+Trop2- cells were primary isolated from the hearts of healthy wild-type (WT) male mice by two-step immunomagnetic microbead-based cell sorting, and cultured in suitable medium to maintain an undifferentiated state.Cell proliferation assayMouse Trop2 short hairpin RNA (shRNA) plasmids were constructed and transfected into Trop2+c-kit+ cells. Western Blot was performed to monitor the change trend of Trop2 expression after transfection, and bromodeoxyuridine (BrdU) incorporation was measured at corresponding time points to assay the proliferation ability.Cell apoptosis assayTo get close to the inflammatory environment cue was comparable to that in vivo, we observed cell apoptosis induced by CM (conditioned medium) derived from LPS (lipopolysaccharides)-stimulated monocytes. Purified Trop2+ and Trop2- c-kit+ cells were both treated with CM at different ratio (volume:volume), respectively, relative to DMEM/F12 medium in vitro, then stained with Annexin V-APC/propidium iodide (PI) and analyzed by flow cytometry following.Investigating the molecular mechanism underlying Trop2 functionWestern Blot and Immune-complex kinase assays were performed to investigate the downstream effectors of Mitogen-activated protein kinase (MAPK) and Akt pathway since we speculated both of them were respond to the potential molecular mechanism underlying the relative biological effect associated with Trop2.Observing the protective role of Trop2 on cardiac c-kit+ cells in vivoTrop2-/- and Trop2+/+ mice born from a single mating of a heterozygote pair were generated. Flow cytometric analysis was performed to compare the number change of c-kit+ cells within cardiomydium between Trop2-/- and Trop2+/+ mice after myocardium infaction, meanwhile, a mortality study was also conducted in the two group.ResultsTrop2 is exclusively expressed in c-kit+ cells within myocardium. The number of Trop2+c-kit+ cells is rare in quiescent but dramatically increased after myocardium infaction, and the change trend of c-kit+Trop2+ cells consisted with the infiltration pattern of inflammatory cell. Increased expression of Trop2 of c-kit+ cells could enhance its ability of proliferation and survival response to the inflammation in vitro, which maybe mediated by activated MAPK signaling pathway. In vivo, it has also been confirmed that Trop2 can enhances the survival of c-kit+ cells. Comparing Trop2-/- mice, Trop2+/+ mice have a higher survival in acute phase of myocardium infaction. ConclusionIn conclusion, this work reveals activation of Trop2 plays an important cardioprotective role after myocardium infaction through promoting proliferation and inhibiting apoptosis of cardiac c-kit+ cells. These observations suggest import of cardiac c-kit+ cells overexpressing Trop2 or manipulation of autogenous cardiac c-kit+ cells using a selective Trop2 agonist may be as potential approaches for management of acute ischemic cardiomyopathy.