The Effect Of Secreted Factors On Cardiomyocyte Proliferation And Cardiac Regeneration

Loss of cardiomyocytes is the main cause of impaired cardiac function in ischemic heart diseases.Cardiomyocytes in adult mammals have limited ability to proliferate.Thus,cardiomyocytes cannot regenerate to replace the missing cardiomyocytes after cardiac injury.Understanding how cardiac tissue regenerates and the molecular mechanism of cardiomyocyte proliferation is important for identifying novel targets and finding new methods to promote cardiac regeneration in patients of ischemic cardiac diseases.Recent studies have found that some ligands secreted by non-cardiomyocytes can bind to receptors on the cardiomyocyte membrane to promote cardiomyocyte proliferation and cardiac regeneration.However,whether there are extracellular signals that inhibit proliferation of cardiomyocytes is still unclear.In recent years,with the development and application of Single-cell RNA-sequencing technology,more and more Single-cell RNA-sequencing data emerged,which can help us understand the various cell groups and their interactions in cardiac tissue.In this dissertation,we analyzed published Single-cell RNA-sequencing data to find potential extracellular signals and receptors on cardiomyocytes that can regulate cardiomyocyte proliferation.Furthermore,we explored their roles in cardiomyocyte proliferation and cardiac regeneration and investigated their downstream mechanisms in the cardiomyocytes.We found that very low-density lipoprotein receptor（Vldlr）is highly expressed in cardiomyocytes through analyzing published Single-cell RNA-sequencing data of mouse hearts.Western Blot indicated the expression level of Vldlr gradually increases after birth.By knocking down and overexpressing Vldlr in primary neonatal rat ventriclular cardiomyocytes（NRVCs）,we found that Vldlr can inhibit cardiomyocyte proliferation.In addition,we found that knocking out of Vldlr can promote cardiomyocyte proliferation in vivo.To search for functional ligands of Vldlr,we analyzed published Single-cell RNA-sequencing data and found that Reelin,a widely characterized ligand for Vldlr,was specifically expressed in Schwann cells and lymphatic endothelial cells in mouse hearts.Adding recombinant Reelin protein to NRVCs can induce cardiomyocyte proliferation.In addition,we found that knocking out Reln inhibit cardiomyocyte proliferation in vivo.The opposite effects of receptor Vldlr and its ligand Reelin on cardiomyocyte proliferation suggest that there may be other ligands that dominate the function of Vldlr.We found that another ligand of Vldlr,TSP-1,has the opposite function compared to Reelin in neural cells,and its encoding gene Thbs1 has a high expression level in mouse heart.Knocking down of Thbs1 and addition of TSP-1 recombinant protein in NRVCs indicated that TSP-1 can inhibit cardiomyocyte proliferation.Using Mesp1-Cre;Thbs1^f/f mice,we found conditionally knocking out of Thbs1 in mouse heart promote cardiomyocyte proliferation in vivo,suggesting that TSP-1 may be the ligand that dominates Vldlr to inhibit cardiomyocyte proliferation.We found the regulation of Reelin and TSP-1 on cardiomyocyte proliferation was dependent on Vldlr by treating cardiomyocytes from Vldlr^-/-mice with Reelin and TSP-1.In addition,we further found that TSP-1 and Reelin compete with each other in regulating cardiomyocyte proliferation.To investigate the mechanism of TSP-1/Reelin-Vldlr signaling in regulating cardiomyocyte proliferation,we performed and analyzed RNA-Seq on NRVCs treated with recombinant Reelin,si-Vldlr,AAV9-c Tn T-Vldlr virus,and si-Thbs1,and found that TSP-1/Reelin-Vldlr signal can regulate small GTPases signal pathway.Further experiments confirmed the small GTPase Rac1 activity was affected by TSP-1/Reelin-Vldlr signal.And functional experiments indicated that the regulation of TSP-1/Reelin-Vldlr on cardiomyocyte proliferation was mediated by Rac1.Further analysis of the RNA-Seq data of NRVCs showed that TSP-1/Reelin-Vldlr signaling regulates Yap signaling.Subsequent experiments demonstrated that Rac1regulate the activation and nuclear translocation of Yap in vitro,and both Rac1 and Yap were involved in the regulation of cardiomyocyte proliferation induced by TSP-1/Reelin-Vldlr signaling.To examine whether TSP-1/Reelin-Vldlr signaling regulate cardiomyocyte proliferation and cardiac regeneration in vivo during cardiac injury repair,apical resection was peformed in the neonatal Reln^-/-mice.We found Reln is required for cardiomyocyte proliferation and cardiac regeneration of neonatal mouse hearts.Peforming myocardial infarction in Mesp1-Cre;Thbs1^f/f mice showed that specificlly knocking out of Thbs1 in the heart promoted cardiomyocyte proliferation and the recovery of cardiac function after myocardial infarction.It demonstrated that Thbs1plays an inhibitory role in cardiomyocyte proliferation and cardiac regeneration.Finally,we performed both apical resection on neonatal Vldlr^-/-mice and myocardial infarction in adult Myh6-Cre;Vldlr^f/fmice.We found Vldlr ablation promoted cardiomyocyte proliferation both after neonatal apical resection injury and myocardial infarction injury in adults,and induced functional recovery after myocardial infarction,suggesting that Vldlr plays an overall negative role in cardiomyocyte proliferation and cardiac regeneration after cardiac injury.TSP-1 may be the dominant ligand of Vldlr in vivo given the similar phenotypes of their knockout mice.In conclusion,we found that the receptor Vldlr on cardiomyocytes mediates both TSP-1 signaling that inhibits cardiomyocyte proliferation and Reelin signaling that promotes cardiomyocyte proliferation.During homestasis,TSP-1-Vldlr signaling plays a dominant role in inhibiting cardiomyocyte proliferation.However,Reelin plays an important role in cardiac regeneration in neonatal mice,which can promote cardiomyocyte proliferation and cardiac regeneration after injury.In addition,we also found that Rac1 and Yap,as downstream effectors of TSP-1/Reelin-Vldlr signaling,mediate the regulatory effect of TSP-1/Reelin-Vldlr on cardiomyocyte proliferation.Thus,we have discovered a new molecular mechanism supressing mammalian cardiomyocyte proliferation in vivo,and have provided a potential therapeutic target for cardiac regeneration.