Induced Differentiation Of Human Cardiac Pacemaker Cells Based On Retinoic Acid Signaling

Objectives and Contents:Cardiac pacemaker cells especially sinoatrial node cells play vital roles in regulating normal heart rhythm.By means of research on direct reprogramming and induced differentiation of cardiac pacemaker cells,this study aimed at finding potential transcription factors and signaling pathways which are important for promoting pacemaker cells specialization,unveiling the mechanisms of subtype cardiomyocytes especially pacemaker cells fate determination,and eventually establishing a method for induced differentiation of human cardiac pacemaker cells in vitro.The main contents of this study includes:screening for transcription factors which promote direct reprogramming of human fibroblasts to pacemaker cells,unveiling the influence of rapamycin on subtype cardiomyocytes specialization and the maturation process of pacemaker cells in monolayer-based cardiac differentiation,studying the effect of retinoic acid(RA)on subtype cardiomyocytes especially pacemaker cells fate determination,and establishing an efficient,cost-effective and non-transgenic method for human cardiac pacemaker cells differentiation.Methods and Results:Using lentivirus-mediated direct reprogramming of human fibroblasts,we found RARG,the retinoic acid receptor gamma,induced the expression of pacemaker cell specific genes.By means of rapamycin induced cardiac differentiation method,meanwhile taking advantage of Western Blot,q PCR,flow cytometry analysis and electrophysiological technology,we first verified that rapamycin and its analogues can all promote cardiac differentiation and share a common mechanism of p53 dependent anti-apoptosis,then we found cardiomyocytes differentiated in later stage are predominantly ventricular subtypes,rapamycin do not disturb the proportion of subtypes.Furthermore,we found a dynamic change of HCN4~+cells throughout monolayer-based differentiation,in early stages,the ratio of HCN4~+cells is higher,also with a higher proportion of atrial and pacemaker like cardiomyocytes,electrophysiological properties of earlier cardiomyocytes are much more naive than later ones.Then the investigation on RA signaling pathway shown that RA can increase the proportion of HCN4~+cells in differentiation process,decrease the proportion of MLC2V~+ventricular cardiomyocytes,in the meantime increase the ratio of atrial and pacemaker subtypes in later stage.We further found that Hedgehog signaling pathway agonist SAG can enhance the expression profile of subtype specific genes induced by RA,and increase the ratio of atrial subtypes in later stage of differentiation.By promoting the proliferation of cardiac precursors,SAG can also further promote ventricular specialization induced by ascorbic acid.At last,we attempted to optimize the method for cardiac pacemaker cells differentiation based on RA signaling pathway.Conclusions:By screening in transdifferentiation studies,we found an inducing effect of RARG on the expression of pacemaker cell specific genes.By investigation on induced differentiation in vitro,we verified that rapamycin and its analogues can all promote cardiac differentiation and proved the unchanged influence of rapamycin on subtype cardiomyocytes specialization;we have proved that there is progressive maturation process of pacemaker cells specialization throughout differentiation;we have found RA can promote pacemaker cells fate determination,SAG can nonspecifically promote differentiation of subtype cardiomyocytes,and accordingly we have optimized the differentiation methods for subtype cardiomyocytes based on RA.