Research On The Molecular Mechanisms Of Differentiation And Apoptosis Of Sca-1 Positive Cardiac Stem Cells

Background and objectiveIschemic heart desease such as acute myocardial infarction is one of the leading cause of mortality in the world today.The hypoxia microenvironment lead to the decrease of cardiomyocytes,eventually leading to heart failure.Therefore,increased the number of cardiomyocytes is the key to treat ischemic heart disease.Mobilize the heart endogenous cardiac stem cells?CSCs?to differentiate into myocardial cells is a new strategy for treatment.However,there are two problems that need to be overcome,such as low differentiation rate and low survival rate of exogenous stem cells.The main reason for the low survival rate of stem cell transplantation is death due to apoptosis of implanted stem cells caused by local hypoxic microenvironment.It is very important to search for efficient differentiation inducer and survival mechanism to provide a theoretical basis and molecular targets for the treatment of myocardial infarction.This paper simply expounds the basic research results of these two aspects.Cyclodextrin?CD?constitute a family of cyclic oligosaccharides comprising repetitive 6,7,or 8 glucose units??-,?-,and ?-CD,respectively?.Among the CDs,?-cyclodextrin??-CD?was got more attentions because the extra roles on the cholesterol.?-CD could remove cholesterol or increase intracellular free cholesterol levels by promoting cholesterol metabolism,thus it had pharmacology effects on atherosclerosis and other metabolic syndrome.Till now,withdraw cholesterol by ?-CD contribute to the differentiation of various cells including chick cardiac cells were reported.However,the broad spectrum of ?-CD on myogenic transition in other species and the related mechanism remains unclear.NR4A2?Nuclear receptor subfamily 4,group A,member 2?also called Nurrl,together with NR4A1,NR4A3 make up the NR4A orphan receptor family.In previous studies,NR4A1 has been proofed to play an important function in the induction of both autophagy and apoptosis in cardiomyocytes,but the role of NR4A2 in the heart is not clear.In this paper,we find the differentiation-inducing agent ?-CD and elucidate the related mechanism of ?-CD induced differentiation,At the same time,the function of NR4A2 in cardiac stem cell apoptosis was revealed,and Mechanism of the NR4A2 was clarified further and our study provide novel drug target for ischemic heart disease therapy.1.?-cyclodextrin induces the differentiation of resident cardiac stem cells to cardiomyocytes through autophagy1.1 P-CD increased the expression of cardiomyocyte markerswe treated CSCs with different concentrations of ?-CD for 4 h,then changes in morphology were observed and the supernatant was collected for LDH detection.p-CD at 5 mM was selected to analyze its roles on differentiation.QPCR and immunofluorescence assay verified the the mRNA and protein levels of cardiac transcription factor,transcriptional enhancer and cardiac marker protein after ?-CD treatment for two weeks.The results showed that RNA level was significantly up-regulated and the increasing of cTnt in protein levels and the translocation of GATA4 to the nucleus.Therefore,?-CD treatment of CSCs for 2 weeks can promote the expression of myocardial cell marker.Therefor,?-CD treatment of CSCs for 2 weeks can promote the expression of cardiomyocyte marker.1.2 ?-CD induced the differentiation of resident Sca-1+ CSCs to cardiomyocytesTo further verify that ?-CD induced the differentiation of CSCs to cardiomyocytes.Flow cytometry and immunofluorescence,Western blot results showed that CSCs treated with ?-CD for 2 weeks,cTnt positive cells and protein levels were significantly up-regulated.EDU proliferation experiments showed that ?-CD inhibits CSCs proliferation and has no effect on H9c2.In vivo experiments show that HP-?-CD has a significant improvement in the cardiac function of MI,cTnt double positive in vivo CSCs increased.Therefore,?-CD induced the differentiation of CSCs into cardiomyocytes.1.3 ?-CD enhanced autophagy during inducing differentiationAutophagy plays an important role in myocardial differentiation.we examined the expressing level of LC3-?,and found that LC3-? levels increased in a time dependent manner in CSCs treated with ?-CD.In order to analyze the reason for the increase of LC3-?,we used two tools of autophagy.Western blot showed LC3-? protein levels were markedly attenuated by 3-methyladenine?3MA?and LC3-? protein levels were increased further by Baf A1,suggesting that ?-CD did not disrupt the autophagy efflux.1.4 P-CD induced the differentiation of resident Sca-1+ CSCs to cardiomyocytes through autophagyIn order to clarify whether autophagy is involved in differentiation,we constructed Atg5 knockdown CSCs and treated Atg5 knockdown CSCs for 2 weeks by ?-CD.QPCR and immunofluorescence showed that the differentiation was significantly inhibited after blocking autophagy.Therefore,?-CD induces cardiac stem cells to differentiate into cardiomyocytes via autophagy1.5 Autophagy regulates ?-CD-induced differentiation through the JNK/STAT3 signaling pathway.It has been reported that JNK/STAT3 signaling pathway involved in cardiomyocyte differentiation process.Western blot results show that phosphorylation of JNK and STAT3 is upregulated.Whether autophagy is related to JNK/STAT3 signaling pathway,CSCs was inhibited autophagy by Baf A1,3MA and Atg5.Western blot results showed that phosphorylation of JNK and STAT3 was inhibited after autophagy inhibition.Therefore,autophagy regulates ?-CD-induced differentiation through the JNK/STAT3 signaling pathway.1.6 Autophagy regulates ?-CD-induced differentiation through the GSK3?/?-catenin signaling pathway.GSK3?/?-catenin is another pathway participated in CSCs differentiation in our previous report.Western blot results show that GSK3?/?-catenin signaling pathway is activated,then the same time,d CSCs was inhibited autophagy by Baf A 1,3MA and Atg5.Western blot results showed that inhibition of autophagy,GSK3P/?-catenin signaling pathway can not be activated by ?-CD.Therefore,autophagy mediates ?-CD-induced differentiation through the GSK3?/?-catenin signaling pathway.1.7 ?-CD might induce the differentiation through regulating cholesterol?-CD can selectively exclude cholesterol from cell membranes to change the membrane composition and structure.We detected the changes of Flot-1 and Cav-1 in CSCs treated with ?-CD.Western blot showed that Flot-1 was not changed and Cav-lwas deduced by ?-CD,which suggested that P-CD impaired the lipid raft.Intracellular free cholesterol test showed that intracellular cholesterol was decreased in the early,at the time,At the same time,Western blot detection showed that ABCA1 protein levels were elevated at 1 and 2 weeks after P-CD treatment of CSCs,which indicated that changes of cholesterol levels might be important for ?-CD to induce differentiation and autophagy.2.The ROS/NF-kB/NR4A2 pathway is involved in H₂O₂ induced apoptosis of resident cardiac stem cells via autophagy2.1 H₂O₂ induced apoptosis of resident cardiac stem cellsCSCs were treated with indicated concentrations of H₂O₂.The morphology changes and LDH detection,determine the apoptosis inducing concentration of 500???without necrosis.Western blot,Hoechst 33258,flow cytometry,Caspase3 activity test results showed that 500???of H₂O₂ can induce apoptosis,Therefore,we chosen a concentration of 500???in the followed mechanism study.2.2 H₂O₂ induced autophagy of resident cardiac stem cellsAutophagy could regulate cell apoptosis,to determine whether H₂O₂ induced autophagy,we treated CSCs with H₂O₂ at various concentrations.Western blot results showed that LC3-II protein levels were significantly up-regulated.To analyze the reason for the increased LC3-?,we co-treated CSCs with 3MA and Baf Al.Western blot results showed that H₂O₂ promoted the initiation of autophagy induction of autophagy.2.3 H₂O₂-induced apoptosis in CSCs was regulated by autophagyTo investigate the functional of autophagy in H₂O₂-caused CSC apoptosis,H₂O₂-treated Atg5 knockdown CSCs and co-treatment of CSCs with 3MA,Rapa.Western blot results showed that apoptosis decreased after autophagy inhibition by 3MA and siAtg5,Apoptosis enhanced apoptosis after autophagy by Rapa.Therefore,H₂O₂ induced apoptosis by autophagy.2.4 NR4A2 was involved in H₂O₂-induced autophagy and apoptosis in CSCsWe then sought to character the mediators implicated in autophagy-dependent apoptosis in CSCs.To evaluate the importance of NR4A2,the mRNA and protein expression were evaluated by QPCR,immunofluorescence,and Western blot respectively.All the results showed that H₂O₂ could enhance the expression of NR4A2,To explore the role of NR4A2 in H202-induced autophagy and apoptosis,we used NR4A2 siRNA to knockdown its expression.Western blot showed that the knockdown of NR4A2 reversed H₂O₂-induced autophagy and apoptosis,and further confirmed the role of NR4A2 by lentivirus interference and overexpression.Therefore,which verified that NR4A2 was involved in H₂O₂-induced autophagy and apoptosis in CSCs.2.5 NR4A2 mediated autophagy-dependent apoptosis in H₂O₂-induced autophagy and apoptosis in CSCsTo clarify the relationship between NR4A2-mediated autophagy and apoptosis,lentiviral-mediated stable ablation of NR4A2 was performed in CSCs with its siRNA combined with two tools of autophagy.As shown in Figure.6A,H₂O₂ stimulated cleavage of caspase 3 and PARP1,and LC3-II levels was attenuated by NR4A2 knockdown?Figure.6A?.Furthermore,Rapamycin increased their changes.Thus,NR4A2 mediated autophagy-dependent apoptosis in H₂O₂-induced autophagy and apoptosis in CSCs.2.6 NF-?B as a direct regulator of NR4A2Transcription factor NF-?B was involved in the apoptosis of various cells treated with H₂O₂.To determine whether NF-?B participated in H₂O₂ induced autophagy and apoptosis in CSCs.The immunofluorescence and Western blot results showed that NF-?B was activated in H₂O₂-treated CSCs.bay11-7082,the specific inhibitor of NF-?B,Bayl 1-7082 was co-treated with H₂O₂.The results showed that bayl 1-7082 could reverse H₂O₂-induced autophagy and apoptosis.Related literature reports,NR4A2 promoter region has NF-KB binding sites,in order to further confirm that NF-?B regulate NR4A2 expression directly,immunofluorescence and Western blot,QPCR confirmed that Bay11-7082 can reverse the expression of NR4A2.At the same time,the dual fluorescent reporter gene assay confirmed that NF-?B directly binds to the promoter region of NR4A2 expression.Therefore,NF-?B is a direct regulator of NR4A2.2.7 ROS was the upstream of NF-?B/NR4A2 in H₂O₂-induced autophagy and apoptosis in CSCsROS increased in the oxidase stress conditions,to investigate whether ROS plays a role in H₂O₂-induced autophagy and apoptosis in CSCs,ROS level was detected by DCFH probe and ROS was significantly up-regulated by H₂O₂,ROS Scavenger NAC treatment,reversed H₂O₂ induced autophagy and apoptosis,we concluded that H₂O₂-induced ROS contributed to autophagy and apoptosis of CSCs.Previous reports showed that ROS could activate NF-?B,we deduced that ROS/NF-?B/NR4A2 pathway existed in H₂O₂-induced CSC autophagy and apoptosis.To confirm our hypotheses,QPCR and Western blot displayed that NAC reversed H₂O₂ stimulated phosphorylation of P65 and NR4A2 expression,The results confirmed that ROS/NF-?B/NR4A2 pathway existed in H₂O₂-induced CSC autophagy and apoptosis.