| Cardiovascular diseases,especially myocardial infarction(MI)is one major cause of deaths in China and methods to treat MI are significant to medical research,while cardiac reprogramming is one potential treatment to supply the lost cardiomyocytes(CMs)during MI.Cardiac reprogramming is a method to reprogram fibroblasts to functional CMs by introducing reprogramming factors like MEF2 C,GATA4,TBX5(MGT).The induced cardiomyocytes(iCMs)generated during cardiac reprogramming have functions like normal CMs,which can be applied to recover the injured myocardium and restore the cardiac function.However,several major problems have restrained the development of cardiac reprogramming field and its potential clinical application,including low reprogramming efficiency and difference between iCMs and native CMs,which makes its important to discover new method to improve the reprogramming efficiency and quality,and uncover the underlying mechanisms of cardiac reprograming.Recent studies have shown that epigenetics regulation plays important role in cardiac reprogramming,and histone acetylation,as a major kind of epigenetics regulation,was involved in post-MI heart protection and recovery.Thus,it is possible that several histone acetylation related factors were also involved in cardiac reprogramming,which will be explored in this study.By overexpressing MGT in mouse embryonic fibroblasts(MEFs)isolated from αMHC-GFP mice to establish an in vitro cardiac reprogramming system,and introducing 33 small-molecule chemicals target histone acetylation respectively to this system for evaluation,BETd-246,a specific small-molecule degrader for bromodomain and extraterminal domain(BET)family proteins,which is the reader of histone acetylation,was identified as a chemical that can significantly improve the efficiency of cardiac reprogramming,while its function was further confirmed with multiple methods and in other in vitro cardiac reprogramming system.Because a method to evaluate the functional maturation of iCMs is lacking in cardiac reprogramming research field,to determine whether the BET degrader could promote the functional maturation of iCMs,a new mouse strain,Myh6-Cre/LSL-GCa MP3,was established.With such new mouse strain,the cardiac reprogramming can be real-timely monitored and the maturation of iCMs can be efficiently evaluated,and it is confirmed that BETd-246 could improve the functional maturation of iCMs.Further investigations were focused on the specific mechanisms how BET degrader can promote cardiac reprogramming,and it is determined that BET degrader promotes cardiac reprogramming by degrades BRD4 to counteract the activation of JAK/STAT pathway by macrophage/OSM.Besides,by applying BET degrader BETd-260 in the in vivo cardiac reprogramming system in mouse MI model,it is determined that BET degrader could improve the recovery of cardiac function of MI mice.The findings described above increase our knowledge about the function of BRD4 in cardiac reprogramming and promote cardiac reprogramming to become a potential method supplying CMs after MI.Chapter 1: BET degrader BETd-246 enhances cardiac reprogrammingBackground:Previous research has proved that epigenetic regulations play a major role in cell reprogramming processes including cardiac reprogramming.Also,histone acetylation,as a major kind of epigenetic regulations,was important in myocardial injury and recovery process.However,whether histone acetylation was important in cardiac reprogramming was not well studied.So,to answer this question and find the method to promote cardiac reprogramming by regulating histone acetylation,which could further became a treatment of MI to recovery the injured myocardium and improve the cardiac function,in this section,the function of histone acetylation in cardiac reprogramming was evaluated by screening the small-molecule chemicals targeting histone acetylation.Aims:Determine the function of histone acetylation related factors in cardiac reprogramming,explore and evaluate potential drugs to regulate cardiac reprogramming.Methods:In vitro cardiac reprogramming system was established by overexpressing MGT in MEFs isolated from αMHC-GFP mice,and 33 histone acetylation related small-molecule chemicals were therefore introduced respectively to this system for evaluation,whose targets include histone acetylation related metabolites,histone acetyltransferases,histone deacetylases,BET family proteins.The function of those chemicals to cardiac reprogramming was evaluated by measuring the αMHC-GFP+ cell percentage with flow cytometry,which implies successfully reprogrammed iCMs.The chemical shows improvement to cardiac reprogramming will be further evaluated with CM-specific and fibroblast-specific genes expression by q PCR and c Tn T and α-actinin expression by immunofluorescence(IF).Those chemicals will also be evaluated in another in vitro cardiac reprogramming system based on NCFs for a comprehensive evaluation.Results:(1)Among 33 histone acetylation related small-molecule chemicals,a BET family protein degrader,BETd-246,shown best improvement to cardiac reprogramming.MGT+BETd-246 could increase the αMHCGFP+ cell percentage to 4 times of it in MGT+DMSO control group.(2)In the in vitro cardiac reprogramming system,BETd-246 achieved best efficiency in promoting cardiac reprogramming by treating the cells 1 h per 2 days.Also,the cardiac reprogramming efficiency improves with more treatment times.Besides,BETd-246 has the best improvement to cardiac reprogramming efficiency among multiple different BET inhibitors or degraders.(3)In the MEF-based in vitro cardiac reprogramming system,BETd-246 promotes CM-specific genes expression and inhibits fibroblastspecific genes expression,while c Tn T+ cells and α-actinin cells number were also increased.In NCF-based in vitro cardiac reprogramming system,BETd-246 can still increase the reprogramming efficiency.Conclusion:By screening histone acetylation related small-molecule chemicals in multiple in vitro cardiac reprogramming systems,BETd-246,a kind of BET degrader,was found to efficiently improve the cardiac reprogramming efficiency.Chapter 2: The development of a new cardiac-reprogrammingrelated mouse strain and its initial application to evaluate BET degrader in cardiac reprogrammingBackground:Although research described above show BETd-246 promotes cardiac reprogramming in multiple aspects,whether BET degrader could influence the function of iCMs is still unknown,which makes it necessary to evaluate the function of iCMs generated during this process.However,the method to evaluate the functional maturation of iCMs is lacking in cardiac reprogramming research field.Regular calcium flux is one specific characteristic of functional CMs,and genetically encoded calcium indicators(GECI)is a method to visualize calcium flux.Thus,a mouse strain whose calcium flux in iCM can be measured with a method based on GECI will be useful to evaluate functional maturation of iCMs,which could help to evaluate the influence of BETd-246 to the functional maturation of iCMs,while the reprogramming can also be monitored in a real-time manner,and finally lead to a better understanding to the function of BET degrader in cardiac reprogramming and promote the development of the cardiac reprogramming research field.Aims:Design a mouse strain based on calcium flux and GECI and establish evaluation methods based on it,including the evaluation of functional maturation of iCMs and real-time monitoring of cardiac reprogramming process,while also evaluating the influence of BET degrader to the function of iCMs.Methods:A new Myh6-Cre/LSL-GCa MP3 mouse strain was established,while a method to reprogram NCFs isolated from such mouse strain and related evaluation system were also developed.In this system,based on the principle of CRISPR technology,this mouse strain can express GCa MP3,a kind of GECI protein,in CMs or CM-like cells,i.e.,Myh6+ cells,and such GCa MP3 reporter protein can visualize the intracellular calcium flow that directly related to the function of iCMs as the green fluorescent of GCa MP3 protein.As Myh6+ promoter is gradually activated during the transdifferentiation process from NCFs to iCMs,such GCa MP3 fluorescence can be used to monitor the reprogramming and evaluate the functional maturation of iCMs.By using NCFs isolated from Myh6-Cre/LSL-GCa MP3 mouse and reprogramming them into iCMs,the principals described above was validated,while the monitoring and evaluating methods were also established.Furthermore,the influence of BETd-246 to the function of iCMs generated by cardiac reprogramming was evaluated with this system.Results:(1)Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCa MP3)Hze/J mouse strain(referred as Myh6-Cre/LSL-GCa MP3)was developed by crossbreeding Tg(Myh6-cre)1Jmk/J and Gt(ROSA)26Sortm38(CAGGCa MP3)Hze/J strain.Myh6-Cre/LSL-GCa MP3 mouse strain can specifically express Cre enzyme in Myh6+ cells,which can delete the LSL(Lox P-StopLox P)sequence in LSL-GCa MP3 sequence to enable the expressing of GCa MP3,and finally this mouse strain will specifically express GCa MP3 in CMs.(2)By comparing neonatal mouse hearts isolated from Myh6-Cre/LSL-GCa MP3 and control strain,it is observed that Myh6-Cre/LSLGCa MP3 hearts show a green fluorescence background and fluorescence flashing synchronized with beating,while no fluorescence was observed in control hearts.(3)By using NCFs isolated from Myh6-Cre/LSL-GCa MP3 mouse for MGT-induced cardiac reprogramming,we found there is no significant difference between this strain and previously reported mouse strain in reprogramming the NCFs to beating iCMs.(4)Real-time cardiac reprogramming monitoring method was further developed based on Myh6-Cre/LSL-GCa MP3 strain.During the cardiac reprogramming,the iCMs differentiated from this strain show green fluorescence flashing under fluorescence microscope,and the reprogramming process can be monitored by evaluate cells with such characteristics.Also,the function of current system in evaluating iCM functional maturation was also investigated by applying it to a previously reported four different chemical compounds IMAP(IGF1,MM589,A83-01,PTC-209)-induced cardiac reprogramming system.Comparing with the control group,IMAP group has more iCMs with fluorescence and the fluorescence is more regular,which shows that Myh6-Cre/LSL-GCa MP3 strain can be used to evaluate iCM functional maturation.(5)Whether BETd-246 could improving iCM functional maturation was evaluated with Myh6-Cre/LSL-GCa MP3 strain.Compared with the control group,BETd-246 treatment increased the number of spontaneous beating iCMs to 8 times after 4 weeks reprogramming,while the numbers of calcium flux positive cells after 2 weeks and 4 weeks reprogramming were also increased to about 7 and 9 times,respectively,which shows BETd-246 could promote the functional maturation of iCM.Conclusion:By applying NCFs isolated from Myh6-Cre/LSL-GCa MP3 mouse for cardiac reprogramming,two major obstacles in cardiac reprogramming research field were overcame,by which the cardiac reprogramming can be real-timely monitored and the maturation of iCMs can be efficiently evaluated.With such evaluation method,it is confirmed that BETd-246 could improve the functional maturation of iCMs.Chapter 3: The mechanistic research of the promoting function of BET degrader in cardiac reprogrammingBackground:Research above has shown the improvement of cardiac reprogramming and functional maturation of iCMs in the in vitro MGTinduced system by treatment of BETd-246,a BET degrader.However,it is still unclear the mechanism how BET degrader promoting cardiac reprogramming,and whether it could still have similar promoting function in the in vivo cardiac reprogramming system.The answer of those questions will be explored in this chapter.Aims:Determine the mechanism how BET degrader promotes cardiac reprogramming and determine its function in the in vivo cardiac reprogramming system.Methods:(1)By measuring the level of each BET protein with WB after BET degrader introduction in cardiac reprogramming system,the major target protein of the degrader will be determined.Also,the BET protein that plays major role in cardiac reprogramming will be determined by applying gene knockdown of each BET protein with CRISPR technology and measuring the reprogramming efficiency.(2)RNA-seq was applied to determine the gene expression level change in both MEF-based and NCF-based cardiac reprogramming system after BETd-246 treatment.Based on the RNA-seq data,further gene ontology(GO)analysis was applied to determine the biological processes,molecular functions and downstream pathways influenced by BETd-246.(3)While macrophages may play an important role in the regulation of cardiac reprogramming,several treatments related to macrophages,including macrophage co-culturing,macrophage supernatant treatment,and cytokines released by macrophages were applied to cardiac reprogramming system with BETd-246 treatment,and multiple methods including WB,IF,q PCR,chromatin immunoprecipitation(Ch IP),etc.was further used for the determination of specific mechanisms.(4)Left anterior descending(LAD)ligation method was used to develop a mouse MI model and MGT-embedded virus was injected directly into the myocardium to initiate in vivo cardiac reprogramming.In this system,BET degrader was further introduced to promote the reprogramming.4 and 8 weeks after the reprogramming,echocardiography was applied to measure the cardiac function recovery,which could help to evaluate the reprogramming efficiency and the function of BET degrader.Results:(1)BET degrader was found to have a great degradation efficiency to multiple BET proteins including BRD2,BRD3,and BRD4 in MEFs.Gene knockdown experiment show that BRD4 knockdown could enhance the cardiac reprogramming efficiency to 3 times of it in the control group,which means BRD4 is the major effector of BET degrader in cardiac reprogramming.(2)Compares with the control group,MGT+BETd-246 treatment led to 52 significantly promoted and 284 significantly inhibited genes expression in both MEFs and NCFs.Further GO analysis results show that multiple biological processes and molecular functions were regulated,while GO pathway analysis show that JAK/STAT pathway is one of the major pathways inhibited by BETd-246 treatment.(3)It is found that co-culturing with macrophages and treatment with macrophage supernatant could repress cardiac reprogramming.The screening of macrophage-released cytokines revealed that OSM is the major inhibitor of cardiac reprogramming among those cytokines,and it could stimulate JAK/STAT pathway to inhibit cardiac reprogramming,while BETd-246 abolishes such inhibition by macrophages/OSM.(4)Further evaluation of the mechanism reveals that BRD4 directly binds to the promotor region of genes of OSMR and IL6 ST,the key receptors of OSM pathway,while sh RNA knockdown of those receptor proteins could enhance the reprogramming.(5)The post-MI cardiac function could be significantly recovered by applying in vivo cardiac reprogramming in mouse MI model and introducing BRD4 degrader BETd-260 to the system.Conclusion:BET degrader protects cardiac reprogramming from macrophage/OSM induced inhibition by degrades BRD4 to inhibit JAK/STAT pathway activation.Meanwhile,BET degrader could promote the recovery of cardiac function in the in vivo cardiac reprogramming system. |
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