Research On NSD2-mediated Metabolic Reprogramming And Its Mechanisms In A Rat Model Of Pulmonary Arterial Hypertension

Chapter 1 IntroductionPulmonary arterial hypertension(PAH)is a cardiovascular disorder with high mortality and multiple factors involved in its development and progression,and lung transplantation or heart-lung transplantation is the only curative treatment for PAH patients in end-stage.So,it’s an urgent task to elucidate the mechanisms underlying PAH for developing novel treatment option in this field.Recent studies have demonstrated that,PAH shares several analogous features with carcinogenesis,including pathological hyperproliferation,apoptotic resistance,and metabolic reprogramming et al.This study used monocrotaline(MCT)-induced rat PAH as experimental model,and focused on nuclear receptor binding SET domain 2(NSD2)and the methylation level of histone lysine(especially H3K36)regulated by NSD2.In order to elucidate the role of NSD2 and methylation of histone lysine in pulmonary arterial pressure,remodeling of pulmonary artery and right ventricle,and function of right ventricle in PAH,western blot was adopted to test the protein expression of NSD2 and methylation level of histone lysine;and adeno-associated virus(AAV)vector was used to downregulate the protein expression of NSD2;and echocardiography was used to examine right ventricle function and hypertrophy;and right ventricular microcatheter was applied to examine pulmonary arterial pressure;and histological analysis was used to evaluate the histological changes in pumlmonary artery and right ventricle;and Immunofluorescence and confocal microscope were adopted to show the protein expresson localization of NSD2 in pulmonary artery.Secondly,gas chromatography-mass spectrometry(GC-MS)was adopted to analyze the metabolites with differential expression and screen the metabolites with most significant difference;and Kyoto Encyclopedia of Genes and Genomes(KEGG)and network analysis were applied to analyze the potential function(s)of these/this metabolite(s)and their/its role in PAH.Lastly,we used Illumina Hiseq2000TMto detect the genes with differential mRNA expression in PAH groups;and gene ontology(GO)was used to analyze the enrichment level of these genes from three levels namely cellular component(CC),molecular function(MF)and biological process(BP);and KEGG was used to analyze the enrichment level of these genes in pathways;and clustering analysis was used to screen out the downstream targets of NSD2 which might participate in NSD2-regulated PAH,which might be helpful for future studies.Conclusively,in this study,we used metabonomics and second generation high throughput sequencing technology as technical supports,focused on NSD2-mediated methylation of H3K36-regulated metabolic reprogramming which might promote the remodeling of pulmonary artery and right ventricle via promoting cell excessive proliferation and apoptotic resistance,and elucidated the mechanisms underlying PAH,hoping to provide experimental evidences for the prevention and treatment for PAH,and provide novel ideas for PAH-related drug discovery.Chapter 2 The role and mechanisms of NSD2 inMCT-induced PAHObjective:To elucidate the influence of downregulation of NSD2 protein expression on pulmonary arterial pressure,right ventricle function and remodeling of pulmonary artery and right ventricle in MCT-induce rat PAH model;and to show the protein expression localization of NSD2 in pulmonary artery;and to verify the regulatory role of NSD2 on methylation of histone lysine;and to examine the influence of downregulation of NSD2 protein expression on mRNA expression of metabolic factors.Methods:Intraperitoneal injection of MCT(60mg/kg body weight/BW)was used to establish rat PAH model,and intraperitoneal injection of saline was used as control.AAV was used to construct the recombinant virus particle of shNSD2(AAV-shNSD2,AAV-Null as control).,and H9C2 infection was used to test the 12 interfering efficiency.Tail vein injection of AAV-shNSD2(5×1012)was used to establish NSD2 interfering model,and AAV-null was used as contol.Rats were divided into 4 groups:rats in group Nu11MCT were injected with AAV-Null and MCT;rats in group NullSham were injected with AAV-Null and saline;rats in group shNSD2MCT were injected with AAV-shNSD2 and MCT;rats in group shNSD2Sham were injected with AAV-shNSD2 and saline.After 3 weeks of injection,right ventricle microcatheter and left carotid artery cannulation were performed for pulmonary arterial pressure and systemic blood pressure respectively.Echocardiography was performed for right ventricle function and hypertrophy.Lungs were harvested,and used to make histological slides,to extract total protein and total RNA.Histological analysis was applied to examine the remodeling of pulmonary artery and right ventricle,and immunofluorescence and confocal microscope examination was performed for the protein expression localization of NSD2,and real time quantitative PCR was performed to detect the mRNA expression.Results:(1)We successfully contructed NSD2-interfering recombinant virus particle,which was verified by the downregulation of NSD2 mRNA in AAV-shNSD2 infected H9C2 cells.(2)Rats were infected by AAV-shNSD2 successfully,which was demonstrated by the downregulation of protein expression of NSD2.(3)MCT can induce PAH(NullMCTvsNullsham;shNSD2MCT vs shNSD2Sham):compared with rats injected with saline,rats injected with MCT showed higher right ventricular mean pressure(RVMP),right ventricular systolic pressure(RVSP),mean pulmonary arterial pressure(mPAP)and total pulmonary resistance(TPR)and lower right ventricular cardiac output(RVCO),whereas there were no significant changes in systemic arterial blood pressures(SBP)and mean systemic arterial pressure(mSAP);Silencing of NSD2 suppressed the development of PAH(shNSD2MCT vs NullMCT),which was indicated by significant decrease in RVMP,RVSP,mPAP and TPR in shNSD2MCT group compared with those in NullMCT group and significant increase in RVCO in shNSD2MCT group compared with that in NullMCT group.(4)MCT-indcued PAH showed right ventricle dysfunction and hypertrophy(NullMCT vs NullSham;shNSD2MCT VS shNSD2Sham):MCT significantly increased right ventricular end diastolic diameter(RVEDD),right ventricular end diastolic volume(RVEDV),right ventricular end systolic diameter(RVESD),right ventricular end systolic volume(RVESV),right ventricular posterior wall thickness(RVPWT)and interventricular septal thickness(IVST)and significantly decreased right ventricular ejection fraction(RVEF),right ventricular fractional shortening(RVFS)and pulmonary artery acceleration time normalized to cycle length(PAAT/CL);NSD2 silencing effectively increased RVEDD,RVEDV,RVESD,RVESV,RVPWT and IVST,and reduced RVEF;RVFS and PAAT/CL in AAV-shRNA-NSD2 infected rats(shNSD2MCTvsNullMCT).(5)MCT can induce the thickening of pulmonary artery media and right ventricle hypertrophy(Nu11MCT vs NullSham;shNSD2MCT vs shNSD2Sham):relative pulmonary arterial wall thickness(PAMT%),the ratio of right ventricle(RV)weight to body weight(BW)(RV/BW)and RV/(LV+S)in MCT treated rats significantly increased compared with those in saline treated rats;NSD2 silencing partially inhibited the increase in PAMT%,RV/BW and RV/(LV+S)(shNSD2MCT vs NullMCT).(6)MCT-induced PAH showed higher methylation level of H3K36(H3K36me2)(NullMCTvs NullSham;shNSD2MCTvsshNSD2Sham),whereas NSD2 silencing markedly decreased H3K36me2(shNSD2MCTvsNullMCT).(7)NSD2 was mainly expressed in pulmonary artery smooth muscle cells(PASMCs)and pulmonary endothelial cells(PAECs)in pulmonary artery.(8)MCT-induced PAH showed significantly decrease in Glutl and PDH mRNA expression(NullMCT vs NullSham;shNSD2MCT vs shNSD2Sham),and markedly increase in LDH,CPT1 and OXCT1 mRNA expression(NullMCTvsNullSham;shNSD2MCTvs shNSD2Sham),whereas NSD2 silencing markedly decreased OXCT1 mRNA(shNSD2MCTvsNullMCT).Conclusions:NSD2 was involved in the development and progression of PAH via catalyzing the di-methylation of H3K36,whereas NSD2 silencing can suppress PAH.Chapter 3 Metabolomics analysis of NSD2-mediated PAHObjective:To screen the metabolites with differential expression,and elucidate the regulatory functions of these metabolites and its potential role in PAH.Methods:Intraperitoneal injection of MCT(60mg/kg)was used to establish rat PAH model,and intraperitoneal injection of saline was used as control.AAV was used to construct the recombinant virus particle of shNSD2(AAV-shNSD2,AAV-Null as control),and H9C2 infection was used to test the interfering efficiency.Tail vein injection of AAV-shNSD2(5×1012)was used to establish NSD2 interfering model,and AAV-null was used as contol..Rats were divided into 4 groups:rats in group NullMCT were injected with AAV-Null and MCT;rats in group NullSham were injected with AAV-Null and saline;rats in group shNSD2MCT were injected with AAV-shNSD2 and MCT;rats in group shNSD2Sham were injected with AAV-shNSD2 and saline.After 3 weeeks of injection,lungs were harvested and used to extract metabolites ana protein.Gas chromatography-mass spectrometry(GC-MS)was adopted to analyze the metabolites with differential expression,and screen the metabolites which might be regulated by NSD2;Kyoto Encyclopedia of Genes and Genomes(KEGG)and network analysis were used to analyze the metabolites function and its potential role in PAH.Western blot was adopted to test the protein expression of autophagy related genes.Results:(1)There were 11 metabolites increased in NullMCT compared with NullSham,and among the 11 upregulated metabolites,trehalose was the only metabolite which was downregulated by NSD2 silencing(shNSD2MCT vs NullMCT;shNSD2Sham vs NullSham).(2)Enrichment analysis of KEGG metabolic pathway indicated that NSD2-regulated trehalose mainly affected ABC transporters,mineral absorption,protein disgestin and absorption,metabolic pathways and aminoacyl-tRNA biosynthesis.(3)Network analysis found that trehalose is closely related to ABC transporters and metabolic pathways in PAH.(4)NSD2 silencing significantly downregulated positive regulators of autophagy including LC3-II,beclin-1 and phos-AMPK,and upregulated autophagy suppressor p62.Conclusions:NSD2-mediated trehalose metabolism might participate in the development and progression of PAH via ABC transporters and metabolic pathways.Trehalose might participate in PAH via regulating autophagy.Chapter 4 Gene expression profile analysis ofNSD2-mediated PAHObjective:To screen the downstream target genes of NSD2,and provide target genes for future study about NSD2-mediated PAH.Methods:Intraperitoneal injection of MCT(60mg/kg)was used to establish rat PAH model,and intraperitoneal injection of saline was used as control.AAV was used to construct the recombinant virus particle of shNSD2(AAV-shNSD2,AAV-Null as control),and H9C2 infection was used to test the interfering efficiency.Tail vein injection of AAV-shNSD2(5×1012)was used to establish NSD2 interfering model,and AAV-null was used as contol..Rats were divided into 4 groups:rats in group NullMCT were injected with AAV-Null and MCT;rats in group NullSham were injected with AAV-Null and saline;rats in group shNSD2MCT were injected with AAV-shNSD2 and MCT;rats in group shNSD2Sham were injected with AAV-shNSD2 and saline.After 3 3 weekw of injection,lungs were harvested and used to extract total RNA.Illumina Hiseq2000TMwas used to screen the genes with differential expression;Gene ontology(GO)and KEGG analysis were performed to analyze the enrichment level of these genes in cellular component,molecular function,biological process and pathways;Clustering analysis was used to screen the downstream targets of NSD2 which might participate in NSD2-regulate PAH.Results:There were many genes with differential expression in MCT-induced PAH rats(NullMCT vs NullSham),among which,NSD2 silencing can significantly decrease the mRNA expression of following genes(shNSD2MCT vs NullMCT;shNSD2Sham vs NullSham):TM4SF1,MID1IP1,PDE3A,MUC13,FOLR1,MTSSILand TXNIP.Conclusions:TM4SF1,MID1IP1,PDE3A,MUC13,FOLR1,MTSS1Land TXNIP might be the downstream of NSD2,and be involved in PAH via mediating NSD2-regulated metabolic reprogramming.