Aberrant Expression Of PSEN1 Is Involved In Pathology Of Vascular Smoonth Muscle And Cardiomyocytes

I.Research backgroundCardiovascular disease is still the main cause of death in China.The occurrence of coronary heart disease is due to endothelial cell injury and abnormal proliferation of vascular smooth muscle,leading to vascular stenosis and myocardial ischemia.Aging is one of the biggest risk factors for the progression of cardiovascular disease.The molecular mechanism of age-related cardiovascular pathophysiology is unclear.The pathophysiological processes related to aging include the phenotypic transformation of vascular smooth muscle and the changes of cardiac function after myocardial ischemia.These changes of pathophysiological activities are closely related to the abnormal expression of many aging related genes.PSEN1 mutation is one of the core genes of familial Alzheimer's disease.PSEN1 is a multiple transmembrane protein,which is widely expressed in various tissues.In this study,we found that PSEN1 is highly expressed in brain and heart.Studies have shown that PSEN1 mutation is associated with genetically related dilated cardiomyopathy.Our research group has also reported that PSEN1 knockout can lead to abnormal cardiac development in mice,including ventricular septal defect,double outlet of right ventricle and pulmonary artery stenosis;In addition,our research group also found that PSEN1 gene knockout can cause abnormal cardiac relaxation.Although PSEN1 plays an important role in the heart,the cardiovascular function of PSEN1 in the process of aging remains to be further studied.II.Experimental model and method1.RNA extractionTotal RNA was extracted from the tissues of humans and mice using TRIzol reagent(Invitrogen,Shanghai,China)and digested with RNase-free DNase to eliminate genomic DNA.2.Real time fluorescence quantitative PCRTotal RNA(500 ng)was used to generate c DNA by M-MLV reverse transcriptase(Promega)with the random 9-nt priming method.Real-time quantitative PCR was performed using 1×SYBR Green Mix(TOYOBO Co,Japan)in an ABI7900HT system(Applied Biosystems).The expression of the target m RNAs was normalized to that of the housekeeping gene GAPDH.The average expression of the target m RNAs in the control group was defined as 1.The relative m RNA level in the experimental group was expressed as the fold-change relative to the control samples.3.Cell line cultureCell lines P19,AC16,H9C2,293T and He La were cultured in DMEM medium containing 10%fetal bovine serum and 100 U/ml streptomycin.RAW264.7 was cultured in RMPI-1640 medium containing 10%fetal bovine serum and 100 U/ml streptomycin.PASMC and AOSMC were cultured in SMCM growth factor medium containing 2%fetal bovine serum and 100 U/ml streptomycin.HCAEC and HUVEC were cultured in ECM growth factor medium containing 5%fetal bovine serum and 100 U/ml streptomycin.4.Isolation and culture of primary cardiomyocytes and myocardial fibroblastsThe ventricles of heart from 3 days old rats were cut into about 1 mm3.Tissue blocks were digested with 0.1%collagenase at 4?overnight.The next day,the tissue block was centrifuged at 1000 rpm for 15 min.Myocardial fibroblasts were isolated after differential adhesion for 2 hours;The supernatant cells were counted and planted.After 24 hours,the medium was replaced with DMEM solution containing 10%calf serum and 0.1 mm Brd U to inhibit the proliferation of non-cardiomyocytes.After 48 hours of incubation,it was replaced with serum-free DMEM solution.5.Smooth muscle phenotype transformation model(1)PASMC and AOSMC cells were cultured in SMCM medium.Half of PASMC and AOSMC cells were cultured in serum-free growth factor free SMCM medium for 24 hours.(2)PASMC and AOSMC cells were inoculated and replaced with serum-free growth factor free SMCM medium.After starvation for 12 hours,half of the cells were not treated,and the other half were added with 20 ng/ml PDGFbb.After continuous culture for 24hours,the phenotype transformation model of smooth muscle cells was constructed.6.Endothelial cell proliferation and injury model(1)Endothelial cell hypoxia model:HCAEC cells were planted into three 6-well plates.Two 6-well plates were cultured in 1%O₂and 5%CO₂cell incubators.RNA was collected after hypoxia treatment for 9 hours and 24 hours respectively.HCAEC cultured in normoxia was harvested for RNA at 24-hour time points.(2)Endothelial cell proliferation model:HUVEC cells were planted into 6-well plates with a density of 70%.Four wells were replaced with serum-free growth factor free ECM medium.After 12 hours,VEGF was added to 2 wells and FGF was added to 2 wells.The protein was collected after continuous treatment for 24 hours.(3)Endothelial cell injury model:HCAEC cells were planted into the cell culture plate,they were treated with serum-free growth factor for 12 hours,treated with homocysteine and ox LDL for 24 hours respectively,and RNA was collected for subsequent analysis.7.Collection of human myocardial samplesHuman heart samples used in this manuscript were collected from patients who had organ transplants in Shanghai Changhai hospital from 2012 to 2018.Seven ICM samples came from heart transplant patients whose failed hearts were abandoned after surgery.Nine normal heart samples from donors for liver transplants or other organ transplants.All samples were obtained with the informed consent of the patient and his family.After obtaining fresh specimens during clinical surgery to take liquid nitrogen freeze storage,place them in an 80-degree refrigerator.The collection of clinical specimens have been approved by the Ethics Committee of the Naval Medical University.8.Western blotForty micrograms of protein were resolved by 12%SDS-PAGE gel and transferred to the PVDF membrane.The membranes were blocked with 5%skimmed milk at room temperature for 2 h and then incubated overnight with one of the following primary antibody at 4?.Washing with TBST for 4 times,the membranes were incubated with secondary monoclonal antibody at room temperature for 1?2 h.After washing with TBST4 times,the blots were visualized with an ECL system.9.Construction of PSEN1 smooth muscle and myocardial specific knockout miceCardiac and smooth muscle PSEN1 knockout mice were obtained by hybridizing PSEN1 floxp mice with Sma22-Cre mice.10.Histological staining and ultrastructural observation of mice(1)Histological staining:mouse tissues were fixed in 4%paraformaldehyde.The tissue was washed with water,dehydrated in turn with concentration gradient ethanol,and removed with xylene to make the tissue block transparent;Immerse the wax for 2-3h,put the soaked sample section down into the embedding device containing paraffin and stand.Paraffin embedded tissue block sections,cut wax strips,flattened in warm water,slide fishing,conventional HE staining.(2)Ultrastructural analysis:the hearts of 1-year-old mice were cut into 1 mm³small pieces,fixed with 4%paraformaldehyde,embedded with conventional electron microscope,sliced and stained with uranium acetate lead citrate.11.The blood pressure of mice was measured by carotid artery intubationAfter isoflurane anesthesia,the external jugular vein and common carotid artery were separated for common carotid artery intubation,and the pressure sensor and biological signal conversion system were connected in turn.Pull out the small plastic plug at the distal end of the common carotid artery intubation(the artery clamp is still clamped)and connect it with the interface of the sensor(filled with normal saline and heparin in advance),and gently open the artery clamp.Blood pressure fluctuations are converted into electrical changes directly through the pressure sensor.After amplification,the amplifier is analyzed and processed by microcomputer through analog-to-digital converter.The display screen can observe the fluctuation of blood pressure.The blood pressure of each mouse was recorded for 10 minutes,and the systolic and diastolic blood pressure were calculated.12.Carotid cannula induced vascular stenosis model and femoral artery intimal injury model(1)Carotid artery stenosis model caused by carotid cannula:after isoflurane anesthesia,the common carotid artery was fully exposed,and a miniature plastic flexible catheter with a length of 1 cm was cut longitudinally.Put the cannula notch into the common carotid artery,fix the cannula with 7-0 silk thread,do not tie tightly on both sides,and then tie tightly after 90%of both ends are fixed.Suture skin wounds.Conventional feeding for 2 weeks,sampling,fixation and histological observation.On the sham operation side,only the common carotid artery was separated,but no plastic soft catheter was placed.(2)Femoral artery intimal injury model:after isoflurane anesthesia,the femoral artery was separated and the distal end was ligated.Put the guide wire into the femoral artery and pull it repeatedly for 10 times.The wound was sutured.After 2 weeks,the materials were taken for histological observation.13.MRNA expression and function enrichment were detected by microarrayRNA was extracted from the aorta and hearts of mice.MRNA microarray analysis was performed after sample mixing.The genes with abnormal expression were determined by using the difference multiple of more than 2 times as the screening condition.The abnormally expressed genes after PSEN1 knockout were analyzed by a series of plug-ins of Cytoscape.Use the default parameters of cytohubba plug-in to obtain hub gene;Gene ontology and KEGG analysis were performed using the cluego plug-in in Cytoscape.14.Detection of cardiac function in adult miceTwelve mice suffered from PSEN1 heart and vascular smooth muscle specific knockout and 12 wild-type mice aged 11 months were anesthetized with isoflurane.The left ventricular end systolic chamber volume,end diastolic chamber volume,shortening fraction,ejection fraction and other indexes were measured by visual sonic ultrasound system.15.Overexpression of ATP2A2 in strip knockout mice by recombinant adenovirusHuman ATP2A2 was amplified by PCR and cloned into p Shuttle CMV plasmid.The recombinant adenovirus was established according to the adeasytm XL adenovirus vector system specification Adenovirus was purified by viratraptm adenovirus Purification Kit.In order to determine the best titer of virus,pfu/ml was estimated.16.Enzymatic separation of single cardiomyocytes and fluorescence detection of Ca2+in cardiomyocytesSingle cardiomyocytes were isolated from the hearts of the adult mice using an enzymatic dissociation method.Briefly,the hearts were removed from the anesthetized mice and were subjected to retrograde perfusion with a modified Langendorff system with Ca²⁺-free Tyrode's solution and subsequently perfused with a collagenase type II solution containing bovine serum albumin(BSA)for approximately 8-10 min.After digestion,the ventricular myocardium was cut into small pieces in a modified Kraft-Bruhe(KB)solution and gently shaken to dissociate the cells.17.Statistical analysisThe data are expressed as mean±standard deviation.For the data conforming to the normal distribution,the experimental group and the control group were tested by t-test.P<0.05 was significant difference.III.Results(1)Abnormal expression of membrane mosaic protein PSEN1 in cardiovascular diseases1.Expression of PSEN1 in tissues and cell linesThe expression level of PSEN1 m RNA in various organs was detected by RT-PCR.The results showed that PSEN1 was widely expressed in 13 tissues.PSEN1 is expressed in cardiovascular related cell lines.PSEN1 has multiple bands in smooth muscle cells,endothelial cells,myocardial fibroblasts and cardiomyocytes,mainly a full-length sequence of about 65 k D,an N-terminal sequence of about 35 k D;and C-terminal sequence of about 20 k D.2.Expression of PSEN1 in cardiovascular disease modelThe phenotypic switching model of smooth muscle cells from contractile to secretory was constructed by PDGFbb.PDGFBB stimulation could reduce the expression of PSEN1.In the model of serum deprivation inhibiting proliferation of smooth muscle cell,the m RNA expression of PSEN1 was increased significantly after serum deprivation.The expression of PSEN1 m RNA was decreased in hypoxia treated endothelial cells.The protein expression level of PSEN1 was not changed in endothelial cell proliferation model constructed by VEGF and FGF.There was no difference in the expression of PSEN1 m RNA in the endothelial cell injury model was constructed by homocysteine and ox LDL.The expression levels of PSEN1 m RNA and protein was decreased significantly to about 60%of normal hearts in human ischemia-induced heart failure samples.(2)Establishment and validation of cardiac and smooth muscle specific PSEN1 knockout mice1.Sma22-Cre can remove loxp flanked stop codon in vascular smooth muscle and myocardiumThrough the hybridization between Sma22-Cre mice and mice having EGFP loxp flanked stop codon,the sections of hearts from offspring mice were observed.It showed that EGFP was mainly in cardiomyocytes and smooth muscle cells.Smooth muscle and myocardial specific knockout mice of PSEN1 were obtained by hybridization between floxp mice of Exon4 and Sma22-Cre mice.2.Knockout validation of Sma22-cre-PSEN1 floxp mice in vascular smooth muscle and myocardiumWestern blot was used to detect the protein expression of PSEN1.The results showed that in sma22-cre-PSEN1 floxp homozygous mice,the RNA and protein expression of PSEN1 decreased significantly in blood vessels and heart,but there was no difference in brain.3.Survival analysis of vascular smooth muscle and myocardial PSEN1 gene knockout miceAt 11 months of age,the survival curves of mice in WT and PSEN1-ko groups showed that Sm22a-PSEN1-ko caused spontaneous death of mice,especially elderly mice.(3)Function and mechanism of membrane mosaic protein PSEN1 in vascular smooth muscle cells1.The histology and blood pressure of PSEN1 knockout vessels were abnormalThrough the fluorescence observation of tissue sections of sma22-cre-PSEN1 floxp EGFP stop floxp mice,it was found that the arrangement of elastic fibers was disordered after sma22 CRE knocked out PSEN1 in smooth muscle cells at the age of 8 weeks.The blood pressure of mice was measured by arterial catheter.It was found that the expression of systolic and diastolic blood pressure increased in both male and female mice after PSEN1 knockout.2.PSEN1 knockout causes vascular inflammation during agingThere was no significant difference in the expression of calp1,calp2,calp3,myh11,ACTA2 and itgb between the blood vessels of 8-week-old wild mice and knockout mice;There was no significant difference in the expression of MCP1 and CRP.Western blot showed that there was no significant difference in the expression of CD45,Ly6C and VCAM1 proteins between the blood vessels of 8-week-old wild mice and knockout mice.The vascular RNA of 8-month-old mice was extracted.Quantitative PCR showed that the expression of ACTA2 and myh11 increased in PSEN1 knockout mice;The expression of CDH1 decreased significantly.Western blot showed that CD45,Ly6C and VCAM1increased significantly in the blood vessels of 8-month-old PSEN1 knockout mice.It was found that PSEN1 knockout promoted the expression of VCAM1,Ly6C and BCL2 in the blood vessels of 11 month old mice.3.Function of PSEN1 knockout in vascular injury modelThe results showed that PSEN1 knockout could significantly promote the process of smooth muscle remodeling caused by arterial constriction.The vascular remodeling model of femoral artery was established through the guide wire injury model.It was found that the vascular remodeling of 12 week old mice in PSEN1 knockout group was significantly higher than that in wild-type control group.4.The effect of PSEN1 knockout on gene expression at transcriptome levelThe abnormally expressed genes in blood vessels were detected by RNA microarry.Functional enrichment analysis of abnormally expressed genes by Cytoscape showed that the genes were enriched in Toll like receptor 4 binding,TCR complex interactions with peptide antigen presenting MHC class I,positive regulation of lymphocyte apoptotic process;Many genes are also enriched in muscle contraction.These abnormally expressed genes are closely related to the occurrence of inflammation and the proliferation of smooth muscle cells in the inflammatory environment.5.Effect of PSEN1 knockout on signal pathway in blood vesselsERK and Akt signaling pathways were detected.It was found that the basic expression and phosphorylation level of ERK were significantly increased in elderly rats.PSEN1 knockout could promote the phosphorylation of ERK.The expression level and phosphorylation level of Akt were detected,which showed that the phosphorylation level of Akt was significantly increased in PSEN1 knockout mice.The results showed that PSEN1 knockout did not affect the expression of tead4 and significantly reduced the expression of Yap1 in young mice;In aged rats,PSEN1 knockout significantly increased the expression of tead4 without affecting the expression of Yap1.The downstream molecules of yap1-tead4 were detected by quantitative PCR,which showed that the expressions of CTGF and cry61 were significantly increased in the blood vessels of PSEN1 knockout mice.The detection showed that the protein expression level and phosphorylation level of SMAD2 increased significantly.The detection showed that compared with young mice,the expression level of ctnnb in blood vessels of old mice decreased significantly.In young mice,PSEN1 knockout did not affect the expression levels of ctnnb and AXIN1,while in old mice,PSEN1 knockout promoted the expression of ctnnb and AXIN1.The expression of HER2 and Hes1 was not affected by PSEN1,but the expression level of HER1 was significantly increased.(4)Function and mechanism of PSEN1 in cardiomyocytes1.Effect of myocardial PSEN1 gene knockout on cardiac function in elderly ratsThe echocardiographic results of 12 WT and 12 Sm22a-PSEN1-ko mice at the age of11 month old showed that the ejection fraction and left ventricular short axis shortening in Sm22a-PSEN1-KO group were significantly lower than those in WT group.The left ventricular diastolic diameter(lvid-d)and systolic phase(lvid-s)were significantly increased in Sm22a-PSEN1-ko group.After the mice were sacrified,the heart weight/body weight ratio of PSEN1-KO mice at the age of 11 months was significantly lower than that of WT mice of the same age.The ultrastructure of heart tissue of WT mice and Sm22a-PSEN1-ko mice was compared.In Sm22a-PSEN1-ko mice,myofibrils decreased,sarcomere Z-line widened and sarcomere length increased.The detection of heart failure marker molecules by q RT-PCR showed that the expression of ANP and BNP in the heart of Sm22a-PSEN1-ko mice was significantly up-regulated compared with WT mice.2.Gene analysis of abnormal cardiac expression in sma22-PSEN1 knockout miceMicroarry was used to screen abnormal genes in the hearts of 3-month-old WT and Sm22a-PSEN1-ko mice.A total of 312 abnormal regulatory genes(difference multiple>2)were detected,including 143 up-regulated genes and 178 down-regulated genes.These genes were analyzed using a string database of protein interactions.Through the cytohubba plug-in,we use the default parameters of Cytoscape software to obtain critical nodes.The top three nodes(TTN,MYBPC3 and NPPA)are used to analyze the network topology of protein-protein interaction.Cluego analysis of Cytoscape software showed that abnormally regulated genes were highly enriched in striated muscle cell development,muscle system process,embryonic heart tube morphogenesis,toll like receptor binding and negative regulation of bone resorption.KEGG analysis showed that the gene enriched pathway was also related to dilated cardiomyopathy,Apelin signal pathway,myocardial contraction,arrhythmogenic right ventricular cardiomyopathy,adrenaline related signal pathway in cardiomyocytes,cell adhesion molecules,regulation of lipolysis of adipocytes,antigen processing and presentation,thyroid hormone synthesis and primary immunodeficiency.3.Sm22a-PSEN1-ko increased the expression of Ry R2 and decreased the expression of ATP2a2 in cardiomyocytesThe expression of Ry R2 and voltage dependent L-type calcium channels in cardiomyocytes of Sm22a-PSEN1-ko mice compared with WT mice?-The expression of1C subunit(cacna1c)was up-regulated.The upregulation of Ry R2 and cacna1c in Sm22a-PSEN1-ko heart was confirmed by QRT PCR.Western blot further confirmed the upregulation of Ry R2 in Sm22a-PSEN1-KO heart;ATPase sarcoplasmic/endoplasmic reticulum Ca2+transport 2(SERCA2,ATP2a2)was significantly down regulated.4.Overexpression of ATP2a2 improved cardiac function in PSEN1-KO aging miceRecombinant adenovirus overexpressed ATP2a2 in PSEN1-ko aging mice.Five mice in each group were successfully transfected with Ad-CTL or Ad-ATP2a2.The expression of ATP2a2 was detected by Western blot.Three weeks later,echocardiography showed that the percentage of EF and FS increased significantly.Compared with Ad-CTL transfected mice,lvidd and lvids of Ad-ATP2a2 transfected mice decreased significantly.SR Ca2+concentration was determined by fluo-5N/AM.Compared with Ad-CTL,overexpression of ATP2a2 significantly increased the concentration of Ca²⁺in SR.Consistent with this,transfection of ATP2a2 down regulated ANP and BNP m RNA levels in PSEN1-KO aging mice.IV.Conclusion(1)PSEN1 is widely expressed in tissues and has a high expression level in smooth muscle cells,endothelial cells,myocardial fibroblasts and cardiomyocytes.(2)The expression of PSEN1 was decreased in the model of smooth muscle cell transformation from contractile to secretory type induced by PDGFbb.In the model of serum deprivation inhibiting proliferation of smooth muscle cell,the m RNA expression of PSEN1 was significantly increased after serum deprivation.After PSEN1 knockout,the levels of systolic and diastolic blood pressure were increased in both male and female mice.PSEN1 knockout causes vascular inflammation during aging.PSEN1 knockout plays an important role in vascular injury model.PSEN1 knockout can significantly promote the process of smooth muscle remodeling caused by arterial constriction.The vascular remodeling model of femoral artery was established through the guide wire injury model.It was found that the vascular remodeling of 12 week old mice in PSEN1 knockout group was significantly higher than that in wild-type control group.PSEN1 knockout resulted in changes signal pathways in blood vessels,such as MAPK-ERK signal pathway,PI3K-AKT signal pathway,YAP1-TEAD4 signal pathway,TGFb-SMAD signal pathway and WNT-CTNNB signal pathway.(3)The expression levels of PSEN1 m RNA and protein were significantly decreased in human ischemia-induced heart failure samples.PSEN1 conditional knockout resulted in age-dependent decrease of cardiac function and up-regulation of ANP and BNP expression.These genes were enriched in striated muscle cell development,muscle system process,embryonic heart tube morphogenesis,toll like receptor binding and negative regulation of bone resorption.The expression of Ry R2 was increased significantly,while the expression of ATP2a2 was decreased significantly.Overexpression of ATP2a2 can improve cardiac function and Ca2+concentration in SR of PSEN1-KO aging mice.