The Effects And Mechanism Of Astaxanthin In High Afterload-induced Mouse Cardiac Dysfunction

BackgroundHeart failure(HF)is a clinical syndrome shown as the loss of pumping energy which is induced by decreased systolic and(or)diastolic function.HF is the end-stage of most cardiac disease,for example,coronary heart disease,hypertension and cardiomyopathy.HF is also the direct reason of death of people who suffer cardiac diseases.Cardiac diseases like hypertension and aortic stenosis can increase afterload,which increase the pressure of heart.The myocardium was hypertrophy at the compensatory stage and then cardiac function decrease at the decompensatory stage.Heart is an organ with high oxygen consumption and the blood supply has significant influence on the cardiac function.Among the vessels,microvessels are the direct ones which exchange with cardiomyocytes.So the normal density and structure of microvessels are very important for cardiac systolic function.According to the previous study,the density of micro vessels decreases during the hypertrophic process,and the structure is damaged due to the changes of physical and chemical circumstance,as well as the increased oxidative stress,which eventually decrease the cardiac function.According to the above,it is of great clinical value to find effective therapy for improving micro vessels.Natural astaxanthin is a kind of xanthophyll carotenoid,has 100?500 fold greater free radical antioxidant activity than do vitamin E.A previous study showed that astaxanthin has the ability of promoting cerebral microvascular proliferation and tube formation.However,the effects of astaxanthin on myocardial microvascular are remain unknown.So,in our present study,we used mice model under pressure overload to investigate the effect of astaxanthin on myocardial microvascular and the effect on cardiac function.Objectives1.To investigate the effect of AST on cardiac dysfunction induced by pressure overload;2.To study the effect of AST on oxidative stress in heart under pressure overload for 12 weeks;3.To observe the effects of AST on the structure and function of microvessels.Methods1.Thoracic aortic coarctation(TAC):Eight weeks old C57 mice were used to model with TAC or SHAM surgery.The surgery was started from the neck incision,at the beginning of truncus brachiocephalicus and left common carotid artery,banded the aorta over a 27G needle,which was removed immediately.The aorta in animal with SHAM surgery was not banded;2.Gavage:TAC mice were randomly distributed to TAC group,TAC+AST-L group and TACC+AST-H group.AST was mixed in physiological saline,the dose of AST in TAC+AST-L group was 75mg/kg/d,and 200mg/kg/d in TAC+AST-H group.Mice in SHAM group were treated with physiological saline;3.Echocardiography:To measure the cardiac function after treatment for 4,8 and 12 weeks,The mice were anaesthetized using a mixture of isoflurane(1%?3%)and 02(2 L/min).Transthoracic echocardiography was performed using a Vevo 770 machine equipped with a 30-MHz transducer;4.Samples:The blood was centrifuged and the serum was used in ELISA to measure BNP level.The tissues were used in the following molecular biology experiments;5.Pathological test:Hematoxylin and eosin(HE)staining was used to observe the pathological changes of myocardium;6.Ultrastructural observation:Transmission Electron Microscope(TEM)was used to observe the ultrastructure of myocardium and the microvessels;7.Immunohistochemical staining:The expression of 4 Hydroxynonenal(4HNE)and 8-hydroxydeoxyguanosine(8-OHdG)was observed;8.Westemblot:The left ventricular myocardium was used to measure the levels of CD31?hypoxia-inducible factor-1?(HIF1?)?vascular endothelial growth factor(VEGF)?vascular endothelial growth factor2(VEGFR2),and the relative expressions were evaluated.9.Statistical analysis:The data was analyzed by using IBM SPSS Statistic 19 software.Differences between multiple groups were determined using t-test or one-way ANOVA analysis.Continuous data are presented as the means±SEM.Normalized data are presented as fold values over the mean of the control.In all comparisons,P<0.05 was considered to indicate statistical significance.Results1.Body weight,heart weight and lung weight of mice:There is no significant difference in body weight(P>0.05).Compared with SHAM group,heart weight,lung weight and the ratios with body weight in TAC group and TAC+AST-L group increased obviously(P<0.05).Compared with TAC group,the values in TAC+AST-H group decreased significantly(P<0.05).The above results showed that AST(200mg/kg/d)treatment inhibited cardiac remodeling2.AST improved pressure overload induced cardiac dysfunction:compared with the SHAM group,the LVEF(%),E/A and Ea/Aa ratios decreased,the BNP level in serum increased;compared with TAC group,the values and ratios in the group with high dose of AST increased,accompanied by decreased BNP level;while the above values and ratios had no significant difference compared with that in the TAC group.Moreover,compared with the SHAM group,the heart size increased in the TAC group,especially the left heart,and treatment with AST at the high dose inhibited the increase,while the low dose AST didn't influence it significantly.3.AST improved the pathological changes in myocardium:Compared with the pathological changes in SHAM group,HE stain showed that the myocardial fibers thickening,disarrangement and even rupture;ultrastructural result showed that part of the myofibril dissolved,Z lines in irregular arrangement or lost,mitochondrial number increased and there is more calcium salt dense particle deposition.The changes in the group with low dose AST were similar with that in TAC group,while the pathological changes were greatly improved by AST treatment with the high dose.4.AST reduced pressure overload induced oxidative injury:4HNE and 8-OHdG are the markers of oxidative injury of lipids and nucleic acids.Immunohistochemical staining showed that the positive area of 4HNE and the positive nuclear number of 8-OHdG are more in TAC group than that in SHAM group(P<0.05).AST treatment with high dose reduced the increase obviously(P<0.05).5.AST improved the injury of myocardial microvessels induced by pressure overload:CD31 stain showed that microvessel density was lower in TAC group compared with SHAM group,and WB showed that CD31,HIF1??VEGF and VEGFR2 protein expression decreased(P<0.05).However,in TAC+AST-H group,the microvessel density and protein expression didn't decrease obviously(P>0.05).Ultrastructural investigation showed that,in TAC group,the vessel wall was irregular,the lumen was narrowed and the cytoplasm of endothelial cells was heterogeneous,more dye appeared out of the vessel revealed increased permeability.The above harmful changes were not obvious in TAC+AST group.6.Survival rate in all groups:Compared with SHAM group,the survival rate in TAC,TAC+AST-L and TAC+AST-H groups all decreased(P<0.05),but there is no significant difference among these three groups(P>0.05).Conclusion1.AST obviously improved the cardiac dysfunction induced by pressure overload;2.AST significantly reduced the oxidative stress injury induced by pressure overload;3.AST increased the density and improved the structure of microvessels.BackgroundMyocardial fibrosis is the pathological process widely happen in most of the heart diseases,and is one of the main reasons which reduced cardiac function.During the pathological process,the fibroblasts transfer to myofibroblasts,excessive extracellular matrix(ECM)is produced and collagen deposited abnormally.The increase of cardiac interstitial fibrosis induces the stiffness and reduces the compliance of left ventricular wall and then results to cardiac function reduction;The increased fibrosis of the perivascular area decreases coronary flow reserve,myocardial ischemia is more easier to be induced and then results to myocardial necrosis;Morever,cardiac fibrosis causes the formation of reentrant pathway and abnormal electrical activity,which is the pathological basement of arrhythmia even the malignant arrhythmia.Transforming growth factor-?(TGF-?)plays a critical role in the process of fibrosis.SMAD protein family is the earliest generally acknowledged downstream molecular of TGF-? signaling,receptor-activated SMADs(R-SMADs),SMAD2 and SMAD3 included,can be phosphorylated after combining with TGF-? receptor directly.They form heterologous polymers with SMAD4,and then transfer the fibrotic signaling into nuclei,followed by regulating the downstream genes expression.In cytoplasm,phosphorylation is the activated form of SMAD2 and SMAD3,is also the acknowledged modified form.Recently,other functional modified forms have been revealed,acetylation for example,previous studies have showed that acetylation level influences the DNA binding capacity of SMAD,and then impacts its transcriptional activity.Studies have showed that inhibiting SMADs acetylation reduced renal fibrosis.However,during the cardiac fibrosis induced by pressure overload,the effect of R-SMADs acetylation remains unclear.Silent information regulator factor 2 related enzyme 1(SIRT1)is a class III histone deacetylase and is the most important protein within the sirtuin family.SIRT1 has been reported to deacetylate lysine residues within many nuclear proteins.Recently,it was also found to participate in apoptosis through deacetylation of the non-histone protein like SMAD7 and to inhibit renal fibrosis by deacetylating SMAD3.Moreover,studies have revealed that SIRT1 expression and activity reduced under oxidative stress.Studies showed that heart-specific overexpression of SIRT1 was protective to hearts under pressure overload.However,this is controversial,some other studies showed that pressure overload-induced ventricular hypertrophy was attenuated in Sirtl(+/-)mice and SIRT1 overexpression reduced cardiac function in mice.So,it's extremely important to do further study to know the effect of SIRT1 on cardiac function,especially the impact on pressure overload induced myocardial fibrosis for long time.In part one,we have shown that AST is protective to cardiac function,in this part,we will do more study to investigate the effect of AST on cardiac fibrosis by using in vivo and vitro models,and further explore the mechanism of SIRT1 in the effect.Objects1.To investigate the effect of AST on cardiac fibrosis by using in vivo and in vitro models;2.To study the impact of AST on SIRTI expression and activity;3.To explore the mechanism of SIRT1 in the effect of AST.Methods1.Animal experiments:We used the same animal models as in part one:SHAM,TAC(12 weeks)and TAC+AST(H);Additionally,TAC+AST+EX-527 group was added,as well as SHAM+AST,SHAM+EX-527 and TAC+EX-527 as control group.According to the result in part one,AST was treated at the dose of 200mg/kg/day.2.Cell experiments:To stimulate human cardiac fibroblasts cells(HCFs)with TGF-?1 and(or)AST.We first test the most suitable dose by setting up various doses.We studied the conversion of HCFs from fibroblasts to myofibroblasts,the collage synthesis and the effect on SIRT1 expression and activity,and further confirmed the effects and mechanisms by using SIRT1 siRNA.3.Masson staining:To observe the fibrosis in all groups;4.Immunohistochemical stain:To obverse the expression of a-SMA,COL I and SIRT1 by in situ staining in all groups;5.Western blot:To measure the protein level of SIRT1,a-SMA,COL I,SMAD2(3)and phosphorylated SMAD2(3);6.Immunoprecipitation:To measure the level of acetylated SMAD2(3)combined with Western blot method;7.SIRT1 activity assay:To measure SIRT1 activity by using a fluorescent assay kit;8.Immunofluorescence staining:To observe the translocation of R-SMADs in myocardium and in HCFs;to observe the conversation of HCFs and the SIRT1 expression by staining a-SMA and SIRT1;9.Real-time fluorescence quantitative polymerase chain reaction(RT-PCR):To value the transcriptional level of TGF-?,a-SMA,COL I,SIRT1,T?RI and T?RII in cardiac tissue and(or)HCFs;10.Dual luciferase reporter gene assay:To test the transcriptional activity of SMADs in HCFs;11.Statistical analysis:The data was analyzed by using IBM SPSS Statistic 19 software.Differences between multiple groups were determined using t-test or one-way ANOVA analysis.Continuous data are presented as the means±SEM.Normalized data are presented as fold values over the mean of the control.In all comparisons,P<0.05 was considered to indicate statistical significance.Results1 Animal experiment1.1 AST slowed the progression of cardiac fibrosis:The Masson staining,immunohistochemical staining of a-SMA and COL I showed that fibrosis increased.in TAC group compared with that in SHAM group(P<0.05),there was no significant difference between SHAM and TAC+AST groups(P>0.05),these showed that AST exert an anti-fibrotic effect in myocardium.1.2 The expression of SIRT1 in hearts after TAC(0,4,12 weeks):Western blot showed that SIRT1 expression increased in hearts after TAC for 4 weeks compared with that for 0 week(P<0.05),while SIRT1 decreased after TAC for 12 weeks compared with that for 4 weeks(P<0.05),even lower than that for 0 week(P<0.05).These changes showed SIRT1 expression was different in different timepoints after TAC surgery.1.3 AST inhibited TAC induced SIRT1 decrease:Compared with the TAC group,SIRT1 expression and activity in TAC+AST group increased(P<0.05).The immunohistochemical results showed that SIRT1 was mainly located in nuclei and the positive nucleus was more in the TAC+AST group than that in the TAC group(P<0.05).1.4 Inhibiting SIRT1 attenuated the anti-fibrotic effect of AST:As shown in Masson staining,immunohistochemical staining of a-SMA and COL ?,cardiac fibrosis in TAC+AST+EX-527 group was more serious than that in TAC+AST group.Among all the groups,the fibrosis in TAC+EX-527 group was the most serious.Moreover,compared with the TAC+AST group,the decreased expression of TGF-?,a-SMA and COL I in TAC+AST+EX-527 group also indicated that inhibiting SIRT1 attenuated the anti-fibrotic effect of AST(P<0.05).1.5 The levels of R-SMADs phosphorylation and acetylation in all groups:Compared with the levels in SHAM group,phosphorylation and acetylation elevated in TAC group(P<0.05),no significant difference in TAC+AST group(P>0.05),while inhibiting SIRT1 increased acetylation level(P<0.05),rather than phosphorylation level in TAC+AST+EX-527 group(P>0.05).1.6 The translocation of R-SMADs(SMAD2 and SMAD3):As shown by immunofluorescence staining,compared with SHAM group,R-SMADs located mainly in the nuclei in TAC group,more in cytoplasm in TAC+AST group,while inhibiting SIRT1 in TAC+AST+EX-527 group didn't significantly influence their location compared with TAC+AST group.2 Cell experiment2.1 The most suitable dose of TGF-?1 for stimulating HCFs and its effect on SIRT1:According to the level of a-SMA,lOng/ml(24 hours)is the just dose for the following experiment,certificated by the obvious increase of a-SMA expression(P<0.05);Meanwhile,the expression and activity of SIRT1 was found decreased significantly at 10ng/ml(P<0.05).2.2 The effect of AST on SIRTI in HCFs:At the time of 24 hours with TGF-?1(10ng/ml),SIRT1 expression and activity were increased by AST treatment at the dose of 80?mol/ml(P<0.05),accompanied by decreased a-SMA and COL I expression(P<0.05);In the cells without TGF-?1 AST treatment at various doses had no diffidence in SIRT1 expression(P>0.05),while SIRT1 activity had already increased at the dose of 40?mol/ml(P<0.05).2.3 The impact of knocking down SIRT1 on AST's effect in HCFs:Compared with TGF-?1+AST group,a-SMA and COL I expression increased in TGF-?1+AST+SIRT1 siRNA group(P<0.05).Moreover,SIRT1 and a-SMA expression changes showed by immunofluorescence staining were similar with that in protein levels(P<0.05).2.4 The levels of R-SMADs phosphorylation and acetylation in HCFs:Compared with CON group,R-SMADs phosphorylation and acetylation levels increased evidently in TGF-?1-stimulated group(P<0.05),AST treatrnent decreased them obviously(P<0.05),while knocking down SIRT1 attenuated the effect of AST on acetylation(P<0.05),but not the effect on phosphorylation(P>0.05).2.5 The translocation of R-SMADs in HCFs:TGF-?1 stimulated the translocation into nuclei,and AST inhibited it,while knocking down SIRT1 had no impact on the effect of AST.Consistant with the translocation,AST still had the ability of inhibiting the transcription level of T?R? and T?R ? after SIRT1 was knocked down.2.6 The transcriptional activity of SMAD3:Compared with CON group,the transcriptional activity of SMAD3 was enhanced by TGF-?1,and AST decreased the enhancement,while AST's effect was attenuated when SIRT1 was knocked down.Conclusions1.AST exerted its anti-fibrotic function in myocardium in vivo and inhibited the conversion of HCFs in vitro by decreasing phosphorylation and acetylation levels;2.SIRT1 expression and activity decreased in TAC hearts and TGF-?1-stimulated HCFs,and AST attenuated the decrease;3.SIRT1 contributed the anti-fibrotic effect of AST by decreasing the acetylation level,rather than the phosphorylation level.