| BackgroundSince the world is heading toward an aging society,the attention to study aging related diseases is particularly important.The cardiovascular system is an important part of the human body to maintain normal life activities,however,the regeneration capacity of the heart is very limited.With the increasing of age,cardiac systolic and diastolic function gradually decline,making it increasingly difficult to maintain its normal pump function.Therefore,prolong the aging heart,maintaining cardiac normal function has become a very meaningful research direction.The pathogenesis of cardiac aging involves multiple molecular mechanisms,including oxidative stress,autophagy disorders,metabolic changes,calcium homeostasis disorder,neuroendocrine pathway activation and so on.Terminal differentiation nature of myocardial cell makes damaged macromolecules and organelles accumulate vulnerably.Hence increasing the activation of autophagy and cleaning up these "garbage" is very important.Unfortunately,the activity of autophagy in myocardial cells declines with age,and the damage of autophagy has multiple adverse consequences,including a reduction in cardiac contractility and increasing risk of arrhythmias.ROS increasing as the time goes by,and mitochondrial is the major source of ROS and also the target ROS attacks.Due to the highly dependent on mitochondrial energy metabolism of myocardial cells,the heart in the whole life cycle are exposed to high load ROS.Therefore,the removal of ROS in a certain extent is an effective measure to delay the aging of the heart.Mitochondrial autophagy is a highly selective process,which can promote the elimination of dysfunctional mitochondrial.PINK1-Parkin pathway is one of mitochondrial autophagy signal pathway,the activation of PINK1 and Parkin can reverse impaired mitochondria to further damage cells.When the mitochondrial membrane potential disappears,leading to the existence of PINK1 in the outer mitochondrial membrane stability,then promotes the recruitment and activation of intracellular E3 ubiquitin ligase Parkin.So through mitochondrial autophagy activation of PINK1 and Parkin can reverse the impairment of mitochondrial injurying on myocardial cells,thus delaying the aging heart.Heat shock protein(HSP)is a kind of highly conserved structure protein,can be induced by endogenous or exogenous stressors.Heat shock protein 27(HSP27)is a member of the small heat shock protein subfamily,which is widely expressed in mammalian with its molecular weight 27 kDa in human,25 kDa in the rodent.Studies demonstrated that HSP27 involves various biological functions:participating signal transduction,apoptosis,regulating cell proliferation,differentiation,and also participating in oxidative stress,heat shock response,regulation hypoxia/ischemia injury.Our study group and other researchers have found that the mechanism of HSP27 on cardiac protection involves antioxidant capacity,suppression of inflammation,improving the survival rate of myocardial cells,and autophagy activation.Therefore,we can reasonably infer that high expression of HSP27 can delay the heart damage caused by aging.ObjectiveIn this study,we constructed cardiac specific transgenic mice expressing HSP27 gene(Tg),and selected wild type mice(WT)with matched age as the research object of aging model to explore the effects of HSP27 on the age induced cardiac senescence and its mechanism may be involved.Material and Methods1.AnimalsTransgenic mice with cardiac specific expression of HSP27 gene(Tg)were created with the help of the Model Animal Research Center of Nanjing University and Animal Laboratory Resource Facility at Nanjing University,In the experiments,24-month-old HSP27 Tg mice and gender matched WT littermates served as old mice.The animal care and experimental protocols were approved by the Nanjing University Committee on Animal Care.All experiments were performed in compliance with the international guidelines on the ethical use of animals.2.EchocardiographyTwo-dimensional ultrasound via Vevo770 device measured echocardiography of mice.2 month old HSP27 Tg mice and gender matched WT mice as young control group.M-mode type ultrasonic model was used to evaluate cardiac function of the mice,and the pulse Doppler evaluated mitral valve function.The experimental data obtained average at least for five cardiac cycles.3.Masson's Trichrome StainingThe heart tissue were collected,then fixed by 4%paraformaldehyde overnight,followed by paraffin embedded in the papillary muscle level to make paraffin sections.Masson trichrome staining method is used to analyze the cardiac fibrosis.The staining was observed under 400 x magnification microscope.By calculating the percentage of fibrosis areas in ventricular myocardium to evaluate the myocardial fibrosis in mice.4.Immunofluorescence StainingHeart tissues were fixed with 4%paraformaldehyde overnight,after using paraffin embedded tissue.Sections will be made after sections of dewaxing,hydration,antigen repair and blocked.Subsequently,the sections were incubated with the appropriate primary antibodies at 4? overnight,next day with FITC(green fluorescence)or Cy3(red fluorescence)were incubated at 37 ? for 60 minutes.With alpha-actinin to represent myocardial cells.The nuclei were counterstained with Hoechst 33342.The section was observed after staining by fluorescence microscopy.5.DCFH AssayROS level was measured by fluorescent indicator DCFH.When the DCFH is added to the tissue homogenate,ROS will make the DCFH oxidation,producing DCF.luM production of fluorescent DCFH and 1 ml 10ug myocardial cytosolic protein were incubated for 1 hour.Then using a fluorometer at an excitation wavelength of 485 nm and an emission wavelength of 535 nm completed the assay.6.Protein CarbonylationThe ventricular tissue homogenate in dissolved liquid(0.3M sucrose,0.03M nicotine and 0.02M EDTA pH7.4),then centrifuged at 4 ?,10000 g for 5 min.A volume of 500ul containing 100 ug of cytosolic proteins was incubated with 2 mM DNPH in the dark for 1 h.Finally The DNPH-treated proteins were used to detect protein carbonylation by Western blotting.7.Western BlottingHearts of 24 month old mice were collected.Protein extracts were prepared for immunoblot analysis against p16,p53,LC3,p62,PINK1,Parkin and so on.The blots against GAPDH served as loading controls.n=3 per group.8.Statistical AnalysisAll data were expressed as mean ? standard deviation,and t test or one-way ANOVA was used to analyze.The data were statistically analyzed by SPSS 13.0 software,P<0.05 was statistically significant.Results1.HSP27 alleviates the aging-induced decline in cardiac function1.1 Detection of cardiac specific expression of HSP27We successfully constructed the transgenic mice expressing human HSP2 7 gene identified by Western blot analysis.The HSP27 expression in the heart of Tg mice was abundant,while not in the control group(WT).(the antibody against HSP27 did not cross-react with murine HSP25)1.2 Effects of HSP27 on cardiac function in aged heartCompared with young WT mice,E/A ratio of old WT mice decreased by 37.5%,EF(40%)and FS(45.9%)decreased at the same time,however,LVVd(67.4%)and LVVs(181%)was significantly increased(P<0.01).Compared with the young Tg mice,E/A ratio of old Tg mice reduced by 15.4%(P<0.05),compared to young mice,EF,FS,and LVVd of the old Tg mice had little change.However,the aging-induced decrease in the E/A ratio was significantly attenuated by 37.4%compared with the old WT mice(P<0.05),and compared with WT mice,EF,FS of Tg mice increased significantly,while LVVd and LVVs were significantly decreasing(P<0.01).2.HSP27 decreases the levels of aging markers in the myocardium of old miceCompared with old WT mice,the levels of p16 and p53 in old Tg mice were significantly decreased by 46.5%and 49.4%(P<0.01).In order to determine the activation of p53,we continue to explore the level of p53 phosphorylation at Ser15(p-p53).P-p53 expression in old Tg mice heart was significantly lower than that in the old WT mice(P<0.01).3.HSP27 attenuates fibrosis in the interstitial myocardium of old miceCompared with the old WT mice,the myocardial interstitial fibrosis was significantly reduced by 76.2%in old Tg mice(P<0.01).4.HSP27 decreases the ROS content in the myocardium of old miceThe ROS content in old WT mice and Tg mice were 2038.6 and 752.4 units,respectively.ROS content in old Tg mice was significantly lower than old WT mice(P<0.01).At the same time,protein carbonyl levels of old Tg mice(46.1%)was significantly decreased compared to old WT mice(P<0.01)5.HSP27 increases the level of autophagy in the heart of aged miceCompared with the old WT mice,the LC3-II protein level in old Tg mice was reduced by 38.7%(P<0.01).The expression level of p62 protein decreased by 59%(P<0.01).Proteins which involved in autophagy increased significantly in old Tg mice:Atgl3(112.7%),Vps34(68.5%),and Rab7(138.4%)(P<0.01).6.HSP27 reduces the content of ubiquitin-conjugated proteinsThe level of ubiquitin-conjugated protein in old Tg mice hearts(76.2%)was significantly lower than that of WT mice(P<0.01).7.HSP27 increases PINK1 and Parkin expression in old heartsCompared with the old WT mice,the expression level of PINK1 and Parkin in old Tg mice were increased by 57.1%and 60.5%(P<0.01).BNIP3 expression level in old Tg mice heart was significantly higher than that in old WT mice(P<0.05).While the expression of Nix in two different genotypes groups had little difference.ConclusionsHSP27 alleviated cardiac aging and this action involved antioxidation and mitophagy activation... |
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