The Regulation And Mechanism Of The TIR/BB-loop Mimetic AS-1 On Pressure Overload Induced Cardiac Remodelling And Heart Failure

Background Heart failure is a severe stage in the development of a variety of heart diseases.So far,the mechanism of heart failure is undefined and lack of effective prevention and treatment.The previous study of our group found that TIR/BB-loop mimetic AS-1 can protect pressure overload-induced cardiac hypertrophy by inhibiting IL-1R mediated My D88 singnaling pathway.Although PO-induced myocardial hypertrophy,myocardial fibrosis and heart failure is a series of sequential pathophysiology process,singnaling pathways participating in regulation during deferent stages of disease are not identical.Therefore,whether AS-1 can protect heart failure,the severe stage of PO-induced pathological myocardial hypertrophy,and its influence on the disease process after compensatory myocardial hypertrophy haven't been reported.In other words,the molecular mechanism of AS-1 mediated PO-induced heart failure is still needed further research.Objective 1.Clarify whether AS-1 has prevention and treatment effcet on PO-induced heart failure under the overall conditions.2.To illuminate whether AS-1 mediates mechanical stretch induced cardiac fibroblasts activation and its paracrine effect.3.Clarify the molecular mechanism of AS-1 involved in mechanical stress mediated cardiac fibroblasts activation and its paracrine effect.Methods?? Models 1?Animal ModelsTo illuminate that the preventive effect of AS-1 on heart failure induced by pressure load,we make model of heart failure in mice though transverse aortic constriction(TAC)operation.6-8 weeks C57/BL6 male mice were randomly divided into four groups,including sham,TAC,TAC+DMSO and TAC+AS-1.AS-1 was administered intraperitoneally(50 mg/kg/day)for 4 weeks 3 days before TAC operation.Monitor the preventive effect of AS-1 on myocardial hypertrophy,myocardial fibrosis and heart failure induced by pressure overload.To clarify that the preventive effect of AS-1 on heart failure induced by pressure load,we make model of heart failure in mice though transverse aortic constriction(TAC)operation.6-8 weeks C57/BL6 male mice were randomly divided into five groups,including sham,TAC,TAC+DMSO,TAC+AS-1 and T2W+AS-1.AS-1 was administered intraperitoneally(50mg/kg/day).Two weeks after surgery,noninvasive echocardiograph were used to confirm that mice were in concentric hypertrophy stage and administered AS-1 for another 2 weeks.Monitor the treatment of AS-1 to heart failure and its regulatory effects on the development of pathological myocardial hypertrophy induced by pressure load.2?Cell models Excessive activation of myocardial fibroblasts and myocardial hypertrophy,apoptosis are the main changes in cellular level.To illuminate the molecular mechanism of AS-1 mediating heart failure induced by pressure load,myocardial fibroblasts produce fibrosis response were stimulated by 15% of mechanical stress for 24 hours in vitro.And the conditioned medium produced by mechanical stress was used to culture cardiomyocyte after filtration and become a stimulus to cardiomyocyte.Observation:(1)The regulatory effect of AS-1 on proliferation,differentiation and collagen synthesis of cardiac fibroblasts;(2)The effect of conditioned medium on cardiomyocyte;(3)The effect of AS-1 to cadiomyocyte hypertrophy and apoptosis induced by conditioned medium;(4)Explore the molecular mechanism of AS-1 involed in mechanical stress mediated cardiac fibroblasts activation and its paracrine effect.??Observation targets To evaluate rat model via altrasonic Doppler.To evaluate myocardial hypertrophy via calculate HW/BW,LVW/TL and echocardiography.Evaluate total collagen content by calculate hydroxyproline.q RT-PCR and Western Blot were used to calculate m RNA and protein level of collagen?,collagen ? and lysyloxidase.To evaluate the degree of myocardial fibrosis,Masson staining and calculation of cross-linked content by restrictive pepsin degradation test were performed.To evaluate heart failure by echocardiography and calculation of LW/BW.To esitimate fibroblast proliferation though EDU staining.To evaluate fibroblast differentiation through calculating the level of ?-SMA by immunofluorescent staining and Western Blot.To evaluate the degree of cardiomyocyte hypertrophy by calculating the level of ANP,BNP,MHC by q RT-PCR and examining cardiomyocyte cross sectional area by immunofluorescent of ?-actinin.To evaluate cardiomyocyte apoptosis by Tunel staining.Results 1.Pre-treated with AS-1 improved pressure overload-induced heart failure apparently.4 weeks after transverse aortic constriction(TAC),the heart weight to body weight ratio(HW/BW)and left ventricular weight to tibial length ratio(LVW/TL)increased significantly,respectively,compared with the age-matched sham control group;H&E staining showed that cross section area of myocardium increased markedly after TAC;echocardiogram showed that interventricular septum diastolic dimension(IVSd),left ventricular posterior wall diastolic dimension(IVPWd)and left ventricular internal diastolic dimension(LVIDd)were significantly multiplied after TAC-treated mice compared with the sham group,indicating that TAC induced cardiac hypertrophy.AS-1 administration notably prevented the increasing of above-mentioned cardiac hypertrophic markers,showing that AS-1 can improve pressure overload-induced cardiac hypertrophy significantly.4 weeks after TAC,the expression of total collagen,collagen?and collagen ? were remarkably increased compared with the age-matched sham control group;Masson dyeing showed that TAC increased fibrous connective tissue deposition,indicating that TAC induced myocardial fibrosis.AS-1 administration notably prevented the increasing of above-mentioned myocardial fibrosis markers,showing that AS-1 can improve pressure overload-induced myocardial interstitial fibrosis markedly.4 weeks after TAC,the ratios of LW/BW increased significantly,ejection fraction(EF)and fractional shortening(FS)decreased apparently compared to the sham goup,while AS-1 administration improved mice heart functions notably,showing that pre-treated with AS-1 improved pressure overload-induced heart failure remarkably.2.AS-1 administration after TAC for 2 weeks efficiently delayed the progression of pressure overload-induced heart failure.2 weeks after TAC,echocardiogram showed that IVSd,IVPWd and LVIDd decreased significantly;while EF and FS increased,indicating that 2 weeks after TAC induced concentric hypertrophy.4 weeks after TAC,echocardiogram showed that IVSd,IVPWd and LVIDd were significantly multiplied;while EF and FS decreased apparently after TAC-treated mice compared with the sham group,indicating that 4 weeks after TAC induced eccentric hypertrophy.AS-1 adminisrtation after TAC for 2 weeks showed no marked change in IVSd and IVPWd compared with mice for 4 weeks after TAC,but significantly decreased LVIDd,while EF and FS increased markedly.Besides,AS-1 adminisrtation after TAC for 2 weeks remarkably decreased the increasing of the ratios of HW/BW,LVW/TL and LW/BW induced by TAC,indicating that AS-1 can efficiently delay pressure overload-induced heart failure.4 weeks after TAC,the expression of total collagen,collagen?,collagen ? and fibrous connective tissue deposition were remarkably increased compared with the age-matched sham control group as we showed above.AS-1 adminisrtation after TAC for 2 weeks remarkably improved the above-mentioned myocardial fibrosis markers except collagen ?,indicating that AS-1 can inhibit the progression from pressure overload-induced compensated cardiac hypertrophy to myocardial fibrosis.3.AS-1 inhibited mechanical stress-induced cardiac fibroblast activity and paracrine secretionCyclic stretch was applied with a peak elongation of 15% for 24 h of cardiac fibroblasts.We found that the numbers of EDU positive cells and ?-SMA positive cells both increased significantly,and the expression of ?-SMA,collagen?and collagen ? were remarkably increased,indicating that mechanical stress can induce cardiac fibroblast activity.AS-1 administration notably suppressed cardiac fibroblast proliferation,differentiation and collagen synthesis increasing which were induced in response to mechanical stress in neonatal rat cardiac fibroblasts(NRCFs)above.With conditioned medium treated for 24 h,q RT-PCR showed that m RNA levels of the hypertrophic markers ANP,BNP and ?-MHC were sinificantly increased in cardiac myocyte;also,the cardiomyocytes size increased markedly;and the number of TUNEL positive cells raised apparently compared with the sham control group. These results indicated that cyclic stretch of cardiac fibroblasts can induce cardiac myocyte hypertrophy and apoptosis via paracrine secretion.AS-1 suppressed the increasing of the markers of myocyte hpertrophy and apoptosis induced by above-mentioned conditioned medium,showing that AS-1 prevent pressure overload-induced HF at least partly by inhibiting mechanical stress-mediated cardiac fibroblast activity and paracrine secretion.4.AS-1 protect mechanical stress-mediated cardiac fibroblast activity and paracrine secretion via LATS1.The results of gene chips screening and q RT-PCR showed that AS-1 up-regulated the downregulation of the expression of LATS1 in failed heart of mice.Besides,the expression of LATS1 decreased in stretched cardiac fibroblasts remarkably.Moreover,AS-1 up-regulated this kind of LATS1 decreasing significantly.Further,AS-1 also up-regulated the downregulation of the expression of YAP,a downstream transcription factor of LATS1,notably.The protecting functions of AS-1 in mechanical stress-induced cardiac fibroblast proliferation,over differentiation and the increasing of the expression of collagen?and collagen ? were partly abolished after si LATS1 administration to knock down the expression of LATS1 in cardiac fibroblasts,indicating that AS-1 regulated mechanical stress-mediated cardiac fibroblast activity at least partly by regulating LATS1.The protecting functions of AS-1 in conditioned medium-induced increasing of cardiomyocytes size and m RNA levels of the hypertrophic markers ANP,BNP and ?-MHC were partly abolished after si LATS1 administration to knock down the expression of LATS1 in cardiac fibroblasts,indicating that AS-1 regulated mechanical stress-mediated cardiac fibroblast paracrine secretion at least partly by regulating LATS1.Conclusion In conclusion,we demonstrated that AS-1 played an important role in preventing and treating pressure overload-induced heart failure.The mechanisms by which AS-1 attenuated heart failure involved interaction with LATS1/YAP signal transduction and regulated mechanical stress-mediated cardiac fibroblast activity and paracrine secretion.Our study provided a vital theory for finding effective biological target points and original new drugs in delaying the progression of heart failure.