Effect Of Cardiac Contractility Modulation On Myocardial Fibrosis And Mechanism In Rabbits With Chronic Heart Failure

Chronic heart failure(CHF)is the end state of a variety of cardiac structural and functional diseases,with causing systolic and(or)diastolic function damage.CHF is kind of pathology state with blood stasis in circulation and insufficient perfusion in organs.The underlying mechanism leading to heart failure is cardiac remodeling.Cardiac remodeling is an adaptive response in myocardial cells,extracellular matrix with respect to myocardial damage,pressure or volume load increasing.Myocardial cells hypertrophy,necrosis,apoptosis and cardiac fibroblasts proliferation,differentiation and extracellular matrix(ECM)collagen deposition are the main performances of cardiac remodeling.Myocardial fibrosis would limit myocardial cell movement,increase myocardial stiffness,reducing ventricular compliance result in falling of myocardial systolic and diastolic function.Myocardial fibrosis show extracellular matrix collagen deposition and cardiac fibroblasts proliferation.Collagen I and collagen III is the main component of myocardial collagen which form the main structural protein of extracellular matrix.Fibroblasts play a important role in the secretion of extracellular matrix collagen fiber,meanwhile it secrete matrix metalloproteinase and its inhibitor,metalloproteinase tissue inhibiting factor.In pathological condition,fibroblasts proliferation and translated into myofibroblasts,the ability of secretion collagen fiber is much stronger.Specific expression of ?-smooth muscle actin identifies myofibroblasts from fibroblasts.The matrix metalloproteinases(MMPs)are an endogenous family of enzymes responsible for extracellular collagen degradation.The metalloproteinase tissue inhibiting factor(TIMPs)could bind to MMPs to regulating MMPs activity.The balance between MMPs and TIMPs maintainthe steady state of extracellular matrix collagen fiber.MMP2 and MMP9 have the most number of publications which show that they play an important part in myocardial fibrosis with respect to pressure overload.TIMP1 can inhabit all MMPs and has greater affinity for MMP9 over MMP2.The transforming growth factors(TGF?s)are multifunction cytokines,which are implicated in a wide variety of cell functions,critically regulating inflammation,extracellular matrix deposition,cell proliferation,differentiation and growth.Four structurally similar isoforms of TGF?(TGF?1,TGF?2,TGF?3 and TGF?1?2)have been identified in mammalian species.Several lines of evidence indicate that TGF?1 is a crucial regulator of ECM metabolism.TGF?1 elicits its biological responses through binding to the receptor of myocardial cell membrane.Smad proteins play act as the downstream factors of TGF?1.The Smad proteins could classify as R-Smads(Smad2 and Smad3),Co-Smads(Smad4)and I-Smads(Smad6 and Smad7)based on their structure and function.The activity TGF?1 binding to binding to the receptor of myocardial cell,Smad proteins were activated through phosphorylation.The activated R-Smads form complexes with Co-Smad and translocation to the nucleus,where they activate or repress gene transcription.Smad3 was critically involved in myofibroblast trans-differentiation and mediated TGF?-induced extracellular matrix synthesis and TIMP up-regulation.I-Smads inhibit TGF-?signaling by interfering with R-Smad action,Smad7 is stronger.Connective tissue growth factor(CTGF)is a secreted multifunctional protein that belongs to the CCN family of growth factors.CTGF has been implicated in damage repair,proliferation in physiological state.CTGF could be up-regulated during the pathogenesis of several disorders.Connective tissue growth factor(CTGF),a downstream mediator of the TGF?1 pathway,plays a major role in myocardial fibrosisCardiac contractility modulation(CCM)is a new mechanical device therapy.CCM signals applied during absolute refractory period of myocardial d can boost the myocardial contractility and improve the heart function.Thebasic research revealed that the action of CCM appears to be mediated by molecular mechanisms that restore the ability of failing myocytes to more normally handle calcium cycling and restore the normal expression of fetal gene.Clinical research suggested the CCM therapy could improve cardiac function,quality of life,exercise capacity.The aim of the present study is to observe the effects and mechanism of CCM on myocardial fibrosis in rabbits with heart failure induced by legating ascending aortic root.Part one Effects of cardiac contractility modulation on myocardial fibr-osis in rabbits with chronic heart failure.Objective: In this study we investigate the effects of cardiac contractility modulation on myocardial fibrosis in rabbits with chronic heart failure.Methods: Thirty healthy New Zealand rabbits were randomly divided into three groups: sham operation group(sham group,n=10);heart failure group(HF,n=10);heart failure+CCM group(HF+CCM,n=10).In the HF group and HF+CCM group,we made constriction in the ascending aorta root distal 1 cm after thoracotomy.In the CCM group,we present a pediatric temporary pacemaker electrode in the anterior left ventricular wall during the operation.After 12 weeks,the heart failure models were established.CCM signal were implied in HF+CCM group lasting 6 hours every day for 4 weeks.UCG indicators,BNP level and myocardial tissue hydroxyproline content were measured.HE staining,Masson staining and Sirius red staining were used to detect to analysis the myocardial fibrosis.Results:1 The general situationNo death in sham group and after 12 weeks.1 rabbit in HF group die of pneumothorax during the surgery.1 rabbit in HF+CCM group die of massive hemorrhage due to ascending aorta injuries.Animals in HF group and HF+CCM group show loss of appetite,shortness of breath,reduction of activities.At last sham group(n=10),HF group(n=9);HF+CCM group(n=9).2 UCG indicatorsThere were no statistical differences among three groups in LVESD,LVEDD,LVEF,LVFS,IVSd,LVPWd,E/A.Compared with sham group,LVESD and LVEDD of HF and HF+CCM group significantly increased,LVEF,LVFS and E/A were significantly decreased after 12 weeks(P<0.05).Compared with HF group,the heart function had improved after CCM in HF+CCM group(P<0.05).3 BNPCompared with sham group,BNP level in HF and HF+CCM group significantly increased after 12 weeks(P<0.05).Compared with HF group,the BNP level has decreased in HF+CCM group(P<0.05).4 Pathological examinations4.1 HE stainingCompared with sham group,HF group showed disordered arrangement of myocardial cells,partial myocardial necrosis,fibrous tissue proliferation and inflammatory cells infiltration.Pathological changes were alleviated in HF+CCM group compared with HF group,as demonstrated by the regularly arranged cardiomyocytes and decreased fibrous tissues4.2 Masson stainingCVF was calculated according to result of Masson's trichrome staining.Heart tissue in the HF group presented a massive and intensive collagen accumulation compared with sham group.CVF in the HF group was significantly increased(P<0.05).After administration with CCM,areas of fibrotic heart tissue were obviously reduced.CVF in CCM group was remarkably decreased(P<0.05).4.3 Sirius red stainingCompared with sham group,Sirius red staining showed increased collagen content,disordered arrangement,increased collagen area in HF group.Pathological changes were alleviated after administration with CCM in HF+CCM group.5 Hydroxyproline quantitative analysisHydroxyproline is unique to collagen and is a well-recognized quanti-tative marker for fibrosis.Compared with sham group,Hydroxyproline content of the HF group was significantly higher than that of the sham group(P < 0.05).After treatment with CCM,the Hydroxyproline content decreased(P < 0.05).Part two Effects of CCM on myocardial collagen metabolism in rabbitswith chronic heart failure.Objective: To observe the effects of cardiac contractility modulation on proteins expression of collagenI,collagenIII,?-SMA,MMP2,MMP9,TIMP1 in rabbits with chronic heart failure induced by legating ascending aortic root and explore the effects of CCM on myocardial collagen metabolism.Methods: Immuno histochemistry and western-blotting technique were used to detect the proteins expression of collagenI,collagenIII,?-SMA,MMP2,MMP9,TIMP1 among three groups.Results:1 ?-SMA is a surrogate maker for differentiation of fibroblast to myofibroblast.The level of ?-SMA by immunostaining assay was used as one way to assess the essential role of CCM treatment in differentiation of fibroblast to myofibroblast.Compared with sham group,the percentage of positive areas of?-SMA was significantly increased by approximately 15% in the HF group(P< 0.05).By contrast,CCM treatment markedly reduced the expression of?-SMA by approximately 8%,as compared to the HF group(P<0.05).2 Western-blot resultsThe results showed that,compared with the sham group,the protein expressions of collagen I,collagen III,MMP2,MMP9,TIMP1,?-SMA increased more significantly in the HF group(P < 0.05).The CCM treatment contributed to markedly reduced protein level of above mentioned(P < 0.05),as compared with the HF group.Part three Effects of CCM on TGF?1/Smad signaling pathway in rabbits with chronic heart failure.Objective: To observe the effects of cardiac contractility modulation on mRNA and proteins expression of TGF?1,Smad3,Smad7,CTGF in rabbitswith chronic heart failure and explore the effects of CCM on TGF?1/Smad signaling pathway.Methods: Real time polymerase chain reaction and western-blotting technique were used to detect the mRNA and proteins expression of TGF?1,Smad3,Smad7 and CTGF in rabbits with chronic heart failure.Results:1 Effects of CCM on TGF?1,Smad3 and Smad7 mRNA and protein expression.Compared with the sham group,the mRNA and protein expression of TGF?1 and Smad3 increased more significantly in the HF group(P<0.05).The CCM treatment markedly reduced mRNA and protein level of TGF?1 and Smad3(P<0.05),as compared with the HF group.The changes of Smad7 mRNA and protein expression were opposite.2 Effects of CCM on CTGF mRNA and protein expression.CTGF act as a downstream mediator of the TGF?1 pathway plays a major role in myocardial fibrosis.Compared with the sham group,the mRNA and protein expression of CTGF increased more significantly in the HF group(P < 0.05).The CCM treatment markedly reduced mRNA and protein level of CTGF(P < 0.05),as compared with the HF group.Conclusion:1 The chronic heart failure model was successful established by legating ascending aortic root.Abmornal expression of Collagen I,collagen III,MMP2,MMP9,TIMP1 protein and incressing differentiation to fibroblast were main performance in heart failure myocardial.TGF?1/Smad signaling pathway played an essential role in myocardial fibrosis.2 CCM could Attenuate myocardial fibrosis in heart failure induced by legating ascending aortic root and improve the myocardial systolic and diastolic function.CCM may be able to reduce the deposition of collagen I and collagen III by down regulation protein expressions of MMP2,MMP9,TIMP1,?-SMA.CCM may be able to improve myocardial fibrosis by inhabit TGF?1/Smad signaling pathway.