| Heart failure(HF)is a clinical syndrome caused by structural and functional defects of the myocardium,characterized by impaired ventricular filling and decreased ejection fraction.There has been nearly 26 million people suffering from heart failure worldwide currently,and the number is still growing at about 2 million people each year,which is severely threatening people's life in the world.With the rising economic burden brought by medical resources consuming and population aging,heart failure will remain as a major medical problem in the world.The key issue of the treatment in heart failure is to improve the cardiac function and the quality of life,as well as relieving the clinical symptoms and extending the life expectancy.Therefore,it is necessary to further explore the pathogenesis of heart failure and seek for new targets and directions of the treatment,which can provide a strong theoretical foundation for further studies of treatment strategies in heart failure.It was shown that heart failure was closely related to the over-activation of the neuro-humoral regulation,the release of inflammatory mediators,apoptosis and autophagy in terms of pathogenesis.Autophagy,as the research focus on the pathogenesis of heart failure rencently,refers to the mechanism of degradation of components in cells mediated by lysosomes and regulated by autophagy related genes,and then the degraded components are recycled for energy supplies or other purposes,which maintains the stability of cells by removing damaged and aged organelles.The classical autophagy procedure involves the formation of a bilayer membrane phagocytic structure to engulf organelles or cytoplasmic components and the degradation by fusion with lysosomes.The formation of autophagosome is a crucial part of autophagy process,which is closely related to the function of autophagy associated gene(Atg).Atg6,namely beclin-1,is one of the atg-expressing proteins.When the expression of Atg family proteins increases such as beclin-1,it is manifested as the increase of autophagosome formation and autophagy activity.Microtuble-associated protein 1 light chain 3(LC3),as the homologous gene of Atg8,is the critical ubiquitin-like pathway protein in the process of autophagy involved in the formation of autophagosomes.In the process of autophagy,water-soluble LC3 I in cytoplasm reacts with phosphatidylethanolamine to form liposoluble LC3 II on the membrane of autophagosome which represents the initiation of autophagy.The content of LC3 II protein or the ratio of LC3 II and LC3 I is positively correlated with the number of autophagosomes,both of which can reflect the activity of autophagy in cells to a certain extent.P62,the substrate of autophagy degradation,interacts with LC3 by recognition sequence of LC3,and transports ubiquitinated proteins to autophagosomes where the degraded compounds were degraded.After that P62 is eliminated accompanied with the clearance of the polymer.The expression of P62 protein decreases when the autophagy activity of the cells enhances,which is negatively correlated with the degree of autophagy.As the low rate of cardiac myocyte regeneration in adults that limited the renewal capacity of cardiac myocyte,the circulation and renewal of intracellular substances play an important role in determining the function and life span of cardiomyocytes.Studies have shown that autophagy is a crucial part in the pathophysiology of heart failure.It is essential to prevent the aggregation of macromolecules and the accumulation of dysfunctional organelles,and to maintain the intracellular environment homeostasis regardless of physiological or pathological state.Autophagy exists in the myocardium of human body involved in regulating the function and life span of cardiomyocytes physiologically.While in the conditions of cardiac hypertrophy,ischemia and hypoxia pathologically,the damage from external factors is reduced by regulating the autophagy process.Researches have shown that the role of autophagy is twofold.Moderate autophagy activation is an adaptive response to stabilize cardiac myocytes and protect the cells from death under external stimulation.Whereas excessive autophagy activation will lead to organelle destruction,and accelerate cardiac cell death.Autophagy is regulated by various signal transduction pathways,such as PI3K/AKT,AMPK,Beclin1,ROS,etc.Phosphoinositide-3-kinase(PI3K)/protein kinase B(PKB,Akt)signal transduction pathway,mainly composed of PI3 K and AKT,is a classical signaling pathway in the process of autophagy.Activation of PI3 K and Akt improves cells survival and cardiac function by regulating a series of downstream proteins such as mammalian target of rapamycin(m TOR),endothelial nitric oxide synthase(e NOS),glycogen synthase kinase(GSK-3?),fork head transcription factor(FOXOs)and certain members of the Bcl-2 family.Studies have found that autophagy activity of cardiomyocytes is effectively regulated by the PI3K/AKT signaling channel affecting cardiac remodeling,which plays a positive role in the occurrence and development of heart failure.Therefore,searching for clinical feasible interventions to regulate cadiomyocytes autophagy and cardiac remodeling and to improve the cardiac function is the new hotspots in the treatment of heart failure.Cardiac contractility modulation(CCM)refers that cardiac tissue receives a high-intensity electrical stimulation within absolute refractory period,which can not result in depolarization of cardiac cells,but enhance the cardiac contractility and improve cardiac function.Previous basic studies have shown that CCM can enhance myocardial contractility by improving the abnormal expression of embryonic genes,and normalizing the expression of sarcoplasmic reticulum calcium cycling genes and stretch response genes,as well as regulating the expression and function of calcium ion transport related proteins such as sarcoplasmic reticulum calcium ATPase and phospholamban.Clinical studies have confirmed that CCM can significantly improve clinical symptoms,exercise tolerance and quality of life,and decrease hospitalization rates in CHF patients.On the basis of an animal model of chronic heart failure in rabbits established by means of ascending aorta constriction,the aim of this study is to observe the effect of cardiac contractility modulation on autophagy of cardiac tissue in rabbits with chronic heart failure and the changes of protein expression levels of AKT,PI3 K,FOXO3,m TOR,TORC2 and GSK-3? in myocardial tissue,so as to provide further theoretical basis for the clinical application of CCM in the treatment of heart failure.Part one Effects of cardiac contractility modulation on cardiac autophagy in rabbits with chronic heart failure.Objective: The model of chronic heart failure in rabbits was established,we conducted the echocardiography and BNP level in blood serum for cardiac function of rabbits,the HE staining for myocardial tissue morphology,and the immunofluorescence staining for the expression of LC3 to explore the effects of cardiac contractility modulation on cardiac function and autophagy in rabbits with chronic heart failure.Methods: Thirty New Zealand white rabbits were randomly assigned to three groups with 10 rabbits in each group including the Sham group,the HF group and the CCM group.Only thoracotomy was performed in the sham group,and animals in the HF group underwent cerclage at the distal 1.0cm of the ascending aortic root to ensure 60% of its original circumference after thoracotomy.In the CCM group,pediatric temporary pacing electrodes were preset in the anterior wall of the left ventricle after the same thoracotomy and cerclage.The animal models of chronic heart failure were established after feeding for 12 weeks,then cardiac function was assessd to screen qualified animals.Additionally,electrical stimulation was given constantly for 4 weeks after successful modeling in the CCM group.The echocardiography was performed to evaluate the cardiac function and the size of left ventricular cavity by parameters of left ventricular ejection fraction,left ventricular short axis reduced rate,left ventricular end systolic and end diastolic diameter before surgery and 12 and 16 weeks after surgery.Venous blood from rabbits was extracted to detect the level of b-type brain natriuretic peptide(BNP)at the same time points.HE staining and LC3 immunofluorescence staining were performed on the myocardial tissues retrieved from the anterior wall of the left ventricle in rabbits sacrificed by air embolism after 16 weeks of operation.The changes of cardiac function,biomarkers,structure of myocardial tissues and the expression of LC3 level in CHF rabbits were compared within three groups to explore the effect of CCM on cadiac function and autophagy in chronic heart failure.Results: 1.Animal models building At 12 weeks after surgery,the echocardiographic parameters of experimental animals in the HF group and the CCM group all met the diagnostic criteria of EF<40%,and animals presented reduced food and water consumption,slow weight gain,slow response,and shortness of breath,which indicates successful models.There were 9 alive animals respectively in each group except for intraoperative death at 16 weeks after surgery.2.Echocardiographic parameters There was no significant difference in preoperative echocardiographic parameters in the three groups(P>0.05).At 12 weeks after surgery,the left ventricular ejection fraction and left ventricular fractional shortening in the HF group and the CCM group significantly decreased compared with those in the sham group(P<0.05),while the left ventricular end systolic and diastolic diameter increased in the HF group and the CCM group(P<0.05).At 16 weeks after the operation,the left ventricular ejection fraction and left ventricular fractional shortening in the CCM group were higher than those in the HF group(P<0.05),inversely the left ventricular systolic and diastolic diameter were significantly lower in the CCM group(P<0.05),which indicated CCM improved the cardiac function of HF rabbits.3.BNP level in venous blood The basal level of BNP in the sham group was 29.79±2.81ng/ml,and there was no significance in three groups(P>0.05).The BNP level of the experimental animals in the HF group and CCM group was higher than that in the sham group at 12 weeks after surgery(P<0.05).At 16 weeks after surgery,the level of BNP in the CCM group decreased compared with that in the HF group(P<0.05)indicating that CCM effectively improved heart failure in rabbits.4.Histopathological examinations(HE staining)The myocardial cells ordely arranged with plump nuclei,uniform cytoplasmic and normal morphology in the sham operation group.In the HF group,ascending aortic constriction caused extensive damage of myocardial cells presented as hypertrophy and edema of cardiomyocytes,disorganized arrangement and rupture of myocardial fibers,disappearance of partial myocardial fibers and inflammatory cells infiltration.After 4 weeks of CCM intervention,the pathologic damage of myocardial tissues in the CCM group was highly improved relative to that in the HF group including improved cellar edema and reduced inflammatory cells infiltration.5.LC3 immunofluorescence staining The results showed that the cardiac tissues in the sham operation group had basic staining presenting red fluorescent spots.The increased density of LC3 immunofluorescent spots in the HF group indicated increasing autophagy activity of myocardial cells in heart failure animals.The density of LC3 immunofluorescent spots in the CCM group decreased compared with that in the HF group,which suggested that CCM intervention inhibited the enhanced autophagy activity in animals with heart failure.Part two Effects of CCM on autophagy-related proteins of cardiac myocytes in chronic heart failure rabbitsObjective: We observed the effects of CCM on the expression of P62,Beclin1 and LC3B(?/?)proteins in myocardial tissues based on the CHF models by ascending aortic constriction,and investigated the effect on autophagy activity of cardiac myocytes in chronic heart failure rabbits.Methods: The animals and their groups were the same as the first part.The animals were sacrificed by air embolization at 16 weeks after ascending aortic constriction or sham operation,and the cardiac tissues were collected from the anterior wall of the left ventricle.Western-blotting technique was used to detect the expression levels of P62,Beclin1 and LC3B(?/?)proteins in cardiac tissues of animals.Results: 1.Expression of Beclin1,P62 and LC3B(?/?)proteins in heart failure The results of Western blot showed that Beclin1,P62 and LC3B(?/?)proteins in the myocardial tissues of the sham operation group had a certain amount of basal expression.Compared with the sham group,Beclin1 and LC3 B proteins expression level significantly increased,as well as the LC3B(?/?)ratio increased,while P62 protein expression level decreased both in the HF group(P<0.05)suggesting that cardiac autophagy activity enhanced in chronic heart failure.2.Effects of CCM on the expression of Beclin1,P62 and LC3B(?/?)proteins After CCM intervention,Beclin1 and LC3 B proteins expression level in the CCM group significantly decreased,as well as the LC3B(?/?)ratio decreased,whereas P62 protein expression level increased compared with those in the HF group(P<0.05),showing that CCM intervention effectively inhibited the enhancement of cardiac autophagy activity caused by heart failure.Part three Effects of CCM on the PI3K/AKT signaling pathway of myocardial tissues in rabbits with chronic heart failureObjective: We observed the expression of protein AKT1,AKT2,AKT3,PI3K(?110),PI3K(?85),FOXO3,m TOR,TORC2 and GSK-3? in myocardial tissues on the basis of CHF models by ascending aortic constriction,and investigated the effects of CCM on the PI3K/AKT signaling pathway and the possible mechanisms of CCM intervention in autophagy regulation of heart failure.Methods: The details of animals and groups were described previously.The cardiac tissues were collected from the anterior wall of the left ventricle at 16 weeks after ascending aortic constriction or sham operation.Western-blotting technique was used to detect the expression of protein AKT1,AKT2,AKT3,PI3K(?110),PI3K(?85),FOXO3,m TOR,TORC2 and GSK-3? in myocardial tissues in each group.Results: 1.Effects of CCM on AKT1,AKT2,AKT3,PI3K(?110),PI3K(?85)proteins expression The results from western blot showed that the AKT1,AKT2,AKT3,PI3K(?110)and PI3K(?85)proteins in the myocardial tissues of the sham group had certain basic expression.There was a higher expression of AKT1,AKT2 and AKT3 proteins and a lower expression of PI3K(?110)and PI3K(?85)proteins in the HF group relative to the sham group(P<0.05).After CCM intervention on heart failure rabbits,the expression levels of AKT1,AKT2 and AKT3 proteins in cardiac tissues significantly decreased,and the expression levels of PI3K(?110)and PI3K(?85)proteins increased(P<0.05)in comparison with those in the HF group.2.Effects of CCM on m TOR,TORC2,FOXO3 and GSK-3? proteins expression The results illustrated that the m TOR,TORC2,FOXO3 and GSK-3? proteins in the myocardial tissues of the sham group had a certain amount of basal expression.Relative to the sham group,the expression levels of m TOR,TORC2,FOXO3 and GSK-3? proteins in the HF group significantly increased(P<0.05).After CCM intervention in heart failure rabbits,the expression levels of m TOR,TORC2,FOXO3 and GSK-3? protein in cardiac tissues had a significant reduction(P<0.05).The above findings suggest that CCM can improve cardiac systolic and diastolic function in rabbits with chronic heart failure and alleviate myocardial autophagy,which may be related to the PI3K/AKT signaling pathway.Conclusions: 1.The ascending aortic constriction method can successfully establish a rabbit model of chronic heart failure.2.The autophagy activity of cardiomyocytes enhance in heart failure,and the expression of AKT,PI3 K,m TOR,GSK-3?,FOXO3 and TORC2 proteins is abnormal.3.CCM can improve myocardial function in rabbits with chronic heart failure and alleviate myocardial autophagy,which may be related to the PI3K/AKT signaling pathway... |
PDF Full Text Request