Correlation And Mechanism Of Obstructive Sleep Apnea And Heart Failure

Background and objective Obstructive sleep apnea(OSA)is common in sleep disordered breathing.Studies demonstrate that OSA could induce and aggravate cardiac insufficiency,as an independent risk factor for the progression of heart failure,however,aggravates heart failure with the increase of apnea-hypopnea index(AHI).Although the coexistence of OSA and heart failure is becoming more and more common,quantitative studies about the readmission rate and prognosis of OSA with heart failure are rare and the mechanism of OSA affecting the readmission rate and prognosis of patients with heart failure is unclear.Therefore,this study is to explore the relationship between OSA and the readmission and poor prognosis of heart failure patients.In addition,microparticles(MPs),the bilayer plasma membrane structure with the diameter of 0.1-1um,released during cell activation or apoptosis,which are usually considered to be a pathological product produced during cell activation and play a harmful role.Endothelial microparticles(EMPs)are released when endothelial cells are activated or apoptotic,which can promote inflammation,coagulating and regulating vascular tension.Studies have shown that EMPs can induce cardiomyocyte apoptosis by promoting oxidative stress.Noteworthily,an increase in EMPs in peripheral blood of OSA patients has also been reported by some studies recently.Therefore,we suggest that OSA is associated with the readmission and poor prognosis of heart failure patients.And the increase of EMPs may be a possible mechanism of it.The focus of this present study was to lay a theoretical foundation for improving the prognosis of OSA patients combined heart failure,we will confirm the effect of OSA on readmission and prognosis of patients with heart failure by clinical studies.At the same time,we explore the effect of EMPs on myocardial cells and its possible mechanism under intermittent hypoxic conditions through animal and cell experiments.Contents:Part Ⅰ: Clinical study to observe the effect of OSA on MPs in peripheral blood,severity and prognosis of patients with heart failure.Part Ⅱ: Animal experiments were conducted to verify the effects of intermittent hypoxia and antioxidant intervention on EMPs in peripheral blood and heart of mice.Part Ⅲ: Cell experiments were conducted to investigate the mechanism of apoptosis of myocardial cells induced by intermittent hypoxia-treated EMPs and the effect of antioxidant intervention.Methods:Part Ⅰ:(1)Elderly patients over 65 years of age were included in the study,who were divided into control group,heart failure withourt OSA group and OSA combined heart failure group according to the incidence of heart failure and OSA.One-year follow-up was conducted and recorded by a specialist.The end point was heart failure readmission and all-cause death.We conducted a cohort study in the three groups and compared the general data,BNP and echocardiography resulrs.(2)We compared the clinical characteristics of MPs level in peripheral blood and the relationship between EMPs and heart function,BNP and prognosis in heart failure patients complicated with OSA.(3)At the same time,we analyzed the relationship between EMPs and oxidative stress index,cardiac function in heart failure patients complicated with OSA.Part Ⅱ:(1)C57BL/6J mice were purchased to establish intermittent hypoxic(IH)model and divided into control group and IH group.Intermittent hypoxia profiles consisted of alternating N2(60s)and compressed air(120s).After exposure to intermittent hypoxia for 2 weeks and4 weeks,we evaluate the changes of cardiac function by echocardiography,assay the levels of EMPs in peripheral blood by flow cytometry analysis,observe the pathological changes of myocardium through HE staining and assay myocardial cells apoptosis rate by TUNEL.(2)To analyze the correlation between indicators of oxidative stress and EMPs in peripheralblood,we observed the changes of cardiac structure,numbers of EMPs in peripheral blood,pathological changes of myocardial tissue,apoptosis of myocardial cells and indicators of oxidative stress(MDA and SOD)after NAC intervention in IH mice.Part Ⅲ:We establised a model of intermittent hypoxia in vitro(hypoxia for 5 minutes,normoxia for 10 minutes,8 hours)and cultured endothelial cells(BEND3)in vitro.At last we extracted the EMPs from endothelial cells which were treated by intermittent hypoxia.Primary cardiomyocytes were cultured in vitro under normal oxygen condition(from the heart tissue of SD suckling mice).EMPs treated with intermittent hypoxia(IH-EMPs)were incubated with myocardial cells for 4,6,8 and12 hours.The activity of myocardial cells was monitored by MTT method.NAC was used to intervene the impaired cardiomyocytes treated by IH-EMPs for 12 hours.Then Western blot was used to monitor the expression of cardiomyocyte apoptotic protein Cleaved Caspase-3.TUNEL were used to monitor the apoptotic rate of cardiomyocytes and Elisa was used to monitor the level of MDA and SOD activity of cardiomyocytes.Results:Part Ⅰ:1.The heart function in patients with heart failure complicated with OSA is worse than that in patients without OSA.A one-year follow-up showed that the readmission rate and mortality rate of heart failure patients complicated with OSA were higher than those of patients without OSA.2.The number of TMPs and EMPs in peripheral blood of heart failure patients combined with OSA was higher than that of patients without OSA and patients in control group.The number of EMPs in peripheral blood was an independent risk factor for the increase of BNP and the decrease of cardiac function.Multivariate COX analysis indicated that the increase of EMPs was associated with the increase of one-year re-admission risk [2.52,(95% CI:1.77-3.61),P=0.016] and mortality risk[3.63,(95%CI:1.81-7.27),P=0.009] in heart failure patients compli--cated with OSA.3.The level of EMPs in peripheral blood of heart failure patients combined with OSA was correlated with cardiac function and oxidative stress index.Part Ⅱ:1.After exposure to intermittent hypoxia for 2 weeks,EMPs numbers in peripheral blood of IH mice increased,but there is no changes in cardiac structure and cardiac function observed by echocardiography.At the end of 4th week,the EMPs numbers in peripheral blood of IH-mice continued to increase,accompanying wih ventricular remodeling by echocardiography indicated in mice.HE staining showed swelling and degeneration of cardiac myocytes,disorder of cell arrange--ment,widening of myocardial intercellular space and ruptured of myocardial fibers.And TUNEL indicated that cardiomyocyte apoptosis increased.Pearson correlation analysis showed that the level of EMPs in peripheral blood of mice was significantly correlated with the systolic function of left ventricular in mice.2.The antioxidant intervention of NAC could attenuate the oxidative stress level of myocardial tissue in mice,reduce the level of EMPs in peripheral blood,change the left ventricula,decerease the cardiomyocyte apoptotic rate of myocardial cells and improve the histopathological changes of myocardial tissue.Part Ⅲ:1.IH-EMPs affected the viability of cardiomyocytes in a time-dependent manner.2.IH-EMPs increased the expression of Cleaved Caspase-3 and the apoptotic rate in Cardiomyocytes.3.IH-EMPs increased MDA level and decreased SOD activity in cardiomyocytes.4.NAC intervention could decrease MDA level,increase SOD activity,downregulate Cleaved Caspase-3 expression and decrease apoptotic rate in myocardial cells induced by IH-EMPs.Conclusions:1.Clinical studies:(1)Heart failure patients with OSA had higher readmission rate and mortality than those without OSA.OSA increased readmission rate and adversed prognostic events of heart failure patients,which was closely related to AHI.This suggested that OSA was an independent risk factor for poor prognosis in patients with heart failure.(2)The Increase of CD144+-EMPs was an independent risk factor and an independent predictor of readmission and death in heart failure patients combined with OSA,suggesting that increased EMPs was a good biomarker for poor prognosis in heart failure patients with OSA.(3)With the deterioration of cardiac function,oxidative stress aggravated in heart failure patients complicated with OSA,the level of EMPs in peripheral blood was correlated with oxidative stress index;their decline of cardiac function was correlated with oxidative stress and the raised level of CD144+-EMPs.2.Animal experiments showed that intermittent hypoxia promoted the increase of CD144+-EMPs in peripheral blood and myocardial injury in mice.Antioxidant intervention could reduce the myocardial injury and decrease the augment of EMPs in peripheral blood in mice.These results indicated that IH can induce myocardial injury and induced oxidative stress which resulted in myocardial injury in mice.Meanwhile,thiese changes were related to the raised numbers of EMPs in peripheral blood of mce.3.Cell experiments showed that the extraction of EMPs from endothelial cells treated by intermittent hypoxia could reduce cardiomyocyte viability by time dependence.IH-EMPs could promote apoptosis and oxidative stress of cardio--myocytes.However,antioxidant intervention could alleviate it and reduce the apoptotic rate of cardiomyocytes which was induced by oxidative stress.