Proteomic Study On The Effect Of Xinfu Kang Oral Liquid On Myocardial Mitochondria In Rats With Heart Failure

Background:Chronic heart failure (CHF) is a clinical syndrome resulting from cardiac structural or functional disorders that impair the heart’s ability to fill with or eject blood. CHF is also the end-result for most cardiovascular diseases and the main cause of human death. With the rapid growth of eldly population, as well as patients with hypertension and (or) coronary heart disease, the incidence and mortality of CHF have increased year by year. Although medical treatment including drug, interventional therapy and surgical operation have made great progress in treating CHF in the past few decades, which have reduced mortality significantly, the overall prognosis of CHF remains very poor. High morbidity, mortality and the resulting high medical costs have made CHF become an important factor which seriously affects both people’s health and the national economic development. Therefore, conducting basic and clinical research actively to find a more effective way to treat CHF is the key of this subject. Recent advances in proteomics technology, and further investigations on mitochondrial protein function and its relationship with human diseases provide a new perspective for CHF research, which is of great significance to explore the molecular mechanism and find new therapeutic targets of CHF.Objective:To investigate the effects of XFK oral liquid on cardiac function, myocardial pathological and ultrastructure changes and mitochondrial proteomic altera-tions in rats with heart failure after myocardial infarction and explore its possible mechanisms.Methods:Heart failure models were established by ligating the left anterior descending coronary artery of SD rats. Rats were randomly divided into sham group, model group, captopril group, trimetazidine group, XFK high dose group and XFK low dose group.8weeks after drug intervention, the left ventricular function (LVIDd、LVIDs、EF、FS、CO)of rats in all groups were evaluated by echocardiography, the pathological and ultrastructure changes of myocardial tissue were observed by optical microscope and electron microscope, the mitochondrial proteomic alterations of myocardial tissue were detected by two-dimensional gel electro-phoresis (2-DE)and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Furthermore, expression levels of part of the differentially expressed proteins were verified by western blot. Results:1. Effects of XFK oral liquid on cardiac function in rats with HF after myocardial infarctionCompared with sham group:LVIDd、LVIDs in model group, captopril group, trimetazidine group, XFK high dose group and XFK low dose group increased (P<0.01), while EF、FS、CO decreased (P<0.01) after establishing models.8weeks after drug intervention, LVIDd in model group, captopril group, trimetazidine group, XFK high dose group and XFK low dose group increased (P<0.01), LVIDs in XFK high dose group and XFK low dose group also increased significantly (P<0.01), EF、FS、CO in model group, captopril group, trimetazidine group, XFK high dose group and XFK low dose group decreased (P<0.05or P<0.01);Compared with model group:LVIDd、LVIDs、EF、FS、CO in captopril group, trimetazidine group, XFK high dose group and XFK low dose group show no obious difference (P>0.05) after establishing models.8weeks after drug intervention, LVIDd、LVIDs in captopril group, trimetazidine group, XFK high dose group and XFK low dose group decreased (P<0.05or P<0.01), while EF、FS、CO increased significantly (P<0.01)2.Effects of XFK oral liquid on myocardial pathomorphology and ultrastruc-ture in rats with HF after myocardial infarction2.1Effects of XFK oral liquid on myocardial pathomorphology in rats with HF after myocardial infarctionSham group:normal myocardial morphology and structure, clear cytoplasm and nucleus, regular myocardial myof ilament arrangement, evenly colored myocardial fiber, no inflammatory cell infiltration in intercellular substance.Model group:wide distribution of myocardial infarcts, nucleus lysis and disappear, disordered myocardial fiber arrangement with part of them fractured, living myocardial cells distributed as islands in myocardial infarcts, inflamma-tory cell infiltration and less fibroblasts in intercellular substance.Captopril group, Trimetazidine group, XFK high dose group and XFK low dose group:reduced myocardial pathological changes, decreased infarct range with part myocardial necrosis, relatively regular myocardial fiber arrangement with regional myocardial fiber broken and fractured, living myocardial cells distri- buted as islands in myocardial infarcts, less inflammatory cell infiltration but more fibroblasts in intercellular substance than in model group.2.2Effects of XFK oral liquid on myocardial ultrastructure in rats with HF after myocardial infarctionSham group:uniform nuclear chromatin distribution, regular myocardial myofilament arrangement, normal sarcomeres, clear cross striation, normal mitochondrial morphology and structure without swelling and vacuole, intact membrane and tight cristae.Model group:disordered myofilament arrangement, a large number of myofila-ment loosed and fractured, irregular sarcomeres, unclear cross striation, and abnormal mitochondrial morphology with swelling and vacuoles, membrane lysis, fuzzy ridge structure, cristae lysis or disappear.Captopril group, Trimetazidine group, XFK high dose group and XFK low dose group:a little sparse myofilament arrangement, relatively regular sarcomeres, slightly fuzzy cross striation, relatively normal mitochondrial morphology and structure with slight swelling and vacuoles, normal matrix density but slightly damaged cristae, a few mitochondrial lysis or disappear.3. Effects of XFK oral liquid on myocardial mitochondrial proteomic in rats with HF after myocardial infarctionCompared with sham group:152differentially expressed protein spots were detected in model group,40of which showed increased protein expression and112decreased. Compared with model group:19differentially expressed protein spots were detected in captopril group,11of which showed increased protein expression and8decreased;18differentially expressed protein spots were detected in trimetazidine group,12of which showed increased protein expression and6decreased;20differentially expressed protein spots were detected in XFK high dose group,13of which showed increased protein expression and7decreased;13differentially expressed protein spots were detected in XFK low dose group,8of which showed increased protein expression and5decreased;9differentially expressed protein spots were all detected in4drug groups (including XFK low dose group or not),5of which showed increased protein expression and4decreased;32differentially expressed protein spots were detected in all4drug groups compared with model group,20of which were successfully identified by MALDI-TOF-MS. Bioinformatics analysis showed that these differential proteins were mainly associated with energy metabolism, stress reaction, oxidative damage, cyto- skeleton, cell differentiation and proliferation.4. Verification of differentially expressed proteins between model group and4drug groupsWestern blot results showed that the expression of Stress-70protein and Nucleophosmin in model group increased, while decreased in all4drug groups, the expression of ATP synthase subunit alpha in model group decreased, while increased in all4drug groups, which were consistent with the results of comparative proteomic analysis. Conclusion:1.XFK oral liquid can obviously reduce LVIDd、LVIDs and increase EF、FS、 CO in HF rats, thus improve cardiac function.2. XFK oral liquid can obviously improve myocardial pathomorphology and ultrastructure in HF rats.3. XFK oral liquid can partly adjust proteomic alterations of myocardial mitochondrial in HF rats, and its intervention mechanism may involve improving energy metabolism, reliving stress reaction and oxidative damage, as well as regulating cell differentiation and proliferation.4. The results of comparative proteomic analysis performed by using2-DE and MALDI-TOF-MS are acurate, stable and reliable.