Efficiency Study Of Xin Fu Kang Oral Liquid On Myocardial Mitochondrial Subpopulations’ Function And Protein Alterations In Mitochondrial Inner Membrane For Heart Failure

Objective:In order to grasp the comprehensive protein expression in cardiac mitochondrial inner membrane of heart failure with the pathological heterogeneity between SSM and IFM, the systematic research based on the inner membrane proteins in cardiac mitochondrial subpupulations should be carried out, therefore providing new perspectives for the pathomechanism in heart failure and updated targets for the therapeutic mechanism resesrch development of traditional Chinese medicine in this disease.Accompanying with the detections of morphology,energy metaboliam associated functions and mitochondrial protein import ability of the cardiac mitochondrial subpopulations,the cardiac SSM and IFM of SD rats with AMI induced heart failure were subjected to the purified inner membrane proteins preparation and the proteomic analysis of mitochondrial inner membrane proteins through iTRAQ to comprehensively and systematically investigate the pathological mechanism of energy metabolic disorders and the effects of XFK oral liquid on energy metabolism targeting cardiac mitochondrial inner membrane in heart failure.Methods:1.The cardiac function (LVSP, LVEDP, ±dp/dtmax, SV, EF, LEVDD, LEVDS, CO) of AMI induced heart failure rats were evaluated by hemodynamic technology and echocardiography respectively;2.The morphology and ultrastructure of cardiac mitochondrial subpopulations in AMI induced heart failure rats were assessed by Mito Tracker deep red 633 labelling flow-cytometry method and electron microscope;3.The proteins on mitochondrial inner membrane mediated biological functions(mitochondrial respiration, mitochondrial membrane potential, membrane permeability transition pore, the enzyme activities of ETC complex Ⅰ,Ⅱ,Ⅲ and phosphorylation apparatus complex Ⅴ) of cardiac mitochondrial subpopulations in AMI induced heart failure rats were evaluated through Clark oxygen electrod, flow cytometry and spectrophotometer respectively;4.The proteomic alterations of cardiac mitochondrial subpopulations’ inner membrane in AMI induced heart failure rats were analysed by iTRAQ, and the expression levels of the differentially expressed proteins were verified by Western Blot;5. The expression levels of mitochondrial protein import mechanism associated proteins in cardiac mitochondrial subpopulations of AMI induced heart failure rats were detected by Western Blot.Results:1.The effects of XFK oral liquid on cardiac function in rats with AMI induced heart failureAfter 8 weeks drug intervention course, Compared with Sham group, SV,EF decreased(P<0.05, P<0.01) while LVIDd,LVIDs increased(P<0.01)after models established in model group. Compared with model group, SV,EF in Captopril group, Trimetazidine group, XFK large dose group and XFK small dose group increased significantly,especially the Trimetazidine group and XFK large dose group (P<0.05). Meanwhile, at the end of 8 weeks drug intervention, compared with Sham group, the rats in model group showed accelerating heart rate and reduced CO, and both of the discomforts were ameliorated in Captopril group, Trimetazidine group, XFK large dose group and XFK small dose group(The differences were not statistically significant).After 8 weeks drug intervention course, Compared with Sham group, LVSP,+dp/dtmax,-dp/dtmax decreased(P<0.01) while LVEDP increased(P<0.01)after models established in model group. Compared with model group, LVSP,+dp/dtmax,-dp/dtmax in Captopril group, Trimetazidine group and XFK large dose group increased(P<0.05), meanwhile,+dp/dtmax in the XFK small dose group increased(P<0.05). LVEDP in Captopril group, Trimetazidine group, XFK large dose group and XFK small dose group decreased inordinately, with the significant decrease in Captopril group, Trimetazidine group and XFK large dose group (P<0.01).2.The effects of XFK oral liquid on the morphology and ultrastructure of cardiac mitochondrial subpopulations in rats with AMI induced heart failureThe results of Mito Tracker deep red 633 labeling mitochondrial integrity assessment through flow cytometry combined with the mitochondrial ultrastructure observation detected by electron microscope proved that the separation procedure of mitochondrial subpopulations was stable and reliable to obtain the intact puried cardiac mitochondrial subpopulations.Using the flow cytometry approach, SSM were larger in size (FSC) and possessed greater internal complexity (SSC) compared with IFM, which were smaller and more compact(the differences were not statistically significant). Mitochondrial size was significantly increased in IFM of rats with heart failure compared with control IFM(P<0.01), whereas SSM showed no significant changes. Mitochondrial complexity in IFM of rats with heart failure was significantly decreased compared with control IFM(P<0.01). No significant differences in SSC were observed in the SSM population. These results indicate that only IFM morphology is impacted as a result of the AMI-induced heart failure. Compact with Model group, mitochondrial size of IFM was significantly decreased in XFK large dose group(P<0.01), and mitochondrial complexity of IFM were significantly increased in Captopril group, Trimetazidine group and XFK large dose group(P<0.01; P<0.05; P<0.01).The transmission electron microscope results showed normal mitochondrial morphology and structure with intact membrane and regular cristae formation in both of the mitochondrial subpopulations, with the consistant size of IFM and random size of SSM.Model group:abnormal mitochondrial morphology with swelling and vacuoles, membrane lysis, fuzzy ridge structure, cristae lysis or disappear in IFM particularly, SSM was almost survived in AMI induced heart failure.Captopril group, Trimetazidine group, XFK large dose group and XFK small dose group:relatively normal mitochondrial morphology and structure with slight swelling and vacuoles, slightly damaged cristae and cristae junctions in IFM of AMI induced heart failure.3.The effects of XFK oral liquid on inner mitochondrial membrane proteins mediated mitochondrial functions of cardiac mitochondrial subpopulations in rats with AMI induced heart failureCompared with Sham group, mitochondrial respiration based on glutamic acid with malic acid and succinic acid with rotenone were all decreased in mitochondrial subpopulations of Model group, with the tendency of IFM respiration injury in NADH oxidation respiratory chain(P<0.01) and SSM respiration damage in FADH oxidation respiratory chain(P<0.01). Compact with Model group, mitochondrial respiration of both subpopulations were increased to various extent in Captopril group, Trimetazidine group, XFK large dose group and XFK small dose group, especially the ATP generation parameters(P/O, OPR).Compared with Sham group, the enzymatic activities of mitochondrial oxidative phosphorylation associated complex Ⅰ、Ⅱ、Ⅳ、Ⅴin model group were inordinately down-regulated, particularly in C I and CIV of IFM(P<0.01),but Captopril and XFK large dose could significantly improve the decreased enzymatic activities of complex Ⅰ、Ⅱ、Ⅳ、 Ⅴin IFM, meanwhile, modulating the pathological reduced △Ψmand increased prolonged mPTP opening(P>0.05, P<0.05).4.The effects of XFK oral liquid on proteomic alterations of mitochondrial inner membrane proteins in cardiac mitochondrial subpopulations of rats with AMI induced heart failureCompared with Sham group:76 differentially expressed proteins were detected in IFM of model group, all of which were down-regulated protein expression. Compared with IFM inner membrane proteomic alterations in model group:3 differentially expressed proteins were detected in Captopril group,2 of which showed increased protein expression and 1 decreased; 9 differentially expressed proteins were detected in Trimetazidine group,1 of which showed increased protein expression and 8 decreased; 76 differentially expressed proteins were detected in XFK large dose group, the whole of which showed increased protein expression; 24 differentially expressed proteins were detected in XFK small dose group, all of which showed increased protein expression.Compared with SSM inner membrane proteomics in Sham group:only 1 differentially expressed protein were detected in model group with increased protein expression.76 differentially expressed proteins in IFM were successfully identified by MALDI-TOF-MS. Bioinformatics analysis showed that these differential proteins were mainly associated with energy metabolism, the maintance of cristae morphology, calcium handling, mitochondrial protein import and mitochondrial dynamics.5.The effects of XFK oral liquid on the expression level of mitochondrial protein import mechanism associated proteins in cardiac mitochondrial subpopulations from rats with AMI induced heart failureThe expression levels of Tom40,Tim22 and TIM23 were all down-regulated in cardiac mitochondrial subpopulations of rats with heart failure, especially the down-regulation of Tim23 expression level in IFM subpopulation. And the Tim23 expression level in Captopril group, Trimetazidine group, XFK large dose group and XFK small dose group were all up-regulated to different extents.Conclusion:1.XFK oral liquid could obviously improve CO and alleviate left ventricular pressure in HF rats, thus improve cardiac function.2.XFK oral liquid could significantly improve mitochondrial pathomorphology and ultrastructure disturbance of IFM in HF rats.3.XFK oral liquid could improve the mitochondrial inner membrane proteins mediated mitochondrial energy metabolism dysfunction in IFM through modulating the ATP generation efficiency, mitochondrial membrane potential and mPTP of IFM.4.XFK oral liquid could remarkably up-regulating the pathologically down-regulated inner membrane protein expression level in IFM, by which means to improve the cardiac function of rats with heart failure through rebuilding the mitochondrial adaptivity for repairment and restoring the oxidative phosphorylation function, mitochondrial membrane potential and mitochondrial protein import.5.XFK oral liquid could modulate the pathological proteomic alterations through improving the mitochondrial inner membrane targeted proteins import ability by elevating the expression level of Tim23, therefore regulate the biological function and protein expression of proteins in mitochondrial inner membrane through improving the critae morphology and IMM-targeted mitochondrial protein import ability.