The Comparative Proteomic Analysis Of The Effects Of Grape Seed Proanthocyanidin Extracts On Reperfusion Arrhythmias In Rats In Vivo

Research backgroundEarly reperfusion is crucial for the survival of ischemic myocardium. However, reperfusion has been referred by Braunwald and Kloner as the "double edged sword" because reperfusion itself may lead to accelerated and additional myocardial injury beyond that generated by ischemia alone. This results in a spectrum of reperfusion-associated pathologies, collectively called "reperfusion injury". Reperfusion injury has been observed in each of reperfusion therapies including percutaneous coronary intervention (PCI), thrombolysis and coronary bypass grafting. The different clinical manifestations of this injury include myocardial necrosis, myocardial apoptosis, reperfusion arrhythmias, myocardial stunning and endothelial-and microvascular dysfunction including the no-reflow phenomenon. However, VT and VF induced by reperfusion remain the most important causes of sudden death following spontaneous restoration of antegrade flow. Therefore, every effort must be made to minimize sudden cardiac death caused by reperfusion arrhythmias. The mechanisms of reperfusion arrhythmias may include heterogeneous recovery of conduction and a refractory period of incomplete reperfusion, reentry, abnormal automaticities, and activities triggered by Ca2+ overload and free radicals. However, the details of the mechanisms remain unclear and reperfusion arrhythmia has not received satisfactory treatment.GSPE is a natural, standardized, water-ethanol extract from red grape seeds which has great free radical scavenging ability. The main pharmacological effective components of GSPE are oligomeric proanthocyanidins. Free radicals and oxidative stress play a crucial role in the pathophysiology of abroad spectrum of cardiovascular diseases including congestive heart failure, valvular heart disease, cardiomyopathy, hypertrophy, atherosclerosis and ischemic heart disease. A series of studies were conducted using GSPE to demonstrate its cardioprotective ability in animals and humans.Proteomics is the large scale study of proteins, usually by biochemical methods. Many studies of cardiovascular disease and cancer have used proteomics techniques. Nonetheless, proteomics has already revolutionized the discovery process for antimicrobial drugs by accelerating target identification and evaluation, assay development and the follow-up support for medicinal chemistry programs. It is important to note that proteomics techniques could discover not only novel biomarkers, but also proteins that may actively participate in cardiovascular process. Proteomic studies of heart tissues can provide new insights into the specific early molecular mechanisms'that underlie the responses to IR injury and therefore may have vital implications for the specificity and efficacy of diagnosis and treatment. Our previous work had shown GSPE reduced reperfusion-induced arrhythmias in rabbits. In isolated rat hearts GSPE had been proved to reduce the incidence of reperfusion-induced VF and VT. However the mechanism of this protection against reperfusion-induced VT, VF by GSPE remained unclear. With the development of proteomic techniques a quantitative proteomics- isobaric tags for relative and absolute quantification (iTRAQ) was introduced. iTRAQ employs primary amine reactive isobaric tags to derivatize peptides at the N-termini and lysine side-chains. Therefore, theoretically, iTRAQ labels most of the peptide fragments in a digested mixture. Although the peptides labeled with any of the iTRAQ reagents are indistinguishable in single MS analysis, tag fragmentation during MS/MS analysis produces reporter ions (m/z=114,115,116, and 117), which provide quantitative information upon integration of the peak areas.An important starting point for drug discovery is to supply a validated therapeutic target, and the proteomic study of GSPE using iTRAQ on reperfusion arrhythmias may yet provide insights into the mechanism of reperfusion-induced ventricular arrhythmias, and provide new starting points for antiarrhythmia drugs.Aims1. We illustrated the ischemia/reperfusion modal in rats in vivo to observe whether GSPE could reduce the accidents of reperfusion-induced VT, VF.2. We applied iTRAQ combined with mass spectrometry to identify alterations in the expression of proteins in myocardial tissue in order to elucidate the potential molecular mechanism of the protective effects of GSPE on reperfusion-induced VT, VF and find potential new drug targets for reperfusion arrhythmias.Materials and methods1. IR Protocol in Rats in vivo Sixty six rats were randomly assigned to three groups:Sham group (n=22), IR group (n=22) and GSPE group (n=22). Twenty two Rats in GSPE group were fed with GSPE (200 mg/kg·d for 4 weeks, intragastricly) while the other 44 rats were given 0.9% NaCl intragastricly only for 4 weeks. Rats (280-340g) were anaesthetized with 20% Urethane(0.5ml/100g, i.p.). Body temperature was routinely monitored via a rectal thermometer using a heated operating platform and appropriate heating lamps. Subdermal electrodes were placed to allow the determination of a leadⅡelectrocardiogram (ECG) which was recorded continuously throughout the experiment by the BL410 multichannel acquisition system (Chengdu Taimeng, China) for physiological signals. The incidence of reperfusion-induced VF and VT during reperfusion was analyzed. A 4-0 polypropylene suture through a 3-mm long polyethylene tube (PE-10) was then placed around the left anterior descending coronary artery (LAD), proximal to its main branching point. Then the LAD was ligated with the 4-0 suture. The coronary artery was occluded by tightening of the occluder. After 30min of ischemia, in the IR and GSPE groups, the suture around the LAD was removed and the occurrence of reperfusion was assessed by the observation of blood flow in the epicardial coronary arteries through a surgical microscope. The ischemic myocardium was reperfused for 120min. Sham group were made following an identical procedure but without the actual tying of the polypropylene suture. At the end of each experiment, the heart was excised. Ultrathin sections cut from the ischemic area were stained with uranylacetate and lead citrate and were examined with an H-800 electron microscope (Hitachi, Japan). With NBT stain, infarct sizes of IR group and GSPE group were assessed.2. iTRAQ ProteomicsThe proteins-enriched fractions were digested by tripsin. The resulting complex peptide mixtures were labeled with iTRAQ reagents (114 for the peptides of Sham group,116 for the peptides of GSPE group, and 118 for the peptides of IR group respectively), and then separated by SCX and C18 fractionation. And 6 SCX fractions were analyzed by MALDI-TOF/TOF and microQ-TOF.Results1. Reduction of the Incidence of Reperfusion-induced VT, VF by GSPE Our results demonstrated that GSPE can significantly reduce the incidence of reperfusion-induced VT, VF in rats in vivo. There was no arrhythmia happened in Sham group. The incidence of reperfusion-induced VF was reduced from 64% in rats without GSPE-treated (IR group) to 17% in rats treated with GSPE (GSPE group) (P< 0.05). The incidence of reperfusion-induced VT was reduced from 91% in rats without GSPE-treated to 33% in rats with GSPE-treated (P<0.01).2. Results of Transmission Electron Microscopic ObservationSpecimens from sham group showed normal features:the cardiac cells were rich in mitochondria arranged between myofibrils in rows with myofibrils arranged evenly and normal intercalated disks. The sarcomere was of the same length. Nuclear membrane was integrated and chromatin was normally distributed. In IR group, the structure abnormalities of nuclear were apparently observed. Disarrangement, breakage and local absence of myofibrils were observed. Numerous disarranged mitochondria substituted for myofibrils. Mitochondria vacuolization and crista loss were observed. In GSPE group there were more apparent differences. The protective effect was acquired in that there were only moderate mitochondrial and nucleolus abnormalities in cardiac myocytes.3. Infarct size Infarct size was noticeably reduced in the GSPE-fed group [13.3%±3.3%) as compared to the IR group (26.7%±2.3%) (P<0.01).4. Identification of ProteinsWe identified 91 differential proteins among which the expression of 53 proteins was significantly changed. The expression of 21 proteins were increased in IR group when compared to sham group, while these were significantly down-regulated in GSPE group and the expression of 43 proteins were decreased in IR group when compared to sham group while these were significantly up-regulated in GSPE group. The function of all 91 proteins consists of ion transport, fatty acid oxygenation, calcium binding, cellular adhesion, regulation of apoptosis and signal transport which indicated that these functions maybe involved in the protective effects of GSPE on ischemia/reperfusion injury.Conclusions1. Our results demonstrated for the first time that GSPE can significantly reduce the incidence of reperfusion-induced VT, VF in rats in vivo. Since ischemia/reperfusion models of rat in vivo mimic clinical myocardial infarction or intracoronary thrombolysis or angioplasty situations, it is expected that GSPE may become effective drugs against reperfusion-induced arrhythmias.2. Comparative proteome-iTRAQ is more accurate and reproducible which can be used to elucidate the potential molecular mechanism of the protective effects of GSPE on reperfusion-induced VT, VF and find potential new drug targets for reperfusion arrhythmias.3. Using we identified 91 differential proteins. The function of all 91 proteins consists of ion transport, fatty acid oxygenation, calcium binding, cellular adhesion, regulation of apoptosis and signal transport which indicated that these functions maybe involved in the protective effects of GSPE on ischemia/reperfusion injury.4. We found that Na+/K+ ATPaseα1 subunit and HSP60 were decreased in IR group while it was significantly increased in GSPE group compared to sham group. These two proteins may be the key proteins involved in the protective effects of GSPE on reperfusion-induced arrhythmias. This change in subunit distribution may lead to the increase of Na+/K+-ATPase activity in GSPE treated rats which would ultimately protect hearts from reperfusion arrhythmia injury. The protection of increased expression of HSP60 in GSPE group maybe involved in against apoptotic cell death which in turn resulted in the reduction of the accidents of reperfusion-induced VT, VF in rats.