Effects And Mechanisms Of Cytochrome P450 Epoxygenase 2J2 Attenuate Cardiac Hypertrophy

Arachidonic acid (arachidonicacid, AA) content is very rich in living bodies, is the precursor of various important biological active substances, which majorly binds to fatty acids in intral surface of cells and is released into cellular plasma to be metabolized when the cells are stimulated by a variety of physiological stimulus. Arachidonic acid by cyclooxygenase and lipoxygenase metabolic pathway has already been extensive research and understanding. Since 1980, scientists have found a third of arachidonic acid metabolic pathway of arachidonic acid that cytochrome P450 (Cytochrome P450, CYP) metabolic pathway, but it is in vivo, especially in biology and in the human body pathophysiological significance of gradually understanding and clarifying. Cytochrome P450 (Cytochrome P450, CYP) epoxygenase form of arachidonic acid metabolism in four different epoxy generation-twenty-three-carbon acid (Epoxyeicosatrienoic acids, EETs), respectively, 5,6-EET,8,9-EET,11,12-EET and 14,15-EET. It has been reported that epoxygenase contain two categories:CYP 2C and 2J,2J2 is the sole epoxygenase in man, is abundant in heart and vascular endothelial cells. Our group and foreign researchers have found that EETs play an important role in regulating homeostasis of cardiovascular system, including regulation of blood pressure, protective effects of heart and vascular endothelial cells and anti-apoptosis.Cardiac hypertrophy plays an important role in the beginning and development of heart failure. Cardiac hypertrophy is a compensatory response for the increased workload of the heart, the initial hypertrophic response is beneficial for the heart, and however, cardiac hypertrophy can lead to lasting heart failure, these cardiovascular events including ischemic heart disease, heart rhythm disorders and independent risk factor of sudden death. The cytochrome P450 epoxygenase-EETs system plays an important role in the cardiovascular system; increased EETs have many protective effects on the cardiovascular system, and may have a role in cardiac hypertrophy. Then, what is the role of cytochrome P450 epoxygenase-EETs system in cardiac hypertrophy and heart failure? There is no coverage.In this study, we assume that overexpression of CYP2J2 gene, as well as the subsequent increased EETs production has an important role in the prevention and reversal of cardiac hypertrophy and heart failure. First of all, we will investigate whether exogenous EETs and endogenous EETs could prevent Ang II-induced cardiomyocyte hypertrophy and its possible molecular mechanism; secondly, we will investigate whether overexpression of CYP2J2 gene could inhibit Ang II-induced cardiac hypertrophy in rats, and investigated the possible molecular mechanisms; the aim of this study is to explore whether cytochrome P450 epoxygenase-EETs system can prevent and reverse cardiac hypertrophy in the whole animal level and cell level, and further defined whether the CYP2J2-EETs can delay the progress of heart failure.A. In Vitro Study Exogenous EETs and endogenous EETs Attenuate Cardiomyocyte HypertrophyMethods:1. H9C2 cells were cultured in DMEM containing 10 % FBS in high glucose medium, when the myocardial cells grew to about 50 % confluence, H9C2 cells were treated with exogenous EETs and endogenous EETs after synchronization.1) exogenous EETs intervention:the cells were incubated with AngⅡ; EET inhibitor,14,15-EEZE; Lorsatan and 14,15-EET for 48 hours; 2) endogenous EETs intervention:before addition of Ang II, the plasmid pcDNA3.1-2J2 and pcDNA3.1-GFP were transfected into myocardial cells by using Fugene HD, while selective inhibition of CYP2J2 agent C26 was added to the pcDNA3.1-2J2 group, the interference persist for 48 hours.2. After treatment, the cells were washed with PBS, and were fixed with paraformaldehyde and were blocked by blocking solution for half an hour,, F-actin antibody was added according to the proportion of 1:200, incubated for 1 hours at room temperature, after washing, sealed solution were added, observed cells under a fluorescence microscope, cell size and cell surface area were calculated using the software.3. After treatment, RNA and protein of the cells were extracted with the Trizol solution, the RNA were reverse transcript to cDNA, the relative amount ofβ-MHC and ANP were analysis using Real-time PCR. Protein concentrations were measured by Brady method, the protein content of each group was compared.4. After synchronization treated H9C2 cells, the EET, EGFR, PI3K, and PPARy inhibitor 14,15-EEZE, AG1478, LY294002 and GW9662, and 14,15-EET were incubated with H9C2 cells for 48 hours, culture medium of cells were collected. After treatment, the cells protein were extracted with three decontamination cell lysate solution, P-EGFR, EGFR, PI3K, P-AKT, AKT, P-CREB, CREB, ANP expression were checked by Western blot. The ANP content in medium and cGMP content of cells were measured by ELISA.5. After synchronization, H9C2 cells were treated by Ang II and 14,15-EET, and ANP antibody were added to culture contain AngⅡand 14,15-EET interference in increasing concentrations for 48 Hours, followed by F-actin staining of cardiomyocytes.6. After synchronization, and subsequently Ang II and 14,15-EET were added to cells, ANP receptor inhibitor A71915 and PKG inhibitor KT5823 were also added to cells respectively, and incubated for 48 Hours, followed by myocardial cells F-actin staining. Total cellular protein was collected, and the cytoplasm and nucleus were extracted protein, Western blot, then the total CnAβexpression and NF-AT nuclear translocation of the situation.Results:1. F-actin staining showed 14,15-EET significantly inhibited Ang II-induced cardiomyocyte hypertrophy (P<0.05); and 14,15-EEZE blocked the protective effect of EET (P<0.05). Transfection with plasmid pcDNA3.1-2J2 also remarkablly inhibited cardiomyocyte hypertrophy, but C26 completely blocked the effect of CYP2J2 (P＜0.05). In addition, inhibited effect of EETs and CYP2J2 on cardiomyocyte hypertrophy were confirmed by cell protein content (P＜0.05). EET and CYP2J2 did not inhibit AngⅡ-induced increase in ANP expression, but increased the expression of ANP, but EET and CYP2J2 inhibit expression of (3-MHC, another marker of cardiac hypertrophy (P<0.05).2. The results of Western blot showed that AG1478 and LY294002 blocked the activation of ANP expression induced by14,15-EET in myocardial cells, the results also showed that 14,15-EET activates EGFR, PI3K/AKT and CREB, which may be signaling pathway EETs prompted the expression of ANP (P＜0.05). The results of ELISA test also showed that 14,15-EET upregulated myocardial ANP, thereby promoted the increase in intracellular cGMP content (P＜0.05).3. The experiments of addition of different concentrations of ANP antibody showed that ANP antibody blocked the anti-hypertrophy effect of EETs in a concentration-dependent manner (P＜0.05).4. The experiments of addition of ANP receptor antagonist and PKG inhibitors showed that ANP receptor antagonist and PKG inhibitors significantly blocked the anti-hypertrophy effect of EETs (P＜0.05). In addition, EETs inhibit CnAβexpression and NF-AT translocation into nuclear, but its effects were significantly blocked by ANP receptor antagonist, and PKG inhibitors (P＜0.05).B. In Vivo Study Effects and Mechanisms of Overexpression of P450 Epoxygenases 2J2 Attenuates Cardiac Hypertrophy in ratsMethods:1. Animals were randomly assigned to different treatment groups and subjected to a one-week adaptation period. Animals were randomly divided into six groups, and each group has 6 rats:saline group, pcDNA3.1 group, pcDNA3.1-2J2 group, Ang II group, AngⅡ+pcDNA3.1 group, and AngⅡ+pcDNA3.1-2J2 group. Before the treatment, the basal blood pressure of all animals was measured by ADI non-inventive blood pressure monitor. 2. Plasmid pcDNA3.1 and pcDNA3.1-2J2 were extracted by alkaline lysis methods and purificated by silica; the target gene pcDNA3.1-2J2 and control gene pcDNA3.1 as well as the equal volume of saline were injected into the bodies of rats via tail veins, and the dose of plasmid injection was 5 mg/kg body weight. In the meantime, Osmotic Pumps (Alzet 2002) infused Ang II were implanted in subcurose, and Ang II was released in a rate of 500ng/kg/min for two weeks. From the beginning of the experiment, blood pressure was measured at 3,7,10,14 days respectively.3.2 weeks after gene delivery and Ang II infused,24-hour urine samples were collected. Echocardiography was measured under anesthia. Before animals were sacrificed, Millar catheter was invented into left ventricle via right caio artery. Blood samples, heart, aorta, liver and kidney were removed and placed in liquid nitrogen immediately, and then transfered into the-80℃refrigerator. The sizes and the weights of heart were recorded, and some of the heart tissues were fixed with formaldehyde solution, embedded in paraffin after dehydration at room temperature.4. Heart 14,15-DHET levels and urinary cGMP levels and ANP levels in serum were measured by ELISA kit. CYP2J2, ANP, P-p38, p38, P-ERK, ERK, CnAβ, NF-AT (nucleus) and NF-AT (cytoplasm) expression in heart were measured by Western blot. Cardiomyodium sections were stained by H&E and Sirius red, the diameter of cardiomyocyte and collagen deposit were recorded.Results:1. The results of blood pressure measurements showed that Ang II significantly increased blood pressure compared to control group, while the plasmid pcDNA3.1-2J2 delivery significantly inhibited increased blood pressure under Ang II (P＜0.05).2. Injection of pcDNA3.1-2J2 induced CYP2J2 overexpression in rat heart tissue; ELISA results showed that 14,15-EET content in heart of pcDNA3.1-2J2 and AngⅡ+ pcDNA3.1-2J2 rats were significantly higher than the control group (P<0.05).3. The results of echocardiography showed that, AngⅡincreased interventricular septum and left ventricular posterior wall significantly, but the pcDNA3.1-2J2 attenuated it (P<0.05). However, both AngⅡand pcDNA3.1-2J2 did not affect the ejection fraction (EF) and fractional shortening rate (FS). Hemodynamic parameters recorded by Millar catheter showed that AngⅡincreased left ventricular end-diastolic pressure (LVEDP) significantly, and decreased the maximum rate of left ventricular pressure (dp/dt min) significantly; pcDNA3.1-2J2 reversed increase of LVEDP and reduction of dp/dt min induced by AngⅡ(P<0.05), but heart rate (HR), left ventricular end systolic pressure (LVESP), cardiac output (CO) and left ventricular pressure maximal rate of rise (dp/dt max) did not show significant changes.4. AngⅡincreased the ratio of heart weight and body weight, but pcDNA3.1-2J2 inhibited the increase of the ratio of heart weight and body weight (P<0.05). H & E staining showed that pcDNA3.1-2J2 remarkably inhibited AngⅡinduced cardiac hypertrophy (P<0.05), Sirius red staining showed that pcDNA3.1-2J2 remarkably improved AngⅡinduced collagen deposition in heart (P<0.05).5. The expression and secretion of ANP in the heart of pcDNA3.1-2J2, AngⅡ, AngⅡ+ pcDNA3.1 and AngⅡ+pcDNA3.1-2J2 groups were increased (P<0.05), while urinary excretion of cGMP was also increased (P<0.05)6. Western blot results showed that, compared with control group, phosphorylation-ERK, CnAβ,nuclear NF-AT levels in myocardial tissue of AngⅡand AngⅡ+pcDNA3.1 group were increased significantly, but pcDNA3.1-2J2 significantly attenuated them (P<0.05), while the myocardial expression of P-p38 was not altered. 1. Exogenous EETs and endogenous EETs can effectively inhibit AngⅡ-induced cardiomyocte hypertrophy, possibly by promoting the expression and secretion of ANP.2. A possible mechanism that EETs promote the expression and secretion of ANP by activating EGFR/PI3K/AKT/CREB signaling pathway.3. A possible mechanism that EETs attenuate cardiac hypertrophy:EETs upregulate ANP, ANP binds to its receptor, and activate the downstream second messenger cGMP, cGMP activates PKG, thereby inhibiting expression of CnAβand NF-AT nuclear translocation.4. In rats, overexpression of CYP2J2 in the heart significantly inhibited AngⅡ-induced cardiac hypertrophy and myocardial remodeling, and its possible mechanism is through increased expression of ANP and inhibiting CnAβexpression and NF-AT nuclear translocation, and the inhibition of ERK phosphorylation.5. CYP2J2-EETs system inhibit the CnAβexpression and NF-AT in nuclear translocation via increasing ANP level, and ultimately attenuate cardiac hypertrophy and myocardial remodeling, thereby delay the progress of heart failure. The findings of this study provide a new research direction, ideas and theoretical basis for future studies of prevention and treatment of cardiac hypertrophy and myocardial remodeling, as well as a new strategy to find new drug targets.