Study On The Regulatory Mechanism Of CYP2E1 On Dilated Cardiomyopathy And Hypertrophic Cardiomyopathy

Objective Cytochrome P450 2E1 (CYP2E1) participats into the metabolism of many exogenous and endogenous materials, such as ethanol, tetrachlormethane, acetone, arachidonic acid and so on. CYP2E1 mainly expresses in liver, in addition, it also has been found in brain, lung, bowels, kidney, heart and other tissues. CYP2E1 participates into lipid peroxidation and apoptosis, and is related to fatty liver, diabetes, cancer, and so on. However, the relationship between CYP2E1 and heart, and the function in the development of cardiomyopathy have not been reported. So we established the heart specific CYP2E1 transgenic mice for analyzing the modulation of CYP2E1 on the cTnTR141w transgene dilated cardiomyopathy (DCM) mice and the cTnTR92Q transgene hypertrophic cardiomyopathy (HCM) mice.Methods The expression of CYP2E1 in both hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) mice were analyzed using gene expression microarray analysis of cTnTR92Q and cTnTR141w myocardium and confirmed by reverse transcriptional polymerase chain reaction (RT-PCR) and western blot. The temporal expression patterns of CYP2E1 in the heart of WT mice were analyzed by western blot. The cDNA of CYP2E1 was amplified by RT-PCR from the mice heart, and then was inserted into the downstream of a-MHC promoter to constructe the CYP2E1 expression vector. The transgenic mice were created by the method of micro injection. And they were crossed with cTnTR141W and cTnTR92Q transgenic mice. The cardiac structure and function were analyzed with M-mode echocardiography. Survival data of the experimental mice were recorded. Pathologic changes were observed by light microscopy and transmission electronic microscopy. The hypertrophy molecular markers, including genes of sarcomeric proteins, cytoskeletal proteins, extracellular matrix proteins and the genes associated with signal transduction were analyzed by RT-PCR and western blot.The contents of hydrogen peroxide (H2O2), malonaldehyde malondialdehyde (MDA), reduced glutathione (GSH) as well as the total anti oxidation capable (T-AOC) were detected by spectrophotometry. The signaling molecules were detected by western blot. Results Gene expression microarray analysis, RT-PCR and western blot revealed that a higher expression of CYP2E1 in HCM mice heart, and a lower expression in DCM mice heart. The expression of CYP2E1 in WT mice heart could not be detected at the embryonic stage and the highest level showed at the birth of mouse. The heart-specific CYP2E1, CYP2ElxcTnTR141w and CYP2El×cTnTR92Qtransgenic mice were established. The heart of CYP2Eltransgenic mice showed DCM phenotype, including the larger ventricular chamber, reduced ventricular wall, and the worse heart function. The CYP2E1 could aggravate the DCM phenotype of cTnTR141w transgenic mice, and reverse the HCM phenotype of cTnTR92Q transgenic mice. The histology results showed the overexpression of CYP2E1 could worse the myocyte disarray and the interstitial fibrosis. The overexpression of CYP2E1 could significantly increase the mortality of cTnTR141w transgenic mice. The expansion of sarcoplasmic reticulum, swell of mitochondrium on CYP2E1 transgenic mice's heart could beobserved under the transmission electronic microscope. The hypertrophy molecular markers on CYP2E1 transgenic mice's heart, such as Myot, Cnnl, Actal, Collal, Co13al, Nppa and Nppb were all up regulation. In addition, the overexpression of CYP2E1 could increase the H2O2 and MDA, and cut down the GSH and T-AOC. The signaling molecules, including ERK1/2, JNK, P38, PI3K, Akt and GSK3?did not change significantly.Conclusion Through the study on CYP2E1 transgenic mice, we firstly found that CYP2E1 transgenic mice displayed the DCM phenotype. CYP2E1 could aggregate the DCM phenotype of cTnTR141W transgenic mice, and lead cTnTR92Q transgenic mice to heart failure. And the oxidative stress induced by CYP2E1 may participate into this cardic remodeling process. Objective Cysteine dioxygenase1 (CDO1) catalyzes the initial step in the biochemical pathway used for oxidation of cysteine to cysteine sulfinic. CDO1 mainly expresses in liver tussie, in addition, it can also be found in lung, kidney, heart and other tussies. However, the relationship study about CDO1 and heart as well cardiomyopathy have not been reported. So we stablished CDO1 transgenic mice and investigate its effect on the development of cardiomyopathy.Methods The expression of CDO1 in both hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) mice were analyzed using gene expression microarray analysis of cTnTR92Q and cTnTR141W myocardium and confirmed by reverse transcriptional polymerase chain reaction (RT-PCR) and western blot. Transgenic mice were generated by the method of microinjection. The genotype of transgenic lines was identified by PCR. The cardiac function and geometry were detected by echocardiography.Results Gene expression microarray analysis, RT-PCR and western blot revealed that a higher expression of CDO1 in HCM mice heart, and a lower expression in DCM mice heart. Seven founders of hearts specific?-MHC-CDO1 transgenic mice were established and two high-level expression line was identified. The transgenic gene itself did not affect the cardiac function and geometry compared with the wild type mice.Conclusion The transgenic mice with cardiac-specific expression of the human CDO1 gene were established successfully and it can be used to cross with the DCM and HCM models to investigate the function of CDO1 gene on the development of cardiomyopathy. Objective To construct heparin-binding epidermal growth factor-like growth factor (HB-EGF) transgenic mice in order to investigate the role of HB-EGF in the fibrosis of tissues.Method HB-EGF gene was cloned form mouse with RT-PCR. The cloned HB-EGF gene was then inserted downstream of Chicken?-actin promoter to construct the vector which could express HB-EGF gene in system. The transgenic mice were produced by microinjection method and the genotype was detected by PCR with specific primers. The expression profiles of HB-EGF in body tissues were illustrated by Western blotting. Massion staining of liver, lung, kidney and bladder from transgenic mice and nontransgenic control mice was carried out.Results Two lines of HB-EGF transgenic mice were established. The expression levels of the HB-EGF gene in liver, lung, kidney and bladder from transgenic mice were obviously higher than those form control mice. The extents of fibrosis in the liver, lung, kidney and bladder of transgenic mice were significantly enhanced by the expression of the transgene.Conclusion Two lines of system-expressing HB-EGF transgenic mice were established successfully. The over-expression of HB-EGF can significantly aggravate tissues fibrosis degrees.