| BackgroundHeart failure (HF) however remains an increasing global problem and the endpoint of a variety of cardiovascular diseases. HF is a serious disease associated with high morbidity and mortality. Allied with the fact of increasingly elderly populations and prior data demonstrating a steep rise in prevalent cases within more elderly populations, it is likely that the increasing burden of disease will continue to pose challenges for modern healthcare. In China, Myocardial remodeling induced by hypertension and coronary heart disease are the leading causes of HF, as they are in many Western nations.The left ventricle can change its size and shape as a result of external load and/or loss of cardiac myocytes. This process, defined as remodeling, can be adaptive, as in compensatory hypertrophy and dilation secondary to diabetes mellitus, or maladaptive in response to long-standing systemic hypertension. In addition to cellular hypertrophy, apoptosis and extensive interstitial collagen deposition also occurs during this remodeling process. The increased fibrous tissue and myocytes of apoptosis can also lead to inappropriate energy production and utilization.The deadly quartet of metabolic syndrome (MS) includes obesity, impaired glucose tolerance, an atherogenic lipid profile and hypertension. The synergistic effects of these comorbidities lead to a combination of functional and structural cardiomyocyte changes and finally result in a global adverse remodeling process. Overall cardiovascular outcome is significantly impaired. Previous studies have shown that single factor, such as glucose/lipid metabolism disorder and hypertension, is contributed to the changes of left ventricular (LV) structure and function. But at present, cardiac remodeling and the relationship between the molecules and cardiac remodeling in MS was still not reported systematically. In the current study, we created a mouse model of MS and these mice thus represent a good model of human MS. Base on this model, the aim of this study is to investigate the changes of left ventricular (LV) structure and function in MS, try to describe the ensuing problems in cardioprotection and discusses strategies that can be taken to divert or circumvent these detrimental changes.Numerous conditions promote oxidative stress, leading to the build-up of reactive aldehydes that cause cell damage and contribute to cardiac diseases. Mitochondrial aldehyde dehydrogenase (ALDH2) is emerging as a key cardioprotective enzyme for its central role in the detoxification of reactive aldehydes. Previous studies have shown that ALDH2 activity strongly correlates to a better cardioprotective effect, and ALDH2 can be activated by several pathways. The active ALDH2 can inhibit opening of the mitochondrial permeability transition pore, reduce myocardial infarct size, and prevent myocardial remodeling. Epidemiological studies in humans carrying an inactive ALDH2, genetic models in mice with altered ALDH2 levels, and small molecule activators of ALDH2 all highlight the role of ALDH2 in cardioprotection and suggest a promising new direction in cardiovascular research and the development of new treatments for cardiovascular diseases. But the cardioprotective roles of ALDH2 in MS are not cleared. Base on the mouse model with MS, this study will discuss the cardioprotective effects of ALDH2 in detail.Our preliminary study showed that ALDH2 overexpression significantly reduced cardiac apoptosis induced by ischemia-reperfusion injury. It is unknown so far whether the cardioprotection of ALDH2 involves in cardiac remodeling in MS.It is anticipated to establish new target point and method for early treatment of myocardial damage with MS, preventing the development of cardiovascular complications. This research will use advanced techniques, such as ALDH2 overexpression, to investigate the effect of ALDH2 on regulating JNK/AP-1 pathway during myocardial remodeling of MS and possible mechanisms in vivo. Molecule biological techniques are also used to determine the expression of JNK/AP-1 pathway, apoptosis and myocardial fibrosis related genes. Furthermore, we will investigate the cardioprotective mechanisms mediated by ALDH2 in terms of myocardial remodeling. This research will contribute to reveal the novel molecular mechanisms for the preventive effect of ALDH2 against myocardial remodeling in mouse of MS, providing new therapeutic strategy for MS cardiomyopathy.Objective1. To establish a mouse model of MS and observe the changes of left ventricular structure and function in MS mice.2. To describe the ensuing problems in cardioprotection with the increasing activation of ALDH2.3. To describe the effect of ALDH2 over-expression on cardiomyocyte apoptosis and myocardial fibrosis.4. To explore the possible mechanism of ALDH2 on left ventricular remodeling in mouse model of MS.Methods90 male C57 BL/6J mice were randomly divided into control group (n=15) and model group (n=75). Control mice were fed with standard chow and tap water. Mice in model group were fed with free access to a high-fat diet (HF-diet). After HF-diet feeding for 10 weeks,55 MS mice were further divided into three groups:MS group (n=15), continuing of HF-diet feeding; GFP Vector group (n=20), continuing of HF-diet feeding plus injection of lentiviral vector not contain ALDH2 by using left ventricle liquid injection; ALDH2 vector group (n=20), continuing of HF-diet feeding plus injection of ALDH2 lentiviral vector by using left ventricle liquid injection. Mice of control and MS groups were injected with the same volume of NS by using left ventricle liquid injection. After lentiviral vector transfection for 4 weeks, all the mice were sacrificed and the hearts were harvested and kept at-80℃The following parameters were measured during the study:(1) Body weight (BW) was measured every two weeks in the morning; (2) Before and after HF-diet feeding and at the end of experiment, samples of venous blood were collected to detect triglycerides (TG), cholesterol (TC), LDL-C, fasting blood glucose (FBG) and insulin (FINS) levels, and calculate insulin resistance; index (HOMA); (3) Ultrasonic cardiogram (UCG) parameters, including left ventricular end-diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD) ejection fraction (EF) and fractional shortening (FS) were measured after HF-diet feeding and at the end of experiment to evaluate the cardiac function of all the mice. At the end of experiment, all the animals were sacrificed, and the hearts were kept for the following study:(1) spectrophotography of mitochondria ALDH2 activation; (2) electron microscopic examination of myocardial ultrastructral changes and pathological study of myocardium, such as Masson and HE staining; (3) TUNEL technique of cardiocyte apoptotic activity; JC-1 of hyperglycemia-induced mitochondrial membrane potential; (4) Intracellular ROS levels were monitored by use of a peroxide-sensitive fluorescent probe, 2’,7’-dichlorofluorescin diacetate (DCFH-DA); (5) 4-HNE content was determined by immunohistochemistry. (6) RT-PCR measurement of collagen Ⅰ/Ⅲ, and TGF-β1 mRNA; (7) Western Blot for p-JNK、AP-1、p-IRS-1(Ser 307)、 IRS-1、p-AKT、AKT、caspase-3 and ALDH2.Statistical Analysis:SPSS 17.0 was used for statistical analysis. Continuous data were expressed as mean±SD and compared by one-way ANOVA, followed by LSD test and independent samples t test. Index relativity was assessed by Pearson tests. A P value < 0.05 was considered statistically significant.Results1. Establishment of MS model with C57 BL/6J miceAfter HF-diet feeding for 10 weeks,55 MS mice were further divided into three groups:MS group (n=15); GFP Vector group (n=20); ALDH2 vector group (n=20). After lentiviral vector transfection,6 mice died in each group of ALDH2 and GFP Vector.1 mouse died in MS group.The MS mice were found to develop insulin resistance, hypercholesterolemia, hyperglycemia, high LDL-C and obesity compared to the NC group (P<0.01). This means the mouse model of MS was achieved by fed with HF-diet for 10 weeks.2. Improvement of metabolic index with over-expression of ALDH2At the end of the experiment, compared with NC group, BW, TG, TC, FINS, FBG and HOMA in MS group were significantly increased (P<0.01). Compared with MS and GFP group, BW, TG, LDL-C, TC, FINS, FBG and HOMA in ALDH2 group were significantly decreased (P<0.05).3. Activation of ALDH2 in different groupsALDH2 activity in heart mitochondria with MS was markedly decreased (P<0.01), by approximately 40%, as compared with the NC group. After transfection with lentiviral vector, mitochondrial ALDH2 activity and expression were significantly improved (P<0.01).4. The effect of over-expression ALDH2 on myocardial ROS and 4-HNEMyocardial ROS content and 4-HNE expression were significantly higher in MS group than in NC group (P<0.01). After transfection with ALDH2 lentiviral vector,4-HNE expression decreased significantly (P<0.01), whereas ROS content were decreased without statistical significance.5. Improvement of cardiac structure and function with over-expression of ALDH2LVEDD and LVESD were increased, further more, EF and FS were decreased significantly in MS group than in NC group (P<0.01). After transfection with ALDH2 lentiviral vector, LVEDD and LVESD were decreased (P<0.05), whereas EF and FS increased significantly (P<0.05).6. Improvement of ultrastructural change of cardiac muscle with over-expression of ALDH2The left ventricular myocytes from NC group arranged regularly. The thick and thin myofilament arranged regularly. The uniformly sized mitochondria were abundant and showed round or oval shape.The left ventricular myocytes from MS group arranged irregularly. The pericellular membrane was interrupted and unclear. The local collagen fiber was disintegrated and increased. The swelling mitochondria increased and accumulated.Compared with MS group, the ultrastructural changes of ALDH2 group were obviously improved. The myocytes from ALDH2 group arrange more regularly than MS group, and the local collagen fiber were integrated and decreased. The mitochondria were more regular than MS group and the swelling mitochondrion was obviously improved.7. Improvement of cardiomyocyte apotosis with over-expression of ALDH2 through JNK/AP-1 and IRS-1/AKT/caspase pathwayTUNEL positive cells were expressed as a percentage of brown nuclei. Compared with the NC group, the number of positive cells was significantly increased in the MS group (P<0.01). Compared with MS and GFP group, the number of positive cells decreased in the ALDH2 group (P<0.01). And mitochondrial membrane potential was decreased greatly in mice with MS (P<0.01). ALDH2 over-expression have a significantly protection effect on mitochondrial membrane potential (P<0.05). At the same time, Compared with MS and GFP group, the protein of p-JNK、AP-1、p-IRS-1(Ser 307)、caspase-3 significantly decreased in the ALDH2 group and p-AKT/ AKT significantly increased (P<0.05).8. Improvement of myocardial fibrosis with over-expression of ALDH2 through JNK/AP-1 and TGF-β1 pathwayThe relative collagen content was markedly increased in the MS group measured by Masson staining (P<0.01). Compared with MS and GFP group, the relative collagen content decreased and collagen fibers arranged regularly in the ALDH2 group (P<0.05). In the ALDH2 group, TGF-β1 and collagen Ⅰ/Ⅲ mRNA were decreased significantly (P<0.05), accompanied by decrease of JNK and AP-1 protein (P<0.05).Conclusion1. A C57 BL/6J mouse model of MS was established by high-fat diet. The animal model was valuable for the study of the mechanism in cardiac remodeling of MS.2. MS mouse showed cardiac remodeling, such as considerable apoptosis of cardiocytes, collagen deposition and left ventricular structure changes. And expression and activity of ALDH2 significantly decreased in this model.3. ALDH2 plays a better cardioprotective role in MS mouse by reducing 4-HNE and improving insulin resistance.4. Myocardial cell apoptosis were reduced by ALDH2 over-expression through JNK/AP-1 and IRS-1/AKT/caspase pathway.5. After transfection with ALDH2 lentiviral vector, myocardial fibrosis was improved through JNK/AP-1 and TGF-β1 pathway. |
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