Effects Of Matrix Metalloproteinase And Tissue Inhibitor Of Metalloproteinase Expression On Myocardial Structural Remodeling

Background and purpose:MMPs is a multi-gene regulation of endogenous zinc-dependent enzyme family which is composition of neutral protease that similar to the structure and function and is secreted by vascular smooth muscle cells, monocytes and endothelial cells, and its function is to degrade all extracellular matrix components(ECM), excluding polysaccharide. MMPs has been found that there are more than 20 kinds, according to its structure and function can be divided into four different categories:①collagenase (MMP-1, MMP-8, MMP-13):the main substrate is hydrolyzed collagen fiber, namely,Ⅰ,Ⅱ,Ⅲcollagen;②gelatinase (MMP-2, MMP-9):digest denatured collagen and gelatin quickly;③stromelysin (MMP-3, MMP-10, MMP-11): hydrolysisⅢ,Ⅳcollagen and proteoglycan, gelatin and sugar protein;⑤The fourth category is the structure and function of more specific MMPs (MMP-7,12,19, 20,23,26, etc.), which can degrade several ECM components, inter alia, but also activate other MMPs. Tissue inhibitors of metalloproteinase (TIMPs) are specific endogenous inhibitor of MMPs, MMPs secreting cells also secrete TIMPs, TIMPs are four kinds:TIMP-1, TIMP-2, TIMP-3 and TIMP-4. TIMPs have two main functions: the inhibition of MMPs activity, TIMPs and activated MMPs are usually to form a high-affinity 1:1 complex and through the closure of the catalytic domain of MMPs to inhibit the degradation of ECM. In addition, TIMPs also promote mitosis, inhibit angiogenesis and inducing cell death role.Two-thirds of myocardial cells in non-cardiac cells, namely, extracellular matrix, its myocardial cells not only has the structural support and protection, and in information transmission between myocardial cells, the coordination of both systolic and diastolic function play an important role. Main functions are:①to provide support to the myocardial cells, blood vessels and lymphatic vessels intercomnected and arranged, determine myocardial stiffness and structure;②to prevent excessive elongation of myocardial cells;③the pressure of cardiac cells will be transmitted to ventricular cavity;④to prevent excessive retraction of myocardial cells;⑤provide resistance when myocardial relaxation;⑥to provide tensile strength to the heart in order to prevent its fracture;⑦to prevent cardiac cells and muscle fibers slippage. Myocardial extracellular matrix of collagen accounts for the main location of the adult myocardial tissue includes at least collagenⅠ,Ⅲ,Ⅳ,ⅤandⅥtype. Two of the most important collagen are the typeⅠ(ColⅠ) and typeⅢ. Collagen fiber network composed by various components of collagen facilitate the integration between myocardial cells, and it can directly influence the structure and function of the heart. In animal models of heart failure and heart failure patient may see the change of the content of cardiac interstitial collagen and the nature, and this change often determines the prognosis, and this prove the importance of myocardial interstitial. Myocardial collagen is also in the ever-changing and metabolism, its daily update rate is about 0.6%. The balance between MMPs and TIMPs plays an important role in the metabolism of dynamic balance for the maintenance of myocardial interstitial collagen synthesis and degradation. In some disease states, such as myocardial infarction, high blood pressure and so on, the number and distribution of myocardial collagen has changed (ie, cardiac remodeling), causing myocardial interstitial fibrosis, limiting cardiac activity and reduce ventricular compliance, impact the systolic and diastolic function of myocardial. It is an important reason for causing heart failure. In addition, collagen deposition may limit the cardiac depolarization waves through the speed of the electrical stability of the myocardium have a negative impact, which can lead to cardiac arrhythmia. Collagen in the adult heart is a continuous synthesis and degradation and to maintain dynamic balance, age-related increase of collagen synthesis and degradation of the balance of offset, a gradual accumulation of collagen. Many enzymes can degrade extracellular matrix components of the collagen protein, therefore, understanding the modification of myocardial extracellular matrix structure of the enzyme system can understand the cardiac remodeling, while the matrix metalloproteinase play an important role on the reconstruction of extracellular matrix.At present, the research of the MMPs in the pathogenesis and treatment of atrial fibrillation and myocardial infarction was few, and research focused on the following aspects:①a special inhibitor of synthetic MMPs, the main focus on the prevention and treatment of invasive and transfer of tumors;②the use of recombinant protein or viral vector gene transfer technology for the endogenous anti-protease agent, or enhance the secretion of TIMPs in vivo;③According to the expression of MMPs and the enzyme activity of regulatory mechanisms, development the drug to control the balance between MMPs and TIMPs. In this study, we collected clinical data and myocardial specimens of 50 cases of rheumatic heart disease patients and 40 cases of coronary heart disease patients, and immunohistochemical staining and fluorescence quantitative PCR reaction for myocardial specimens, to study the MMPs and TIMPs associated with myocardial remodeling, and analysis of the pathophysiology mechanisms of myocardial remodeling and the clinical features of myocardial remodeling.Materials and methods:1. Patients and sample collection:Selection 50 cases of rheumatic heart disease patients for artificial mitral valve replacement surgery from September 2007 to May 2009 in our hospital,28 males and 22 females, aged from 26 to 66 years old, the 50 cases is divided into 20 cases of sinus rhythm group (SR) and 30 cases of atrial fibrillation group (AF). One of the following conditions are not included in this study:①younger than 20 years old or more than 66 years of age;②complicated with infective endocarditis;③complicated with hypertension, diabetes, coronary heart disease, hyperthyroidism, myocardial disease, chronic pulmonary heart disease or severe liver and kidney dysfunction;④non-rheumatic heart disease undergoing heart valve replacement surgery;⑤rheumatic heart disease were non-mitral valve replacement surgery;⑥The clinical signs of rheumatism activity or fever. And over the same period selected 40 cases of coronary heart disease patients who received coronary artery bypass surgery,25 males and 15 females, aged from 40 to 65 years old, those were divided into 22 cases of myocardial infarction group(MI) and 18 cases of unstable angina group(UA). Preoperative coronary angiography, imaging results showed that in the MI group,9 cases of left main coronary vascular disease more than 50% (41%),8 cases of three coronary vascular disease (36%),5 cases of double coronary vascular disease (23%); UA group,8 cases of left main coronary vascular disease more than 50% (44%),6 cases for three coronary vascular disease (33%),4 cases of severe proximal left anterior descending artery stenosis (23%). All patients were consistent with coronary artery bypass graft surgery to testify. One of the following conditions are not included in this study:merger liver, kidney dysfunction, pulmonary fibrosis, bone metabolic diseases, severe diabetes, systemic autoimmune disease, infection, rheumatic diseases and malignant tumors and so on. Registration clinical data of all research subjects, including age, sex, height, weight, combined with other diseases, drug use, and electrocardiogram, echocardiography and chest X-ray in preoperatively. All patients cardiac function wereⅡtoⅢgrade (NYHA classification). All patients were preoperatively to sign informed consent. Taken 100mg specimens from the right atrial appendage before cardiopulmonary bypass surgery, quickly placed it in liquid nitrogen to save after removed blood and fatty tissue.2. Immunohistochemical staining:using streptavidin peroxidase conjugated method(SP immunohistochemical staining method), dropping amount of dilution of mouse anti-human MMP-1, MMP-3, MMP-7, MMP-9 and TIMP-1, TIMP-2, TIMP-3, TIMP-4 monoclonal antibody with specific antigen to form antigen-antibody complex, followed by the use of biotin-labeled anti-2, combined with the DAB color reagent, the positive results appear brown cytoplasmic granules, Showing that for the diffuse positive expression of myocardial. To use the light microscope observation and take digital photographs in the same lighting conditions,and then using Image-pro plus image analysis software to calculate the PU values.3. Fluorescence quantitative PCR reactions:using Trizol one-step method extracting organizations RNA, measuring OD260 and OD280 of the RNA samples, and calculating the concentration of RNA, then RNA reverse transcription synthesis of cDNA, reverse transcription steps in strict accordance with TaKaRa SYBR(?) Premix Ex TaqTM RT reagent Kit (Perfect Real Time) instructions to carry out,and primers were designed and synthesized, and then use of restricted reference materials (GAPDH) primers and the various indicators primers to carry out conventional PCR reaction to test the template cDNA and primers, and then do fluorescence quantitative PCR reaction,the parameters according TaKaRa SYBR(?) Premix Ex TaqTM (Perfect Real Time) kit instructions. Analysis of the data in accordance with the following formula to calculate each target relative to the percentage of GAPDH levels in each sample, namely, (target/GAPDH)* 100%4. Statistical Analysis:All data applied SPSS 13.0 statistical package for analysis, Indicators to mean±standard deviation expressed, difference between the groups used t tests,p<0.05 mean the significant difference.Results:1. Rheumatic heart disease:Clinical data such as age, height and weight were no significant difference between the SR group and AF group, the left atrial diameter and right atrial diameter of the AF group were significantly greater than those of the SR group (p<0.01), the difference was significant, but the left ventricular diastolic diameter and left ventricular ejection fraction were no significant differences. Immunohistochemical staining:positive results was that the cytoplasm appeared brown granules. SR group atrial cytoplasm can be seen evenly distributed sparse brownish particles, AF groups atrial cytoplasm can be seen the irregular distribution of thick brown-yellow granules. Using Image-proplus image analysis software to calculate the PU values, it shown that the expression of MMP-3, MMP-7, MMP-9 and TIMP-1, TIMP-2, TIMP-3, TIMP-4 were significantly increased in AF group (all p<0.01), excluding MMP-1, the difference was significant. The difference of MMP-1 of the two groups was not statistically significant, but the level of AF group was more than that of the SR group. FQ-PCR reaction:the AF group atrium expression of MMP-3, MMP-7, MMP-9 and TIMP-1, TIMP-2, TIMP-3, TIMP-4 were significantly increased (all p<0.01), the difference was significant. The difference of MMP-1 of the two groups was not statistically significant, but the level of AF group was more than that of the SR group.2. Coronary heart disease:Clinical data such as age, height and weight were no significant difference between the MI group and UA group, Two groups of patients with echocardiography shown that the left atrial diameter and right atrial diameter and left ventricular diameter of the MI group were significantly greater than those of the UA group (p<0.01), but the left ventricular ejection fraction was significantly less than that of the UA group (p<0.05). Immunohistochemical staining:positive results was that the cytoplasm appeared brown granules. UA group atrial cytoplasm can be seen evenly distributed sparse brownish particles, MI group atrial cytoplasm can be seen the irregular distribution of thick brown-yellow granules. Using Image-proplus image analysis software to calculate the PU values, it shown that the expression of MMP-3, MMP-9 and TIMP-1 TIMP-2,TIMP-3,TIMP-4 were significantly increased in MI group(respectively, p<0.01, p<0.01, p<0.05, p<0.05,p<0.01, p<0.01), excluding MMP-1 and MMP-7. The difference of MMP-1 and MMP-7 of the two groups was not statistically significant, but the level of MI group was more than those of the UA group. FQ-PCR reaction:The MI group myocardial expression of MMP-3, MMP-9 and TIMP-1 TIMP-2,TIMP-3,TIMP-4 were significantly increased (all p<0.01), The difference of MMP-1 and MMP-7 of the two groups was not statistically significant, but the level of MI group was more than those of the UA group.Discussion:Cardiac extracellular matrix is composed by collagen fiber, matrix membrane protein, proteoglycan, and the signaling molecules with biological activity, it is an important component of myocardial structure and has important physiological function such as cell information transmission and coordination the role of systolic and diastolic function. It can maintain the myocardial cells and myocardial fibers arranged, to limit the role of myocardial cells over-stretched. Myocardial extracellular matrix can also cause changes in cardiac contraction and ejection of effective coordination, and lost the support of the role of the heart geometry, resulting in impaired pump function.MMPs are a zinc binding protease family with the decomposition of extracellular matrix, it has been found more than 20 kinds, according to the substrate, it is divided into four types including collagenase, gelatinase, matrix soluble enzymes and membrane-type enzymes. It is involved in normal embryonic development and tissue remodeling and involved in many of the body pathological and physiological processes. TIMPs are specific inhibitors of endogenous MMPs, it is divided into four types, the interaction and the dynamic balance between TIMPs and MMPs play an important role to maintain the normal heart shape and heart function. There studies have shown that expression and abnormalities activity of MMPs and TIMPs involved in the atrial structural remodeling.At present, different levels of MMPs and TIMPs expression in cardiovascular disease have been proposted at home and abroad.Most studies have focused on the expression of both serum levels or the use of animal models to speculate MMPs and TIMPs in human heart disease in patients with myocardial expression. In our study, we use the way of human myocardial biopsy.This study selected four types of MMPs and all kinds of TIMPs to explore the expression of matrix metalloproteinases and their inhibitors in patients with atrial fibrillation and myocardial infarction, so the study was comprehensive and general. Due to the different and the degree of heart failure of rheumatic heart valve disease will affect the degree of atrial fibrosis and the incidence of atrial fibrillation, for which the election in this study were all patients with mitral valve disease, and valvular disease in all groups was no significant difference nature of cases in each group and no significant difference in left ventricular ejection fraction. The results shown that the right and left atrial diameter of rheumatic heart disease patients with atrial fibrillation expanded significantly compared to sinus rhythm patients, the difference was significant. To show that atrial fibrillation leading to myocardial structural changes have taken place, the atrial cavity expansion increased myocardial stiffness.The right and left atrial diameter and left ventricular diameter of myocardial infarction patients expanded significantly compared to angina pectoris patients, the difference was significant. These indicated myocardial structural remodeling has taken place of myocardial infarction patients and the cavity expansion and myocardial stiffness increased, which leading to-impair the cardiac function. This study used immunohistochemical staining and the PU values which can quantitatively calculat the expression of MMPs and TIMPs, and then used fluorescence quantitative PCR method to accurate measure the expression of MMPs and TIMPs. The results shown that the expression of MMP-3, MMP-7, MMP-9 and TIMP-1, TIMP-2, TIMP-3, TIMP-4 of atrial fibrillation patients of Rheumatic heart disease were significantly increased, while MMP-1 expression in the two groups was no significant difference, but the overall value was still an increasing trend, and the expression of MMP-3, MMP-9 and TIMP-1,TIMP-2,TIMP-3,TIMP-4 of myocardial infarction patients were significantly increased, although the expression of MMP-1 and MMP-7 in the two groups were no significant difference, but the overall value was still an increasing trend, these suggest that the increase expression of MMP-1, MMP-3, MMP-7, MMP-9 and TIMP-1, TIMP-2, TIMP-3, TIMP-4 cause myocardial extracellular matrix degradation, myocardial cell stretch, atrial wall thinning, atrial expansion, normal cell components decreased, matrix components increased and myocardial extensive fibrosis, and then promote the occurrence of atrial remodeling and maintain it. It shows increased activity of MMPs expression can imbalance of ECM synthesis and degradation, both involved in cardiac and vascular remodeling and ventricular dilatation. It plays an important role in the pathogenesis of cardiovascular disease. Therefore, regulating the activity of MMPs may be the treatment of cardiovascular disease, a new direction for further research will contribute to its clinical application as soon as possible.In summary, the change of the level of MMPs and TIMPs determines varying degrees of myocardial remodeling, and directly affect and change of collagen metabolism. By regulating expression and activity of the MMPs and TIMPs to prevent or even reverse the cardiac remodeling may be an important clinically mean of treatment and prevention.