Extracellular Matrix in Hypertensive Heart Disease & Transition to Heart Failure

 描述(由申请人提供)：高血压性心脏病(HHD)患者发生心肌重构和舒张期功能异常是一件非常重要的事情。导致舒张期功能异常并进展为射血分数保留的心力衰竭(HFpEF)的机制尚不清楚。这项建议将检验原纤维胶原蛋白变化的因果贡献。我们假设：1-基质金属蛋白酶(MMPs)和内源性MMPs组织抑制物(TIMPs)之间的化学计量平衡是决定舒张期僵硬和胶原含量的主要因素，2-HHD患者发生HFpEF时这种化学计量平衡发生了关键变化，3-HFpEF患者TIMP-1增加，导致间质蛋白酶活性降低，胶原降解减少，胶原含量增加，胶原依赖僵硬增加，4-TIMP-1和间质MMP活性的变化反映了成纤维细胞功能的变化；在发生HFpEF的HHD患者中，成纤维细胞转变为更倾向于纤维化的表型，5-TIMP-1缺失将防止和逆转压力超负荷对TIMP-1的依赖效应。我们开发了新的方法，首次允许在体内测量聚集的间质蛋白水解酶活性(使用微透析和淬灭的荧光肽底物)和测量胶原依赖的心肌舒张期硬度(使用顺序提取技术治疗的左心室活检中测量的心肌应力与应变)。这些方法将应用于HHD患者和临床相关压力超负荷的新型小鼠手术和转基因模型(他莫昔芬诱导的、成纤维细胞特异性的TIMP-1基因敲除小鼠的横动脉收缩)。具体目的一：证明在高血压性心脏病患者向心力衰竭的过渡过程中，TIMP-1的增加和体内间质蛋白水解酶活性的降低导致胶原含量增加和胶原依赖的僵硬增加。具体目的二：利用原代成纤维细胞培养，在体外证明HFpEF患者心脏成纤维细胞功能发生根本性变化，这是由于TIMP-1诱导的蛋白水解酶活性降低和胶原降解减少所致。具体目标三：在体内确定在压力超负荷所致心力衰竭小鼠模型中TIMP-1的表达/丰度、间质蛋白水解酶活性的变化与胶原依赖僵硬增加之间是否存在因果关系。

 DESCRIPTION (provided by applicant): The development of myocardial remodeling and abnormal diastolic function is a critical event in patients with hypertensive heart disease (HHD). The mechanisms that contribute to the development of abnormal diastolic function and progression to heart failure with a preserved ejection fraction (HFpEF) are poorly defined. This proposal will examine the causal contribution made by changes in fibrillar collagen. We hypothesized that 1- the stoichiometric balance between matrix metalloproteinases (MMPs) and endogenous tissue inhibitors of MMPs (TIMPs) is a primary determinant of diastolic stiffness and collagen content, 2- there is a critical change in this stoichiometric balance in HHD patients that develop HFpEF, 3- HFpEF patients have an increase in TIMP-1 that results in decreased interstitial protease activity, decreased collagen degradation, increased collagen content and increased collagen-dependent stiffness, 4- changes in TIMP-1 and interstitial MMP activity reflect a change in fibroblast function; fibroblasts shift to a more profibrotic phenotype in HHD patients that develop HFpEF, and 5- TIMP-1 deletion will prevent and reverse TIMP-1 dependent effects of pressure overload. We developed novel methods that allow, for the first time, in vivo measurements of aggregate interstitial protease activity (using microdialysis and a quenched fluorogenic peptide substrate) and measurement of collagen-dependent myocardial diastolic stiffness (myocardial stress vs. strain measured in LV biopsies treated with sequential extraction techniques). These methods will be applied to both patients with HHD and novel murine surgical and transgenic models of clinically relevant pressure-overload (transverse aortic constriction in tamoxifen-inducible, fibroblast-specific, TIMP-1 knock-out mice). Specific Aim One: Demonstrate that in patients with hypertensive heart disease, the transition to heart failure is characterized by an increase in TIMP-1 and a decrease in in vivo interstitial protease activation leading to an increase in collagen content and an increase in collagen-dependent stiffness. Specific Aim Two: Demonstrate in vitro, using primary fibroblast cultures that fundamental changes in cardiac fibroblast function occur in patients with HFpEF that result from a TIMP-1 induced decrease in protease activation and decreased collagen degradation. Specific Aim Three: Determine in vivo whether there is a cause and effect relationship between a change in TIMP-1 expression/abundance, interstitial protease activity, and the development of increased collagen- dependent stiffness in a murine model of pressure-overload induced heart failure.