Endothelial Myocyte Matrix in Cardiac Remodeling

描述（由申请人提供）：本项目的总体目标是了解慢性心力衰竭中内皮细胞-心肌细胞（E-M）功能障碍的机制。上一个资助期的研究表明，内皮细胞内皮功能障碍与氧化基质蓄积（纤维化）增加、潜在驻留心肌基质金属蛋白酶（MMP）激活和继发于氧化和蛋白水解应激的心肌组织金属蛋白酶抑制剂（TIMP-4）失活相关。TIMP-4的施用改善了活性氧簇（ROS，氧化应激）和MMP活化（蛋白水解应激）的形成。此外，我们还发现了蛋白酶激活受体-1（PAR-1）的诱导作用。然而，PAR-1在纤维化和E-M解偶联中的作用仍然不清楚。H2S气体是缓解氧化应激的最有效的抗氧化剂，最近的研究表明H2S具有心脏保护作用。这种竞争性更新提议的中心假设是，在慢性心力衰竭期间，氧化和蛋白水解应激诱导PAR-1，导致分别产生线粒体（mt）ROS和活性氮物质（RNS）以及线粒体一氧化氮合酶（mtNOS），从而激活潜在的常驻心脏MMPs。这些事件破坏MMP/TIMP轴，引起内皮细胞和肌细胞之间的纤维化。用H2S处理可减轻纤维化并减轻E-M解偶联。因此，本提案的具体目的是：#1：确定慢性左心室（LV）容量超负荷是否通过诱导NADPH氧化酶（p47亚基）、mtNOS和PAR-1引起线粒体氧化应激（ROS和RNS），以及H2S是否引起线粒体氧化应激。二：为了确定慢性LV容量超负荷是否通过增加胶原/弹性蛋白比率引起心脏纤维化，MMP-2、-9、-13、TIMP-1、-3、降低TIMP-4和诱导PAR-1和H2S减轻心脏纤维化。#3：确定慢性LV容量超负荷是否通过诱导PAR-1和H2S降低E-M解偶联而引起E-M功能障碍和LVH。在野生型（WT）、PAR-1-/+、iNOS-/-、MMP-9-/-、TIMP-3-/-和TIMP-4++/++小鼠中，通过左心室-腔静脉瘘（AVF）的LV容量超负荷产生慢性心力衰竭，用或不用NaHS（H2S供体）处理。公共卫生相关性：只有当肌细胞通过细胞外基质（ECM）连接时，肌细胞收缩和舒张才同步。我们创造了术语内皮-肌细胞（E-M）偶联，因为内皮细胞（EE）细胞衍生的心脏活性剂调节心肌细胞在收缩期的收缩和在舒张期的舒张。心脏中的内皮是研究最少的系统，因为与管道动脉不同，内皮细胞内皮被埋在肌肉中，并且难以分离其在肌细胞功能中的作用。我们开发了一种新的技术，“心脏环”，其中内皮依赖或独立的心脏收缩和舒张可以分别在LV和RV检查。该提议有两个新的方面：1）所提议的实验将证明PAR-1在心力衰竭中纤维化和内皮-肌细胞解偶联的潜在机制中的作用;和2）将具有H2S在治疗舒张性心力衰竭中的临床转化分支。

DESCRIPTION (provided by applicant): The overall goal of this project is to understand the mechanism of endocardial endothelial-myocyte (E-M) dysfunction in chronic heart failure. Studies from the previous funding period suggested that endocardial endothelial dysfunction is associated with increased oxidized-matrix accumulation (fibrosis), activation of latent resident myocardial matrix metalloproteinases (MMPs) and inactivation of cardiac tissue inhibitor of metalloproteinase (TIMP-4) secondary to oxidative and proteolytic stresses. Administration of TIMP-4 ameliorated both the formation of reactive oxygen species (ROS, oxidative stress) and MMP activation (proteolytic stress). In addition, we discovered the induction of proteinase activated receptor-1 (PAR-1). However, the role of PAR-1 in fibrosis and E-M uncoupling remains poorly defined. H2S gas is the most potent antioxidant in mitigating oxidative stress and recent studies have implicated a cardioprotective role of H2S. The central hypothesis of this competitive renewal proposal is that during chronic heart failure the oxidative and proteolytic stresses induce PAR-1, leading to generate mitochondrial (mt) ROS and reactive nitrogen species (RNS) and mitochondrial nitric oxide synthase (mtNOS), respectively, thus activating the latent resident cardiac MMPs. These events disrupt the MMP/TIMP axis, causing fibrosis between endothelium and myocyte. Treatment with H2S alleviates fibrosis and mitigates E-M uncoupling. Therefore, the specific aims of this proposal are: #1: To determine whether chronic left ventricle (LV) volume overload causes mitochondrial oxidative stress (ROS and RNS) by inducing NADPH oxidase (p47 subunit), mtNOS and PAR-1, and H2S alleviates mitochondrial oxidative stress. #2: To determine whether chronic LV volume overload causes cardiac fibrosis by increasing collagen/elastin ratio, MMP-2, -9, -13, TIMP-1, -3, decreasing TIMP-4, and inducing PAR-1, and H2S mitigates cardiac fibrosis. #3: To determine whether chronic LV volume overload causes E-M dysfunction and LVH by inducing PAR-1 and H2S decreases E-M uncoupling. Chronic heart failure will be created by LV volume overload by aorta-venacava fistula (AVF) in wild type (WT), PAR-1-/+, iNOS-/-, MMP-9-/-, TIMP-3-/-, and TIMP-4++/++ mice, treated with or without NaHS, a H2S donor. PUBLIC HEALTH RELEVANCE: The myocyte contraction and relaxation are synchronized only when myocytes are connected by extracellular matrix (ECM). We coined the term endothelial-myocyte (E-M) coupling because endocardial endothelial (EE) cells derived cardio-active agents modulate myocyte contraction in systole and relaxation in diastole. The endothelium in the heart is the least studied system, because unlike conduit arteries, the endocardial endothelium is buried in the muscle and it is difficult to separate its role in myocyte function. We developed a new technique, "cardiac rings," in which endothelium dependent or independent cardiac contraction and relaxation can be examined separately in LV and RV. There are two novel aspects of this proposal: 1) the proposed experiments will demonstrate the role of PAR-1 in the underlying mechanism of fibrosis and endothelial-myocyte uncoupling in heart failure; and 2) will have clinical translational ramifications of H2S in treating diastolic heart failure.