Endothelial Myocyte Matrix in Cardiac Remodeling

描述(申请人提供)：本项目的总体目标是了解慢性心力衰竭患者心内膜内皮细胞功能障碍的机制。前一资金时期的研究表明，心内膜内皮功能障碍与氧化基质堆积(纤维化)增加、潜伏的驻留心肌基质金属蛋白酶(MMPs)激活以及继发于氧化和蛋白分解应激的心脏组织金属蛋白酶抑制物(TIMP-4)失活有关。给予TIMP-4可改善ROS(氧化应激)和MMPs(蛋白水解性应激)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解偶联中的作用仍不清楚。在减轻氧化应激方面，硫化氢气体是最有效的抗氧化剂，最近的研究表明，硫化氢具有心脏保护作用。这一竞争性更新方案的中心假设是，在慢性心力衰竭过程中，氧化应激和蛋白分解应激诱导PAR-1，导致分别产生线粒体(Mt)ROS、活性氮(RNS)和线粒体一氧化氮合酶(MtNOS)，从而激活潜在的驻留心肌MMPs。这些事件破坏了基质金属蛋白酶/TIMP轴，导致内皮和心肌细胞之间的纤维化。硫化氢治疗减轻纤维化，减轻E-M解偶联。因此，该方案的具体目的是：1.确定慢性左心室容量超负荷是否通过诱导NADPH氧化酶(P47亚单位)、mtNOS和PAR-1而导致线粒体氧化应激(ROS和RNS)，以及H_2S是否减轻线粒体氧化应激。#2：为了确定慢性左心室容量超负荷是否通过增加胶原/弹性蛋白比率，增加基质金属蛋白酶-2，-9，-13，组织金属蛋白酶-1，-3，降低组织金属蛋白酶-4，并诱导PAR-1，以及H_2S减轻心肌纤维化，确定慢性左室容量超负荷是否通过增加胶原/弹性蛋白比率而导致心肌纤维化。#3：确定慢性LV容量超负荷是否通过诱导PAR-1和H_2S减少E-M解偶联而导致E-M功能障碍和LVH。在野生型(WT)、PAR-1-/+、iNOS-/-、MMP9-/-、TIMP-3-/-和TIMP-4+/++小鼠中，通过主动脉-下腔静脉瘘(AVF)造成的左室容量超负荷将导致慢性心力衰竭，无论是否接受硫化氢供体NaHS的治疗。公共卫生相关性：只有当肌细胞通过细胞外基质(ECM)连接时，心肌细胞的收缩和松弛才是同步的。我们创造了内皮-心肌细胞(E-M)偶联这一术语，因为心内膜内皮细胞(EE)衍生的心脏活性物质调节收缩和舒张期的心肌细胞收缩。心脏内皮细胞是研究最少的系统，因为与管道动脉不同，心内膜内皮细胞埋藏在肌肉中，很难分离它在心肌细胞功能中的作用。我们开发了一种新技术，“心环”，在这种技术中，可以分别检测左室和右室的内皮依赖性或非内皮依赖性的心脏收缩和松弛。这项提议有两个新的方面：1)拟议的实验将证明PAR-1在心力衰竭纤维化和内皮-肌细胞解偶联的潜在机制中的作用；2)硫化氢在治疗舒张性心力衰竭方面的临床翻译分支。

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.