Beta-1 Integrin and Caveolin-3 in Cardiac Mechanotransduction

描述(由申请人提供)： 导致心力衰竭的许多疾病的一个共性是心肌细胞对正常机械感觉的紊乱。尽管进行了广泛的研究，但心肌细胞感知机械变化、将这些变化传递到细胞并改变心肌细胞功能的确切机制尚不清楚。此外，心脏是如何在机械挑战的情况下保持代偿状态的，以及是什么改变了这种状态并使心肌向衰竭过渡，目前尚不清楚。心肌细胞的两个区域似乎对正确的机械转导至关重要，这两个区域是腔隙和腔隙。这些区域的关键蛋白质是整合素和小凹蛋白。整合素是细胞表面黏附受体的大家族，也是几乎所有细胞中的信号分子。小窝是包括心肌细胞在内的细胞质膜上的烧瓶状凹陷。这些结构与许多细胞功能有关，包括内吞作用、信号微域的建立和脂类代谢。已发现小窝蛋白的表达对小窝的形成是必不可少的。重要的是，这两个家族成员的突变都与骨骼和心脏肌病有关。我们的初步数据显示，免疫沉淀实验表明，b1和Caveolin-3在成年心肌细胞中共定位，并且这些蛋白相互作用。重要的是，我们制造了可以用于功能丧失和功能获得的小鼠，以评估这些蛋白质在心肌细胞和整个心脏中的作用。其中包括b1心脏特异性基因敲除(B1cKO)和Caveolin-3KO，以及各种整合素亚基和Caveolin-3以心肌细胞特异性方式增加表达的转基因小鼠。使用这些小鼠模型，我们还表明，在Caveolin-3KO中，b1整合素的胞间定位丢失，其在细胞亚组分中的分布发生了变化。同样，在b1cKO中，Caveolin-3的亚细胞排列也发生了变化。此外，心肌细胞中b1整合素和小窝蛋白-3的丢失最终会导致心力衰竭。相反，我们已经开始证明，在心肌细胞中增加这些蛋白的表达可以起到保护作用。基于这些数据，这一提议的总体假设是：B1整合素和小窝蛋白-3在心肌细胞中具有作为机械转导和膜稳定蛋白的协同性质。由此得出的推论是，这些蛋白质一起在心脏适应机械负荷的过程中发挥重要作用，并可能改变心脏从代偿状态向心力衰竭转变的方式。为此，我们提出了3个目标：1)通过单独或联合应用Caveolin-3缺失和b1整合素基因敲除小鼠，确定b1整合素和小窝蛋白-3在心肌细胞机械传递反应中的协同作用。2)明确指导心肌细胞β-1整合素和小窝蛋白-3协同机械信号传导的相互作用。3)利用小鼠模型，确定小窝蛋白-3和/或AB1整合素的单一或双重过表达如何改变机械反应，以及如何延迟或保护面对肥厚诱导的心力衰竭。临床意义：在大量VA患者中发现各种原因的心力衰竭，需要经常住院和注意门诊护理。确定心脏功能不全的根本原因，以及重要的是，可能导致这种常见疾病的新疗法的研究，是必不可少的，也将是本提案的重点。

DESCRIPTION (provided by applicant): A commonality of many of the diseases which lead to heart failure is a derangement in normal mechanosensing by the cardiac muscle cell. Despite extensive investigation, the precise mechanisms by which the cardiomyocyte senses mechanical changes, transmits these changes into the cell, and alters myocyte function, is poorly understood. Further, how the heart is maintained in a compensated state despite mechanical challenges, and what alters this state and transitions the myocardium towards failure, is not known. Two areas of the cardiomyocyte that appear critical for proper mechanotransduction are the costamere and caveolae. Key proteins of these areas are the integrins and caveolins. Integrins are a large family of cell- surface adhesive receptors that are also signaling molecules in virtually all cells. Caveolae are flask-shaped invaginations in the plasma membrane of cells, including cardiac myocytes. These structures have been linked to numerous cellular functions including endocytosis, establishment of a signaling microdomain and lipid metabolism. Caveolin protein expression has been found essential for caveolae formation. Importantly, mutations in members of both these families have been linked to skeletal and cardiac myopathies. Our preliminary data show that b1 and Caveolin-3 co-localize in the adult myocyte and that these proteins interact with each, as shown by immunoprecipitation experiments. Importantly, we have produced mice which can be used for loss and gain of function approaches to assess the role of these proteins in the myocyte, and whole heart. These include b1 cardiac-specific knockout (KO) (b1cKO) and Caveolin-3KO, as well as transgenic mice in which various integrin subunits and Caveolin-3 expression are increased in a cardiac myocyte specific manner. Using these mouse models we have also shown that in the Caveolin-3KO, costameric localization of b1 integrin is lost and that its distribution in cellular sub-fractions is altered. Likewise in the b1cKO, the sub-cellular arrangement of Caveolin-3 is changed. Further, loss of b1 integrin as well as Caveolin-3 from the cardiac myocyte can ultimately lead to heart failure. In contrast, we have begun to show that increased expression of these proteins in myocytes can protect. Based on this data, the overall hypothesis for this proposal is that: b1 integrins and caveolin-3 have cooperative properties as mechanotransducers and membrane stabilizing proteins in the cardiac myocyte. A corollary that follows from this is that together, these proteins will have important roles in the process of cardiac adaptations to mechanical load and may modify how the heart transitions from a compensated state towards heart failure. To investigate this, 3 aims are proposed: 1) Determine the cooperative roles of b1 integrins and caveolin-3 in mechanotransductive responses of the myocardium by use of Caveolin-3 null and b1 integrin cardiac myocyte specific knockout mice alone or in combination. 2) Define the interactions which direct cooperative mechanical signaling of cardiomyocyte beta-1 integrin and caveolin-3. 3) Determine how single or dual overexpression of caveolin-3 and/ or ab1 integrins alters mechanical responses, and delays or protects from cardiac failure in the face of hypertrophic induction using mouse models. Clinical Significance: Heart failure of varied causes is found in a large number of VA patients, necessitating frequent hospitalizations and attention to outpatient care. Identification of root causes of cardiac dysfunction and importantly, studies which could lead to novel therapeutics of this common disease, are essential, and will be the focus of this proposal.