Dynamic limits on contraction of gastrointestinal smooth muscle

描述(申请人提供)：平滑肌行为的一个特点是它能够通过重塑适应功能需求的变化，如糖尿病和胃肠道梗阻性疾病，如巨结肠的先天性巨结肠，这是本项目的主要重点。重塑可能包括平滑肌的生长(肥大、增生)以及或不伴随细胞外基质的改变。平滑肌缩短取决于(A)外加载荷和(B)细胞外结缔组织基质提供的内部阻力，该基质将平滑肌细胞连接在一起，并与正在施力的相邻细胞相连。这两个因素也控制着长度-力关系和缩短范围，它们可能会因重塑而发生不同的变化。更复杂的是，最近的研究发现，刺激启动了细胞皮质中肌动蛋白细丝的长度依赖聚合，表面上增强了细胞骨架和增强力，预计这将提供内部负荷并阻止缩短。严格分析细胞骨架重塑对完整肌肉力学性能的影响是有必要的。我们对G.I.的结构研究。平滑肌肉提出了我们的中心假设，即在完整的肌肉中，由于细胞外基质松弛、收缩细丝错位以及可能形成僵硬的细胞骨架而导致的交叉桥事件的传递失败，所以整体的力量输出和缩短是有限的。迫切需要一种关于结构极限如何在平滑肌肉中发挥作用的综合观点。我们将研究(1)致死性斑点巨结肠小鼠模型的结肠重塑，(A)巨结肠的致死性斑点小鼠模型，它显示短肌肉的力量产生增强和顺应性增加，有利于广泛缩短；(B)糖尿病大鼠模型，它显示静息时僵硬，短肌肉长度的力量产生增强，缩短的能力有限；以及(2)两个正常的胃肠平滑肌，它们代表了长度、力量产生和缩短能力之间的极端关系：兔带绦虫结肠和大鼠肛尾肌。我们的长期目标是确定其机制，生理和结构，以及缩短能力。限制力输出和平滑肌肉的缩短，从而确定疾病重塑后可能的治疗干预对象。具体目的1是确定肌动蛋白-肌球蛋白相互作用的程度和机械事件通过细胞内和细胞外基质传递的程度，以及伴随着结构取向的变化，如何作为肌肉长度的函数来管理平滑肌力输出。具体目标2是确定控制细胞骨架肌动蛋白聚合和解聚的力学因素，以及这些动态转变如何限制完整肌肉中的力生产、缩短和功生产。具体目标3是定义和量化细胞外基质的组成，它解释了在糖尿病结肠和先天性巨结肠重塑后发生的静息顺应性的剧烈和不同的变化。

DESCRIPTION (provided by applicant): A hallmark of smooth muscle behavior is its ability to adapt to changes in functional demand by remodeling, as in diabetes and in obstructive disorders of the gastrointestinal tract such as megacolon of Hirschsprung's disease, which are a major focus of this project. Remodeling may include smooth muscle growth (hypertrophy, hyperplasia) together with or without concomitant changes in extracellular matrix. Smooth muscle shortening depends on (a) external applied loads and (b) internal resistances provided by the extracellular connective tissue matrix which links smooth muscle cells together and to neighboring cells that are exerting force. Both factors also govern the length-force relationships and shortening range, which may be differentially changed as a result of remodeling. Complicating this further is the recent finding that stimulation initiates a length- dependent polymerization of actin filaments in the cellular cortex, ostensibly strengthening the cytoskeleton and enhancing force, which would be expected to provide an internal load and impede shortening. A rigorous analysis of the effects of the cytoskeletal remodeling on the mechanical properties of intact muscle is warranted. Our structural studies on g.i. smooth muscles prompted our central hypothesis that in intact muscles, overall force output and shortening are limited due to the failure of transmission of events occurring at the crossbridge, resulting from floppiness of the extracellular matrix, contractile filament misalignment and possibly also formation of a stiff cytoskeleton. There is a critical need for an integrated view on how structure limits function in smooth muscle. We will study (1) remodeling in the colon of (a) the lethal spotted murine model of Hirschsprung's megacolon which shows enhanced force production at short muscle lengths and increased compliance, favoring extensive shortening, (b) a rat model of diabetes (streptozotocin), which shows stiffening at rest, enhanced force production at short muscle lengths, and a limited capacity to shorten, and (2) two normal gastrointestinal smooth muscles representing the extremes in their relationships between length and force production and ability to shorten: the rabbit taenia coli and rat anococcygeus m. Our long term goal is to define the mechanisms, physiological and structural, limiting force output and shortening in smooth muscles and, thereby, identify likely candidates for therapeutic intervention following remodeling in disease. Specific Aim 1 is to determine how the extent of actin-myosin interaction and transmission of mechanical events through intra- and extracellular matrices, concomitantly with changes in structural orientation, govern force output of smooth muscle as a function of muscle length. Specific Aim 2 is to determine the mechanical factors that control the polymerization and depolymerization of cytoskeletal actin, and how these dynamic transitions limit force production, shortening and work production in intact muscles. Specific Aim 3 is to define and quantify the composition of the extracellular matrix that account for the drastic and disparate changes in resting compliance that occur following remodeling in the diabetic colon and in the Hirschsprung's megacolon.