Dynamic limits on contraction of gastrointestinal smooth muscle

描述（由申请人提供）：平滑肌行为的一个标志是其通过重塑来适应功能需求变化的能力，如糖尿病和胃肠道阻塞性疾病，如巨结肠，这是本项目的主要重点。重塑可包括平滑肌生长（肥大、增生），伴有或不伴有细胞外基质的改变。平滑肌的缩短取决于(a)外部施加的载荷和(b)细胞外结缔组织基质提供的内阻，细胞外结缔组织基质将平滑肌细胞连接在一起，并与施加力的邻近细胞相连。这两个因素也决定了长度-力关系和缩短范围，这可能会因重塑而不同地改变。更复杂的是，最近的研究发现，刺激启动了细胞皮层中肌动蛋白丝的长度依赖性聚合，表面上增强了细胞骨架并增强了力，这将提供内部负载并阻碍缩短。对细胞骨架重塑对完整肌肉力学性能的影响进行严格的分析是必要的。我们对g.i.平滑肌的结构研究提示了我们的中心假设，即在完整的肌肉中，由于细胞外基质的松弛、可收缩丝的错位以及可能形成的僵硬的细胞骨架，在交叉桥上发生的事件传递失败，导致整体力输出和缩短受到限制。对于结构如何限制平滑肌的功能，迫切需要一个完整的观点。我们将研究：(1)结肠的重塑：(a)巨结肠的致死斑点小鼠模型，它显示出短肌肉长度时增强的力量产生和增强的顺应性，有利于广泛缩短；(b)糖尿病大鼠模型（链脲佐菌素），它显示静止时僵硬，短肌肉长度时增强的力量产生，以及有限的缩短能力。(2)两个正常的胃肠道平滑肌，代表它们在长度和力量产生和缩短能力之间的极端关系：兔大肠带绦虫和大鼠无尾肌。我们的长期目标是确定机制，生理和结构，限制平滑肌的力量输出和缩短，从而确定疾病重塑后治疗干预的可能候选人。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的传递，伴随着结构取向的变化，如何作为肌肉长度的函数控制平滑肌的力输出。具体目标2是确定控制细胞骨架肌动蛋白聚合和解聚的机械因素，以及这些动态转变如何限制完整肌肉的力产生、缩短和功产生。具体目标3是定义和量化细胞外基质的组成，细胞外基质解释了糖尿病结肠和巨结肠重塑后静息顺应性发生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.