Physiology of Respiratory Muscle Micromechanics

描述(申请人提供)：肌营养不良的发病机制是由于细胞骨架和细胞外基质之间的机械和/或信号连接断裂。在这笔赠款的第一个资助期，我们专注于确定特定模型的肌营养不良症骨骼肌的机械、结构和功能完整性的变化。在这个项目即将到来的阶段，我们的目标是了解在特定的肌营养不良小鼠模型的呼吸泵中可能涉及的促炎信号通路异常激活的机制。我们将使用两种互补的肌营养不良小鼠模型，MDX和Alpha？整合素空鼠标。我们的具体目标是：特定目标1：评估Rho家族的小GTP酶蛋白，如RhoA、Rac1和CDC42在正常骨骼肌机械牵张诱导的信号转导中的作用。假设1：小的GTP酶蛋白RhoA、Rac1和CDC42参与了横隔膜的机械信号转导；这些蛋白中的每一个都有不同的信号作用，这可能取决于所施加的机械应力的方向。每种蛋白都参与启动特定的信号转导途径，影响骨骼肌细胞的增殖、分化和基因表达。特定目的#2；检测在缺乏dystrophin或A7整合素的肌肉纤维中，机械拉伸诱导的机械敏感转录因子如AP-1、C/EBP、NFKB的激活和Rho家族GTP酶的活性是否发生改变假设2：机械拉伸导致营养不良小鼠骨骼肌MAP激酶和机械敏感转录因子的异常激活，可能是通过对Rho家族GTP结合蛋白的异常调节。具体目的#3：检测Dystrophin或A7整合素的缺失是否会导致机械敏感转导通路的激活，从而导致肌肉损耗细胞因子(如TNF-α、IL-1p)和蛋白酶(如基质金属蛋白酶MMP9)的表达改变。假设3：机械应激诱导MDX和alpha7整合素缺失小鼠骨骼肌中核因子-KB和AP-1的激活可能导致肌肉损耗细胞因子和组织降解酶水平的增加。这些促炎分子的高度激活可能会导致这些营养不良小鼠结构完整性的丧失和收缩功能障碍。具体目的#4；确定机械敏感转录因子NF-KB、C/EBP和AP-1在MDX和A7整合素缺失小鼠肌肉变性发病机制中的作用。假设4：抑制核因子-KB和/或AP-1将减少炎症基因如肿瘤坏死因子-α、白介素6、白介素1B和基质金属蛋白酶的表达，因此可能减少MDX和α7整合素缺失小鼠骨骼肌结构完整性的丧失和可能的功能获得。这些目标的完成将大大有助于从根本上理解呼吸泵中的机械信号转导。

DESCRIPTION (provided by applicant): Muscular dystrophy pathogenesis results from a broken mechanical and/or signaling linkage between the cytoskeleton and the extracellular matrix. In the first funding period of this grant we focused on determining the alteration in the mechanical, structural and functional integrity in skeletal muscles of specific models of muscular dystrophies. In the upcoming period of this project our objective is to understand the mechanisms involved with possible aberrant activation of pro-inflammatory signaling pathways in the respiratory pump of specific mouse models of muscular dystrophy. We will use the 2 complementary mouse models of muscular dystrophy, the mdx and alpha? integrin null mouse. Our specific aims are: Specific Aim # 1: To evaluate the role of the small GTPase proteins of Rho family such as RhoA, Racl and cdc42 in the mechanical stretch-induced signal transduction in normal skeletal muscles. Hypothesis 1: The small GTPase proteins RhoA, Racl and Cdc42 are involved in mechanical signal transduction in the diaphragm; each of these proteins has a distinct signaling role, which may depend on the direction of applied mechanical stress. Each of the proteins is involved in the initiation of a specific signal transduction pathway that affects the proliferation, differentiation and the gene expression in skeletal muscle cells. Specific Aim # 2; To examine whether mechanical stretch-induced activation of mechanosensitive transcription factors such as AP-1, C/EBP, NFKB and the activity of the Rho family GTPases are altered in muscle fibers lacking either dystrophin or a7 integrin Hypothesis 2: Mechanical stretching of skeletal muscles from dystrophic mice leads to aberrant activation of MAP kinases and mechanosensitive transcription factors, possibly through the anomalous regulation ofGTP binding proteins of Rho family. Specific Aim # 3: To test whether loss of either dystrophin or a7 integrin results in the activation of mechanosensitive transduction pathways leading to altered expression of muscle wasting cytokines such as TNF-a, IL-1p and proteases such as matrix metalloproteinase MMP9. Hypothesis 3: Mechanical stressinduced activation ofNF-KB and AP-1 in skeletal muscle of the mdx, and alpha7 integrin null mice may lead to an augmented level of muscle wasting cytokines and tissue degrading proteases. Higher activation of these proinflammatory molecules could lead to loss of structural integrity and contractile dysfunction in these dystrophic mice. Specific Aim # 4; To determine the role of mechanosensitive transcription factors NF-KB, C/EBP and AP-1 on the pathogenesis of muscular degeneration in mdx and a7 integrin null mice. Hypothesis 4: Inhibition of NF-KB and/or AP-1 will reduce the expression of inflammatory genes such as TNF-a, IL-6, IL-1B and MMPs, and therefore may reduce loss of structural integrity and cause possible gain in function in skeletal muscles of the mdx, and the alpha7 integrin null mice. The completion of these aims will significantly contribute to the fundamental understanding of mechanical signal transduction in the respiratory pump.