A Model Multi-systems Approach for Understanding the Role of the PIX Pathway in Cardiac Muscle and Cardiomyopathy

理解 PIX 通路在心肌和心肌病中作用的模型多系统方法

• 批准号: 10532707
• 负责人: GUY Martin BENIAN
• 金额: $ 57.56万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10532707
• 关键词: Adhesions; Age Months; Animals; Automobile Driving; Biological Models; Caenorhabditis elegans; Cardiomyopathies; Cell membrane; Cell-Matrix Junction; Cells; Classification; Complex; DH Domain; Defect; Dilated Cardiomyopathy; Drosophila genus; Dystrophin; Extracellular Matrix; Focal Adhesions; GTP Binding; GTPase-Activating Proteins; Genes; Genetic; Genetic Screening; Glycoproteins; Heart; Hereditary Disease; Human; Inherited; Integral Membrane Protein; Integrins; Knockout Mice; Learning; Location; Locomotion; Mammals; Mitochondria; Modeling; Molecular; Mus; Muscle; Muscle Cells; Muscle Contraction; Muscular dystrophy cardiomyopathy; Mutation; Myocardium; Myofibrils; Nature; Nematoda; Organism; Orthologous Gene; PAK-1 kinase; PIX protein; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Population; Prevalence; Protein Kinase; Proteins; Publishing; Reporting; Role; Sarcolemma; Sarcomeres; Skeletal Muscle; Striated Muscles; System; Testing; Ventricular; analog; autosome; cell type; congenital myopathy; fly; human disease; improved; insight; knock-down; loss of function; member; mortality; muscular structure; mutant; overexpression; protein complex; scaffold; transmission process

Myofibrils located at the periphery of the muscle cell are attached to the sarcolemma via costameres, muscle- specific integrin adhesion complexes (IACs), that transmit the force of muscle contraction to the outside of the cell. IACs consist of the transmembrane protein integrin and hundreds of proteins in a complex both in the ECM and especially intracellularly. Although much is known about the composition of IACs and mechanisms that initiate their assembly, we do not know how the composition of an IAC is determined, and what determines where an IAC will form. We exploit the conservation of muscle structure/function and the power of genetics in C. elegans. From a genetic screen, we identified a gene, pix-1, which is required for the formation of IACs at muscle cell boundaries (MCBs), but not the other IACs (M-lines and dense bodies). PIX-1 is the nematode ortholog of human b-PIX, which contains a RhoGEF domain known to be an activator of Rac1 and Cdc42. pix- 1 mutants show decreased whole animal locomotion. As compared to wild type, a pix-1 null mutant, shows 50% reduction in the level of activated (GTP bound) Rac in muscle. In addition to deficiency of PIX-1, overexpression of wild type PIX-1 protein results in decreased locomotion and disrupted MCBs. PIX-1 localizes to all 3 IACs in muscle—M-lines, dense bodies and MCBs. Loss of function mutants in each of the known PIX pathway proteins (PIX, Rac, GIT) result in disrupted MCBs. The protein kinase activity of PAK-1 is essential for its MCB function: a kinase-dead PAK-1 mutant has disrupted MCBs. Although PIX proteins are known to be important in several cell types in mammals and nematodes, our results are the first to demonstrate that a PIX protein is required for assembly of an IAC, and is required in striated muscle. A GAP protein (inactivates Rac) for this pathway has not been reported for any cell type or organism, but we now have two candidate GAPs. Remarkably, we found that heart specific knockout of the mouse ortholog b-PIX results in dilated cardiomyopathy at 8 months of age. Similarly, heart-specific knockdown of the Drosophila ortholog, dPix, also results in cardiomyopathy. We hypothesize that PIX proteins, through their RacGEF activity, direct assembly of IACs by activating PAK kinases to phosphorylate key substrate(s) in muscle. We will leverage the unique advantages of 3 model systems to learn new conserved functions of the PIX pathway in striated and cardiac muscle. Aim 1: Exploiting the power of studying muscle in C. elegans we will: (a) identify the GAP protein(s); (b) determine the localization, function and substrates of PAK kinases, and (c) test the hypothesis that PIX-1 is only required at MCBs because there are additional RacGEF proteins at the other IAC locations. Aim 2: We will determine how b-PIX functions in the mouse heart and how its absence leads to cardiomyopathy. Aim 3: We will take advantage of the power of genetic modifier screens in Drosophila to identify new members of the PIX pathway in muscle, and more specifically, cardiac muscle. There is a need to identify additional cardiomyopathy genes in humans and genes encoding members of the PIX pathway may be new candidates.

位于肌细胞外围的肌原纤维通过肋节、肌纤维附着在肌膜上。 特定的整合素粘附复合物（IAC），将肌肉收缩的力量传递到外部 细胞。 IAC 由跨膜蛋白整合素和数百种蛋白质组成，位于复合体中 ECM，尤其是细胞内。尽管人们对 IAC 的组成和机制了解很多 我们不知道 IAC 的组成是如何确定的，以及是什么决定了 IAC 将在此成立。我们利用肌肉结构/功能的保守性和遗传学的力量 C.线虫。通过基因筛选，我们鉴定出了一个基因 pix-1，它是 IAC 形成所必需的。 肌细胞边界 (MCB)，但不是其他 IAC（M 线和致密体）。 PIX-1是线虫 人类 b-PIX 的直系同源物，包含已知为 Rac1 和 Cdc42 激活剂的 RhoGEF 结构域。像素- 1突变体显示整个动物运动能力下降。与野生型相比，pix-1 无效突变体显示 肌肉中活化的（GTP 结合）Rac 水平降低 50%。除了PIX-1缺乏之外， 野生型 PIX-1 蛋白的过度表达会导致运动减少和 MCB 破坏。 PIX-1 本地化 肌肉中的所有 3 个 IAC——M 线、致密体和 MCB。每个已知 PIX 中的功能丧失突变体 途径蛋白（PIX、Rac、GIT）会导致 MCB 破坏。 PAK-1 的蛋白激酶活性对于 它的 MCB 功能：激酶死亡的 PAK-1 突变体破坏了 MCB。尽管已知 PIX 蛋白 PIX 在哺乳动物和线虫的多种细胞类型中很重要，我们的结果首次证明 PIX 蛋白质是 IAC 组装所必需的，也是横纹肌所必需的。 GAP 蛋白（使 Rac 失活） 因为尚未报道任何细胞类型或生物体的该途径，但我们现在有两个候选 GAP。 值得注意的是，我们发现小鼠直系同源物 b-PIX 的心脏特异性敲除会导致扩张 8个月大时患心肌病。同样，果蝇直系同源物 dPix 的心脏特异性敲低也 结果导致心肌病。我们假设 PIX 蛋白通过其 RacGEF 活性直接组装 通过激活 PAK 激酶磷酸化肌肉中的关键底物来控制 IAC。我们将利用独特的 3个模型系统的优势来学习纹状体和心脏中PIX通路的新保守功能 肌肉。目标 1：利用研究秀丽隐杆线虫肌肉的能力，我们将： (a) 识别 GAP 蛋白； (b) 确定 PAK 激酶的定位、功能和底物，以及 (c) 检验 PIX-1 的假设 仅 MCB 需要，因为其他 IAC 位置还有额外的 RacGEF 蛋白。目标2：我们 将确定 b-PIX 在小鼠心脏中的功能以及它的缺失如何导致心肌病。目标 3： 我们将利用果蝇基因修饰筛选的力量来识别果蝇的新成员 肌肉中的 PIX 通路，更具体地说是心肌中的 PIX 通路。需要确定额外的 人类心肌病基因和编码 PIX 通路成员的基因可能是新的候选者。