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.

位于肌肉细胞周围的肌原纤维通过服装，肌肉 - 特定的整联蛋白依从性复合物（IACS），将肌肉收缩的力传递到外部 细胞。 IACS由跨膜蛋白整合素和数百种蛋白质组成 ECM，尤其是细胞内。虽然对IAC和机制的组成知之甚少 启动他们的组装，我们不知道如何确定IAC的组成以及什么决定 IAC会形成的地方。我们利用肌肉结构/功能的保护以及遗传学的力量 秀丽隐杆线。从遗传筛选中，我们确定了一个基因PIX-1，这是IAC在 肌肉细胞边界（MCB），但没有其他IAC（M线和密集体）。 pix-1是线虫 人类B-Pix的直系同源物，其中包含一个已知是Rac1和Cdc42激活剂的Rhogef域。 pix- 1个突变体显示整个动物运动减少。与野生型相比，pix-1 null突变体显示 肌肉中激活（GTP结合）RAC的水平降低了50％。除了pix-1的不足之外， 野生型Pix-1蛋白的过表达导致运动减少和MCB中断。 PIX-1本地化 到肌肉中的所有3个IAC-M线，密集的身体和MCB。每个已知像素中功能突变体的损失 途径蛋白（PIX，RAC，GIT）导致MCB的破坏。 PAK-1的蛋白激酶活性对于 它的MCB函数：激酶死亡的PAK-1突变体破坏了MCB。尽管已知Pix蛋白是 在哺乳动物和线虫中的几种细胞类型中很重要，我们的结果是第一个证明PIX的结果 蛋白质是IAC组装所必需的，并且需要在条纹的肌肉中进行蛋白质。间隙蛋白（灭活RAC） 对于这种途径尚未报告任何细胞类型或生物体，但我们现在有两个候选差距。 值得注意的是，我们发现小鼠直系同源B-Pix的心脏特异性敲除导致扩张 8个月大的心肌病。同样，果蝇直系同源物的心脏特异性敲低也 导致心肌病。我们假设Pix蛋白通过其RACGEF活动直接组装 通过激活pak激酶以磷酸化的肌肉中磷酸化的键底物来进行IACS。我们将利用独特的 3个模型系统的优点，用于学习Strated和Cardiac中PIX途径的新配置功能 肌肉。目标1：利用在秀丽隐杆线虫中研究肌肉的力量，我们将：（a）识别GAP蛋白； （b）确定PAK激酶的定位，功能和底物，（c）检验Pix-1是 仅在MCBS需要，因为其他IAC位置还有其他RACGEF蛋白。目标2：我们 将确定B-Pix在小鼠心脏中的功能以及其缺失如何导致心肌病。目标3： 我们将利用果蝇中遗传修饰的筛查的力量来确定新成员 肌肉，更具体地说是心脏肌肉的PIX途径。有必要确定其他 人类的心肌病基因和编码PIX途径成员的基因可能是新的候选者。