In vivo Structure-Function relationships of GDF11 and GDF8

GDF 11和GDF 8是细胞外基质转化生长因子β（TGFβ）超家族的成员。 配体和最初被认为是服务相似或冗余的作用，由于高序列同一性（90%）， 相同的）在其成熟的信号传导结构域中。我们的实验和其他人的实验表明， GDF 11和GDF 8信号传导的变化可以调节心肌细胞的大小。最重要的是我们的 实验室的工作刺激了其他人将该系统作为心脏病患者预后的生物标志物进行研究。 疾病来自两个队列的新的长期临床数据，每个队列有近1000名患者，表明低血 与密切相关的蛋白质GDF 8（也称为肌肉生长抑制素）一起测量的GDF 11水平 有力地预测了心脏病患者随后8年的死亡率。更以 在患有心脏病的患者中，GDF 11和GDF 8的水平随着年龄的增长而下降。这 这条途径引起了人们的兴趣和争议，争议的主要来源之一是是否 成熟配体GDF 11和GDF 8在生物学上是相同的。通过结构和生物化学 实验，我们最近确定了两个配体的关键区域负责显着差异 在这些配体之间。然而，目前还不清楚GDF 11和GDF 8配体的差异是否与GDF 11和GDF 8配体之间的差异有关。 分子水平转化为体内不同的功能结果和途径激活。解决 在这场争论中，我们使用Crispr技术产生了新的小鼠，这些小鼠的结构变化是由 GDF 11和GDF 8的生物化学。我们提出的目标是假设驱动的，可以在这些新的 小鼠：目标1.为了验证将GDF 11的成熟结构域引入肌生长抑制素中 GDF 8基因座调节心脏大小和功能。使用CRISPR/Cas9系统，我们已经取代了 完整的成熟GDF 8结构域与成熟GDF 11结构域。使用这些小鼠，我们将表征心脏- 以及肌肉生长和对损伤的反应，以确定增强的效力是否 与GDF 8相比，GDF 11可以激活独特的信号通路来调节肌肉生长。目标二。测试 具有来自GDF 11的两个特定氨基酸的GDF 8中的效力的获得调节两者的假设 心脏和骨骼肌生长。我们将研究我们新产生的嵌合小鼠，以确定是否不同 表型差异出现在GDF 11残基交换到GDF 8成熟结构域的小鼠中。这些 实验将基于强结构生物化学数据来确定这些配体的生理差异。 目标3。为了检验GDF 11效力是正常胚胎发育所需的假设， 使用具有特定氨基酸的嵌合小鼠在体内维持心脏和骨骼肌功能 从GDF 8引入成熟的GDF 11。使用具有GDF 8氨基酸残基的嵌合小鼠 交换到GDF 11成熟结构域中，我们将确定GDF 11效力的要求， 发展到成年。

GDF11 and GDF8 are members of the transforming growth factor β (TGFβ) superfamily of extracellular ligands and were initially thought to serve similar or redundant roles due to high sequence identity (90% identical) within their mature signaling domains. Our experiments and the experiments of others suggest that changes in the GDF11 and GDF8 signaling may regulate cardiomyocyte size. Most importantly, our laboratory’s work stimulated others to study this system as a biomarker for outcome in humans with heart disease. New long-term clinical data from two cohorts, each with almost 1000 patients, indicate that low blood levels of GDF11 measured together with the closely-related protein GDF8 (also known as myostatin) powerfully predict subsequent mortality in patients with heart disease over the ensuing 8 years. Furthermore, in patients with heart disease, the levels of GDF11 and GDF8 measured together declined with age. This pathway has generated interest and controversy, and one of the major sources of controversy is whether the mature ligands GDF11 and GDF8 are biologically identical. Through structural and biochemical experiments, we recently identified key regions of the two ligands responsible for significant differences between these ligands. However, it is not yet understood if differences in the GDF11 and GDF8 ligands at the molecular level translate to distinct functional outcomes and pathway activation in vivo. To address this controversy, we generated new mice using Crispr technology with changes guided by the structural biochemistry of GDF11 and GDF8. Our proposed Aims are hypothesis-driven and can be tested in these new mice: Aim 1. To test the hypothesis that introducing the mature domain of GDF11 into the myostatin (GDF8) locus regulates cardiac size and function. Using the CRISPR/Cas9 system, we have replaced the entire mature GDF8 domain with the mature GDF11 domain. Using these mice, we will characterize cardiac— as well as skeletal—muscle growth and response to insult to determine whether the enhanced potency of GDF11 can activate unique signaling pathways compared to GDF8 regulating muscle growth. Aim 2. To test the hypothesis that gain of potency in GDF8 with two specific amino acids from GDF11 regulates both cardiac and skeletal muscle growth. We will study our newly generated chimeric mice to determine if distinct phenotypic differences arise in mice with GDF11 residues swapped into the GDF8 mature domain. These experiments will define physiological differences in these ligands based on strong structural biochemistry data. Aim 3. To test the hypothesis that GDF11 potency is required for normal embryonic development and to maintain cardiac and skeletal muscle function in vivo using chimeric mice with specific amino acids from GDF8 introduced into the mature GDF11. Using chimeric mice with GDF8 amino acid residues swapped into the GDF11 mature domain, we will determine the requirement of GDF11 potency during development and into adulthood.