Myocardial Physiology of Growth Differentiation Factor Signaling

生长分化因子信号传导的心肌生理学

基本信息

批准号：
10711086
负责人：
RICHARD T LEE
金额：
$ 60.85万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-01 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10711086
关键词：
Address Affect Age Aging American Amino Acid Sequence Amino Acids Biochemical Biochemistry Biological Markers Blood CRISPR/Cas technology Cardiac Myocytes Cardiovascular Diseases Cardiovascular system Cessation of life Clinical Data Collaborations Coronary Coronary heart disease Data Differentiation and Growth Disease Event Future GDF11 gene GDF8 gene Genetically Engineered Mouse Growth Healthcare Heart Heart Diseases Heart Hypertrophy Hospitals Human Human Biology In Vitro Laboratories Ligands MADH2 gene Maintenance Mass Spectrum Analysis Measurement Measures Molecular Mus Muscle Development Muscle function Myocardial Myocardium Outcome Pathway interactions Patients Phenotype Physiological Physiology Proteins Publishing Receptor Signaling Role Signal Pathway Signal Transduction Skeletal Development Structural Biochemistry Structure System Systems Development Techniques Testing Transforming Growth Factor beta Translating Universities Woman X-Ray Crystallography cell growth cohort experimental study extracellular functional outcomes genomic locus heart dimension/size heart function human data human disease in vivo insight interest laboratory experiment loss of function mutation member morphogens mortality outcome prediction pressure prospective

项目摘要

SUMMARY: Myocardial Physiology of Growth Differentiation Factor Signaling GDF11 and the closely related protein GDF8 (also known as myostatin) can regulate cardiac hypertrophy. We now have new prospective data in a large cohort of coronary heart disease patients showing that low blood levels of subforms of GDF8 and GDF11 powerfully predict future all-cause mortality. These new data point to specific forms of GDF11 and GDF8 as critical factors in heart disease. Furthermore, human loss- of-function mutations in GDF11 have now been identified that cause multi-system disease, including cardiovascular disease, showing the importance of GDF11 in human biology. 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 protein sequence identity (90% identical) within their mature signaling domains. We recently collaborated with multiple other laboratories to determine that mature GDF11 is a significantly more potent activator of SMAD2/3 dependent signaling than GDF8 in vitro, likely due to better utilization of key signaling receptors. Moreover, through x-ray crystallography-guided biochemical experiments, we identified key amino acids of the two ligands responsible for their differences in potency. These findings support the concept that GDF11 and GDF8 are likely not functionally equivalent, especially when ligand concentrations are low, as exist in vivo. However, it is not yet understood if differences in GDF11 and GDF8 at the molecular level translate to distinct functional outcomes and pathway activation in vivo. Defining the roles of these ligands in vivo can best be addressed by genetically engineered mice. Using CRISPR technology, we have now generated three new lines of mice with specific changes guided by our structural and biochemical studies on GDF11 vs. GDF8 to address this Project’s three Aims. This project will uncover the biochemistry of these ligands in vivo while retaining regulatory structure of the endogenous genomic loci. Importantly, we have already used Targeted Locus Amplification to prove that we have edited only the intended amino acids in all three of the new lines of mice. Using these newly generated mice, we will pursue the following Aims: Aim 1. To test the hypothesis that introducing the mature domain of GDF11 into the myostatin (GDF8) locus regulates cardiac size and function using Gdf8Gdf11swap mice. Aim 2. To test the hypothesis that gain of potency in GDF8 with two specific amino acids from GDF11 regulates cardiac muscle growth in mice (Gdf8G89D/E91Q mice). Aim 3. To test the hypothesis that GDF11 potency is required to maintain cardiac muscle function in vivo under pressure overload using chimeric mice with specific amino acids from GDF8 introduced into mature GDF11 (Gdf11D89G/Q91E mice).

摘要：生长分化因子信号传导的心肌生理学 GDF11和密切相关的蛋白质GDF8（也称为肌生长抑制素）可以调节心脏我们现在有大量冠心病患者的新前瞻性数据显示。 GDF8 和 GDF11 亚型的低血液水平有力地预测了未来的全因死亡率。数据表明，特定形式的 GDF11 和 GDF8 是导致心脏病的关键因素。现已发现 GDF11 的功能性突变会导致多系统疾病，包括心血管疾病，显示了 GDF11 在人类生物学中的重要性 GDF11 和 GDF8 都是成员。细胞外配体转化生长因子 β (TGFβ) 超家族的成员，被认为由于其成熟信号传导中的蛋白质序列同一性（90% 相同），因此发挥相似或冗余的作用我们最近与多个其他实验室合作，确定成熟的 GDF11 是一个在体外，SMAD2/3 依赖性信号传导的激活剂明显比 GDF8 更有效，这可能是由于更好的此外，通过X射线晶体学引导的生化实验，我们确定了两种配体的关键氨基酸，这些氨基酸导致了它们的效力差异。支持 GDF11 和 GDF8 可能在功能上不等价的概念，特别是当配体浓度较低，与体内存在的情况一样。然而，尚不清楚 GDF11 和 GDF11 是否存在差异。 GDF8 在分子水平上转化为不同的功能结果和体内通路激活。定义这些配体在体内的作用最好通过使用基因工程小鼠来解决。 CRISPR 技术，我们现在已经培育出了三个新的小鼠品系，它们在我们的指导下发生了特定的变化对 GDF11 与 GDF8 进行结构和生化研究，以实现该项目的三个目标。揭示这些配体在体内的生物化学，同时保留内源性的调节结构重要的是，我们已经使用靶向基因座扩增来证明我们已经编辑过。我们将使用这些新产生的小鼠，仅使用所有三个新小鼠系中的氨基酸。追求以下目标：目的 1. 检验将 GDF11 的成熟结构域引入肌肉生长抑制素 (GDF8) 的假设使用 Gdf8Gdf11swap 小鼠调节心脏大小和功能。目标 2. 检验 GDF8 与 GDF11 中的两种特定氨基酸的效力增强这一假设调节小鼠（Gdf8G89D/E91Q 小鼠）的心肌生长。目标 3. 检验以下假设：GDF11 效力是维持心肌功能所必需的使用将来自 GDF8 的特定氨基酸引入成熟体内的嵌合小鼠，在压力超负荷的体内 GDF11（Gdf11D89G/Q91E 小鼠）。