Myocardial Physiology of Growth Differentiation Factor Signaling

生长分化因子信号传导的心肌生理学

• 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
• 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

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).

生长分化因子信号传导的心肌生理学 GDF 11和密切相关的蛋白质GDF 8（也称为肌肉生长抑制素）可以调节心脏 肥厚我们现在有一个大型冠心病患者队列的新的前瞻性数据， GDF 8和GDF 11亚型的低血液水平有力地预测了未来的全因死亡率。这些新 数据表明GDF 11和GDF 8的特定形式是心脏病的关键因素。此外，人类的损失- 现已确定GDF 11中的功能缺失突变可导致多系统疾病，包括 心血管疾病，显示了GDF 11在人类生物学中的重要性。GDF 11和GDF 8是成员 转化生长因子β（TGFβ）超家族的细胞外配体，最初被认为是 由于蛋白质序列同一性（90%相同），在其成熟信号传导中发挥相似或冗余的作用 域.我们最近与其他多个实验室合作，确定成熟的GDF 11是一种 在体外SMAD 2/3依赖性信号传导的激活剂比GDF 8显著更有效，可能是由于更好的 关键信号受体的利用。此外，通过X射线晶体学引导的生化实验， 我们鉴定了两种配体的关键氨基酸，这是它们效力不同的原因。这些发现 支持GDF 11和GDF 8可能在功能上不等同的概念，特别是当配体 浓度低，如体内存在的。然而，目前还不清楚GDF 11和 GDF 8在分子水平上转化为体内不同的功能结果和途径激活。 确定这些配体在体内的作用最好通过基因工程小鼠来解决。使用 CRISPR技术，我们现在已经产生了三种新的小鼠品系，这些小鼠在我们的指导下发生了特定的变化。 GDF 11与GDF 8的结构和生物化学研究，以解决该项目的三个目标。该项目将 揭示这些配体在体内的生物化学，同时保留内源性配体的调控结构， 基因座重要的是，我们已经使用靶向基因座扩增来证明我们已经编辑了 在所有三种新品系的小鼠中只含有预期的氨基酸。使用这些新产生的小鼠，我们将 追求以下目标： 目标1.为了验证将GDF 11的成熟结构域引入肌生成抑制素（GDF 8）中， 使用Gdf 8 Gdf 11 swap小鼠研究基因座调节心脏大小和功能。 目标2.为了检验GDF 8中具有来自GDF 11的两个特定氨基酸的效价增加的假设， 调节小鼠（Gdf 8 G89 D/E91 Q小鼠）的心肌生长。 目标3.为了检验GDF 11效力是维持心肌功能所必需的假设， 使用具有来自GDF 8的特异性氨基酸的嵌合小鼠在压力超负荷下体内引入成熟 GDF 11（Gdf 11 D89 G/Q91 E小鼠）。