Myocardial Physiology of Growth Differentiation Factor Signaling

生长分化因子信号传导的心肌生理学研究进展 GDF11和密切相关的蛋白质GDF8(也称为肌肉生长抑制素)可以调节心脏 肥大。我们现在有了新的前瞻性数据，在一大群冠心病患者中显示 血液中GDF8和GDF11亚型的低水平可以很好地预测未来的全因死亡率。这些新的 数据表明，特定形式的GDF11和GDF8是心脏病的关键因素。此外，人的损失- 现在已经确定GDF11的功能突变会导致多系统疾病，包括 心血管疾病，表明GDF11在人类生物学中的重要性。GDF11和GDF8是成员 转化生长因子β(转化生长因子β)超家族的细胞外配体，最初被认为是 由于成熟信号中的蛋白质序列相同(90%相同)，因此起到相似或冗余的作用 域名。我们最近与多个其他实验室合作，确定成熟的GDF11是一种 在体外，Smad2/3依赖信号的激活剂明显比GDF8更有效，可能是因为更好的 关键信号受体的利用。此外，通过X射线结晶学指导的生化实验， 我们确定了两个配体的关键氨基酸，这些氨基酸是导致它们效力差异的原因。这些发现 支持GDF11和GDF8可能在功能上不相等的概念，特别是当配体 浓度很低，就像在体内存在的那样。然而，目前还不清楚GDF11和GDF11的差异是否 在体内，GDF8在分子水平上翻译成不同的功能结果和途径激活。 通过基因工程小鼠来确定这些配体在体内的作用是最好的。vbl.使用 CRISPR技术，我们现在已经产生了三个新的小鼠品系，它们在我们的 GDF11和GDF8的结构和生化研究，以满足该项目的三个目标。这个项目将 在体内揭示这些配体的生物化学，同时保留内源性 基因组基因座。重要的是，我们已经使用了目标基因扩增来证明我们已经编辑了 只有在所有三个新品系的小鼠中都有预期的氨基酸。利用这些新产生的小鼠，我们将 追求以下目标： 目的1.验证将GDF11成熟结构域引入肌肉生长抑制素(GDF8)的假说。 利用Gdf8Gdf11交换小鼠，Locus调节心脏的大小和功能。 目的2.验证GDF11的两个特定氨基酸对GDF8细胞效力增强的假设 调节小鼠(Gdf8G89D/E91Q小鼠)的心肌生长。 目的3.验证GDF11活性是维持心肌功能所必需的假设。 携带GDF8特定氨基酸的嵌合小鼠在压力超负荷下进入成熟体内 GDF11(Gdf11D89G/Q91E小鼠)。

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).