In vivo Structure-Function relationships of GDF11 and GDF8

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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.