Dynamic tissue-specific modulation of redox stress using chemogenetics

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This R33 research program proposes to characterize and validate novel transgenic mouse lines that exploit chemogenetics to create powerful models that will be used to identify new targets for the prevention and treatment of heart failure and aortic aneurysms. Most current animal models of heart failure and aortic aneurysm formation are limited by methodological complexities, and many models have limited relevance to human disease pathophysiology. Nearly all human heart failure and most arterial disease states are associated with high levels of reactive oxygen species (ROS) in affected tissues. We therefore developed a novel chemogenetic approach that dynamically modulates redox stress in vivo by exploiting a yeast D-amino acid oxidase (DAAO) that generates the ROS hydrogen peroxide (H2O2). DAAO is activated by D-amino acids, but not by the L-amino acids found in mammalian cells. We infected mice or rats with a recombinant cardiotropic adeno-associated virus isotype 9 (AAV9) that expresses DAAO. When animals are provided with D-amino acids, DAAO generates H2O2 in the heart, and the animals rapidly develop heart failure– which can be reversed by drug treatments. Although this approach is informative, a major limitation is the need to infect animals one at a time with the virus. Moreover, virus-based methods are constrained by the limited tissue selectivity of most viral vectors in vivo. Therefore, we generated new transgenic mouse lines designed to express DAAO in selected cardiovascular tissues. We have developed rigorous Performance Measures to quantitatively assess our progress in the proposed studies of these new transgenic lines. Aim 1 proposes experiments studying three independent transgenic founder lines expressing DAAO in the heart (driven by the MYH6 promoter) that will create a robust and cost-effective model to identify new pharmacological targets and new drugs to prevent and/or treat heart failure. In Aim 2, we propose to expand this approach to study redox stress in the vascular wall. We recently produced two new transgenic founder mouse lines containing DAAO in which a stop codon flanked by LoxP sites was cloned into the coding region of DAAO. By breeding these “floxed” DAAO transgenic lines with selected commercially-available lines expressing Cre recombinase under control of tissue-specific promoters, we will be able to dynamically regulate redox stress in different mouse tissues, permitting the study of the broad range of disease states in which redox stress is associated with pathogenesis. Here we will focus on DAAO expression in vascular endothelial cells (using VE-cadherin-Cre) and vascular smooth muscle cells (using MYH11-Cre) in order to identify the mechanisms whereby oxidative stress causes aortic aneurysms and to identify new drug targets to prevent or treat aneurysms. In both heart and aorta, we will perform state-of-the-art single cell RNA sequencing in order to track the changes in cell populations and transcriptional profiles that accompany development and regression of redox stress. The proposed studies will lead to the generation of powerful new model systems that will lead to the identification of novel targets and new drugs for the treatment and prevention of heart failure and aortic aneurysms.