This R21 application proposes experiments that will develop and validate new models of aging-related cardiac pathology by using chemogenetic approaches to generate redox stress in the heart and other tissues. The development of an ?age-related cardiomyopathy?? associated with both systolic and diastolic cardiac dysfunction, redox stress, and a high mortality rate? has been extensively documented. We have recently developed a new chemogenetic approach that leads to the development of cardiomyopathy that is directly and specifically caused by inducing an increase in redox stress in the heart. In the proposed studies, we will examine this new cardiomyopathy model in the context of aging. We used a cardiotropic adeno-associated virus serotype 9 (AAV9) to express a yeast D-amino acid oxidase (DAAO) that selectively generates ROS in cardiac myocytes in vivo, causing redox stress in the heart and leading to dilated cardiomyopathy. DAAO is a stereospecific enzyme that generates hydrogen peroxide (H2O2) only when D-amino acids are available as substrate. Since most mammalian tissues contain only L-amino acids, the yeast DAAO is inactive until D-amino acids are provided. We injected recombinant DAAO-AAV9 virus into mice (or rats), and used cellular imaging methods to show that H2O2 is robustly generated in cardiac myocytes only after adding D-alanine to myocytes. We then developed an in vivo chemogenetic model to generate chronic redox stress in the heart: we added D-alanine to the drinking water of mice that had been infected with recombinant DAAO-AAV9. Compared to control animals, the DAAO-AAV9-infected mice develop a dilated cardiomyopathy within 3 weeks of D-alanine treatment. We propose to extend these approaches to study the roles of redox stress in age-associated cardiomyopathy by infecting middle-aged and elderly mice with recombinant DAAO-AAV9, and then analyzing physiological, metabolic, biochemical, transcriptomic, and proteomic characteristics using both in vivo and ex vivo approaches. These experiments are likely to identify new therapeutic targets to prevent or treat the age-associated cardiomyopathy. We also propose to develop DAAO-TGLoxP transgenic mouse lines that will permit analyses of tissue-specific redox stress in aging using Cre-Lox methodologies to express DAAO in the heart and other tissues. We anticipate that tissue-specific transgenic expression of DAAO will provide new mechanistic insights into the broad range of disease states in which age-associated tissue pathology is associated with redox dysfunction.
The proposed research program is relevant to public health because heart failure is a major cause of morbidity and mortality in this country, and is increasing in prevalence as the population ages. There is an urgent need to develop new drugs to prevent and to treat heart failure. The proposed research will lead to the development of new animal models of heart failure and to the identification of effective new drugs and therapeutic approaches to prevent and treat heart failure in the elderly.