PROJECT SUMMARY The goal of this study is to elucidate novel mechanisms underlying exercise training (ExT)-induced sympatho- inhibition in chronic heart failure (CHF) (Aim 1) and cardioprotection following acute coronary ischemia/reperfusion (I/R) (Aim 2), based on an innovative concept of inter-organ transfer of antioxidant enzymes. It is well established that exercise generates muscle-derived reactive oxygen species (ROS) which activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2), resulting in upregulation of a panel of antioxidant enzymes in skeletal muscle per se. We hypothesize that these antioxidants can be transported from skeletal muscle to remote tissues through circulating extracellular vesicles (EVs), providing recipient cells with a second and enhanced line of antioxidant defense. In the rostral ventrolateral medulla (RVLM) of mice with CHF, these antioxidants restore redox homeostasis of pre-sympathetic neurons, contributing to ExT-sympatho- inhibition. Furthermore, in the heart of mice subjected to coronary I/R, these antioxidants reduce free radical damage and salvage ischemic myocardium, thus playing a critical role in ExT-cardioprotection. To address these hypotheses, we developed three skeletal muscle-specific transgenic mouse lines. The MS-mG line is a reporter model, which allows us to track, capture, and analyze EVs released specifically from skeletal muscle. This model will be used to assay cargo proteins of skeletal muscle-derived EVs and their distribution in brain and heart following ExT. The iMS-Nrf2flox/flox and iMS-Keap1flox/flox lines will enable us to delete skeletal muscle Nrf2 (i.e. Nrf2 deficiency) and Keap1 (i.e. Nrf2 overexpression), respectively. These two models will be used to demonstrate a causal relationship between the Nrf2/antioxidant system and ExT-sympatho-inhibition in Aim 1 and ExT-cardioprotection in Aim 2. Interdisciplinary methods of EV biology, proteomics, bioinformatics, electrophysiology, and cardiovascular physiology will be utilized to characterize skeletal muscle-derived EVs following ExT, determine the effects of ExT-EVs on central neuron discharge and peripheral sympathetic nerve activity, and explore the mechanisms underpinning cardioprotection of ExT-EVs against coronary I/R injury. Upon completion of this project, we expect to provide novel mechanistic insights on ExT-cardiovascular protection, paving a new avenue to translate the beneficial effects of regular physical activity into clinical practice to prevent and treat acute and chronic ischemic heart diseases.