SUMMARY Reactive oxygen species (ROS) play both physiological and pathological roles in the heart and critically regulate survival and death of cardiomyocytes (CMs). NADPH oxidases purposefully produce ROS, including superoxide and hydrogen peroxide, by transferring electrons from NADPH or NADH to molecular oxygen. We have been focusing on NADPH oxidase 4 (Nox4), one of the major NADPH oxidase isoforms expressed in intracellular membranes in the heart, and we have discovered its key features. Excessive activation of Nox4 in response to hypertrophic stimuli, aging, and ischemia/reperfusion (I/R) leads to increases in oxidative stress, mitochondrial dysfunction, CM cell death, and cardiac dysfunction. On the other hand, basal production of ROS by either Nox2 or Nox4 during ischemia and reperfusion acts as a physiological second messenger to induce hypoxic adaptation. These studies suggest that the activity of Nox4 is supposed to be kept at appropriate levels in order to avoid accumulation of ROS either above or below physiological levels. How the activity of Nox4 is controlled in CMs under various pathophysiological conditions is poorly understood. Nox4 is believed to be regulated primarily at the level of transcription/translation. However, using yeast two-hybrid screening, we have identified that Nox4 physically associates with Fyn, a non-receptor type tyrosine kinase and that Fyn- mediated phosphorylation of Nox4 negatively regulates Nox4 and serves as an important negative feedback mechanism to limit excessive oxidative stress in the heart. We here test our hypotheses 1) that Fyn-mediated phosphorylation of Nox4 at Y566 inhibits interaction between Nox4 and p22phox and negatively regulates ROS production by Nox4, and 2) that activation of endogenous Fyn in response to PO and ischemic preconditioning (IPC) induces phosphorylation/suppression of Nox4 at Y566, thereby attenuating the NADPH oxidase activity of Nox4 and protecting the heart from PO and I/R injury. Using both molecular signaling and in vivo physiology approaches, we will show the molecular mechanism of the Fyn-mediated suppression of Nox4 and the functional significance of Fyn-mediated regulation of Nox4 during PO and IPC in vivo. Our study will elucidate a novel and direct PTM mechanism by which Nox4, the major ROS-producing enzyme in the heart, is regulated by stress and its functional significance during cardiac conditions frequesntly associated with aging. Our study will provide an important clue to better control the activity of Nox4 in the heart during cardiac stress in cardiovascular patients.