Summary/Abstract This application describes chemical approaches for determining the role of protein cysteine glutathionylation in the sarcomere. Sarcomere is a basic unit of myofibrils in muscle. Sarcomere contains numerous sarcomeric proteins, including titin, actin and myosin, that form a highly organized structure for continuous contraction of muscle. In muscle cell, the reactive oxygen species (ROS) are emerging as critical signaling molecules that strongly contribute to physiology and pathology associated with heart function. However, the precise molecular target proteins of ROS and their redox-based regulatory mechanisms that affect sarcomere stability and integrity remain unknown. Glutathionylation is one of the major protein cysteine oxidative modifications that mediate the role of ROS in redox signaling and oxidative stress. This application is based on our recently developed chemical approach, i.e. clickable glutathione for identification and characterization of glutathionylation. SET and MYND domain-containing protein 2 (SMYD2) is an abundant protein in heart and skeletal muscle. With clickable glutathione, we found that SMYD2 is selectively glutathionylated at C13, and SMYD2 C13 glutathionylation is a crucial mechanism by which ROS induce sarcomere destabilization in cardiomyocytes. The main goal of application is to identify sarcomeric proteins, including SMYD2, that are susceptible to glutathionylation in response to ROS and to characterize functional roles of protein glutathionylation in regulating sarcomere stability. There are three specific aims. First, we plan to couple clickable glutathione with mass analysis to identify glutathionylation of SMYD2 and other sarcomeric proteins in response to ischemic conditions. Clickable glutathione approach will be used in H9c2 cell line with isotopic-labelled azido-Ala and cleavable biotin-alkyne for quantitative mass analysis of glutathionylated proteins under oxygen-glucose-deprivation. Second, we will determine sarcomere stability and integrity resulting from SMYD2 C13 glutathionylation. We will determine sarcomere stability in myocytes in response to ROS by fluorescence imaging of sarcomeric proteins, including myosin and actin. Also, we will couple clickable glutathione with proximity ligation for visualizing localization of glutathionylated SMYD2. Third, we plan to determine the molecular mechanism by which SMYD2 glutathionylation leads to sarcomere destabilization. We will synthesize site-specifically glutathionylated SMYD2, which will be used for characterizing structural and functional changes of SMYD2 C13 glutathionylation with subsequent cellular studies. Taken together, these studies will uncover the key molecular target protein and molecular mechanisms by which ROS contribute to muscle dysfunction.