Nitric oxide and cardiobiology-methods for intact hearts and isolated myocytes

The cross talk between reactive oxygen species (ROS) and reactive nitrogen species (RNS) plays a pivotal role in the regulation of myocardial and vascular function. Both nitric oxide and redox-based signaling involve the posttranslational modification of proteins through S-nitrosylation and oxidation of specific cysteine residues. Disruption of this cross talk between ROS and RNS contributes to the pathogenesis of heart failure. Therefore, the elucidation of these complex chemical interactions may improve our understanding of cardiovascular pathophysiology. This chapter discusses the significant role of spatial confinement of nitric oxide synthases, NADPH oxidase, and xanthine oxidoreductase in the regulation of myocardial excitation-contraction coupling. This chapter describes techniques for assessing oxidative and nitrosative stress. A variety of assays have been developed that quantify S-nitrosylated proteins. Among them, the biotin-switch method directly evaluates endogenously nitrosylated proteins in a reproducible way. Identification of the biotinylated or S-nitrosylated proteins subjected to the biotin-switch assay are described and evaluated with a one-dimensional gel (Western blot) or with the newly developed two-dimensional fluorescence difference gel electrophoresis proteomic analysis. Quantifying the number of free thiols with the monobromobimane assay in a protein of interest allows estimation of cysteine oxidation and, in turn, the state of nitroso-redox balance of effector molecules. In summary, this chapter reviews the biochemical methods that assess the impact of nitroso/redox signaling in the cardiovascular system.