In vivo imaging of nitric oxide and hydrogen peroxide in cardiac myocytes

Nitric oxide (NO) and hydrogen peroxide (H2O2) are synthesized within cardiac myocytes, and both molecules play key roles in modulating cardiovascular responses. However, the interconnections between NO and H2O2 in cardiac myocyte signaling have not been properly understood. Adult mouse cardiac myocytes represent an informative model for the study of receptor-modulated signaling pathways involving reactive oxygen species and reactive nitrogen species. However, these cells typically survive for only 1-2 days in culture, and the limited abundance of cellular protein undermines many biochemical analyses. We have exploited chemical sensors and biosensors for use in in vivo imaging studies of H2O2 and NO in adult cardiac myocytes. Here we describe detailed methods for the isolation of cardiac myocytes suitable for imaging studies. We also present our methods for the generation of recombinant lentiviral preparations encoding the H2O2 biosensor HyPer2 that permit analysis of intracellular H2O2 levels using fluorescence microscopy in living cardiac myocytes following tail vein injection and in cultured endothelial cells following infection. We also describe our protocols for using the NO chemical sensor Cu2(FL2E) in living adult mouse cardiac myocytes to study the effects of agonist-modulated H2O2 production on NO synthesis. Using these techniques, we have demonstrated that receptor-stimulated increases in intracellular H2O2 modulate NO levels in living cardiac myocytes. These and similar approaches may facilitate a broad range of studies in other terminally differentiated cells that involve the interaction of NO- and H2O2-regulated signaling responses.