Abstract 3581: Global Cardiac Phosphoproteome Analysis in Nitric Oxide-Induced Heart Failure
The inducible nitric oxide synthase (iNOS) is only expressed during pathophysiological conditions such as ischemia-reperfusion, septicaemia and heart failure. End-targets and signaling pathways of nitric oxide (NO) in the modulation of cardiac function are not fully understood. For a global gel-free phosphoproteome analysis, isolated hearts of mice with cardiac specific iNOS overexpression and lack of myoglobin (iNOS+/myo−/−) were co-perfused either with or without the iNOS substrate L-arginine (200μM). After 1 min L-arginine, contractile force was decreased by 30% to a new steady state, at which point hearts were freeze clamped. For MS-based quantitative analysis, stable isotope dimethyl labeling was applied which introduced a 4Da mass difference per labelled primary amino group. To reduce sample complexity peptides were fractionated on a strong cation exchange column, followed by phosphopeptide enrichment (titanium dioxide). Fractions were further separated by nano flow reverse phase liquid chromatography online coupled with a tandem mass spectrometer (nanoRP-LC-MS/MS). Two biological and two technical replicates were measured of each membrane and cytosolic fraction (n=8). Overall, 826 phosphorylation sites (246 novel) were identified corresponding to 790 peptides which relate to 475 proteins. In NO-induced heart failure 50 phosphorylation sites were downregulated (31 novel) and 74 were upregulated (16 novel) in proteins involved in calcium homeostasis (e.g. L-type calcium channel, phospholam-ban, ryanodine receptor, sarcalumenin), cardiac contractility (e.g. myosin-6, myosin binding protein C), energetics (e.g. pyruvate dehydrogenase, ATP citrate lyase) mitochondrial respiration (e.g. NADH dehydrogenase, ATP synthase) and transcription (e.g. histone deacetylase 4, glucocorticoid receptor). Additionally, kinases and phosphatases (e.g. PKCα, MAP kinase kinase, protein phosphatase 2A) were also regulated. In summary, this study provides the first global account of the cardiac phosphoproteome in NO-induced heart failure. Many novel phosphorylation sites were discovered which can be linked to depressed contractility, cardiac energetics and remodeling and are likely to have important novel regulatory functions.