title: Molecular mechanisms of myocardial nitroso-redox imbalance during on-pump cardiac surgery.
abstract: BACKGROUND
  The mechanism responsible for left ventricular dysfunction after cardiac surgery is only partly understood. In isolated rat hearts subjected to an ischaemia-reperfusion protocol, left ventricular dysfunction was associated with uncoupling of endothelial nitric oxide synthase (NOS) activity secondary to oxidation of the NOS cofactor, tetrahydrobiopterin (BH4). Here we investigated the effect of cardiopulmonary bypass and reperfusion on myocardial nitroso-redox balance in patients undergoing cardiac surgery.
  
  
  METHODS
  From 116 patients who underwent elective cardiac surgery on cardiopulmonary bypass, paired samples of the right atrial appendages were obtained before venous cannulation of the right atrium and after myocardial reperfusion. Superoxide production from atrial samples was measured by lucigenin (5 μmol/L) enhanced chemiluminescence and 2-hydroxyethidium (2-OHE) detection by high-performance liquid chromatography (HPLC). BH4, oxidised biopterins, GTP-cyclohydrolase 1 (GTPCH-1, the rate-limiting enzyme in BH4 synthesis), and NOS activity ((14)C L-arginine to L-citrulline conversion) were measured by HPLC.
  
  
  RESULTS
  Atrial superoxide production increased significantly after reperfusion (from mean 37·83 relative light units per s per mg [SE 3·71] before cannulation to 65·02 [6·01] after reperfusion, p<0·0001; n=46 samples from 23 patients) due to increased mitochondrial and NOX2 oxidase activity (by 309% and 149%; p=0·002 and p=0·0002, respectively) and uncoupling of NOS activity. Atrial content of BH4 after perfusion was reduced (by 32%, p=0·001), as was activity of GTPCH1 (50%, p<0·0001). NOS activity decreased significantly after reperfusion (60%, p=0·0005) and this reduction was not affected by BH4 supplementation (10 μM) or NOX2 inhibition ex vivo. Instead, we identified increased endothelial NOS s-glutathionylation as the main mechanism for NOS uncoupling after reperfusion. Reversing NOS s-glutathionylation with dithiothreitol (100 μmol/L) completely restored NOS activity after reperfusion (p=0·34).
  
  
  CONCLUSIONS
  Our findings suggest that NOS s-glutathionylation, rather than BH4 depletion, accounts for NOS dysfunction in patients after cardiac surgery and cardiopulmonary bypass.
  
  
  BACKGROUND
  British Heart Foundation.
authors: Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
authors: Raja
authors: R
authors: Jayaram
authors: Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
authors: Nicky
authors: N
authors: Goodfellow
authors: Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
authors: Mei Hua
authors: MH
authors: Zhang
authors: Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
authors: Svetlana
authors: S
authors: Reilly
authors: Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
authors: Mark
authors: M
authors: Crabtree
authors: Cardiothoracic Surgery, Oxford University Hospital NHS Trust, Oxford, UK.
authors: Ravi
authors: R
authors: De Silva
authors: Cardiothoracic Surgery, Oxford University Hospital NHS Trust, Oxford, UK.
authors: Rana
authors: R
authors: Sayeed
authors: Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK. Electronic address: Barbara.casadei@cardiov.ox.ac.uk.
authors: Barbara
authors: B
authors: Casadei