Abstract 13082: On-Pump Cardiac Surgery in Humans Induces Myocardial Nitric Oxide Synthase Dysfunction via S-Glutathionylation of eNOS
Background and Aims: The mechanism responsible for LV dysfunction after cardiac surgery is only partially understood. In isolated rat hearts subjected to a prolonged ischemia-reperfusion protocol, LV dysfunction was associated with uncoupling of endothelial nitric oxide synthase (eNOS) activity secondary to oxidation of the NOS co-factor, tetrahydrobiopterin (BH4). Here we investigated the effect of cardiopulmonary bypass (CPB) and reperfusion on myocardial NOS activity and ROS production in 116 patients who underwent elective cardiac surgery.
Results: Paired samples of the right atrial appendages (RAA) were obtained prior to venous cannulation of the right atrium (PRE) and after myocardial reperfusion (POST). Atrial superoxide (O2-) release increased significantly after reperfusion (in RLU/sec/mg: from 37.83 ± 3.71 to 65.02 ± 6.01 in the POST samples; p < 0.001, n = 46 samples from 23 patients) due to uncoupling of NOS activity (D-NAME - L-NAME = ΔO2- in RLU/sec/mg: from - 31.47 ± 9.15 PRE to 40.75 ± 11.84 POST; p < 0.001 in n = 36) and increased mitochondrial and NOX2 oxidase activity (by 309% and 149%, respectively, P<0.001). POST atrial content of BH4 was reduced by 32% (P=0.001), as was the activity of the rate-limiting enzyme in BH4 synthesis, GTP-cyclohydrolase 1 (GTPCH1, by 50%, P<0.001). NOS activity (HPLC detection of 14C L-Arginine to L-Citrulline conversion) decreased significantly after reperfusion (by 60%; p < 0.001) whereas the protein content of NOS isoforms remained unchanged. Surprisingly, the PRE-POST difference in NOS activity was not affected by BH4 supplementation (10 μM) or NOX2 inhibition with the gp-91-ds tat peptide. Instead, we identified increased eNOS s-glutathionylation as the main mechanism responsible for NOS uncoupling after reperfusion. Reversing NOS s-glutathionylation with dithiothreitol (DTT: 100 μM; n=18) completely restored NOS activity after reperfusion (p =0.34).
Conclusions: Our findings indicate that NOS s-glutathionylation, rather than BH4 depletion, accounts for NOS dysfunction in patients after cardiac surgery and CPB. Although myocardial NOX2 activity is increased after reperfusion, NOX2 do not appear to be the source of oxidative stress responsible for NOS dysfunction under these conditions.
Author Disclosures: R. Jayaram: None. N. Goodfellow: None. M. Zhang: None. S. Reilly: None. M. Crabtree: None. R. DeSilva: None. R. Sayeed: None. B. Casadei: Research Grant; Significant; British Heart Foundation.
- © 2014 by American Heart Association, Inc.