Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
Cardiothoracic Surgery, Oxford University Hospital NHS Trust, Oxford, UK.
Lancet. 2015 Feb 26;385 Suppl 1:S49. doi: 10.1016/S0140-6736(15)60364-6.
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.
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.
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).
Our findings suggest that NOS s-glutathionylation, rather than BH4 depletion, accounts for NOS dysfunction in patients after cardiac surgery and cardiopulmonary bypass.
British Heart Foundation.
心脏手术后左心室功能障碍的机制尚不完全清楚。在经历缺血再灌注方案的离体大鼠心脏中，左心室功能障碍与内皮型一氧化氮合酶 (NOS) 活性的解偶联有关，这是由于 NOS 辅助因子四氢生物蝶呤 (BH4) 的氧化。在这里，我们研究了体外循环和再灌注对接受心脏手术的患者心肌硝基-氧化还原平衡的影响。
从 116 名在体外循环下接受择期心脏手术的患者中，在右心房静脉插管前和心肌再灌注后获得右心房心耳的配对样本。通过荧光素（5 μmol/L）增强化学发光法测量心房样本中超氧化物的产生，并通过高效液相色谱法（HPLC）检测 2-羟基乙啶（2-OHE）。通过 HPLC 测量 BH4、氧化生物蝶呤、GTP-环化水解酶 1（GTPCH-1，BH4 合成的限速酶）和 NOS 活性（（14）C L-精氨酸到 L-瓜氨酸转化）。
再灌注后心房中超氧化物的产生显著增加（从插管前的平均 37.83 相对光单位/秒/毫克[SE 3.71]增加到再灌注后的 65.02 [6.01]，p<0.0001；n=23 名患者的 46 个样本），这是由于线粒体和 NOX2 氧化酶活性增加（分别增加 309%和 149%；p=0.002 和 p=0.0002）和 NOS 活性解偶联。灌注后心房 BH4 含量减少（减少 32%，p=0.001），GTPCH1 活性也减少（减少 50%，p<0.0001）。再灌注后 NOS 活性显著降低（60%，p=0.0005），并且 BH4 补充（10 μM）或体外 NOX2 抑制均不能影响 NOS 活性的降低。相反，我们发现，再灌注后内皮型 NOS 的 s-谷胱甘肽化是 NOS 解偶联的主要机制。用二硫苏糖醇（100 μmol/L）逆转 NOS s-谷胱甘肽化后，再灌注后 NOS 活性完全恢复（p=0.34）。
我们的研究结果表明，在心脏手术后和体外循环中，NOS 的 s-谷胱甘肽化而不是 BH4 耗竭导致患者的 NOS 功能障碍。
英国心脏基金会。
