Atrial nitroso-redox balance and refractoriness following on-pump cardiac surgery: a randomized trial of atorvastatin

Abstract

Aims: Systemic inflammation and increased activity of atrial NOX2-containing NADPH oxidases have been associated with the new onset of atrial fibrillation (AF) after cardiac surgery. In addition to lowering LDL-cholesterol, statins exert rapid anti-inflammatory and antioxidant effects, the clinical significance of which remains controversial.

Methods and results: We first assessed the impact of cardiac surgery and cardiopulmonary bypass (CPB) on atrial nitroso-redox balance by measuring NO synthase (NOS) and GTP cyclohydrolase-1 (GCH-1) activity, biopterin content, and superoxide production in paired samples of the right atrial appendage obtained before (PRE) and after CPB and reperfusion (POST) in 116 patients. The effect of perioperative treatment with atorvastatin (80 mg once daily) on these parameters, blood biomarkers, and the post-operative atrial effective refractory period (AERP) was then evaluated in a randomized, double-blind, placebo-controlled study in 80 patients undergoing cardiac surgery on CPB. CPB and reperfusion led to a significant increase in atrial superoxide production (74% CI 71-76%, n = 46 paired samples, P < 0.0001) and a reduction in atrial tetrahydrobiopterin (BH4) (34% CI 33-35%, n = 36 paired samples, P < 0.01), and in GCH-1 (56% CI 55-58%, n = 26 paired samples, P < 0.001) and NOS activity (58% CI 52-67%, n = 20 paired samples, P < 0.001). Perioperative atorvastatin treatment prevented the effect of CPB and reperfusion on all parameters but had no significant effect on the postoperative right AERP, troponin release, or NT-proBNP after cardiac surgery.

Conclusion: Perioperative statin therapy prevents post-reperfusion atrial nitroso-redox imbalance in patients undergoing on-pump cardiac surgery but has no significant impact on postoperative atrial refractoriness, perioperative myocardial injury, or markers of postoperative LV function.

Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT01780740.

Keywords: Atorvastatin; Atrial refractory period; Clinical trial; Ischaemia-Reperfusion Injury; Nitric Oxide Synthase; Oxidant stress; Postoperative Atrial fibrillation.

Graphical abstract

Figure 1

Superoxide (${\mathrm{O}}_2^{-}$) production in right atrial samples obtained before (PRE) and after CPB and reperfusion (POST) measured by lucigenin-enhanced chemiluminescence (n = 46 paired samples from 23 patients; A) and by HPLC detection of 2-OH ethidium (n = 26 paired samples from 13 patients; B), respectively. ****P < 0.0001 vs. PRE, by Wilcoxon matched-pairs signed rank test (A) or the paired Student’s t-test (B). The contribution of mitochondrial complex I (n = 20 paired samples from 10 patients; C) or NOX2 oxidase (n = 22 paired samples from 11 patients; D) expressed as the rotenone-inhibitable fraction of lucigenin-enhanced chemiluminescence and the gp91 tat-inhibitable fraction of 2OH-ethidium by HPLC, respectively, were increased in POST atrial samples. **P < 0.01 and ***P < 0.001 vs. PRE, by the Wilcoxon matched-pairs signed rank test (C) or the paired Student’s t-test (D). Data are shown as median and IQR (A and C) or mean and SD (B and D). RLU, relative light units.

Figure 2

Atrial NOS activity significantly decreased in POST samples (n = 20 paired samples from 10 patients; A). ***P < 0.001 vs. PRE, by paired Student’s t-test. Atrial nNOS and eNOS protein content was unaltered after CPB and reperfusion (B and C) while iNOS protein was absent (D) and mRNA below the detection threshold (E, n = 14–40 samples from 7 to 20 patients). There were no significant differences between PRE and POST (Band C) by paired Student’s t-test. Data are shown as mean and SD (A–D). nNOS positive control (+) was murine brain tissue; iNOS positive control (+) were primary murine macrophages treated with LPS; Negative control (−) were primary murine macrophage in the absence of LPS stimulation. eNOS positive control (+) was human saphenous vein homogenate.

Figure 3

Atrial BH₄ content decreased after CPB and reperfusion (A), whereas BH₂ (C) and biopterin levels (D) were unaltered resulting in a reduction in the ratio of BH₄ to BH₂ + Biopterin (B), n = 36 paired samples from 18 patients. **P < 0.01, *P < 0.05 vs. PRE in A and B by Wilcoxon matched pairs sign rank test. C and D PRE-POST differences were not significant by paired Student’s t -test (C) or the Wilcoxon matched pairs sign rank test (D). Data are shown as median and IQR (A–D) or mean and SD (C).

Figure 4

(A) Atrial GCH-1 activity decreased after CPB and reperfusion, n = 26 paired samples from 13 patients. ***P < 0.001 vs. PRE, by paired Student’s t test. (B) The atrial protein level of GCH-1 was unaltered whereas atrial GFRP (C) was significantly increased after CPB and reperfusion, n = 16—20 samples from 8 to 10 patients. **P < 0.01 vs. PRE by paired Student’s t-test. Data are shown as mean and SD.

Figure 5

(A) The reduction in atrial NOS activity in POST samples (n = 18 paired samples from 9 patients) is not abolished by pre-treatment with BH₄, **P < 0.01 vs. PRE by Wilcoxon matched pairs sign rank test. (B) Treatment with the reducing agent, dithiothreitol (DTT) restored NOS activity in POST samples, n = 12 paired samples from 6 patients; ****P < 0.0001, by analysis of covariance (ANCOVA) adjusted for PRE measurements, whereas (C) NOX2 inhibition with gp91ds tat vs. scrambled peptide did not, n = 16 paired samples from 8 patients by ANCOVA adjusted for PRE measurements. Data are expressed as median and IQR (A) or mean and SD (B and C).

Figure 6

Differences between right atrial superoxide production, NOS activity, BH₄ content and GCH-1 activity in PRE and POST samples from patients allocated to atorvastatin (80 mg od) or placebo. Treatment with atorvastatin prevented the increase in superoxide production (A) in POST atrial samples vs. placebo (n = 60 paired samples from 30 patients in each group; **P < 0.01, by ANCOVA) and the reduction in NOS activity (B, n = 24 paired samples from 12 patients; *P < 0.05 vs. placebo, by ANCOVA). Atorvastatin prevented the reduction in atrial BH₄ content (n = 28 paired samples from 14 patients; C) and GCH-1 activity (n = 18 paired samples from 9 patients; D) in POST samples vs. placebo. **P < 0.01, *P < 0.05 vs. placebo, by ANCOVA. Data are shown as median and IQR (A, B, and D) or mean and SD (C).

Figure 7

Average right atrial effective refractory period on first four postoperative days at three pacing cycle lengths. (A) pacing cycle length 500 ms, 14 patients allocated to atorvastatin and 15 patients allocated to placebo; (B) pacing cycle length 600 ms, 14 patients allocated to atorvastatin and 15 patients allocated to placebo; (C) pacing cycle length 700 ms, 10 patients allocated to atorvastatin, and 12 patients allocated to placebo; P  = 0.83, P  = 0.87 and P  = 0.75 by mixed model linear regression analysis after log transformation. Back transformed data are shown as geometric mean ± 95% confidence interval. AERP, atrial effective refractory period; PCL, pacing cycle length.