Reduced post-operative DPP4 activity associated with worse patient outcome after cardiac surgery

Abstract

Cardiac surgery with cardiopulmonary bypass (CPB) triggers myocardial ischemia/reperfusion injury contributing to organ dysfunction. Preclinical studies revealed that dipeptidyl peptidase (DPP4) inhibition is protective during myocardial infarction. Here, we assessed for the first time the relation of peri-operative DPP4-activity in serum of 46 patients undergoing cardiac surgery with patients' post-operative organ dysfunction during intensive care unit (ICU) stay. Whereas a prior myocardial infarction significantly reduced pre-operative DDP4-activity, patients with preserved left ventricular function showed an intra-operative decrease of DPP4-activity. The latter correlated with aortic cross clamping time, indicative for the duration of surgery-induced myocardial ischemia. As underlying mechanism, mass-spectrometry revealed increased DPP4 oxidation by cardiac surgery, with DPP4 oxidation reducing DPP4-activity in vitro. Further, post-operative DPP4-activity was negatively correlated with the extent of post-operative organ injury as measured by SAPS II and SOFA scoring, circulating levels of creatinine and lactate, as well as patients' stay on the ICU. In conclusion, cardiac surgery reduces DPP4-activity through oxidation, with low post-operative DPP4-activity being associated with organ dysfunction and worse outcome of patients during the post-operative ICU stay. This likely reflects the severity of myocardial ischemia/reperfusion injury and may suggest potential beneficial effects of anti-oxidative treatments during cardiac surgery.

Figure 1

Flowchart of the present study. From initially 60 screened patients, 46 patients were enrolled.

Figure 2

Perioperative analysis of DPP4 activity, CXCL12 and GLP-1. Serum samples were collected before (pre-OP) or at different time points after surgery (0, 6, 12, 24 h post-OP). (a) DPP4 activity in serum samples. n = 6 (controls); 29 ≤ n ≤ 44 (patients). Statistical significance is indicated relative to pre-OP values. (b) CXCL12 serum levels before and at different time points after surgery. 26 ≤ n ≤ 42. Statistical significance is indicated relative to ‘0 h post-OP’ values. (c) Serum levels of total GLP-1 before and at different time points after surgery. n = 9. (a–c) Shown are means ± SD. One-way ANOVA (Kruskal-Wallis) with Dunn’s post-test. *P < 0.05; **P < 0.01; ***P < 0.001. n.s. = not significant.

Figure 3

A larger intra-operative decrease in DPP4 activity is observed for patients with better heart function and increased time of aortic cross clamping. (a) Relative pre-OP DPP4 activity in patients that did vs. did not experience myocardial infarction (MI) within the 90 days prior to cardiac surgery. 12 ≤ n ≤ 32. T-test. (b) Relative DPP4 activity in serum of patients with different LVEF, before (pre-OP) and directly after OP (post-OP). 32 ≤ n ≤ 33 (LVEF > 50%); 4 ≤ n ≤ 8 (other). Two-way ANOVA with Sidak’s post-test for comparison of pre- vs. post-OP values. (c,d) Intra-operative decrease in relative DPP4 activity (pre-OP minus 0 h post-OP): (c) in serum of patients according to LVEF and occurrence of a prior MI within the 90 days prior to cardiac surgery. n = 24 (LVEF > 50% no prior MI); 5 ≤ n ≤ 8 (other); one-way ANOVA (Kruskal Wallis) with Dunn’s post-test; (d) in function of aortic cross clamping time. a-c, Shown are means ± SD. (d) Data are depicted as linear regression (black line) with 95% confidence intervals (dashed lines). r = Spearman correlation coefficient; two-tailed P-value. (a–d) *P < 0.05; **P < 0.01; ***P < 0.001; n.s. = not significant. LVEF = left ventricular ejection fraction; MI = myocardial infarction.

Figure 4

DPP4 activity levels negatively correlate with post-operative morbidity scores. Correlations between relative serum DPP4 activity levels 12 h post-OP and SAPS II score (a) or SOFA score (b) on the first post-operative day. Data are depicted as linear regression (black line) with 95% confidence intervals (dashed lines). r = Spearman correlation coefficient; two-tailed P-value; *P < 0.05.

Figure 5

DPP4 activity levels negatively correlate with post-operative organ morbidity and ICU stay. (a,b) Correlations between relative serum DPP4 activity levels 12 h post-OP and circulating creatinine levels (a) and lactate levels (b) on the first post-operative day. (c) Correlations between relative serum DPP4 activity levels 12 h post-OP and ICU stay. (a–c) Data are depicted as linear regression (black line) with 95% confidence intervals (dashed lines). r = Spearman correlation coefficient; two-tailed P-value; *P < 0.05; **P < 0.01.

Figure 6

DPP4 oxidation is induced during cardiac surgery and decreases DPP4 activity. (a) Quantification of oxidized amino acids of DPP4 peptides from serum of patients before (Pre-OP) vs. after (Post-OP) cardiac surgery (oxidized amino acids: lysine, histidine, methionine, tryptophane) using MALDI mass-spectrometry (n = 10). Shown are means ± SD. Two-tailed t-test. **P < 0.01. (b) Characteristic MALDI-TOF mass-spectrum of trypsinized DDP4 protein isolated from serum of a patient after cardiac surgery. The arrow at 809.2 Da indicates the mass signal of the oxidized DDP4fragment NTYRLK^*, with K* the oxidized amino acid lysine. (c) Characteristic mass fingerprint spectra of trypsinized recombinant human DPP4, before (left) and after (right) in vitro oxidation with H₂O₂ (representative for 3 experiments). The arrow in the right panel indicates the mass signal of the oxidized DDP4 fragment NTYRLK^* at 809.2 Da, which was not detected (n.d.) before oxidation (left panel). Mass signals were normalized to the internal standard (C¹³ Ang II). (d) Quantification of the activity of in vitro oxidized DPP4 (ox. DPP4) versus native recombinant human DPP4. To evaluate potential effects of residual H₂O₂ after extensive dialysis on DPP4 activity, a “H₂O₂ control sample” lacking DPP4 was dialyzed in the same way, after which DPP4 was added to the same final concentration as the oxidized DPP4 after dialysis (“control”). n = 3. Shown are means ± SD. One-way ANOVA (Kruskal Wallis) with Dunn’s post-test. *P < 0.05; n.s. = not significant.

Figure 7

Schematic representation of the main findings of this study. Cardiac surgery increases DPP4 oxidation (DPP4-ox) through the production of reactive oxygen species (ROS) and reduces DPP4 activity in the patients’ serum, with low post-operative DPP4 activity levels associated with worse outcome during ICU stay.