Activation of poly(ADP-ribose) polymerase by myocardial ischemia and coronary reperfusion in human circulating leukocytes

Abstract

Reactive free radical and oxidant production leads to DNA damage during myocardial ischemia/reperfusion. Consequent overactivation of poly(ADP-ribose) polymerase (PARP) promotes cellular energy deficit and necrosis. We hypothesized that PARP is activated in circulating leukocytes in patients with myocardial infarction and reperfusion during primary percutaneous coronary intervention (PCI). In 15 patients with ST segment elevation acute myocardial infarction, before and after primary PCI and 24 and 96 h later, we determined serum hydrogen peroxide concentrations, plasma levels of the oxidative DNA adduct 8-hydroxy-2'-deoxyguanosine (8OHdG), tyrosine nitration, PARP activation, and translocation of apoptosis-inducing factor (AIF) in circulating leukocytes. Plasma 8OHdG levels and leukocyte tyrosine nitration were rapidly increased by PCI. Similarly, poly(ADP-ribose) content of the leukocytes increased in cells isolated just after PCI, indicating immediate PARP activation triggered by reperfusion of the myocardium. In contrast, serum hydrogen peroxide concentrations and the translocation of AIF gradually increased over time and were most pronounced at 96 h. Reperfusion-related oxidative/nitrosative stress triggers DNA damage, which leads to PARP activation in circulating leukocytes. Translocation of AIF and lipid peroxidation occurs at a later stage. These results represent the first direct demonstration of PARP activation in human myocardial infarction. Future work is required to test whether pharmacological inhibition of PARP may offer myocardial protection during primary PCI.

Figure 1

Determination of the oxidative imbalance in patients with stable angina pectoris and acute ST-segment elevation myocardial infarction. (A) Total plasma hydrogen peroxide concentration measurements. (B) Serum 8OHdG level measurements. Results are expressed as mean (represented by squares) ± SEM (represented by boxes) and ± SD (represented by bars). Lane 1 indicates peroxide levels in stable angina patients; lanes 2–5 show peroxide concentration in patients with acute myocardial infarction before coronarography (lane 2), just after the successful primary PCI (lane 3), 24 ± 4 h after reperfusion of the ischemic myocardium (lane 4), and 96 ± 4 h after PCI (lane 5). Primary PCI itself did not affect total peroxide levels; gradual increase of hydrogen peroxide concentration was observed at 24- and 96-h time points after myocardial infarction. In contrast, serum 8OHdG levels showed a significant, rapid increase after the primary PCI, and were normalized by 96 h. *P < 0.05, **P < 0.005; NS, nonsignificant.

Figure 2

Rapid activation of PARP-1 in peripheral leukocytes induced after recanalization of the infarct-related coronary artery by primary PCI. (A) Densitometry analysis of PAR Western blots performed on patient leukocytes. (C) Immunohistochemical PAR-score analysis of patient leukocytes. In both figures, lane 1 indicates densitometry units (A) or PAR-score values (C) in stable angina patients before coronarography; lanes 2–5 show PAR content in control patients with elective PCI before coronarography (lane 2), immediately after the successful PCI (lane 3), 24 ± 4 h after (lane 4), and 96 ± 4 h after PCI (lane 5); lanes 6–9 indicate PAR content in patients with acute myocardial infarction before coronarography (lane 6), immediately after the successful PCI (lane 7), 24 ± 4 h after reperfusion of the ischemic myocardium (lane 8), and 96 ± 4 h after PCI (lane 9). Primary PCI leads to a significant increase in leukocyte cellular PAR content, reflecting rapid activation of PARP during reperfusion. A gradual decrease of PARP activity can be observed at 24- and 96-h time points after myocardial infarction. PARP activity is not affected during elective PCI. Results are expressed as mean (represented by squares) ± SEM (represented by boxes) and ± SD (represented by bars). (B) Representative examples of PAR Western blots from 3 stable angina patient leukocytes as controls (first panel) and 3 STEMI patients (second panel). Time points are indicated. Commercially available PARP enzyme served as a positive control. *P < 0.05, NS, nonsignificant.

Figure 3

Immunohistochemical analysis of tyrosine nitration, PAR content, and AIF translocation. Representative examples indicating gradual increase of the NT positive cell numbers (arrows) in peripheral leukocyte preparations after STEMI. The second row demonstrates increased PAR content in leukocytes immediately after the primary PCI. In the third row, AIF staining was performed and positive cells are depicted by arrows; AIF translocation increased by 96 h. Leukocytes from a stable angina patient are shown as negative controls in the first column.

Figure 4

Tyrosine nitration (A) and AIF translocation (B) determined by immunohistochemistry scores. After staining, NT-positive cells were counted in peripheral leukocyte smears. Results are expressed as mean (represented by squares) ± SEM (represented by boxes) and ± SD (represented by bars). Lane 1 indicates control samples from stable angina patients; lanes 2–5 show NT-positive cell counts or AIF translocation–positive cell counts in patients with acute myocardial infarction before coronarography (lane 2), immediately after the successful primary PCI (lane 3), 24 ± 4 h after reperfusion of the ischemic myocardium (lane 4), and 96 ± 4 h after PCI (lane 5). Primary PCI induced an immediate increase in tyrosine nitration, whereas a gradual increase of AIF translocation was observed at 24- and 96-h time points after reoxygenation of the ischemic myocardium. *P < 0.05; NS, nonsignificant.