Level of Complement Activity Predicts Cardiac Dysfunction after Acute Myocardial Infarction

Treated with Primary Percutaneous Coronary Intervention

ABSTRACT: Background. The positive effect of reperfusion after ST-elevation myocardial infarction (STEMI) can be reduced by ischemic/reperfusion (I/R) injury. Mannose-binding-lectin (MBL) and soluble C5b-9 (membrane-attack-complex) are involved in complement-driven cell lysis and may play a role in human myocardial I/R injury. We evaluated the potential association between MBL and sC5b-9 in plasma and subsequent cardiac dysfunction in patients with STEMI treated with primary percutaneous coronary intervention (pPCI). Methods. The study included 74 STEMI-patients with acute occlusion of the left anterior descending coronary artery who were successfully treated with pPCI. Cardiac dysfunction was defined as left ventricular ejection fraction LVEF Methods Patients. The Gentofte University Hospital serves a catchment population of 1.2 million citizens (with respect to PCI, i.e., more than 20% of the total Danish population) referred directly or via noninvasive centers. More than 650 primary PCIs are performed annually. Inclusion criteria in the present study were: significant (minimum 2 mm) ST-segment elevation in at least 2 contiguous precordial leads (V1–V6) of the electrocardiogram; significant increase in cardiac markers (troponin I > 0.5 µg/L and CKMB > 5 µg/L); 4.5 mmol/L. Patients were categorized as being diabetic if they received antidiabetic treatment or if fasting plasma-glucose ≥ 7 mmol/L or non-fasting plasma-glucose ≥ 11.1 mmol/L was measured. Anamnestic information regarding smoking status was obtained. The study was approved by the local scientific ethical committee and The Danish Data Protection Agency and complied with the Declaration of Helsinki. All patients gave informed consent. Measurement of plasma MBL. Peripheral arterial blood was drawn from the femoral sheath at the beginning of the procedure, thus avoiding contamination with contrast fluid. Blood was allocated to different containers including 4 ml EDTA containers and was hour centrifuged within a half hour at 10,000 RPM for 10 minutes. Plasma was stored in nunc cryo tubes at -80°C. Plasma MBL concentration was measured using an in-house time-resolved monoclonal immunofluorometric assay, as described elsewhere.23 In brief, mannan-coated microtitre wells were incubated with plasma samples and bound MBL was detected with europium-labeled anti-MBL antibody. The lower detection level was 5 µg/L, and the intra-assay and interassay CVs were below 10%. Measurement of plasma sC5b-9. Plasma sC5b-9 was measured by a newly-developed in-house immunoassay. Microtitre wells were coated with antihuman sC5b-9 (Quidel, San Diego, California) (1 µg/ml PBS) overnight at 4°C, and blocked with 1 mg BSA /ml PBS for 1 hour at room temperature. After wash in PBS containing 0.05% Tween 20 (PBS/Tw), EDTA plasma samples were diluted 4-fold in PBS/Tw containing 10 mM EDTA and incubated overnight at 4°C. A standard was made from normal human serum and activated by incubation with human ig-sepharose for 1 hour at 37°C. The concentration of sC5b-9 was quantified by comparison with recombinant sC5b-9 (Quidel). Two EDTA plasma samples were used as internal controls and wells receiving only buffer as negative controls. Bound sC5b-9 was detected with 0.05 µg biotinylated antihuman C6 antibody (Quidel) for 2 hours at room temperature, and was subsequently incubated with 10 ng Eu3+-labeled streptavidin (Perkin Elmer) in 100 µl PBS/Tw containing 25 µm EDTA for 1 hour at room temperature. Bound europium was detected by the addition of 200 µl of enhancement solution (Perkin Elmer) and reading the time-resolved fluorescence on a DELFIA fluorometer (Victor3, Perkin Elmer). The limit of detection for the sC5b-9 assay was 1 µg/L. The intra- and interassay variations (%CV) were below 5 and 12%, respectively. Echocardiography. Within 48 hours after primary PCI, a transthoracic 2-dimensional echocardiography (Vingmed, Vivid7, GE Medical, Norway) was performed in the left lateral decubitus position. All images were recorded in second harmonic imaging. Parasternal long-axis and short-axis and apical 4-chamber, 2-chamber and long-axis projections were obtained. The 16 standard segments model suggested by the American Society of Echocardiography was used for evaluation of regional function. Left ventricular systolic dysfunction was defined as LVEF Results Among the 74 included patients mean LVEF was 34%. Thirty-six patients (49%) ended up with LVEF Discussion Our study suggests that high activity in the complement system, measured by MBL and sC5b-9, is associated with increased risk of cardiac dysfunction in STEMI patients treated with pPCI. This is an important and clinically relevant finding because of its potential prognostic value and as a supplement to the ongoing exploration of complement activity during I/R injury. The role of complement activity in I/R injury — particularly MBL and sC5b-9 — has previously been demonstrated in experimental studies, but only in a limited number of clinical studies.10–14,16, 26–33 Accordingly, the present study is the first to measure plasma levels of MBL and sC5b-9 in STEMI patients treated with primary PCI. These patients are common in the industrialized world and have a high potential risk of I/R injury, leading to increased mortality and left ventricular cardiac dysfunction. Several attempts have been made to reduce I/R injuries through different cardioprotective strategies including modulation of the complement system.9,34–36 However, the results are still inconclusive, probably because of the complexity of I/R injuries and difficulties in targeting the mechanisms leading to I/R injury. The aim of this study was to investigate whether complement activity, measured by plasma levels of MBL and sC5b-9 in pPCI-treated STEMI patients, predicts left ventricular cardiac dysfunction. Only patients with STEMI due to LAD occlusion and no prior MI or heart failure was included. Thus, we studied a homogenous group of patients who ended up with a broad range of left ventricular cardiac dysfunction due to acute coronary occlusion and without any significant preconditioning of the myocardium due to the development of collaterals. Furthermore, only patients who underwent a successful pPCI (TIMI flow = 3) within 12 hours after the onset of chest pain were included since “no-flow” or “slow-flow” after pPCI would offer an adequate explanation of the myocardial dysfunction. Finally, patients with ongoing infectious or inflammatory disease were excluded in order to avoid activation of the complement system due to other causes than STEMI. The prognostic value of MBL.The damaging effect of MBL during I/R injury is probably due to an upregulation of tissue-specific ligands on the myocytes induced by the oxidative stress during hypoxia.16 Experimental attempts have been made to manipulate the lectin pathway to improve outcome after myocardial injury. In animal models of ischemia and reperfusion, it has been possible to reduce myocardial injury by neutralization of MBL.16,30 Studies from Busche et al15,16 have demonstrated that diabetic MBL knockout mice were protected from myocardial I/R injury measured by LVEF. Furthermore, studies have shown that patients who are genetically determined for low MBL levels and undergo surgery for thoracoabdominal aortic aneurysm, experience less complement activation and inflammatory response and thus less I/R injury.27,29,32 We found that MBL was significantly higher in patients with a LVEF Conclusion Our study suggests that high plasma levels of MBL and low plasma levels of sC5b-9 are associated with an increased risk of cardiac dysfunction in STEMI patients treated with pPCI, probably due to increased complement activity during the ischemic /reperfusion process. This result may be of importance in future risk stratification after STEMI and adds to the knowledge about complement activity during I/R injury as a potential target of treatment in patients with MI. Acknowledgements. This study was financially supported by The Danish Heart Foundation, The Murermester Lauritz Peter Christensen and Wife Kirsten Sigrid Christensen Foundation, The Danish Medical Research Council and The Danish Diabetes Association. Dr. Steen Rasmussen, Dr. Flemming Pedersen and Dr. Klaus Klausen are thanked for valuable discussions. Dorte E Wulff, Kurt Svarre Jensen and Grete Haahr are thanked for their excellent technical assistance.

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