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. 2010 Sep 8:8:Doc20.
doi: 10.3205/000109.

Role of the complement components C5 and C3a in a mouse model of myocardial ischemia and reperfusion injury

Marc N Busche  1 , Gregory L Stahl
Affiliations

Role of the complement components C5 and C3a in a mouse model of myocardial ischemia and reperfusion injury

Marc N Busche et al. Ger Med Sci. .

Abstract
in English, German

Objective: Ischemic heart disease is the leading cause of death worldwide. The complement system plays a major role in inflammation and tissue injury following myocardial ischemia and reperfusion (MI/R) injury. Systemic C5 inhibition in clinical studies has resulted in mixed results and the role of earlier complement components (e.g., C3a), upstream from C5 cleavage, has not been elucidated for MI/R injury. Therefore, we evaluated the role of C5 or C3a in a mouse model of MI/R injury.

Methods: We performed experimental MI/R with 30 min of ischemia and 4 hr of reperfusion in 8-12 wk old C57BL/6 (WT) mice. Systemic C5 or C3a inhibition was performed with an anti-C5 monoclonal antibody (BB5.1) 30 min prior to reperfusion or with a C3a receptor antagonist (C3aRA). Since the C3aRA induces neutropenia that resolves within 120 min, we administered C3aRA at two different time points in two separate groups: 30 min prior to reperfusion within the neutropenic time frame and 120 min prior to reperfusion, when the neutropenia had resolved, but C3aRA remained active. Following MI/R, cardiac function was assessed via echocardiography, serum troponin I concentrations were measured as an index of myocardial cell death and myocardial inflammation was determined via myocardial polymorphonuclear leukocyte (PMN) infiltration.

Results: In wild type mice, MI/R significantly decrease myocardial ejection fraction and increased serum troponin I levels and myocardial PMN infiltration compared to sham-operated animals. Systemic C5 inhibition, 30 min prior to reperfusion, significantly protected mice from MI/R injury, confirming an important role for C5 in murine MI/R injury.Treatment with the C3aRA, 30 min prior to reperfusion (i.e., within the neutropenic time frame), protected mice significantly from MI/R related injury. In contrast, administration of the C3aRA 120 min prior to reperfusion, when the neutropenia had resolved, but C3aRA remained active, did not prevent MI/R injury.

Conclusions: These results confirm an important role for C5 cleavage in murine MI/R injury. At the same time, they suggest a minimal role for C3a, since neutropenia rather than C3a receptor antagonism appears to be responsible for C3aRA related amelioration in MI/R injury. While C5 inhibition in the clinical setting of MI/R does not appear to be therapeutic, our results raise the possibility that inhibition of either C5a or C5b-9 may be more advantageous than inhibition of C3a or complete inhibition of C5 in humans.

Einleitung: Die koronare Herzerkrankung ist weltweit die führende Todesursache. Das Komplementsystem spielt eine wichtige Rolle bei der Entzündungsreaktion und dem Gewebeschaden nach myokardialer Ischämie und Reperfusion (MI/R). Die Inhibition des Komplementfaktors C5 hatte in klinischen Studien unterschiedliche Ergebnisse gezeigt, und die Rolle von Komplementfaktoren, die oberhalb der C5-Spaltung in der Komplementkaskade liegen (z.B. C3a), wurde für MI/R nicht erforscht. Daher untersuchten wir die Rolle von C5 und C3a in einem MI/R-Mausmodell.

Methoden: Wir führten in 12 Wochen alten C57BL/6 (WT)-Mäusen experimentell MI/R mit 30 min Ischämie und 4 h Reperfusion durch. Systemische Inhibition der Komplementfaktoren C5 oder C3a wurde mittels eines anti-C5 monoklonalen Antikörpers (BB5.1) 30 min vor der Reperfusion oder mit einem C3a-Rezeptorantagonist (C3aRA) durchgeführt. Da der C3aRA eine Neutropenie induziert, die innerhalb von 120 min abgeklungen ist, verabreichten wir den C3aRA in zwei unterschiedlichen Versuchsgruppen zu zwei Zeitpunkten: 30 min vor der Reperfusion, innerhalb der Neutropenie, und 120 min vor der Reperfusion, wenn die Neutropenie abgeklungen war, aber der C3aRA noch aktiv war. Nach MI/R untersuchten wir die kardiale Funktion mittels Echokardiographie, bestimmten die Serumkonzentration von Troponin I als Zeichen myokardialen Zelluntergangs und die myokardiale Infiltration mit Polymorphonukleären Zellen (PMN) als Maß myokardialer Inflammation.

Ergebnisse: WT-Mäuse hatten nach MI/R im Vergleich zu sham-operierten Mäusen signifikant reduzierte Ejektionsfraktionen, während Troponin I und die myokardiale PMN-Infiltration signifikant erhöht waren. Systemische C5-Inhibierung 30 min vor der Reperfusion schützte Mäuse signifikant vor MI/R-Schädigung und bestätigt damit eine wichtige Rolle von C5 in MI/R im Mausmodell. Eine Behandlung mit dem C3aRA 30 min vor der Reperfusion, während der neutropenischen Phase, schützte die Mäuse signifikant vor MI/R-Schädigung. Eine Verabreichung des C3aRA 120 min vor der Reperfusion, wenn die Neutropenie abgeklungen war, aber der C3aRA noch aktiv war, verhinderte allerdings keine MI/R-Schädigung.

Fazit: Diese Ergebnisse bestätigen eine wichtige Rolle für C5 bei MI/R im Mausmodell. Die durch den C3aRA verursachte Neutropenie, und nicht der C3a-Rezeptorantagonismus, scheint für die Abschwächung des MI/R-Schadens verantwortlich zu sein. Damit scheint C3a bei MI/R im Mausmodell nur eine untergeordnete Rolle zu spielen. Da die Inhibition des Komplementfaktors C5 in klinischen Studien nicht erfolgreich war, sprechen unsere Ergebnisse dafür, dass die Inhibition von C5a oder C5b-9 in klinischen Studien erfolgversprechender sein könnte als die Inhibition von C3a oder eine komplette Inhibition von C5.

Keywords: C3a; C5; I/R; cardiac; complement; heart; ischemia; ischemic heart disease; myocardial; reperfusion.

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Figures

Figure 1
Figure 1. Complement pathways
Diagram outlining the three complement pathways. MBL = mannose-binding lectin; MASP = MBL-associated serine protease; TCC = terminal complement complex; MAC = membrane attack complex.
Figure 2
Figure 2. Experimental MI/R
2A: Diagram outlining the location of left anterior descending artery (LAD)-ligation and direction of myocardial sections/slices. 2B: Intraoperative picture of surgical approach through third intercostal space to the heart and LAD-ligation. 5-0 black-braided silk sutures are used to retract third and fourth rib. Myocardial ischemia is induced by LAD-ligation using 8-0 black-braided silk suture over a 1- to 2-mm piece of 0-0 suture placed for protection on the LAD to prevent the 8-0 suture from cutting into the LAD and myocardium.
Figure 3
Figure 3. Typical M-mode recordings
M-mode measurements were performed by echocardiography. In the upper row: sham-operated mice (Sham vehicle) and mice that underwent MI/R with 30 of ischemia and 4 hr reperfusion (MI/R vehicle). The lower row represents animals after MI/R treated with C5 monoclonal antibody (antiC5) 30 min prior to reperfusion (R) or with C3a receptor antagonist (C3aRA) 120 min and 30 min prior to R, respectively.
Figure 4
Figure 4. Summary of ejection fraction data
Echocardiography was performed and ejection fraction (%) was calculated from the M-mode measurements as described in Methods. Groups: vehicle-treated sham-operated mice (sham vehicle), vehicle-treated mice following 30 min of ischemia and 4 hr of reperfusion (vehicle) and mice undergoing MI/R that were treated with C5 monoclonal antibody (antiC5) 30 min prior to reperfusion (30preR) or with C3a receptor antagonist (C3aRA) 120 min and 30 min prior to R, respectively (120preR or 30preR). All data are mean ± SE of 4–6 animals per group. * p<0.05 compared to sham-operated animals (sham). ** p<0.05 compared to untreated mice undergoing MI/R with 30 min of ischemia and 4 hr of reperfusion.
Figure 5
Figure 5. Serum troponin I concentrations after MI/R
Serum troponin I concentrations after MI/R were measured as described in Methods. The columns represent mice that were vehicle-treated, mice injected with C5 monoclonal antibody (antiC5) 30 min prior to reperfusion (30preR) or with C3a receptor antagonist (C3aRA) 120 min and 30 min prior to R, respectively (120preR or 30preR). All data are mean ± SE of 4–6 animals per group. * p<0.05 compared to untreated mice following MI/R.
Figure 6
Figure 6. PMN infiltration into the myocardium following MI/R
6A: Representative sections. Heart sections were stained and PMN infiltration into the myocardium was measured using an infrared imaging system as described in Methods. Representative sections are shown from 4–6 animals per group. Upper row: 1.+2. Ab (primary and secondary antibody). Lower row: control group with 2. Ab only (secondary only antibody). Groups: vehicle-treated sham-operated mice (sham vehicle), vehicle-treated mice that underwent MI/R with 30 min of I and 4 hr of R (vehicle), mice undergoing MI/R and treated with C5 monoclonal antibody (antiC5) 30 min prior to R (30preR) or with C3a receptor antagonist (C3aRA) 120 min and 30 min prior to R respectively (120preR or 30preR). 6B: Quantitatvie analysis of sections by pixel count. Pictures from heart sections stained for PMNs from 4-6 animals per group were quantitatively analyzed by pixel counting using ImageJ software. * p<0.05 compared to sham-operated animals (sham). ** p<0.05 compared to untreated mice undergoing MI/R (MI/R vehicle).
See this image and copyright information in PMC

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