Myocardial ischemia and reperfusion injury is dependent on both IgM and mannose-binding lectin

Abstract

Complement activation has been shown to play an important role in the inflammation and tissue injury following myocardial ischemia and reperfusion (MI/R). Several recent studies from our laboratory demonstrated the importance of mannose-binding lectin (MBL) as the initiation pathway for complement activation and the resulting pathological effects following MI/R. However, other studies from the past suggest an important role of the classical pathway and perhaps natural antibodies. In the present study, we used newly generated genetically modified mice that lack secreted IgM (sIgM), MBL-A, and MBL-C (sIgM/MBL null) in a plasma reconstitution mouse model of MI/R. Following 30 min of ischemia and 4 h of reperfusion, left ventricular ejection fractions were significantly higher in sIgM/MBL null mice reconstituted with MBL null or sIgM/MBL null plasma compared with reconstitution with wild-type (WT) plasma or WT mice reconstituted with WT plasma following MI/R. Serum troponin I concentration, myocardial polymorphonuclear leukocyte infiltration, and C3 deposition were dependent on the combined presence of sIgM and MBL. These results demonstrate that MI/R-induced complement activation, inflammation, and subsequent tissue injury require both IgM and MBL. Thus MBL-dependent activation of the lectin pathway may not be completely antibody independent in I/R models.

Fig. 1.

Left ventricular function following myocardial ischemia and reperfusion (MI/R). Echocardiography was performed following MI/R with 30 min of ischemia and 4 h of reperfusion and ejection fraction (in %) was calculated from the M-mode measurements as described in materials and methods. A: representative M-mode recordings. A, top: wild-type (WT) mice reconstituted with WT plasma sham-operated (WT/WT sham) or following MI/R (WT/WT). A, bottom: secreted IgM (sIgM)/mannose-binding lectin (MBL) null (Triple) mice reconstituted with WT (Triple/WT), MBL null [Triple/MBL knockout (KO)], or sIgM/MBL null plasma (Triple/Triple) following MI/R. B: summary of ejection fraction data. All data are means ± SE of 4–6 animals per group. *P < 0.001 compared with sham-operated WT mice following reconstitution with WT plasma (WT/WT sham). **P < 0.001 compared with WT mice and sIgM/MBL null mice following MI/R after reconstitution with WT plasma (WT/WT or Triple KO/WT).

Fig. 2.

Serum troponin I concentrations following MI/R. Serum troponin I concentrations following MI/R were measured as described in materials and methods. The groups were as follows: WT mice reconstituted with WT plasma (WT/WT) following MI/R and sIgM/MBL null mice reconstituted with WT (Triple KO/WT), MBL null (Triple KO/MBL KO), or sIgM/MBL null plasma (Triple KO/Triple KO). All data are means ± SE of 4–6 animals per group. *P < 0.05 compared with WT mice reconstituted with WT plasma (WT/WT). **P < 0.05 compared with WT/WT mice and sIgM/MBL null mice reconstituted with WT plasma (Triple KO/WT).

Fig. 3.

Myocardial polymorphonuclear leukocyte (PMN) infiltration following MI/R. A: representative sections. Myocardial sections were stained, and PMN infiltration was measured using an infrared imaging system (LI-COR) as described in materials and methods. Representative sections are shown from 3 animals per group. A, top: 1.+ 2. antibody (Ab; primary and secondary antibody). A, bottom: control group with 2. Ab only (secondary only antibody). The groups were as follows : WT mice reconstituted with WT plasma in either sham-operated (WT/WT sham) or following MI/R (WT/WT) and sIgM/MBL null mice reconstituted with WT (Triple KO/WT), MBL null (Triple KO/MBL KO), or sIgM/MBL null plasma (Triple KO/Triple KO). B: quantitative analysis of sections via pixel counting. Myocardial sections stained for PMN from 3 animals per group were quantitatively analyzed by pixel counting using ImageJ software [National Institutes of Health (NIH)]. *P < 0.05 compared with sham-operated WT mice following reconstitution with WT plasma (WT/WT sham). **P < 0.05 compared with WT mice and sIgM/MBL null mice undergoing MI/R following reconstitution with WT plasma (WT/WT and Triple KO/WT, respectively).

Fig. 4.

Myocardial deposition of C3. A: representative sections. Myocardial sections were stained and C3 deposition was measured using an infrared imaging system (LI-COR) as described in materials and methods. Representative sections are shown from 3–6 animals per group. A, top: 1.+ 2. Ab (primary and secondary antibody). A, bottom: control group with 2. Ab only (secondary only antibody). The groups were as follows: WT mice reconstituted with WT plasma in either sham-operated (WT/WT sham) or following MI/R (WT/WT) and sIgM/MBL null mice reconstituted with WT (Triple KO/WT), MBL null (Triple KO/MBL KO), sIgM/MBL null plasma (Triple KO/Triple KO) or sIgM null plasma (Triple KO/IgM KO). B: quantitative pixel count analysis. Myocardial sections stained for C3 deposition from 3–6 animals per group were quantitatively analyzed by pixel counting using ImageJ software (NIH). *P < 0.05 compared with sham-operated WT mice following reconstitution with WT plasma (WT/WT sham). **P < 0.05 compared with WT mice and sIgM/MBL null mice undergoing MI/R after reconstitution with WT plasma (WT/WT and Triple KO/WT, respectively).