The role of natural IgM in myocardial ischemia–reperfusion injury
Ming Zhanga,b,1, Lloyd H. Michaelc, Sandrine A. Grosjeand, Ralph A. Kellyd,e,
Michael C. Carrolla,b,f, Mark L. Entmanc,⁎
aThe CBR Institute for Biomedical Research, Inc., Harvard Medical School, Boston, MA 02115, USA
bThe Department of Pathology, Harvard Medical School, Boston, MA 02115, USA
cSection of Cardiovascular Sciences, The Methodist Hospital and the DeBakey Heart Center, Baylor College of Medicine, Houston, TX 77030, USA
dCardiovascular Division, Brigham and Women’s Hospital, Boston, MA 02115, USA
eGenzyme Corporation, Framingham, MA 01701, USA
fThe Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
Received 8 February 2006; accepted 24 February 2006
Available online 16 June 2006
Myocardial ischemia–reperfusion injury represents a combination of factors, namely the intrinsic cellular response to ischemia and the extrinsic
acute inflammatory response. Recent studies in mesenteric and skeletal muscle reperfusion models identified natural IgM as a major initiator of
pathology through the activation of the complement system and inflammatory cells. To determine whether a similar mechanism is involved in
myocardial tissues, mice bearing an altered natural IgM repertoire (Cr2−/−) were examined in a murine model of coronary artery ischemia. Notably,
these mice were significantly protected based on the reduced infarct size, limited apoptosis of cardiomyocytes, and decreased neutrophil infiltration.
Protection was IgM-dependent as reconstitution of these mice with wild-type IgM restored myocardial reperfusion injury. These results support a
model in which natural IgM initiates the acute inflammatory response in the myocardium following ischemia and reperfusion.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Natural antibody; Complement; Myocardial ischemia–reperfusion injury; Infarction; Apoptosis; Neutrophil
Acute myocardial ischemia and myocardial infarction are a
leading cause of mortality in today's society. Restoration of
blood flow (reperfusion) is essential for survival of the
myocardium, but, paradoxically, it also induces an acute
inflammatory response referred to as ischemia–reperfusion
injury (I/R). Full injury represents a manifestation of both the
intrinsic cellular response of myocardium and the extrinsic
acute inflammatory response [1–3].
While numerous studies have revealed the decisive
contribution of active cellular responses to injury resulting
from ischemic insults [4–10], evidence is emerging that acute
inflammatory responses also play an important role in
myocardial I/R [2,11]. Studies on the serum complement
system, a major component of natural immunity, indicate that
it may be a major factor [2,12–15]. Early observations that
transient depletion of the central complement, i.e. C3, reduced
inflammation in a rat model of myocardial infarction
suggested a role for complement in I/R . Weisman et al.
 subsequently demonstrated in a rat myocardial model that
I/R injury was dramatically reduced by pretreatment with a
modified form of a natural inhibitor of complement, soluble
CR1 (sCR1). Since then, a similar approach has shown that I/
R injury in multiple tissues could be reduced or blocked by
pretreatment with sCR1 [16,17]. These studies led to the
examination of how complement was activated during the
early stages of I/R injury.
Three pathways leading to activation of the complement
system have been identified, i.e. the classical, lectin, and
alternative pathways. Each is activated by different initiators,
but all converge to complement protein C3 and are followed by
a common cascade . Although antibody–antigen interaction
Journal of Molecular and Cellular Cardiology 41 (2006) 62–67
⁎Corresponding author. Tel.: +1 713 798 4188.
E-mail address: email@example.com (M.L. Entman).
1Current address: Department of Anesthesiology, SUNY-Downstate Medical
Center, Brooklyn, NY 11203, USA.
0022-2828/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
is traditionally recognized as activating via the classical
pathway, new evidence suggests that certain antibodies also
activate the lectin pathway [19,20].
The first indication that serum antibody may be involved in
complement-mediated I/R injury came from studies of hind
limb I/R by Weiser et al. . Using knockout mice deficient in
complement proteins C3 or C4, they found that an intact
pathway of complement was required for full I/R injury in hind
limb. Thus, mice deficient in C4, the protein which is activated
in the classical and lectin pathways, were protected to a degree
similar to C3-deficient mice or to wild-type mice (WT) treated
with sCR1. The finding that C4 was involved suggested that the
complement system might be activated by serum antibody.
Subsequent experiments confirmed a role for serum immuno-
globulin and identified IgM as the important isotype. Thus,
mice bearing a genetic deficiency in the Recombination
Activation Gene-1 (RAG-1−/−), and thus do not develop mature
B and T lymphocytes, failed to develop full injury, but
reconstitution with fresh pooled serum  or with pooled
WT IgM  restored full injury.
The suggestion that specific, pre-existing, or naturalIgM was
important in I/R came from studies using mice bearing a genetic
deficiency in the Cr2 locus which encode complement receptors
CD21 and CD35 [23,24]. Cr2−/−mice have an impaired
humoral response to T-dependent and T-independent antigens
due to both an intrinsic defect in B cell activation and reduced
antigen trapping on FDC [23,24]. Although the mice have
normal levels of circulating IgM, it was proposed that their
repertoire mightbe limited given theimpaired responsiveness of
their B cells. Examination of the mice in the intestinal I/R model
revealed a significant reduction in injury that was reversed by
reconstitution with pooled IgM prepared from WT mice but not
Cr2−/−mice [25,26]. By contrast, reconstitution with pooled
WT IgG did not cause histological injury, although it did
augment neutrophil infiltration when combined with IgM .
Thus, it was suggested that injury is mediated by specific clone
(s) of natural IgM [25,26]. More recently, a single IgM clone
isolated from a panel of natural IgM-producing hybridoma cells
was identified that was sufficient to reconstitute I/R injury in
both the intestinal and hind limb I/R models [27,28].
To determine if specific natural IgM mediates the acute
inflammatory response in the myocardial I/R, we examined
Cr2−/−and RAG-1−/−mice in a left anterior descending (LAD)
coronary artery model [29,30]. Significantly, myocardial infarct
size, frequency of apoptotic cardiomyocytes and number of
infiltrating neutrophils relative to the WTcontrols were found to
be dependent on WT IgM. Reconstitution of Cr2−/−and RAG-
1−/−mice prior to ischemia with WT IgM restored full injury.
These results suggest a pathogenic mechanism by which
myocardial I/R injury is mediated by naturally occurring IgM.
Cr2−/−mice were constructed as described using the
approach of homologous recombination in embryonic stem
cells which introduces a frameshift mutation and a stop codon
disrupting the coding for Cr2 coding region and bred onto the
C57BL/6 background for at least 10 generations . RAG-
1−/−mice (C57BL/6 background) which were purchased from
Jackson Laboratory (Bar Harbor, ME) were originally devel-
oped by homologous recombination as described , as a
result a 1356 bp deletion which occurred in the 5′ end of RAG-1
gene coding sequence. Animals were maintained in the animal
facilities of Harvard Medical School and Baylor College of
Medicine according to Federal and State regulations.
2.2. Mouse model of myocardial ischemia–reperfusion injury
An established murine myocardial model was used in this
injection of pentobarbital sodium, 40 mg/kg weight. Reconsti-
prior to surgery. Midline sternotomy followed by sternal
retraction is performed to permit the visualization and ligation
of the left anterior descending (LAD) coronary artery under a
microscope (Stemi 2000-C, Carl Zeiss, Thornwood, NY) as
described by Michael et al. . Reperfusion of the previously
occluded coronary bed is confirmed by visual inspection and
the animal allowed to recover under a heating lamp. Physiologic
variables, includingheart rate, corebody temperature,and ECG,
a 22-gauge Luer stub, and LAD is re-occluded before 1% Evans
Blue is perfused into the aorta and coronary arteries with
distribution throughout the ventricular wall proximal to the
coronary artery ligature. After this procedure, the heart is
sectioned transversely into four sections, with one section being
made at the site of the ligature. Sections of the ventricle are then
incubated in 1.5% triphenyltetrazolium chloride (TTC). After
TTC staining, viable myocardium is brick red and the infarct
appears pale white. The sections are weighed. The apical side of
each slice is imaged, and the area of infarction for each slice is
software program (Image Tools, Houston, TX). The size of
infarction is determined by the following equations:
Weight of infarction ¼ ðA1? Wt1Þ þ ðA2? Wt2Þ þ ðA3
? Wt3Þ þ ðA4? Wt4Þ
where A is percent area of infarction by planimetry and Wt is the
weight of each section. Percentage of infarcted LV = (weight of
infarction/weight of LV) × 100. Area at risk (AAR) as a
percentage of LV = (weight of LV − weight of LV stained blue)/
weight of LV × 100).
2.3. Immunohistochemistry for neutrophils
Neutrophils in the infarcts were analyzed after 24 h re-
perfusion as previous studies showed neutrophil infiltration
peak after 24 h reperfusion . Tissues are fixed in 10%
63 M. Zhang et al. / Journal of Molecular and Cellular Cardiology 41 (2006) 62–67
paraformaldehyde or Z-fix (Anatech, Battle Creek, MI) for at
least 3 h, dehydrated with ethanol (50–100%), cleared with
xylene, and embedded in paraffin. The sections (5 μm thick)
were rehydrated with deionized water in preparation for
immunohistochemistry. Staining for neutrophils was performed
as described previously . Briefly, rat anti-mouse neutrophil
monoclonal antibody AMU-0021 (Biosource, Camarillo, CA)
was used as a primary antibody and anti-rat IgG biotinylated
monoclonal antibody as a secondary antibody (Vectastain, ABC
Kit Peroxidase Rat IgG PK-4004, Vector Laboratories, Burlin-
game, CA) . Neutrophils were counted manually using an
Axioskop microscope (Carl Zeiss) at a power of 400×.
Neutrophil numbers in each section were expressed per 400×
power field of section.
2.4. Assessment of cardiomyocyte apoptosis
To determine the prevalence of apoptosis after LAD
occlusion, an in situ DNA ligase technique (Serologicals,
Norcross, GA) was used that identifies only double-stranded
DNA breaks with single 3′ base pair overhangs that are more
characteristic of DNA breaks that occur during apoptosis .
Studies were performed in myocardial sections (n = 10/heart)
obtained 6 h after LAD ligation. Previous studies showed that
the maximal frequency of apoptosis occurred at 6 h in the WT
mice, and there was no significant difference in the frequency of
apoptosis between 6 and 24 h [34,35]. Apoptotic cell nuclei
detected by the ligase assay were stained with fluorescein.
Sections were counterstained with the nucleic acid binding dye
DAPI (4′,6-diamidino-2-phenylindole) to visualize the entire
population of cell nuclei within each myocardial section. Two
different sections per slice and per heart were analyzed for each
experiment. To determine the fraction of myocyte nuclei that
were labeled, we determined the total number of myocyte nuclei
per unit area of the myocardium (10,000 μm2) by enumerating
the number of DAPI (4,6-diamidino-2-phenylindole)-stained
per 10,000 μm2) × 100%.
To examine the role of natural IgM in myocardial infarction,
1−/−) in IgM were examined in a myocardial I/R model .
Previous study of peripheral blood leukocytes in Cr2−/−mice
ratios of peripheral mononuclear cells to granulocytes compared
1a cells reduce 50% in Cr2−/−mice . In the present
myocardial I/R study, infarct size was significantly reduced
two-fold in Cr2−/−mice versus WT controls after 1 h LAD
16.8 + 3.8 respectively, P < 0.05, n = 6 per group) (Fig. 1). A
similar reduction was observed in RAG-1−/−mice (% infarct/
AAR: 8.7 + 2.3, P < 0.01 compared with WTcontrols, n = 6 per
surgery (i.v. 400 μg WT IgM). Notably, infarct sizes similar to
IgM = 24.4 + 5.5; P = 0.171 compared with WTcontrols; RAG-
1−/−with WT IgM = 24.3 + 2.7; P = 0.054 compared with WT
controls; WT mice = 34.8 + 4; n = 6 per group) (Fig. 2). Thus,
similar to the intestinal and hind limb models, natural IgM can
directly mediate myocardial damage in infarction.
Myocardial I/R injury in this model is characterized by both
necrosis and apoptosis . To determine if natural IgM is
involved in apoptosis of cardiac myocytes, an in situ DNA
ligase assay was performed to assess the frequency of apoptotic
cells in the AAR at 6 h post-reperfusion [34,36]. A two-fold
reduction in the frequency of apoptotic cardiomyocytes was
observed in hearts prepared from Cr2−/−mice relative to that of
WT controls (% Positive Ligase Nuclei: 2.5 ± 0.4 vs. 5.2 ± 1.1,
respectively, P < 0.05, number of WT mice = 11, number of
Cr2−/−mice = 7) (Fig. 3). Reconstitution of Cr2−/−mice with
WT IgM restored apoptosis to the WT level (4.9 ± 1.3, n = 4)
A hallmark of myocardial I/R injury is neutrophil infiltration
[2,11,37,38]. To determine if natural IgM operates upstream of
neutrophil infiltration, heart sections were characterized by
immunohistochemistry after LAD occlusion of 1 h and reperfu-
sionfor 24h.Approximatelytwo-foldreduction ofthe neutrophil
number in the infarct zone was observed in hearts prepared from
Cr2−/−mice (neutrophil number/power field: WT = 101 ± 36;
Cr2−/−= 55 ± 24, P < 0.05; number of WT mice = 11, number of
Cr2−/−mice = 7) (Fig. 4). Furthermore, reconstitution of Cr2−/−
mice with WT IgM prior to ischemia restored neutrophil
infiltration to that of WT level (102 ± 26, n = 4) (Fig. 4).
Our results identified a pathogenic role for natural IgM in
myocardial reperfusion injury. We found more than two-fold of
Fig. 1. Decreased myocardial infarct size in Cr2−/−and RAG−/−mice. Animals
were subjected to LAD occlusion for 1 h followed by 24 h reperfusion.
Myocardial infarcts were delineated by Evans Blue and TTC staining. Infarct
size is expressed as a percentage infarct of area at risk (AAR). Results represent
means ± SEM (*P < 0.05, **P<0.01 compared with WT control, n = 6 per
64M. Zhang et al. / Journal of Molecular and Cellular Cardiology 41 (2006) 62–67
reduction in infarct size in both Cr2−/−and RAG1−/−mice
compared with WT controls in the LAD occlusion model (Fig.
1). Moreover, neutrophil infiltration and apoptosis of cardio-
myocytes were reduced in Cr2−/−versus WT mice (Figs. 3 and
4). The limited injury is due to IgM as reconstitution of the IgM
deficient mice with pooled WT IgM restored full injury (Fig. 2).
One interpretation of the results is that neoepitope(s) are
expressed or exposed during myocardial ischemia and recog-
nized by specific natural IgM following reperfusion. This
possibility is supported by similar findings in our studies of I/R
injury in Ig deficient mice in both intestinal and hind limb
The molecular events leading to the exposure of self-antigen
during I/R are unknown. One possibility is that organ ischemia,
thus, hypoxia to cells, leads to cell membrane disturbance
revealing intracellular components. It was reported that, during
the earliest phase of reperfusion (minutes), cardiomyocytes
could develop hypercontracture and concomitantly rupture the
cellular membrane . In addition, the generation of reactive
oxygen species (ROS) duringischemia followed by a ROS burst
during reperfusioninduces lipid peroxidation  and alteration
of cytoskeletal structures , thus, possibly exposes specific
self-antigen. Alternatively, apoptosis during ischemia could
expose self-antigen resulting in binding of IgM in reperfusion.
How natural IgM induces direct apoptosis in reperfused
myocardium remains unclear; however, IgM-dependent apo-
ptosis was also observed in normal and tumor epithelial cells
[41,42]. Presumably in the latter two cases, cell death pathway
was triggered by direct IgM binding to a neoepitope on the cell
Acute inflammation could be induced by IgM–antigen
interaction through activation of the complement system which
produces the potent anaphylatoxins C3a and C5a, activating
mast cells , and neutrophils [2,11]. In a recent report,
Krijnen et al. showed that IgM colocalizes with complement
and C reactive protein in infarcted human myocardium,
indicating that IgM targets complement locally to jeopardized
cardiomyocytes after acute myocardial infarction . Evi-
dence of complement activation in myocardial I/R includes
deposition of terminal complement complex within reperfused
heart tissues [45,46] and reduction in myocardial injury in
complement deficient animals  and in animals treated with
complement inhibitors [48,49].
Recent studies also suggested a role lectin pathway of
complement in both intestinal  and myocardial  I/R
models. In those reports, inhibition of mannose binding
lectin (MBL) blocked complement activation and led to a
reduction in I/R injury. Given our results with IgM and the
observation that MBL binds IgM [19,20], a unifying model
explaining both sets of results is that IgM first recognizes
altered cell surface providing a binding site for MBL which
subsequently leads to activation of the lectin-complement
pathway. Indeed, preliminary results identifying IgM binding
to ischemic intestinal tissue in MBL-a/c double knockout
mice support this speculation (Zhang and Carroll, unpub-
In summary, the current study supports a novel mechanism
of myocardial reperfusion injury mediated by natural IgM, and
Fig. 3. Reduced apoptosis of cardiac myocytes in Cr2−/−mice and restoration of
apoptosis by reconstitution of WT IgM. Apoptosis frequencies of cardiac
myocytes were examined after 6 h of reperfusion. Numbers of experimental
animals: WT = 11, Cr2−/−= 7, Cr2−/−+ IgM = 4 (*P < 0.01 comparing Cr2−/−
with WT mice, **P < 0.05 comparing Cr2−/−mice with or without WT IgM).
Results represent means ± SEM.
Fig. 4. Decreased neutrophil infiltration in Cr2−/−mice and restored by WT IgM
reconstitution. Neutrophil density in the infarct was quantified after 24 h
reperfusion. Numbers of experimental mice: WT = 11, Cr2−/−= 7, Cr2−/−+ IgM =
4 (*P < 0.01 comparing Cr2−/−with WTmice, **P < 0.05 comparing Cr2−/−mice
with or without WT IgM). Results represent means ± SEM.
Fig. 2. Restoration of myocardial reperfusion injury in Cr2−/−and RAG−/−mice
by WT IgM. Animals were reconstituted with 400 μg WT IgM by intravenous
injection 30 min prior to myocardial I/R surgery. Infarct size was assessed as
described in Fig. 1 (n = 6 per group). Results represent means ± SEM.
65 M. Zhang et al. / Journal of Molecular and Cellular Cardiology 41 (2006) 62–67
further understanding of this mechanism may help develop
clinical strategies for ischemic heart disease.
We thank Jennifer Pocius for excellent scientific support;
Isaac Chiu for helpful discussions and critical comments.
Research was supported by grants from NIH: P50 GM52585
(MCC) and HL-42550 (MLE).
 Garcia-Dorado D. Myocardial reperfusion injury: a new view. Cardiovasc
 Frangogiannis NG, Smith CW, Entman ML. The inflammatory response in
myocardial infarction. Cardiovasc Res 2002;53:31–47.
 Eltzschig HK, Collard CD. Vascular ischaemia and reperfusion injury. Br
Med Bull 2004;70:71–86.
 Piper HM, Abdallah Y, Schafer C. The first minutes of reperfusion: a
window of opportunity for cardioprotection. Cardiovasc Res 2004;61:
 Schulz R, Kelm M, Heusch G. Nitric oxide in myocardial ischemia/
reperfusion injury. Cardiovasc Res 2004;61:402–13.
 Eefting F, Rensing B, Wigman J, Pannekoek WJ, Liu WM, Cramer MJ,
et al. Role of apoptosis in reperfusion injury. Cardiovasc Res 2004;61:
 Becker LB. New concepts in reactive oxygen species and cardiovascular
reperfusion physiology. Cardiovasc Res 2004;61:461–70.
 Halestrap AP, Clarke SJ, Javadov SA. Mitochondrial permeability
transition pore opening during myocardial reperfusion—A target for
cardioprotection. Cardiovasc Res 2004;61:372–85.
 Armstrong SC. Protein kinase activation and myocardial ischemia/
reperfusion injury. Cardiovasc Res 2004;61:427–36.
 Szabo G, Liaudet L, Hagl S, Szabo C. Poly(ADP-ribose) polymerase
activation in the reperfused myocardium. Cardiovasc Res 2004;61:
 Vinten-Johansen J. Involvement of neutrophils in the pathogenesis of
lethal myocardial reperfusion injury. Cardiovasc Res 2004;61:481–97.
 Hill JH, Ward PA. The phlogistic role of C3 leukotactic fragments in
myocardial infarcts of rats. J Exp Med 1971;133:885–900.
 Weisman HF, Bartow T, Leppo MK, Marsh Jr HC, Carson GR, Concino
MF, et al. Soluble human complement receptor type 1: in vivo inhibitor of
complement suppressing post-ischemic myocardial inflammation and
necrosis. Science 1990;249:146–51.
 Park JL, Lucchesi BR. Mechanisms of myocardial reperfusion injury. Ann
Thorac Surg 1999;68:1905–12.
 Hart ML, Walsh MC, Stahl GL. Initiation of complement activation
following oxidative stress. In vitro and in vivo observations. Mol Immunol
 Hill J, Lindsay TF, Ortiz F, Yeh CG, Hechtman HB, Moore Jr FD. Soluble
complement receptor type 1 ameliorates the local and remote organ injury
after intestinal ischemia–reperfusion in the rat. J Immunol 1992;149:
 Lindsay TF, Hill J, Ortiz F, Rudolph A, Valeri CR, Hechtman HB, et al.
Blockade of complement activation prevents local and pulmonary albumin
leak after lower torso ischemia–reperfusion. Ann Surg 1992;216:677–83.
 Carroll MC. The role of complement and complement receptors in
induction and regulation of immunity. Annu Rev Immunol 1998;16:
 Nevens JR, Mallia AK, Wendt MW, Smith PK. Affinity chromatographic
purification of immunoglobulin M antibodies utilizing immobilized
mannan binding protein. J Chromatogr 1992;597:247–56.
 Arnold JN, Wormald MR, Suter DM, Radcliffe CM, Harvey DJ, Dwek
RA, et al. Human serum IgM glycosylation: identification of glycoforms
that can bind to mannan-binding lectin. J Biol Chem 2005;280:29080–7.
 Weiser MR, Williams JP, Moore FD, Kobzik L, Ma M, Hechtman HB,
et al. Reperfusion injury of ischemic skeletal muscle is mediated by natu-
ral antibody and complement. J Exp Med 1996;183:2343–8.
 Williams JP, Pechet TT, Weiser MR, Reid R, Kobzik L, Moore FD, Carroll
MC, Hechtman HB. Intestinal reperfusion injury is mediated by IgM and
complement. J Appl Physiol 1999;86:938–42.
 Ahearn JM, Fischer MB, Croix D, Goerg S, Ma M, Xia J, et al. Disruption
of the Cr2 locus results in a reduction in B-1a cells and in an impaired B
cell response to T-dependent antigen. Immunity 1996;4:251–62.
 Molina H, Holers VM, Li B, Fung Y, Mariathasan S, Goellner J, et al.
Markedly impaired humoral immune response in mice deficient in
 Fleming SD, Shea-Donohue T, Guthridge JM, Kulik L, Waldschmidt TJ,
Gipson MG, et al. Mice deficient in complement receptors 1 and 2 lack a
tissue injury-inducing subset of the natural antibody repertoire. J Immunol
 Reid RR, Woodcock S, Shimabukuro-Vornhagen A, Austen Jr WG,
Kobzik L, Zhang M, et al. Functional activity of natural antibody is altered
in Cr2-deficient mice. J Immunol 2002;169:5433–40.
 Zhang M, Austen Jr WG, Chiu I, Alicot EM, Hung R, Ma M, et al.
Identification of a specific self-reactive IgM antibody that initiates
intestinal ischemia/reperfusion injury. Proc Natl Acad Sci USA
 Austen Jr WG, Zhang M, Chan R, Friend D, Hechtman HB, Carroll MC,
et al. Murine hindlimb reperfusion injury can be initiated by a self-
reactive monoclonal IgM. Surgery 2004;136:401–6.
 Michael LH, Entman ML, Hartley CJ, Youker KA, Zhu J, Hall SR, et al.
Myocardial ischemia and reperfusion: a murine model. Am J Physiol
 Nossuli TO, Lakshminarayanan V, Baumgarten G, Taffet GE, Ballantyne
CM, Michael LH, et al. A chronic mouse model of myocardial ischemia–
reperfusion: essential in cytokinestudies. Am J Physiol, Heart Circ Physiol
 Mombaerts P, Iacomini J, Johnson RS, Herrup K, Tonegawa S,
Papaioannou VE. RAG-1-deficient mice have no mature B and T
lymphocytes. Cell 1992;68:869–77.
 Dewald O, Ren G, Duerr GD, Zoerlein M, Klemm C, Gersch C, et al. Of
mice and dogs: species-specific differences in the inflammatory response
following myocardial infarction. Am J Pathol 2004;164:665–77.
 BriaudSA, Ding ZM, Michael LH, Entman ML, Daniel S, Ballantyne CM.
Leukocyte trafficking and myocardial reperfusion injury in ICAM-1/P-
selectin-knockout mice. Am J Physiol, Heart Circ Physiol 2001;280:
 Kurrelmeyer KM, Michael LH, Baumgarten G, Taffet GE, Peschon JJ,
Sivasubramanian N, et al. Endogenous tumor necrosis factor protects the
adult cardiac myocyte against ischemic-induced apoptosis in a murine
model of acute myocardial infarction. Proc Natl Acad Sci USA
 Misra A, Haudek SB, Knuefermann P, Vallejo JG, Chen ZJ, Michael LH,
et al. Nuclear factor-kappaB protects the adult cardiac myocyte against
ischemia-induced apoptosis in a murine model of acute myocardial
infarction. Circulation 2003;108:3075–8.
 Engel D, Peshock R, Armstong RC, Sivasubramanian N, Mann DL.
Cardiac myocyte apoptosis provokes adverse cardiac remodeling in
transgenic mice with targeted TNF overexpression. Am J Physiol, Heart
Circ Physiol 2004;287:H1303–11.
 Engler RL, Dahlgren MD, Peterson MA, Dobbs A, Schmid-Schonbein
GW. Accumulation of polymorphonuclear leukocytes during 3-h experi-
mental myocardial ischemia. Am J Physiol 1986;251:H93–H100.
 Jolly SR, Kane WJ, Hook BG, Abrams GD, Kunkel SL, Lucchesi BR.
Reduction of myocardial infarct size by neutrophil depletion: effect of
duration of occlusion. Am Heart J 1986;112:682–90.
 Nanobashvili J, Neumayer C, Fugl A, Punz A, Blumer R, Prager M, et al.
Ischemia/reperfusion injury of skeletal muscle: plasma taurine as a
measure of tissue damage. Surgery 2003;133:91–100.
 VanWinkle WB, Snuggs M, Miller JC, Buja LM. Cytoskeletal alterations
in cultured cardiomyocytes following exposure to the lipid peroxidation
product, 4-hydroxynonenal. Cell Motil Cytoskelet 1994;28:119–34.
66M. Zhang et al. / Journal of Molecular and Cellular Cardiology 41 (2006) 62–67
 Vollmers HP, Brandlein S. Death by stress: natural IgM-induced apoptosis. Download full-text
Methods Find Exp Clin Pharmacol 2005;27:185–91.
 Li CX, Wan YH, Chi SM, Wang G, Sun LC, Zhang YG, et al. Purification
of natural antikeratin autoantibodies from normal human serum and their
effect on human keratinocytes cultured in vitro. Br J Dermatol
 Abonia JP, Friend DS, Austen Jr WG, Moore Jr FD, Carroll MC, Chan R,
et al. Mast cell protease 5 mediates ischemia–reperfusion injury of mouse
skeletal muscle. J Immunol 2005;174:7285–91.
 Krijnen PA, Ciurana C, Cramer T, Hazes T, Meijer CJ, Visser CA, et al.
IgM colocalises with complement and C reactive protein in infarcted
human myocardium. J Clin Pathol 2005;58:382–8.
 Schafer H, Mathey D, Hugo F, Bhakdi S. Deposition of the terminal C5b-9
complement complex in infarcted areas of human myocardium. J Immunol
 Robert-Offerman SR, Leers MP, van Suylen RJ, Nap M, Daemen MJ,
Theunissen PH. Evaluation of the membrane attack complex of
complement for the detection of a recent myocardial infarction in man. J
 Kilgore KS, Park JL, Tanhehco EJ, Booth EA, Marks RM, Lucchesi
BR. Attenuation of interleukin-8 expression in C6-deficient rabbits
after myocardial ischemia/reperfusion. J Mol Cell Cardiol 1998;30:
 Vakeva AP, Agah A, Rollins SA, Matis LA, Li L, Stahl GL. Myocardial
infarction and apoptosis after myocardial ischemia and reperfusion: role of
the terminal complement components and inhibition by anti-C5 therapy.
 Roos A, Ramwadhdoebe TH, Nauta AJ, Hack CE, Daha MR. Therapeutic
inhibition of the early phase of complement activation. Immunobiology
 Hart ML, Ceonzo KA, Shaffer LA, Takahashi K, Rother RP, Reenstra WR,
et al. Gastrointestinal ischemia–reperfusion injury is lectin complement
pathway dependent without involving C1q. J Immunol 2005;174:
 Walsh MC, Bourcier T, Takahashi K, Shi L, Busche MN, Rother RP,
et al. Mannose-binding lectin is a regulator of inflammation that
accompanies myocardial ischemia and reperfusion injury. J Immunol
67M. Zhang et al. / Journal of Molecular and Cellular Cardiology 41 (2006) 62–67