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Cardiovascular Research 2003 57(2):523-534; doi:10.1016/S0008-6363(02)00697-1
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Copyright © 2003, European Society of Cardiology

Cardiac overexpression of monocyte chemoattractant protein-1 in transgenic mice mimics ischemic preconditioning through SAPK/JNK1/2 activation

Alessandra Martirea,*, Borja Fernandeza, Alexandra Buehlera, Claudia Strohma, Jutta Schapera, René Zimmermannb, Pappachan E Kolattukudyc and Wolfgang Schapera

aDepartment of Experimental Cardiology, Max-Planck-Institute, Benekestrasse 2, D-61231 Bad Nauheim, Germany
bKerckhoff-Clinic, Bad Nauheim, Germany
cNeurobiotechnology Center, The Ohio State University, Columbus, OH, USA

* Corresponding author. Tel.: +49-6032-705-402; fax: +49-6032-705-419. a.martire{at}kerckhoff.mpg.de

Received 22 March 2002; accepted 13 September 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 
Objective and methods: Although a beneficial association between innate immunity and ischemic preconditioning has recently been proposed, the mechanisms responsible for this link are poorly understood. To test the hypothesis that pro-inflammatory cytokines have a beneficial role in the activation of the cell survival pathway mediated by ischemic preconditioning, we have studied transgenic mice with cardiac myocyte specific overexpression of murine monocyte chemoattractant protein-1 (MCP-1). The resistance to ischemia was studied by performing 45-min (with or without injection of the SAPK/JNKs inhibitor D-JNKI1) and 3-day left coronary artery occlusions as well as 45-min left coronary artery occlusion followed by 3 days of reperfusion. In addition, quantitative Western blot analyses for TNF-{alpha}, and SAPK/JNK1/2, ERK1/2 and p38 activity were performed. Results: Infarct size, expressed in percent of either the risk area or the left ventricle, was reduced in transgenic mice when compared with control after both, 45-min (14.7±2.6% vs. 52.0±2.4%; P<0.05) and 45-min occlusion followed by 3 days of reperfusion (23.2±1.8% vs. 30.0±1.8%; P<0.05) but it was not significantly different for 3-day occlusion. Western blot analyses showed significantly increased levels of TNF-{alpha} (1.8-fold) and phosphorylated-SAPK/JNK1/2 (1.5-fold) in transgenic hearts. Phosphorylated-ERK1/2, and phosphorylated-p38 levels were unchanged. Immunohistochemistry revealed that in transgenic mice monocytes/macrophages, lymphocytes, and fibroblasts are the source of TNF-{alpha}, whereas myocytes have increased phosphorylated-SAPK/JNK1/2 levels. In addition, injection of the SAPK/JNKs inhibitor D-JNKI1 partially abrogated the cardioprotective effect observed in untreated transgenic mice. Conclusion: Overexpression of MCP-1 by cardiomyocytes causes chronic infiltration and activation of leukocytes, resulting in elevated TNF-{alpha} secretion and SAPK/JNK1/2 activation. The activation of this pathway is in part responsible for the preconditioning effect of MCP-1 overexpression. These results show a possible beneficial link between innate immunity and ischemic preconditioning through MAP-kinase activation.

KEYWORDS Cytokines; Ischemia; Leukocytes; Preconditioning; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Material and methods
 3. Results
 4. Discussion
 References
 
The innate immune system is a preprogrammed nonspecific host defense mechanism against pathogens. It is based on activation of myeloid-derived cells that secrete pro-inflammatory cytokines and chemokines, which control recruitment and activation of leukocytes at the sites of infection [1]. Recently, it has emerged that TNF-{alpha}, a component of the innate immune system, plays a role in the protection of the myocardium against viral myocarditis [2] and ischemic injury [3,4].

Ischemic preconditioning (PC) is the endogenous mechanism of the myocardium to protect itself against infarction. PC consists of short transient periods of ischemia that confer resistance against cardiomyocyte death after a subsequent prolonged coronary occlusion [5–7]. Currently, the search for endogenous or pharmacological substances able to mimic PC is one of the most important challenges in cardiovascular research.

Recently, Smith et al. have proposed a beneficial link between innate immunity and PC [8]. These authors suggest that the recruitment and activation of inflammatory cells and the production of cytokines play a role not only in the healing and remodeling process after myocardial infarction but also in the cardioprotective effects promoted and initiated by PC. This group and others have proposed an association between TNF-{alpha} production and PC in different animal models [8–10]. The possible cardioprotective pathway activated by TNF-{alpha} during ischemia or ischemia/reperfusion is still poorly understood but it has been suggested to be mediated by MAP-kinases (MAPKs) activation [11–13].

The MAPKs are a superfamily of Pro-directed Ser/Thr cytoplasmic protein kinases involved in signal transduction pathway from extracellular stimuli to the nucleus [14]. The MAPKs include at least three different protein kinase subfamilies: the ERKs, the SAPK/JNKs, and the p38. MAPKs are activated by different stimuli. The ERKs are mostly activated by growth factors such as FGF, IGF, EGF, Ang II, and ET-1 [15,16]. SAPK/JNKs and p38 are activated by cellular stress such as heat shock, ultraviolet radiation, protein synthesis inhibitors, ischemia, ischemia/reperfusion, or pro-inflammatory cytokines such as TNF-{alpha} and IL-1 [17,18]. Activated leukocytes are the major source of TNF-{alpha} and IL-1 [19,20]. In recent years, several groups have shown that different members of the MAPKs are activated during PC [21–24].

Recently, a transgenic (TG) strain of mice that overexpress murine MCP-1 in the heart has been generated [25]. Kolattukudy et al. showed that young TG mice exhibit focal accumulations of monocytes/macrophages in the heart leading to ischemic cardiomyopathy [25,26] and a gradual loss of myocytes in elderly [26].

Therefore, with this background we have postulated the hypothesis that activated leukocytes expressing TNF-{alpha} in the heart of MCP-1 TG mice might confer myocardial protection against ischemia by activating the MAPKs pathway. To test this hypothesis we investigated the cardiac resistance to ischemia in TG and control (WT) (non-transgenic mice from the same litter) mice with and without injection of the SAPK/JNKs inhibitor D-JNKI1. In addition, histopathological examinations of MCP-1 TG hearts were performed, as well as quantitative analysis of TNF-{alpha}, phosphorylated-SAPK/JNK1/2, phosphorylated-ERK1/2, and phosphorylated-p38 protein levels.


   2. Material and methods
Top
 Abstract
 1. Introduction
 2. Material and methods
3. Results
 4. Discussion
 References
 
2.1 Animals
The generation of MCP-1 TG mice from FVB/N mice has been previously described [25]. In summary, the construct consists of the cDNA of the murine MCP-1 (JE) gene, which is linked downstream to the {alpha}-cardiac myosin heavy chain promoter in order to confer cardiac specific expression. WT and TG mice were selected by Southern blot and PCR analyses. The total number of mice used for all the experiments was 39 WT, 3–10 months old, and 43 TG, 3–9 months old.

Housing and handling of the animals was in accordance with the American Physiological Society guidelines for animal welfare and the Bioethical Committee of the District of Darmstadt, Germany.

2.2 Coronary artery occlusion (CAO)
We performed 45-min and 3-day CAO, and 45-min CAO followed by 3 days of reperfusion (45-min CAO + 3-day R) in WT (n=12) and TG (n=14) mice (~4 months old).

The mice were anaesthetized with an intraperitoneal injection of 10% Ketamin (0.1 mg/g body weight) and 2% Xylazin (0.005 mg/g body weight) and the trachea was intubated with a 1.1-mm steel tube connected to an animal ventilator. Heparin solution (125 IU) was injected intraperitoneally to prevent microthrombus formation during occlusion and reperfusion. The body temperature was kept at 37 °C. Heart function was continuously recorded with a 3-lead surface electrocardiogram (ECG). After opening the skin overlying the left part of the thoracic cage the pectoral muscles were prepared free and retracted with a 6–0 silk suture. Subsequently, the chest was opened through the third or fourth intercostal space by dissecting the intercostal muscles. Lungs were retracted with a moistened sponge and a retractor was introduced to gain access to the thoracic cage. Ligation was performed proximal to the main branch of the left coronary artery, using a 6–0 prolene suture (for 45 min of CAO and 45-min CAO + 3-day R, a PE-10 tube was also used to allow removal of the ligation). When perfusion was stopped by ligation, changes in the ECG and tissue color were assessed.

In animals subjected to short-term CAO, 45 min after the ligation the tube was removed and the reperfusion of the coronary artery visually assessed and confirmed by the changes in the ECG. Propidium iodide (Sigma, 0.05% in 0.9% NaCl solution) was perfused via a catheter into the right carotid artery and allowed to circulate for 10 min. After 10 min, the coronary artery was re-occluded and Thioflavin S (Sigma, 4% in 0.9% NaCl solution) was injected into the carotid artery for a few seconds. The heart was then removed, the right ventricle excised, and the left ventricle cryopreserved with nitrogen-cooled methylbutane.

Mice receiving 3-day CAO and 45-min CAO + 3-day R were assessed for coronary ligation efficiency, and coronary ligation and reperfusion efficiency, respectively. The chest was then closed in layers using a 4–0 silk suture. The animal ventilator was disconnected when the mice started to wake up, and the mice were kept warm with a heat lamp until complete recovery.

2.3 Experimental protocol for the SAPK/JNKs inhibitor D-JNKI1
A 500-µl solution of a sub-lethal dose of the JNKs inhibitor D-JNKI1 (Alexis Biochemicals, 0.25 mg/body weight in 0.9% NaCl solution/DMSO) or 500 µl solution of DMSO (0.5% in 0.9% NaCl solution) were injected in the tail artery in TG (n=7) and WT (n=6) mice 30 min before CAO. After the injections, we performed 45-min CAO as described above.

2.4 Determination of infarct size
Two methods were employed to determine infarct size: propidium iodide (for 45 min CAO) [27,28] and triphenyl tetrazolium chloride (TTC) (for 3-day CAO and 45-min CAO + 3-day R).

In experiments using propidium iodide, the endothelial cell marker thioflavin S was employed to quantify the non-risk area of the left ventricle. The risk area was defined and measured as thioflavin S-negative. The infarct area was defined as the propidium iodide-positive regions. Left ventricular cryosections 14-µm thick were cut and photographed under fluorescent light with a DM-RB Leica microscope. The total left ventricle, risk area and infarcted areas were determined using computer assisted planimetry software (NIH Image). Finally, three ratios were obtained: infarct area/risk area (IA/RA), infarct area/left ventricle (IA/LV), and risk area/left ventricle (RA/LV).

In experiments with 3-day CAO and 45-min CAO + 3-day R, after 3 days the animals were killed by an anesthetic overdose (10% Ketamin and 2% Xylazin injected intraperitoneally), the heart was excised, the right ventricle removed, and the left ventricle was frozen for 5–10 min and cut in slices. The slices were weighed and incubated at 37 °C in 1% TTC in phosphate saline buffer (PBS), pH 7.0, for 20 min. After incubation with TTC, the slides were photographed and the pictures used for planimetric analysis. The left ventricular area and infarct size were determined using computer assisted planimetry software (NIH Image). These measurements provided the IA/LV ratio.

2.5 Western blot analysis
The hearts of WT (n=14) and TG (n=14) animals were excised, snap-frozen in liquid nitrogen and stored at –80 °C until use. The tissue was homogenized with ice-cold buffer containing 20 mM Tris–HCl, 250 mM sucrose, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, 0.1 mM sodium orthovanadate, 10 mM NaF, and 0.5 mM PMSF, pH 7.4. The homogenate was centrifuged at 4 °C and 13,000 g for 30 min and the supernatant was kept for protein analysis. Bio-Rad Protein Assay (Bio-Rad Laboratories) was used to assess the equivalent amount of proteins for immunoblot analysis. We used Laemmli Sample Buffer (Bio-Rad Laboratories) and 5 min denaturation by heating at 95 °C for electrophoresis protein preparation. Electrophoresis was performed in 10% and 18% Tris–HCl ready gel (Bio Rad), and proteins were transferred onto nitrocellulose membranes and blocked overnight with 5% nonfat dry milk. After blockage, the membranes were incubated overnight with the first antibody. Polyclonal antibodies against JNK1 (Santa Cruz Biotechnology), phosphorylated-SAPK/JNK1/2 (Cell Signaling Technology), ERK2 (Santa Cruz Biotechnology), phosphorylated-ERK1/2 (Cell Signaling Technology), p38 (Santa Cruz Biotechnology), phosphorylated-p38 (Cell Signaling Technology), and TNF-{alpha} (Santa Cruz Biotechnology) were used. After washing, the membranes were incubated 2 h with peroxidase-conjugated anti-rabbit IgG (Amersham). The ECL-system (Amersham Pharmacia Biotech) was used for signal detection and quantification was performed on STORM 860 (Amersham Pharmacia Biotech), using ImageQuan software.

2.6 Terminal dUTP deoxynucleotidyltransferase nick end-labeling (TUNEL) assay
TUNEL staining was performed in three randomly chosen cryosections from the hearts of WT (n=3) and TG (n=5) mice. The In Situ Cell Death detection Kit Fluorescein (Roche Diagnostics) was used according to the manufacturer's instructions. Cryosections of duodenum were used as positive control.

2.7 Electron microscopy
The hearts from additional WT (n=2) and TG (n=3) animals were perfusion-fixed with a mixture of 2% paraformaldehyde and 1% glutaraldehyde in PBS, immersed in 3% glutaraldehyde, post-fixed with osmium tetroxide, dehydrated in a series of ethanol, and embedded in epoxy resin following routine methods. Ultramicrotome sections were counterstained with uranyl acetate and Reynolds lead citrate and photographed with a Philips CM10 electron microscope.

2.8 Histology and immunohistochemistry
For the analysis of specific structural changes due to MCP-1 overexpression, additional animals (WT, n=5; TG, n=5) were killed by anesthetic overdose (10% Ketamin, 2% Xylazin) injected intraperitoneally. The hearts were dissected and cryopreserved with nitrogen-cooled methylbutane and stored at –80 °C until use. Alternatively, the hearts were perfusion fixed, first with 0.01% adenosine in PBS, and then with 4% paraformaldehyde in PBS. The hearts were embedded in paraffin, cut in 5-µm sections and stained with hematoxilin and eosin (H&E).

For immunohistochemistry three primary antibodies were used. A FITC-conjugated monoclonal antibody against the transferrin receptor CD 71 (Ancell), a polyclonal antibody against TNF-{alpha} (Santa Cruz Biotechnology), and a polyclonal antibody against phosphorylated-SAPK/JNK1/2 (Cell Signaling Technology). Cryosections, 5-µm thick, were air dried and fixed with 4% paraformaldehyde or ice-cold acetone for 10 min. In addition, the sections used for TNF-{alpha} and phosphorylated-SAPK/JNK1/2 antibodies were incubated with 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. The sections used for phosphorylated-SAPK/JNK1/2 antibody were then immersed in citrate buffer solution (pH 6) and cooked in a microwave for 15 min. After several washing steps in PBS, the slides were incubated in a blocking solution containing 0.1% bovine serum albumin and 0.4% glycine for 30 min. After overnight incubation with the primary antibodies, the sections were washed with PBS. The sections treated with CD 71 antibody were incubated with the nuclear marker 4',6-diamidino-2-phenylindole dilactate (DAPI) (Molecular Probes) for 10 min, and those treated with TNF-{alpha} and phosphorylated-SAPK/JNK1/2 antibodies were incubated with peroxidase-conjugated anti-rabbit IgG (Amersham). Peroxidase was detected by incubation with 3,3'-diaminobenzidine (Sigma), and the nuclei were counterstained with hematoxilin. Omission of the first antibodies was used as negative control. Finally, the sections were evaluated and photographed under transmitted or fluorescent light with a DM-RB Leica microscope.

2.9 Quantitative immunohistochemical analysis by densitometry
Three randomly chosen cryosections from WT (n=3) and TG (n=4) hearts were immunolabeled for phosphorylated-SAPK/JNK1/2 as described above. All sections were stained and photographed using the same conditions. Twenty-four randomly selected phosphorylated-SAPK/JNK1/2-positive myocytes were measured from each section. Quantitative intensity level analysis was performed using computer assisted planimetry software (NIH Image).

2.10 Statistical analysis
Results are reported as mean±S.E.M. Unpaired t-test or the Mann–Whitney U-test were used to estimate significant differences between groups. The accepted minimum level of statistical significance was P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
4. Discussion
 References
 
3.1 Infarct size
After 45 min of CAO, the infarct size was 3.5-fold reduced in TG mice when compared to WT (Fig. 1).


Figure 1
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  		Fig. 1 (A) and (B) Transversal section (14 µm) of the left ventricle from WT (A) and TG (B) animals after 45-min CAO. Dead myocytes are stained red with propidium iodide. Endothelial cells from the non-risk area are stained blue with thioflavin S. The risk area is not stained and appears dark; x50. (C) Quantification of the ratios IA/RA, IA/LV, RA/LV, in WT (n=5) and TG (n=5) animals after 45-min CAO.

 
Infarct size did not differ between TG and WT animals after 3 days of CAO (Fig. 2A–C) but it was significantly reduced in TG mice as compared with WT after 45-min CAO + 3-day R (Fig. 2D).


Figure 2
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  		Fig. 2 (A) and (B) Left ventricular slices from WT (A) and TG (B) animals after 3-day CAO. Viable myocardium is stained red by the reaction with TTC. Infarct area appears pale due to lack TTC staining. (C) Quantification of the ratio IA/LV in WT (n=3) and TG (n=4) animals after 3-day CAO. No significant difference in the infarct size can be detected between WT and TG animals at this time point after CAO. (D) Quantification of the ratio IA/LV in WT (n=4) and TG (n=4) after 45-min CAO + 3-day R.

 
3.2 D-JNKI1 and infarct size
Injection of the SAPK/JNKs inhibitor D-JNKI1 increased significantly infarct size in TG mice as compared with untreated TG mice but did not increase significantly the infarct size in WT mice as compared with untreated WT animals (Fig. 3). Despite the fact that infarct size was still significantly different between TG treated and WT untreated mice, the infarct size of TG treated mice was not significantly different from WT treated mice (Fig. 3). Application of NaCl/DMSO without D-JNKI1 had no influence on infarct size in TG and WT animals (data not shown).


Figure 3
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  		Fig. 3 Infarct size determination after injection of the SAPK/JNKs inhibitor D-JNKI1 followed by 45-min CAO in WT (n=4) and TG (n=5) mice. WT and TG treated mice (+inh in the figure) were compared to each other and with WT and TG untreated (45-min CAO without inhibitor) mice.

 
3.3 Western blot analysis
A significant (1.5-fold) increase of the phosphorylated (activated) SAPK/JNK1/2 was found in the hearts of TG animals when compared to WT (Fig. 4A,C). Western blot of JNK1 (inactivated SAPK/JNK1/2) showed similar amounts of non-phosphorylated proteins in WT and TG hearts (Fig. 4B).


Figure 4
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  		Fig. 4 (A) Western blot assay of hearts from WT and TG animals using a polyclonal antibody against phosphorylated-SAPK/JNK1/2 (p-SAPK/JNK1/2 in the figure). The activation is induced in both, p46 and p54 respectively isoforms of SAPK/JNK1/2. (B) Western blot assay of the same WT and TG hearts used in A, using a polyclonal antibody against JNK1 (inactivated SAPK/JNK1/2). (C) Quantification of phosphorylated-SAPK/JNK1/2 proteins in WT (n=4) and TG (n=4) animals. The differences in the phosphorylated-SAPK/JNK1/2 levels between WT and TG mice are significant for both isoforms.

 
In the heart of TG and WT mice no differences were found in the protein levels of phosphorylated-ERK1/2 (Fig. 5A), and phosphorylated-p38 (Fig. 5C). Antibodies against inactivated ERK2 (Fig. 5B) and inactivated p38 (Fig. 5D) revealed equal amounts of non-phosphorylated proteins in WT and TG hearts.


Figure 5
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  		Fig. 5 (A) Western blot analysis of hearts from WT and TG mice using a polyclonal antibody against phosphorylated-ERK1/2 (p44 and p42 isoforms, respectively). No statistical differences in the protein levels can be detected between WT (n=5) and TG (n=5) animals. (B) Western blot analysis of the same WT and TG hearts used in A using a polyclonal antibody against ERK2 (inactivated ERK2). (C) Western blot analysis of hearts from WT and TG mice using a polyclonal antibody against phosphorylated-p38. No statistical by significant differences in the protein levels can be detected between WT (n=5) and TG (n=5) animals. (D) Western blot analysis of the same WT and TG hearts used in C using a polyclonal antibody against p38 (inactivated p38).

 
TNF-{alpha} expression was significantly (1.8-fold) increased in the myocardium of TG mice when compared to WT (Fig. 6).


Figure 6
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  		Fig. 6 (A) Western blot analysis of hearts from WT and TG animals, using a polyclonal antibody against TNF-{alpha}. The band at 26 kDa probably corresponds to a transmembrane precursor form of TNF-{alpha}, the band at 17 kDa corresponds to the active form of TNF-{alpha}. (B) Quantification of TNF-{alpha} protein in WT (n=4) and TG (n=4) hearts. The heart samples used for TNF-{alpha} protein level quantification were the same used for phosphorylated-SAPK/JNK1/2 protein quantification by Western blot.

 
3.4 Apoptosis
In order to investigate the possibility that activation of SAPK/JNK1/2 might cause apoptosis in the heart of TG mice, we used an apoptosis detection system. TUNEL-positive cardiomyocytes were detected neither in TG nor in WT animals (Fig. 7). TUNEL-positive interstitial cells were occasionally detected in TG hearts (Fig. 7A). In the duodenum, used as positive control, many TUNEL-positive cells were found (not shown).


Figure 7
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  		Fig. 7 TUNEL staining (red) in TG (A) and WT (B) mice. No TUNEL-positive myocytes are present in (A) and (B). Arrow, TUNEL-positive infiltrating cells; green, phalloidin; blue, DAPI; yellow spots, lipofuscin; x250.

 
3.5 Histomorphology, immunohistochemistry, and ultrastructure
The myocardium of WT animals was of normal histological appearance (Fig. 8A), whereas TG hearts showed accumulation of infiltrating cells in the ventricular myocardial interstitium, surrounding the vessel walls, and in the subepicardial space (Fig. 8B). The cells were identified as monocytes/macrophages, lymphocytes, granulocytes, and fibroblasts (Fig. 8C). In the atrial myocardium of TG mice the infiltrating cells observed were mainly granulocytes (not shown).


Figure 8
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  		Fig. 8 H&E staining. (A) WT heart; x250. (B) TG heart. Arrows, foci of interstitial cells; E, epicardium; x250. (C) TG heart. High magnification of an interstitial cell accumulation. Big arrow, lymphocyte; arrowhead, monocyte/macrophage; small arrow, fibroblast; x630.

 
Immunofluorescence using an antibody against the transferrin receptor (CD71), a marker of activated lymphocytes, showed a high proportion of activated lymphocytes in both the left and right ventricles, surrounding the vessel walls and in the subepicardial space of TG hearts (Fig. 9A). In WT hearts, isolated positive cells were only occasionally detected (not shown). Electron microscopic analysis also revealed the presence of activated monocytes/macrophages in the myocardial interstitium of TG animals (Fig. 9B).


Figure 9
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  		Fig. 9 (A) Immunofluorescence for CD 71 (green). TG heart. The interstitial accumulations are composed of many CD 71-positive cells. E, epicardium; V, coronary vein; x400. (B) TG heart. Electron microscopy. Myocardial interstitium. M, activated monocyte/macrophage; x7000.

 
In TG mice, TNF-{alpha} immunostaining was detected in the myocardial interstitium (Fig. 10B) and in the media of coronary arteries (not shown). TNF-{alpha}-positive interstitial cells were identified as monocytes/macrophages, lymphocytes, and fibroblasts (Fig. 10C). In WT hearts, weak TNF-{alpha} immunostaining was found in the arterial media and in the myocardial interstitium (Fig. 10A).


Figure 10
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  		Fig. 10 Immunohistochemistry for TNF-{alpha}. (A) WT heart. TNF-{alpha} staining was found mainly in the media of the vessels (arrows) and weakly in the myocardial interstitium; x250. (B) TG heart. TNF-{alpha} staining is located in the myocardial interstitium; E, epicardium; x250. (C) TG heart. High magnification of leukocyte accumulations showing TNF-{alpha}-positive interstitial cells. The nuclei were counterstained with hematoxilin. Big arrow, monocyte/macrophage; arrowhead, fibroblast; small arrow, lymphocyte; x630.

 
Phosphorylated-SAPK/JNK1/2-positive cells were found in the heart of TG mice (Fig. 11A). The cells were identified as myocytes as well as leukocytes (Fig. 11A, B). In the heart of WT mice only phosphorylated-SAPK/JNK1/2-positive myocytes were detected (Fig. 11C,D). In order to identify the cell type responsible for the increased levels of phosphorylated-SAPK/JNK1/2 found in TG hearts by Western blot analysis, we performed intensity quantification of phosphorylated-SAPK/JNK1/2-positive cardiomyocyte in TG and WT mice. The analysis revealed a statistical difference in the optical density of the staining between TG and WT cardiomyocytes (Fig. 11E).


Figure 11
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  		Fig. 11 Immunohistochemistry for phosphorylated-SAPK/JNK1/2. (A) TG heart. Distribution of phosphorylated-SAPK/JNK1/2 in the left ventricle; x250. (B) TG heart. Phosphorylated-SAPK/JNK1/2 staining was found in the nuclei of myocytes (arrow) and leukocytes (arrowhead); x1000. (C) WT heart. Distribution of phosphorylated-SAPK/JNK1/2 in the left ventricular myocardium; x250. (D) WT heart. Phosphorylated-SAPK/JNK1/2 staining was detected in the nuclei of myocytes (arrow); x1000. (E) Quantification of the phosphorylated-SAPK/JNK1/2 intensity level in WT (n=3) and TG (n=4) mice. The sections used in this figure were stained and photographed under the same conditions. The difference in the optical density between TG and WT cardiomyocyte nuclei is evident.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
References
 
Our results indicate that MCP-1 overexpression exerts myocardial protection against short-term (45 min) vs. longer term (3 days) of ischemia. In addition, these studies show that some of the salvaged myocardium dies during reperfusion but another part was rescued by reperfusion. We can conclude that MCP-1 overexpression induces a permanent reduction in infarct size that is not caused by angiogenic and/or arteriogenic mechanisms but by a short-acting mechanism that mimics PC. Our data also show an activation of the SAPK/JNK1/2 pathway but not ERK1/2 and p38 in TG hearts. Moreover, the injection of the SAPK/JNK1/2 inhibitor D-JNKI1 increased significantly infarct size in treated TG mice when compared with untreated TG mice, demonstrating that the cardioprotective effect of MCP-1 overexpression is partially attenuated after injection of the SAPK/JNKs inhibitor. Recent studies reported evidence for the activation of different MAPKs subfamilies during ischemia, ischemia/reperfusion and PC suggesting MAPKs importance as signaling components in the protection of the myocardium against ischemia [21–24,29,30]. The activation of the SAPK/JNKs pathway was originally associated with the induction of cell death [31–33], however, recent studies have indicated that the functions of SAPK/JNKs can vary and even oppose each other, depending on the cell type, stimuli, and model system used [22,24,29–34]. Sato et al. [24] demonstrated that activation of SAPK/JNKs is obligatory for PC in isolated rabbit hearts. In addition, stimulation of SAPK/JNKs with okadaic acid and anisomycin reduced infarct size in a pig model of coronary artery occlusion [22]. We suggest that a permanent activation of the SAPK/JNK1/2 pathway in MCP-1 TG mice could be involved in the development of cardiac resistance against ischemia.

In MCP-1 TG hearts we found increased levels of TNF-{alpha}. Immunohistochemical analysis revealed that leukocytes and fibroblasts accumulating in the interstitium of TG hearts are the source of TNF-{alpha}. We suggest that activated leukocytes secreting TNF-{alpha} serve as stimulus for the activation of the SAPK/JNK1/2 pathway in TG cardiomyocytes, since the SAPK/JNKs pathway can be activated by TNF-{alpha} [35,36]. An activation of the TNF-receptor 1 (TNFR1) has been shown to stimulate apoptosis by activating the caspase-8 cascade through a member of FADD/MORT-proteins, which selectively induces SAPK/JNK1/2 phosphorylation [36]. On the other hand, the caspase-8 independent TNFR1 pathway includes activation of SAPK/JNK1/2 through a member of TRAF-proteins, to selectively mediate cell survival [36]. In our model, the caspase-8 independent pathway seems to be responsible for SAPK/JNK1/2 activation leading to cell survival and not to apoptosis. This is confirmed by the fact that in TG hearts neither apoptotic TUNEL-positive myocytes nor activation of the pro-apoptotic factor p38 could be observed. We suggest that in our TG model TNF-{alpha} activates a signaling cascade downstream of TNFR leading to cytoprotective and not to cell death programs.

Kolattukudy et al. [25] described the development of cardiomyopathy in MCP-1 TG mice from neonatal to 2 months old. At this early age the mice show accumulations of monocytes/macrophages in the myocardial interstitium, but no signs of leukocyte activation was detected [25]. The MCP-1 TG animals described here are from 3 to 9 months old. Our results confirm the finding of Kolattukudy et al. [25], which showed moderate autoimmune myocarditis in the heart of TG mice. In addition, the present results indicate that lymphocytes, together with monocytes/macrophages and fibroblasts, constitute the principal cell types in the ventricular interstitial cell accumulations in TG mice and that an important fraction of the leukocytes were activated. Although it is established that MCP-1 in vivo is a chemoattractant for monocytes/macrophages and lymphocytes [37,38], it is unclear whether MCP-1 may activate these cells [37–39]. The present results provide in vivo evidence for the capacity of MCP-1 not only for chemotaxis but also for activation of monocytes/macrophages and lymphocytes. However, we cannot exclude that the moderate secondary autoimmune myocarditis in TG mice may additionally induce attraction and activation of lymphocytes.

Interestingly, the MCP-1 mice have a short life span (~10 months) due to the development of inflammatory cardiomyopathy and pulmonary edema, which start approximately at 3–4 months after birth and increase until the death of the animals. These data support the hypothesis proposed by Mann [40] that an appropriate and moderate production of cytokines has beneficial effects (cardioprotection) whereas an excessive and prolonged synthesis has detrimental effects (cardiomyopathy, organ failure, and death).

In summary, we showed activated leukocytes in the hearts of MCP-1 TG mice that secrete TNF-{alpha}. We also showed activation of the SAPK/JNK1/2 pathway in this model, together with cardioprotection against short-term ischemia. We suggest that overexpressed MCP-1 attracts and activates leukocytes, which in turn secrete TNF-{alpha} that could be involved in the activation of the SAPK/JNK1/2 pathway in cardiomyocytes with a final cardioprotective effect in TG hearts. These results may constitute in vivo evidence for the link between innate immunity and PC through MAPKs activation.

Time for primary review 31 days.


   Acknowledgements
 
We would like to thank Dr. Sawa Kostin for his invaluable advice, Dr. Keisuke Suzuki for the discussions, Beate Grohmann for her kind technical help, and Gerhard Staemmler and Gunther Schuster for their patient and constant computer assistance.


   References
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 

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H. Morimoto, M. Hirose, M. Takahashi, M. Kawaguchi, H. Ise, P. E. Kolattukudy, M. Yamada, and U. Ikeda
MCP-1 induces cardioprotection against ischaemia/reperfusion injury: role of reactive oxygen species
Cardiovasc Res, June 1, 2008; 78(3): 554 - 562.
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