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Departments of 1Surgery, 2Pathology, and 3Medicine, College of Physicians and Surgeons, Columbia University, New York, New York
Submitted 18 October 2007 ; accepted in final form 24 January 2008
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ABSTRACT |
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receptor for advanced glycation end-products; glycation end-products; myocardial infarction; left anterior descending coronary artery
B, and several proapoptotic pathways. We have previously reported the beneficial effect of RAGE blockade in the isolated perfused heart model of I/R injury (1). In the present study, we probed the consequences of the RAGE-ligand interaction during myocardial I/R injury in an in vivo left anterior descending coronary artery (LAD) occlusion model wherein humoral mediators and inflammatory cells in flowing blood may impact on I/R-mediated injury. Using pharmacological blockade of the ligand-RAGE interaction and genetic modulation of RAGE strategies, we demonstrate that RAGE-ligand interaction leads to key cell death signaling events in myocardial infarction (MI). |
METHODS |
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Surgical procedures.
The mouse was placed in a supine position, intubated with 20-gauge plastic cannula, and ventilated with room air using a rodent miniventilator (Harvard Apparatus, Hollston, MA). The ventilator was set at a stroke volume of 0.2 ml and at 120 strokes/min. Surgery was performed under x8 magnification using a Leica surgical microscope. The skin was dissected, the left pectoralis major muscles were refracted toward the right shoulder, and the left rectus thoracic and serratus anterior muscles were reflected toward the left with two hooked microretractors. The third intercostal space was exposed and delicately dissected 3 mm from the sternocostal junction, avoiding injury to the left internal mammary artery. Thoracotomy proceeded laterally on the upper border of the fourth rib to avoid damaging the intercostal nerves and vessels on the lower border of the third rib. Self-retaining microretractors were then used to separate the third and fourth rib sufficiently to gain adequate exposure of the operating region yet preserve rib integrity. The pericardium was cut, the heart was mobilized, and the LAD component of the left ventricle (LV) was visualized. An 8/0 prolene suture (Sharpoint) was then passed under the LAD at 1 mm distal to left atrial appendage, immediately after the bifurcation of the major left coronary artery. A short piece of sterile 20-gauge plastic cannula was placed above the stitch, and the LAD was ligated including the cannula for 30 min. After 30 min of ischemia, the prolene suture was cut, and the LAD blood flow was restored. Care was taken to avoid contact with the lungs during the surgery. The chest wall was closed by approximating the third and fourth ribs with two interrupted stitches using a 5/0 silk suture (Ethicon, Johnson & Johnson, Brussels, Belgium). The left rectus thoracis, serratus anterior, and left pectoralis major muscles were then returned to their original positions, and the skin was closed with 5/0 silk continuous sutures. The mouse was gently disconnected from the ventilator, and spontaneous breathing resumed almost immediately. The entire procedure usually required 
15–20 min. During recovery from the anesthesia, the mouse was kept in a humidified 37°C incubator. Heart samples from the ischemic area were further evaluated at different time points of reperfusion (30 min to 48 h). Note that sham-treated animals were subjected to the identical anesthesia procedure and surgical interventions except that there was no occlusion of the LAD.
Echocardiographic measurements of cardiac function were performed as previously described (23). Briefly, on the day before surgery and just prior to the euthanization of the mice after surgery, mice were treated with isoflurane anesthesia (1.5%) and then subjected to two-dimensional echocardiography to assess cardiac function using the PHILIPS 5500 ultrasound system (Philips Medical, Andover, MA) with a 15-MHz transducer. Two-dimensional echocardiographic images were obtained and recorded in a digital format. Images were then analyzed offline by a researcher blinded to the murine genotype. The LV end-diastolic dimension (LVDd) and left ventricular end-systolic dimension (LVDs) were measured. The percent fractional shortening (FS) was calculated as follows: percent FS = [(LVDd – LVDs)/LVDd] x 100.
Immunohistochemistry and histology. Consecutive sections (5 µm) from paraffin-embedded hearts were prepared for hematoxylin-eosin (H&E) staining, TUNEL, and immunohistochemical evaluation. Slices of 5 µm thickness were produced for 10 short-axis slices (each 500 µm) covering the entire LV. Slices were stained with H&E and scanned with x25 magnification using a microscope and mounted digital camera. Axiovison 4.0 software was used for the evaluation of the infarcted area. The infarction area was calculated as a percentage of the total LV area.
Western blot analysis. Extracts from mouse hearts were prepared by tissue homogenization in cell lysis buffer (Cell Signaling) or for cytochrome c measurement, tissue was fractioned using the centrifugation method as previously described (19), and the cytosolic fraction was subjected to further analysis. The protein concentration was determined using a DC Protein Assay kit (Bio-Rad). Equal amounts of protein were separated by SDS-PAGE (4–20% gradient gels), and proteins were transferred to a polyvinylidene difluoride membrane (Invitrogen), which was probed with primary antibodies according to the manufacturer's instructions. The antibodies were diluted at 1:1,000 with the exception of anti-mouse/rat RAGE IgG (1:400). The primary antibodies used were anti-caspase-8 p-20 subunit IgG (Santa Cruz Biotechnology), anti-mouse/rat RAGE IgG (R&D Research), anti-cleaved caspase-3 IgG (Cell Signaling), anti-cytochrome c IgG (Santa Cruz Biotechnology), anti-β-actin IgG (Sigma), and anti-phospho-STAT3/total STAT3, anti-phospho-STAT5/total STAT5, and anti-phospho-JNK/total JNK IgGs (Cell Signaling). Blots were visualized with a Phototop-Horseradish Peroxidase Western Blot Detection System (Cell Signaling), and quantitative analysis was performed using Image Quant TL software (Amersham).
Primary antibodies were also used for immunocytochemical analysis at dilutions suggested by the manufacturers. Anti-mouse IgG conjugated with Alexa fluor 488 and anti-rabbit IgG conjugated with Alexa fluor 594 (Molecular Probes) were used as secondary antibodies in the immunocytochemical analysis, and stained cells were observed under a laser scanning confocal microscope (Bio-Rad) or regular fluorescent microscope (Axio Carl Zeiss).
To detect apoptotic cells, a TUNEL assay was performed using the Apoptag kit (Intergen) following the manufacturer's protocols. Cardiomyocyte nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI), and the percentage of TUNEL-positive cells was calculated accordingly.
Statistical analysis. All data are reported as numbers of experiments or samples (n) and means ± SD for each experiment. Student's t-test was used to compare the two groups, and the multiple-group comparison was performed by ANOVA followed by post hoc analysis using Tukey's procedure (JMP statistical analysis software). A probability value of P < 0.05 indicated statistical significance.
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RESULTS |
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2.0- and 2.5-fold compared with baseline or sham treatment, as assessed by Western blot analysis (P < 0.05; Fig. 1A).
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-sarcomeric actin, localizing RAGE to cardiomyocytes (Fig. 1B, top); -smooth muscle actin, localizing RAGE to smooth muscle cells (Fig. 1B, middle); and CD31, localizing RAGE expression to certain endothelial cells as well (Fig. 1B, bottom). In the basal state, we observed low-level RAGE expression in the distribution of -sarcomeric actin and in some capillary endothelium of perimyocyte interstitial capillaries. However, we did not observe staining for RAGE in the distribution of arterial endothelium (whose walls are outlined with smooth muscle -actin stain). As I/R resulted in increased expression of RAGE in the heart, we next sought to identify if LAD occlusion and reperfusion resulted in increased generation of RAGE ligands. First, we performed HPLC on heart tissue extracts to determine if levels of a central precursor of AGEs, methylglyoxal (MG), were modulated. After 30 mins ischemia, an approximately sixfold increase in MG was observed in heart tissue compared with baseline (P < 0.05; Fig. 1C). To determine if increased levels of AGE precursors were linked to AGE levels, we measured AGEs in the heart and found that at 1 h of reperfusion, a greater than twofold increase in AGE levels was noted compared with baseline (P < 0.05; Fig. 1D). MG peaked during ischemia (Fig. 1C), whereas AGEs peaked during the reperfusion phase (Fig. 1D). Interestingly, these data are consistent with the likely lag in AGE synthesis from a central precursor. These data placed RAGE and its ligands (AGEs) in LAD ligation-reperfusion injury and prompted us to test if RAGE contributed to injury to the heart in this model.
Genetic deletion of RAGE improves contractile recovery after ischemia and alleviates damage to the myocardium.
To assess the involvement of RAGE in myocardial I/R injury, WT or RAGE–/– mice were subjected to 30 min of ischemia induced by LAD occlusion; coronary flow was then restored, and myocardial functional recovery during reperfusion was assessed. H&E staining revealed significantly smaller infarction areas in RAGE–/– animals compared with WT infarcted myocardium at 48 h (Fig. 2A). Quantification of multiple mouse heart sections was performed and revealed significantly lower infarction areas within the LAD area at risk in RAGE–/– versus WT murine hearts (12% vs. 33% infarct area/total myocardium, respectively, P < 0.05; Fig. 2B). Plasma creatine kinase (CK) release, a marker of ischemic injury, was significantly higher in the plasma of WT mice at 30 min of ischemia and at 1 h of reperfusion versus baseline (P < 0.05; Fig. 2C). However, CK release was attenuated in RAGE–/– mice compared with WT hearts after both 30 min of ischemia and 1 h of reperfusion (P < 0.05; Fig. 2C). Echocardiography was performed and revealed no differences in FS at baseline between WT and RAGE–/– mouse hearts (Table 1). However, at 48 h after reperfusion, the percent FS was significantly greater in RAGE–/– than WT mouse hearts (P < 0.05; Table 1). These data demonstrate that RAGE–/– mice are significantly protected from injury provoked by LAD occlusion followed by reperfusion and that RAGE–/– hearts exhibit improved functional recovery compared with WT hearts.
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3.2 vs. 0.1%, respectively, P < 0.05; Fig. 3A) Caspase-3 activation (Fig. 3B) and cytochrome c release (Fig. 3C), two proapoptotic stress stimuli, were determined in WT and RAGE–/– hearts. RAGE–/– hearts exhibited significantly reduced activation of caspase-3 versus WT hearts at 48 h after reperfusion (P < 0.05). Similarly, cytochrome c release in the cytosol was significantly reduced in RAGE–/– versus WT hearts after 30 min of ischemia and 40 min after reperfusion (P < 0.05).
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RAGE modulates I/R stress signaling machinery in the heart. In view of the significant impact of deletion of RAGE on infarct size and functional recovery of the heart after occlusion and reperfusion of the LAD, we next sought to examine the potential impact of RAGE in ischemic myocardium on three major early signal transduction pathways linked to the recruitment of cell death pathways. We first explored the expression of STAT3 and STAT5 and their phosphorylated forms in reperfused hearts (Fig. 4, A and B). At 30 min and 1 h of reperfusion, significantly higher levels of phospho-STAT3/total STAT3 were noted in RAGE–/– versus WT hearts (P < 0.05; Fig. 4A). In contrast, examination of phospho-STAT5/total STAT5 revealed significantly lower levels of phospho-STAT5/total STAT5 in RAGE–/– versus WT hearts at 30 min and 1 h of reperfusion (P < 0.05; Fig. 4B).
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Pharmacological blockade of RAGE attenuates I/R injury in the mouse heart.
These experiments in RAGE–/– mice strongly pointed to roles for RAGE in LAD occlusion and reperfusion injury. As our data revealed that I/R generated RAGE ligands (AGEs), it was logical to investigate if blockade of the ligand-RAGE interaction in MI suppressed injury. Thus, we complemented the above experiments in RAGE–/– mice with the administration of sRAGE, a ligand-binding decoy, to test the impact of binding RAGE ligands and preventing their interaction with RAGE in myocardial injury. H&E staining revealed significantly smaller infarction areas in sRAGE-treated animals compared with vehicle-treated WT infarcted myocardium at 48 h (23% vs. 35%, respectively, P < 0.05; Fig. 5, A and B). CK release during ischemia (30 min) and 1 h reperfusion was significantly attenuated in sRAGE-treated hearts compared with WT hearts (P < 0.05; Fig. 5C). Evaluation of myocardial function by echocardiography demonstrated that FS recovery at 48 h after reperfusion was significantly greater in sRAGE-treated mouse hearts than in vehicle-treated WT mouse hearts (37% vs. 27%, respectively, P < 0.05; Fig. 5D).
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We next investigated if treatment of WT mice with sRAGE modulated phosphorylation of STAT and JNK pathways. In sRAGE-treated mouse hearts, STAT3 phosphorylation was significantly increased after 1 h of reperfusion compared with vehicle-treated WT mouse hearts (P < 0.05; Fig. 6A). Although no differences were observed in STAT5 phosphorylation in I/R between sRAGE and vehicle-treated mice (data not shown), significantly decreased JNK phosphorylation was noted in sRAGE-treated versus vehicle-treated WT hearts at 1 h of reperfusion (P < 0.05; Fig. 6B).
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DISCUSSION |
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RAGE and pathogenic roles in myocardial I/R injury. In transient occlusion and reperfusion of the LAD, in addition to cells innate to the heart, RAGE-expressing cells that infiltrate the injured heart, such as monocytes, lymphocytes, and neutrophils, may contribute to a range of mechanisms linked to injury. Both homozygous RAGE–/– and WT mice treated with the ligand-binding decoy sRAGE revealed reduced injury in the myocardium compared with their respective controls. Interestingly, our data revealed that LAD occlusion itself produced the pre-AGE MG, followed by significant AGE production in reperfusion, thus underscoring a specific mechanism by which I/R may activate RAGE. These data are consistent with our earlier study (1) in the isolated perfused heart demonstrating the role of RAGE in mediating I/R injury and the study by Tsoporis et al. (24), who demonstrated roles for a distinct RAGE ligand, S100b (9), in modulating LV function recovery after LAD occlusion. As neutrophils, lymphocytes, and monocytes express RAGE, it is thus not surprising that in this in vivo model, contributions of RAGE may be evoked from multiple cellular sources both innate and exogenous to the heart.
RAGE and signal transduction in the heart subjected to I/R injury. By what mechanisms does RAGE exert its damaging effects in the myocardium in I/R? Several studies have implicated the STAT pathway as a key player in myocardial I/R injury. In a rat model of LAD ligation, phosphorylation of JAK2, STAT1, STAT3, STAT5a, and STAT6 was observed as early as 5 min post-MI (14). STAT1, STAT3, and STAT5a remained activated for 7 days post-MI (14). Blockade of the upstream regulator of STATs, JAK2, by AG490 effectively inhibited the functional activation of STATs and resulted in improved cardiac function in hearts after I/R (14). Studies of relevance to RAGE and the STAT pathway come from isolated perfused heart studies that have linked changes in glucose flux via the polyol pathway to JAK-STAT signaling in ischemic hearts (10). It has been shown that the AGE precursor-generating polyol pathway mediates I/R injury by activating JAK2 and STAT5 phosphorylation (10).
Consistent with key roles for STAT3 in myocardial I/R injury, in contrast to WT mice, STAT3-deficient mice exhibited increased infarct size, apoptosis, and vulnerability to developing heart failure after I/R (8, 12). Together, these studies demonstrate that the activation of STAT3 protects the myocardium from ischemic injury. The data presented here demonstrating increased phosphorylation of STAT3 in RAGE–/– and sRAGE-treated mice versus their respective controls provide strong support for the premise that RAGE mediates ischemic injury, in part via modulation of STAT phosphorylation.
In addition to STAT phosphorylation, our experiments also revealed that RAGE-dependent mediation of I/R injury likely ensues in part via JNK. Interestingly, the role of JNK in I/R appears to be dependent on the particular model under study. In contrast to the discordant results on the role of the JNK pathway in mediating the cardioprotective effects of ischemic preconditioning (7, 11, 17, 22), the role of JNK pathway in nonpreconditioned hearts appears to be more clear. In an in vivo model of I/R (such as that employed in our study), JNK–/– mice were protected from injury (13) compared with WT littermates, and the protection in JNK–/– mice was associated with a reduction in caspase-3 activation. Furthermore, it has been shown that the administration of specific JNK inhibitors also imparted cardioprotection (16), both in the isolated perfused heart model and in vivo, upon ligation and reperfusion of the LAD. Here, in our I/R study, we show that blockade of RAGE using sRAGE and genetic deletion of RAGE (RAGE–/– mice) significantly attenuated JNK activation. The present study thus illustrates a link between RAGE, JNK activation, and ischemic injury in the murine LAD ligation model and suggests that RAGE may be a viable therapeutic target to protect the myocardium against I/R impact.
We observed that in RAGE–/– mice and sRAGE-treated mice, protection from injury due to LAD occlusion was also associated with reductions in cytochrome c release and caspase-3 activation, in parallel with increased Bcl-xL expression. The involvement of caspase-3 suggests that mitochondrial apoptotic pathways are being influenced by RAGE during I/R. These experiments are consistent with earlier studies that have linked cardioprotection with reduced apoptosis in mice models of I/R (3, 15, 26).
Taken together, these data illustrate key roles for RAGE in the pathogenesis of myocardial injury induced by transient occlusion and reperfusion of the LAD. The generation of RAGE ligands (AGEs) in this setting demonstrates that in vivo, rapid generation of these species upon I/R signals cues for the recruitment of RAGE signaling. As recent studies have highlighted cardioprotective roles for the modulation of STAT and JNK signaling in myocardial I/R, our findings identify RAGE as a major upstream regulator of these I/R injury-provoking signaling pathways in the heart.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* A. Aleshin and R. Ananthakrishnan contributed equally to this work. 
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