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(Circulation. 2003;108:3075-3078.)
© 2003 American Heart Association, Inc.

Brief Rapid Communication

Nuclear Factor-B Protects the Adult Cardiac Myocyte Against Ischemia-Induced Apoptosis in a Murine Model of Acute Myocardial Infarction

Arunima Misra, MD; Sandra B. Haudek, PhD; Pascal Knuefermann, MD; Jesus G. Vallejo, MD; Zhijian J. Chen, PhD; Lloyd H. Michael, PhD; Natarajan Sivasubramanian, PhD; Eric N. Olson, PhD; Mark L. Entman, MD; Douglas L. Mann, MD

From the Winters Center for Heart Failure Research, Medical Care Service Line, Houston Veterans Administration Medical Center; The DeBakey Heart Center and Cardiovascular Sciences (Z.J.C., E.N.O.), Methodist Hospital, Baylor College of Medicine, Houston; and Department of Molecular Biology (L.H.M., M.L.E.), University of Texas Southwestern Medical Center, Dallas, Tex.

Correspondence to Douglas L. Mann, MD, Winters Center for Heart Failure Research, 6565 Fannin, MS 524, Houston, TX 77030. E-mail dmann{at}bcm.tmc.edu

Received April 2, 2002; de novo received September 15, 2003; revision received October 27, 2003; accepted October 28, 2003.

	Abstract

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Background— Previous studies have shown that tumor necrosis factor (TNF) confers cytoprotective responses in cardiac myocytes. However, the mechanisms for the cytoprotective effects of TNF remain unknown. Given that TNF signals through nuclear factor B (NF-B) and given that NF-B mediates cytoprotective responses, we asked whether NF-B activation conferred cytoprotective responses in acute myocardial ischemia/infarction.

Methods and Results— We examined infarct size and the prevalence of apoptosis in transgenic mice harboring cardiac-restricted expression of a mutated IB protein (IBN) that prevents nuclear translocation of NF-B in cardiac myocytes. Triphenyltetrazolium chloride staining showed that infarct size was 50% greater (P<0.02) in the IBN mice compared with littermate controls at 24 hours. The prevalence of cardiac myocyte apoptosis was significantly greater (P<0.008) in the IBN mice compared with the littermate control mice 3 and 6 hours after left anterior descending occlusion. To explore the mechanism for these findings, we examined protein levels of c-IAP1, c-IAP2, and Bcl-2 as well as manganese superoxide dismutase and c-Jun NH2-terminal kinase activity. These studies showed that protein levels of c-IAP1 and Bcl-2 were significantly lower in the IBN mice, whereas there was no change in c-IAP2 levels, manganese superoxide dismutase, or c-Jun NH2-terminal kinase activity.

Conclusions— Transgenic mice with a defect in activation of NF-B have increased susceptibility to tissue injury after acute left anterior descending occlusion. These studies suggest that the cytoprotective effects of NF-B are mediated, at least in part, by Bcl-2 or c-IAP1.

Key Words: myocytes • apoptosis • myocardial infarction

	Introduction

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The extant literature suggests that the adult mammalian myocardium responds to hemodynamic overloading or myocardial infarction/ischemia by synthesizing a portfolio of proteins that are in turn responsible for initiating and modulating the processes of cardiac growth, repair, and remodeling. Recently, we and others have suggested that phylogenetically conserved cytokines belonging to the innate immune system play an important role in the myocardial adaptation to environmental stress by modulating cytoprotective responses in the adult heart.^1,2 However, the mechanisms for the cytoprotective effects of these cytokines remain unknown. Germane to this discussion is the recent observation that mice deficient in both type 1 and type 2 tumor necrosis factor (TNF) receptor developed accelerated cardiac myocyte apoptosis and increased infarct size after acute coronary artery ligation.¹ Noting that nuclear factor-B (NF-B) was common to the type 1 and type 2 TNF receptor, we hypothesized that NF-B activation might be responsible for mediating the cytoprotective effects of TNF. To begin to test this hypothesis, we examined infarct size and cardiac myocyte apoptosis in a line of transgenic mice that have defective nuclear translocation of NF-B by virtue of cardiac-restricted overexpression of a dominant-negative IB (IBN) construct that retains NF-B in the cytoplasm and thus prevents NF-B activation in response to tissue injury.³

	Methods

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Characterization of the IB Dominant-Negative (IBN) Mice
For these studies, we used a previously reported line of mice with cardiac-restricted expression of a nondegradable form of IB (referred to as IBN), in which the critical phosphorylation (serine-32 and serine-36) and ubiquitination sites (lysine-21 and lysine-22) were removed, rendering the mutant protein resistant to proteolytic degradation.³ The IBN construct was linked to an -myosin heavy chain promoter, thereby restricting expression of this dominant-negative construct to the cardiac myocyte. These mice have defective nuclear translocation of NF-B after provocation with lipolysaccharide.³ The IBN mice were maintained on a C57BL/6JxC3B6 hybrid background. Age-matched littermate controls lacking the transgene were used as appropriate controls.

Left ventricular structure was characterized in the IBN and littermate control mice using 2D directed M-mode echocardiography and light microscopy, as described.¹

Effect of Acute Coronary Artery Occlusion on Infarct Size
Experimental Model
Ligation of the left anterior descending (LAD) coronary artery was performed in 12- to 16-week-old mice, as described.¹ These studies were performed in accordance with the animal care and use guidelines at Baylor College of Medicine.

Assessment of Infarct Size
Twenty-four hours after LAD ligation, the mice were killed and their hearts removed. Hearts were stained with 2% Evans blue to delineate the area at risk and then sliced in sections and incubated in 1% triphenyltetrazolium chloride (TTC) for 15 minutes to define the area of infarction. Infarct size and area at risk were determined using computerized planimetry, as described.¹

Mechanisms Responsible for Infarct Size
Assessment of Cardiac Myocyte Apoptosis
To determine the prevalence of apoptosis after LAD occlusion, we used an in situ DNA ligase technique method that identifies only double-stranded DNA breaks with single 3' base pair overhangs that are more characteristic of DNA breaks that occur during apoptosis.¹ The in situ DNA ligase technique was performed as described (see the online data supplement),⁴ using biotin-labeled hairpin oligonucleotides. Apoptotic cell nuclei detected by the ligase assay were stained with fluorescein (excitation/transmission wavelength, 495/525 nm). Sections were counterstained with the nucleic acid binding dye (excitation/transmission wavelength, 310/353 nm) DAPI (4',6-diamidino-2-phenylindole) to visualize the entire population of cell nuclei within each myocardial section. 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 myocyte nuclei; final results were expressed as follows: (number of positively labeled nuclei/total number of DAPI stained nuclei per 10 000 µm²)x100%. Studies were performed in myocardial sections (n=10/heart) obtained from IBN and littermate control mice 3 and 6 hours after LAD ligation.

Mechanisms for NF-B–Mediated Cytoprotection
To determine whether cytoprotective proteins downstream from NF-B–mediated signaling were altered in the IBN mice, we examined protein levels of c-IAP1, c-IAP2, and Bcl-2 as well as the functional activity of manganese superoxide dismutase (MnSOD) in cytosolic extracts obtained from wild-type and IBN mice at 1 and 3 hours after occlusion. Given that NF-B suppresses c-Jun NH2-terminal kinase (JNK) activity⁵ and that excessive JNK activity has been linked to apoptosis, we also examined JNK activity at 1 and 3 hours after LAD occlusion.

Protein levels for c-IAP1-1, c-IAP2, and Bcl-2 were measured by Western blot analysis as described,⁶ using a monoclonal mouse anti-mouse Bcl-2 antibody, a polyclonal rabbit anti-mouse c-IAP1 antibody, or a polyclonal rabbit anti-mouse c-IAP2 (see the online data supplement). The membranes were then stripped and reprobed with a monoclonal mouse anti-mouse GAPDH. After blocking, the blots were incubated with an appropriate peroxidase-labeled secondary antibody. The membranes were incubated with luminol using a commercially available kit (ECL, Amersham) and were exposed to chemiluminescent film for 2 to 5 minutes. The films were scanned on a personal densitometer, and band intensities were evaluated using ImageQuaNT 4.2a (Molecular Dynamics) software. The resulting band intensities were normalized to the corresponding GAPDH levels.

MnSOD Activity
MnSOD activity was measured using the RanSOD kit (Randox, Crumlin), according to the manufacturer’s protocol (see the online data supplement).

JNK Activity
JNK activity was determined by measuring the degree of phosphorylation of c-Jun, a specific substrate for JNK (see the online data supplement). Band intensities were determined as described above.

Statistical Analysis
Data are expressed as mean±SEM. An unpaired Student t test was used to evaluate mean differences in echocardiographic indices, infarct size, and area at risk between littermate control and IBN mice. Two-way ANOVA was used to assess differences in prevalence of apoptosis between the 2 groups. Post hoc ANOVA testing was performed using the Tukey test. Significant differences were said to exist at P<0.05.

	Results

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Characterization of the IB Dominant-Negative (IBN) Mice
As shown in the Table, there was no significant difference in LV end-diastolic and end-systolic dimensions, LV posterior wall thickness, percent LV fractional shortening, and ratio of LV mass to body weight in the IBN and littermate control mice. Furthermore, there was no difference in the area at risk between the 2 groups. Finally, histological analysis of the hearts from the littermate control and IBN mice using light microscopy revealed no obvious qualitative differences in the morphological appearance of the myocardium sections between the 2 groups (data not shown).

View this table: [in this window] [in a new window]   Characterization of the IBN Mice

Effect of Acute Coronary Artery Occlusion on Infarct Size: Assessment of Infarct Size
Figure 1 depicts a representative Evans blue/TTC staining pattern in sequential myocardial sections obtained from a littermate control mouse (Figure 1A) and a IBN mouse (Figure 1B). As shown, the extent of TTC staining was less in the IBN mouse compared with littermate control mouse. Figure 1C summarizes the results of the group data with respect to the infarct size at 24 hours in the littermate control mice (n=9) and the IBN mice (n=8). Infarct size was 50% greater (P<0.02) in the IBN mice compared with the littermate control mice.

View larger version (37K): [in this window] [in a new window]   Figure 1. Assessment of infarct size in IBN and littermate control mice. Myocardial sections from littermate control mice (A) (area at risk, 55.9%) and IBN mice (B) (area at risk, 55.7%) were stained with Evans blue and TTC 24 hours after acute LAD occlusion, and the hearts were serially sectioned. C, Results of group data for infarct size/area at risk (24 hours) for littermate control mice and IBN mice. D, Prevalence of cardiac myocyte apoptosis within the area at risk at 3 and 6 hours in the littermate control mice and IBN mice (*P<0.05 compared with littermate control). There was no survival difference at 24 hours between the littermate control and IBN mice.

Mechanisms Responsible for Infarct Size
Assessment of Cardiac Myocyte Apoptosis
To determine whether increased cardiac myocyte apoptosis contributed to the increase in infarct size observed in the IBN mice, we examined the prevalence of cardiac myocyte apoptosis at 3 and 6 hours after LAD occlusion. Figure 1D shows that the prevalence of cardiac myocyte apoptosis was significantly greater in the IBN mice (n=4 hearts) compared with littermate controls (n=4 hearts). Two-way ANOVA indicated that there was a significant overall difference between the groups (P<0.008), whereas post hoc analysis testing showed that the prevalence of apoptosis was significantly (P<0.01) greater at 3 and 6 hours in the IBN mice compared with littermate controls.

Mechanisms for NF-B–Mediated Cytoprotection
Figure 2 shows representative Western blots for myocardial levels of Bcl-2, c-IAP1, and c-IAP2 at 1 and 3 hours after LAD ligation in the littermate control and IBN mice (n=6 to 7 hearts/time per group); hearts from naive animals were used as the appropriate controls (n=4 hearts/time per group). Figure 2B shows that there was a significant decrease in the expression of Bcl-2 (P<0.03) and c-IAP1 (P<0.04) in the hearts of the IBN mice at 1 hour after infarction compared with wild-type control mice. However, there was no significant difference in the levels of c-IAP1, c-IAP2, and Bcl-2 at 3 hours. As shown in Figures 2C and 2D, respectively, there was no difference in the levels of MnSOD activity or JNK activity in the IBN and littermate control mice 1 and 3 hours after acute coronary artery ligation.

View larger version (29K): [in this window] [in a new window]   Figure 2. Cytoprotective proteins in wild-type and IBN mice. A, Representative Western blots of levels of c-IAP1, c-IAP2, Bcl-2, and GAPDH in naive, sham-operated, and infarcted littermate control wild-type and IBN mice at 1 and 3 hours. B through D, Results of group data for protein levels of c-IAP1, c-IAP2, and Bcl-2 normalized by GAPDH, respectively. E, MnSOD activity. F, JNK activity in wild-type and IBN mice at 1 and 3 hours (*P<0.05 compared with littermate control).

	Discussion

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The results of this study suggest that activation of NF-B gives rise to one or more cytoprotective signals that prevent the development of cardiac myocyte apoptosis after acute ischemic injury. Two major sets of experimental observations support this statement. First, there was a significant 50% increase in infarct size in the IBN mice compared with littermate control mice subjected to identical coronary artery occlusions (Figures 1A through 1C). The observed increase in infarct size in the IBN mice was not secondary to differences in cardiac structure (Table), genetic strain, or differences in area at risk (Table) between the 2 groups. Second, there was an increase in the prevalence of cardiac myocyte apoptosis in the IBN mice (Figure 1D). This latter observation suggests (but does not prove) that the increase in infarct size in the IBN mice was secondary to an increase in cardiac myocyte apoptosis. These findings are consistent with studies that have suggested an important role for NF-B in terms of mediating ischemic preconditioning,⁷ as well as a previous report from our laboratory in mice lacking TNF-mediated signaling, in which deficient NF-B activation was suggested as a mechanism for the accelerated apoptosis and increased tissue injury observed in the mice lacking TNF signaling.¹ However, the results of the present study conflict with studies that have shown that antisense decoys directed against NF-B attenuate infarct size after ischemia reperfusion injury.⁸ Although the reason for this discrepancy is not apparent from this study, it may relate to differences in experimental models (ischemia/reperfusion versus complete coronary occlusion), differences in the strain of mice, as well as spatial differences in NF-B activation between the 2 models. That is, the use of oligonucleotide decoys would be expected to inhibit NF-B activation in cardiac myocytes, fibroblasts, smooth muscle cells, as well as endothelial cells, whereas the cardiac restricted expression of IBN would be expected to inhibit NF-B activation in cardiac myocytes alone. Thus, activation of NF-B in the vascular compartment during ischemia/reperfusion injury might result in increased influx of inflammatory cells and hence increased tissue damage that offsets the cytoprotective effects mediated by NF-B in cardiac myocytes.

Although this study was not designed to identify the complete portfolio of biological mechanisms that were responsible for the cytoprotective effects of NF-B, the findings reported herein suggest a role for Bcl-2 and c-IAP1 as potential NF-B–regulated proteins that confer cytoprotection in the heart, either through stabilization of mitochondrial function or delimiting caspase activation after acute ischemic injury. This point of view is consistent with a prior study that showed that forced expression of Bcl-2 reduced cardiac myocyte apoptosis in a Langendorff model of ischemia/reperfusion injury.⁹ Additional studies will be necessary to address these interesting possibilities.

	Acknowledgments

 
This research was supported by research funds from the Veterans Administration and the NIH (P50 HL-O6H and RO1 HL-58081-01, RO1 HL-61543-01, HL-42250-10/10, and NIH RO1 GM-59203-03).

	Footnotes

 
An online-only Data Supplement is available at http://circulationaha.org.

Guest Editor for this article was Carlin Long, MD, Denver Medical Health Center, Denver, Colo.

	References

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This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 108/25/3075    most recent 01.CIR.0000108929.93074.0Bv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Misra, A. Articles by Mann, D. L. Search for Related Content PubMed PubMed Citation Articles by Misra, A. Articles by Mann, D. L. Related Collections Acute myocardial infarction Apoptosis Growth factors/cytokines