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J Thorac Cardiovasc Surg 2008;136:1274-1279
© 2008 The American Association for Thoracic Surgery

Cardiopulmonary Support and Physiology

Inhibitory kappa B kinase-β is a target for specific nuclear factor kappa B-mediated delayed cardioprotection

^a Department of Surgery, University of North Carolina, Chapel Hill, North Carolina
^b Department of Pathology, University of North Carolina, Chapel Hill, North Carolina
^c Department of Biology, University of North Carolina, Chapel Hill, North Carolina

Received for publication April 1, 2008; revisions received June 25, 2008; accepted for publication July 26, 2008.

^_* Address for reprints: Craig H. Selzman, MD, Division of Cardiothoracic Surgery, University of North Carolina at Chapel Hill, 3040 Burnett-Womack Bldg, CB 7065, Chapel Hill, NC 27599-7065. (Email: Selzman{at}med.unc.edu).

	Abstract

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Objective: Myocardial ischemia/reperfusion injury remains a vexing problem. Translating experimental strategies that deliver protective agents before the ischemic insult limits clinical applicability. We targeted 2 proteins in the nuclear factor-B pathway, inhibitory kappa B kinase-β, and 26S cardiac proteasome to determine their cardioprotective effects when delivered during reperfusion.

Methods: C57BL/6 mice underwent left anterior descending artery occlusion for 30 minutes. An inhibitory kappa B kinase-β inhibitor (Compound A), a proteasome inhibitor (PS-519), or vehicle was administered at left anterior descending artery release or 2 hours afterward. Infarct size was analyzed 24 hours later. Pressure-volume loops were performed at 72 hours. Serum and left ventricular tissue were collected 1 hour after injury to examine protein expression by enzyme-linked immunosorbent assay and Western blot.

Results: Inhibitory kappa B kinase-β and proteasome inhibition significantly attenuated infarct size and preserved ejection fraction compared with the vehicle groups. When delivered even 2 hours after reperfusion, Compound A, but not PS-519, still decreased infarct size in mice. Finally, when delivered at reperfusion, successful inhibition of phosphorylated-p65 and decreased interleukin-6 and tumor necrosis factor- levels occurred in mice given the inhibitory kappa B kinase-β inhibitor, but not in mice with proteasome inhibition.

Conclusion: Although inhibitory kappa B kinase-β and proteasome inhibition at reperfusion attenuated infarct size after acute ischemia/reperfusion, only inhibitory kappa B kinase-β inhibition provided cardioprotection through specific suppression of nuclear factor-B signaling. This feature of highly targeted nuclear factor-B inhibition might account for its delayed protective effects, providing a clinically relevant option for treating myocardial ischemia/reperfusion associated with unknown periods of ischemia and reperfusion as seen in cardiac surgery and acute coronary syndromes.

	Introduction

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Nuclear factor-kappa B (NF-B) is a transcription factor that regulates proinflammatory cytokine expression and is activated in cardiac tissue after ischemia/reperfusion (I/R) injury.¹ Several common cardiovascular drugs that have nonspecific inhibitory NF-B activity are a routine part of our armamentarium, including statins, aspirin, and adenosine.^2-4 Although strategies aimed at NF-B inhibition have been shown to decrease infarct size and preserve function in animal models of myocardial I/R,^5,6 the majority of experimental models study interventions before the actual ischemic insult. A treatment targeting NF-B that is efficacious when delivered before injury may not be effective or may function differently when administered at a later point in time, because periods of ischemia are not predictable (ie, patients with acute coronary syndrome). Furthermore, patients with controlled I/R associated with cardiac surgery can have disparate ischemic times. The only physiologic phenomenon clinicians can reproducibly rely on is reperfusion. Thus, NF-B inhibition before an ischemic insult creates a temporal disconnect between injury and therapy.⁷

NF-B, in its heterodimer form of the p50 and p65 subunits, remains inactive in the cell cytoplasm while bound to the repressor protein, inhibitory kappa-B alpha (IB). After cardiac reperfusion injury, reactive oxygen species unleash a kinase cascade that ultimately activates the inhibitory kappa-B kinase (IKK) complex. In particular, activation of the IKKβ subunit results in phosphorylation of IB with its subsequent polyubiquitination and degradation by the 26S cardiac proteasome. This releases the p65/p50 heterodimer, which translocates to the nucleus and transcribes target genes, including cytokines and cell adhesion molecules (Figure 1, A ).⁸

View larger version (30K): [in this window] [in a new window]   Figure 1. A, Diagram of NF-B activation. Compound A inhibits the ability of IKKβ to phosphorylate IB, thus preventing liberation of the p65/p50 heterodimer. PS-519 acts at the proteasome, preventing polyubiquitination and breakdown of phosphorylated IB. B, Experiment design. The inhibitors, Compound A or PS-519, were given at 0 minutes or 2 hours of reperfusion, with infarct size measured 24 hours later. Separate groups of animals received the inhibitors at 0 minutes and either sacrificed at 1 hour of reperfusion for protein analysis or at 3 days for functional analysis. IKK, Inhibitory kappa-B kinase; CK-MB, creatine kinase muscle-brain fraction; IL, interleukin; TNF, tumor necrosis factor.

	Materials and Methods

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Surgical Protocols
All experimental protocols were preapproved by the University of North Carolina's Animal Care and Use Committee, and mice received humane care in compliance with the Guide for the Care and Use of Laboratory Animals. C57BL/6 mice 8 to 10 weeks of age were obtained from Charles River Laboratories (Wilmington, Mass) and housed in Division of Laboratory Animal Medicine-approved facilities with light cycling and food and water access ad libitum at least 7 days before surgery. Our experimental groups and general protocol are shown in Figure 1, B. With the animals under general anesthesia with ketamine (100 mg/kg) and xylazine (5 mg/kg) and inhaled isoflurane, myocardial I/R was induced by 30-minute occlusion of the left anterior descending (LAD) artery using a polyethelene-10 tubing tied down to the LAD as previously described.¹⁰ After 30 minutes of ischemia, the tubing was removed. Sham animals had an air knot placed. Immediately after LAD release, the proteasome inhibitor, the IKKβ inhibitor, or a normal saline vehicle was delivered intraperitoneally to the animal. We measured infarct size in a subset of those animals and in animals given the inhibitors 2 hours after reperfusion. Infarct size was analyzed 24 hours after LAD release because gross alterations and irreversible ischemic damage occurs within the first 12 to 24 hours after ischemia.¹³ To analyze infarct size 24 hours after LAD occlusion, mice were reanesthetized and hearts were excised after systemic heparinization. The LAD was ligated at the same site as the previous occlusion (as noted by the suture that remained after tubing removal), and the coronary arteries were perfused with fluorescent polymer microspheres (Duke Scientific, Palo Alto, Calif) to determine the area at risk. Hearts were then serially sectioned in 1-mm intervals along the short axis and stained with 1% triphenyltetrazolium chloride to determine the area of infarct. Image J (National Institutes of Health, Bethesda, Md) was used to calculate areas of interest.

Functional parameters were obtained 3 days after I/R injury. Mice that had undergone in vivo I/R and given inhibitors or vehicle at reperfusion were reanesthetized and a 1F Millar catheter (PVR-1045; Millar Instruments, Houston, Tex) was placed into the left ventricle using a closed chest technique as previously described.¹⁰ In vivo pressure-volume loops were recorded with Labview 7.1 software (National Instruments, Austin, Tex) and interpreted with PVAN (Millar Instruments). We focused on ejection fraction and dP/dt, calculated through PVAN, as measurements of myocardial function (Figure 1, B).

Reagents
The proteasome inhibitor, PS-519, was dissolved in polypropylene glycol before use and delivered at a dose of 1 mg/kg delivered intraperitoneally. This dose and method of delivery has been shown to decrease proteasome activity by approximately 80% in murine studies.^14,15 Compound A, the IKKβ inhibitor, was dissolved in 10% Cremophor in water and subsequently administered at a dose of 5 mg/kg delivered intraperitoneally. Compound A has been shown to have a desirable pharmacokinetic profile,¹⁶ and this dose has been effective in our previous experiments.¹⁰

Serum Markers of Injury and Cytokine Expression
A subset of mice were sacrificed 1 hour after LAD release for serum and left ventricular tissue collection. At this time, approximately 0.4 mL of blood was collected from animals into serum separator tubes (Becton Dickinson, Franklin Lakes, NJ) using the facial vein puncture technique. The blood was then centrifuged for 10 minutes at 2000g to obtain serum. Serum creatine kinase muscle-brain fraction (CK-MB) was measured using a Vitros 250 Chemistry System (Ortho-Clinical Diagnostics, Raritan, NJ). Serum cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)- were measured with respective enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, Minn) and quantified with a spectrophotometer (Wallac 1420 VICTOR, PerkinElmer, Waltham, Mass). Concentration of cytokines was determined according to the respective standard curve and expressed in picograms/milliliter.

Left Ventricular Protein Expression
Left ventricles obtained 1 hour after LAD release were homogenized in whole cell lysis buffer (Cell Signaling Technology, Danvers, Mass). Homogenates were then centrifuged for 30 minutes at 18,000g. The Bradford assay (Bio-Rad Laboratories, Hercules, Calif) was used to quantify protein concentration. Western blots were then run on polyvinylidene membranes as previously described.⁹ Membranes were incubated with phosphorylated (phospho) p65 (Ser 536), total endogenous p65 (Sigma-Aldrich, St Louis, Mo), or GAPDH (Chemicon International, Temecula, Calif) primary antibodies. Visualization of protein bands was performed with an Invitrogen kit (Invitrogen, Carlsbad, Calif), and densitometry was performed with Image J software.

Statistical Analysis
Comparisons between experimental infarct groups were made using 1-way analysis of variance with subsequent Dunnett's test using the statistical package Prism 4 (GraphPad, San Diego, Calif). Two-tailed Student t test was used for specific comparisons between other end-points in the experiment. Data are expressed as mean ± standard error of the mean. Statistical significance was accepted at the 95% confidence interval.

	Results

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Temporal Differences in Proteasome and IKKβ Inhibition Affect Reperfusion Injury
Myocardial damage was assessed enzymatically 1 hour after I/R. Animals that underwent LAD occlusion followed by vehicle administration had significantly higher levels of serum CK-MB than sham animals. Compared with the vehicle group, animals that received proteasome inhibition at the time of reperfusion had significantly lower serum CK-MB levels. Similarly, IKKβ inhibition also resulted in lower serum CK-MB levels. No significant difference exists between the PS-519 group and the Compound A group (Table 1 ).

Myocardial Function Improves After Delivery of Proteasome and IKKβ Inhibitors at Reperfusion
Pressure-volume loops recorded 3 days after I/R document the improvement of cardiac function after administration of both proteasome and IKKβ inhibitors at reperfusion. Body weight, heart rate, and left ventricular end-diastolic function were not significantly different among all groups (data not shown). Loops were shifted to the right in those animals that received vehicle, whereas proteasome and IKKβ inhibition preserved pressure-volume loops close to baseline (Figure 2, A ). After I/R, animals administered vehicle had significantly lower ejection fraction and dP/dt (Figure 2, B) than mice without I/R injury. Proteasome and IKKβ inhibition delivered after acute I/R maintained ejection fraction similar to baseline levels. On analysis of dP/dt, a parameter that is less load-dependent and more indicative of myocardial contractility than ejection fraction,¹⁷ only IKKβ inhibition provided significant functional protection above the vehicle group. This suggests that although both inhibitors allow for recovery of global left ventricular function after reperfusion, inhibition of IKKβ better protects from intrinsic contractile dysfunction after I/R.

View larger version (28K): [in this window] [in a new window]   Figure 2. Functional assessment of cardiac reperfusion injury. A, Sample pressure-volume loops from animals administered vehicle, PS-519, or Compound A at 0 minutes of reperfusion and a baseline animal that had not undergone surgery. Hemodynamic parameters were measured 3 days after I/R in 4 groups of mice: baseline (n = 7), vehicle (n = 7), PS-519 (n = 7), and Compound A (n = 5). B, Mice treated with proteasome and IKKβ inhibitors at reperfusion had improved function as measured by ejection fraction; however, proteasome inhibition failed to significantly preserve dP/dt. * P < .05 versus baseline. ** P < .05 versus vehicle.

Delayed IKKβ Inhibition, but not Proteasome Inhibition, Decreases Expression of NF-B

View larger version (21K): [in this window] [in a new window]   Figure 3. Myocardial NF-B activation after reperfusion. Expression of the NF-B subunit, p65, as well as its activated form, phosphorylated-p65, was analyzed in left ventricular homogenates 1 hour after treatment at 0 minutes of reperfusion. A mouse administered lipopolysaccharide (LPS), a potent stimulant of NF-B, is a positive control for p65 phosphorylation. GAPDH serves as a loading control. Densitometry was performed, and the ratios of phospho-p65 to p65 and phosph-p65 to GAPDH are graphically represented. N = 3 for sham, vehicle, PS-519, and Compound A groups. * P < .05 versus sham. ** P < .05 versus vehicle. LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

View larger version (10K): [in this window] [in a new window]   Figure 4. NF-B–related cytokine expression. Serum cytokines TNF- (A) and IL-6 (B) in animals treated with PS-519 and Compound A after reperfusion. TNF-: sham n = 3; vehicle n = 6; PS-519, Compound A n = 4. IL-6: sham, PS-519, Compound A n = 4; vehicle n = 7. * P < .05 versus sham. ** P < .05 versus vehicle. TNF, Tumor necrosis factor.

	Discussion

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This is an important observation in the context of transforming basic science findings into daily clinical practice. For various cancer models, NF-B inhibitors, including proteasome inhibitors, have successfully translated from the cell and animal model to human clinical trials.¹⁸ The introduction of specific NF-B inhibitors for cardioprotection, however, has failed to materialize. Matsuki and colleagues¹⁹ recently described the cardioprotective effects of fluvastatin when administered to rats 2 weeks before I/R. However, the beneficial effects of this statin were lost on delivery at the time of ischemia or the start of reperfusion. Furthermore, the mechanism of protection was not mediated via antioxidant properties as expected, but via increased nitric oxide production. The proteasome inhibitor, like fluvastatin, can alter several pathways activated after myocardial I/R. Although PS-519 decreased infarct size in our model of myocardial I/R when administered after ischemia, the mechanism is not NF-B mediated. Furthermore, any cardioprotective effects of proteasome inhibition were lost after late delivery 2 hours after myocardial reperfusion. Therapeutic timing is a critical determinant of mechanism, therefore, especially regarding relatively nonspecific targets, such as the cardiac proteasome, that can ultimately modify multiple cellular mechanisms.

We previously demonstrated that delivery of PS-519 before myocardial I/R was cardioprotective through inhibition of NF-B.^6,9 The results of this study demonstrate, however, that proteasome antagonism immediately after ischemia does not attenuate phosphorylated-p65 levels or downstream cytokines regulated by NF-B. Rather the decrease in myocardial damage at this time point could be mediated through other proteasome-regulated pathways. Part of the ubiquitin-proteasome system, the proteasome regulates the proteolysis and elimination of proteins involved in myriad cellular mechanisms, from apoptosis and differentiation to inflammation and proliferation.²⁰ For example, destabilization of the apoptosis inhibitor, apoptosis repressor with caspase recruitment domain (ARC), after oxidative stress is mediated by the ubiquitin-proteasomal pathway.²¹ Although ARC is expressed at the start of reperfusion in mouse models of myocardial I/R, its expression is minimal 2 hours after reperfusion.²¹ Proteasome inhibition at the onset of reperfusion may prevent the destabilization of ARC, protecting myocardium from reperfusion-induced apoptosis. In fact, ARC-deficient mice had 50% larger infarcts after myocardial I/R than their wild-type controls.²² In addition to altering apoptotic pathways, proteasome inhibition has also been shown to induce heat-shock proteins in cardiomyocytes, conferring protection after oxidative stress.^23,24 Thus the effects of proteasome inhibition can be multifactorial and are not restricted to the NF-B pathway alone.

Unlike the proteasome, IKKβ is specific to the NF-B pathway. IKKβ knockout mice die in utero of liver failure from massive hepatocyte apoptosis and have complete disruption of the NF-B signaling pathway.²⁵ Pharmacologic inhibition of IKKβ with Compound A is a specific process that directly prevents the phosphorylation of the NF-B repressor protein, IB. Downstream expression of phosphorylated p65 and cytokines IL-6 and TNF- is repressed after administration of Compound A. This pattern of NF-B disruption is similar when IKKβ is inhibited before¹⁰ and after the period of cardiac ischemia. Furthermore, IKKβ inhibition appears to be even more effective after 2 hours of reperfusion. Late protection is rational, because previous studies have documented the persistence of NF-B levels for several hours after reperfusion.^26,27 Although this smaller infarct size is not significantly different from delivery at reperfusion, this trend emphasizes the distinct advantage specific NF-B inhibition through the IKKβ subunit provides in a late setting compared with the relatively nonspecific proteasome.

	Conclusions

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Despite 3 decades of research investigating methods to reduce myocardial reperfusion injury, few experimental therapies have translated successfully to our patients. Although hundreds of pharmacologic inhibitors have been tested in animal models of I/R, our review reveals a dearth of studies that have focused on delayed interventional therapies for cardiac reperfusion injury. To our knowledge, this is the first study both directly comparing 2 applicable NF-B inhibitors and investigating delayed delivery of NF-B inhibition after the reperfusion event has occurred. This approach is especially important, because successful NF-B–mediated cardioprotection is not only temporally dependent but also dependent on the specificity of the mechanism of NF-B inhibition.

	Acknowledgments

 
The authors thank Peter Charles, PhD, for statistical assistance and Margaret Cloud for editorial assistance.

	Footnotes

 
The Thoracic Surgery Foundation for Research and Education (W.E.S.), National Institutes of Health (A.S.B.), and American College of Surgeons (C.H.S.) provided funding.

Presented at the C. Walton Lillehei Resident Forum, 88th Annual Meeting of the AATS, San Diego, California, May 10 to 14, 2008.

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This Article Abstract Full Text (PDF) 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 Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Craig H. Selzman Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Moss, N. C. Articles by Selzman, C. H. Search for Related Content PubMed Articles by Moss, N. C. Articles by Selzman, C. H. Related Collections Cardiac - pharmacology Cardiac - physiology Coronary disease Myocardial infarction