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Ann Thorac Surg 2007;84:120-125
© 2007 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Proteasome Inhibition Attenuates Infarct Size and Preserves Cardiac Function in a Murine Model of Myocardial Ischemia-Reperfusion Injury

^a Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
^b Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Accepted for publication February 20, 2007.

^_* Address correspondence to Dr Selzman, Division of Cardiothoracic Surgery, University of North Carolina, 3040 Burnett-Womack Bldg., CB #7065, Chapel Hill, NC 27599-7065 (Email: selzman{at}med.unc.edu).

Presented at the Basic Science Forum of the Fifty-third Annual Meeting of the Southern Thoracic Surgical Association, Tucson, AZ, Nov 8–11, 2006.

	Abstract

Top Abstract Introduction Material and Methods Results Comment Footnotes Acknowledgments References

 
Background: Despite improvements in protection, myocardial ischemia-reperfusion remains an important cause of cardiac dysfunction. Multiple strategies exist experimentally; few are clinically accessible. Nuclear factor kappa-B (NF-B) is a transcription factor central to the inflammatory response and is implicated in reperfusion injury. Its activation relies on the degradation of its inhibitory molecule, IB, by the 20S proteasome. We hypothesized that proteasome inhibition would decrease the extent of infarction after temporary coronary occlusion.

Methods: C57Bl6 mice received a specific proteasome inhibitor (PS-519) and were subjected to 30 minutes of transient occlusion of the left anterior descending artery. After 24 hours of reperfusion, echocardiography was performed to evaluate ventricular function and hearts were excised and analyzed for infarct size, areas at risk, and molecular markers of injury and NF-B activation.

Results: Compared with controls, PS-519 delivered before left anterior descending (coronary artery) ligation reduced the area of infarct without a change in the area at risk. Similar results were seen with PS-519 delivered at reperfusion. Echocardiography demonstrated a relative reduction in fractional shortening in the vehicle group of 9.8% versus only 2.7% in the PS-519 group. Markers of myocardial stress and injury were accordingly suppressed with PS-519. These physiologic findings were associated with PS-519 decreasing p65 and TNF expression while preserving IB expression.

Conclusions: In this murine infarct model PS-519 significantly preserved regional myocardial function, reduced the size of infarction, and attenuated expression of myocardial inflammatory response genes. These data demonstrate that a currently available and well-tolerated inhibitor of NF-B can decrease the risk of myocardial injury associated with ischemia-reperfusion.

	Introduction

Top Abstract Introduction Material and Methods Results Comment Footnotes Acknowledgments References

 
Myocardial ischemia and reperfusion (IR) is a complex set of events that is associated with significant alterations in cellular metabolism, oxidant stress, and triggering of proinflammatory molecules [1]. As oxygenated blood is supplied to ischemic tissue, these factors paradoxically predispose myocardium to further injury, ultimately leading to larger infarcts resulting in impaired ventricular function [2]. Although many agents have been used experimentally to curtail IR injury, few have proven clinical efficacy [3, 4]. Thus, identifying effective and clinically accessible methods of limiting myocardial injury associated with IR remains an important and challenging issue.

Nuclear factor kappa B (NF-B) is a transcription factor that regulates expression of several genes involved in inflammation, the immune response, apoptosis, and cell proliferation [5]. Many of these same genes are activated during IR injury and likely contribute to its severity [6, 7]. Nuclear factor kappa B exists in the cytoplasm as a heterodimer of its two principle subunits (p65 and p50) and its active inhibitory subunit, IB. When stimulated by a broad array of physiologic substrates, membrane receptors invoke signals that converge at IB kinase (IKK) which subsequently phosphorylates IB. Phospho-IB becomes polyubiquitinated and is targeted for degradation by the 20S proteasome. Nuclear factor kappa B is thus liberated to translocate to the nucleus, where it binds DNA and activates transcription of numerous downstream inflammatory products such as TNF, IL-1ß, and leukocyte adhesion molecules [5].

Currently, numerous strategies exist that variably inhibit NF-B activation and modulate IR injury including antisense and overexpression vectors [8, 9]. Unfortunately, many of these approaches are experimentally appealing but clinically inaccessible. Conversely, proteasome inhibition is a viable method of preventing NF-B activation that has been clinically tested. We are particularly interested in the compound PS-519. A synthetic analogue of the naturally occurring compound lactacystin, PS-519 is approximately 2.5 times as potent, and rapidly inactivates breakdown of phospho-IB [10]. This allows IB to stabilize the NF-B complex and prevent its nuclear translocation. PS-519 has already undergone phase one clinical trials for safety in acute dosing regimens and has not been observed to have any significant side effects when administered daily to mice over a period of several months [11]. The proteasome inhibitor PS-341, which similarly acts to inhibit NF-B, has already gained clinical approval for use in patients with advanced multiple myeloma [12]. Because NF-B is an important feature of the injury response and because it can be regulated by the proteasome, we hypothesized that proteasome inhibition would provide cardioprotection after murine myocardial IR.

	Material and Methods

Top Abstract Introduction Material and Methods Results Comment Footnotes Acknowledgments References

 
Experimental Protocol
Our experimental protocol is depicted in Figure 1. Ten-week-old C57Bl6 mice were split into four experimental groups as further detailed below: sham (n = 29), control left anterior descending artery (LAD) ligation (n = 34), ligation with pretreatment with PS-519 (n = 30), and ligation with delayed PS-519 (n = 5). PS-519 (generously given by Albert Baldwin, PhD, University of North Carolina) is known to inhibit proteasome-mediated degradation of the inhibitory protein IB. This prevents NF-B activation and subsequent nuclear translocation. The dose of 1 mg/kg per day, administered intraperitoneally, has been previously shown to result in a rapid and stable decrease in chymotryptic proteasome activity to approximately 80 to 85% of baseline in the mouse model [11]. The vehicle used was a 1:1 mix of propylene glycol and normal saline.

View larger version (14K): [in this window] [in a new window]   Fig 1. Experimental design. Proteasome inhibitor PS-519 was administered either 30 minutes prior to left anterior descending coronary artery ligation (A) or at reperfusion (B). (RT-PCR = real time polymerase chain reaction.)

Determination of Area-at-Risk and Area-of-Infarct
After 24 hours of reperfusion, animals were sacrificed and hearts were excised (sham = 8, control = 10, PS-519 = 5, delayed PS-519 = 5). The LAD was suture-ligated at its prior occlusion site, and the heart was perfused with green fluorescent beads (Duke Scientific, Fremont, CA) to illuminate all areas outside of the area at risk (AAR). The heart was subsequently sliced into 1 mm sections along the short axis. These sections were incubated at 37°C for 20 minutes with triphenyl tetrazolium chloride (TTC) (Sigma-Aldrich, St. Louis, MO) to stain viable heart tissue bright red and infarcted tissue white. Respective areas were determined immediately after staining by tracing the infarct and perfused areas. Fluorescent beads outlining the AAR were visualized using a 450 nm UV lamp. Areas were analyzed using ImageJ analysis software.

Echocardiography
Prior to surgery and prior to sacrifice at the 24-hour time point, animals underwent transthoracic cardiac echocardiography using a Vevo 660 ultrasound system (VisualSonics, Inc, Toronto, ON, Canada) (sham = 3, control = 4, PS-519 = 6). Animals were lightly anesthetized with standardized heart rates using isoflurane in the range of 0.5% to 2% to facilitate accuracy of measurement.

Creatinine Kinase Measurement
Serum samples were collected by facial vein bleeding prior to sacrifice into serum separator tubes (Becton Dickinson, Franklin Lakes, NJ) and then stored at –20°C (n = 5 for each group). Samples were evaluated for creatine kinase MB fraction (CK-MB) using a Vitros 250 Chemistry System (Ortho-Clinical Diagnostics, Raritan, NJ).

Gene Expression
Frozen left ventricular (LV) tissue taken 60 minutes after reperfusion (sham = 5, control = 5, PS-519 = 4) was thawed in Trizol reagent (Sigma, St. Louis, MO) and homogenized using a bead-mill homogenizer (Qiagen, Valencia, CA). Ribonucleic acid (RNA) was then extracted using standard Trizol technique, with an additional DNA precipitation step. The RNA concentration and purity were evaluated using a Nano-Drop 1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). Equal amounts of RNA from each sample were used to produce complementary (c)DNA using the High Capacity cDNA Archive Kit (ABI, Applied Biosystems, Foster City, CA). The cDNA and taq-man primers and probes (ABI, Applied Biosystems) were also used for p65 with an 18S ribosomal (r)RNA as the endogenous loading control. Data are depicted as mean fold changes (MFC) versus the internal control. As a surrogate for early myocardial injury, we examined the expression of early growth response gene-1 (Egr1) that has been implicated as a marker of ischemic myocardial damage [13]. To test for apoptosis, we probed for Bax and Bcl2 expression. Samples were run on a 9700 ABI real-time polymerase chain reaction (PCR) machine and quantified using SDS 2.2 software (ABI, Applied Biosystems).

Protein Extraction
Samples were homogenized in ice-cold lysis buffer (20 mM tris-HCl [pH7.5], 150 mM NaCl, 1mM Na₂ EDTA, 1 mM EGTA, 1% Triton, 2.5 mM pyrophosphate, 1 mM beta-glycerophosphate, 1 mM sodium orthovanadate, 1 ug/mL leupeptin) (Cell Signaling Technology, Inc, Danvers, MA) with additional phosphatase and protease inhibitors including the following: 1 mM sodium meta-vanadate (Sigma), 5 mM sodium fluoride (Sigma), 10 mM PnPP (Sigma), 10 uM sodium molybdate (Sigma), and 2X Complete Protease Inhibitor tablets (Roche Diagnostics, Pleasanton, CA). Samples were clarified by centrifugation at 18,000g for 30 minutes at 4°C. Protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA).

Western Blot Analysis
Whole cell tissue homogenates from frozen LV tissue at 30 and 60 minutes of reperfusion (sham = 8, control = 12, PS-519 = 13) were separated using SDS-PAGE in phosphate-buffered saline containing 1 mmol/L EDTA, 0.5% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, and protease inhibitors. Samples were transferred to polyvinylidene fluoride membranes, incubated with 5% milk tris-buffered saline-Tween (TBST) buffer and the appropriate antibody. Polyclonal antibodies were incubated with membranes for one hour at 4°C while monoclonals were incubated overnight at 4°C. After washing with TBST buffer, goat antirabbit IgG secondary antibody was then used to identify primary antibody binding. Specific protein bands were visualized using an Invitrogen kit (Invitrogen, Carlsbad, CA). Antibodies included phospho-p65 (ser 536), IB, and TNF (Cell Signaling Technology, Inc).

Statistical Methods
All data are presented as mean ± standard error of the mean. Comparisons among groups were made using type 3, two-tailed, t tests. Gene expression fold changes were log base 2 transformed to enable t test comparison. Statistical significance was accepted within 95% confidence interval. All analysis was performed using the statistical package Prism 4 (GraphPad, San Diego, CA).

	Results

Top Abstract Introduction Material and Methods Results Comment Footnotes Acknowledgments References

 
PS-519 Decreases the Size of Myocardial Infarction
As depicted in Figure 2, ligation of the LAD for 30 minutes followed by 24 hours of reperfusion results in a substantial transmural infarction. The infarct was near complete at the apex and decreased in extent toward the base. At the midpapillary level (Fig 2A), a clear histologic distinction is observed in mice pretreated with PS-519. Across multiple sections, PS-519 decreased the area of infarction by nearly 50% (38.5 ± 2.4% vs 21.5 ± 3.5%, p < 0.05). There were no differences among groups in the area at risk. The ratio of infarct area to area at risk was profound in control animals (Fig 2C). This ratio was reduced in PS-519 animals (70.7 ± 3.4% vs 49.7 ± 6.0%, p < 0.05), albeit not to the level observed in shams.

View larger version (45K): [in this window] [in a new window]   Fig 2. Infarct analysis 24 hours after ischemia and reperfusion in control and proteasome inhibitor PS-519 treated mice. (A) Cross-sectional photomicrograph (5x) of triphenyl tetrazolium chloride-stained (white) infarcted regions. (B) Percent infarct area relative to ventricular cross-sectional area in sham, control, and PS-519 treated mice (n 8 for all groups). (C) Percent infarct area relative to area at risk. (*p < 0.05, sham versus control; **p < 0.05 control versus PS-519.)

PS-519 Improves Myocardial Function After Ischemia-Reperfusion Injury
Transthoracic echocardiography was performed serially in animals prior to ligation and 24 hours after reperfusion. Ventricular dimensions were plotted and fractional shortening was calculated (Table 1). Compared with sham animals, ligated mice had significantly decreased fractional shortening which was preserved in animals pretreated with PS-519. There were no differences between sham and PS-519 treated animals.

PS-519 Decreases NF-B Pathway Activation
Mechanistically, we hypothesized that the cardioprotective influence of proteasome inhibition was related to inhibition of NF-B. As such, whole cell heart lysates were probed for several NF-B related antibodies. When activated, p65 exists in a phosphorylated form. Early after ligation (30, 60 minutes), phospho-p65 protein and p65 message were upregulated compared with both sham animals, and those animals that were treated with PS-519 (Fig 3A). The proteasome is responsible for degradation of ubiquinated proteins, including IB. To verify our mechanism, a time course for IB expression was performed. As a corollary to p65 expression, IB decreased after ligation compared with control (Fig 3B). The PS-519 successfully maintained levels of IB, thus preventing p65 translocation. Finally, we examined expression of TNF, a well-known downstream effector of NF-B activation and the inflammatory response. As demonstrated in Figure 3C, TNF was markedly upregulated after ligation compared with sham or PS-519 treated animals.

View larger version (36K): [in this window] [in a new window]   Fig 3. Effect of proteasome inhibitor PS-519 on NF-B related proteins after ischemia-reperfusion. (A) Western blot analysis of myocardial homogenates probed for phospho-p65 after 30 minutes of reperfusion. Real-time polymerase chain reaction of p65 gene expression relative to 18S at the same time points (*p < 0.05, sham versus control; **p < 0.05 control versus PS-519). Western blots for (B) IB protein and (C) TNF at 30 and 60 minutes of reperfusion.

	Comment

Top Abstract Introduction Material and Methods Results Comment Footnotes Acknowledgments References

Our results corroborate several previous studies targeting NF-B as therapy for IR injury. Nearly a decade ago, Morishita and colleagues [15] demonstrated that myocardial transfection of a cis element "decoy" for NF-B decreased the size of myocardial infarction in a rat model of IR injury. Subsequent approaches have included animals treated with antisense oligonucleotides for NF-B [16], with vectors overexpressing IB [17], and with pharmacologic inhibitors [18–20]. To the later point, the antioxidant diethyldithiocarbamic acid was administered in a rabbit IR model and subsequently decreased NF-B activation by 80% and decreased infarct sized by 30% [21]. Interestingly, a number of drugs commonly used in the treatment of acute coronary syndromes have intrinsic anti-NF-B activity including aspirin [22] and statin compounds [23]. Moreover, adenosine, which is one of only three agents that have proven successful in clinical trials for myocardial IR [24], is also a potent blocker of NF-B. Cumulatively, these studies reinforce the rationale for targeting NF-B as a therapy for IR. However, adenosine and other pharmacologic compounds are nonspecific inhibitors of NF-B. Conversely, the more specific genetic approaches are not yet clinically accessible.

Proteasome inhibition as a means of NF-B blockade has been increasingly advanced in research and clinical models related to both cancer and IR injury. The proteasome inhibitor PS-341 (bortezomib/velcade) was approved in 2005 for use as a chemotherapy sensitizing agent for patients with advanced multiple myeloma [12]. Although the proteasome inhibitor PS-519 similarly decreases activation of NF-B, it has been developed as an antiinflammatory agent for use in acute organ IR injury, particularly for neuroprotection in several animal stroke models. In 2003, Berti and colleagues [25] demonstrated that administration of PS-519 immediately after transient occlusion of the middle cerebral artery resulted in decreased expression of several inflammatory genes known to be regulated by NF-B, such as ICAM-1, Il-1ß, and TNF [25]. Subsequent studies extended these results to show that both the area of infarction and long-term neurological function of the subject rats were significantly improved with PS-519 treatment [26, 27]. Our present study in murine hearts indeed corroborates the protective influence of PS-519 as initially demonstrated in cerebral IR.

More recently, other investigators have examined the effects of proteasome inhibition in myocardial IR using the proteasome inhibitor PR-39, which similarly functions to block degradation of IB [28]. In a rat model of myocardial injury, PR-39 and its derivative PR-11, were both cardioprotective [29]. Unfortunately, delivery of the inhibitor necessitated intramyocardial injection. An important benefit to PS-519 is that it has been closely studied in humans and is effective and well-tolerated. PS-519 is easily administered and has rapid systemic distribution by intravenous and intraperitoneal injection. When taken together with our results, these studies add strength to our hypothesis that proteasome mediated inhibition of NF-B is a powerful pharmacologic strategy for alleviating myocardial IR injury.

NF-B is well-known to have antiapoptotic functions. In our study, we examined apoptosis by measuring expression of the apoptosis regulating transcripts, Bax and Bcl2, and found no significant difference among sham, control, and treatment groups at any time point in the experiment. This corroborates existing evidence that apoptosis contributes to the IR event in a limited fashion in the immediate post injury period [30] as did a previous study examining proteasome inhibition and myocardial infarction [31]. A recent study by Trescher and colleagues [9] demonstrated improved outcomes in a rat model of myocardial IR when NF-B was inhibited through continuous overexpression of IB. This study served two purposes. First, it strengthened the evidence for the targeting of IB in the regulation of NF-B during IR injury. Second, it showed that even over a nearly two-month period, the net effect of continued myocardial NF-B inhibition is positive with regard to myocardial function, further reinforcing the importance of interrupting the inflammatory action of NF-B.

We recognize that our experimental data must be interpreted with certain caveats. Most importantly, we acknowledge that while PS-519 acts effectively to block the activation of NF-B, it may have other important and not yet described functions. Although more specific inhibitors of NF-B are available, we selected PS-519 because of its rapid onset of action, moderate duration of action (approximate half-life 24 hours), facile delivery, and relative clinical accessibility. In addition, white blood cell measurements in other studies have demonstrated that the dose of PS-519 can be adjusted to provide an 80% inhibition of proteasome activity, thus allowing some endogenous NF-B activity, while significantly blunting the stress response [10]. Second, despite good evidence that we successfully inhibited activation of NF-B, we were only able to achieve a 50% decrease in the relative infarct area with PS-519 treatment. This favorably compares with reported infarct areas in other successful IR studies [15, 16, 21, 28, 29]. For example, in a recent multicenter phase 2 clinical trial of adenosine for patients undergoing reperfusion therapy for ST-segment elevation myocardial infarction, the best treatment subgroup was in the range of a 60% decrease in infarct size [24]. These findings reinforce the multifactorial nature of IR injury, and indicate that the most successful treatments will likely be combination therapies.

In this series of experiments, we pretreated animals with PS-519 prior to the onset of ischemia as did a previous porcine study [31]. While this scenario is relevant for cardiac surgery, it does not meet the reality of patients with acute coronary syndromes where reperfusion is unpredictable. Recently, Williams and colleagues [32] demonstrated significant protection from IR injury when PS-519 was administered as late as 10 hours after the onset of reperfusion in a rat model of middle cerebral artery occlusion. Similarly, we demonstrate equivalently decreased infarct size when PS-519 was given prior to ischemia or at the time of reperfusion. Quite possibly, with longer periods of ischemia, this rescue capability might not exist. Nonetheless, this suggests a wide therapeutic window for the use of proteasome inhibition in the treatment of IR injury, and greatly expands its potential use for this therapeutic strategy.

	Acknowledgments

Top Abstract Introduction Material and Methods Results Comment Footnotes Acknowledgments References

 
This work was supported by grants from the University of North Carolina, the American College of Surgeons, and the Thoracic Surgery Foundation for Research and Education. We appreciate the assistance provided by Margaret Cloud, Mauricio Rojas, MD, and Susan Smyth, MD.

	Footnotes

Top Abstract Introduction Material and Methods Results Comment Footnotes Acknowledgments References

 
^_* Drs Stansfield and Moss contributed equally to this work.

	References

Top Abstract Introduction Material and Methods Results Comment Footnotes Acknowledgments References

 

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