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Original Article

Macrophage Inhibitor, Semapimod, Reduces Tumor Necrosis Factor-Alpha in Myocardium in a Rat Model of Ischemic Heart Failure

Kherani, Aftab R. MD*; Moss, Garrett W. BSE, MBA*; Zhou, Hua PhD; Gu, Anguo MD; Zhang, Geping MD; Schulman, Allison R. BA*; Fal, Jennifer M. BA*; Sorabella, Robert*; Plasse, Terry MD; Rui, Liu MD; Homma, Shunichi MD; Burkhoff, Daniel MD, PhD; Oz, Mehmet C. MD*; Wang, Jie MD, PhD

Author Information
Journal of Cardiovascular Pharmacology: December 2004 - Volume 44 - Issue 6 - p 665-671
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Abstract

In 1990, the potential role of cytokines in heart failure was revealed by Levine et al.1 They found that heart failure patients had increased serum levels of tumor necrosis factor-alpha (TNF-α).1 They also demonstrated a direct correlation between TNF-α level and both degree of cachexia and extent of heart failure. Subsequent studies further established possible roles of other cytokines, such as interleukin (IL)-12 and IL-63 in heart failure. Cytokines became attractive targets for novel medical therapies aiming to improve cardiac function by inhibition of cytokine production or by blocking cytokine receptors. TNF-α is believed to contribute in various ways to adverse myocardial remodeling.4 For instance, it potentiates the activity of matrix metalloproteinase (MMP), an enzyme that plays an important role in left ventricular structure remodeling.5 Furthermore, the negative inotropic effects of TNF-α are directly related to changes in myocyte calcium homeostasis, as treatment with TNF-α leads to attenuated peak intracellular calcium levels during systole.6

Two commercially available TNF-α receptor blockers have been clinically tested in patients with heart failure, but the results of these clinical trials caused safety concerns and were halted due to lack of efficacy on the morbidity and mortality endpoints.7,8 Therefore, whether a more broad-based anti-cytokine strategy would be safe and more efficacious for treatment of heart failure remained unknown. Semapimod (formerly known as CNI-1493) is a synthetic guanylhydrazone that inhibits macrophage activation and the production of several inflammatory cytokines.9 Semapimod has shown beneficial activity in several preclinical models of diseases that are characterized by elevated cytokine levels. These include sepsis,10 pancreatitis,11 stroke,12 and collagen-induced arthritis.13,14 Preliminary clinical trials have shown that Semapimod can inhibit the rise in plasma TNF-α caused by high-dose IL-2 administration,15 that the drug is well tolerated, and that it may have therapeutic effects in Crohns disease.16

The rat model of myocardial infarction has been used as a standard model for studying myocardial remodeling, heart failure, and inflammation during heart failure, but whether this model is suitable for assessing the therapeutic effects of cytokine inhibitors on cardiac function has not been reported. However, it has been reported that the myocardial tissue levels of cytokines were increased in this animal model. For example, when administration was started on the day of myocardial infarction, β-blockers have been shown to reduce expression of TNF-α and IL-1 in myocardium.17 This was associated with improved ventricular function and reduced ventricular dilation. The purposes of this study were: (1) to study the effects of Semapimod on myocardial TNF-α production and (2) to determine if a decrease in inflammatory cytokine was associated with improved cardiac function.

METHODS

Heart Failure Model and Experimental Groups

All studies were performed in compliance with the Guide for the Care and Use of Laboratory Animals (NRC 1996) and were approved by the Institutional Animal Care and Use Committee of the College of Physicians and Surgeons of Columbia University. Sprague-Dawley rats weighing 250 to 300 g were anesthetized with intraperitoneal ketamine (75 mg/kg) and xylazine (5 mg/kg) and supported by rodent ventilator (Model AH-55-3438, Harvard Apparatus, Inc., Holliston, MA). A left thoracotomy was performed, and the left anterior descending artery (LAD) was ligated proximally. A chest tube (16 g Angiocath) was placed, the incision was closed in 2 layers, and the chest tube was removed immediately after closure. To exclude small infarctions, animals were maintained on protocol only if an echocardiograph 2 weeks after the ligation surgery results for fractional shortening (FS, as defined in the next paragraph) were less than 30%. Then, Semapimod or vehicle was administered daily for 5 days via tail vein injection. The appropriate Semapimod dosage was diluted with vehicle (2.5% mannitol in water) to create a volumetrically consistent injection of 2 mL/kg/d during the drug administration period. The animals were observed for 9 weeks in 1 of 4 groups: (1) rats with myocardial infarct receiving a high dose of Semapimod, 10 mg/kg/d (MI-H, N = 13); (2) rats with myocardial infarct receiving a low dose of Semapimod, 3 mg/kg/d (MI-L, N = 9); (3) rats with myocardial infarct receiving vehicle treatment, 2.5% mannitol in water (MI-0, N = 9); and (4) control rats with sham operation and the vehicle treatment (Sham, N = 10).

Echocardiography and Serum Collection

Under mild isoflurane anesthesia, 2D echocardiography (Sonos-5500, Agilent Technologies, Palo Alto, CA) was performed 2, 5, 8, and 12 weeks after surgery. Also at 2 weeks, prior to any Semapimod administration, serum was collected from 5 random rats and TNF-α level was determined by a core laboratory using a commercially available ELISA kit (Catalog #RTA00, R&D System Inc., Minneapolis, MN). The sensitivity of this assay was 12.5 pg/mL. For each echo, left ventricular (LV) anteroposterior diameter (D) and short-axis area (A) at the papillary muscle level were measured at end diastole (ED) and end systole (ES). Fractional area change (FAC, %) was calculated as [(LVEDA − LVESA)/LVEDA] × 100 and fraction shortening (FS, %) was calculated as [(LVEDD − LVESD)/LVEDD] × 100.

Hemodynamic Measurements and Tissue Harvest

Twelve weeks after operation (9 weeks after treatment with Semapimod), the rats were anesthetized with 5% inhaled isoflurane in oxygen. A Millar catheter (Millar Instruments, Inc., Houston, TX) was inserted into the right carotid artery and advanced to the left ventricle. Using MacLab (ADInstruments Pty Ltd., Castle Hill, Australia), left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), mean aortic pressure (MAoP), heart rate, and left ventricular dP/dtmax were determined. Subsequently, a median sternotomy was performed. The heart was quickly excised and a short-axis section of the heart was fixed in a 4% paraformaldehyde solution for immunohistochemistry. The remaining heart tissue was flash-frozen in liquid nitrogen and stored at −80°C.

Immunohistochemistry

Harvested short-axis heart sections were initially fixed in 4% paraformaldehyde solution for 12 hours. Sections were subsequently embedded in paraffin and 5-μm sections underwent TNF-α staining, using a Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA). The primary antibody used was anti-rat TNF-α antibody (0.2 μg/mL; R&D Systems, Minneapolis, MN; AF-510-NA) diluted to a 50:1 ratio in PBS. Immunoreactivity was evaluated by light microscopy and graded blindly on a semiquantitative scale. Myocyte staining was assessed and separately graded at both the infarction and non-infarction zones. The grading scale was as follows: (0) no visible myocyte staining; (1) mild staining; (2) moderate staining; and (3) marked staining. Additionally, trichrome staining was performed on short-axis heart sections to determine the amount of LV infarction.

Statistical Analysis

All statistical analysis was performed using SPSS 11.5 software (SPSS, Inc., Chicago, IL). Comparisons between treatment groups were made using analysis of variance (ANOVA) method with Bonferroni correction for multiple groups. When the myocardial infarct group was compared in aggregate against the sham group, the independent sample t test was used, with equal variances assumed. Comparisons of initial versus final echocardiogram data within treatments groups were made using the paired t test. Mortality data was analyzed using the χ2 method and Kaplan-Meier curves. For all tests, a value of P < 0.05 was considered significant. All data are expressed as mean ± SEM.

RESULTS

Animals Qualifying for the Study

Perioperative (within 48 hours following LAD ligation) mortality was 52% (87 surviving animals of 180 total surgeries). Of the 87 animals that survived surgery, 31 had FS < 30% and qualified for the study.

Serum Levels of TNF-α and Echocardiographic Data

Serum taken from 5 randomly selected rats (all of which satisfied the study requirement of FS < 30%) showed undetectable levels of TNF-α in each of the 5 animals (ie, < 12.5 pg/ml). Table 1 shows short-axis echocardiographic data from the 4 groups at prior to, 2, 5, and 9 weeks after treatment with Semapimod. Two weeks after MI, there was a significant decrease in FS in the MI groups (MI-0: 23.5 ± 1.5, n = 9; MI-L: 19.5 ± 1.77, n = 9; MI-H: 20.6 ± 1.92, n = 13) versus sham (35.9 ± 1.9, n = 10). While differences in FS (%) between each of the MI groups and the sham group were significant (P < 0.001 for each MI group versus sham), differences among the individual MI groups were not significant. This echocardiogram analysis was confirmed with FAC results for MI groups versus sham (P < 0.01 for all MI groups versus sham), as shown in Table 1. After 12 weeks, an increased end-diastolic diameter (EDd) was noted, compared with baseline. Though dilatation occurred in all groups, the percentage increase was greater in MI groups versus sham group (19.1% ± 2.3 versus 11.8% ± 2.4; P = 0.077). Within the MI groups, no significant differences were observed in LV dysfunction or dilatation 12 weeks after surgery.

T1-7
TABLE 1:
Echocardiographic Data by Treatment Groups

Hemodynamic Data

The hemodynamic data collected at 9 weeks after the treatment demonstrated a significant difference in dP/dtmax between the sham group (3451 ± 294 mm Hg/s) and each of the MI groups (MI-0: 2240 ± 256 mm Hg/s, P = 0.009; MI-L (2358 ± 217, P = 0.023; (MI-H: 2251 ± 217, P = 0.010). No significant differences in hemodynamic data were observed among different MI treatment groups. All hemodynamic results are summarized in Table 2.

T2-7
TABLE 2:
Hemodynamic Data, 9 Weeks After Semapimod Treatment

Histology and Immunohistochemistry

Infarction size was determined using trichrome staining, and percentage of LV circumference infarcted was calculated under light microscopy (blinded to group assignment). Figure 1 shows representative examples of the trichrome and TNF-α staining for each of the 4 groups. Differences in LV infarction percentage among the 3 MI groups were not significant (MI-0: 18.3 ± 4.4; MI-L: 20.6 ± 3.9; MI-H: 21.7 ± 4.3; P = 0.851), and infarction sizes among these groups were comparable. No signs of infarction were evident in the sham group.

F1-7
FIGURE 1:
Trichrome and TNF-α immunohistochemical staining of short-axis heart sections for each of the 4 treatment groups. TNF-α staining was performed and assessed in both the infarction and non-infarction zones. Percentage of infarcted tissue, measured by trichrome, was comparable among the 3 MI groups. TNF-α staining in the noninfarction region is substantially attenuated in the MI-H versus the MI-0 group (0.33 ± 0.14 versus 1.19 ± 0.32; P = 0.03, based on a semiquantitative grading scale) and moderately attenuated in the MI-L group (0.39 ± 0.22; P = 0.05 versus MI-0). Differences in staining in the infarction zone were not significant among MI groups (Magnification = 5× for Trichrome; Magnification = 40× for TNF-α staining).

TNF-α staining in the non-infarcted region was evident only in the MI groups. The sham groups had no visible staining in any of the individual short-axis heart sections. When blindly compared on a semiquantitative scale (ie, 0 = no visible staining to 3 = marked staining), a significant difference in non-infarction zone staining was observed between MI-0 versus MI-H (1.19 ± 0.32 versus 0.33 ± 0.14; P = 0.03) and between MI-0 and MI-L (1.19 ± 0.32 versus 0.39 ± 0.22; P = 0.05).

TNF-α staining in the infarction zone for the aggregate MI group was significantly higher than in the non-infarction zone (1.35 ± 0.17 versus 0.64 ± 0.15; P < 0.01; n = 27), and this significance held when disaggregated into treatment groups. Looking only at the infarction zone, TNF-α staining is slightly attenuated in the MI-L group versus the MI-0 group (0.97 ± 0.28 versus 1.75 ± 0.33; P = 0.213), though the difference was not significant. Figure 2 summarizes immunohistochemistry results in both the infarction and non-infarction regions.

F2-7
FIGURE 2:
TNF-α myocyte staining in short-axis heart sections. TNF-α levels were assessed blindly on a semiquantitative scale from 0 (no visible myocyte staining) to 3 (marked myocyte staining). There was significant attenuation (P = 0.03) of TNF-α in the non-infarction zone between MI-H (high Semapimod treatment group) and MI-0 (no treatment group). There was borderline significance (P = 0.05) in TNF-α staining between the MI-L (low Semapimod treatment group) and MI-0. No significant differences in TNF-α staining were observed in the infarction zones.

Mortality

Four rats expired during the experiment, excluding perioperative deaths within 48 hours of surgery. All belonged to the MI-H groups (log-rank P value = 0.075 for both the comparison between MI-0 and MI-H and for the comparison between MI-L and MI-H). Kaplan-Meier survival curves are shown in Figure 3.

F3-7
FIGURE 3:
Kaplan-Meier survival curve. This figure compares survival probabilities for the high treatment group (MI-H) group versus the other MI groups (MI-L and MI-0) in aggregate. A log rank P value of 0.012 shows a significant decrease in survival probability for the high treatment group.

DISCUSSION

The concentration of circulating TNF-α is directly related to the extent of left-ventricular dysfunction18 and these levels of proinflammatory cytokines increase in patients as their functional heart failure classification clinically worsens,19 although concentration and density of cytokine receptors are independent predictors of mortality in the setting of heart failure.20 Therefore, therapies targeting cytokines, especially TNF-α, were the subject of great interest. Animal studies and initial clinical trials demonstrated promise with an anti-TNF-α monoclonal antibody and soluble TNF-α receptor, biologic agents targeting TNF-α specifically.21-23 However, placebo-controlled phase 3 studies of these agents were halted due to adverse results, raising the question of whether animal models and early patient studies demonstrating some hemodynamic benefit reflected clinical reality.

It has been hypothesized that cytokines may be involved in normal defense and healing processes mechanisms, and it has been reported that TNF-α plays a protective role in acute viral myocarditis in mice.24 These investigators believed that the protective mechanism of TNF-α against viral infection was via participation in the healing process even in situations in which no infectious agent was present. This observation suggested the protective role of TNF-α in a variety of other traumatic situations such as myocardial infarction. In a murine model, Kurrelmeyer et al25 found that endogenous tumor necrosis factor protects myocytes against ischemia-induced apoptosis. TNF-α stimulates hypertrophic growth following MI, an effect that may be important in maintaining myocyte homeostasis,26 and it provides resistance to myocyte injury caused by hypoxia.27 Rathi et al28 also demonstrated that TNF-α in low concentration may exert a cardioprotective effect on the heart by reducing the occurrence of intracellular calcium overload. Though these experiments show the potentially beneficial effects of moderate TNF-α levels, excessive levels of cytokines may cause harmful effects. Therefore, a therapeutic strategy should perhaps not totally block cytokine production. Also, perhaps the pharmaceutical agent should target production of multiple inflammatory cytokines, without having any cytotoxicity of its own, to treat congestive failure producing and exacerbated by TNF-α. These were the rationale behind testing Semapimod as a potential therapy for heart failure.

Semapimod inhibits production of several inflammatory cytokines. In this study we assessed its inhibitory effects on TNF-α production using a rat heart failure model due to coronary artery ligation-induced myocardial infarction, as evidenced by deteriorated echocardiograph parameters such as decreased FS and FAC, elevated LVEDP, and decreased LVdP/dt. Our results showed that in rats with heart failure, myocardial tissue levels of TNF-α were significantly increased in both infarcted areas and remote, non-infarcted areas, but the levels of TNF-α in infarcted myocardium were much higher than in non-infarcted myocardium. These increases were noted at the time of sacrifice, 12 weeks after infarction. However, serum levels of TNF-α taken 2 weeks after MI were not increased. Birks et al29 reported that the mean TNF-α level was around 12 + 1.9 pg/ml in patients with Left Ventricular Assist Device support and was 4.0 + 0.4 pg/ml in heart failure patients waiting for heart transplantation, indicating significantly increased TNF-α levels were only observed in overt heart failure status. Our results showed that the animals did not have a detectable rise in circulating TNF-α levels.

While there have been previous reports of elevated systemic TNF-α levels in the acute setting of ischemia-reperfusion status,30-32 the elevated serum levels of TNF-α appear to peak 1 week after MI,33 whereas in this experiment serum measurements were taken 2 weeks after MI. Additionally, Francis et al34 demonstrated increases in serum TNF-α as early as 30 minutes after MI to levels on the order of 10 pg/ml. Given that the sensitivity of the ELISA TNF-α assay used for this experiment was only 12 pg/ml, it is possible that TNF-α levels were increased significantly as compared with those found in healthy rats, but were not detectable with our methods 2 weeks after MI.

After administration of Semapimod, TNF-α levels in noninfarcted myocardium were significantly decreased 9 weeks after treatment, indicating a prolonged effect. In addition there was a trend toward decreased myocardial TNF-α in animals treated with the low dose of Semapimod as compared with vehicle controls, though no such trend was noted in surviving animals in the high-dose group. The inability to distinguish between the high- and low-dose categories was probably due to the sensitivity of the test, and we believe that a more sensitive method of measurement may be needed in the future.

While the TNF-α levels in infarcted myocardium did show a slight attenuation with the low-dose Semapimod as compared with vehicle control, the differences were not significant. This may have been due to the inability of the ligated coronary artery to deliver Semapimod to the infarcted area. Despite the reduction in myocardial TNF-α levels at the end of the study, treatment with Semapimod did not improve myocardial function, measured either by echocardiographic or hemodynamic parameters.

Alternatively, TNF suppression after myocardial infarction and in the setting of chronic CHF may have a deleterious rather than a beneficial effect. The effects of cytokine inhibitors on cardiac function might be determined by whether myocardium was exposed to acute or to chronic TNF-α elevations. Haudek et al35 showed differential expression of NFκB, a transcription factor in unstimulated cells that exists as a latent cytoplasmic complex bound to its inhibitor protein, in response to either an acute or chronic exposure to TNF-α. They found that chronic exposure of myocardium to TNF-α led to production of p50 homodimers of NFκB, while acute TNF exposure stimulated myocardial p65-p50 heterodimers of NFκB as well as p50 homodimers. They hypothesized that the differentiation of NFκB represents a protective adaptation of myocardium to chronic TNF-α production. It is noteworthy that experiments showing negative effects of TNF-α on myocardial contractility using either myocyte culture or isolated papillary muscles were all performed in the acute setting. Taken together, it may suggest that inhibition of TNF-α production or activity may acutely improve cardiac function. In the chronic setting, however, prolonged blockade of TNF production (eg, in the case of the high-dose Semapimod group in this study or the clinical studies of infliximab) may inhibit the effects of TNF-α on healing or regeneration of myocardial tissue and lead to worsening of heart failure. This may explain why all 4 deaths in the study occurred in the MI-H group, as well as the increased mortality in clinical studies of infliximab. Future studies should focus on a different clinical regimen of Semapimod, focusing on treatment in the acute MI setting, to delineate whether global macrophage inhibition serves any therapeutic role in cardiac patients.

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Keywords:

macrophage inhibitor; cytokine; heart failure; TNF; rat; Semapimod; CNI-1493

© 2004 Lippincott Williams & Wilkins, Inc.