AJP - Regu Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 285: R463-R469, 2003. First published May 15, 2003; doi:10.1152/ajpregu.00153.2003
0363-6119/03 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
285/2/R463    most recent
00153.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aker, S.
Right arrow Articles by Schulz, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aker, S.
Right arrow Articles by Schulz, R.

INFLAMMATION, CYTOKINES, AND TEMPERATURE REGULATION

Serum but not myocardial TNF-{alpha} concentration is increased in pacing-induced heart failure in rabbits

Stephanie Aker, Sergej Belosjorow, Ina Konietzka, Alexej Duschin, Claus Martin, Gerd Heusch, and Rainer Schulz

Institute of Pathophysiology, University of Essen Medical School, 45122 Essen, Germany

Submitted 26 March 2003 ; accepted in final form 8 May 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In animals and patients with severe heart failure (HF), the serum tumor necrosis factor-{alpha} (TNF-{alpha}) concentration is increased. It is, however, still controversial whether or not such increased serum TNF-{alpha} originates from the heart itself or is of peripheral origin secondary to gastrointestinal congestion and increased endotoxin concentration. We therefore now examined TNF-{alpha} in serum, myocardium, and liver of sham-operated and HF rabbits. In nine rabbits in which HF was induced by left ventricular (LV) pacing at 400 beats/min for 3 wk, LV end-diastolic diameter was increased and systolic shortening fraction (9.4 ± 1.0 vs. 28.5 ± 1.3%, echocardiography, P < 0.05) was reduced. Serum TNF-{alpha} was higher in HF than in sham-operated rabbits (240 ± 24 vs. 150 ± 22 U/ml, WEHI-cell assay, P < 0.05). In the heart, TNF-{alpha} was located mainly in the vascular endothelium (immunohistochemistry), and TNF-{alpha} protein (920 ± 160 vs. 900 ± 95 U/g) did not differ between groups. In the liver of HF rabbits, hepatocytes expressed TNF-{alpha}, and TNF-{alpha} protein was increased compared with sham-operated rabbits (2,390 ± 310 vs. 1,220 ± 135 U/g, P < 0.05) and correlated to the number of hepatic leukocytes (r = 0.85) and serum TNF-{alpha} (r = 0.69). The intestinal endotoxin concentration was 24.5 ± 1.2 vs. 17.0 ± 3.1 endotoxin units/g wet wt (P < 0.05) in HF compared with sham-operated rabbits. In this HF model, serum but not myocardial TNF-{alpha} is increased. The increased serum TNF-{alpha} originates from peripheral sources.

liver; inflammation

EXOGENOUS tumor necrosis factor-{alpha} (TNF-{alpha}) depresses ventricular function in dogs (20, 33) and rats (10), and transgenic mice overexpressing TNF-{alpha} develop dilated cardiomyopathy (12). The endogenous myocardial TNF-{alpha} concentration is increased during aortic banding (6), acute myocardial ischemia-reperfusion (22), and after myocardial infarction (25, 37) and coronary microembolization (20, 46). During ischemia, the increased myocardial TNF-{alpha} concentration is causally involved in the development of ventricular dysfunction (20) and myocardial infarction (7).

In patients with severe heart failure (5, 15, 28, 32, 38, 45, 47), particularly in those with signs of cachexia (2, 28, 31), the serum TNF-{alpha} concentration is increased, and TNF-{alpha} concentration is an independent predictor of mortality in patients with advanced heart failure (17, 21, 40). The origin of the increased serum TNF-{alpha} concentration is, however, not clearly identified yet. While in some experimental (41) and clinical (32, 38) studies, the TNF-{alpha} concentrations did not differ in paired arterial and coronary sinus blood samples, suggesting that TNF-{alpha} is of peripheral origin, in other experimental (29) and clinical (18, 26, 44) studies the myocardial TNF-{alpha} protein expression was increased. These results suggest that even though the myocardial TNF-{alpha} concentration might be increased in failing hearts, other peripheral organs must contribute to the increased serum TNF-{alpha} concentration.

Critically ill patients often suffer from congestion of gastrointestinal organs, possibly leading to elevated endotoxin levels (39), a hypothesis originally introduced by Anker and coworkers (3). Endotoxemia, in turn, is associated with an increased concentration of circulating cytokines (23, 35). Experimentally, however, no data on the endotoxin and cytokine concentrations in gastrointestinal organs have been obtained in animals with heart failure.

We therefore measured the serum, myocardial, and hepatic TNF-{alpha} concentrations and the intestinal endotoxin concentration in conscious rabbits with pacing-induced heart failure.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental model. This study was approved by the bio-ethical committee of the district of Düsseldorf, Germany, and the experiments were in accordance with the guidelines of the American Physiological Society.

Instrumentation. The experiments were performed in 20 male Chinchilla bastard rabbits (specific pathogen-free, Charles River, Kisslegg, Germany) weighing 3.4 ± 0.1 kg. The animals were anesthetized initially with a subcutaneous injection of a mixture of ketamine (50 mg/kg) and xylazine (3 mg/kg). After tracheal intubation, anesthesia was maintained with 12–25 ml/h of propofol and a bolus injection of fentanyl (0.005 mg/kg) intravenously. Ventilation was maintained using a Dräger UV-2 ventilator (Dräger, Lübeck, Germany) with 70% room air-30% oxygen. Body temperature was continuously monitored, and hypothermia was prevented using a heating pad. After left thoracotomy, a pacing lead was sutured onto the apical region of the left ventricle. The pacing lead was connected to a pacemaker (Medtronic, Düsseldorf, Germany), which was implanted subcutaneously. The chest was closed in layers and evacuated with a Bülau-Drainage. The tracheal tube was removed after spontaneous breathing was ensured. The rabbits were placed on an antibiotic regimen (enrofloxacin 12 mg/kg) for 3 days, and postoperative analgesia was achieved by administration of buprenorphine (0.03 mg/kg). After instrumentation, the rabbits were allowed to recover for 7–10 days.

Experimental protocol. When the rabbits had fully recovered from surgery, heart failure was induced in nine rabbits by rapid LV pacing at a rate of 400 beats/min for 3 wk (13, 30). Heart failure was evident from clinical signs, such as ascites, and echocardiographic parameters, such as an increase in LV end-diastolic diameter and a reduction of LV systolic shortening fraction. Eleven sham-operated rabbits served as controls.

At the end of each study, rabbits were anesthetized and arterial blood samples were collected, centrifuged, and serum stored in liquid nitrogen. After euthanasia of the rabbits, two to three tissue samples were taken from the LV free wall and the liver and frozen in liquid nitrogen and stored at -70°C for later analysis. Further samples were fixed in formalin and embedded in paraffin.

Echocardiography. LV function was measured in the short axis view at baseline and after 3 wk of pacing, with the rabbits in conscious state and the pacemaker turned off for at least 60 min. Echocardiography in two-dimensional real time and M-mode acquisition (Supervision 7000, Toshiba, Neuss, Germany) was performed using a 10-MHz sector phase array transducer. Fractional shortening was assessed [(end-diastolic diameter - end-systolic diameter)/end-diastolic diameter x 100]. Additionally, LV wall thickness was measured and LV weight calculated (14). Echocardiographic determination of LV weight closely correlated to anatomic LV weight as assessed in 10 separate rabbits (echocardiographic LV weight = 1.02 x anatomic LV weight - 0.25, r = 0.72).

Histology. The extent of myocardial fibrosis was determined using Masson-Goldner staining and expressed as percentage of field of view (3 fields of 0.075 mm2 each). Apoptosis was determined using TdT-mediated dUTP nick end labeling (TUNEL) technique (In Situ Cell Death Detection Kit, La Roche Diagnostics, Mannheim, Germany), counterstaining with bisbenzimide (HOE-33342) and phalloidin (both Sigma, Taufkirchen, Germany) (20). TUNEL-positive cardiomyocyte nuclei were counted using fluorescence microscopy (Leica DMLB, Bensheim, Germany) and calculated per square millimeter. Leukocytes were quantified in hematoxylin and eosin-stained tissue sections in myocardium and liver (19). Ten randomized fields (0.193 mm2 each) were counted, and the number of leukocytes was calculated per square millimeter. For analysis of intestinal edema, a part of the small intestine (10.1 ± 0.7 g) was dried over 3 wk at 80°C. Fresh and dry weights were measured, and the percent change was calculated.

TNF-{alpha} concentration (WEHI cell assay). Serum, myocardial, and hepatic TNF-{alpha} concentrations were determined using a cytotoxic activity assay (WEHI cells) (8, 20, 46). The TNF-{alpha} concentration was expressed in units per milliliter for serum and in units per gram for myocardium and liver (1 U is the reciprocal of the dilution necessary to cause 50% cell destruction) (8). The WEHI cell assay was calibrated using purified rabbit TNF-{alpha} (Pharmingen, San Diego, CA), which was added at given concentrations to the WEHI cell assay. The regression analysis was as follows: rabbit TNF-{alpha} = 0.0564 x WEHI cell assay + 2.7419, n = 66, r = 0.99, P < 0.001. Using the WEHI cell assay, the detection limit was ~3 pg/ml rabbit TNF-{alpha}, which is far below the detection limit of the TNF-{alpha} ELISA [~25 pg/ml (4)].

Immunohistochemistry. Paraffin-embedded specimens from heart and liver were cut into 4-µm sections, dewaxed and rehydrated with graded alcohol, and rinsed in PBS. Sections were pretreated with pepsin for antigen retrieval and then incubated with a mouse monoclonal anti-TNF-{alpha} antibody (Santa Cruz sc-7317, Santa Cruz, CA) for 1 h at 37°C diluted in 1:10 antibody diluent (Dako Kopenhagen, Denmark). Several rinsing steps with PBS followed. A secondary FITC-conjugated goat anti-mouse antibody (sc-2010) (1:100, 37°C for 1 h) was added. Negative controls included omission of primary antibodies. The samples were cover-slipped in Vectashield (H-1000, Vector Laboratories, Burlingame, CA) and examined by laser scan microscopy at x630 magnification (Zeiss LSM, Oberkochem, Germany).

Myocardial TNF-{alpha} receptors 1 and 2. Cytosolic proteins were prepared using a modification of the method described by Schreiber et al. (43). The myocardial samples were weighed, diluted with buffer [1:10, 20 mM Tris·HCl, 0.33 M sucrose, 5 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.005% leupeptin; pH 7.4], and homogenized. The homogenates were then centrifuged for 20 min at 20,000 g. The supernatant was saved for cytosolic protein analysis. For preparation of membrane extracts the pellet was rehomogenized with buffer (1:5, 20 mM Tris·HCl, 0.33 M sucrose, 5 mM EDTA, 0.5 mM EGTA, 1 mM PMSF, 0.005% leupeptin, and 1% Triton X-100; pH 7.4), incubated on ice for 30 min, and centrifuged for 45 min at 20,000 g. The supernatant was saved for membrane protein analysis. Protein concentrations were determined using the bicinchoninic acid protein assay (Pierce, Rockford, IL).

Cytosolic (60 µg) and membrane (50 µg) proteins were loaded on 10% PAGE-SDS gels. The proteins were separated by electrophoresis (25 µA, for 2 h at 4°C), and the separated proteins were transferred to nitrocellulose membranes by electroblotting (40 V, overnight at 4°C). The membranes were incubated in Tris-buffered saline (TBS; 20 mM Tris and 120 mM NaCl at 25°C) containing 5% nonfat dry milk for 90 min followed by incubation with either specific polyclonal anti-TNF receptor 1 or specific polyclonal anti-TNF receptor 2 antibodies (StressGen Biotechnologies, Victoria, BC, Canada) overnight at 4°C. The blots were then washed four times with TBS containing 0.05% Tween 20 (TTBS) and were then incubated for 1 h with a secondary antibody (anti-rabbit immoglobulin linked to horseradish peroxidase; Santa Cruz). After four washes with TTBS, detection was performed by enhanced chemiluminescence. The resulting autoradio-graphs were analyzed by quantitative two-dimensional densitometry using commercially available software (Herolab, Wiesloch, Germany). The two-dimensional band intensity of cytosolic and membrane TNF receptors was expressed as percentage of the total TNF receptor content.

Endotoxin concentrations. Arterial blood samples were collected in sterile, endotoxin-free tubes and diluted with LAL Reagent Water (1:1). Tissue samples (intestine and liver) were weighed, collected in sterile, endotoxin-free tubes, diluted with LAL Reagent Water (1:10), and homogenized. The samples were centrifuged at 14,000 g for 10 min, and the supernatants were collected for the measurements of endotoxin. Inactivation of inhibitors was obtained by heating all samples for 15 min at 75°C. A chromogenic kinetic limulus amebocyte lysate assay (LAL assay QCL-1000, BioWhit-taker, Walkersville, MD) was used to measure the endotoxin concentration (9). The concentration of endotoxin was calculated from a standard curve (Esherichia coli endotoxin 0111: B4) and expressed in endotoxin units (EU) per milliliter for serum and EU per gram for intestinal and hepatic tissues.

Statistical analysis. Values are expressed as means ± SE. Statistical comparison of all data before and after 3 wk of pacing between the two groups was performed by two-way ANOVA. When a significant overall effect was detected, individual mean values were compared using Bonferroni's method. TNF-{alpha} concentration, TNF-{alpha} receptors 1 and 2, extent of fibrosis, and number of TUNEL-positive cells and leukocytes were compared between sham-operated and heart failure rabbits using an unpaired t-test. Statistical significance was accepted for a P value <0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hemodynamics. Heart rate did not change significantly from baseline to 3 wk in sham-operated (260 ± 8 to 236 ± 9 beats/min) and heart failure rabbits (265 ± 15 to 260 ± 14 beats/min) and also did not differ between groups. Heart failure was characterized by an increase of LV end-diastolic diameter from 14.7 ± 0.6 to 18.4 ± 0.6 mm and a reduction of LV systolic shortening fraction from 32.6 ± 1.5 to 9.4 ± 1.0% (both P < 0.05) from baseline to 3 wk of pacing. In the sham-operated group, LV end-diastolic diameter (14.9 ± 0.5 to 15.2 ± 0.3 mm) and LV systolic shortening fraction (30.1 ± 1.4 to 28.5 ± 1.3%) remained unchanged over the time course of the protocol. There was no difference in LV weight from baseline to 3 wk in sham-operated (5.1 ± 0.5 vs. 5.4 ± 0.3 g) and heart failure rabbits (5.0 ± 0.4 vs. 5.3 ± 0.4 g). Histology. The extent of fibrosis (31.4 ± 1.8 vs. 7.0 ± 0.8% of analyzed area, P < 0.05, Fig. 1) and the number of TUNEL-positive cardiomyocyte nuclei (0.033 ± 0.004 vs. 0.002 ± 0.001/mm2, P < 0.05) was greater in failing than in sham-operated hearts. The number of leukocytes in myocardium was not different between failing and sham-operated hearts (174 ± 20 vs. 142 ± 9 cells/mm2). In the liver, the number of leukocytes was significantly greater in heart failure than in sham-operated rabbits (108 ± 13 vs. 52 ± 3 cells/mm2, P < 0.05, Fig. 1). The percentage of dry vs. fresh small intestinal weight was not different between groups (19.0 ± 1.1 vs. 18.2 ± 0.8%).



View larger version (126K):
[in this window]
[in a new window]
  		Fig. 1. Myocardial fibrosis (Masson-Goldner trichrome staining) in a heart failure (A) and a sham-operated (B) rabbit. Scale bar, 50 µm. Liver (hematoxylin and eosin staining) in a heart failure (C) and a sham-operated (D) rabbit. Scale bar, 50 µm.

 

TNF-{alpha} concentration (WEHI cell assay). Serum TNF-{alpha} concentration (240 ± 24 vs. 150 ± 22 U/ml, P < 0.05) and hepatic TNF-{alpha} concentration (2,390 ± 310 vs. 1,220 ± 135 U/g, P < 0.05) were increased in heart failure compared with sham-operated rabbits (Fig. 2). In contrast, the myocardial TNF-{alpha} concentration (920 ± 160 vs. 900 ± 95 U/g, Fig. 2) did not differ between groups and did not correlate to the serum TNF-{alpha} concentration (Fig. 3A). In contrast, both the hepatic TNF-{alpha} concentration and the number of leukocytes (Fig. 3B) and the serum and hepatic TNF-{alpha} concentrations (Fig. 3C) were correlated.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 2. Tumor necrosis factor-{alpha} (TNF-{alpha}) concentrations in serum, myocardium, and liver of heart failure and sham-operated rabbits. TNF-{alpha} concentration in serum is expressed as U/ml, whereas TNF-{alpha} concentrations in myocardium and liver are expressed as U/g wet wt. Values are means ± SE. *P < 0.05 vs. sham operated.

 


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 3. A: correlation between the myocardial and serum TNF-{alpha} concentrations. B: correlation between the hepatic leukocyte count and the hepatic TNF-{alpha} concentration. C: correlation between the hepatic and the serum TNF-{alpha} concentrations.

 

Immunohistochemistry. In the heart, TNF-{alpha} was almost entirely localized in the vascular endothelium in heart failure and sham-operated rabbits. In the liver of sham-operated rabbits, TNF-{alpha} was again localized in the vascular endothelium; however, in heart failure rabbits, TNF-{alpha} was also expressed in hepatocytes (Fig. 4).



View larger version (67K):
[in this window]
[in a new window]
  		Fig. 4. TNF-{alpha} localization in heart and liver (immunohistochemistry) in a sham-operated (A, C) and in a heart failure (B, D) rabbit.

 

TNF-{alpha} receptors 1 and 2. Both the cytosolic [333,772 ± 45,024 vs. 293,878 ± 27,858 arbitrary units (AU)] and the membrane protein concentrations (692,434 ± 93,926 vs. 575,047 ± 41,959 AU) of TNF-{alpha} receptor 1 did not differ between heart failure and sham-operated rabbits. Also, the cytosolic (99,731 ± 12,868 vs. 97,498 ± 5,825 AU) and the membrane protein concentrations (188,016 ± 26,768 vs. 201,297 ± 15,587 AU) of TNF-{alpha} receptor 2 were similar between groups.

Endotoxin concentration. The intestinal endotoxin concentration was increased in heart failure compared with sham-operated rabbits (24.5 ± 1.2 vs. 17.0 ± 3.1 EU/g wet wt, P < 0.05). Serum and hepatic endotoxin concentrations did not differ between heart failure (1.7 ± 0.3 EU/ml, 66.7 ± 11.3 EU/g wet wt, respectively) and sham-operated rabbits (1.6 ± 0.2 EU/ml, 73.4 ± 7.9 EU/g wet wt, respectively).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The main finding of the present study is that the increased serum TNF-{alpha} is of peripheral origin and not derived from the myocardium in this heart failure model.

About one-half of all patients with severe heart failure have increased serum TNF-{alpha} concentration (5, 15, 28, 32, 38, 45, 47). Particularly in those with signs of cachexia (2, 28, 31), the serum TNF-{alpha} concentration is increased, and the serum TNF-{alpha} concentration is an independent predictor of mortality in patients with advanced heart failure (17, 21, 40).

In the present study, we as others before (6, 25) could detect TNF-{alpha} within the heart mainly localized to the vascular endothelium. However, the myocardial TNF-{alpha} protein concentration was not elevated in failing hearts, suggesting that the increased serum TNF-{alpha} concentration was not of cardiac origin.

The finding of an unchanged myocardial TNF-{alpha} concentration in the pacing-induced heart failure model, in which baseline transmural blood flow is only minimally affected and signs of ischemia are absent (34), contrasts with results obtained in models of LV dysfunction secondary to aortic banding (6), coronary microembolization (20, 46), or myocardial infarction (25, 37). During ischemia, the myocardial TNF-{alpha} concentration is rapidly increased within the area at risk (22, 24, 25). With prolongation of ischemia and development of cardiomyocyte necrosis, the TNF-{alpha} concentration increases also in the surrounding viable portions of the myocardium (20, 37, 46). Indeed, in the scenario of myocardial ischemia-reperfusion, treatment with TNF-{alpha} antibodies reduced the extent of myocardial infarction (7) and attenuated the contractile dysfunction after coronary microembolization (20, 46). In the latter studies, the serum TNF-{alpha} concentration remained unaltered, thereby supporting the notion of a direct action of TNF-{alpha} on the level of the cardiomyocytes during ischemia-reperfusion.

In heart failure, the increased serum TNF-{alpha} concentration could result from the peripheral congestion and potential endotoxemia (3, 39) rather than being released from the heart itself. Indeed, endotoxin when administered to whole blood samples taken from heart failure patients increased the serum TNF-{alpha} concentration (23), indicating that blood constituents could produce or release TNF-{alpha}. Further supporting the view that TNF-{alpha} is of peripheral origin, the TNF-{alpha} concentration in the arterial and coronary sinus blood remained unchanged in dogs with pacing-induced heart failure (41) and in patients with severe heart failure (32, 38), although the serum TNF-{alpha} concentration was increased.

Although congestion of the small intestine was not detected in the present study, the endotoxin concentration of the small intestine was increased in heart failure compared with sham-operated rabbits. In contrast, serum and hepatic endotoxin concentrations were comparable between groups. Two potential explanations for the increased serum TNF-{alpha} concentration could be envisaged: first, the liver clears endotoxin from the intestinal blood draining into the portal vein. Such increased endotoxin clearance is then associated with increased hepatic leukocyte infiltration and TNF-{alpha} concentration in heart failure rabbits. Indeed, in heart failure rabbits TNF-{alpha} was expressed in hepatocytes (Fig. 4). The increased hepatic TNF-{alpha} concentration correlated to the increased serum TNF-{alpha} concentration (Fig. 3C). Second, the increased endotoxin concentration in the intestinal blood might stimulate TNF-{alpha} production or release from blood constituents (23); such TNF-{alpha} is subsequently cleared in part by the liver. The increased hepatic TNF-{alpha} concentration then causes an increase in the hepatic leukocyte concentration, as recently demonstrated in rats in vivo (48). In the present study, however, we could not distinguish between these two possibilities.

One explanation for the progression of LV dysfunction in heart failure relates to an increased level of apoptosis (1, 36). Also, in the present study the extent of apoptosis was increased in failing hearts. Although an increased TNF-{alpha} concentration induces apoptosis in skeletal muscle (16), the primarily vascular localization of TNF-{alpha} and the unchanged myocardial TNF-{alpha} concentration in the present study do not support the idea that TNF-{alpha}-induced apoptosis contributed to the induction or progression of LV dysfunction in this heart failure model. Similarly, in explanted failing human hearts cardiomyocyte apoptosis was increased (0.041 vs. 0.007%) without a correlation to the highly variable TNF-{alpha} concentration (42).

In mice lacking the myocardial TNF-{alpha} receptors 1 and 2, the ischemia-induced cardiomyocyte apoptosis was increased (27). Thus a decrease of the myocardial TNF-{alpha} receptor density, in the presence of an unchanged myocardial TNF-{alpha} concentration, could partially be responsible for the increased number of apoptotic cardiomyocytes observed in failing hearts. Indeed, a reduction in the density of TNF-{alpha} receptors 1 and 2 was measured in explanted failing human hearts (47). In the present study, however, the myocardial TNF-{alpha} receptor density was not different between failing and sham-operated hearts, thus excluding an important role of TNF-{alpha} and its receptors for the observed morphological alterations in the failing heart.

From the results of the present study, we can rule out a direct action of TNF-{alpha} on the level of the cardiomyocytes. However, the increased circulating TNF-{alpha} concentration might still contribute to the progression of heart failure. Circulating TNF-{alpha} activates angiotensinogen gene expression in hepatocytes (11), thereby potentially contributing to the increased circulating ANG II concentration measured in heart failure, and also activates neurons in the paraventricular nucleus of the hypothalamus, leading to an increased cardiac sympathetic nerve activity (49), thereby potentially contributing to the increased myocardial norepinephrine concentration measured in heart failure. Thus, despite an unchanged myocardial TNF-{alpha} concentration, the increased circulating TNF-{alpha} concentration might contribute to the depression of LV function during the progression of heart failure.

Summarizing the data, the changes in the serum and myocardial TNF-{alpha} concentrations in heart failure appear to be independent from each other. The increased serum TNF-{alpha} concentration is of peripheral origin and most likely secondary to LV dysfunction.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Schulz, Institute of Pathophysiology, Univ. of Essen Medical School, Hufelandstraße 55, 45122 Essen, Germany (E-mail: rainer_schulz{at}uni-essen.de).

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Agnoletti L, Curello S, Bachetti T, Malacarne F, Gaia G, Comini L, Volterrani M, Bonetti P, Parrinello G, Cadei M, Grigolato G, and Ferrari R. Serum from patients with severe heart failure downregulates eNOS and is proapoptotic: role of tumor necrosis factor-{alpha}. Circulation 100: 1983–1991, 1999.[Abstract/Free Full Text]
  2. Anker SD, Chua TP, Ponikowski P, Harrington D, Swan JW, Kox WJ, Poole-Wilson P, and Coats AJ. Hormonal changes and catabolic/anabolic imbalance in chronic heart failure and their importance for cardiac cachexia. Circulation 96: 526–534, 1997.[Abstract/Free Full Text]
  3. Anker SD, Egerer KR, Volk HD, Kox WJ, Poole-Wilson P, and Coats AJ. Elevated soluble CD14 receptors and altered cytokines in chronic heart failure. Am J Cardiol 86: 1054–1055, 1997.
  4. Arras M, Höche A, Bohle R, Eckert P, Riedel W, and Schaper J. Tumor necrosis factor-{alpha} in macrophages of heart, liver, kidney, and in the pituitary gland. Cell Tissue Res 285: 39–49, 1996.[ISI][Medline]
  5. Aukrust P, Ueland T, Lien E, Bendtzen K, Muller F, Andreassen AK, Nordoy I, Aass H, Espevik T, Simonsen S, Froland SS, and Gullestad L. Cytokine network in congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. Am J Cardiol 83: 376–382, 1999.[ISI][Medline]
  6. Baumgarten G, Knuefermann P, Kalra D, Gao F, Taffet GE, Michael L, Blackshear PJ, Carballo E, Sivasubramanian N, and Mann DL. Load-dependent and -independent regulation of proinflammatory cytokine and cytokine receptor gene expression in the adult mammalian heart. Circulation 105: 2192–2197, 2002.[Abstract/Free Full Text]
  7. Belosjorow S, Bolle I, Duschin A, Heusch G, and Schulz R. TNF-{alpha} antibodies are as effective as ischemic preconditioning in reducing infarct size in rabbits. Am J Physiol Heart Circ Physiol 284: H927–H930, 2003.[Abstract/Free Full Text]
  8. Belosjorow S, Schulz R, Dörge H, Schade FU, and Heusch G. Endotoxin and ischemic preconditioning: TNF-{alpha} concentration and myocardial infarct development in rabbits. Am J Physiol Heart Circ Physiol 277: H2470–H2475, 1999.[Abstract/Free Full Text]
  9. Berzofsky RN. Endotoxin detection in pharmaceuticals and medical devices with kinetic-QCL, a kinetic-quantitative chromogenic limulus amebocyte lysate assay. ALTEX 12: 93–97, 1995.[Medline]
  10. Bozkurt B, Kribbs SB, Clubb FJ, Michael LH, Didenke VV, Hornsby PJ, Seta Y, Oral H, Spinale FG, and Mann DL. Pathophysiologically relevant concentrations of tumor necrosis factor-{alpha} promote progressive left ventricular dysfunction and remodeling in rats. Circulation 97: 1382–1391, 1998.[Abstract/Free Full Text]
  11. Brasier AR and Wimbish KA. Tumor necrosis factor activates angiotensinogen gene expression by the Rel A transactivator. Hypertension 27: 1009–1017, 1996.[Abstract/Free Full Text]
  12. Bryant D, Becker L, Richardson J, Shelton J, Franco F, Peshock R, Thompson M, and Giroir B. Cardiac failure in transgenic mice with myocardial expression of tumor necrosis factor-{alpha}. Circulation 97: 1375–1381, 1998.[Abstract/Free Full Text]
  13. Coleman HN III, Taylor RR, Pool PE, Whipple GH, Covell JW, Ross J Jr, and Braunwald E. Congestive heart failure following chronic tachycardia. Am Heart J 81: 790–798, 1971.[ISI][Medline]
  14. Collins KA, Korcarz CE, Shroff SG, Bednarz JE, Fentzke RC, Lin H, Leiden JM, and Lang RM. Accuracy of echocardiographic estimates of left ventricular mass in mice. Am J Physiol Heart Circ Physiol 280: H1954–H1962, 2001.[Abstract/Free Full Text]
  15. Cugno M, Agostoni P, Brunner HR, Gardinali M, and Agostoni A. Plasma bradykinin levels in human chronic congestive heart failure. Clin Sci 99: 461–466, 2000.[Medline]
  16. Dalla Libera L, Sabbadini R, Renken C, Ravara B, Sandri M, Betto R, Angelini A, and Vescovo G. Apoptosis in the skeletal muscle of rats with heart failure is associated with increased serum levels of TNF-{alpha} and sphingosine. J Mol Cell Cardiol 33: 1871–1878, 2001.[ISI][Medline]
  17. Deswal A, Petersen NJ, Feldman AM, Young JB, White BG, and Mann DL. Cytokines and cytokine receptors in advanced heart failure. An analysis of the cytokine database from the vesnarinone trial (VEST). Circulation 103: 2055–2059, 2001.[Abstract/Free Full Text]
  18. Doyama K, Fujiwara H, Fukumoto M, Tanaka NM, Fujiwara Y, Oda T, Inada T, Ohtani S, Hasegawa K, Fujiwara T, and Sasayama S. Tumor necrosis factor is expressed in cardiac tissues of patients with heart failure. Int J Cardiol 54: 217–225, 1996.[ISI][Medline]
  19. Dörge H, Neumann T, Behrends M, Skyschally A, Schulz R, Kasper C, Erbel R, and Heusch G. Perfusion-contraction mismatch with coronary microvascular obstruction: role of inflammation. Am J Physiol Heart Circ Physiol 279: H2587–H2592, 2000.[Abstract/Free Full Text]
  20. Dörge H, Schulz R, Belosjorow S, Post H, van de Sand A, Konietzka I, Frede S, Hartung T, Vinten-Johansen J, Youker KA, Entman ML, Erbel R, and Heusch G. Coronary microembolization: the role of TNF{alpha} in contractile dysfunction. J Mol Cell Cardiol 34: 51–62, 2002.[ISI][Medline]
  21. Ferrari R, Bachetti T, Confortini R, Opasich C, Febo O, Corti A, Cassani G, and Visioli O. Tumor necrosis factor soluble receptors in patients with various degrees of congestive heart failure. Circulation 92: 1479–1486, 1995.[Abstract/Free Full Text]
  22. Frangogiannis NG, Lindsey ML, Michael LH, Youker KA, Bressler RB, Mendoza LH, Spengler RN, Smith CW, and Entman ML. Resident cardiac mast cells degranulate and release preformed TNF-{alpha}, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion. Circulation 98: 699–710, 1998.[Abstract/Free Full Text]
  23. Genth-Zotz S, von Haehling S, Bolger AP, Kalra PR, Wensel R, Coats AJS, and Anker SD. Pathophysiologic quantities of endotoxin-induced tumor necrosis factor-alpha release in whole blood from patients with chronic heart failure. Am J Cardiol 90: 1226–1230, 2002.[ISI][Medline]
  24. Gurevitch J, Frolkis I, Yuhas Y, Paz Y, Matsa M, Mohr R, and Yakirevich V. Tumor necrosis factor-{alpha} is released from the isolated heart undergoing ischemia and reperfusion. J Am Coll Cardiol 28: 247–252, 1996.[Abstract]
  25. Irwin MW, Mak S, Mann DL, Qu R, Penninger JM, Yan A, Dawood F, Wen WH, Shou Z, and Liu P. Tissue expression and immunolocalization of tumor necrosis factor-{alpha} in postinfarction dysfunctional myocardium. Circulation 99: 1492–1498, 1999.[Abstract/Free Full Text]
  26. Kubota T, Miyagishima M, Alvarez RJ, Kormos R, Rosenblum WD, Demetris AJ, Semigran MJ, Dec GW, Holubkov R, McTiernan CF, Mann DL, Feldman AM, and McNamara DM. Expression of proinflammatory cytokines in the failing human heart: comparison of recent-onset and end-stage congestive heart failure. J Heart Lung Transplant 19: 819–824, 2000.[ISI][Medline]
  27. Kurrelmeyer KM, Michael LH, Baumgarten G, Taffet GE, Peschon JJ, Sivasubramanian N, Entman ML, and Mann DL. Endogenous tumor necrosis factor protects the adult cardiac myocyte against ischemic-induced apoptosis in a murine model of acute myocardial infarction. Proc Natl Acad Sci USA 97: 5456–5461, 2000.[Abstract/Free Full Text]
  28. Levine B, Kalman J, Mayer L, Fillit HM, and Packer M. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med 323: 236–241, 1990.[Abstract]
  29. Marin-Garcia J, Goldenthal MJ, and Moe GW. Abnormal cardiac and skeletal muscle mitochondrial function in pacing-induced cardiac failure. Cardiovasc Res 52: 103–110, 2001.[ISI][Medline]
  30. Masaki H, Imaizumi T, Ando S, Hirooka Y, Harada S, Momohara M, Nagano M, and Takeshita A. Production of chronic congestive heart failure by rapid ventricular pacing in the rabbit. Cardiovasc Res 27: 828–831, 1993.[ISI][Medline]
  31. McMurray J, Abdullah I, Dargie HJ, and Shapiro D. Increased concentrations of tumor necrosis factor in "cachectic" patients with severe chronic heart failure. Br Heart J 66: 356–358, 1991.[Abstract/Free Full Text]
  32. Munger MA, Johnson B, Callahan KS, and Gilbert EM. Circulating concentrations of proinflammatory cytokines in mild or moderate heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. Am J Cardiol 77: 723–727, 1996.[ISI][Medline]
  33. Murray DR and Freeman GL. Tumor necrosis factor-{alpha} induces a biphasic effect on myocardial contractility in conscious dogs. Circ Res 78: 154–160, 1996.[Abstract/Free Full Text]
  34. Neumann T and Heusch G. Myocardial, skeletal muscle, and renal blood flow during exercise in conscious dogs with heart failure. Am J Physiol Heart Circ Physiol 273: H2452–H2457, 1997.[Abstract/Free Full Text]
  35. Niebauer J, Volk HD, Kemp M, Dominguez M, Schumann RR, Rauchhaus M, Poole-Wilson PA, Coats AJS, and Anker SD. Endotoxin and immune activation in chronic heart failure: a prospective cohort study. Lancet 353: 1838–1842, 1999.[ISI][Medline]
  36. Olivetti G, Abbi R, Quaini F, Kajstura J, Cheng W, Nitahara J, Quaini E, Di Loreto C, Beltrami CA, Krajewski S, Reed JC, and Anversa P. Apoptosis in the failing human heart. N Engl J Med 336: 1131–1141, 1997.[Abstract/Free Full Text]
  37. Ono K, Matsumori A, Shioi T, Furukawa Y, and Sasayama S. Cytokine gene expression after myocardial infarction in rat hearts. Circulation 98: 149–156, 1998.[Abstract/Free Full Text]
  38. Petretta M, Gian L, Condorelli MD, Spinelli L, Scopacasa F, de Caterina M, Leosco D, Vicario MLE, and Bonaduce D. Circulating levels of cytokines and their site of production in patients with mild to severe chronic heart failure. Am Heart J 140: e28, 2000.
  39. Rauchhaus M, Coats AJ, and Anker SD. The endotoxin-lipoprotein hypothesis. Lancet 356: 930–933, 2000.[ISI][Medline]
  40. Rauchhaus M, Doehner W, Francis DP, Davos C, Kemp M, Liebenthal C, Niebauer J, Hooper J, Volk HD, Coats AJS, and Anker SD. Plasma cytokine parameters and mortality in patients with chronic heart failure. Circulation 102: 3060–3067, 2000.[Abstract/Free Full Text]
  41. Recchia FA, Bernstein RD, Seghal PB, Ferreri NR, and Hintze TH. Cytokines are not a requisite part of the pathophysiology leading to cardiac decompensation. Proc Soc Exp Biol Med 223: 47–52, 2000.[Abstract/Free Full Text]
  42. Saraste A, Voipio-Pulikki LM, Heikkila J, Laine P, Nieminen MS, and Pulkki K. Soluble tumor necrosis factor receptor levels identify a subgroup of heart failure patients with increased cardiomyocyte apoptosis. Clin Chim Acta 320: 65–67, 2002.[ISI][Medline]
  43. Schreiber E, Matthias P, Muller MM, and Schaffner W. Rapid detection of octamer binding proteins with "mini-extracts," prepared from a small number of cells. Nucleic Acids Res 11: 6419, 1989.
  44. Sigusch HH, Lehmann ME, Schnittler U, Reinhardt D, and Figulla HR. Tumour necrosis factor-{alpha} expression in idiopathic dilated cardiomyopathy: correlation to myocardial inflammatory activity. Cytokine 12: 1261–1266, 2000.[ISI][Medline]
  45. Testa M, Yeh M, Lee P, Fanelli R, Loperfido F, Berman JW, and LeJemtel TH. Circulating levels of cytokines and their endogenous modulators in patients with mild to severe congestive heart failure due to coronary artery disease or hypertension. J Am Coll Cardiol 28: 964–971, 1996.[Abstract]
  46. Thielmann M, Dörge H, Martin C, Belosjorow S, Schwanke U, van de Sand A, Konietzka I, Büchert A, Krüger A, Schulz R, and Heusch G. Myocardial dysfunction with coronary microembolization: signal transduction through a sequence of nitric oxide, tumor necrosis factor-{alpha} and sphingosine. Circ Res 90: 807–813, 2002.[Abstract/Free Full Text]
  47. Torre-Amione G, Kapadia S, Lee J, Durand JB, Bies RD, Young JB, and Mann DL. Tumor necrosis factor-{alpha} and tumor necrosis factor receptors in the failing human heart. Circulation 93: 704–711, 1996.[Abstract/Free Full Text]
  48. Yee SB, Hanumegowda UM, Hotchkiss JA, Ganey PE, and Roth RA. Role of neutrophils in the synergistic liver injury from monocrotaline and bacterial lipopolysaccharide exposure. Toxicol Sci 72: 43–56, 2003.[Abstract/Free Full Text]
  49. Zhang ZH, Wei SG, Francis J, and Felder RB. Cardiovascular and renal sympathetic activation by blood-borne TNF-{alpha} in rat: the role of central prostaglandins. Am J Physiol Regul Integr Comp Physiol 284: R916–R927, 2003.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
E. A. Jankowska, P. Ponikowski, M. F. Piepoli, W. Banasiak, S. D. Anker, and P. A. Poole-Wilson
Autonomic imbalance and immune activation in chronic heart failure - Pathophysiological links
Cardiovasc Res, June 1, 2006; 70(3): 434 - 445.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
S. Lecour, N. Suleman, G. A. Deuchar, S. Somers, L. Lacerda, B. Huisamen, and L. H. Opie
Pharmacological Preconditioning With Tumor Necrosis Factor-{alpha} Activates Signal Transducer and Activator of Transcription-3 at Reperfusion Without Involving Classic Prosurvival Kinases (Akt and Extracellular Signal-Regulated Kinase)
Circulation, December 20, 2005; 112(25): 3911 - 3918.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
S. Aker, A. K Snabaitis, I. Konietzka, A. van de Sand, K. Bongler, M. Avkiran, G. Heusch, and R. Schulz
Inhibition of the Na+/H+ exchanger attenuates the deterioration of ventricular function during pacing-induced heart failure in rabbits
Cardiovasc Res, August 1, 2004; 63(2): 273 - 282.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
A. Skyschally, R. Schulz, P. Gres, I. Konietzka, C. Martin, M. Haude, R. Erbel, and G. Heusch
Coronary microembolization does not induce acute preconditioning against infarction in pigs--the role of adenosine
Cardiovasc Res, August 1, 2004; 63(2): 313 - 322.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
285/2/R463    most recent
00153.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aker, S.
Right arrow Articles by Schulz, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aker, S.
Right arrow Articles by Schulz, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2003 by the American Physiological Society.