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Basic Science Aspects

IL-1β AND IL-6 ACT SYNERGISTICALLY WITH TNF-α TO ALTER CARDIAC CONTRACTILE FUNCTION AFTER BURN TRAUMA

Maass, David L.; White, Jean; Horton, Jureta W.

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Abstract

INTRODUCTION

Stressful and pathologic insults such as burn trauma elicit coordinated immunologic responses directed to neutralize the initial insult. Despite these innate mammalian protective responses to traumatic injury, progressive organ dysfunction occurs frequently, contributing to burn-related mortality (1–4). Our previous studies have shown that burn trauma promotes a rise in systemic TNF-α levels (5), paralleled by increased myocardial synthesis of TNF-α (6–8). Although several studies have suggested that the local or organ-specific synthesis and secretion of cytokines shape the body's immunologic defenses in an effort to contain infection, the escape of inflammation-related molecules such as cytokines from specific compartments into the systemic circulation is counter-regulated by the secretion of anti-inflammatory molecules designed to limit the systemic response (9). Complex interaction between inflammatory and anti-inflammatory molecules as well as between cytokines, hormones, and neurotransmitters likely occurs in burn trauma; however, these interactions are not clearly understood, and may vary in different cell types (10,11).

Although considerable attention has focused on the role of the inflammatory cytokine TNF-α in mediating the cardiac contractile dysfunction associated with burn trauma, sepsis, ischemic heart disease, congestive heart failure, and cardiopulmonary bypass (12–17), recent attention has focused on the synergistic effects of TNF-α with IL-1β (18). Several studies have described that TNF-α and IL-1β, administered independently in suboptimal amounts, produced no hemodynamic derangements, whereas the combined administration of suboptimal concentrations of these cytokines produced significant cardiovascular derangements (19–21). Other studies have described a rise in circulating levels of IL-6 after inflammation and injury; the biological functions of IL-6 include upregulation of acute phase protein responses, immunoglobulin production, and neutrophil emigration and activation, and other studies have described anti-inflammatory effects of IL-6 (22–27). Despite a wealth of information regarding the proinflammatory and anti-inflammatory functions of IL-6, the effects of this cytokine on cardiac performance after traumatic injury such as burn trauma have not been studied.

We recently described that cardiomyocytes isolated 24 h after major burn trauma secreted significant IL-1β, IL-6, and TNF-α (7); however, the contribution of myocardial-derived IL-1β alone, IL-6 alone, or these inflammatory interleukins in concert with TNF-α to postburn cardiac contractile deficits has not been studied. Thus, this present study was designed to examine the time course of burn-mediated cardiomyocyte secretion of IL-1β and IL-6 as well as TNF-α; parallel studies examined the independent effects of each inflammatory interleukin alone or in concert with TNF-α on cardiomyocyte viability and morphology as well as on cardiac contractile function.

MATERIALS AND METHODS

Experimental animals

Adult Sprague-Dawley rats (Harlan Laboratories, Houston, TX) weighing 325–360 g were used throughout the study. Animals were allowed 5–6 days to acclimate to their surroundings. Commercial rat chow and tap water were available at will throughout the experimental protocol. All work described herein was approved by The University of Texas Southwestern Medical Center Institutional Animal Care and Research Advisory Committee, and performed according to the guidelines outlined in the “Guide for the Care and Use of Laboratory Animals” as published by American Physiological Society.

Catheter placement

Rats were anesthetized with methoxyflurane 18 h before the burn experiment. Body hair was closely clipped, the neck region was treated with a surgical scrub (Betadine), and a polyethylene catheter (PE-50 tubing) was inserted into the left carotid artery with the tip advanced toward the aortic arch. Catheter position was confirmed at the time of harvesting the hearts. In addition, a polyethylene catheter (PE-50) was placed in the right external jugular vein for administration of fluids. The catheters were filled with heparinized saline, exteriorized at the nape of the neck, and the skin was closed. After recovery from anesthesia and catheter placement, animals were housed in individual cages, and body temperature was maintained throughout the experimental period with a heating lamp.

Burn procedure

Animals were anesthetized with methoxyflurane, and secured in a constructed template device as described previously (2,5,8,28). The surface area of the skin exposed through the template device was immersed in 100°C water for 12 s on each side; using this technique, full-thickness dermal burns comprising 40% of the total body surface area were obtained. This burn technique produces complete destruction of the underlying neural tissue and a transient (less than 45 s) increase in internal body temperature of 1°C to 3°C. Sham burn rats were subjected to identical preparation, except that they were immersed in room temperature water (28). After immersion, the rats were immediately dried and burned rats were given lactated Ringer's solution (4 mL/kg per percentage of burn area, with one-half of the calculated volume given during the first 8 h postburn, and the remaining volume was given during the next 16 h postburn). Subsets of rats (n = 4–5/group/time period) were sacrificed at several different times after burn trauma (1, 2, 4, 8, 12, 18, and 24 h) and cardiomyocytes were prepared to determine the time course of burn-mediated IL-1β, IL-6, and TNF-α secretion by this cell population. In addition, subsets of rats were sacrificed at identical time points and hearts were perfused in vitro to determine the time course of postburn cardiac function. Additional control rats were included to provide naive myocytes and naive hearts for in vitro cytokine challenge.

Cardiomyocyte isolation

To prepare cardiac myocytes, animals from each experimental group were heparinized, decapitated, and the heart was removed through a medial sternotomy using sterile techniques. The isolated heart was immediately placed in ice-cold calcium-free Tyrodes solution (in millimoles: 136 NaCl, 5 KCl, 0.57 MgCl2, 0.33 NaH2PO4, 10 Hepes, and 10 glucose). The aorta was cannulated within 60 s, and the excised heart was perfused with calcium-free Tyrodes solution using a Langendorff perfusion apparatus. Perfusion was maintained for 5 min and then switched to a collagenase solution that contained 80 mL of calcium-free Tyrodes, 40 mg of collagenase A (0.05%, Boehringer Mannheim, Indianapolis, IN), and 4 mg of protease (Polysaccharide XIV; Sigma Chemical Company, St. Louis, MO) with continuous oxygenation (95% O2 and 5% CO2). After this enzymatic digestion over a 10-min period, the heart was removed from the cannula and the ventricular tissue was separated from the base of the heart. This tissue was placed in a petri dish containing Tyrodes solution with 100 μM calcium and was gently minced to increase cell dispersion over 6 min. The myocyte suspension was then filtered and the cells were allowed to settle. This rinsing and settling step was repeated three times with 10 min between each step and with gentle swirling between each step to allow myocyte separation. The calcium concentration of the rinsing solution was gradually increased during these steps from 100 to 200 μM and finally to 1.8 mM. The cell viability was measured, and cell suspensions with greater than 85% viability were used for subsequent studies. Myocytes with a rod-like shape, clear defined edges, and sharp striations were prepared with a final cell count of 5 × 104 cells/mL (5,7).

Cytokine secretion by myocytes

Myocytes were pipetted into microtiter plates at 5 × 104 cell/mL/well (12-well cell culture cluster, Corning Inc., Corning, NY) and were incubated for 18 h (CO2 incubator at 37°C). Supernatants were collected to measure myocyte-secreted IL-1β (rat ELISA; Endogen, Woburn, MA), IL-6 (rat ELISA; BioSource, Camarillo, CA), and TNF-α (rat ELISA; Endogen). We previously examined the contribution of contaminating cells (non-myocytes) in our cardiomyocyte preparations using flow cytometry, cell staining (hematoxylin and eosin), and light microscopy. We confirmed that less than 2% of the total cell number in a myocyte preparation was non-cardiomyocytes (7). Because our cardiomyocyte preparations are 98% pure, we concluded that a majority of the cytokines measured in the cardiomyocyte supernatant was indeed cardiomyocyte derived.

The effects of interleukins alone or in combination with TNF-α on cardiomyocyte viability and morphology

Two groups of experiments were included to examine the effects of IL-1β and IL-6 alone or in combination with TNF-α on cardiomyocyte integrity in the absence of burn trauma. Myocytes were prepared from naive control Sprague-Dawley rats as described above. Cardiomyocytes were plated 5 × 104 cells/mL/well; myocytes were subsequently challenged with either diluent alone, or one of several concentrations (100, 200, 300, or 400 pg/mL) of either IL-1β (recombinant IL-1β; Roche Molecular Biochemical, Indianapolis, IN), IL-6 (recombinant human IL-6; Roche Molecular Biochemical), or TNF-α (rat recombinant TNF-α; ZeptoMetrix Corp., Buffalo, NY). Cells were exposed to inflammatory cytokine for either 1, 2, or 3 h; concentration-dependent as well as time-dependent effects were examined with regard to cell viability, myocyte morphology, and creatine kinase (CK) secreted into the supernatant. A total of 10 cell preparations/cytokine concentration/time period were included. Diluent consisted of standard culture media (medium 199 diluted in a balanced salt solution and supplemented with penicillin, streptomycin, and glutamine), and incubation conditions included 5% CO2 incubator at 37°C for several time periods as described above (1, 2, or 3 h). After the designated time of cardiomyocyte exposure to cytokine, microliter plates were removed from the incubator, and supernatants were harvested to measure CK in the supernatant; cell morphology was assessed and cell viability was determined. These experiments provided information regarding the concentration- and time-dependent effects of interleukins and TNF-α in the absence of burn trauma and in the absence of the complex inflammatory milieu that occurs in vivo. In addition, these preliminary experiments allowed us to select concentrations of IL-1β and IL-6 for subsequent use in combination with TNF-α to assess the effects of cytokine challenge on cardiomyocyte integrity as well as cardiac contractile performance.

Morphology and determination of cardiomyocyte injury from supernatant CK

The morphology of cardiomyocytes was classified; normal myocytes were rod shaped with clear cross striations and they lacked evidence of sarcolemma blebbing. Nonviable or injured cells were contracted or rounded with webbed or unclear striations and did have cell membrane blebbing. In each experiment, 20 to 40 individual cells were examined, and cell viability was expressed as a percentage of the total cells examined. CK activity in the supernatant was measured using enzymatic methods (CK kit, procedure #47-UV; Sigma), and activity was expressed as units per milliliter per 5 × 104 cells.

Perfused hearts

To examine the effects of inflammatory cytokines alone or in combination on cardiac contraction and relaxation, awake control Sprague-Dawley rats were anticoagulated with sodium heparin (1000 U; Elkins-Sinn, Inc., Cherry Hill, NJ) and decapitated with a guillotine. The heart was rapidly removed and placed in ice-cold (4°C) Krebs-Henseleit bicarbonate buffered solution (in millimoles: NaCL 118, KCl 4.7, NaHCO3 21, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, and glucose 11). All solutions were prepared on the day of experimental performance and were bubbled with 95% O2–5% CO2 (pH 7.4; pO2, 550 mmHg; pCO2, 38 mmHg). A 17-gauge cannula placed in the ascending aorta was connected to a buffer-filled reservoir for perfusion of the coronary circulation at a constant flow rate of 7 mL/min. Hearts were suspended in a temperature-controlled chamber maintained at 38°C, and a constant flow pump (Holter, model 911; Critikon, Tampa, FL) was used to maintain perfusion of the coronary arteries by retrograde perfusion of the aortic stump cannula. Coronary perfusion pressure was measured and effluent was collected to confirm coronary flow rate. Contractile function was assessed by measuring intraventricular pressure with a saline-filled latex balloon placed in the left ventricular chamber. Peak systolic left ventricular pressure (LVP) was measured with a Statham pressure transducer (model P23ID; Gould Instruments, Oxnard, CA) attached to the balloon cannula; the rate of LVP rise (+dP/dt max) and fall (−dP/dt max) were obtained using an electronic differentiator (model 7P20C; Grass Instruments, Quincy, MA) and recorded (model 7DWL8P; Grass Recording Instruments).

A Frank-Starling relationship for each heart was determined by plotting left ventricular developed pressure (peak systolic pressure minus LV end diastolic pressure) and ±dP/dt max responses to increases in preload (LVEDV). Hearts were paced at twice the minimum capture voltage through an electrode attached to the right atrium (3–4 Hz, 2–10 watts for a 4-ms duration, Grass Stimulator; Grass Instruments) In vitro heart rates were similar in all experimental groups, and differences in cardiac performance could not be attributed to differences in heart rate.

Synergistic effects of IL-1β, IL-6, and TNF-α on cardiac contractile function

The next set of experiments determined the effects of the inflammatory interleukins (either IL-1β, IL-6, or TNF-α) alone or in combination on cardiac contractile function using a Langendorff preparation. Initial studies described herein confirmed that burn trauma per se increased cardiomyocyte secretion of IL-1β (35 ± 2 vs. 1.0 ± 0.5 pg/5 × 104 myocytes, P < 0.05), IL-6 (143 ± 18 vs. 6 ± 1 pg/5 × 104 myocytes, P < 0.05), and TNF-α 175 ± 10 vs. 3.1 ± 0.8 pg/5 × 104 cells, P < 0.05) compared with cytokine concentrations secreted by myocytes from sham-burned rats. The doses of cytokines used alone or in combination were within the range of myocardial cytokine levels measured in sepsis alone or sepsis complicated by a previous burn injury. Higher concentrations of these cytokines impaired myocyte integrity and produced overwhelming myocardial depression (5). After perfusing the isolated hearts for 15 to 20 min, either IL-1β, IL-6, or TNF-α alone (400 pg of each cytokine and n = 6 hearts per cytokine) was added to the perfusate (total volume of 250 mL) via a side-arm port above the heart, ensuring thorough mixing of the cytokine with coronary perfusate. For the combined challenge, the three cytokines were injected as a combined bolus via the side-arm port. Coronary flow rate was maintained constant, and the hearts were perfused with cytokine-containing-buffer in a recirculating manner for 40 to 45 min. Additional hearts were included to serve as control (n = 6); in these hearts, an identical volume of diluent was added to the side-arm port above the heart, and perfusion was maintained for an identical time period as described for the cytokine-challenged hearts. The effect of each cytokine alone or in combination on cardiac performance was examined by measuring LVDP responses to incremental increases in left ventricular diastolic volume.

Statistical analysis

All values are expressed as mean ± SEM. Analysis of variance (ANOVA) was used to assess an overall difference among the groups for each of the variables. Levene's test for equality of variance was used to suggest the multiple comparison procedure to be used if the ANOVA was significant. If equality of variance among the groups was suggested, multiple comparison procedures were performed (Bonferroni). If inequality of variance was suggested, Tamhane multiple comparisons were performed. Relative changes in contractile performance to altered cardiomyocyte cytokine secretion were also compared. Multiple regression analysis of best-fitting curves with test evaluation were included. Probability values less than 0.05 were considered statistically significant (Analysis was performed using SPSS for Windows, version 7.5.1).

RESULTS

Hemodynamic-metabolic function after burn trauma

Burn trauma in adult rats produced a hemodynamic and metabolic profile that is characteristic of major thermal injury. Blood pressure and heart rate were unchanged over the 24-h study period in sham-burn controls. Twenty-four hours after burn trauma, blood pressure fell (from 145 ± 5 to 110 ± 7 mmHg, P < 0.05) and heart rate rose (from 400 ± 20 to 450 ± 10 bpm, P < 0.05) despite fluid resuscitation. Compared with values measured in sham burns, arterial pH (7.49 ± 0.01 vs. 7.5 ± 0.02), pCO2 (31 ± 0.3 vs. 28 ± 3 mmHg), and PO2 (99 ± 5 vs. 102 ± 4 mmHg) were unchanged after burn trauma; hematocrit fell (from 40 ± 1 to 29 ± 2, P < 0.05), likely due to fluid resuscitation and hemodilution. Twenty-four hours after burn trauma, serum cytokine levels were significantly elevated compared with values measured in sham burns (IL-1β: 697 ± 139 vs. 10 ± 2 pg/mL, P < 0.01; TNF-α: 51 ± 12 vs. 5 ± 1 pg/mL, P < 0.01). All animals survived the experimental protocol.

Time course of cardiomyocyte secretion and cardiac dysfunction after burn

Our initial studies were designed to examine the time course of burn-induced cardiomyocyte secretion of the inflammatory interleukins IL-1β, IL-6, and TNF-α. As shown in Figure 1, cardiomyocyte secretion of IL-1β (upper panel) and IL-6 (middle panel) increased progressively after burn trauma (P < 0.05), and are consistent with the time course of TNF-α secretion by cardiomyocytes (lower panel). Myocardial contractile abnormalities were evident 12, 18, and 24 h after burn injury, times when cardiomyocyte cytokine secretion of IL-1β, IL-6, and TNF-α were highest (Fig. 2). Twenty-four hours after burn trauma, LV developed pressure (63 ± 4 mmHg), +dP/dt max (1309 ± 59 mmHg/s) −dP/dt max (1025 ± 98 mmHg/s) were significantly less than values measured in time-matched sham burns (LVP, 96 ± 3 mmHg; +dP/dt max, 2104 ± 56 mmHg/s; −dP/dt max, 1750 ± 37 mmHg/s, P < 0.05). These changes in left ventricular contraction and relaxation after burn injury occurred despite fluid resuscitation.

Fig. 1
Fig. 1:
Time course of cardiomyocyte secretion of IL-1β (upper panel), IL-6 (middle panel), and TNF-α (lower panel) after major burn trauma. Each bar represents data from four to five rats at each time point. All values are mean ± SEM. An asterisk indicates a significant increase in cytokine levels secreted by myocytes from burns compared with cytokine levels secreted by myocytes from control rats at P < 0.05.
Fig. 2
Fig. 2:
Time course of burn-mediated changes in left ventricular developed pressure (LVP, upper panel), in the rate of LVP rise (+dP/dt max, middle panel), and LVP fall (−dP/dt max, lower panel). Each bar represents data from six to seven rats at each time point. All values are mean ± SEM. An asterisk indicates a significant difference from values measured in hearts prepared from control (nonburned) rats at P < 0.05.

Effects of inflammatory cytokines on myocyte integrity

To determine whether IL-1β, IL-6, or TNF-α could independently alter myocyte integrity, separate aliquots of cardiomyocytes isolated from naive rats were challenged with each cytokine (IL-1β, IL-6, or TNF-α) and the time-dependent and concentration-dependent changes in cell viability and myocyte CK release were examined. Cell viability decreased significantly as either the concentration or the time of IL-1β exposure (Fig. 3, upper panel) or IL-6 exposure (Fig. 3, middle panel) was increased; similarly, cell viability decreased as the concentration of TNF-α was increased (P < 0.05;Fig. 3, lower panel). As shown in this figure, exposure of cardiomyocytes to IL-1β (400 pg/5 × 104 cells) for 1 h decreased cell viability to 60%, whereas a 3-h exposure decreased cell viability to 42%. Similarly, exposure of cardiomyocytes (5 × 104) to 400 pg of IL-6 for 1 h decreased cell viability to 42%, whereas exposure for 3 h decreased viability to 22%. Finally, exposure of myocytes to TNF-α (400 pg/5 × 104 cells) for 1 h decreased cell viability to 50%; and after a 3-h exposure to TNF-α, myocyte viability was 45%.

Fig. 3
Fig. 3:
Effects of IL-1β (upper panel), IL-6 (middle panel), or TNF-α challenge (lower panel) on cardiomyocyte cell viability. All values are mean ± SEM. An asterisk indicates a dose-related effect of cytokine challenge on viability compared with viability measured in control, unchallenged myocytes at P < 0.05. A dagger indicates a time-related effect at each level of cytokine challenge at P < 0.05.

Myocytes exposed to diluent alone for 3 h retained normal morphology; cell striations were clear and there was no evidence of membrane blebbing. Cytokine challenge altered several aspects of myocyte morphology, and striations were webbed or lost; membrane blebbing was obvious in most cells after cytokine challenge.

CK measured in the supernatants from myocytes incubated in buffer alone for 3 h ranged from 96 ± 3 to 194 ± 18 U/L (indicated as control in Fig. 4). Challenge of cardiomyocytes with either IL-1β, IL-6, or TNF-α for 3 h produced a significant rise in supernatant CK levels. Combined cytokine challenge of cardiomyocytes produced greater cellular injury than that seen with each cytokine challenge alone or after IL-1β + TNF-α in the absence of IL-6 (Fig. 5, A and B). As shown in Figure 5A, combined cytokine challenge (IL-1β + IL-6 + TNF-α) for 3 h reduced myocyte viability to less than 10% (P < 0.05), whereas IL-1β + TNF-α in the absence of IL-6 decreased cell viability to 39% (Fig. 5B) compared with myocyte viability of 45% after a 3 h exposure to TNF-α alone. Similarly, combined inflammatory cytokine challenge (Fig. 5A) produced a significantly greater increase in supernatant CK (P < 0.05) compared with that measured after either IL-1β, IL-6, or TNF-α alone (Fig. 4) or after IL-1β + TNF-α (Fig. 5B).

Fig. 4
Fig. 4:
Cytokine challenge (IL-1β, upper panel; IL-6, middle panel; TNF-α, lower panel) of cardiomyocytes produced a significant rise in supernatant CK levels, indicating cytokine-mediated cellular injury. All values are mean ± SEM. An asterisk indicates a significant difference from values measured in control at P < 0.05.
Fig. 5
Fig. 5:
A, Synergistic effects of IL-1β, IL-6, and TNF-α on cardiomyocyte viability (upper panel) and cardiomyocyte CK release (lower panel). All values are ± SEM. An asterisk indicates a significant difference from control at P < 0.05. B, Synergistic effects of IL-1β and TNF-α (in the absence of IL-6) on cardiomyocyte viability and myocyte CK release. All values are mean ± SEM. An asterisk indicates a significant difference from control at P < 0.05.

Effects of inflammatory cytokines on cardiac contractile function

The next set of studies examined the cardiac contraction and relaxation responses to either IL-1β alone, IL-6 alone, TNF-α alone, IL-1β + TNF-α, or IL-6 + IL-1β + TNF-α combined; concentration of inflammatory cytokines selected for the Langendorff study were based on the above cardiomyocyte studies. As shown in Figure 6, either IL-1β or TNF-α alone impaired cardiac responsiveness to increases in preload. In contrast, IL-6 challenge in isolated hearts produced no significant cardiac contraction and relaxation deficits. These differences in cardiac contraction and relaxation occurred despite identical levels of coronary flow rate and heart rate in all hearts. Combined cytokine challenge (IL-1β, IL-6, and TNF-α) in Langendorff perfused hearts (Fig. 6, left panel) exacerbated the cardiac contractile deficits seen with each cytokine alone; in addition, all three cytokines in combination produced greater contractile defects than those seen with IL-1β + TNF-α in the absence of IL-6 (Fig. 6, right panel). These studies confirmed that although either IL-1β or TNF-α independently altered cardiac contractile performance, challenge of ventricular muscle preparations with a combined inflammatory cytokine regimen (similar to that expected in the myocardium after a major traumatic injury such as burn trauma or after overwhelming sepsis) produced significantly greater cardiac contractile deficits than that produced by any cytokine alone.

Fig. 6
Fig. 6:
Ventricular function (LVP) was studied as left ventricular end diastolic volume (shown on thexaxis) was increased. Cytokine challenge in hearts isolated from control rats produced significant cardiac contraction and relaxation defects. As shown in the left panel, either IL-1β alone or TNF-α alone (but not IL-6 alone) impaired cardiac function; however, the combined cytokine challenge (IL-1β/TNF-α/IL-6) produced greater contractile defects than any single cytokine. As shown in the right panel, this combined cytokine regimen produced greater contractile dysfunction that than seen with IL-1β/TNF-α challenge in the absence of IL-6. All values are mean ± SEM. An asterisk indicates a significant difference among groups at P < 0.05 (ANOVA and multiple comparison procedure).

DISCUSSION

Although we have shown previously that major burn trauma promotes cardiomyocyte secretion of several inflammatory mediators (7), the time course of cardiomyocyte cytokine secretion and the precise contribution of each cytokine to cardiomyocyte injury and myocardial contractile abnormalities remain unclear. In this present study, TNF-α secretion by cardiomyocytes was significant 1 h postburn, occurring earlier than secretion of either IL-1β or IL-6. These data suggest that TNF-α may serve as a more proximal mediator of the inflammatory cascade that occurs after burn injury. The progressive increase in cardiomyocyte IL-1β, IL-6, and TNF-α secretion over the 24 h after burn injury was paralleled by a progressive increase in cardiac abnormalities. The direct contribution of each proinflammatory cytokine to cardiomyocyte injury and to cardiac contraction and relaxation defects has remained difficult to distinguish in the intact burn subject; cytokines are only one facet of the overall postburn response, which includes neurohumoral and endocrine modulation of function, altered metabolic function, the release of numerous peptide and lipid mediators, as well as significant coagulopathies. Our use of cytokine challenge in either isolated cardiomyocytes or isolated hearts allowed us to examine the direct cardiodepressive effects of each cytokine in an environment free of the neurohumoral, metabolic, and endocrine responses that occur in the intact subject with major burn injury.

Challenge of naive cardiomyocytes with concentrations of either IL-1β, IL-6, or TNF-α previously shown to occur in either burn trauma or in burn complicated by sepsis produced significant cardiomyocyte injury as evidenced by a decrease in cell viability and a rise in cardiomyocyte supernatant CK levels. Either IL-1β or TNF-α challenge altered cardiac contraction and relaxation in naive hearts, whereas IL-6 challenge produced no measurable contractile abnormalities. The failure of IL-6 to alter ventricular performance may be related to the time period for IL-6 challenge in the isolated heart. Hearts were perfused in a Langendorff mode, and each cytokine was added to the perfusate in a recirculating mode, resulting in myocardial exposure to cytokine containing buffer for approximately 45 min. Direct exposure of isolated myocytes to IL-6 for 1 to 3 h produced significant injury. However, the cardiomyocyte isolation procedure removes all connective tissue, myocardial matrix, as well as non-myocyte cells. The lack of IL-6-related cardiac contractile defects in the perfused hearts may be related to failure of IL-6 to permeate the myocardial tissue and to gain direct access to the myocytes.

Of particular importance was our finding that combined inflammatory cytokine challenge (IL-1β, IL-6, and TNF-α) produced significantly greater cardiomyocyte injury and greater cardiac contractile deficits than the defects observed after challenge with a single cytokine. Our current data support previous reports of synergistic effects of inflammatory cytokines on myocardial contractile function (19–21), and our data are consistent with previous reports that inflammatory cytokines have overlapping proinflammatory activities with regard to acute phase responses, neutrophil activation, and induction of PGE2 (23–26). Finally, the overlapping and synergistic effects of these inflammatory cytokines may provide one explanation for the failure of anti-cytokine strategies to decrease morbidity and mortality in models of sepsis and traumatic injury (29,30).

Although numerous previous studies have implicated cytokines in the systemic inflammatory response syndrome, systemic levels of proinflammatory mediators such as TNF-α and IL-1β frequently remain low or are undetectable in experimental and clinical trauma in the absence of sepsis (31–33). However, this present study as well as previous studies by us and others have shown that the heart produces considerable TNF-α, IL-1β, and IL-6 in response to either burn trauma, burn complicated by sepsis, endotoxin challenge, or after hemodynamic pressure overload (7,8,34–37). TNF-α mRNA and protein have been also described in cardiac tissue from patients with dilated cardiomyopathy and ischemic heart disease (37). These data suggest that the secretion of inflammatory cytokines within the myocardium may produce higher local concentrations than observed in the systemic circulation. In addition, the compartmentalized secretion of inflammatory cytokines within the myocardium may not be readily buffered by soluble receptors or endogenous scavaging mechanisms that constitute a counter-regulatory system.

This present study provided the first direct evidence that cytokines produce cardiomyocyte injury and impair cardiac function in the absence of neurohumoral responses, metabolic aberrations, coagulopathies, and protein and lipid mediators that occur in vivo. IL-1β and TNF-α have been shown to bind to distinct cell surface receptors, and cardiomyocytes have been shown to possess functional TNF-α and IL-1β receptors. Although the biological functions of these inflammatory cytokines include upregulation of acute phase protein synthesis and neutrophil activation, it is unlikely that the cardiac contractile deficits produced by proinflammatory cytokines in our study were related to these mechanisms. Exposure of cardiomyocytes to inflammatory cytokines produced significant loss of cellular integrity after a relatively brief of time (within 1 h). Although our cardiomyocyte preparations have been shown to be extraordinarily pure (containing less than 2% of non-cardiomyocyte cell types), it is clearly recognized that emigrated neutrophils and other non-myocyte cell populations were present in the perfused heart. However, the period of cytokine challenge in the isolated perfused hearts was extraordinarily short (45–50 min), providing limited time for neutrophil stimulation and activation, and a neutrophil-mediated cascade that include de novo protein synthesis.

Our finding that IL-6 challenge in the isolated hearts produced no measurable cardiac contractile deficits was somewhat surprising; however, Munford and colleagues (27) have proposed that IL-6 produces anti-inflammatory effects under some circumstances. We considered that in the perfused heart, IL-6 may serve a counter-regulatory role, balancing transient changes in the myocardial matrix to preserve mechanical performance. Thus, we initially attributed the greater contractile dysfunction with the combined cytokine challenge (IL-1β + IL-6 + TNF-α) to the effects of TNF-α alone or to the synergistic effects of IL-1β and TNF-α (19). In our study, the addition of IL-1β to TNF-α treatment of cardiomyocytes decreased cell viability from the 45% viability seen with TNF-α treatment alone to 39% (IL-1β plus TNF-α. Although addition of IL-1β to TNF-α perfusion of isolated hearts shifted LV function curves to the right, these effects on mechanical performance did not disallow that TNF-α was the primary mediator of contractile dysfunction. When we compared the cardiodepressive effects of either IL-1β or TNF-α alone or IL-1β plus TNF-α with the effects of all three cytokines, we noted that the addition of IL-6 significantly exacerbated the effects of IL-1β/TNF-α on myocyte viability and on LV function. The greater myocardial deficits observed with combined cytokine challenge suggest that these mediators act synergistically to modulate organ function, but further studies are warranted.

In summary, our studies confirmed that IL-1β and TNF-α independently impaired cardiac myocyte viability and morphology and produced cardiac contractile abnormalities. Combined cytokine challenge (IL-1β plus IL-6 plus TNF-α) exacerbated the independent effects of any cytokine challenge alone and suggested that these inflammatory cytokines act synergistically to produce significantly greater loss of cardiomyocyte integrity and greater cardiac contraction and relaxation deficits than deficits produced by any one inflammatory cytokine. The present study confirmed that cardiomyocytes themselves secrete inflammatory cytokines in a time-dependent and concentration-dependent manner after major burn trauma, and this study also extended our previous studies that showed that burn trauma produced nuclear translocation of the transcription factor NF-κB and promoted cardiomyocyte secretion of TNF-α (5). The present study provides a mechanism for the previously reported failure of anti-cytokine strategies to improve morbidity and mortality in the setting of traumatic injury. The complex inflammatory cascade evoked by burn trauma produces a host of cytotoxic mediators with overlapping and synergistic activities.

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Keywords:

Inflammatory cytokines; cardiac contraction and relaxation; cardiomyocyte injury; cytokine-mediated cardiac contractile dysfunction

© 2002 Lippincott Williams & Wilkins, Inc.