Severe burn injury results in organ dysfunction and failure (1). Burn-induced changes in cardiac function have been shown to occur independent of intravascular plasma loss (2-4). Therefore, other mechanisms such as stress-induced alterations in cellular signaling pathways and generation of potentially harmful mediators such as proinflammatory cytokines, reactive oxygen intermediates, and complement anaphylatoxins (C3a, C5a) likely play a central role in the pathogenesis of postburn cardiomyopathy. Our knowledge about the effects of burn injury and concomitant infection or sepsis at the cardiac myocyte functional level is limited. This study was therefore designed to evaluate the effects of burn injury and in vitro lipopolysaccharide (LPS) exposure on single-cell sarcomere contractile properties.
Endotoxin derived from gram-negative bacteria can induce leukocytes to secrete cytokines, leading to fever, coagulation defects, pulmonary dysfunction, kidney failure, and circulatory collapse. Infectious complications are the major cause of late death in severe burn injury (5, 6). Changes in vascular permeability induce postburn fluid shifts which, together with burn-induced cardiac dysfunction, cause hypoperfusion of vital end-organ tissues. The hypoperfused gastrointestinal tract is the source of translocated gram-negative bacteria and endotoxemia (7). This sets up a cycle of burn injury, infection, increased proinflammatory cytokine release, worsening cardiac dysfunction, and end-organ hypoperfusion. One end result is multiorgan dysfunction syndrome that can become severe and irreversible, leading to eventual demise of the patient. As early as 1966, Baxter et al. (8) postulated the presence of a myocardial depressant factor in the serum of burn patients. Ferrara et al. (9) demonstrated that lymph fluid isolated from the scald burn-injured hind limb of animals impairs the regional myocardial blood flow and contractile function. Subsequently, it was shown that diversion of mesenteric lymph fluid can prevent burn-induced myocardial contractile dysfunction (10). These data support the hypothesis that burn injury results in production of inflammatory mediators that can exert a negative effect on cardiac function.
Transient cardiac contractile abnormalities appear as early as 2 h after burn injury and resolve by 72 h after burn injury (11). Studies by Giroir et al. (12) and White et al. (13) suggest that an inflammatory response that includes the direct local cardiac synthesis of tumor necrosis factor α (TNF-α), interleukin (IL) 1β, and IL-6 mediates myocardial dysfunction following burn trauma. The local myocardial levels of cytokines produced may exceed those measured in the systemic circulation. In a murine model of burn injury, cardiomyocytes from burned animals secreted significantly greater amounts of TNF-α when exposed to in vitro LPS than cardiomyocytes from animals subjected to sham burn (13). It was shown previously that cardiac secretion of TNF-α correlates with cardiac contraction and relaxation deficits, and TNF-α has therefore been proposed to mediate cardiac function deficits in burn trauma, ischemia/reperfusion, and hemorrhagic shock (14). This is interesting because the myocardial stress response to thermal injury seems to include mechanisms-for example, TNF-α, IL-1β, and IL-6 production-that themselves, in a presumably autocrine fashion, have been shown to induce myocyte necrosis (15). Highlighting the possible central role of local TNF-α in the induction of myocardial dysfunction, it was shown in transgenic mice capable of overexpressing TNF-α exclusively in cardiac myocytes that the local production of TNF-α caused dilated cardiomyopathy and severe congestive heart failure (16).
In this study, we sought to determine whether the potential detrimental effect of LPS exposure on cardiomyocyte single-cell sarcomere contractile parameters is additive or synergistic with burn injury. Our studies of cardiomyocyte function were carried out under in vitro conditions at the single-cell level. The time course of cardiomyocyte dysfunction after burn injury was studied. We also investigated the effect of burn injury and LPS exposure on cardiomyocyte secretion of inflammatory mediators such as TNF-α, IL-1β, and IL-6.
MATERIALS AND METHODS
Adult male Sprague-Dawley rats (Harlan, Inc., Indianapolis, Ind) weighing 300 to 350 g were used in all experiments. Before use, the animals were housed in a specific pathogen-free environment and allowed to acclimate to their surroundings for 1 week. Standard rat chow and water were available to the animals ad libitum. All experiments were performed in accordance with the guidelines set forth by the National Institutes of Health for care and use of animals. The experimental protocol was approved by the University Committee on Use and Care of Animals at the University of Michigan.
Rats were anesthetized with ketamine hydrochloride 100 mg/kg (Ketaset, Fort Dodge Inc., Fort Dodge, Iowa) and xylazine hydrochloride 5 mg/kg (AnaSed, Lloyd Laboratory, Shenandoah, Iowa) by i.p. injection. Dorsal hair was closely clipped and removed using Nair depilatory cream (Church & Dwight Inc., Princeton, NJ). The animals were then placed in a prefabricated mold device with a rectangular opening which exposed the dorsal skin surface while protecting the remaining skin from burn exposure. The exposed skin surface was immersed in 60°C water for 40 s. This technique produces a full-thickness dermal burn over 30% of the total body surface area (TBSA). Sham burn animals underwent an identical procedure, except that they were immersed in room temperature water (28°C). After immersion, the animals were immediately dried and resuscitated with lactated Ringer solution at a dose of 4 mL/kg per percent TBSA burn. One half of the calculated resuscitation volume was given intraperitoneally, and the remaining volume was given as divided dose s.c. injections immediately after burn. The animals also received buprenorphine hydrochloride 0.1 mg (Buprenex Injectable, Reckitt Benckiser Pharmaceuticals, Richmond, Va) by s.c. injection every 8 h for the first 24 h after burn injury. Sham burn animals received the same resuscitation and analgesia treatment.
To prepare cardiac myocyte suspensions for study of single-cell function and cytokine secretion, animals were anticoagulated with 1,000 U of heparin sodium i.p. (Abbott Laboratories, North Chicago, Ill) and anesthetized with pentobarbital sodium 65 mg/kg (Nembutal, Abbott Laboratories) at time 1, 6, 12, and 24 h after burn or sham injury. A summary of the experimental timeline is illustrated in Figure 1. A bilateral thoracotomy flap was created, and the heart was rapidly excised and rinsed in ice-cold Krebs-Henseleit buffer (containing [in mmol/L] 118 NaCl, 4.7 KCl, 21 NaHCO3, 1.8 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 11 glucose) supplemented with 5 mmol/L taurine (Sigma Corp., St. Louis, Mo).
The heart perfusion and the cardiomyocyte isolation procedures were performed using sterile technique. All of the perfusate solutions were prepared on the day of use and equilibrated with 95% O2 to 5% CO2 (carbogen) by continuous gas bubbling for at least 30 min before heart perfusion. The pH of the perfusate solution was measured and adjusted to 7.4 using either concentrated HCl or NaOH solution. The distal aortic arch was cannulated and secured to the cannula with 4-0 silk sutures. The cannula with the attached heart was mounted on a Langendorff perfusion system (ADInstruments Inc., Colorado Springs, Colo) connected to a PC-based signal transduction/amplification system (PowerLab, ADInstruments Inc.). Cannulation and initiation of heart perfusion took place within 60 s of removal of the organ from the animal. The perfusion system allowed continuous measurement and control of system parameters (perfusate flow [mL/min], perfusion pressure [mmHg], perfusate temperature [°C]). Retrograde coronary artery perfusion was initiated with calcium-containing (1.8 mmol/L) Krebs-Henseleit buffer for 5 min. The flow rate was set to 7.5 mL/min, and temperature was kept at 38°C (±0.5°C). After 5 min, the perfusate solution was switched to calcium-free Krebs-Henseleit buffer, and perfusion was continued for 3 min. The flow rate was increased to 9.0 mL/min. Enzyme digestion was then performed by switching to a calcium-free Krebs-Henseleit perfusion solution containing collagenase type II 0.5 mg/mL (lot no. 42P5948, Worthington Biochemical Corp., Lakewood, NJ) and hyaluronidase 0.2 mg/mL (lot no. 013K1194, Sigma Corp.), and perfusion continued for 10 min. The calcium concentration was gradually increased to 1.0 mmol/L by adding three 600 μL aliquots of 100 mmol/L CaCl2 solution at 30-s intervals. Heart perfusion was then continued for an additional 10 min or terminated early if the measured perfusion pressure decreased acutely. The heart was removed from the cannula, gently minced, and resuspended in 10 mL of calcium-containing enzyme solution. The tissue solution was triturated for 2 min to increase cell dispersion using prefabricated sterile silanized glass pipettes. The supernatant was pipetted off and centrifuged at 500 rpm for 15 s, and the pellet was resuspended in 10 mL of Ca2+-Krebs-Henseleit buffer supplemented with 6% bovine serum albumin. This cycle was repeated for three to five times. All of the collected single-cell suspensions were combined, filtered through 100-μm nylon mesh and then allowed to gravity-settle for 12 min. This cardiomyocyte pellet was resuspended in 10 mL of Dulbecco modified Eagle medium (GIBCO Corp., Grand Island, NY) supplemented with 200 mmol/L l-glutamine, 10% fetal calf serum (Invitrogen Corp., Carlsbad, Calif), and antibiotic solution at a concentration of 1 mL/500 mL of medium (Primocin 100 μg/mL, InvivoGen Corp., San Diego, Calif). Cardiomyocyte concentration was determined using a hemacytometer; cell viability was assessed by trypan blue dye exclusion and cell morphology. Myocytes with a rodlike shape, clearly defined edges, and sharp striations were counted as viable cells, whereas cells with membrane blebbing and loss of striation pattern and rounded cells were classified as nonviable. Cell suspensions with a viability of more than 75% were used for all subsequent experiments. Purity of the cell suspension and grade of contamination with leukocytes (neutrophil granulocytes, lymphocytes, and macrophages) was assessed with FACScan Flow Cytometry System (Becton Dickinson, San José, Calif). Using the size of a known reference population of leukocytes for comparison, cardiomyocytes were gated by size with the typical forward and side light scatter profiles revealing a purity of more than 99% cardiomyocytes.
LPS stimulation of cardiomyocytes
Bacterial endotoxin (LPS, lot no. 013K4148, derived from E. coli 0111:B4, Sigma Corp.) was prepared by dissolving 25 mg of the lyophilized powder in 25mL M199 medium. This suspension was then sonicated and boiled for 10 min. The solution was separated into 1-mL aliquots and stored at −20°C. The cardiomyocytes were plated onto sterile 22 × 22-mm glass coverslips precoated with 40 μg/mL natural mouse laminin (Invitrogen Corp.) at a density of 5 × 104 cells per coverslip per well. The coverslips were incubated in six-well tissue culture plates (37°C, 5% CO2) for 3 h to allow for cell attachment and adhesion to the laminin matrix. The fetal calf serum-containing DMEM was then carefully pipetted off and replaced with 2 mL per well serum-free M199 medium (Sigma Corp.) supplemented with 10 mmol/L glutathione (Sigma Corp.), 0.2 mg/mL BSA (GIBCO Corp.), 15 mmol/L HEPES (Sigma Corp.), 26 mmol/L NaHCO3 (Sigma Corp.), and the desired experimental concentration of LPS. Separate aliquots of the plated cardiomyocytes were exposed to one of the following LPS concentrations: 0, 1, 10, 25, and 50 μg/mL. The plates containing single-cell suspensions of cardiomyocytes and reagents were placed in an incubator (37°C, 5% CO2) for 18 h. All media and other reagents used for the cardiomyocyte isolation were certified endotoxin-free by the manufacturers. At the end of the incubation period, supernatants were collected from the wells, or the cells underwent single-cell sarcomere contraction analysis.
Single-cell sarcomere contraction and relaxation analysis
Plated cardiomyocytes that had been incubated for 18 h under experimental conditions underwent single-cell sarcomere contraction and relaxation analysis using a variable-rate CCD video camera system (MyoCam, IonOptix Corp., Milton, Mass) equipped with sarcomere length detection software (IonWizard, IonOptix Corp.). Microscopic imaging was performed using an inverted microscope system (Eclipse TE-2000S, Nikon Corp., Melville, NY) connected to the CCD video camera system. A coverslip with the plated cardiomyocytes was placed in a prefabricated chamber, which was filled with warm (37°C) M199 and mounted on the microscope system. The chamber system was connected to a Grass stimulator system (Grass Inc., West Warwick, RI). Electrical pacing stimulation was performed using a 100-mV stimulus of 4-ms duration and a frequency of 1 Hz. The measurement of sarcomere contraction at the different experimental conditions (LPS dose) and the selection of the cardiomyocytes from each coverslip were performed in a randomized fashion. For each measured cardiomyocyte, a rectangle-shaped region of interest was defined, and sarcomeres within the focused region were selected for analysis. Typically, this region included approximately 15 to 20 sarcomeres. Sarcomere contractions were recorded for 75 s. Raw data for sarcomere shortening and relaxation was collected and stored using the IonWizard software. Analysis of values at each experimental condition was performed by averaging normalized values for four to eight cardiomyocytes per coverslip, ×three wells, × three animals for each of the experimental conditions. Parameters measured included relative peak sarcomere shortening, departure velocity, and return velocity (Fig. 2).
Cytokine secretion analysis
Cell culture supernatants were collected from the 12-well, laminin-precoated plates after 18 h of exposure to the reagent mixtures and stored at −80°C for later measurement of myocyte-secreted TNF-α, IL-1β, and IL-6. The previously collected and frozen supernatants were thawed to room temperature. A commercially available ELISA kit (DuoSet, R&D Systems, Minneapolis, Minn) was used, and the assay was performed in duplicates as specified in the manufacturer‘s instructions. Optical density was assessed using an automated plate reader set at a wavelength of 450 nm with a correction reading of 540 nm (Synergy HT-1 automated fluorescent plate reader, Bio-Tek Instruments, Winooski, Vt), and cytokine concentrations were determined from the standard curve.
Limulus amebocyte assay
A chromogenic limulus amebocyte assay (QCL-1000, Cambrex Corp., Baltimore, Md) was performed to confirm endotoxin-free conditions in the cardiomyocyte suspensions which received no LPS stimulation. Briefly, samples of the cell culture supernatants were mixed with the limulus amebocyte assay reagent and chromogenic substrate reagent over a short incubation period (16 min) and read on a spectrophotometer at a wavelength of 405 nm. The assay has a sensitivity range of 0.1 to 1.0 EU/mL.
Stimulation of RAW 267.4 cells with increasing doses of LPS
The LPS (E. coli 0111:B4, lot no. 013K4148, Sigma Corp.) used in all experiments was checked for its ability to stimulate cytokine production from murine RAW 267.4 cells. RAW 267.4 cells were plated at 1 × 105 cells per well on 96-well plates and incubated with increasing LPS concentrations (0, 0.1, 1, 10, and 100 ng/mL media) for 6 h. Supernatants were collected and subjected to standard sandwich ELISA for TNF-α (DuoSet, R&D Systems). Four experiments were conducted per LPS concentration, and each of the supernatants was analyzed in triplicate.
All statistical analysis was performed using STATA Statistics/Data Analysis 8.0 software (Stata Corp., College Station, Tex). Results are expressed as the mean value ± SEM unless otherwise noted. Analysis of variance (ANOVA) and multiple linear regression analysis were used to assess for measured differences among the groups for each of the grouping variables (burn injury vs. sham injury, LPS stimulation vs. no LPS stimulation, and LPS dose). Student t test was used to analyze the cytokine data for the two different groups. Statistical significance was defined as a P value ≤ 0.05.
Cardiac proinflammatory cytokine secretion after burn (TNF-α, IL-1β, IL-6)
IL-1β, IL-6, and TNF-α levels were elevated in the supernatants from burn compared with sham cardiomyocytes isolated 1 h after burn injury (Fig. 3), A-C). For cardiomyocyte cells isolated 6, 12, and 24 h after burn, the cytokine levels of IL-1β, IL-6, and TNF-α were decreased compared with those seen at 1 h after burn. There was no difference between the levels of IL-1β, IL-6, and TNF-α demonstrated in supernatants from the burn versus sham cardiomyocyte cells isolated 6, 12, and 24 h after burn (Fig. 4), A-C). Cell culture with escalating concentrations of LPS did not produce a proportional dose-response increase in cytokine production from the burn group cardiomyocytes. This was true for all of the postinjury time points tested (1, 6, 12, and 24 h).
Cardiomyocyte viability before culture with LPS was more than 75% for all experiments. The mean percentage of viable cardiomyocytes after the 18-h incubation period was as follows: 0 μg/mL LPS, 79.4% (±3.2%); 1 μg/mL LPS, 78.0% (±2.9%); 10μg/mL LPS, 75.2% (±5.1%); 25 μg/mL LPS, 74.2% (±4.2%); 50 μg/mL LPS, 72.6% (±5.6%). We also evaluated the supernatants from non-LPS-exposed cultured cardiomyocytes to determine potential endotoxin contamination using a limulus colorimetric endotoxin assay. No endotoxin contamination was detected in all samples (endotoxin threshold detection level = 0.1 EU/mL).
TNF-α production in RAW 267.4 cell cultures after LPS stimulation
To check the activity of the endotoxin used in our experiments, we stimulated RAW 267.4 cells in culture and measured TNF-α production. Figure 5 depicts the measured TNF-α levels in RAW 267.4 cell culture supernatants 6 h after stimulation with LPS from lot no. 013K4148. A robust dose-dependent response for TNF-α production was demonstrated with increasing doses of LPS.
Burn injury decreases cardiomyocyte sarcomere shortening
At all of the postburn injury time points investigated, the cardiomyocytes isolated from burned rats demonstrated a significant inhibition of peak sarcomere shortening when compared with sham controls (Fig. 6). It was interesting to observe that a significant decrease in peak sarcomere shortening was observed in burn cardiomyocytes compared with sham control cardiomyocytes harvested as early as 1 h after burn. A progressive decrease in peak sarcomere shortening was observed at 6 and 12 h after burn injury followed by complete recovery of contractile function in cells harvested 24 h after the thermal insult.
Endotoxin exposure inhibits cardiomyocyte sarcomere contractility in a dose-dependent fashion
Using cardiomyocytes isolated from burn and sham control animals, we investigated the effect of in vitro LPS exposure on single-cell sarcomere contraction and relaxation. Cardiomyocytes were isolated at 1, 6, 12, and 24 h after injury. After plating and exchange of media, the cardiomyocytes were then cultured for 18 h in the presence of 0, 1, 10, 25, or 50 μg/mL LPS in the absence of serum. At the end of the 18-h incubation period, sarcomere contractility was significantly lower in burn and sham cardiomyocyte cells exposed to LPS (Fig. 7, A-D). Increasing the concentration of LPS in the culture medium resulted in a dose-dependent reduction of peak sarcomere shortening in cardiomyocytes from both burn and sham animals 1 h after injury (Fig. 7, A). Burn injury coupled with LPS exposure did not have a synergistic effect but did appear to have an additive effect toward decreasing peak sarcomere shortening. The concentration of LPS required to produce a significant decrease in peak sarcomere shortening in cardiomyocytes from burn animals decreased as time at which the myocytes were harvested after burn injury increased. For cells isolated 1 h postinjury, a concentration of 50 μg/mL LPS induced a significant depression of peak sarcomere shortening when compared with the burn/no LPS group (Fig. 7A), P = 0.04), whereas at 6, 12, and 24 h after burn injury, lower LPS concentrations induced significant changes in peak sarcomere shortening versus the burn/no LPS group (Fig. 7), B-D, 6 h, P = 0.002 for LPS 25 μg/mL; 12 h, P = 0.02 for LPS 10 μg/mL; 24 h, P = 0.04 for LPS 1 μg/mL, multiple linear regression).
Cardiomyocytes from burn animals respond to injury early with a decrease in contraction departure velocity
Cardiomyocytes isolated from animals which received a burn injury showed a decreased normalized contraction departure velocity (1/s), defined as the maximal velocity measured during the contraction portion of the sarcomere contraction-relaxation cycle normalized to the peak shortening length. A significant difference in departure velocity between burn and sham cardiomyocytes were seen at one h post thermal injury, P < 0.05, ANOVA (Fig. 8). The difference between departure velocities for cells following burn or sham injury was abolished in cardiomyocytes isolated at the 6, 12 and 24 h time points after injury (data not shown). Cell culture with increasing concentrations of LPS in vitro did not produce an additive or synergistic change in the measured departure velocity difference over the difference already seen for burn versus sham cardiomyocytes harvested 1 h post thermal injury.
Cardiomyocytes from burn animals respond to injury early with a decrease in relaxation velocity
Cardiomyocytes isolated from animals which received a burn injury showed a decreased relaxation velocity, which is the maximal velocity measured during the relaxation portion of the sarcomere contraction-relaxation cycle. This difference in relaxation velocity between burn and sham cardiomyocytes was seen at 1 h after thermal injury, P < 0.03, ANOVA (Fig. 9). The difference between relaxation velocities for cells after burn or sham injury was abolished in cardiomyocytes isolated at the 6, 12, and 24 h time points after injury (data not shown). Culture with increasing concentrations of LPS in vitro did not produce an additive or synergistic change in the measured relaxation velocity difference over the difference already seen for burn versus sham cardiomyocytes harvested 1 h after thermal injury (Fig. 9), not significant). The only LPS effect seen was a decrease in relaxation velocity for burn versus sham cardiomyocytes harvested 24 h after thermal injury and incubated with 50 μg/mL LPS (P = 0.012, multiple linear regression).
In the clinical setting, severe burn injury is acutely accompanied by dysfunction of multiple organs, including the heart. Previous studies by Horton et al. have shown that cardiac function as measured by maximum left ventricular pressure and maximum rate of left ventricular pressure rise and fall (±dP/dt maximum) in isolated, Langendorff-perfused, whole hearts is substantially impaired by burn injury (11, 14). These effects are seen as early as 2 h after thermal insult. Our current study extends beyond the organ level to the subcellular level and finds evidence of suppressed sarcomere shortening and also impaired contractile function as early as 1 h after burn injury. Using a single-cell analysis system, this study demonstrates that a 30% TBSA full-thickness scald burn results in severely decreased cardiomyocyte contractile function. These experiments also show that exposure of the isolated cardiomyocytes to endotoxin in vitro results in a dose-dependent inhibition of peak sarcomere shortening during at least the first 24 h after burn injury (Fig. 7). Burn injury and LPS exposure were additive, and the concentration of LPS at which a significant depression of contractility was seen decreased as time elapsed from burn injury increased before cardiomyocyte isolation.
The molecular mechanisms behind burn-induced myocardial suppression remain undetermined. There has been experimental evidence to suggest that inflammatory cytokines such as TNF-α, IL-1β, and IL-6 may play an important role in suppression of cardiomyocyte function after burn injury (11, 15). We assayed cardiomyocyte culture supernatants for TNF-α, IL-1β, and IL-6 production. Cardiomyocytes isolated from the burn versus sham group produced significantly more TNF-α, IL-1β, and IL-6 when isolated 1 h after burn. Elevations in all three of these cytokines were accompanied by observed decreases in sarcomere shortening. However, in cardiomyocyte cultures harvested at 6, 12, and 24h after burn injury, we failed to see differences in cytokine levels despite differences in sarcomere shortening between the burn and sham groups.
Furthermore, we did not observe a difference in the measured cytokine levels after LPS stimulation of burn or sham cardiomyocytes. Even at the 1-h time point, we saw no effect of LPS treatment on cytokine production, although we noted a difference in baseline cytokine production between burn and sham cardiomyocytes. In fact, at 6, 12, and 24 h after burn injury, the production of IL-1β and TNF-α was barely above the detection threshold for each of the two groups. Only IL-6 showed a small increase, with maximum levels achieved in cells harvested 12 h after thermal injury and a return to levels less than the 1-h values by 24 h after burn. Although LPS treatment did not alter cytokine production, we observed significant inhibition of cardiomyocyte sarcomere shortening with increasing doses of LPS.
In our system, cardiomyocyte cells showed significant depression in sarcomere contractility after burn injury and exposure to LPS. This was in the absence of generation of large amounts of proinflammatory cytokines as the time from burn injury increased. Based on our results, it is conceivable that that the cardiomyocyte contractility depression seen after LPS exposure may not be due to intrinsic proinflammatory cytokine expression alone. Instead, it may be mediated by an immunosuppressed state and LPS tolerance after burn injury. The initial surge of proinflammatory gene expression in response to injury or infection is brief and replaced by a prolonged expression of anti-inflammatory mediators (i.e., compensatory anti-inflammatory response syndrome). This down-regulation of the proinflammatory response and continued production of anti-inflammatory proteins can lead to an overall immunosuppressed state that is characterized by LPS tolerance that can increase the risk of secondary bacterial infection (17-19). It is unlikely that our cell preparations are affected by contamination with macrophages or neutrophils as the presence of these cells should lead to a high level of cytokine production when cocultured with LPS. It is possible that the enzymatic isolation technique renders some cell-surface receptors on cardiomyocytes inactive because of degradation. However, these cardiac myocytes continued to show significant LPS dose-dependent suppression of sarcomere shortening without evidence of differential inflammatory cytokine production in tissue culture when treated with increasing doses of LPS.
In light of our finding of a minimal difference in cytokine production by cardiomyocytes after burn injury and LPS exposure despite large observed differences in cardiomyocyte contractility, we postulate that factors other than locally produced cytokines can also mediate the myocardial suppression seen after burn injury and sepsis. In this context, Schirmer et al. (20) have reported that animals with an intact complement system exhibit cardiac output depression and deterioration of hemodynamic parameters such as mean arterial pressure and systemic vascular resistance as early as 15 min after burn injury, whereas the hemodynamics of complement depleted animals are unaffected by burn injury. Acute thermal injury to the skin has been proven to initiate local complement activation, and the progressive increase in vascular permeability is linked to complement activation and histamine release (21). Increased levels of the anaphylotoxin C5a have been detected in serum and lung homogenates after thermal injury (22). Treatment of animals with a polyclonal neutralizing antibody to C5a before burn injury resulted in diminished vascular permeability changes and reduced tissue buildup of myeloperoxidase within the lung. C5a blockade in the setting of sepsis has a protective effect and results in decreased levels of bacteremia, preserved the H2O2-producing capacity of neutrophils, and increased 10-day survival from 9.5% to 50% in a rat cecal ligation and puncture model (23). We have also recently found in a standardized rat CLP model that pretreatment with specific anti-C5a antibody at the time of injury attenuated septic cardiomyocyte contractility deficits significantly (data not yet published). Other experiments in our laboratory using a mouse burn model showed significant up-regulation of hepatic complement anaphylotoxin C5a.
A recent study by Tavener et al. (24) demonstrated that LPS-treated C57Bl/6 mice showed reduced cardiomyocyte shortening and calcium transients as compared with myocytes from untreated mice. LPS injected into chimeric mice with TLR4-positive leukocytes and TLR4-deficient myocytes showed reduced shortening in response to LPS. Myocytes from chimeric mice with TLR4-deficient leukocytes and TLR4-positive cardiac myocytes had no response to treatment with LPS. They concluded that TLR4 presence on leukocytes, and not on cardiac myocytes, was important for cardiac myocyte impairment during endotoxemia. Conversely, Dunzendorfer et al. (25) have shown in monocytes and endothelial cells of CD14 and TLR4 knockout mice that LPS uptake is independent of TLR4 expression. Comstock et al. (26) have recently shown a direct CD14-mediated effect of LPS on cardiomyocytes in culture; however, TLR4 contribution to LPS signaling was not evaluated. They conclude that cardiodepressant effects may not only be due to negative inotropic effects of TNF-α, but may also be complicated by TNF-mediated apoptosis. In this context, Maass et al. (15) reported a substantial reduction in cardiomyocyte viability when the cells were stimulated in vitro with various doses of IL-1β, IL-6, and TNF-α, either alone or in combination. The mechanisms of LPS-binding and TLR4 involvement in this process are still not entirely clear, and further mechanistic experiments are warranted. Our current studies using isolated cardiomyocytes in the absence of leukocytes show a clear LPS effect on contractility. There are also other receptors besides TLR4 capable of mediating LPS effects, including CXCR4, Nod1, Nod2, and Hsp70/Hsp90. It has to be appreciated that the multifactorial pathogenesis of stress-induced cardiac contractility deficits is reflected by a multitude of stress response and host defense systems which are activated in concert after thermal injury (complement activation, generation of reactive oxygen intermediates, neuroimmunologic mechanisms, etc.).
In summary, our study clearly demonstrates an early depression of cardiomyocyte sarcomere contractility from burn injury. This effect was evident as early as 1 h after burn.
In addition, we demonstrated on a single-cell level that LPS exposure has a dose-dependent inhibitory effect on cardiomyocyte sarcomere contraction without significant induction of proinflammatory cytokines. Further experiments to explore and clarify the mechanisms of cardiac LPS signaling are warranted.
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Burn trauma; cardiac function; endotoxin; langendorff preparation; sarcomere shortening analysis; IL-1β; IL-6; TNF-α