Jureta W. Horton

IL-1beta and IL-6 act synergistically with TNF-alpha to alter cardiac contractile function after burn trauma.

Although numerous studies have provided evidence that the inflammatory cytokines TNF-alpha and IL-1beta have significant negative inotropic effects, the role of the interleukins in burn-mediated cardiac dysfunction has not been defined. Furthermore, most studies examining the cardiotoxic effects of inflammatory cytokines have ignored the complex inflammatory milieu that occurs in the intact subject with trauma, sepsis, or ischemic heart disease. Therefore, this study examined the time course of IL-1beta and IL-6 secretion by cardiomyocytes after burn trauma, and additional studies examined the effects of these cytokines alone or in combination with TNF-alpha on cardiac contractile performance (Langendorff). Sprague-Dawley rats were given a full thickness burn injury over 40% of the total body surface area; fluid resuscitation was lactated Ringers solution, 4 mL/kg per burn percentage of burn area. Sham burn animals received identical anesthesia and handling, but no burn injury. Rats were sacrificed at several different times postburn, and isolated hearts (n = 4-5 rats/group/time period) were perfused with collagenase-containing buffer to prepare cardiomyocytes or were perfused in vitro to examine cardiac contractile function (n = 5-6 rats/group/time period). Additional naive control rats (n = 10) were included to prepare cardiomyocytes that, in turn, were challenged with different concentrations of either IL-1beta, IL-6, or TNF-alpha alone or in combination for several time periods (CO2 incubator at 37 degrees C for 1-3 h). Finally, inflammatory cytokines alone or in combination were added to the perfusate of hearts isolated from additional control rats (n = 6-7/group) to assess the cardiac contraction and relaxation effects of cytokine challenge. Despite aggressive fluid resuscitation, burn trauma produced a time-related increase in cardiomyocyte secretion of IL-1beta, IL-6, and TNF-alpha. Exposure of naive cardiomyocytes prepared from control rats to each cytokine alone or combined cytokine challenge produced a time-dependent and concentration-dependent decrease in cell viability and an increase in supernatant creatine kinase levels. Either IL-1beta or TNF-alpha produced greater cardiac defects than IL-6 when added separately to Langendorff-perfused hearts; dysfunction was maximal with combined cytokine challenge (IL-1beta plus TNF-alpha plus IL-6). The data confirm that burn trauma upregulates inflammatory cytokine secretion by cardiomyocytes and suggest that these inflammatory cytokines act in concert to produce burn-mediated cardiac contractile dysfunction.

The time course of cardiac NF-kappaB activation and TNF-alpha secretion by cardiac myocytes after burn injury: contribution to burn-related cardiac contractile dysfunction.

Previous studies have suggested that cardiac synthesis of TNF-&agr; contributes to myocardial dysfunction in several models of trauma, sepsis and ischemia. Therefore, it is likely that myocyte secretion of TNF-&agr; occurs early after major burn trauma, contributing to progressive cardiac contractile dysfunction that is characteristic of thermal injury. This study examined the time course of nuclear translocation of the transcription factor NF-&kgr;B, the time course of TNF-&agr; secretion by cardiomyocytes after burn trauma, and the development of cardiac contractile defects. Rats were given burn injury over 40% TBSA (sham burns included for controls), and fluid resuscitation included lactated Ringers solution, 4 mL/kg/%burn. Subsets of rats were sacrificed at several times postburn (1, 2, 4, 8, 12, 18 and 24 h), hearts were harvested to prepare cardiomyocytes (N = 4 rats/group/time period), to prepare nuclear fractions to measure burn-induced NF-&kgr;B activation (N = 3–4 rats/group/time period), or to examine the time course of postburn cardiac contractile dysfunction (N = 6–7 rats/group/time period). Despite aggressive fluid resuscitation, burn trauma activated NF-&kgr;B 4 h postburn, and this activation persisted over the 24 h study period. In addition, burn trauma produced a time-related increase in TNF-&agr; secretion by cardiac myocytes with cytokine secretion evident 1 h postburn. Cardiac dysfunction occurred 8 h postburn and persisted over the 24 h study period. Administration of a strategy designed to inhibit NF-&kgr;B activation (N-acetyl-leucinyl-leucinyl-norleucinal, ALLN, 50 mg/kg, in additional groups of burn rats) inhibited TNF-&agr; secretion by cardiac myocytes and improved myocardial function. This study confirms that burn trauma activates myocardial NF-&kgr;B and promotes cardiomyocyte secretion of TNF-&agr;. This inflammatory cascade preceded the appearance of cardiac dysfunction, suggesting that cardiac myocyte derived TNF-&agr; contributes, in part, to postburn cardiac contractile deficits.

Antioxidant Vitamin Therapy Alters Burn Trauma-mediated Cardiac Nf-κb Activation and Cardiomyocyte Cytokine Secretion

BACKGROUND This study examined the effects of antioxidant vitamins A, C, and E on nuclear transcription factor-kappa B (NF-kappaB) nuclear translocation, on secretion of inflammatory cytokines by cardiac myocytes, and on cardiac function after major burn trauma. METHODS Adult rats were divided into four experimental groups: group I, shams; group II, shams given oral antioxidant vitamins (vitamin C, 38 mg/kg; vitamin E, 27 U/kg; vitamin A, 41 U/kg 24 hours before and immediately after burn); group III, burns (third-degree scald burn over 40% total body surface area) given lactated Ringers solution (4 mL/kg/% burn); and group IV, burns given lactated Ringers solution plus vitamins as described above. Hearts were collected 4, 8, 12, and 24 hours after burn to assay for NF-kappaB nuclear translocation, and hearts collected 24 hours after burn were examined for cardiac contractile function or tumor necrosis factor-alpha secretion by cardiomyocytes. RESULTS Compared with shams, left ventricular pressure was lower in burns given lactated Ringers solution (group III) (88 +/- 3 vs. 64 +/- 5 mm Hg, p < 0.01) as was +dP/dt max (2,190 +/- 30 vs. 1,321 +/- 122 mm Hg/s) and -dP/dt max (1,775 +/- 71 vs. 999 +/- 96 mm Hg, p < 0.01). Burn injury in the absence of vitamin therapy (group III) produced cardiac NF-kappaB nuclear migration 4 hours after burn and cardiomyocyte secretion of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 by 24 hours after burn. Antioxidant therapy in burns (group IV) improved cardiac function, producing left ventricular pressure and +/-dP/dt (82 +/- 2 mm Hg, 1,880 +/- 44 mm Hg, and 1,570 +/- 46 mm Hg/s) comparable to those measured in shams. Antioxidant vitamins in burns inhibited NF-kappaB nuclear migration at all times after burn and reduced burn-mediated cytokine secretion by cardiomyocytes. CONCLUSION These data suggest that antioxidant vitamin therapy in burn trauma provides cardioprotection, at least in part, by inhibiting translocation of the transcription factor NF-kappaB and interrupting cardiac inflammatory cytokine secretion.

Major Burn Trauma In Rats Promotes Cardiac And Gastrointestinal Apoptosis

The hypothesis that cardiac functional abnormalities that occur after major burn trauma are paralleled by an increased incidence of apoptosis in cardiac myocytes was examined. Adult Sprague-Dawley rats were given a full thickness scald burn comprising 43+/-1% of the total body surface area or were manipulated identically but not exposed to burn injury (sham burn); burned rats were fluid resuscitated with lactated Ringers solution. Tissues from burn and sham burn animals were then examined by the TUNEL (TdT-mediated dUTP nick end labeling) assay and light microscopy to determine the presence of apoptosis 24 and 48 h after burn trauma. In parallel, the mechanical function of the heart was assayed in separate groups of rats. Tissues harvested from the hearts of sham-treated animals showed essentially no apoptosis, whereas a small number of apoptotic cells were noted in the intestinal villi and liver of sham-treated animals. Twenty-four hours after burn trauma, there was a marked increase in apoptotic cells in the left ventricle (+916%), and the number of apoptotic cells remained increased by eightfold 48 h postburn. Apoptosis was noted predominately in the subendocardial tissue of the left ventricle. The appearance of apoptotic cells was paralleled by a decrease in cardiac mechanical function with significant decreases in left ventricular pressure and +/-dP/dt(max). Burn injury also increased apoptosis in the small intestine significantly, whereas apoptosis in the liver did not increase with burn trauma. These data suggest that the apoptosis of the cardiac myocytes that occurs after burn trauma may contribute, in part, to postburn cardiac mechanical dysfunction.

Left ventricular contractile dysfunction as a complication of thermal injury.

As early as 1931, Blalock (1) proposed that impaired cardiovascular function was a major component of organ dysfunction and failure after major burn injury. Burn-related alterations in cardiac function were initially attributed to fluid shifts that occurred as a result of plasma loss into the burn area, producing a fall in venous return and a decrease in preload (2, 3). Numerous studies during the early 1960s described that cardiac output fell precipitously after burn injury, but this change in cardiac output occurred before the loss of plasma into the burn wound and in the presence of a normal blood volume. Studies in animals and man described that cardiac output improved rapidly with aggressive fluid resuscitation from burn injury, whereas others showed that volume resuscitation, adequate to replace or exceed intravascular fluid loss, did not always restore left ventricular stroke work (4, 5). The burn-related fall in cardiac output was attributed to changes in the microcirculation and coagulopathies that produced “sludging” of the blood (4) and peripheral vasoconstriction (5–7). Others proposed that the mismatch between the fall in cardiac output and alterations in circulating plasma volume was related to burn-mediated myocardial injury (8, 9). The finding of myocardial lesions after burn injury in humans (10, 11) led Baxter and colleagues (12) to study the presence of a “myocardial depressant factor” in serum from burn patients. In 1984, Adams and colleagues (13–15) established a model of burn injury in adult guinea pigs and examined the effects of burn injury on left ventricular contractility and compliance. These studies described that burn injury over 47% of the total body surface area (TBSA) slowed isovolumic relaxation, decreased diastolic compliance, and impaired left ventricular contractility. The authors concluded that intrinsic changes in contraction and relaxation impaired filling of the left ventricle, reducing ejection fraction and contributing to the postburn decrease in cardiac output described in experimental and clinical burn subjects. Our studies over the past 25 years have confirmed this early work by Baxter, Adams, and others. In addition, we have addressed an initial concern with Baxter’s work, which determined the myocardial depressant capabilities of burn serum by adding human burn serum to isolated perfused guinea pig hearts (12). Our approach included collecting serum from rats given burn over 40% TBSA and adding the rat burn serum to the perfusate of a control naïve rat heart, eliminating confounding effects of species-related differences (15). A study by Ferrara and colleagues (17) also extended Baxter’s initial observation of burn-related myocardial depressant factors, showing that lymph isolated from the scalded hind limb of animals impaired regional myocardial blood flow, coronary vascular reactivity and contractile performance. More recently, Sambol and associates (18) described that mesenteric lymph diversion prevented burn-related myocardial contractile depression. Collectively, these data have suggested that burn injury promotes the mobilization or release of potent factors that exert negative inotropic effects on the myocardium. Postburn myocardial contractile dysfunction has been confirmed by numerous experimental approaches, and a variety of animal models have been used to examine burn-related myocardial contractile responses to inotropic stimuli. Animal species used to examine contractile responses to burn injury include mice, rats, hamsters, guinea pigs, rabbits, dogs, pigs, and sheep. The time course of myocardial contraction and relaxation defects appear to be species-related (8, 12, 13, 16–23). However, there is remarkable similarity in the degree of myocardial contractile depression across species. As shown in Figure 1, postburn myocardial contractile deficits were evident in all hearts harvested 24 h after burn injury regardless of species. These data are consistent with numerous reports of burn-related decreases in ventricular responsiveness to increases in either cardiac filling, coronary flow rate, perfusate calcium, or isoproterenol challenge (14, 18–21). The decreased ventricular responsiveness to inotropic stimuli has been shown to correlate with a rise in serum troponin I levels, a biochemical indicator of significant cardiac injury (21). Recent data confirmed that burn trauma in healthy adults produces a rise in serum troponin I levels that were remarkably similar to the rise in troponin levels measured in burned animals. This recent clinical study indicated that serum troponin I was not detected in patients with burns smaller than 10% TBSA but was Address reprint requests to Dr. Jureta W. Horton, Department of Surgery, University of Texas Southwestern Medical Center, Dallas, TX 75390. E-mail: jureta.horton@utsouthwestern.edu. This work was supported by the National Institutes of Health (grants NIH P50 GM21681-38 and 2 RO1 GM57054-05). DOI: 10.1097/01.shk.0000145205.51682.c3 SHOCK, Vol. 22, No. 6, pp. 495–507, 2004

Burn-induced impairment of cardiac contractile function is due to gut-derived factors transported in mesenteric lymph

Neither the source nor the cause of burn-induced myocardial dysfunction is known. Because scald burns have been shown to cause cardiac contractile dysfunction, the purpose of this study was to test the hypothesis that gut-derived myocardial depressant factors were responsible for burn-induced cardiac contractile dysfunction. Male rats were subjected to laparotomy with or without mesenteric lymph duct ligation (LDL). After LDL or sham-LDL, the rats were randomized to receive sham or scald burn (43% TBSA full thickness) after which they were resuscitated for 24 h with 4 mL/kg/%burn of Ringers lactate solution, and then killed, and the hearts removed. Cardiac function was assessed by measuring the left ventricular pressure (LVP) and maximal rate of LVP rise and fall (±dP/dt) in response to increases either in 1) preload, 2) coronary flow rate, or 3) perfusate calcium. At 24 h after burn or sham burn and before killing, the mean arterial pressure of the burn group was less than the burn + LDL or the sham burn groups (P < 0.05). Pre-burn LDL significantly prevented burn-induced depression in LVP and ±dP/dt (P < 0.05). In addition, the hearts harvested from the burn group showed a significant impairment in contraction and relaxation when preload, coronary flow, or perfusate calcium was increased compared with the burn + LDL and sham groups (P < 0.05). Burn-induced cardiac dysfunction, manifested by impaired contraction and relaxation, is prevented by pre-burn lymph duct ligation. These results indicate that gut-derived myocardial depressant factors transported in mesenteric lymph contribute to burn-induced impairment of cardiac contractile function, because burn-induced cardiac dysfunction can be totally abrogated by pre-burn mesenteric lymph duct ligation.

Cardiomyocyte intracellular calcium and cardiac dysfunction after burn trauma.

Objective To examine the effects of pharmacologic agents designed to limit burn-mediated calcium overload on cardiomyocyte [Ca2+] and cardiac contractile function. Design Experimental, comparative study. Setting Cellular biology and physiology laboratory. Subjects Adult Sprague Dawley rats. Interventions Rats were given third-degree burn injury over 40% of the total body surface area, were fluid resuscitated, and then were divided randomly to receive one of five treatments: vehicle (normal saline); amiloride (50 mg/kg) to inhibit H+-Na+ exchange and subsequent Na+-Ca2+ exchange; dantrolene (10 mg/kg, 30 mins, 6 and 22 hrs postburn) to inhibit sarcoplasmic reticulum Ca2+ release; diltiazem (10 mg/kg given over first 6 hrs postburn); or amlodipine (0.07 mg/kg, 24 hrs preburn and 30 mins postburn) to block calcium slow channels. Appropriate controls (sham burns given the appropriate pharmacologic agent) were included in each group. Twenty-four hrs postburn, left ventricular function (Langendorff), cardiomyocyte [Ca2+]i and [Na+]i measured by fura-2-AM or sodium-binding benzofurzan isophthalate loading of cardiomyocytes, and myocyte secretion of tumor necrosis factor-&agr; (enzyme-linked immunosorbent assay) were assessed in shams and burns from each experimental group. This time point was selected based on our previous work confirming maximal ventricular contractile defects and maximal cytokine secretion 24 hrs postburn. Measurements and Main Results Burn trauma increased myocyte [Ca2+]i and [Na+]i, promoted tumor necrosis factor-&agr; secretion by cardiomyocytes, and impaired left ventricular function. All pharmacologic agents reduced the burn-mediated Ca2+/Na+ accumulation in cardiomyocytes and ablated burn-mediated tumor necrosis factor-&agr; secretion by myocytes; in contrast, dantrolene and amiloride provided significantly greater cardioprotection than pharmacologic agents that specifically targeted Ca2+ slow channels (diltiazem and amlodipine). Conclusion Our data suggest that the calcium antagonists used in this study provide cardioprotection by modulating several aspects of the overall inflammatory cascade rather than solely limiting cardiomyocyte accumulation of calcium.

Activation of Stress-responsive Pathways by the Sympathetic Nervous System in Burn Trauma

We have shown previously that burn trauma activates the stress responsive proteins, p38 mitogen-activated protein kinase (MAPK), c-jun NH2-terminal kinase (JNK), and NF-&kgr;B, and we have shown further that p38 MAPK is an important mediator of cardiomyocyte TNF-&agr; secretion and cardiac dysfunction in burn trauma. Since burn trauma causes a rise in circulating catecholamine levels, we hypothesized that this increased sympathetic activity may function as an upstream activator of the p38 MARK pathway in burn trauma. This study determined whether the &agr;1-adrenergic receptor ligand phenylephrine could mimic burn trauma activation of p38 MAPK, JNK, and NF-&kgr;B nuclear translocation; and the effect of the &agr;1-adrenergic receptor antagonist prazosin on either phenylephrine or burn-mediated activation of the stress response pathway was examined. Sprague Dawley rats were divided into seven groups: Group 1, controls; Group 2, phenylephrine-treated (2 &mgr;g/kg, i.v.) control rats; Group 3, phenylephrine-treated plus prazosin-treated (1 mg/kg, i.v.) control rats; additional rats were given burn over 40% total body surface area (TBSA) and received vehicle (1 mL of 2% sucrose, PO) plus fluid resuscitation (Group 4), while in Group 5, burn rats were given prazosin (1 mg/kg, PO) plus fluid resuscitation. In Groups 6 and 7, sham-burned rats were given either vehicle (1 mL of 2% sucrose, PO) or prazosin (1 mg/kg, PO) to provide appropriate controls. Administration of phenylephrine to rats caused a significant activation of cardiac p38 MAPK/JNK activities (Western blot) and cardiac NF-&kgr;B nuclear translocation (electrophoretic mobility shift assay, EMSA). Prazosin blocked phenylephrine mediated changes in p38 MAPK/JNK activities. Burn trauma activated cardiac p38 MAPK/JNK and NF-&kgr;B, increased TNF-&agr; secretion by cardiomyocytes, and impaired cardiac function. Prazosin treatment in burns interrupted the burn-mediated signaling cascade, decreasing TNF-&agr; secretion by cardiomyocytes and preventing post-burn cardiac contractile dysfunction. Thus, burn trauma-related sympathetic activity likely activates the stress-responsive cascade, which regulates myocardial TNF-&agr; transcription/translation and culminates in cardiac contraction and relaxation defects.

Increasing percent burn is correlated with increasing inflammation in an adult rodent model.

Burn injury has been associated with systemic/compartmental inflammatory responses and myocardial dysfunction. We hypothesized that burn size correlates with the extent of cardiac inflammatory response/contractile dysfunction. Adult male Sprague-Dawley rats were divided to receive anesthesia, a 3-degree burn covering 20%, 30%, 40%, or 60% total body surface area (TBSA) plus fluid resuscitation (lactated Ringer, 4 mL/kg per percent burn); sham burn animals were included as controls. There were seven rats in each group. Rats were euthanized Twenty-four h postburn, and TNF-&agr;, IL-1&bgr;, and IL-6 were measured in the plasma and in supernatant from isolated cardiac myocytes by enzyme-linked immunosorbent assay. In addition, left ventricular function (Langendorff) was studied in vitro, and troponin levels were measured by enzyme-linked immunosorbent assay. There were progressive, statistically significant increases in plasma and myocyte inflammatory cytokine levels, as well as plasma troponin with increasing burn size. Similarly, left ventricular pressure (in millimeters of mercury) and ±dP/dtmax (in millimeters of mercury per second) progressively fell with increasing burn size. However, myocardial contractile depression induced by 60% TBSA burn was similar to that produced by 40% TBSA burn. These data suggest that the degree of inflammatory response, cardiac tissue injury, and myocardial contractile depression were correlated directly with the percent TBSA burn. However, unlike inflammation and cardiac tissue damage, myocardial contractile depression reached a plateau, with maximal myocardial contraction and relaxation defects observed at 40% TBSA burn, which were not further aggravated by a larger (60%) burn.

Myocardial Inflammatory Responses to Sepsis Complicated by Previous Burn Injury

BACKGROUND It is generally accepted that an initial injury such as burn trauma alters immune function such that a second insult increases the morbidity and mortality over that observed with each individual insult. We have shown previously that either burn trauma or sepsis promotes cardiomyocyte secretion of TNF-alpha and IL-1beta, cytokines that have been shown to produce myocardial contractile dysfunction. This study determined whether a previous burn injury (given eight days prior to sepsis) (1) provides a preconditioning phenomenon, decreasing inflammatory responses to a second insult or (2) exacerbates inflammatory response observed with either injury alone. METHODS Anesthetized Sprague-Dawley rats were given either burn injury over 40% total body surface area, sepsis alone (intratracheal S. pneumoniae, 4 x 10(6) colony forming units) or sepsis eight days after burn; all rats received lactated Ringers solution. Hearts harvested 24 h after onset of sepsis alone or sepsis plus eight-day burn were used to (1) isolate cardiomyocytes (collagenase) or (2) assess contractile function (Langendorff). Cardiomyocytes loaded with 2 microg/mL Fura-2AM or sodium-binding benzofuran isophthalate were used to measure intracellular calcium and sodium concentrations (Nikon inverted microscope, Grooney optics, InCyt Im2 Fluorescence Imaging System). Additional cardiomyocytes were used to measure myocyte-secreted TNFalpha, IL-1, IL-6, IL-10 (pg/ml, ELISA). RESULTS Either burn trauma alone or sepsis alone promoted TNF-alpha, IL-1beta, nitric oxide, IL6 and IL-10 secretion by cardiomyocytes (p < 0.05). Producing aspiration-related pneumonia eight days postburn produced myocardial pro- and anti-inflammatory responses and increased myocyte Ca2+/Na+ concentrations to a significantly greater degree than the responses observed after either insult alone. CONCLUSIONS A previous burn injury alters myocardial inflammatory responses, predisposing the burn-injured subject to exaggerated inflammation, which correlates with greater myocardial dysfunction.