Jean White

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.

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.

The role of oxygen-derived free radicals in burn-induced myocardial contractile depression.

The release of oxygen free radicals from ischemic myocardium has been implicated as a causative factor of cardiac dysfunction after thermal injury. In this study, isolated coronary perfused guinea pig hearts were used to determine if free radical scavengers improve left ventricular (LV) intrinsic contractile response to burn shock. Parameters measured included peak isovolumic LV pressure (LVP) and maximal rate of LVP rise (+dP/dtmax) and fall (-dP/dtmax) at a constant preload. Control animals were immersed in body temperature water and divided into four groups: Group 1, untreated N = 10; Group 2, control animals treated with unbound superoxide dismutase (SOD), N = 5; Group 3, control animals treated with ficoll-SOD, N = 5; and Group 4, control animals treated with PEG-SOD, N = 5. Scald burn equivalent to 45% of total body surface area was produced in 64 animals. Fluid resuscitation was initiated immediately after burn in all animals, and animals were then divided into seven burn experimental groups. In Group 5, 10 animals were treated with fluid alone, lactated Ringers, 4 mL/kg/% burn. Burned animals in Group 6 (N = 10) received a reduced volume of Ringers 2 mL/kg/% burn plus unbound-SOD, 50 mg/kg; 10 animals in Group 7 received this volume of Ringers plus ficoll-SOD, 50 mg/kg. In groups 8, 9, and 10 animals were given fluid, lactated Ringers, 2 mL/kg/% burn plus varying doses of PEG-SOD (Group 8: N = 9, 1,000 U; Group 9: N = 10, 6,000 U; Group 10: N = 5, 12,000 U). In Group 11 (N = 10), animals received SOD-PEG, 6,000 U, plus catalase, CAT-PEG, 6,000 U, given with 4 mL/kg/% burn lactated Ringers solution. Hypotension, hypothermia, and hemoconcentration were similar in all animals after thermal injury, regardless of treatment regimen. Burn hearts showed significantly lower LVP, +dP/dt max, and -dP/dt max than control hearts (P less than 0.05). Compared to controls, coronary pressure and coronary vascular resistance were significantly higher in all treated burn groups. There was no significant difference in heart rate or time to peak pressure or time to maximal contraction or relaxation among the groups. Left ventricular function curves for burned hearts were shifted downward and to the right of curves obtained from control hearts (P less than 0.01), regardless of scavenger treatment. PEG-SOD, 6,000 U, improved left ventricular contractility (+dP/dt) at maximal levels of end-diastolic pressure but deficits in left ventricular pressure and relaxation persisted.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Sodium/hydrogen exchange activity in sepsis and in sepsis complicated by previous injury: 31P and 23Na NMR study

Objective:Sepsis or septic shock occurs frequently in sick and injured patients and is associated with a significant mortality. Myocardial contractile dysfunction has been proposed to be a major determinant of sepsis-related mortality. This study was directed to examine the role of Na+/H+ exchange activity in myocardial defects after sepsis or after sepsis complicated by a previous burn injury. Design:Laboratory study. Setting:University research laboratory. Subjects:Sprague-Dawley rats (300–350 g, males). Interventions:Cardiac function, cellular Na+ and Ca2+, myocardial pH, and high-energy phosphates were examined in perfused hearts harvested after sepsis alone (intratracheal Streptococcus pneumoniae, 0.4 mL of 1 × 107 CFU/mL), after sepsis complicated by previous burn injury (40% total body surface area), and after amiloride (a selective inhibitor of Na+/H+ exchange) treatment of either sepsis alone or sepsis plus burn. Measurements and Results:The ratio of Na+ signal from the intracellular compartment (Na+i) compared with an external standard (monitored by 23Na-NMR spectroscopy, TmDOTP−4 shift reagent) increased by 70% in sepsis alone and by 41% in sepsis complicated by previous burn injury compared with shams. Cardiac adenosine triphosphate and intracellular pH (31P nuclear magnetic resonance spectroscopy) were unchanged by sepsis or sepsis plus burn. Left ventricular pressure and maximal change in pressure over time were reduced after sepsis or after sepsis plus burn injury. Amiloride treatment in either sepsis or sepsis complicated by a previous burn injury prevented myocardial Na+ and Ca2+ accumulation, attenuated sepsis-related lactic acidosis, and improved left ventricular function. Conclusion:Our results suggest that sepsis-related cardiac dysfunction is mediated, in part, by Na+/H+ exchange activity, and inhibition of Na+/H+ exchange activity improves cardiac function after sepsis alone or sepsis complicated by a previous injury.

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.

Protein Kinase C Inhibition Improves Ventricular Function after Thermal Trauma

OBJECTIVE To examine the effect of protein kinase C (PKC) inhibition on cardiac performance and intracellular Ca2+ homeostasis. DESIGN Previous studies have shown that trauma impairs cardiac mechanical function, and recent studies suggest that PKC activation and subsequent perturbations in Ca2+ sequestration/release contribute to this cardiac dysfunction. In this study, anesthetized guinea pigs were given third-degree scald burns over 43 +/- 1% of the total body surface area and resuscitated with lactated Ringers solution (LR) 4 mL/kg per percent of burn, Parkland formula. Animals with sham burns served as controls (n = 18). Burns were randomly divided into two groups: LR alone (N = 18) or LR + PKC inhibitor, calphostin C (0.1 mg/kg, intravenous bolus), given 30 minutes and 3, 6, and 21 hours after burn (n = 18). MATERIALS AND METHODS Cardiac function was assessed by Langendorff preparation 24 hours after burn in 8 to 12 animals per group. Intracellular calcium concentration ([Ca2+]i) was measured in cardiac myocytes (collagenase digestion) from additional animals in each experimental group (n = 5-9 per group) after Fura-2 AM loading of myocytes; fluorescence ratios were measured with a Hitachi spectrofluorometer. RESULTS Cardiac dysfunction occurred 24 hours after burn in LR burns as indicated by lower left ventricular pressure and a reduced rate of left ventricular pressure rise and fall, +/-dP/dt (61 +/- 3 mm Hg, 1,109 +/- 44 mm Hg/s, and 880 +/- 40 mm Hg/s, respectively) compared with values measured in sham-burned animals (86 +/- 2 mm Hg, 1365 +/- 43 mm Hg/s, and 1183 +/- 30 mm Hg/s, respectively; p < 0.05). Ventricular function curves confirmed significant postburn contractile depression despite aggressive fluid resuscitation. Cardiac injury in burned animals was indicated by an increase in perfusate creatine kinase and lactate dehydrogenase, and Ca2+ dyshomeostasis was confirmed by increased myocyte [Ca2+]i (sham 151 +/- 6 vs. burn 307 +/- 20 nmol/L, p < 0.05). PKC inhibition improved all indices of cardiac performance, producing left ventricular pressure (82 +/- 3 mm Hg), +/-dP/dt (1,441 +/- 48 and 1,294 +/- 32 mm Hg/s), and left ventricular function curves that were comparable with those of sham-burned animals. In addition, [Ca2+]i in calphostin-treated burned animals (154 +/- 11 nmol/L) was identical to values in sham-burned animals. CONCLUSION Our data suggest that PKC may serve as a final common pathway in signal transduction events mediating postburn cardiac dysfunction.