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Cardiac apoptosis in burned rats with delayed fluid resuscitation
Burns 27 (2001) 250– 253 www.elsevier.com/locate/burns
Cardiac apoptosis in burned rats with delayed fluid resuscitation Guang-Qing Wang, Zhao-Fan Xia *, Bao-Jun Yu, Shi-Chu Xiao, Yu-Lin Chen Burns Centre, Changhai Hospital, Shanghai 200433, People’s Republic of China Accepted 26 May 2000
Abstract Clinical and experimental studies have shown that delayed fluid resuscitation postburn decreases heart function. We hypothesized that apoptosis occurs in the cardiomyocyte in this condition. To investigate this hypothesis, rats were burned, fluid resuscitation was delayed, and the integrity of cardiac genomic DNA in the burned rats was determined with an LM-PCR Ladder Assay kit. DNA fragmentation shown as DNA ladders on gels, the hallmark of apoptosis, was found in the heart tissue of these rats. In the early phase of delayed fluid resuscitation, the nuclear factor kappa B (NF-kappa B) was examined using an electrophoretic mobility shift assay and was found to be activated. In comparison with burned rats with immediate fluid resuscitation, nitric oxide levels in hearts from burned rats with delayed fluid resuscitation were significantly lower (PB0.01). These results suggest that apoptosis may be an important pathway for cardiac injury, which may result from the activation of NF-kappa B and decreased nitric oxide levels. © 2001 Elsevier Science Ltd and ISBI. All rights reserved. Keywords: Burn; NF-kappa B; Nitric oxide; Apoptosis
1. Introduction Severe burn injury is associated with cardiac dysfunction both in clinical and experimental studies [1,2]. Reynolds et al. [1] observed 28 pediatric patients with more than 60% TBSA, all of whom had depressed left ventricular function, with lower left ventricular stroke work index. Murphy et al. [2] also reported cardiac injury in patients with burn trauma. Burn patients given delayed fluid resuscitation suffered from more heart injury than patients given immediate fluid resuscitation [3]. This heart injury may be due to the overproduction of oxygen metabolites, calcium overload in the myocardium or a change of membrane lipid metabolism. However, it is clear that cell injury remains the most important cause of the complication. Necrosis and apoptosis are two types of cell death. Recently, more attention has been paid to apoptosis, by which the organism eliminates unwanted cells in many pathological processes. Apoptosis has been proposed as an important type of cell death in ischemia/reperfusion injury [4]. We hypothesized that apoptosis may be one * Corresponding author. Fax: +86-21-65585829. E-mail address: [email protected] (Z.-F. Xia).
type of cardiomyocyte death after burn trauma with delayed fluid resuscitation. This study provided evidence of cardiac apoptosis following delayed fluid resuscitation in burned rats.
2. Material and methods Male Sprague –Dawley rats (200 –250 g) were purchased from Animal Center of Shanghai, and randomly divided into three groups, which were sham burn with no fluid resuscitation (SB), burn with immediate fluid resuscitation (BE), and burn with delayed fluid resuscitation (BD). Each group was further divided into three subgroups for examination at 8, 16 and 24 h following burn injuries. All groups were given standard laboratory rat chow and water ad libitum for 1 week to allow them to adapt to the laboratory conditions. There were 10 rats in each group.
2.1. Burn models [5] Rats were anesthetized with sodium pentobarbital i.p. (30 mg/kg), then the dorsal surfaces were shaved the day before experiment; 24 h later, the rats in burned
0305-4179/01/$20.00 © 2001 Elsevier Science Ltd and ISBI. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 0 0 ) 0 0 1 0 5 - 4
G.-Q. Wang et al. / Burns 27 (2001) 250–253
groups (BE and BD) were anesthetized with sodium pentobarbital again and placed in molds that exposed the dorsal surface to a 98°C water bath for 12 s. All were quickly dried after each exposure to avoid additional injury. This process produced a 30% TBSA fullthickness scald burn. Ringer’s lactate (1 ml/kg per %TBSA) was administered i.p. to the early resuscitation group at 30 min and again at 90 min after burn injury and to the delayed resuscitation groups at 6 and 7 h after injury. Sham-burned animals underwent identical anesthetic and handling procedures, but were immersed in 37°C water. At the indicated times, rats were anesthetized as above to excise hearts, which were quickly frozen in liquid nitrogen and stored at − 70°C for further detection.
2.2. Analysis of DNA fragmentation The genomic DNA from 10 rats in each group was obtained. Freshly isolated or frozen myocardium (50 mg) was frozen in liquid nitrogen then pestled to powder. The tissue was dissolved in cell lysis solution (10 mmol/l Tris –Cl, pH 8.0; 0.1 mol/l EDTA, pH 8.0; 0.5% SDS) at the concentration of 30 ml/mg and incubated at 65°C for 60 min, then 12 ml of proteinase K solution (20 mg/ml H2O) was added and incubated at 55°C overnight. After treatment with RNase A (2 mg/mg) and protein precipitation, soluble DNA was subsequently precipitated with isopropanol, washed with 75% ethanol, and suspended in PCR-grade H2O to detect DNA fragmentation using ApoAlert™ LM-PCR Ladder Assay Kit (Clontech, USA). The genomic DNA was mixed with ligation mix (approximately 70 ml of ligation mix per 1 mg of genomic DNA), which was heated to 55°C for 10 min then cooled to 10°C over approximately 1 h to allow the adaptor oligonucleotides to anneal, then 0.5 ml of T4 DNA ligase was added and the solution was kept at 16°C for 12 –16 h. These adaptor-ligated DNA could be used as templates for PCR at the following conditions: 72°C 8 min, 94°C 1 min, 72°C 3 min, 30 cycles; 72°C 15 min (PCR mixture: 10× LM-PCR mix, adaptor-ligated DNA, PCR-grade H2O and 50× advantage cDNA polymerase mix). Finally, 10 m1 of each reaction was processed on a 1.2% agarose/EtBr gel at 6 V/cm for approximately 2.5 h.
2.3. Electrophoretic mobility shift assays (EMSA) The frozen myocardium (100 – 200 mg) was frozen in liquid nitrogen, pestled to powder, and DNA-binding protein was extracted according to the method of Andrews and Faller [6]. Briefly, the powder was re-suspended in 400 ml cold Buffer A (10 mM Hepes – KOH, pH 7.9 at 4°C, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM PMSF) by flicking the tube and
251
allowing the cells to swell on ice for 10 min, then vortexed for 10 s. The pellet was re-suspended in 50 ml of cold Buffer C (20 mM Hepes –KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF) and incubated on ice for 20 min for high-salt extraction. Cellular debris was removed by centrifugation (10 000× g) for 2 min at 4°C, and the supernatant fraction containing DNA binding proteins was stored at − 70°C for EMSA using a EMSA system kit (catalogue number: E3300, Promeaga, USA). For supershifts, 1 mg of protein extracts was incubated with oligonucleotides, which was labelled using [g-32P]ATP (3000 Ci/mmol) with T4 polynucleotide kinase. DNA – protein complexes were separated from unbound DNA probe on native 4.5% polyacrylamide gels at 20 mA in 1% TBA. Gels were vacuum-dried and exposed to Kodak X-film at − 80°C using intensifier screens. The sequences of oligonucleotide probes were as follows: kB: 5%-AGTTGAGGGGACTTTCCCAGGC-3% 3%-TCAACTCCCCTGAAAGGGTCCG-5%
2.4. Nitric oxide assay [7] The hearts from different groups 8 h after burn injury were collected and immediately homogenized at 4°C. Tissue protein concentrations were measured at a wavelength of 595 nm with an ultraviolet absorbent detector using the Bradford method [8]. Samples (tissue protein concentration 0.1 mg/ml) were deproteinized by addition of 1 ml saturated sodium tetroborate solution, 1 ml 30% zinc sulfate solution and 9.9 ml distilled water to 100 ml sample, incubated on 100°C water for 15 min and centrifuged at 1500 rev./min, and 5 ml of supernatant was filtered. The filtrate was reacted with coppercoated cadmium particles for 90 min to reduce nitrate to nitrite. After reaction, the samples were mixed with 1 vol. Griess’s reagent (5% sulfanilamide and 0.02% Nnaphthyl ethylenediamine), and read at a wavelength of 545 nm. Results were expressed as nmol per mg tissue protein.
2.5. Statistical methods Data were expressed as the mean9 SEM. Student’s t-test and 2-test for multiple samples were used. PB 0.05 was considered significant. 3. Results
3.1. DNA fragmentation detection PCR ladder assay was used to detect the development of apoptosis following burn trauma. Fig. 1 clearly
G.-Q. Wang et al. / Burns 27 (2001) 250–253
252
Fig. 1. Apoptosis of cardiac myocytes from the burned rats with delayed fluid resuscitation. Lanes 1, 2, and 3 are the results of PCR with the templates of genomic DNA from burned rats with delayed fluid resuscitation at 24, 8 and 6 h after burn injury, respectively. Lane 4 is the PCR ladder marker.
shows the DNA ladder in the delayed fluid resuscitation group. In the immediate fluid resuscitation group, only two heart tissues had the DNA ladder among the 10 burned rats after 24 h. However in the delayed fluid resuscitation group, the number of hearts with DNA ladders increased significantly (P B0.05) (Table 1).
3.2. Acti6ation of NF-kappa B in the heart from BD group In rat heart tissue, NF-kappa B activation was identified following burn trauma with delayed fluid resuscitation (Fig. 2). In the sham burn group and burn group with immediate fluid resuscitation, there was essentially no detectable NF-kappa B activation.
Fig. 2. The effect of burn trauma on the DNA-binding activity of NF-kappa B. Total cell extracts were prepared from myocardia of rats following sham burn (lane 1), burn trauma (8 h) with immediate fluid resuscitation (lane 2) and burn trauma (8 h) with delayed fluid resuscitation (lane 3). Nuclear extracts were incubated with [g32 P]ATP-labeled oligonucleotides containing an NF-kappa B consensus motif and analyzed by EMSA. A section of a fluorograph from a native gel is shown, The filled arrowhead indicates the position of the specific complex (p65/p50).
3.3. Change of nitric oxide le6el in the heart tissue The content of NO in the heart of the rats from the sham burned group was 7989 51 nmol/mg, which was significantly lower (PB0.01) than that from the burn with immediate fluid resuscitation group (BE) or with delayed fluid resuscitation group (BD). Ten rats were analyzed in each group. In comparison with the BE group (26349 257 nmol/mg), the content of NO in the heart of rats from BD group (1654972 nmol/mg) was significantly lower (PB 0.01).
4. Discussion Table 1 Appearance of DNA ladders in the hearts of burned rats with immediate or delayed fluid resuscitationa Time (h postburn)
BD group (no. with BE group (no. with DNA ladders/total no. DNA ladders/total no. examined) examined)
8 16 24
0/10 3/10 6/10b
0/10 0/10 2/10
a BD, burn injury with delayed fluid resuscitation; BE, burn injury with immediate fluid resuscitation. b PB0.05, significantly different from early fluid resuscitation group at 24 h postburn.
The present study shows that burned rats either with immediate fluid resuscitation or with delayed fluid resuscitation have the phenomena of apoptosis in their myocardia. But cardiomyocytes from the burned rats with delayed fluid resuscitation tended to have more apoptosis, This increased apoptosis may be one factor that partially explains why patients with delayed fluid resuscitation have a higher rate of heart failure. Our results partially concur with the previous observation of Lightfoot et al. [9], who reported that apoptosis in cardiomyocytes paralleled depressed ventricular function. In general, cells undergoing apoptosis show a sequence of cardinal morphological and biochemical
G.-Q. Wang et al. / Burns 27 (2001) 250–253
features. One important biochemical change associated with apoptosis is the activation of endonuclease, which cleaves genomic DNA into multiples of internucleosomal fragments. The DNA fragments yielded are discrete multiples of a 180-bp subunit, detected as a ‘DNA ladder’ on agarose gels after extraction and separation of the fragmented DNA [10]. Although many pathways have been proposed to explain the mechanism of apoptosis, there is still no clear picture about it. Nitric oxide (NO) is one of the important factors in regulation of apoptosis. Arginine supplementation coupled with fluid resuscitation to burn injured rats can improve cardiac performance and protect cardiomyocyte membranes through NO [11,12]. In the present study, decreased NO in the myocardium of the rats with burn plus delayed fluid resuscitation might have had deleterious effects on myocytes. Studies have shown that NO from exogenous NO donors, as well as NO endogenously derived from inducible NO synthase, substantially suppress Fas which trigger cell death [13] and inhibit stress-induced endothelial cell apoptosis [14]. However some authors report that increased expression of NO synthase contributes to apoptosis [15]. Based on the fact that NO can induce the expression of genes related to either apoptosis or anti-apoptosis, Brune et al. [16] suggested that the cross-talk between destructive and protective responses would determine the role of NO in cell injury [17]. The transcription factor, nuclear factor kappa B, regulates the expression of a large number of genes including cytokines, adhesion molecules, and some molecules related to apoptosis. Burn trauma could activate the nuclear factor kappa B in the myocardia of rats with delayed fluid resuscitation as shown in Fig. 2. The activation of NF kappa B leads to the production of molecules such as TNF-a, Fas ligand [18], NO, and others, which promote apoptosis. In previous studies, a high level of TNF-a was demonstrated in myocardia from burned rats, and myocardial dysfunction can be prevented by decreasing the production of tumor necrosis factor [20]. In an endotoxin model, the diminished NF-kappa B activation attenuated myocardial TNF-alpha production and improved cardiac contractility [19]. We hypothesized that the activation of NF-kappa B has deleterious effects on the myocardia from burned rats with delayed fluid resuscitation, by producing inflammatory mediators that induce the apoptosis of cardiomyocytes. In summary, the presence of apoptosis in the cardiomyocytes from burned rats with delayed fluid resuscitation may be one important pathway for cardiac injury. Both activation of NF-kappa B and decreased
.
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production of NO might lead to the apoptosis in the rat heart observed in this model. References [1] Reynolds EM, Ryan DP, Sheridan RL, Doody DP. Left ventricular failure complicating severe pediatric burn injuries. J Pediatr Surg 1995;30:264– 9. [2] Murphy JT, Horton JW, Purdue GF, Hunt JL. Evaluation of troponin-I as an indicator of cardiac dysfunction after thermal injury. J Trauma 1998;45:700– 4. [3] Yang ZC, Li G. Postburn multiple organ failure. Chin J Plastic Surg Burns 1985;1:21– 3. [4] Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ Res 1996;79:949– 56. [5] Xia ZF, He F, Barrow RE, Broemeling LD, Herndon DN. Reperfusion injury in burned rats after delayed fluid resuscitation. J Burn Care Rehabil 1991;12:430– 6. [6] Andrews NC, Faller DV. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res 1991;19:2499. [7] Cortas NK, Wakld NW. Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method. Clin Chem 1990;36:1440– 3. [8] Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Ann Biochem 1976;76:248– 56. [9] Lightfoot E, Jr, Horton JW, Maass DL, White DJ, McFarland RD, Lipsky PE. Major burn trauma in rats promotes cardiac and gastrointestinal apoptosis. Shock 1999;11:29– 34. [10] Clutton S. The importance of oxidative stress in apoptosis. Br Med Bull 1997;53:662– 8. [11] Horton JW, White J, Maass D, Sanders B. Arginine in burn injury improves cardiac performance and prevents bacterial translocation. J Appl Physiol 1998;84:695– 702. [12] Garcia NM, Horton JW. L-arginine improves resting cardiac transmembrane potential after burn injury. Shock 1994;6:354–8. [13] Dimmeler S, Haendeler J, Sause A, Zeiher AM. Nitric oxide inhibits APO-1/Fas-mediated cell death. Cell Growth Differ 1998;9:415– 22. [14] DeMeester SL, Qiu Y, Buchman TG, et al. Nitric oxide inhibits stress-induced endothelial cell apoptosis. Crit Care Med 1998;26:1500– 9. [15] Stein B, Eschenhagen T, Rudiger J, Scholz H, Forstermann U, Gath I. Increased expression of constitutive nitric oxide synthase III, but not inducible nitric oxide synthase II, in human heart failure. J Am Coll Cardiol 1998;32:1179– 86. [16] Brune B, Sandau K, von Knethen A. Apoptotic cell death and nitric oxide: activating and antagonistic transducing pathways. Biochem Mosco 1998;63:817– 25. [17] Wink DA, Mitchell JB. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med 1998;25:434– 56. [18] Bauer MKA, Vogt M, Los M, Siegel J, Wesselborg S, SchulzeOsthoff K. Role of reactive oxygen intermediates in activation-induced CD95 ligand expression. J Biol Chem 1998;273:8048–55. [19] Shames BD, Meldrum DR, Selzman CH, et al. Increased levels of myocardial IkappaB-alpha protein promote tolerance to endotoxin. Am J Physiol 1998;275:H1084– 91. [20] Giroir BP, Horton JW, White DJ, McIntyke KL, Lin CQ. Inhibition of tumor necrosis factor prevents myocardial dysfunction during burn shock. Am J Physiol 1994;267:H118–24.
Cardiac apoptosis in burned rats with delayed fluid resuscitation Guang-Qing Wang, Zhao-Fan Xia *, Bao-Jun Yu, Shi-Chu Xiao, Yu-Lin Chen Burns Centre, Changhai Hospital, Shanghai 200433, People’s Republic of China Accepted 26 May 2000
Abstract Clinical and experimental studies have shown that delayed fluid resuscitation postburn decreases heart function. We hypothesized that apoptosis occurs in the cardiomyocyte in this condition. To investigate this hypothesis, rats were burned, fluid resuscitation was delayed, and the integrity of cardiac genomic DNA in the burned rats was determined with an LM-PCR Ladder Assay kit. DNA fragmentation shown as DNA ladders on gels, the hallmark of apoptosis, was found in the heart tissue of these rats. In the early phase of delayed fluid resuscitation, the nuclear factor kappa B (NF-kappa B) was examined using an electrophoretic mobility shift assay and was found to be activated. In comparison with burned rats with immediate fluid resuscitation, nitric oxide levels in hearts from burned rats with delayed fluid resuscitation were significantly lower (PB0.01). These results suggest that apoptosis may be an important pathway for cardiac injury, which may result from the activation of NF-kappa B and decreased nitric oxide levels. © 2001 Elsevier Science Ltd and ISBI. All rights reserved. Keywords: Burn; NF-kappa B; Nitric oxide; Apoptosis
1. Introduction Severe burn injury is associated with cardiac dysfunction both in clinical and experimental studies [1,2]. Reynolds et al. [1] observed 28 pediatric patients with more than 60% TBSA, all of whom had depressed left ventricular function, with lower left ventricular stroke work index. Murphy et al. [2] also reported cardiac injury in patients with burn trauma. Burn patients given delayed fluid resuscitation suffered from more heart injury than patients given immediate fluid resuscitation [3]. This heart injury may be due to the overproduction of oxygen metabolites, calcium overload in the myocardium or a change of membrane lipid metabolism. However, it is clear that cell injury remains the most important cause of the complication. Necrosis and apoptosis are two types of cell death. Recently, more attention has been paid to apoptosis, by which the organism eliminates unwanted cells in many pathological processes. Apoptosis has been proposed as an important type of cell death in ischemia/reperfusion injury [4]. We hypothesized that apoptosis may be one * Corresponding author. Fax: +86-21-65585829. E-mail address: [email protected] (Z.-F. Xia).
type of cardiomyocyte death after burn trauma with delayed fluid resuscitation. This study provided evidence of cardiac apoptosis following delayed fluid resuscitation in burned rats.
2. Material and methods Male Sprague –Dawley rats (200 –250 g) were purchased from Animal Center of Shanghai, and randomly divided into three groups, which were sham burn with no fluid resuscitation (SB), burn with immediate fluid resuscitation (BE), and burn with delayed fluid resuscitation (BD). Each group was further divided into three subgroups for examination at 8, 16 and 24 h following burn injuries. All groups were given standard laboratory rat chow and water ad libitum for 1 week to allow them to adapt to the laboratory conditions. There were 10 rats in each group.
2.1. Burn models [5] Rats were anesthetized with sodium pentobarbital i.p. (30 mg/kg), then the dorsal surfaces were shaved the day before experiment; 24 h later, the rats in burned
0305-4179/01/$20.00 © 2001 Elsevier Science Ltd and ISBI. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 0 0 ) 0 0 1 0 5 - 4
G.-Q. Wang et al. / Burns 27 (2001) 250–253
groups (BE and BD) were anesthetized with sodium pentobarbital again and placed in molds that exposed the dorsal surface to a 98°C water bath for 12 s. All were quickly dried after each exposure to avoid additional injury. This process produced a 30% TBSA fullthickness scald burn. Ringer’s lactate (1 ml/kg per %TBSA) was administered i.p. to the early resuscitation group at 30 min and again at 90 min after burn injury and to the delayed resuscitation groups at 6 and 7 h after injury. Sham-burned animals underwent identical anesthetic and handling procedures, but were immersed in 37°C water. At the indicated times, rats were anesthetized as above to excise hearts, which were quickly frozen in liquid nitrogen and stored at − 70°C for further detection.
2.2. Analysis of DNA fragmentation The genomic DNA from 10 rats in each group was obtained. Freshly isolated or frozen myocardium (50 mg) was frozen in liquid nitrogen then pestled to powder. The tissue was dissolved in cell lysis solution (10 mmol/l Tris –Cl, pH 8.0; 0.1 mol/l EDTA, pH 8.0; 0.5% SDS) at the concentration of 30 ml/mg and incubated at 65°C for 60 min, then 12 ml of proteinase K solution (20 mg/ml H2O) was added and incubated at 55°C overnight. After treatment with RNase A (2 mg/mg) and protein precipitation, soluble DNA was subsequently precipitated with isopropanol, washed with 75% ethanol, and suspended in PCR-grade H2O to detect DNA fragmentation using ApoAlert™ LM-PCR Ladder Assay Kit (Clontech, USA). The genomic DNA was mixed with ligation mix (approximately 70 ml of ligation mix per 1 mg of genomic DNA), which was heated to 55°C for 10 min then cooled to 10°C over approximately 1 h to allow the adaptor oligonucleotides to anneal, then 0.5 ml of T4 DNA ligase was added and the solution was kept at 16°C for 12 –16 h. These adaptor-ligated DNA could be used as templates for PCR at the following conditions: 72°C 8 min, 94°C 1 min, 72°C 3 min, 30 cycles; 72°C 15 min (PCR mixture: 10× LM-PCR mix, adaptor-ligated DNA, PCR-grade H2O and 50× advantage cDNA polymerase mix). Finally, 10 m1 of each reaction was processed on a 1.2% agarose/EtBr gel at 6 V/cm for approximately 2.5 h.
2.3. Electrophoretic mobility shift assays (EMSA) The frozen myocardium (100 – 200 mg) was frozen in liquid nitrogen, pestled to powder, and DNA-binding protein was extracted according to the method of Andrews and Faller [6]. Briefly, the powder was re-suspended in 400 ml cold Buffer A (10 mM Hepes – KOH, pH 7.9 at 4°C, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM PMSF) by flicking the tube and
251
allowing the cells to swell on ice for 10 min, then vortexed for 10 s. The pellet was re-suspended in 50 ml of cold Buffer C (20 mM Hepes –KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF) and incubated on ice for 20 min for high-salt extraction. Cellular debris was removed by centrifugation (10 000× g) for 2 min at 4°C, and the supernatant fraction containing DNA binding proteins was stored at − 70°C for EMSA using a EMSA system kit (catalogue number: E3300, Promeaga, USA). For supershifts, 1 mg of protein extracts was incubated with oligonucleotides, which was labelled using [g-32P]ATP (3000 Ci/mmol) with T4 polynucleotide kinase. DNA – protein complexes were separated from unbound DNA probe on native 4.5% polyacrylamide gels at 20 mA in 1% TBA. Gels were vacuum-dried and exposed to Kodak X-film at − 80°C using intensifier screens. The sequences of oligonucleotide probes were as follows: kB: 5%-AGTTGAGGGGACTTTCCCAGGC-3% 3%-TCAACTCCCCTGAAAGGGTCCG-5%
2.4. Nitric oxide assay [7] The hearts from different groups 8 h after burn injury were collected and immediately homogenized at 4°C. Tissue protein concentrations were measured at a wavelength of 595 nm with an ultraviolet absorbent detector using the Bradford method [8]. Samples (tissue protein concentration 0.1 mg/ml) were deproteinized by addition of 1 ml saturated sodium tetroborate solution, 1 ml 30% zinc sulfate solution and 9.9 ml distilled water to 100 ml sample, incubated on 100°C water for 15 min and centrifuged at 1500 rev./min, and 5 ml of supernatant was filtered. The filtrate was reacted with coppercoated cadmium particles for 90 min to reduce nitrate to nitrite. After reaction, the samples were mixed with 1 vol. Griess’s reagent (5% sulfanilamide and 0.02% Nnaphthyl ethylenediamine), and read at a wavelength of 545 nm. Results were expressed as nmol per mg tissue protein.
2.5. Statistical methods Data were expressed as the mean9 SEM. Student’s t-test and 2-test for multiple samples were used. PB 0.05 was considered significant. 3. Results
3.1. DNA fragmentation detection PCR ladder assay was used to detect the development of apoptosis following burn trauma. Fig. 1 clearly
G.-Q. Wang et al. / Burns 27 (2001) 250–253
252
Fig. 1. Apoptosis of cardiac myocytes from the burned rats with delayed fluid resuscitation. Lanes 1, 2, and 3 are the results of PCR with the templates of genomic DNA from burned rats with delayed fluid resuscitation at 24, 8 and 6 h after burn injury, respectively. Lane 4 is the PCR ladder marker.
shows the DNA ladder in the delayed fluid resuscitation group. In the immediate fluid resuscitation group, only two heart tissues had the DNA ladder among the 10 burned rats after 24 h. However in the delayed fluid resuscitation group, the number of hearts with DNA ladders increased significantly (P B0.05) (Table 1).
3.2. Acti6ation of NF-kappa B in the heart from BD group In rat heart tissue, NF-kappa B activation was identified following burn trauma with delayed fluid resuscitation (Fig. 2). In the sham burn group and burn group with immediate fluid resuscitation, there was essentially no detectable NF-kappa B activation.
Fig. 2. The effect of burn trauma on the DNA-binding activity of NF-kappa B. Total cell extracts were prepared from myocardia of rats following sham burn (lane 1), burn trauma (8 h) with immediate fluid resuscitation (lane 2) and burn trauma (8 h) with delayed fluid resuscitation (lane 3). Nuclear extracts were incubated with [g32 P]ATP-labeled oligonucleotides containing an NF-kappa B consensus motif and analyzed by EMSA. A section of a fluorograph from a native gel is shown, The filled arrowhead indicates the position of the specific complex (p65/p50).
3.3. Change of nitric oxide le6el in the heart tissue The content of NO in the heart of the rats from the sham burned group was 7989 51 nmol/mg, which was significantly lower (PB0.01) than that from the burn with immediate fluid resuscitation group (BE) or with delayed fluid resuscitation group (BD). Ten rats were analyzed in each group. In comparison with the BE group (26349 257 nmol/mg), the content of NO in the heart of rats from BD group (1654972 nmol/mg) was significantly lower (PB 0.01).
4. Discussion Table 1 Appearance of DNA ladders in the hearts of burned rats with immediate or delayed fluid resuscitationa Time (h postburn)
BD group (no. with BE group (no. with DNA ladders/total no. DNA ladders/total no. examined) examined)
8 16 24
0/10 3/10 6/10b
0/10 0/10 2/10
a BD, burn injury with delayed fluid resuscitation; BE, burn injury with immediate fluid resuscitation. b PB0.05, significantly different from early fluid resuscitation group at 24 h postburn.
The present study shows that burned rats either with immediate fluid resuscitation or with delayed fluid resuscitation have the phenomena of apoptosis in their myocardia. But cardiomyocytes from the burned rats with delayed fluid resuscitation tended to have more apoptosis, This increased apoptosis may be one factor that partially explains why patients with delayed fluid resuscitation have a higher rate of heart failure. Our results partially concur with the previous observation of Lightfoot et al. [9], who reported that apoptosis in cardiomyocytes paralleled depressed ventricular function. In general, cells undergoing apoptosis show a sequence of cardinal morphological and biochemical
G.-Q. Wang et al. / Burns 27 (2001) 250–253
features. One important biochemical change associated with apoptosis is the activation of endonuclease, which cleaves genomic DNA into multiples of internucleosomal fragments. The DNA fragments yielded are discrete multiples of a 180-bp subunit, detected as a ‘DNA ladder’ on agarose gels after extraction and separation of the fragmented DNA [10]. Although many pathways have been proposed to explain the mechanism of apoptosis, there is still no clear picture about it. Nitric oxide (NO) is one of the important factors in regulation of apoptosis. Arginine supplementation coupled with fluid resuscitation to burn injured rats can improve cardiac performance and protect cardiomyocyte membranes through NO [11,12]. In the present study, decreased NO in the myocardium of the rats with burn plus delayed fluid resuscitation might have had deleterious effects on myocytes. Studies have shown that NO from exogenous NO donors, as well as NO endogenously derived from inducible NO synthase, substantially suppress Fas which trigger cell death [13] and inhibit stress-induced endothelial cell apoptosis [14]. However some authors report that increased expression of NO synthase contributes to apoptosis [15]. Based on the fact that NO can induce the expression of genes related to either apoptosis or anti-apoptosis, Brune et al. [16] suggested that the cross-talk between destructive and protective responses would determine the role of NO in cell injury [17]. The transcription factor, nuclear factor kappa B, regulates the expression of a large number of genes including cytokines, adhesion molecules, and some molecules related to apoptosis. Burn trauma could activate the nuclear factor kappa B in the myocardia of rats with delayed fluid resuscitation as shown in Fig. 2. The activation of NF kappa B leads to the production of molecules such as TNF-a, Fas ligand [18], NO, and others, which promote apoptosis. In previous studies, a high level of TNF-a was demonstrated in myocardia from burned rats, and myocardial dysfunction can be prevented by decreasing the production of tumor necrosis factor [20]. In an endotoxin model, the diminished NF-kappa B activation attenuated myocardial TNF-alpha production and improved cardiac contractility [19]. We hypothesized that the activation of NF-kappa B has deleterious effects on the myocardia from burned rats with delayed fluid resuscitation, by producing inflammatory mediators that induce the apoptosis of cardiomyocytes. In summary, the presence of apoptosis in the cardiomyocytes from burned rats with delayed fluid resuscitation may be one important pathway for cardiac injury. Both activation of NF-kappa B and decreased
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production of NO might lead to the apoptosis in the rat heart observed in this model. References [1] Reynolds EM, Ryan DP, Sheridan RL, Doody DP. Left ventricular failure complicating severe pediatric burn injuries. J Pediatr Surg 1995;30:264– 9. [2] Murphy JT, Horton JW, Purdue GF, Hunt JL. Evaluation of troponin-I as an indicator of cardiac dysfunction after thermal injury. J Trauma 1998;45:700– 4. [3] Yang ZC, Li G. Postburn multiple organ failure. Chin J Plastic Surg Burns 1985;1:21– 3. [4] Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ Res 1996;79:949– 56. [5] Xia ZF, He F, Barrow RE, Broemeling LD, Herndon DN. Reperfusion injury in burned rats after delayed fluid resuscitation. J Burn Care Rehabil 1991;12:430– 6. [6] Andrews NC, Faller DV. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res 1991;19:2499. [7] Cortas NK, Wakld NW. Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method. Clin Chem 1990;36:1440– 3. [8] Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Ann Biochem 1976;76:248– 56. [9] Lightfoot E, Jr, Horton JW, Maass DL, White DJ, McFarland RD, Lipsky PE. Major burn trauma in rats promotes cardiac and gastrointestinal apoptosis. Shock 1999;11:29– 34. [10] Clutton S. The importance of oxidative stress in apoptosis. Br Med Bull 1997;53:662– 8. [11] Horton JW, White J, Maass D, Sanders B. Arginine in burn injury improves cardiac performance and prevents bacterial translocation. J Appl Physiol 1998;84:695– 702. [12] Garcia NM, Horton JW. L-arginine improves resting cardiac transmembrane potential after burn injury. Shock 1994;6:354–8. [13] Dimmeler S, Haendeler J, Sause A, Zeiher AM. Nitric oxide inhibits APO-1/Fas-mediated cell death. Cell Growth Differ 1998;9:415– 22. [14] DeMeester SL, Qiu Y, Buchman TG, et al. Nitric oxide inhibits stress-induced endothelial cell apoptosis. Crit Care Med 1998;26:1500– 9. [15] Stein B, Eschenhagen T, Rudiger J, Scholz H, Forstermann U, Gath I. Increased expression of constitutive nitric oxide synthase III, but not inducible nitric oxide synthase II, in human heart failure. J Am Coll Cardiol 1998;32:1179– 86. [16] Brune B, Sandau K, von Knethen A. Apoptotic cell death and nitric oxide: activating and antagonistic transducing pathways. Biochem Mosco 1998;63:817– 25. [17] Wink DA, Mitchell JB. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med 1998;25:434– 56. [18] Bauer MKA, Vogt M, Los M, Siegel J, Wesselborg S, SchulzeOsthoff K. Role of reactive oxygen intermediates in activation-induced CD95 ligand expression. J Biol Chem 1998;273:8048–55. [19] Shames BD, Meldrum DR, Selzman CH, et al. Increased levels of myocardial IkappaB-alpha protein promote tolerance to endotoxin. Am J Physiol 1998;275:H1084– 91. [20] Giroir BP, Horton JW, White DJ, McIntyke KL, Lin CQ. Inhibition of tumor necrosis factor prevents myocardial dysfunction during burn shock. Am J Physiol 1994;267:H118–24.