Role Of Heme Oxygenase-1 In Early Myocardial Damage After Severe Burn

Background and Research StrategySevere burn causes cardiac injury and dysfunction, because of acute ischemia,anoxyaemia, variety of cytokine releasing, shock and sepsis, which was namedwith post-burn cardiac shock. Heme oxygenase enzyme (HO) is ubiquitous inmammalian cells, which catalyzes the first and rate-limiting step in the degradationof heme to yield equimolar quantities of biliverdin, carbon monoxide, and iron.HO may play a vital function in oxidative stress, inflammatory reaction,maintaining cellular homeostasis and angiotasis. HO-1 is highly inducible isoformsof HO. Recent studies have demonstrated that HO-1 was obvious induced inkidney, lung, and liver in post-burn rats, and provided protection against post-burndamage. The express of HO-1 increased highly in myocardium in ischemiareperfusion heart, and the level of HO-1 was relative with the area of myocardialinfarction. Gene polymorphism of HO-1 has a close relationship with stentrestenosis in PTCA and stenting patients. HO-1 inhibited the secretion ofendothelin-1(ET-1) in EC in vitro. All of these studies showed that HO-1provided an important role in myocardial injury.We hypothesized that HO-1 play an important role in myocardial injury inacute post-burn rats, and act as an endogenous protective substance againstthe first and second attack on heart in post-burn.To investigate the hypothesis and approach the possible mechanism, a serialexperiment was designed.At first, the degree of cardiac damage and the changes of HO-1 were examinedwith ECG, biochemical indicator, pathology analysis, and the level of HO-1mRNA and protein in myocardium in well-described 30%TBSAⅢ°burn ratsmodel in vivo. Then, hearts were isolated and perfused according to the Langendorff techniqueto evaluate the degree of injury and the role of HO-1 in myocardial injury withexutory hemin and specific inhibitor zinc protoporphyrin Ⅸ(ZnPP) in post-burnrats. The purpose was to study whether HO-1 directly protect against themyocardium injury in post-burn rats on the level of isolated heart organ. At last, cardiomyocyte was cultured under thermal and endotoxin stress modelto mimic effects of thermal and sepsis on myocardium in the early and later periodof burn. Role of oxidative stress and inflammatory factor was investigated throughdetection malondialdehyde, superoxide dismutase, intracellular Ca2+ concentration,nuclear factor NF-κB,and tumor necrosis factor-α(TNF-α) and so on. And therole of HO-1 on oxidative stress and inflammatory reaction was studied withhemin and specific inhibitor ZnPP on cellular and molecular level.Part ⅠChanges of cardiac damage and HO-1 expression in acute post-burnrats.Objective: To understand the pattern of cardiac damage in severe burn and heat stresspretreatment, changes of electrocardiogram, biochemical indicator, pathologyanalysis, and ultramicrostructure were examined during 24 h follow severe burn.Some molecular biology methods such as reverse transcription-polymerase chainreaction (RT-PCR) and so on were used to detect the changes of HO-1 inmyocardium. The purpose was to investigate the variation pattern of myocardiuminjury, the cardiac protection of heat stress pretreatment, and the variation of HO-1in post-burn rat in vivo.Methods: Rats were randomized into control group (n=6), burn group(n=30), and heatpretreatment group(n=30). Heat stress was done through holding rats up 42℃for30 min, then rats were fed normally 24h, followed a procedure to construct awell-described 30% TBSA Ⅲ°burn rats model. Changes of arrhythmia, STsegment, creatine kinase-MB isoenzyme (CK-MB) concentrate in serum, andpathological section in optical microscope and electronmicroscope were analyzedat 1h, 3h, 6h, 12h, and 24h post-burn with rats sacrifice. And the level of HO-1mRNA and protein was detected with RT-PCR and Western blotting method inmyocardium, respectively.Results: Cardiac damage: Compared with control rats, except for increasing heart rate,atrial premature beats and premature ventricular contraction (PVB) at 1h and 3hpost-burn, the burn rats showed no obvious arrhythmia at other time. The majorchange of ECG was the elevation of ST segment. The amplitude became obviouswith the course of extension, and emerges peak value during 6h to 12h. Theactivity of CK-MB increased significantly at 3h post-burn (p<0.05) , and achievedhighest value at 12h post-burn (p<0.01) with lasting to 24 h in burn rats. Comparedwith burn group rats, there were no arrhythmias in pretreatment group rats. Thedegree of ST segment elevation and CK-MB decreased markedly at eachcorresponding time (p<0.05), and the peak was delayed. Diversity damages such aswavilness change of myofilament, interstitial edema, intercellular gap widen, andmyofilament alinement disorder, collapse, dissolve and so on were shown atdifferent phase of post-burn. The worst phase stay between 6h to 12h post-burnin burn group rats. The mitochondria swell and dysmorphia and ultramicro-structure of cardiocyte collapsed at 24h. While pretreatment amelioratedmyocardium damage in pretreatment group rats, and the worst phase delay to 12hpost-burn. The level of HO-1: The quantity of HO-1mRNA and protein was at low levelin control group rats. At 1h, 3h, 6h, 12h, 24h post-burn in burn group rats, Thequantity of HO-1mRNA was 1.24±0.15,1.93±0.41,2.81±0.57,2.24±0.52,1.55±0.24 fold of that in myocardium in control group rats, respectively. Thequantity began to increase at 1h, significantly increased at 3 h (p<0.05), andachieved highest at 6~12h (p<0.01). The quantity of HO-1mRNA was 1.55±0.18,2.77±0.54, 4.22±0.76, 3.34±0.55, 2.06±0.21 fold of that in control group ratsin myocardium in pretreatment group rats, respectively. Compared with that inburn group rats, the quantity of HO-1mRNA increased at 1h post-burn, andsignificantly at 3h, 6h, 12h, (p<0.05). The quantity was no significant difference at24h between burn group and pretreatment group. The expression of HO-1 proteinincreased 2.6±0.58 fold of that in control rats at 3h in myocardium in burn rats(p<0.5), and markedly increased at 6h, 12h and 24h (6.3±1.13, 9.6±1.8, and 3.2±0.72 fold, respectively, p<0.01). The phase of peak was between 6h to 12h. Theexpression of HO-1 protein was 4.4±1.2,5.1±1.3,11.4±2.1, and 16.7±2.5 foldof that in control rats at 1h, 3h, 6h, 12h post-burn, respectively. There wassignificantly different at each corresponding time between burn group andpretreatment group rats (p<0.05), except for 24h post-burn.Discussion and conclusion The renal and respiratory function injury had been well understood, whilecardiac injury was little understood and caught little interest caused by severe burnfor a long time. There were not only some factors which inhibited cardiac functionafter severe burn, such as decrease of effect circulation blood volume, increase ofmyocardium inhibition factor and so on, and severe burn had direct effects onmyocardium injury. The exactly mechanism of interaction was not clear. Someprotective substance for example HSP70 express increase, and some injury factorfor example TNF-α, interleukin-1β(IL-1β) express up-regulate after burn. Thisstudy documented that there was visible myocardial damage in the early of severeburn, especially at 12h post-burn. The damage showed ST segment elevation inECG, myocardium injury marker CK-MB and troponin (data not show) increase inserum, and pathology structure collapse such as wavilness change of myofilament,interstitial edema, intercellular gap widen, and myofilament alinement disorder,collapse, dissolve and so on. Heat stress pretreatment ameliorated and delayedmyocardium damage caused by severe burn, and provided a cardiac protection.Data had demonstrated that HO-1, one of HSP, ameliorated myocardium injury insome ischemia-reperfusion animal model. HO-1 was induced in lung and liver inburn animal, and played an important role in anti-injury. Our study first discoveredthat the level of HO-1mRNA and protein increased significantly in myocardium inpost-burn rat. Heat stress pretreatment induced HO-1 expression more markedly.The phase of HO-1 expression peak was ahead of the phase of damage peak, whichhinted that HO-1 involve in the cardiac protection of the heat stress pretreatmentafter severe burn. In order to clear that, further study was carried out.PartⅡRole of HO-1 in cardiac reperfusion injury in burn ratObjective: Cardiac ischemia-reperfusion injury was evaluated through the exam of infarctsize and cardiac function by the Langendorff technique after severe burn, and therole of HO-1 was evaluated with exutory hemin and specific inhibitor ZnPP in theprocess. The study investigated whether HO-1 directly protect against themyocardium injury in post-burn rats.Methods: Rats were randomized into burn group(n=6), heat pretreatment group(n=6), heatpretreatment+ ZnPP group(n=6), and heat pretreatment+ZnPP+Hemin group (n=6).Burn and heat pretreatment were done according to the description ahead, thenhearts were isolated and perfused according to the Langendorff technique. Heartswere perfused with Kerbs buffer containing ZnPP(10μM) and Hemin(25μM)respectively, following baseline perfusion 30 min, then ischemia 30 min andreperfusion 45 min at constant pressure. Stock solutions of both ZnPP and Heminwere prepared by dissolving the compounds in 0.1 M NaOH and then adjusting thepH to 7.4 by the addition of 0.01M PBS. ECG, LVDP,±dp/dtmax, heart rate (HR),coronary flow (CF), coronary resistance (CR), and CK were measured throughinstrumentation of multichannel physiologic recorder and accompanying software.Infarct size was measured with 2,3,5-Triphenyl-tetrazolium chloride solutionsdyeing at the end of each experimental protocol. The level of HO-1 mRNA andprotein was detected respectively in myocardium.Results The level of HO-1: HO-1mRNA was easily detected in myocardium byRT-PCR in burn group rats. Perfusion with ZnPP down-regulated the level ofHO-1mRNA (0.85±0.11 fold of that in burn rats), which was significantlydifferent from that in pretreatment rats (1.21±0.15 fold of that in burn rats,p<0.05). This effect was suppressed by perfusion with Hemin. The level ofHO-1mRNA recovered to 1.16±0.12 fold. The level of HO-1 protein was 2.5 foldin pretreatment group than that in burn group. The level markedly decreased afterperfusion with ZnPP, and was significantly different from that without perfusionwith ZnPP. This effect was also suppressed by perfusion with Hemin, and the levelof HO-1 protein return to high level in myocardium. ECG: Arrhythmia PVB and paroxysmal ventricular tachycardia (PVT) andelevation of ST segment were recorded during phase of ischemia in burn group rats.Then HR stepped down, and reperfusion arrhythmia PVB, PVT, and ventricularfibrillation (VF) were recorded frequently during phase of reperfusion. Elevationof ST segment and irrhythmia were recorded during phase of ischemia, andfrequency and duration of reperfusion arrhythmia such as PBV, VT and VFdecreased during phase of reperfusion in heat pretreatment group (p<0.05). ZnPPinhibited the anti-arrhythmia protection of heat pretreatment, and arrhythmia VTand VF became worse. Hemin suppressed the inhibition of ZnPP, and markedlyameliorated the reperfusion arrhythmia. Myocardium injury: There was high level of CK in coronary effluent andinfarct size in the heart of burn group rats. Pretreatment degraded the level of CK,and minified the infarct size significantly (p<0.01), compared with those in burngroup rats. ZnPP inhibited this protection, and the level of CK and infarct size wasup-regulated prominently (p<0.05). Hemin suppressed the inhibition of ZnPP, andrestored the protection of pretreatment from ischemia-reperfusion cardiac injury. Cardiac function: The decrease of CF and increase of CR were shown beforethe procession of ischemia-reperfusion in the burn group. The decrease of LVDP,dp/dtmax, CF, and the increase of CR were noticeable after reperfusion in the burngroup. Heat pretreatment increased LVDP, dp/dtmax, CF, and decreased CRmarkedly after reperfusion (p<0.01), compared with those in burn group. ZnPPinhibited the cardiac protection of pretreatment, attenuated myocardial contractility,and increased CR significantly (p<0.05). Hemin restored pretreatment effect oncardiac dysfunction, which caused by ischemia-reperfusion injury, decreased CRand ameliorated myocardial contractility.Discussion and conclusion: Severe burn, a severe trample behavior of organism , caused obviousmyocardium injury in the early. Negligent myocardial protection antishockmanagement would result in ischemia-reperfusion injury and multi organdysfunction syndrome (MODS). Some endogenous protective substances wereup-regulated to help the adaptation of stress. We have observed that the level ofHO-1 increased significantly in myocardium post-burn in the partⅠ. The cardiacischemia-reperfusion injury was investigated by perfusion heart with Langendorfftechnique after sever burn in this part. And whether HO-1 plays a protection roleon the cardiac injury. Data displayed that heat pretreatment ameliorated cardiacischemia-reperfusion injury, minified the infarct size, and improved myocardialcontractility, compared with that of burn group rats. Perfusion with ZnPP inhibitedthe production of HO-1, which aggratated the reperfusion injury, followed withaugmentation of infarct size, frequency of server reperfusion arrhythmia, andcontractile dysfunction. While Perfusion with Hemin induced expression of HO-1,which suppress the inhibition of ZnPP on cardiac protection of pretreatment,ameliorated cardiac reperfusion injury, and improved myocardial contractility.These data demonstrated that HO-1 involve in the protection of heat pretreatmenton attenuation cardiac injury, and play direct protection against theischemia-reperfusion injury post-burn pro and con on the level of isolated heartorgan. In fact, HO-1 might play protection through inhibition activation ofMitogen-activated protein kinase (MAPK), anti-oxidation, and anti-inflammatorypathways, and so on. While the exact pathway of the protection which observedfrom this part study need further investigation.Part ⅢThe investigation of protection Mechanisms of HO-1 againstMyocardial injury past-burnObjective Oxidative stress and intracellular calcium overload are the substantial element inthe course of ischemia-reperfusion injury. Bacterial infection, endotoxin releaseand systemic inflammatory reaction syndrome (SIRS) are major factors in thecourse of further injury and MODS in the second attack post-burn. Ratcardiomyocyte was cultured under thermal injury and endotoxin stress model tomimic effects of thermal and sepsis on myocardium in the early and later period ofburn. The possible mechanism of HO-1 on cardiac protection post-burn wasinvestigated by using of ZnPP and Hemin.1 The protection mechanism of HO-1 on cardiomyocyte thermal injuryMethods: The primary neonatal rat myocardiocyte was cultured in 42℃for two hours toconstruct a thermal injury cell model. Cells were randomized into control, thermalinjury, ZnPP: thermal injury + ZnPP, and Hemin group: thermal injury + Hemin.ZnPP and Hemin were added into solution before thermal injury, and theconcentration was 3μM and 5μM, respectively. The degree of cell injury wasevaluated by measure of cardiocyte morphosis, beat rate, activity of LDH,cardiocyte survival rate with MTS method, and apoptosis by flow cytometer. Thelevel of HO-1mRNA and protein was detected with RT-PCR and Western blottingin cardiocyte, respectively. The role of oxidative stress, calcium overload, andeffects of HO-1 were studied by measure of the level of MDA in supernatant,activity of SOD in supernatant, and calcium concentration in cardiocyte bymeasure of mean fluorescence magnitude by flow cytometer.Results: Cardiocyte injury: Noticeable enhancement of cell beat rate and amplitudewere observed after thermal injury, but the rhythm was not regularity. The activityof LDH and apoptosis increased significantly (p<0.05), cardiocyte survival ratedecreased markedly (p<0.01) after thermal injury. The HO-1 inhibition of ZnPPmade the cardiocyte injury more significant: cell beat rate slowed down, beatamplitude attenuated, the rhythm disturbed, the activity of LDH increased to 176.1±25.3 IU/L, the cardiocyte survival rate decreased to about 40 percent, and therate of apoptosis reached 18.2±3.4%, which had a significant difference comparedwith that in control group (p<0.01) and burn group (p<0.05). Hemin markedlysuppressed the increase of LDH and apoptosis, and improved the cardiocytesurvival rate through inducement of HO-1 expression. The level of HO-1: The level of HO-1mRNA was very low, and the proteinwas difficult to detect in normal cardiocyte. The level of HO-1mRNA and proteinwas increased in thermal injury cell (2.83±0.33 and 2.13±0.28 fold respectively,p<0.05). ZnPP definitively blocked the production of HO-1, and markedlydown-regulated the level of HO-1mRNA and protein (1.72±0.28 and 1.52±0.22fold of that in control). The difference was significant between ZnPP and thermalinjury group. Hemin up-regulated the level of HO-1mRNA and protein (5.61±0.46 and 3.82±0.41 fold, respectively), which increased significantly than that ofthermal injury group (p<0.05). Oxidative stress and calcium: The level of MDA increased significantly insupernatant in thermal injury and ZnPP group than that in control (1.95±0.55,3.52±0.73 vs. 0.87±0.25 nmol/L, respectively, p<0.01). And the difference wasalso significant between thermal injury and ZnPP group (p<0.05). The activity ofSOD decreased markedly than that in control, 14.6±3.2, 10.9±2.6 and 24.3±4.1U/ml, respectively (P<0.05). the decrease of MDA and increase of SOD wereshown in Hemin group, which were significant difference, compared with that ofthermal injury group. The level of [Ca2+]i increased significantly than that ofcontrol in cardiocyte after thermal injury (2.53±0.32 fold, p<0.01). Hemininduced expression of HO-1 and significantly decreased the elevation of [Ca2+]i inthermal injury (1.56±0.18, p<0.05). ZnPP blocked the production of HO-1 andmarkedly increased the concentration of [Ca2+]i than that of thermal injury group( reach 5.42±0.68 fold of that in control, p<0.01).2 The protection mechanism of HO-1 on cardiomyocyte LPS stressMethods: The primary neonatal rat myocardiocyte was culture in DMEM with 30μMLPS, to construct a LPS stress cell model. LPS was dissolved in DMEM mediumbefore. Cells were randomized into control, LPS stress, ZnPP: LPS stress + ZnPP,and Hemin group: LPS stress + Hemin. ZnPP and Hemin were added into solutionbefore LPS stress, and the concentration was 3μM and 5μM, respectively. Thedegree of cell injury was evaluated by measure of cardiocyte morphosis, beat rate,activity of LDH, cardiocyte survival rate, and apoptosis rate. The level of HO-1,MDA and SOD was detected. The protection mechanism of HO-1 on thecardiocyte injury caused by LPS stress was investigated by measure of the level ofTNF-αprotein with Western blotting, and NF-κB by measure of meanfluorescence magnitude by flow cytometer in cardiocyte.Results: Cardiocyte injury: An enhancement of cell beat rate and amplitude wasobserved after LPS stress, but the rhythm was not regularity. The activity of LDHand apoptosis increased significantly (p<0.05), cardiocyte survival rate decreasedmarkedly (p<0.05) in LPS group. The cardiocyte injury was more severer in ZnPPgroup than that in LPS group: cell beat rate slowed down, beat amplitudeattenuated, the rhythm disturbed, the activity of LDH increased, the cardiocytesurvival rate decreased, and the rate of apoptosis reached high level. Which had asignificant difference compared with that in LPS group (p<0.05). Hemin markedlysuppressed the increase of LDH and apoptosis, and attenuated the cardiocyteinjury. HO-1 and oxidative stress: LPS induced the production of HO-1 incardiocyte, and the level of HO-1mRNA and protein was increased significantly(3.42±0.43 and 2.94±0.38 fold of that in control respectively, p<0.05). ZnPPmarkedly down-regulated the level of HO-1mRNA and protein. The difference wassignificant between ZnPP and LPS group (p<0.05). The level of HO-1mRNA andprotein increased significantly in Hemin group than that of LPS group (p<0.05).The elevation of MDA and degradation of SOD were significant in supernatant inLPS group. ZnPP enhanced the change of MDA and SOD caused by LPS throughblocked the production of HO-1 in cardiocyte. There was significant differencebetween LPS and ZnPP group (p<0.05). Hemin inhibited the elevation of MDAand degradation of SOD caused by LPS through inducing the production of HO-1in cardiocyte (p<0.05). NF-κB and TNF-α: The level of TNF-αprotein and NF-κB activationwere low in normal cardiocyte. LPS enhanced the activation of NF-κB (2.84±0.35 fold, p<0.01), and induced the high expression of TNF-αin cardiocyte (reach4.53±0.66 fold, p<0.01 ). ZnPP promoted the enhancement of LPS on theactivation of NF-κB and inducement of TNF-αthrough the blockage ofHO-1.The activity of NF-κB reached 3.55±0.54 fold of that in control, andincreased significantly than that of LPS group. Hemin significantly inhibited theactivation of NF-κB caused by LPS (1.62±0.21 fold of that in control), anddown-regulated the expression of TNF-αin cardiocyte, which were significantlydifferent from that of LPS group (p<0.05).Discussion and conclusion: Studies showed that LPS could induced the secretion of some inflammatoryfactor, such as TNF-αand IL-1 βetc in EC, VSMC, and mononuclearmacrophage, and initiated directly the cellular injury. LPS, the essential componentof endotoxin, played an important role in the pathogenesis of SIRS and MODSpost-burn. Our investigations have demonstrated that HO-1 attenuated the cardiacinjury post-burn as an endogenous cardiac protective substance in the past two partof the study. In this part, the protection mechanisms of HO-1 were studied throughthe construction of thermal injury and LPS stress cellular model to mimic thedirect myocardium injury of post-burn and secondary infection. The data showedthat there were visible cardiocyte injury in the two models, and HO-1 providedcardiac protection and ameliorated the cardiocyte injury. While the mechanism wasdifferent in the two models. In the thermal injury, HO-1 played cardiac protectionthrough inhibition of oxidative stress, calcium overload and apoptosis. HO-1ameliorated the cardiocyte injury through inhibition of oxidative stress, apoptosis,the activation of NF-κB and expression of TNF-αin LPS stress model. Whichmeans that HO-1 plays cardiac protection through different mechanism in thedifferent phase of burn.