Rapid expression of heat shock protein in the rabbit after brief cardiac ischemia.

The effect of brief myocardial ischemia on the expression of heat shock protein (HSP 70) was examined in an in vivo rabbit model of myocardial ischemia using Northern blotting. Functional studies were carried out in the open-chested anesthetized rabbit. The large marginal branch of the left circumflex was occluded four times for 5 min. Using piezoelectric crystals implanted midwall in the ischemic zone, end-diastolic length, end-systolic length, and percent segmental shortening were assessed. Expression of HSP 70 was measured by Northern blotting. A single 5-min coronary occlusion doubled the expression of HSP 70 whereas four cycles of 5 min of ischemia/5 min of reperfusion resulted in a threefold increase in HSP 70 mRNA (P less than 0.001). Measurements with the piezoelectric crystals showed mild myocardial dysfunction concomitant with the increase in HSP 70. This increase in HSP 70 mRNA after repetitive brief ischemia was transient, occurring as early as 1 h and returning to baseline by 24 h after ischemia. Western blot analysis with a monoclonal antibody to HSP 70 was used to compare sham and postischemic myocardial HSP 70 levels. Changes in the amount of HSP 70 were evident as early as 2 h and were even more striking at 24 h.     

Stretch-induced VEGF expression in the heart.

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen involved in vascular development and angiogenesis. Recently we have observed increased VEGF expression in the normal myocardium after myocardial infarction in a rat heart. This study was designed to explore the mechanism responsible for this increase in VEGF expression. Induction of myocardial stretch in an isolated perfused Langendorff preparation by inflation of an intraventricular balloon to an end-diastolic load of 35 mmHg for 30 min resulted in a nearly sixfold increase in VEGF message level not only in the chamber subjected to stretch (left ventricle) but also in the unstretched right ventricle, thus raising the possibility of a soluble factor mediating stretch- induced induction of VEGF expression. This was further confirmed by demonstrating that coronary venous effluent collected from the stretched heart and used to perfuse isolated hearts in which no balloon was present was able to induce VEGF expression in these normal hearts. Inhibition of TGF-beta activity using a neutralizing antibody, but not antagonists/inhibitors of endothelin and angiotensin II, eliminated stretch-induced increase in VEGF expression. Staurosporine, a protein kinase C inhibitor, also blocked stretch-induced increase of VEGF expression. Measurement of TGF-beta concentration in the perfusate demonstrated increased amounts of the cytokine after myocardial stretch, and addition of TGF-beta protein to the perfusion buffer resulted in increased VEGF expression in control hearts. These results suggest that stretch-induced increase of VEGF expression in the heart is mediated at least in part by TGF-beta.     

Protection against myocardial dysfunction after a brief ischemic period in transgenic mice expressing inducible heat shock protein 70.

Brief ischemic periods lead to myocardial dysfunction without myocardial infarction. It has been shown that expression of inducible HSP70 in hearts of transgenic mice leads to decreased infarct size, but it remains unclear if HSP70 can also protect against myocardial dysfunction after brief ischemia. To investigate this question, we developed a mouse model in which regional myocardial function can be measured before and after a temporary ischemic event in vivo. In addition, myocardial function was determined after brief episodes of global ischemia in an isolated Langendorff heart. HSP70-positive mice and transgene negative littermates underwent 8 min of regional myocardial ischemia created by occlusion of the left descending coronary artery, followed by 60 min of reperfusion. This procedure did not result in a myocardial infarction. Regional epicardial strain was used as a sensitive indicator for changes in myocardial function after cardiac ischemia. Maximum principal strain was significantly greater in HSP70-positive mice with 88+/-6% of preischemic values vs. 58+/-6% in transgene-negative mice (P < 0.05). Similarly, in isolated Langendorff perfused hearts of HSP70-positive and transgene-negative littermates exposed to 10 min of global ischemia and 90 min of reperfusion, HSP70 transgenic hearts showed a better-preserved ventricular peak systolic pressure. Thus, we conclude that expression of HSP70 protects against postischemic myocardial dysfunction as shown by better preserved myocardial function.     

In vivo gene transfection with heat shock protein 70 enhances myocardial tolerance to ischemia-reperfusion injury in rat.

Heat shock protein 70 (HSP70) has been reported to be involved in the myocardial self-preservation system. To obtain the evidence that HSP70 plays a direct role in the protection from myocardial ischemia-reperfusion injury, rat hearts were transfected with human HSP70 gene by intracoronary infusion of hemagglutinating virus of Japan (HVJ)-liposome containing human HSP70 gene. The control hearts were infused with HVJ-liposome without the HSP70 gene. The hearts from whole-body heat-stressed or nontreated rats were also examined. Western blot and immunohistochemical analysis showed that apparent overexpression of HSP70 occurred in the gene transfected hearts and that gene transfection might be more effective for HSP70 induction than heat stress. In Langendorff perfusion, better functional recovery as well as less creatine phosphokinase leakage after ischemia were obtained in the gene transfected hearts with HSP70 than in the control or nontreated hearts. Furthermore, the gene transfected hearts showed better functional recovery than the heat-stressed hearts. These results indicated that overexpressed HSP70 plays a protective role in myocardial injury, suggesting the possibility that gene transfection with HSP70 may become a novel method for myocardial protection through enforcing the self-preservation systems.     

急性心肌梗死猝死患者心肌热休克蛋白70和血红素氧合酶1的改变及其意义

目的 探讨急性心肌梗死(AMI)猝死者梗死区心肌热休克蛋白70(HSP70)、血红素氧合酶1(HO-1)的mRNA和蛋白表达改变与临床意义.方法 收集18例AMI猝死者的尸体解剖梗死区心肌为研究组,17例因交通事故快速死亡者的尸体解剖正常心肌为对照组.采用逆转录-聚合酶链反应(RT-PCR)和免疫组化法检测两组心肌组织HSP70、HO-1的mRNA和蛋白表达,并观察心肌组织病理变化.结果 研究组梗死区心肌HSP70 mRNA(0.841±0.058)、HO-1 mRNA(0.918±0.161)和HSP70蛋白(3 556.68±574.19)、HO-1蛋白(4 336.68±865.30)表达均显著高于对照组(0.105±0.034、0.086±0.053、289.21±68.51、1 556.78±506.26,均P＜0.01).研究组梗死区心肌细胞内HSP70、HO-1蛋白呈阳性或弱阳性表达;对照组心肌细胞内HSP70表达阴性,而HO-1蛋白有弱阳性表达.研究组梗死区心肌HSP70与HO-1的mRNA及蛋白表达均呈显著正相关(r1=0.865,r2=0.816,均P＜0.01).结论 HSP70、HO-1可能共同参与了AMI的病理生理过程;HO-1在心肌细胞内表达可能具有普遍性,但在梗死区心肌表达更明显.     

氟桂利嗪对脑缺血再灌损伤HSP70基因表达减少神经细胞损伤的影响

Asakindofselectivecalciumantagonist,Flunarizine(FNZ)iswidelyusedtotreattheischemiccerebrovasculardiseaseinclinic.WewonderwhetherFNZcanaffecttheexpressionofHSP70andapoptoticgeneornotwhileitisabletoprotectcellsfromtheischemiccerebralinjuries.Ourresearchisdescribedindetailasbelow.1Materialsandmethods1.1Thegroupingandpreparationofanimals60healthymaleGerbils(providedbyShanghaimedicalexperimentanimaladminis-trationcommittee),weightwithintherangeof(100±10)g,wererandomlydividedintoseveralgroups.…     

Increased rat cardiac angiotensin converting enzyme activity and mRNA expression in pressure overload left ventricular hypertrophy. Effects on coronary resistance, contractility, and relaxation.

We compared the activity and physiologic effects of cardiac angiotensin converting enzyme (ACE) using isovolumic hearts from male Wistar rats with left ventricular hypertrophy due to chronic experimental aortic stenosis and from control rats. In response to the infusion of 3.5 X 10(-8) M angiotensin I in the isolated buffer perfused beating hearts, the intracardiac fractional conversion to angiotensin II was higher in the hypertrophied hearts compared with the controls (17.3 +/- 4.1% vs 6.8 +/- 1.3%, P less than 0.01). ACE activity was also significantly increased in the free wall, septum, and apex of the hypertrophied left ventricle, whereas ACE activity from the nonhypertrophied right ventricle of the aortic stenosis rats was not different from that of the control rats. Northern blot analyses of poly(A)+ purified RNA demonstrated the expression of ACE mRNA, which was increased fourfold in left ventricular tissue obtained from the hearts with left ventricular hypertrophy compared with the controls. In both groups, the intracardiac conversion of angiotensin I to angiotensin II caused a comparable dose-dependent increase in coronary resistance. In the control hearts, angiotensin II activation had no significant effect on systolic or diastolic function; however, it was associated with a dose-dependent depression of left ventricular diastolic relaxation in the hypertrophied hearts. These novel observations suggest that cardiac ACE is induced in hearts with left ventricular hypertrophy, and that the resultant intracardiac activation of angiotensin II may have differential effects on myocardial relaxation in hypertrophied hearts relative to controls.     

Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery.

Heat shock treatment induces expression of several heat shock proteins and subsequent post-ischemic myocardial protection. Correlations exist between the degree of stress used to induce the heat shock proteins, the amount of the inducible heat shock protein 70 (HSP70) and the level of myocardial protection. The inducible HSP70 has also been shown to be protective in transfected myogenic cells. Here we examined the role of human inducible HSP70 in transgenic mouse hearts. Overexpression of the human HSP70 does not appear to affect normal protein synthesis or the stress response in transgenic mice compared with nontransgenic mice. After 30 min of ischemia, upon reperfusion, transgenic hearts versus nontransgenic hearts showed significantly improved recovery of contractile force (0.35 +/- 0.08 versus 0.16 +/- 0.05 g, respectively, P < 0.05), rate of contraction, and rate of relaxation. Creatine kinase, an indicator of cellular injury, was released at a high level (67.7 +/- 23.0 U/ml) upon reperfusion from nontransgenic hearts, but not transgenic hearts (1.6 +/- 0.8 U/ml). We conclude that high level constitutive expression of the human inducible HSP70 plays a direct role in the protection of the myocardium from ischemia and reperfusion injury.     

Cardiac UCP2 expression and myocardial oxidative metabolism during acute septic shock in the rat

Septic shock decreases cardiac hydraulic work relative to the rate of myocardial oxygen consumption, causing decreased mechanical efficiency (hydraulic work/myocardial oxygen consumption). This study tested whether the mitochondrial uncoupling protein UCP2 was responsible for decreased cardiac mechanical efficiency after polymicrobial septic shock. Sepsis was initiated in ketamine/xylazine-anesthetized rats by cecal ligation and puncture (CLP). Steady-state mRNA content was quantified by Northern blot analysis, and protein content was estimated by western blot. Additional hearts were removed after 12 h and perfused in working mode to measure work (mmHg x mL/min/100 g dry wt) and efficiency (CE = work/oxygen consumption, %). The 72-h mortality rate was 80%, and deaths occurred between 12-32 h. Cardiac work (152 +/- 15, shock vs. 235 +/- 16, control; P < 0.05) and cardiac efficiency (4.0 +/- 0.4 vs. 5.6 +/- 0.3; P < 0.05) were significantly decreased when hearts were isolated 12 h after CLP. Myocardial UCP2 mRNA expression was increased by 52% (12 h) compared with control hearts; however, there was no detectable UCP2 protein in mitochondria isolated from either control or septic hearts. Conclusions: Although polymicrobial sepsis decreased cardiac mechanical efficiency and increased UCP-2 expression coincident with premortal hypothermia, we did not detect any evidence of UCP-2 protein in septic heart muscle. These data argue against the hypothesis that UCP-2 causes decreased cardiac mechanical efficiency in septic shock.     

8Hz次声作用后大鼠脑热休克蛋白70的表达

目的探讨次声作用后大鼠脑的热休克蛋白７０（ｈｅａｔｓｈｏｃｋｐｒｏｔｅｉｎ７０,ＨＳＰ７０）的表达。方法大鼠暴露于８Ｈｚ的次声,２ｈ／ｄ,点杂交检测次声作用１次后多器官ＨＳＰ７０ＭＲＮＡ平;免疫组织化学方法检测次声作用１、３、７、１４、２１ｄ后脑ＨＳＰ７０的表达和分布。结果次声作用后脑、心、肝、肺等器官ＨＳＰ７０ｍＲＮＡ明显增加,脑中有２４个部位出现ＨＳＰ７０阳性神经元,且随声压增强、作用时间延长,阳性表达增强。结论这些部位对次声敏感,次声的作用效应与声压、作用时间有关。     

Synthesis of stress proteins in rat cardiac myocytes 2-4 days after imposition of hemodynamic overload.

Isolated adult myocytes incubated with [35S]methionine were used to study the expression of proteins in the rat heart during the first 2 wk after either pressure or volume overload. In both models an early (2-4 d) and transient expression of three major stress proteins (heat shock protein [HSP] HSP 70, HSP 68, and HSP 58) was observed together with an increased synthesis of putative ribosomal proteins. Only traces of 35S-labeled HSPs were detected in controls and sham-operated animals. The three stress proteins were identified by their migration in two-dimensional gels, by comigration with HSPs, which had been induced in myocytes by incubation at 41 degrees C and immunoblot analysis using antisera directed against the 70-kD protein. Immunohistochemical staining of HSP 70 in rod-shaped myocytes and detection by immunoblot showed that HSP 70 was equally present and distributed in both sham-operated and overloaded hearts, and provided no evidence for a subpopulation of myocytes acutely involved in the increased expression of HSP 70. It is suggested that the transient expression of HSPs that occurs during the early adaptation of the myocardial cells to overload could confer some degree of protection to the actively growing myocytes.     

Alteration of growth responses in established cardiac pressure overload hypertrophy in rats with aortic banding.

We examined the acute effects of elevated wall stress, norepinephrine, and angiotensin II on cardiac protein synthesis as well as protooncogene expression in hearts with established pressure overload left ventricular hypertrophy. Isolated rat hearts with chronic hypertrophy (LVH) were studied 12 wk after ascending aortic banding when systolic function was fully maintained. New protein synthesis (incorporation of [3H]phenylalanine [Phe]) was analyzed in isolated perfused rat hearts after a 3-h protocol; c-fos, c-jun, c-myc, and early growth response gene-1 (EGR-1) mRNA levels (Northern blot) were studied over a time course from 15 to 240 min of perfusion. Under baseline conditions (i.e., before mechanical or neurohormonal stimulation), [3H]-Phe-incorporation (280 nmoles/gram protein/h) and protooncogene mRNA levels were similar in age-matched control and LVH hearts. However, hearts with chronic LVH were characterized by a markedly blunted or absent [3H]-Phe-incorporation after acute imposition of isovolumic systolic load (90 mmHg/gram left ventricle), as well as norepinephrine (10(-6)M), or angiotensin II infusion (10(-8)M plus prazosin 10(-7)M) compared with nonhypertrophied control hearts. Similarly, stimulation of LVH hearts with acute systolic load or norepinephrine was associated with a significantly blunted increase of protooncogene mRNA levels relative to control hearts. The blunted induction of c-fos mRNA in LVH hearts was not due to feedback inhibition, since cycloheximide perfusion of hearts exposed to elevated wall stress further increased the differences between age-matched control and LVH hearts. The data suggest that acute molecular growth responses to mechanical or neurohormonal stimulation are altered in rat hearts with established LVH relative to nonhypertrophied control hearts. This alteration of molecular adaptations in hearts with compensatory hypertrophy may prevent inappropriate excess cardiac growth in response to mechanical and neurohormonal stimuli.     

严重烧伤早期肠黏膜组织热休克蛋白70的表达规律

目的探讨大鼠烧伤后早期肠黏膜组织热休克蛋白70(HSP70)的表达变化规律及其意义。方法采用大鼠烫伤模型,通过逆转录-聚合酶链反应(RT-PCR)、蛋白质免疫印迹(Westernblot)及免疫组化等方法,检测伤后3、6、12、24和48h不同时间点肠黏膜组织内HSP70及热休克因子1(HSF1)的表达分布情况。结果烫伤后3h肠黏膜组织内HSP70mRNA及蛋白表达均显著增加,分别在伤后6h和12h达高峰,伤后48h仍高于正常对照组(P均<0.01);伤后3h大鼠肠黏膜组织HSF1出现一过性降低,伤后6h其表达显著高于正常对照组,并呈逐渐增加的趋势直至持续到伤后48h(P均<0.01)。结论严重烧伤早期肠黏膜组织HSP70及HSF1表达均显著增加,提示严重烧伤早期即可引起肠黏膜组织细胞的应激反应,可能与细胞的自我保护机制启动有关。     

Protection against ischemia/reperfusion injury and myocardial dysfunction by antisense-oligodeoxynucleotide directed at angiotensin-converting enzyme mRNA.

Angiotensin-converting enzyme (ACE) activity in the myocardium and angiotensin-II (Ang-II) levels in plasma increase after myocardial ischemia, which lead to exacerbation of myocardial injury and cardiac dysfunction. We examined the protective role of novel antisense-oligodeoxynucleotide (AS-ODN) directed at ACE mRNA in myocardial ischemic injury. Sprague-Dawley rats were treated with ACE-AS-ODN (200 microg per rat, n = 8, i.v.) or inverted-ODN (IN-ODN, 200 microg per rat, n = 8, i.v.), given with 600 microg per rat of liposome DOTAP/DOPE. Hearts from AS-ODN- or IN-ODN-treated rats were excised, perfused in vitro, and subjected to 25 min of global ischemia followed by 30 min of reperfusion. Parallel groups of rats were given ACE inhibitor captopril (5 mg/kg, n = 8) or saline (n = 8) before excising the hearts. Ischemia/reperfusion resulted in myocardial dysfunction (increase in coronary perfusion pressure and LV end-diastolic pressure and a decrease in developed LV pressure) in the saline-treated rats. Myocardial dysfunction was associated with evidence of lipid peroxidation and enzyme leakage (MDA and LDH levels in the myocardium) and up-regulation of ACE protein expression. Administration of AS-ODN or captopril, but not IN-ODN, reduced Ang-II levels in the plasma, decreased ischemia/reperfusion-mediated cardiac functional deterioration and lipid peroxidation, and preserved LDH in the myocardium (all P < 0.05 versus the saline group). AS-ODN and captopril had equipotent effects on cardiac dynamics. ACE protein expression (western blot) was decreased in the hearts of the AS-ODN-treated group, but not in IN-ODN-treated rat hearts. In contrast, ACE protein expression was significantly increased in captopril-treated rat hearts. These observations suggest that AS-ODN directed at ACE mRNA can ameliorate myocardial dysfunction and injury after ischemia/reperfusion, and its use is associated with decreased expression of ACE protein in the ischemic myocardium.     

Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury.

Myocardial protection and changes in gene expression follow whole body heat stress. Circumstantial evidence suggests that an inducible 70-kD heat shock protein (hsp70i), increased markedly by whole body heat stress, contributes to the protection. Transgenic mouse lines were constructed with a cytomegalovirus enhancer and beta-actin promoter driving rat hsp70i expression in heterozygote animals. Unstressed, transgene positive mice expressed higher levels of myocardial hsp70i than transgene negative mice after whole body heat stress. This high level of expression occurred without apparent detrimental effect. The hearts harvested from transgene positive mice and transgene negative littermates were Langendorff perfused and subjected to 20 min of warm (37 degrees C) zero-flow ischemia and up to 120 min of reflow while contractile recovery and creatine kinase efflux were measured. Myocardial infarction was demarcated by triphenyltetrazolium. In transgene positive compared with transgene negative hearts, the zone of infarction was reduced by 40%, contractile function at 30 min of reflow was doubled, and efflux of creatine kinase was reduced by approximately 50%. Our findings suggest for the first time that increased myocardial hsp70i expression results in protection of the heart against ischemic injury and that the antiischemic properties of hsp70i have possible therapeutic relevance.     

Protective role of heat stress in burn trauma

OBJECTIVE: This study was designed to determine whether cutaneous burn injury up-regulated expression of myocardial heat shock protein (HSP)70 and to determine a potential cardioprotective role of inducible HSP70 (iHSP70) in postburn myocardial contractile function. DESIGN: Experimental study. SETTING: Research laboratory. SUBJECTS: Adult Hartley guinea pigs. INTERVENTIONS: The first set of studies determined whether heat stress (increasing body temperature to 42 degrees C for 20 mins) in adult Hartley guinea pigs would increase expression of myocardial iHSP70. MEASUREMENTS AND MAIN RESULTS: Our model of heat stress increased expression of inducible HSP in the myocardium (Western blot), and this response persisted 1, 2, 4, and 24 hrs after the initial heat stress. We then determined whether burn trauma over 40% total body surface area (TBSA) increased myocardial expression of iHSP70. Time-matched sham and burned guinea pigs were killed 1, 2, 4, 12, 18, or 24 hrs postburn, and hearts were used either to examine myocardial iHSP70 expression by Western blot or to determine myocardial contractile function (Langendorff). Burn trauma produced a two-fold increase in myocardial iHSP70 that was evident as early as 1 hr postburn and persisted 24 hrs postburn; increased iHSP70 expression occurred despite only a modest and transient increase in body temperature after burn trauma. We then determined whether heat shock stress before burn trauma provided a protective or detrimental effect on cardiac function. Body temperature was increased to 42 degrees C for 20 mins, animals were allowed to recover, and body temperature returned to baseline; burn trauma was then produced (40% TBSA) either 1, 2, 4, or 24 hrs after the initial heat stress. Myocardial contraction and relaxation deficits were evident after burn trauma alone; however, heat stress 1 hr before burn trauma improved left ventricular developed pressure and positive or negative maximum change in pressure in time and shifted left ventricular function curves upward and leftward from those calculated for burn in the absence of heat stress, indicating improved ventricular performance. Increasing the time between the initial heat stress and burn injury decreased the cardioprotective effects of heat stress. Thus, organ protection was evident only when the time period between the initial heat stress and the second insult was brief (1 hr). CONCLUSIONS: Our finding that the amount of myocardial iHSP70 remained constantly elevated after heat stress while the cardio-protective effect afforded by a prior heat stress declined with time suggested that the initial heat stress evoked several compensatory/adaptive mechanisms that may include modulation of autonomic nervous system responses, changes in metabolic function, modulation of pro/anti-inflammatory cytokine responses, and heat stress-related alterations in antioxidant capacity.     

热休克蛋白70及pim-1基因在白血病骨髓单个核细胞的表达特点及临床意义

本研究观察热休克蛋白70（HSP70）基因／蛋白，pim-1基因在白血病患者骨髓单个核细胞（BMMNC）中的表达，确定其表达是否与白血病类型、肿瘤负荷程度及预后有规律性联系。用RT—PCR技术检测40例白血病患者和10例对照BMMNCHSPT0mRNA及pim-1mRNA的表达并半定量；用Westernblot检测34例白血病患者和10例对照BMMNCHSP70蛋白表达并半定量，并结合白血病类型、肿瘤负荷程度及对治疗的反应进行分析。结果表明：白血病组与对照组BMMNC均存在HSP70mRNA和蛋白表达，在白血病组显著高于对照组；CML组、AML组HSP70mRNA／蛋白表达的平均ODR值均显著高于ALL组；肿瘤重度负荷的急性白血病患者组HSP70mRNA／蛋白表达显著高于肿瘤轻度负荷组；白血病患者化疗后HSP70mRNA／蛋白表达显著高于化疗前；白血病组及对照组BMMNC均表达pim-1mRNA，在白血病组显著高于对照组；ALL组pim-1mRNA表达显著高于AML组和CML组：经相关性分析，白血病患者HSP70mRNA表达与pim-1mRNA表达成正相关（r=0．327，P〈0．05）。结论：白血病患者BMMNC存在HSP70、pim-1高表达，其中pim-1mRNA表达与HSP70mRNA表达呈正相关，HSP70与pim-1基因／蛋白过表达亦与白血病肿瘤负荷程度有关。     

Role of myocardial nitric oxide in diabetic ischemia-reperfusion dysfunction: studies in mice with myocyte-specific overexpression of endothelial nitric-oxide synthase

We investigated the role of nitric oxide (NO) in myocardial ischemia-reperfusion injury of diabetic mice with myocyte-specific overexpression of endothelial NO synthase (NOS). Four weeks after diabetes induction with streptozotocin (blood glucose approximately 29 mM), isolated isovolumic heart function and cellular NO metabolites in response to brief normothermic ischemia-reperfusion were determined. Under normoxic conditions transgenic (TG) hearts from nondiabetic and diabetic animals generated less left-ventricular developed pressure compared with wild-type (WT) control hearts, and this abnormality was unaffected by NOS inhibition. During ischemia, the rise in end-diastolic pressure was less in the TG than WT group of nondiabetic hearts, whereas the transgene had no effect in the diabetic group. Similarly, the transgene also improved reperfusion systolic and diastolic function in nondiabetic but not in diabetic hearts. NOS inhibition worsened reperfusion function in diabetic hearts. Postischemic nitrite and cGMP formation were higher in nondiabetic TG than WT hearts, but in diabetic hearts cGMP was no longer elevated. The formation of reactive oxygen species (superoxide and peroxynitrite) during early reperfusion, measured by electron spin resonance spectroscopy, was similar in nondiabetic WT and TG hearts, but it was significantly higher in diabetic TG hearts. Stimulating endogenous NO production with 10 microM bradykinin more strongly reduced myocardial O(2) consumption in diabetic TG than diabetic WT hearts perfused in normoxia, whereas there was no difference after ischemia-reperfusion. Thus, providing additional endogenous NO is sufficient to protect nondiabetic hearts against ischemia-induced injury, but for a similar protection in diabetic hearts, effective scavenging of reactive oxygen species is also important.