Effect of the myosin light chain kinase inhibitor ML-7 on the proteome of hearts subjected to ischemia-reperfusion injury

In the development of ischemia/reperfusion (I/R) injury, the role of the myosin light chain (MLC) phosphorylation has been given increased consideration. ML-7, a MLC kinase inhibitor, has been shown to protect cardiac function from I/R, however the exact mechanism remains unclear. Isolated rat hearts were perfused under aerobic conditions (controls) or subjected to I/R in the presence or absence of ML-7. Continuous administration of ML-7 (5 μM) from 10 min before onset of ischemia to the first 10 min of reperfusion resulted in significant recovery of heart contractility. Analysis of gels from two-dimensional electrophoresis revealed eight proteins with decreased levels in I/R hearts. Six proteins are involved in energy metabolism:ATP synthase beta subunit, cytochrome b-c1 complex subunit 1, 24-kDa mitochondrial NADH dehydrogenase, NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, cytochrome c oxidase subunit, and succinyl-CoA ligase subunit. The other two proteins with decreased levels in I/R hearts are: peroxiredoxin-2 and tubulin. Administration of ML-7 increased level of succinyl-CoA ligase, key enzyme involved in the citric acid cycle. The increased level of succinyl-CoA ligase in I/R hearts perfused with ML-7 suggests that the cardioprotective effect of ML-7, at least partially, also may involve increase of energy production.     

Proteomics of ischemia and reperfusion injuries in rabbit myocardium with and without intervention by an oxygen-free radical scavenger

A brief period of ischemia followed by timely reperfusion may lead to prolonged, yet reversible, contractile dysfunction (myocardial stunning). Damage to the myocardium occurs not only during ischemia, but also during reperfusion, where a massive release of oxygen-free radicals (OFR) occurs. We have previously utilized 2-DE and MS to define 57 protein spot changes during brief ischemia/reperfusion (15 min ischemia, 60 min reperfusion; 15I/60R) injury in a rabbit model (White, M. Y., Cordwell, S. J., McCarron, H. C. K., Prasan, A. M. et al., Proteomics 2005, 5, 1395-1410) and shown that the majority of these occur because of physical and/or chemical PTMs. In this study, we subjected rabbit myocardium to 15I/60R in the presence of the OFR scavenger N-(2-mercaptopropionyl) glycine (MPG). Thirty-seven of 57 protein spots altered during 15I/60R remained at control levels in the presence of MPG (15I/60R + MPG). Changes to contractile proteins, including myosin light chain 2 (MLC-2) and troponin C (TnC), were prevented by the addition of MPG. To further investigate the individual effects of ischemia and reperfusion, we generated 2-DE gels from rabbit myocardium subjected to brief ischemia alone (15I/0R), and observed alterations of 33 protein spots, including 18/20 seen in both 15I/60R-treated and 15I/60R + MPG-treated tissue. The tissue was also subjected to ischemia in the presence of MPG (15I/0R + MPG), and 21 spot changes, representing 14 protein variants, remained altered despite the presence of the OFR scavenger. These ischemia-specific proteins comprised those involved in energy metabolism (lactate dehydrogenase and ATP synthase alpha), redox regulation (NADH ubiquinone oxidoreductase 51 kDa and GST Mu), and stress response (Hsp27 and 70, and deamidated alpha B-crystallin). We conclude that contractile dysfunction associated with myocardial stunning is predominantly caused by OFR damage at the onset of reperfusion, but that OFR-independent damage also occurs during ischemia. These ischemia-specific protein modifications may be indicative of early myocardial injury.     

Proteomic analysis of hepatic ischemia/reperfusion injury and ischemic preconditioning in mice revealed the protective role of ATP5β

Hepatic ischemia/reperfusion (I/R) injury is an inevitable consequence during liver surgery. Ischemic preconditioning (IPC) has been shown to protect the livers from I/R injury, partially mediated by preservation of hepatic ATP contents. However, the precise molecular mechanisms of these events remain poorly elucidated. In this study, liver proteomes of the mice subjected to I/R injury pretreated with or without IPC were analyzed using 2‐DE combined with MALDI‐TOF/TOF mass analysis. Twenty proteins showing more than 1.5‐fold difference were identified in the livers upon I/R injury. Among these proteins, four proteins were further regulated by IPC when compared with nonpretreated controls. One of these proteins, ATP synthase β subunit (ATP5β) catalyzes the rate‐limiting step of ATP formation. The expression level of ATP5β, which was further validated by Western blot analysis, was significantly decreased upon I/R injury while turned over by IPC pretreatment. Change pattern of hepatic ATP corresponded with that of ATP5β expression, indicating that increasing hepatic ATP5β expression might be a reason for ATP‐preserving effect of IPC. In summary, this study provided new clues for understanding the mechanisms of IPC against I/R injury. The protective role of ATP5β might give evidences for developing new therapeutic approaches against hepatic I/R injury.     

Diabetic animal fed with high‐fat diet prevents the protective effect of myocardial ischemic preconditioning effect in isolated rat heart perfusion model

Diabetic heart (diabetes mellitus [DM]) has been shown to attenuate the beneficial effect of ischemic preconditioning (IPC) in rat heart. But the effect of IPC on diabetic rat heart that develops myopathy remains unclear. This study was designed to test the impact of IPC on diabetic cardiomyopathy (DCM) rat heart. Male Wistar rats were grouped as (a) normal, (b) DM (streptozotocin: 65 mg/kg; fed with normal diet), and (c) DCM (streptozotocin: 65 mg/kg; fed with high‐fat diet). Isolated rat hearts from each group were randomly subjected to (a) normal perfusion, (b) ischemia‐reperfusion (I/R), and (c) IPC procedure. At the end of the perfusion experiments, hearts were analyzed for injury, contractile function, mitochondrial activity, and oxidative stress. The results obtained from hemodynamics, cardiac injury markers, and caspase‐3 activity showed that DCM rat displayed prominent I/R‐associated cardiac abnormalities than DM rat heart. But the deteriorated physiological performance and cardiac injury were not recovered in both DM and DCM heart by IPC procedure. Unlike normal rat heart, IPC did not reverse mitochondrial dysfunction (determined by electron transport chain enzymes activity, ATP level, and membrane integrity, expression levels of genes like PGC‐1ɑ, GSK3β, complex I, II, and V) in DCM and DM rat heart. The present study demonstrated that IPC failed to protect I/R‐challenged DCM rat heart, and the underlying pathology was associated with deteriorated mitochondrial function.     

肠缺血再灌注时肠内给予不同营养物对肠粘膜ATP含量的影响

将SD大鼠分组 ,先制作空肠袋 ,分别向袋内注射不同营养物 :10mmol/L丙氨酸 ,10mmol/L葡萄糖 ,10mmol/L甘露醇或 5mmol/L丙氨酸 +5mmol/L葡萄糖的混合液 ,用动脉夹阻断肠系膜上动脉血流 6 0min后 ,再恢复灌流 6 0min。分别于阻断血流 6 0min和恢复灌注 6 0min测定肠粘膜ATP含量。研究结果显示 ,缺血再灌注能显著降低肠粘膜ATP含量 ,给予丙氨酸或葡萄糖 /丙氨酸混合液使肠粘膜ATP含量进一步降低 (P <0 .0 1) ,而给予葡萄糖能显著增加肠粘膜ATP含量 (P <0 .0 1)。结论 :缺血再灌注过程中 ,肠内给予葡萄糖能改善肠粘膜ATP含量 ,对缺血再灌注损伤的肠道提供保护作用     

Proteomic analysis of right and left cardiac ventricles under aerobic conditions and after ischemia/reperfusion

Ischemia/reperfusion (I/R) injury is a major consequence of a cardiovascular intervention. The study of changes of the left and right ventricle proteomes from hearts subjected to I/R may be a key to revealing the pathological mechanisms underlying I/R-induced heart contractile dysfunction. Isolated rat hearts were perfused under aerobic conditions or subjected to 25 min global ischemia and 30 min reperfusion. At the end of perfusion, right and left ventricular homogenates were analyzed by 2DE. Contractile function and coronary flow were significantly reduced by I/R. 2DE followed by mass spectrometry identified ten protein spots whose levels were significantly different between aerobic left and right ventricles, eight protein spots whose levels were different between aerobic and I/R left ventricle, ten protein spots whose levels were different between aerobic and I/R right ventricle ten protein spots whose levels were different between the I/R groups. Among these protein spots were ATP synthase beta subunit, myosin light chain 2, myosin heavy chain fragments, peroxiredoxin-2, and heat shock proteins, previously associated with cardiovascular disease. These results reveal differences between proteomes of left and right ventricle both under aerobic conditions and in response to I/R that contribute to a better understanding of I/R injury.     

PKCbeta modulates ischemia-reperfusion injury in the heart

Protein kinase C-betaII (PKCbetaII) is an important modulator of cellular stress responses. To test the hypothesis that PKCbetaII modulates the response to myocardial ischemia-reperfusion (I/R) injury, we subjected mice to occlusion and reperfusion of the left anterior descending coronary artery. Homozygous PKCbeta-null (PKCbeta(-/-)) and wild-type mice fed the PKCbeta inhibitor ruboxistaurin displayed significantly decreased infarct size and enhanced recovery of left ventricular (LV) function and reduced markers of cellular necrosis and serum creatine phosphokinase and lactate dehydrogenase levels compared with wild-type or vehicle-treated animals after 30 min of ischemia followed by 48 h of reperfusion. Our studies revealed that membrane translocation of PKCbetaII in LV tissue was sustained after I/R and that gene deletion or pharmacological blockade of PKCbeta protected ischemic myocardium. Homozygous deletion of PKCbeta significantly diminished phosphorylation of c-Jun NH(2)-terminal mitogen-activated protein kinase and expression of activated caspase-3 in LV tissue of mice subjected to I/R. These data implicate PKCbeta in I/R-mediated myocardial injury, at least in part via phosphorylation of JNK, and suggest that blockade of PKCbeta may represent a potent strategy to protect the vulnerable myocardium.     

Mitochondrial Src tyrosine kinase plays a role in the cardioprotective effect of ischemic preconditioning by modulating complex I activity and mitochondrial ROS generation

Abstract

While ischemic preconditioning (IPC) and other cardioprotective interventions have been proposed to protect the heart from ischemia/reperfusion (I/R) injury by inhibiting mitochondrial complex I activity upon reperfusion, the exact mechanism underlying the modulation of complex I activity remains elusive. This study was aimed to test the hypothesis that IPC modulates complex I activity at reperfusion by activating mitochondrial Src tyrosine kinase, and induces cardioprotection against I/R injury. Isolated rat hearts were preconditioned by three cycles of 5-min ischemia and 5-min reperfusion prior to 30-min index ischemia followed by 2 h of reperfusion. Mitochondrial Src phosphorylation (Tyr⁴¹⁶) was dramatically decreased during I/R, implying inactivation of Src tyrosine kinase by I/R. IPC increased mitochondrial Src phosphorylation upon reperfusion and this was inhibited by the selective Src tyrosine kinase inhibitor PP2. IPC's anti-infarct effect was inhibited by the selective Src tyrosine kinase inhibitor PP2. Complex I activity was significantly increased upon reperfusion, an effect that was prevented by IPC in a Src tyrosine kinase-dependent manner. In support, Src and phospho-Src were found in complex I. Furthermore, IPC prevented hypoxia/reoxygenation-induced mitochondrial reactive oxygen species (ROS) generation and cellular injury in rat cardiomyocytes, which was revoked by PP2. Finally, IPC reduced LDH release induced by both hypoxia/reoxygenation and simulated ischemia/reperfusion, an effect that was reversed by PP2 and Src siRNA. These data suggest that mitochondrial Src tyrosine kinase accounts for the inhibitory action of IPC on complex I and mitochondrial ROS generation, and thereby plays a role in the cardioprotective effect of IPC.     

Reduction of asymmetric dimethylarginine involved in the cardioprotective effect of losartan in spontaneously hypertensive rats

Previous studies have indicated that nitric oxide synthase (NOS) inhibitors can induce an increase of blood pressure and exacerbate myocardial injury induced by ischemia and reperfusion, whereas angiotensin II receptor antagonists protect the myocardium against injury induced by ischemia and reperfusion. Isolated hearts from male spontaneously hypertensive rats (SHR) or male Wistar-Kyoto rats (WKY) were subjected to 20 min global ischemia and 30 min reperfusion. Heart rate, coronary flow, left ventricular pressure, and its first derivatives (+/-dP/dt(max)) were recorded, and serum concentrations of asymmetric dimethylarginine (ADMA) and NO and the release of creatine kinase in coronary effluent were measured. The level of ADMA was significantly increased and the concentration of NO was decreased in SHR. Ischemia and reperfusion significantly inhibited the recovery of cardiac function and increased the release of creatine kinase, and ischemia and reperfusion-induced myocardial injury in SHR was aggravated compared with WKY. Vasodilation responses to acetylcholine of aortic rings were decreased in SHR. Treatment with losartan (30 mg/kg) for 14 days significantly lowered blood pressure, elevated the plasma level of NO, and decreased the plasma concentration of ADMA in SHR. Treatment with losartan significantly improved endothelium-dependent relaxation and cardiac function during ischemia and reperfusion in SHR. Exogenous ADMA also aggravated myocardial injury induced by ischemia and reperfusion in isolated perfused heart of WKY, as shown by increasing creatine kinase release and decreasing cardiac function. The present results suggest that the protective effect of losartan on myocardial injury induced by ischemia and reperfusion is related to the reduction of ADMA levels.     

Hypothermia-induced cardioprotection using extended ischemia and early reperfusion cooling

Optimal timing of therapeutic hypothermia for cardiac ischemia is unknown. Our prior work suggests that ischemia with rapid reperfusion (I/R) in cardiomyocytes can be more damaging than prolonged ischemia alone. Also, these cardiomyocytes demonstrate protein kinase C (PKC) activation and nitric oxide (NO) signaling that confer protection against I/R injury. Thus we hypothesized that hypothermia will protect most using extended ischemia and early reperfusion cooling and is mediated via PKC and NO synthase (NOS). Chick cardiomyocytes were exposed to an established model of 1-h ischemia/3-h reperfusion, and the same field of initially contracting cells was monitored for viability and NO generation. Normothermic I/R resulted in 49.7 +/- 3.4% cell death. Hypothermia induction to 25 degrees C was most protective (14.3 +/- 0.6% death, P < 0.001 vs. I/R control) when instituted during extended ischemia and early reperfusion, compared with induction after reperfusion (22.4 +/- 2.9% death). Protection was completely lost if onset of cooling was delayed by 15 min of reperfusion (45.0 +/- 8.2% death). Extended ischemia/early reperfusion cooling was associated with increased and sustained NO generation at reperfusion and decreased caspase-3 activation. The NOS inhibitor N(omega)-nitro-L-arginine methyl ester (200 microM) reversed these changes and abrogated hypothermia protection. In addition, the PKCepsilon inhibitor myr-PKCepsilon v1-2 (5 microM) also reversed NO production and hypothermia protection. In conclusion, therapeutic hypothermia initiated during extended ischemia/early reperfusion optimally protects cardiomyocytes from I/R injury. Such protection appears to be mediated by increased NO generation via activation of protein kinase Cepsilon; nitric oxide synthase.     

Netrin‐1 Pretreatment Protects Rat Kidney against Ischemia/Reperfusion Injury via Suppression of Oxidative Stress and Neuropeptide Y Expression

Netrin‐1 has been found to protect kidneys from ischemia/reperfusion injury. In this study, we aimed to address whether the protective effects were mediated through suppression of oxidative stress and neuropeptide Y. Compared to sham‐operated animals, animals after ischemia/reperfusion showed marked kidney damage and significantly increased levels of serum creatinine, blood urea nitrogen, malondialdehyde, and neuropeptide Y. Renal myeloperoxidase activity was elevated in animals with ischemia/reperfusion relative to sham‐operated animals, whereas renal superoxide dismutase activity was reduced. Netrin‐1 pretreatment attenuated ischemia/reperfusion‐induced functional and pathological changes in the kidney. Moreover, the ischemia/reperfusion‐induced changes in the oxidative stress biomarkers and neuropeptide Y were significantly counteracted by prior administration of netrin‐1. Taken together, our data showed that netrin‐1 pretreatment prevented renal ischemia/reperfusion injury, at least partially through reduction of oxidative stress and neuropeptide Y expression. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:231‐236, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21474     

Overexpression of the Na+/H+ exchanger and ischemia-reperfusion injury in the myocardium

In the myocardium, the Na(+)/H(+) exchanger isoform-1 (NHE1) activity is detrimental during ischemia-reperfusion (I/R) injury, causing increased intracellular Na(+) (Na(i)(+)) accumulation that results in subsequent Ca(2+) overload. We tested the hypothesis that increased expression of NHE1 would accentuate myocardial I/R injury. Transgenic mice were created that increased the Na(+)/H(+) exchanger activity specifically in the myocardium. Intact hearts from transgenic mice at 10-15 wk of age showed no change in heart performance, resting intracellular pH (pH(i)) or phosphocreatine/ATP levels. Transgenic and wild-type (WT) hearts were subjected to 20 min of ischemia followed by 40 min of reperfusion. Surprisingly, the percent recovery of rate-pressure product (%RPP) after I/R improved in NHE1-overexpressing hearts (64 +/- 5% vs. 41 +/- 5% in WT; P < 0.05). In addition, NMR spectroscopy revealed that NHE1 overexpressor hearts contained higher ATP during early reperfusion (levels P < 0.05), and there was no difference in Na(+) accumulation during I/R between transgenic and WT hearts. HOE642 (cariporide), an NHE1 inhibitor, equivalently protected both WT and NHE1-overexpressing hearts. When hearts were perfused with bicarbonate-free HEPES buffer to eliminate the contribution of HCO(3)(-) transporters to pH(i) regulation, there was no difference in contractile recovery after reperfusion between controls and transgenics, but NHE1-overexpressing hearts showed a greater decrease in ATP during ischemia. These results indicate that the basal activity of NHE1 is not rate limiting in causing damage during I/R, therefore, increasing the level of NHE1 does not enhance injury and can have some small protective effects.     

Chronic type-I diabetes could not impede the anti-inflammatory and anti-apoptotic effects of combined postconditioning with ischemia and cyclosporine A in myocardial reperfusion injury

It has been shown that diabetes modifies the myocardial responses to ischemia/reperfusion (I/R) and to cardioprotective agents. In this study, we aimed to investigate the effects of combined treatment with ischemic postconditioning (IPostC) and cyclosporine A (CsA) on inflammation and apoptosis of the diabetic myocardium injured by I/R. Eight weeks after induction of diabetes in Wistar rats, hearts were mounted on a Langendorff apparatus and were subsequently subjected to a 30-min regional ischemia followed by 45-min reperfusion. IPostC was induced at the onset of reperfusion, by 3 cycles of 30-s reperfusion/ischemia (R/I). The concentration of creatine kinase (CK), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were determined; the levels of total and phosphorylated glycogen synthase kinase 3 beta (p-GSK3β) and B-cell lymphoma 2 (Bcl-2) were quantified by western blotting, and the rate of apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. Administration of either IPostC or CsA alone in nondiabetic animals significantly reduced CK, TNF-α, IL-1β, and IL-6 concentrations, increased the p-GSK3β and Bcl-2, and decreased the level of apoptosis (P < 0.05) but had no effect on diabetic hearts. However, in diabetic animals, after administration of CsA, the cardioprotective effects of IPostC in increasing the p-GSK3β and Bcl-2 and decreasing apoptosis and inflammation were restored in comparison with nonpostconditioned diabetic hearts. IPostC or CsA failed to affect apoptosis and inflammation and failed to protect the diabetic myocardium against I/R injury. However, combined administration of IPostC and CsA at reperfusion can protect the diabetic myocardium by decreasing the inflammatory response and apoptosis.     

中药抗心肌缺血再灌注损伤的信号通路研究进展

冠心病发生率、致死率高,严重危害人类健康。心肌缺血再灌注损伤是加重心肌损伤的主要病理机制,干预再灌注损伤挽救激酶、 单磷酸腺苷激酶、蛋白激酶 C 等信号传导通路保护心肌,成为减轻心肌损伤的重要途径之一。综述近 3 年国际期刊收录的中药有效成分、 提取物及复方制剂调节相关信号传导通路, 减轻心肌再灌注损伤的研究进展, 以期为阐释中药的作用特点, 有效防治心血管疾病提供参考。     

Effect of duration of ischemia on myocardial proteome in ischemia/reperfusion injury

Ischemia/reperfusion (I/R) injury is a serious problem resulting from clinical setting of coronary revascularization. Despite extensive studies on I/R injury, the molecular bases of cardiac dysfunction caused by I/R are still unknown, but are likely to result from alterations in protein expression. Isolated rat hearts were subjected to 15-30 min of no-flow ischemia without (Ischemia protocol) or with 30 min of reperfusion (I/R protocol). 2-DE analysis of heart proteins from both experimental protocols showed wide-ranging changes in protein levels. In the Ischemia protocol, 39 protein spots were changed in ischemic groups and those changes correlated with duration of ischemia. Ninety percent of the affected proteins were increased. In contrast to increased protein levels, the total messenger RNA (mRNA) level decreased approximately two fold. Compared to the Ischemia protocol, changes in protein levels in the I/R protocol did not correlate with the duration of ischemia and the degree of recovery of mechanical function. The decrease of affected protein from I/R protocol was associated with the increase in total protein level in reperfusate. Our studies show that the protein increase is correlated with the mechanical function of the I/R hearts and the increase is not likely associated with an increase in protein synthesis.     

Bcl-xL gene transfer protects the heart against ischemia/reperfusion injury

Ischemia and reperfusion (I/R) injury causes the progression of cardiac dysfunction. The prevention of cardiomyocyte-loss due to I/R injury is important for the treatment of heart failure. Therefore, we employed antiapoptotic Bcl-xL protein to prevent I/R injury in the heart and evaluated the cardioprotective effect of Bcl-xL transduction by adenoviral vector (Adv) after I/R injury. Adv with Bcl-xL gene was injected in the rat heart 4 days prior to I/R. The prevention of cardiac performance-loss and the reduction of cardiac apoptosis, after 30min ischemia and 30min reperfusion of global I/R, were demonstrated in the heart with adenoviral Bcl-xL transduction. Also, significant reductions of the infarct size and serum creatine kinase levels were observed in the heart transduced with Bcl-xL gene compared with control after 30min ischemia and 24h reperfusion of the left anterior coronary artery. Thus, Bcl-xL may serve as a potential therapeutic tool for cardioprotection.     

In vivo hyperoxic preconditioning protects against rat-heart ischemia/reperfusion injury by inhibiting mitochondrial permeability transition pore opening and cytochrome c release

In vivo hyperoxic preconditioning (PC) has been shown to protect against ischemia/reperfusion (I/R) myocardial damage. Mitochondrial permeability transition pore (MPTP) opening is an important event in cardiomyocyte cell death occurring during I/R and therefore a possible target for cardioprotection. We tested the hypothesis that in vivo hyperoxic PC, obtained by mechanical ventilation of animals, could protect heart against I/R injury by inhibiting MPTP opening and cytochrome c release from mitochondria. Mechanically ventilated rats were first exposed to a short period of hyperoxia and isolated hearts were subsequently subjected to I/R in a Langendorff apparatus. Hyperoxic PC significantly improved the functional recovery of hearts on reperfusion, reduced the infarct size, and decreased necrotic damage as shown by the reduced release of lactate dehydrogenase. Mitochondria from hyperoxic PC hearts were less sensitive than mitochondria from reperfused heart to MPTP opening. In addition, hyperoxic PC prevented mitochondrial NAD(+) depletion, an indicator of MPTP opening, and cytochrome c release as well as cardiolipin oxidation/depletion associated with I/R. Together, these results demonstrate that hyperoxic PC protects against heart I/R injury by inhibiting MPTP opening and cytochrome c release. Thus, in vivo hyperoxic PC may represent a useful strategy for the treatment of cardiac I/R injury and could have potential applications in clinical practice.     

Urotensin II protects ischemic reperfusion injury of hearts through ROS and antioxidant pathway

Urotensin II (UII) is a vasoactive peptide which is bound to a G protein-coupled receptor. UII and its receptor are upregulated in ischemic and chronic hypoxic myocardium, but the effect of UII on ischemic reperfusion (I/R) injury is still controversial. The aim of the present study was to investigate whether UII protects heart function against I/R injury. Global ischemia was performed using isolated perfused Langendorff hearts of Sprague-Dawley rats. Hearts were perfused with Krebs-Henseleit buffer for 20min pre-ischemic period followed by a 20min global ischemia and 50min reperfusion. Pretreatment with UII (10nM) for 10min increased recovery percentage of the post-ischemic left ventricular developed pressure and ±dp/dt, and decreased post-ischemic left ventricular end-diastolic pressure as compared with I/R group. UII decreased infarct size and an increased lactate dehydrogenase level during reperfusion. Cardioprotective effects of UII were attenuated by pretreatment with UII receptor antagonist. The hydrogen peroxide activity was increased in UII-treated heart before ischemia. The Mn-SOD, catalase, heme oxygenase-1 and Bcl-2 levels were increased, and the Bax and caspase-9 levels were decreased in UII-treated hearts. These results suggest that UII has cardioprotective effects against I/R injury partly through activating antioxidant enzymes and reactive oxygen species.     

Cardioprotective effect of MMP‐2‐inhibitor‐NO‐donor hybrid against ischaemia/reperfusion injury

Hypoxic injury of cardiovascular system is one of the most frequent complications following ischaemia. Heart injury arises from increased degradation of contractile proteins, such as myosin light chains (MLCs) and troponin I by matrix metalloproteinase 2 (MMP‐2). The aim of the current research was to study the effects of 5‐phenyloxyphenyl‐5‐aminoalkyl nitrate barbiturate (MMP‐2‐inhibitor‐NO‐donor hybrid) on hearts subjected to ischaemia/reperfusion (I/R) injury. Primary human cardiac myocytes and Wistar rat hearts perfused using Langendorff method have been used. Human cardiomyocytes or rat hearts were subjected to I/R in the presence or absence of tested hybrid. Haemodynamic parameters of heart function, markers of I/R injury, gene and protein expression of MMP‐2, MMP‐9, inducible form of NOS (iNOS), asymmetric dimethylarginine (ADMA), as well as MMP‐2 activity were measured. Mechanical heart function, coronary flow (CF) and heart rate (HR) were decreased in hearts subjected to I/R Treatment of hearts with the hybrid (1‐10 µmol/L) resulted in a concentration‐dependent recovery of mechanical function, improved CF and HR. This improvement was associated with decreased tissue injury and reduction of synthesis and activity of MMP‐2. Decreased activity of intracellular MMP‐2 led to reduced degradation of MLC and improved myocyte contractility in a concentration‐dependent manner. An infusion of a MMP‐2‐inhibitor‐NO‐donor hybrid into I/R hearts decreased the expression of iNOS and reduced the levels of ADMA. Thus, 5‐phenyloxyphenyl‐5‐aminoalkyl nitrate barbiturate protects heart from I/R injury.