Negative regulation of vascular smooth muscle cell migration by blood shear stress

Urocortin (UCN) protects hearts against ischemia and reperfusion injury whether given before ischemia or at reperfusion. Here we investigate the roles of PKC, reactive oxygen species, and the mitochondrial permeability transition pore (MPTP) in mediating these effects. In Langendorff-perfused rat hearts, acute UCN treatment improved hemodynamic recovery during reperfusion after 30 min of global ischemia; this was accompanied by less necrosis (lactate dehydrogenase release) and MPTP opening (mitochondrial entrapment of 2-[(3)H]deoxyglucose). UCN pretreatment protected mitochondria against calcium-induced MPTP opening, but only if the mitochondria had been isolated from hearts after reperfusion. These mitochondria also exhibited less protein carbonylation, suggesting that UCN decreases levels of oxidative stress. In isolated adult and neonatal rat cardiac myocytes, both acute (60 min) and chronic (16 h) treatment with UCN reduced cell death following simulated ischemia and re-oxygenation. This was accompanied by less MPTP opening as measured using tetramethylrhodamine methyl ester. The level of oxidative stress during reperfusion was reduced in cells that had been pretreated with UCN, suggesting that this is the mechanism by which UCN desensitizes the MPTP to reperfusion injury. Despite the fact that we could find no evidence that either PKC-epsilon or PKC-alpha translocate to the mitochondria following acute UCN treatment, inhibition of PKC with chelerythrine eliminated the effect of UCN on oxidative stress. Our data suggest that acute UCN treatment protects the heart by inhibiting MPTP opening. However, the mechanism appears to be indirect, involving a PKC-mediated reduction in oxidative stress.     

Intermittent hypoxia attenuates ischemia／reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2／Bax expression

Intermittent hypoxia has been shown to provide myocardial protection against ishemiaJreperfusion-induced injury.Cardiac myocyte loss through apoptosis has been reported in ischemia/reperfusion injury. Our aim was to investigate whether intermittent hypoxia could attenuate ischemia/reperfusion-induced apoptosis in cardiac myocytes and its potential mechanisms. Adult male Sprague-Dawley rats were exposed to hypoxia simulated 5000 m in a hypobaric chamber for 6 h/day, lasting 42 days. Normoxia group rats were kept under normoxic conditions. Isolated perfused hearts from both groups were subjected to 30 min of global ischemia followed by 60 min reperfusion.Incidence of apoptosis in cardiac myocytes was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DNA agarose gel electrophoresis. Expressions of apoptosis related proteins,Bax and Bcl-2, in cytosolic and membrane fraction were detected by Western Blotting. After ischemia/reperfusion,enhanced recovery of cardiac function was observed in intermittent hypoxia hearts compared with normoxia group.Ischemia/reperfusion-induced apoptosis, as evidenced by TUNEL-positive nuclei and DNA fragmentation, was significantly reduced in intermittent hypoxia group compared with normoxia group. After ischemia/reperfusion,expression of Bax in both cytosolic and membrane fractions was decreased in intermittent hypoxia hearts comparedwith normoxia group. Although ischemia/reperfusion did not induce changes in the level of Bcl-2 expression in cytosolic fraction between intermittent hypoxia and normoxia groups, the expression of Bcl-2 in membrane fraction was upregulated in intermittent hypoxia group compared with normoxia group. These results indicated that the cardioprotection of intermittent hypoxia against ischemia/reperfusion injury appears to be in part due to reducemyocardial apoptosis. Intermittent hypoxia attenuated ischemia/reperfusion-induced apoptosis via increasing the ratio of Bcl-2/Bax, especially in membrane fraction.     

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.     

Baicalein attenuates oxidant stress in cardiomyocytes

Flavonoids within Scutellaria baicalensis may be potent antioxidants on the basis of our studies of S. baicalensis extract. To further this work, we studied the antioxidative effects of baicalein, a flavonoid component of S. baicalensis, in a chick cardiomyocyte model of reactive oxygen species (ROS) generation during hypoxia, simulated ischemia-reperfusion, or mitochondrial complex III inhibition with antimycin A. Oxidant stress was measured by oxidation of the intracellular probes 2',7'-dichlorofluorescin diacetate and dihydroethidium. Viability was assessed by propidium iodide uptake. Baicalein attenuated oxidant stress during all conditions studied and acted within minutes of treatment. For example, baicalein given only at reperfusion dose dependently attenuated the ROS burst at 5 min after 1 h of simulated ischemia. It also decreased subsequent cell death at 3 h of reperfusion from 52.3 +/- 2.5% in untreated cells to 29.4 +/- 3.0% (with return of contractions; P < 0.001). In vitro studies using electron paramagnetic resonance spectroscopy with the spin trap 5-methoxycarbonyl-5-methyl-1-pyrroline-N-oxide revealed that baicalein scavenges superoxide but does not mimic the effects of superoxide dismutase. We conclude that baicalein can scavenge ROS generation in cardiomyocytes and that it protects against cell death in an ischemia-reperfusion model when given only at reperfusion.     

Urocortin protects the heart from reperfusion injury via upregulation of p42/p44 MAPK signaling pathway

Reperfusion of ischemic myocardium is essential for tissue salvage but paradoxically contributes to cell death. We hypothesized that activation of potential survival pathways such as p42/p44 MAPK may prevent lethal reperfusion injury. Urocortin is a peptide factor that affects the p42/p44 MAPK signaling pathway. Both isolated and in vivo rat heart models were used to examine the potential for urocortin to prevent reperfusion injury. Isolated rat hearts underwent 35-min regional ischemia and 2-h reperfusion, with urocortin perfused for 20 min from the onset of reperfusion. In the in vivo study, urocortin was administered as an intravenous bolus 3 min before reperfusion with a protocol of 25-min regional ischemia and 2-h reperfusion. Blockade of the p42/p44 MAPK pathway with the inhibitor PD-98059 was used in both models. Urocortin attenuated lethal reperfusion-induced injury both in vitro and in vivo via a p42/p44 MAPK-dependent mechanism. Furthermore, Western blot analysis demonstrated the ability of urocortin to directly upregulate this signaling pathway. In conclusion, we believe that the p42/p44 MAPK-dependent signaling pathway represents an important survival mechanism against reperfusion injury.     

Activation of the JNK pathway is important for cardiomyocyte death in response to simulated ischemia

Multiple signaling pathways, including the c-Jun N-terminal kinase (JNK) pathway, are activated in myocardial ischemia and reperfusion (MI/R) and correlate with cell death. However, the role of the JNK pathway in MI/R-induced cell death is poorly understood. In a rabbit model, we found that ischemia followed by reperfusion resulted in JNK activation which could be detected in cytosol as well as in mitochondria. To address the functional role of the JNK activation, we examined the consequences of blockade of JNK activation in isolated cardiomyocytes under conditions of simulated ischemia. The JNK activity was stimulated approximately sixfold by simulated ischemia and reperfusion (simulated MI). When a dominant negative mutant of JNK kinase-2 (dnJNKK2), an upstream regulator of JNK, and JNK-interacting protein-1 (JIP-1) were expressed in myocytes by recombinant adenovirus, the activation of JNK by simulated MI was reduced 53%. Furthermore, the TNFalpha-activated JNK activity in H9c2 cells was completely abolished by dnJNKK2 and JIP-1. In correlation, when dnJNKK2 and JIP-1 were expressed in cardiomyocytes, both constructs significantly reduced cell death after simulated MI compared to vector controls. We conclude that activation of the JNK cascade is important for cardiomyocyte death in response to simulated ischemia.     

Oxidant stress during simulated ischemia primes cardiomyocytes for cell death during reperfusion

Ischemia-reperfusion injury induces oxidant stress, and the burst of reactive oxygen species (ROS) production after reperfusion of ischemic myocardium is sufficient to induce cell death. Mitochondrial oxidant production may begin during ischemia prior to reperfusion because reducing equivalents accumulate and promote superoxide production. We utilized a ratiometric redox-sensitive protein sensor (heat shock protein 33 fluorescence resonance energy transfer (HSP-FRET)) to assess oxidant stress in cardiomyocytes during simulated ischemia. HSP-FRET consists of the cyan and yellow fluorescent protein fluorophores linked by the cysteine-containing regulatory domain from bacterial HSP-33. During ischemia, ROS-mediated oxidation of HSP-FRET was observed, along with a decrease in cellular reduced glutathione levels. These findings were corroborated by measurements using redox-sensitive green fluorescent protein, another protein thiol ratiometric sensor, which became 93% oxidized by the end of simulated ischemia. However, cell death did not occur during ischemia, indicating that this oxidant stress is not sufficient to induce death before reperfusion. However, interventions that attenuate ischemic oxidant stress, including antioxidants or scavengers of residual O(2) that attenuate/prevent ROS generation during ischemia, abrogated cell death during simulated reperfusion. These findings reveal that, in isolated cardiomyocytes, sublethal H(2)O(2) generation during simulated ischemia regulates cell death during simulated reperfusion, which is mediated by the reperfusion oxidant burst.     

间歇性低压低氧方式对发育大鼠心脏缺血/再灌注损伤的影响

本研究旨在探讨两种不同形式的间歇性低压低氧（intermittent hypobaric hypoxia,IHH）对发育大鼠心脏缺血,再灌注损伤的影响。雄性Sprague-Dawley（SD）新生大鼠72只,随机分为三组：对照组、IHH3000in组（IHH3000）、IHH5000m组（IHH5000）。低氧组大鼠出生后立即于低压氧舱分别接受28d、42d和56d（海拔5000m、每天6h：海拔3000m、每天5h）的低压低氧处理。应用Langendorff离体心脏灌流技术,给予心脏缺血（停灌30min）／再灌注（复灌60min）处理,分别在缺血前5min及复灌后l、5、10、20、30、60min记录心功能和冠状动脉流量变化,并测定乳酸脱氢酶（1actate dehydrogenase,LDH）活性。实验结束时测定心脏重量。结果显示：（1）IHH3000组大鼠体重增长与对照组无明显差异;IHH5000组大鼠体重增长明显慢于对照组及IHH3000组大鼠（P〈0．01）。（2）IHH3000组人鼠表现明显的心脏保护效应。与对照组相比较,在心脏停灌,再灌注60min时,心功能（LVDP、±LVdp／drmax）恢复增强（P〈0．05）、LDH活性降低（P〈0．05）、冠状动脉流量增多（P〈0．05）;心脏重量与对照组大鼠无差异;IHH42d处理的大鼠心功能恢复明显好于IHH28d处理的大鼠（P〈0．05）。（3）IHH5000组大鼠表现出明显的心脏损伤效应,各项心功能指标（LVDP、±LVdp／dtmax）的恢复均低于对照组（P〈0．05）,复灌过程中LDH活性明显高于相应对照组（P〈0．05）,右心室重量明显高于对照组大鼠（P〈0．05）。结果表明,适当的IHH增强发育大鼠心脏对缺血,再灌注损伤的抵抗能力;间歇性低氧方式是影响其心脏保护作用的重要因素。     

间歇低压低氧预处理对心肌缺血／再灌注损伤及ZFP580表达的影响

目的：探讨间歇性低压低氧（IHH）预处理对大鼠心肌缺血／再灌注（I/R）损伤后血清中心肌酶、心肌梗死的影响及锌指核转录因子ZFP580发挥的作用。方法：32只雄性Wistar大鼠随机分为IHH预处理组和常氧对照组（n=16）。IHH组大鼠置于模拟海拔高度为5000m的低压氧舱中,每天6h,持续42d。两组大鼠经结扎冠状动脉左前降支建立心肌I/R损伤模型后,检测血清中乳酸脱氢酶（LDH）活性及肌酸磷酸肌酶同功酶（CK-MB）浓度,并利用Western blot方法观察各组大鼠心肌组织中ZFP580的表达情况。每组另外8只大鼠经心肌酞菁蓝-TIC染色后比较心肌梗死面积。培养大鼠H9c2心肌细胞,利用慢病毒介导的基因转染实验获得高表达ZFP580的心肌细胞,并进行心肌细胞模拟缺血／再灌注（SI/）损伤实验。利用Annexin V-PE／7-AAD柒色及流式细胞术检测H9c2心肌细胞的凋亡情况。结果：IHH预处理能明显减少心肌I／R损伤后IDH、CK-MB漏出至血清,并明显缩小心肌梗死面积。大鼠经IHH预处理后心肌组织中ZFP580的表达上调,IHH预处理明显上调心肌L／R损伤后心肌组织中ZFP580的表达。高表达ZFP580的H9c2心肌细胞在STIR损伤后细胞凋亡率明显下降。结论：IHH预处理对于心肌I／R损伤具有明显细胞保护作用,其上调的ZFPS80表达具有减少心肌细胞凋亡的作用,ZFP580可能作为心肌细胞内源性抗凋亡分子之一,参与IHH预处理抗心肌I／R损伤的过程。     

Placental growth factor signaling regulates isoform splicing of vascular endothelial growth factor A in the control of lung cancer cell metastasis

The omega-3 fatty acid, alpha linolenic acid (ALA) found in plant-derived foods induces significant cardiovascular benefits when ingested. ALA may be cardioprotective during ischemia; however, the mechanism(s) responsible for this effect is unknown. Isolated adult rat cardiomyocytes were exposed to medium containing ALA for 24 h and then exposed to non-ischemic (control), simulated ischemia (ISCH), or simulated ischemia/reperfusion (IR) conditions. Cardiomyocyte phospholipids were extracted and analyzed by an HPLC/electrospray ionization tandem mass spectrometry system. Pre-treatment of cells with ALA resulted in a significant incorporation of ALA within cardiomyocyte phosphatidylcholine. Cell death, DNA fragmentation and caspase-3 activity increased during ischemia and ischemia/reperfusion. Two pro-apoptotic oxidized phosphatidylcholine (OxPC) species, 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) were significantly increased during both ischemia and ischemia/reperfusion. Pre-treatment of the cells with ALA resulted in a significant reduction in cell death during ischemia and ischemia/reperfusion challenge. Apoptosis was also inhibited during ischemia and ischemia/reperfusion as shown by reduced DNA fragmentation and decreased caspase activation. ALA pre-treatment significantly decreased the production of POVPC and PGPC during ischemia and ischemia/reperfusion. ALA pre-treatment also significantly increased in resting Ca²⁺ during ischemia or ischemia/reperfusion but did not improve Ca²⁺ transients. ALA protects the cardiomyocyte from apoptotic cell death during simulated ISCH and IR by inhibiting the production of specific pro-apoptotic OxPC species. OxPCs represent a viable interventional target to protect the heart during ischemic challenge.     

Impact of hypoxia, simulated ischemia and reperfusion in HL-1 cells on the expression of FKBP12/FKBP12.6 and intracellular calcium dynamics

AimsTo establish a cardiac cell culture model for simulated ischemia and reperfusion and in this model investigate the impact of simulated ischemia and reperfusion on expression of the calcium handling proteins FKBP12 and FKBP12.6, and intracellular calcium dynamics.MethodsHL-1 cell cultures were exposed to normoxia (as control), hypoxia, simulated ischemia (HEDA) or HEDA + reactive oxygen species (ROS) for up to 24 h and after HEDA, with or without ROS, followed or not by simulated reperfusion (REPH) for 6 h. Viability was analyzed with a trypan blue exclusion method. Cell lysates were analyzed with real-time PCR and Western blot (WB) for FKBP12 and FKBP12.6. Intracellular Ca²⁺measurements were performed using dual-wavelength ratio imaging in fura-2 loaded cells.ResultsA time-dependent drop in viability was shown after HEDA (P < 0.001). Viability was not further influenced by addition of ROS or REPH. The general patterns of FKBP12 and FKBP12.6 mRNA expression showed upregulation after hypoxia, downregulation after ischemia and normalization after reperfusion, which was partially attenuated if ROS was added during HEDA. The protein contents were unaffected after hypoxia, tended to increase after ischemia and, for FKBP12.6, a further increase after reperfusion was shown. Hypoxia or HEDA, with or without REPH, resulted in a decreased amplitude of the Ca²⁺ peak in response to caffeine. In addition, cells subjected to HEDA for 3 h or HEDA for 3 h followed by 6 h of REPH displayed irregular Ca²⁺ oscillations with a decreased frequency.ConclusionA threshold for cell survival with respect to duration of ischemia was established in our cell line model. Furthermore, we could demonstrate disturbances of calcium handling in the sarcoplasmic reticulum as well as alterations in the expressions of the calcium handling proteins FKBP12 and FKBP12.6, why this model may be suitable for further studies on ischemia and reperfusion with respect to calcium handling of the sarcoplasmic reticulum.     

Overexpression of the NHE1 isoform of the Na+/H+ exchanger causes elevated apoptosis in isolated cardiomyocytes after hypoxia/reoxygenation challenge

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH in the myocardium and other tissues. NHE1 is an important mediator of myocardial damage that occurs after ischemia–reperfusion injury. It has also been implicated in apoptotic damage in many tissues and its expression and activity are elevated in disease states in the myocardium. In this study, we examined the effect of additional exogenous NHE1 expression on isolated cardiomyocytes susceptibility to ischemia/reperfusion damage. Exogenous NHE1 elevated Na⁺/H⁺ exchanger expression and activity when introduced into isolated cardiomyocytes through an adenoviral system. Isolated cardiomyocytes were subjected to simulated ischemia and reperfusion after infection with either control or NHE1-containing adenovirus. Cells were placed into an anaerobic chamber and effects of NHE1 expression after hypoxia/reoxygenation were examined. Hypoxia/reoxygenation increased caspase-3-like activity in controls, and the effect was greatly magnified in cells expressing NHE1 protein. It also elevated the percentage of apoptotic cardiomyocytes, which was also aggravated by expression of NHE1 protein. Hypoxia/reoxygenation also increased phospho-ERK levels. Elevated NHE1 expression was coincidental with increased expression of the ER stress protein, protein disulfide isomerase (PDI) and calreticulin (CRT). Our results demonstrate that increased NHE1 protein expression makes cells more susceptible to damage induced by hypoxia/reoxygenation in isolated cardiomyocytes. They suggest that elevated NHE1 in cardiovascular disease could predispose the human myocardium to enhanced apoptotic damage.     

Activation of corticotropin releasing factor receptor type 2 in the heart by corticotropin releasing factor offers cytoprotection against ischemic injury via PKA and PKC dependent signaling

Corticotrophin-releasing factor receptor 2β (CRFR2β) is expressed in the myocardium. In the present study we explore whether acute treatment with the neuropeptide corticotrophin-releasing factor (CRF) could induce cytoprotection against a lethal ischemic insult in the heart (isolated murine neonatal cardiac myocytes and the isolated Langendorff perfused rat heart) by activating CRFR2. In vitro, CRF offered cytoprotection when added prior to lethal simulated ischemic stress by reducing apoptotic and necrotic cell death. Ex vivo, CRF significantly reduced infarct size from 52.1±3.1% in control hearts to 35.3±3.1% (P<0.001) when administered prior to a lethal ischemic insult. The CRF peptide did not confer cytoprotection when administered at the point of hypoxic reoxygenation or ischemic reperfusion. The acute effects of CRF treatment are mediated by CRF receptor type 2 (CRFR2) since the cardioprotection ex vivo was inhibited by the CRFR2 antagonist astressin-2B. Inhibition of the mitogen activated protein kinase-ERK1/2 by PD98059 failed to inhibit the effect of CRF. However, both protein kinase A and protein kinase C inhibitors abrogated CRF-mediated protection both ex vivo and in vitro. These data suggest that the CRF peptide reduces both apoptotic and necrotic cell death in cardiac myocytes subjected to lethal ischemic induced stress through activation of PKA and PKC dependent signaling pathways downstream of CRFR2.     

间歇性低氧处理大鼠心肌的抗心律失常与抗氧化效应

利用结扎在体大鼠冠脉方法研究不同时间间歇性低氧处理对血、再灌注心律失常以及心肌超氧化物歧化酶（ＳＯＤ）、丙二醛（ＭＤＡ）的影响,并与连续性低氧相比较。实验结果如下：⑴间歇性低氧（ｉｎｔｅｒｍｉｔｔｅｎｔ ｈｙｐｏｘｉａ ｅｘｐｏｓｕｒｅ）２８ｄ（ＩＨ２８）、４２ｄ（ＩＨ４２）、间歇性低氧２８ｄ后１周（ＰＩＨ２８－２Ｗ）和连续性低氧（ｃｏｍｔｉｎｕｅｄ ｈｙｐｏｘｉａ ｅｘｐｏｓｕｒｅ）２８ｄ（ＣＨ     

The role of Coptidis Rhizoma extract in a renal ischemia–reperfusion model

The effect of Coptidis Rhizoma extract on ischemia–reperfusion in rats was examined. The blood levels of urea nitrogen and creatinine increased significantly more in rats subjected to 24-h reperfusion than those subjected to 6-h reperfusion following 1-h ischemia, indicating functional kidney damage was more severe after the longer reperfusion time. These parameters were reduced by oral administration of Coptidis Rhizoma extract. Greater activity was found in rats given the extract for 30 days than in rats given the extract for 10 days prior to ischemia–reperfusion. In addition, the serum malondialdehyde level was lower, while the glutathione/glutathione disulfide ratio and the activities of the antioxidation enzymes, superoxide dismutase and catalase, were higher in rats given Coptidis Rhizoma extract orally for 30 consecutive days prior to 1-h ischemia and 24-h reperfusion in comparison with control rats given water. These results indicate that Coptidis Rhizoma has a protective action against the renal dysfunction caused by the ischemia and reperfusion process. Furthermore, renal DNA of rats given Coptidis Rhizoma extract orally showed a significantly lower DNA fragmentation rate, which was dose-dependent, implying that the extract afforded the kidneys protection against oxidative stress-mediated apoptosis during the process and ameliorated renal function impairment.     

Zinc pyrithione salvages reperfusion injury by inhibiting NADPH oxidase activation in cardiomyocytes

Zinc pyrithione (ZPT), has a strong anti-apoptotic effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether ZPT can reduce the production of reactive oxygen species during reoxygenation in cultured neonatal rat cardiac myocytes and evaluated the role of NADPH oxidase in hypoxia/reoxygenation (H/R) injury. The cells were subjected to 8 h of simulated ischemia, followed by either 30 min or 16 h of reoxygenation. ZPT when started just before reoxygenation significantly reduced superoxide generation, LDH release and improved cell survival compared to H/R. Attenuation of the ROS production by ZPT paralleled its capacity to prevent pyknotic nuclei formation. In addition, ZPT reversed the H/R-induced expression of NOX2 and p47^phox phosphorylation indicating that ZPT directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates NADPH oxidase mediated intracellular oxidative stress.     

Reperfusion,not simulated ischemia,initiates intrinsic apoptosis injury in chick cardiomyocytes

Although ischemia-reperfusion (I/R) can initiate apoptosis, the timing and contribution of the mitochondrial/cytochrome c apoptosis death pathway to I/R injury is unclear. We studied the timing of cytochrome c release during I/R and whether subsequent caspase activation contributes to reperfusion injury in confluent chick cardiomyocytes. One-hour simulated ischemia followed by 3-h reperfusion resulted in significant cell death, with most cell death evident during the reperfusion phase and demonstrating mitochondrial cytochrome c release within 5 min after reperfusion. By contrast, cells exposed to prolonged ischemia for 4 h had only marginally increased cell death and no detectable cytochrome c release into the cytosol. Caspase activation could not be detected after ischemia only, but it significantly increased after reperfusion. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, Ac-Asp-Gln-Thr-Asp-H, or benzyloxycarbonyl-Leu-Glu (Ome)-His-Asp-(Ome)-fluoromethyl ketone given only at reperfusion significantly attenuated cell death and resulted in return of contraction. Antixoxidants decreased cytochrome c release, nuclear condensation, and cell death. These results suggest that reperfusion oxidants initiate cytochrome c release within minutes, and apoptosis within hours, significant enough to increase cell death and contractile dysfunction.