C-phycocyanin protects against ischemia-reperfusion injury of heart through involvement of p38 MAPK and ERK signaling

We previously showed that C-phycocyanin (PC), an antioxidant biliprotein pigment of Spirulina platensis (a blue-green alga), effectively inhibited doxorubicin-induced oxidative stress and apoptosis in cardiomyocytes. Here we investigated the cardioprotective effect of PC against ischemia-reperfusion (I/R)-induced myocardial injury in an isolated perfused Langendorff heart model. Rat hearts were subjected to 30 min of global ischemia at 37 degrees C followed by 45 min of reperfusion. Hearts were perfused with PC (10 microM) or Spirulina preparation (SP, 50 mg/l) for 15 min before the onset of ischemia and throughout reperfusion. After 45 min of reperfusion, untreated (control) hearts showed a significant decrease in recovery of coronary flow (44%), left ventricular developed pressure (21%), and rate-pressure product (24%), an increase in release of lactate dehydrogenase and creatine kinase in coronary effluent, significant myocardial infarction (44% of risk area), and TdT-mediated dUTP nick end label-positive apoptotic cells compared with the preischemic state. PC or SP significantly enhanced recovery of heart function and decreased infarct size, attenuated lactate dehydrogenase and creatine kinase release, and suppressed I/R-induced free radical generation. PC reversed I/R-induced activation of p38 MAPK, Bax, and caspase-3, suppression of Bcl-2, and increase in TdT-mediated dUTP nick end label-positive apoptotic cells. However, I/R also induced activation of ERK1/2, which was enhanced by PC treatment. Overall, these results for the first time showed that PC attenuated I/R-induced cardiac dysfunction through its antioxidant and antiapoptotic actions and modulation of p38 MAPK and ERK1/2.     

Reversal of the postischaemic suppression of coronary function in perfused guinea pig heart by ischaemic preconditioning.

In the isolated guinea pig hearts suppression of endothelium-dependent (Acetylcholine, Substance P, postocclusive hyperaemia) and endothelium-independent (Sodium nitroprusside, PGE1) responses after 30 min subglobal ischaemia (reduction of coronary flow to 5%) were analysed in hearts which were not preconditioned or preconditioned by various protocols. Preconditioning consisted of single 5 min ischaemia (IP5) or single 10 min ischaemia (IP10) or double 5 min ischaemia (IP5 + 5). Thirty minutes of ischaemia followed by reperfusion reduced both endothelium-dependent and endothelium-independent responses approximately by 30-50% and slightly suppressed basal coronary flow by 10%. IP5 and IP5 + 5 protected against postischaemic suppression of responses to NaNP but not against postischaemic impairment of SP, ACh, and POH responses. The endothelium-dependent responses and postischaemic suppression of basal coronary flow were protected by IP10 only. In summary, in the isolated guinea pig heart the 30-min ischaemia impairs vasodilator responses to both endothelium-dependent and endothelium-independent agents. Ischaemic preconditioning protects both endothelial and smooth muscle cells function against this impairment, though endothelial cells require a more extensive preconditioning to put in motion protective mechanisms than smooth muscle cells do. Independent mechanisms of IP in endothelial cells and in smooth muscle cells are suggested.     

Postconditioning for salvage of ischemic skeletal muscle from reperfusion injury: efficacy and mechanism

We tested our hypothesis that postischemic conditioning (PostC) is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP). In bilateral 8x13 cm pig latissimus dorsi muscle flaps subjected to 4 h ischemia, muscle infarction increased from 22+/-4 to 41+/-1% between 2 and 24 h reperfusion and remained unchanged at 48 (38+/-6%) and 72 (40+/-1%) h reperfusion (P<0.05; n=4 pigs). PostC induced by four cycles of 30-s reperfusion/reocclusion at the onset of reperfusion after 4 h ischemia reduced muscle infarction from 44+/-2 to 22+/-2% at 48 h reperfusion. This infarct protective effect of PostC was mimicked by intravenous injection of the mPTP opening inhibitor cyclosporin A or NIM-811 (10 mg/kg) at 5 min before the end of 4 h ischemia and was abolished by intravenous injection of the mPTP opener atractyloside (10 mg/kg) at 5 min before PostC (P<0.05; n=4-5 pigs). PostC or intravenous cyclosporin A injection at 5 min before reperfusion caused a decrease in muscle myeloperoxidase activity and mitochondrial free Ca2+ concentration and an increase in muscle ATP content after 4 h ischemia and 2 h reperfusion compared with the time-matched controls. These effects of PostC were abolished by intravenous injection of atractyloside at 5 min before PostC (P<0.05; n=6 pigs). These observations support our hypothesis that PostC is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mPTP.     

Glycogen turnover and anaplerosis in preconditioned rat hearts

Using (13)C NMR, we tested the hypothesis that protection by preconditioning is associated with reduced glycogenolysis during ischemia. Preconditioned rat hearts showed improved postischemic function and reduced ischemic damage relative to ischemic controls after 30 min stop-flow ischemia and 30 min reperfusion (contractility: 30+/-10 vs. 2+/-2%; creatine kinase release: 41+/-4 vs. 83+/-15 U/g; both P<0.05). Preconditioning decreased preischemic [(13)C]glycogen by 24% (a 10% decrease in total glycogen), and delayed ischemic [(13)C]glycogen consumption by 5-10 min, reducing ischemic glycogenolysis without changing acidosis relative to controls. Upon reperfusion, glycogen synthesis resumed only after preconditioning. Glutamate (13)C-isotopomer analysis showed recovery of Krebs cycle activity with higher anaplerosis than before ischemia (23+/-4 vs. 11+/-3%, P<0.05), but in controls reperfusion failed to restore flux. Compared to control, preconditioning before 20 min ischemia increased contractility (86+/-10 vs. 29+/-14%, P<0.05) and restored preischemic anaplerosis (13+/-3 vs. 39+/-9%, P<0.05). Preconditioning is associated with reduced glycogenolysis early during ischemia. However, protection does not rely on major variations in intracellular pH, as proposed earlier. Our isotopomer data suggest that preconditioning accelerates metabolic and functional recovery during reperfusion by more efficient/active replenishment of the depleted Krebs cycle.     

A1 adenosine receptor overexpression attenuates ischemia-reperfusion-induced apoptosis and caspase 3 activity

We tested the hypothesis that myocardial ischemia-reperfusion (I/R)-induced apoptosis is attenuated in transgenic mice overexpressing cardiac A(1) adenosine receptors. Isolated hearts from transgenic (TG, n = 19) and wild-type (WT, n = 22) mice underwent 30 min of ischemia and 2 h of reperfusion, with evaluation of apoptosis, caspase 3 activity, function, and necrosis. I/R-induced apoptosis was attenuated in TG hearts. TG hearts had less I/R-induced apoptotic nuclei (0.88 +/- 0.10% vs. 4.22 +/- 0.24% terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells in WT, P < 0.05), less DNA fragmentation (3.30 +/- 0.38-fold vs. 4.90 +/- 0.39-fold over control in WT, P < 0.05), and less I/R-induced caspase 3 activity (145 +/- 25% over nonischemic control vs. 234 +/- 31% in WT, P < 0.05). TG hearts also had improved recovery of function and less necrosis than WT hearts. In TG hearts pretreated with LY-294002 (3 microM) to evaluate the role of phosphosinositol-3-kinase in acute signaling, there was no change in the functional protection or apoptotic response to I/R. These data suggest that cardioprotection with transgenic overexpression of A(1) adenosine receptors involves attenuation of I/R-induced apoptosis that does not involve acute signaling through phosphoinositol-3-kinase.     

NO produced by endothelial NO synthase is a mediator of delayed preconditioning-induced endothelial protection

Preconditioning with brief periods of ischemia-reperfusion (I/R) induces a delayed protection of coronary endothelial cells against reperfusion injury. We assessed the possible role of nitric oxide (NO) produced during prolonged I/R as a mediator of this endothelial protection. Anesthetized rats were subjected to 20-min cardiac ischemia/60-min reperfusion, 24 h after sham surgery or cardiac preconditioning (1 x 2-min ischemia/5-min reperfusion and 2 x 5-min ischemia/5-min reperfusion). The nonselective NO synthase (NOS) inhibitor l-NAME, the selective inhibitors of neuronal (7-nitroindazole) or inducible (1400W) NOS, or the peroxynitrite scavenger seleno-l-methionine were administered 10 min before prolonged ischemia. Preconditioning prevented the reperfusion-induced impairment of coronary endothelium-dependent relaxations to acetylcholine (maximal relaxation: sham 77 +/- 3; I/R 44 +/- 6; PC 74 +/- 5%). This protective effect was abolished by l-NAME (41 +/- 7%), whereas 7-NI, 1400W or seleno-l-methionine had no effect. The abolition of preconditioning by l-NAME, but not by selective nNOS or iNOS inhibition, suggests that NO produced by eNOS is a mediator of delayed endothelial preconditioning.     

Phalloidin attenuates postischemic neutrophil infiltration and increased microvascular permeability.

The aim of this study was to determine whether phalloidin (1 microM) or antamanide (1 microM), cyclic peptides that stabilize dense peripheral band and stress fiber F-actin in endothelium, would attenuate the increase in microvascular permeability induced by 4 h of ischemia and 30 min of reperfusion (I/R) in the isolated canine gracilis muscle. Changes in microvascular permeability (1 - sigma) were assessed by determining the solvent drag reflection coefficient for total plasma proteins (sigma) in muscles subjected to 4.5 h of continuous perfusion (nonischemic controls), I/R alone, I/R + phalloidin, or I/R + antamanide. Muscle neutrophil content was assessed by determination of myeloperoxidase (MPO) activity in tissue samples obtained at the end of the experiments. Fluorescent detection of nitrobenzoxadiazole-phallicidin in endothelial cell monolayers confirmed that phalloidin enters these cells. I/R was associated with marked increases in microvascular permeability and muscle neutrophil content (1 - sigma = 0.45 +/- 0.07; MPO = 8.9 +/- 0.5 units/g) relative to control (4.5 h continuous perfusion) preparations (1 - sigma = 0.12 +/- 0.03; MPO = 0.5 +/- 0.8 unit/g). These I/R-induced changes were largely prevented by administration of phalloidin (1 - sigma = 0.19 +/- 0.02; MPO = 0.8 +/- 0.4 U/g) or antamanide (1 - sigma = 0.07 +/- 0.11; MPO = 0.9 +/- 0.3 unit/g) at reperfusion. Similar results were obtained when phalloidin was administered before ischemia (1 - sigma = 0.24 +/- 0.04; MPO = 1.2 +/- 1.0 units/g). Although antamanide decreased superoxide production (by approximately 60%) and adherence to plastic (by approximately 75%) by activated neutrophils in vitro, phalloidin failed to alter these aspects of granulocyte function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of MMP-2 activation and release as a novel mechanism for HDL-induced cardioprotection

High density lipoproteins (HDL) protect the heart against ischemia/reperfusion (I/R) injury, and matrix metalloproteinase-2 (MMP-2) directly contributes to cardiac contractile dysfunction after I/R. To investigate the possible involvement of MMP-2 inhibition in HDL-mediated cardioprotection, isolated rat hearts underwent 20 min of low-flow ischemia and 30 min of reperfusion. Plasma-derived and synthetic HDL attenuated the I/R-induced cardiac MMP-2 activation and release in a dose-dependent way. The attenuation of I/R-induced MMP-2 activation by HDL correlated with the reduction of post-ischemic contractile dysfunction and cardiomyocyte necrosis. These results indicate prevention of MMP-2 activation as a novel mechanism for HDL-mediated cardioprotection.     

Reperfusion injury in skeletal muscle is reduced in inducible nitric oxide synthase knockout mice.

Inducible nitric oxide synthase (iNOS) participates in many pathological events, and selective inhibition of iNOS has been shown to reduce ischemia-reperfusion (I/R) injury in different tissues. To further confirm its role in this injury process, I/R injury was observed in denervated cremaster muscles of iNOS-deficient (iNOS-/-) and wild-type mice. After 3-h ischemia and 90-min reperfusion, blood flow in reperfused muscle was 80 +/- 8.5% (mean +/- SE) of baseline at 10-min reperfusion and completely returned to the preischemia baseline after 20 min in iNOS-/- mice. In contrast, blood flow was 32 +/- 7.4% at 10 min and increased to 60 +/- 20% of the baseline level at 90 min in wild-type mice (P < 0.001 vs. iNOS-/- mice at all time points). The increased muscle blood flow in iNOS-/- mice was associated with significantly less vasospasm in all three sizes of arterial vessel size categories. The weight ratio to the contralateral muscle not subjected to I/R was greater in wild-type mice (173 +/- 11%) than in iNOS-/- mice (117 +/- 3%; P < 0.01). Inflammation and neutrophil extravasation were also more severe in wild-type mice. Western blot analysis demonstrated an absence of iNOS protein band in iNOS-/- mice and upregulation of iNOS protein expression in wild-type mice. Our results confirm the importance of iNOS in I/R injury. Upregulated iNOS exacerbates I/R injury and appears to be a therapeutic target in protection of tissues against this type of injury.     

Amiloride和Ni2+抑制缺血/复灌引起的大鼠心肌细胞内钙的增加

本文旨在研究Na+/H+交换以及Na+/Ca2 +交换对模拟缺血 /复灌引起的大鼠心肌细胞内游离钙水平变化的调节作用。分别利用模拟缺血液和正常台氏液对大鼠心肌细胞进行缺血 /复灌处理 ,在缺血期间分别应用Na+/H+交换抑制剂阿米洛利 (amiloride)、Na+/Ca2 +交换抑制剂NiCl2 以及无钙液 ,观察它们对细胞内游离Ca2 +浓度变化的影响。利用Zeiss LSM 5 10激光共聚焦显微镜检测、采集细胞内游离Ca2 +的指示剂Fluo 3 AM的荧光信号 ,计算出相对于正常(缺血前 )的相对荧光强度 ,以表示胞内游离Ca2 +浓度的变化。结果显示 ,模拟缺血引起大鼠心肌细胞内游离Ca2 +持续上升 ,缺血前的相对荧光强度值为 10 0 % ,模拟缺血 5min后为 140 3± 13 0 % (P <0 0 5 ) ,复灌 15min后为 142 8±15 5 % (P <0 0 5 )。经 10 0 μmol/Lamiloride、5mmol/LNiCl2 和无钙液分别预处理 ,模拟缺血 5min后的相对荧光强度分别为 10 1 4± 16 3 % (P <0 0 5 )、110 4± 11 1% (P <0 0 5 )和 10 7 1± 10 8(P <0 0 5 ) ;复灌 15min后则分别为 97 8±14 3 % (P <0 0 5 )、10 6 2± 14 5 % (P <0 0 5 )和 10 6 6± 15 7(P <0 0 5 )。另外 ,与对照组细胞相比 ,再灌注期间NiCl2和无钙液处理的细胞钙振荡的产生幅度明显减弱 ,amilorid     

Effects of recombinant human extracellular-superoxide dismutase type C on myocardial reperfusion injury in isolated cold-arrested rat hearts.

The efficacy of recombinant human extracellular-superoxide dismutase type C (EC-SOD C) on myocardial reperfusion injury was explored in hypothermically arrested rat hearts, as was its site of action. Forty isolated working rat hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. The hearts were arrested by the administration of 10 mL of cold perfusate at the onset of ischemia. At the same time, they were randomly assigned to one of five groups; A: cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate+bovine CuZn-SOD 7.5 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L + heparin 50,000U/L; E: cold perfusate + heparin 50,000 U/L. Heparin was given to prevent binding of EC-SOD C to endothelial cell surfaces. Left ventricular function was studied before ischemia and at the end of reperfusion. Percent recovery of maximal left ventricular dP/dt after reperfusion was more pronounced in group B (109 +/- 24%; p less than .05) than in groups A (42 +/- 40%), C (47 +/- 36%), D (44 +/- 33%) and E (58 +/- 25%). Likewise, percent recovery of the double product (heart rate x systolic left ventricular pressure) was better in group B (104 +/- 18%; p less than .05) than in the other groups (A: 47 +/- 37%, C: 49 +/- 36%, D: 50 +/- 35%, E: 69 +/- 31%). Compared to the preischemic level, creatine kinase increased significantly in the coronary effluent after reperfusion in groups A, C, D, and E, but not in group B. The results suggest that EC-SOD C, which attaches to the endothelial cell surfaces, might be particularly effective as protection against myocardial reperfusion injury when given together with cardioplegic solution.     

Pathophysiological role of platelets in acute experimental pancreatitis: influence of endothelin A receptor blockade

The potential pathophysiological role of platelet-endothelium interactions was investigated during ischemia/reperfusion (I/R), and the effect of a selective endothelin(A) receptor antagonist (ET(A)-RA) was evaluated in an acute pancreatitis model. Acute pancreatitis was induced by warm ischemia (60 min) in Wistar rats, and its effects with and without antagonist treatment were investigated. Equivalent sham-operated animals were also studied. Microcirculatory changes were assessed by in vivo microscopy, and serum levels for lipase/amylase and histological specimens were investigated. Capillary constriction to 83.7 +/- 6.7% of sham-operated diameters was observed after 60 min of ischemia. A capillary density of 56.8 +/- 9.3% of the sham-operated group (396.3 +/- 15.8 mm(-1)) was measured after reperfusion. Stagnant leukocytes (329.5 +/- 30.4%) and platelets (337.5 +/- 32.3%) increased in postcapillary venules (P < 0.05). Administration of the ET(A)-RA significantly reduced I/R injury. Capillary diameters were maintained (101.4 +/- 4.5%), and capillary density was improved to 73.3 +/- 7.6% of sham-operated animals (P < 0.05). Stagnant leukocytes (152.3 +/- 10.6%) and platelets (207.1 +/- 19.8%) in sinusoids and postcapillary venules were reduced (P < 0.05). The extent of acute pancreatitis was reduced in the therapy group as indicated by serum lipase/amylase values and histological tissue damage (P < 0.05). Thus, ET(A)-RA therapy was effective in reducing I/R-induced pancreatitis in this experimental model.     

Erythropoietin affords additional cardioprotection to preconditioned hearts by enhanced phosphorylation of glycogen synthase kinase-3 beta

The aim of this study was to determine whether erythropoietin (EPO) affords additional cardioprotection to the preconditioned myocardium by enhanced phosphorylation of Akt, STAT3, or glycogen synthase kinase-3beta (GSK-3 beta). Preconditioning (PC) with 5-min ischemia/5-min reperfusion and EPO (5,000 U/kg iv) reduced infarct size (as % of area at risk, %IS/AR) after 20-min ischemia in rat hearts in situ from 56.5 +/- 1.8% to 25.2 +/- 2.1% and to 36.2 +/- 2.8%, respectively. PC-induced protection was significantly inhibited by a protein kinase C inhibitor, chelerythrine (5 mg/kg), and slightly blunted by a phosphatidylinositol-3-kinase inhibitor, wortmannin (15 microg/kg). The opposite pattern of inhibition was observed for EPO-induced protection. The combination of PC and EPO further reduced %IS/AR to 8.9 +/- 1.9%, and this protection was inhibited by chelerythrine and wortmannin. The additive effects of PC and EPO on infarct size were mirrored by their effects on the level of phosphorylated GSK-3 beta at 5 min after reperfusion but not their effects on the level of phospho-Akt or phospho-STAT3. To mimic phosphorylation-induced inhibition of GSK-3 beta activity, SB-216763 (SB), a GSK-3 beta inhibitor, was administered before ischemia or 5 min before reperfusion. Infarct size was significantly reduced by preischemic injection (%IS/AR = 40.4 +/- 2.2% by 0.6 mg/kg SB and 34.0 +/- 1.8% by 1.2 mg/kg SB) and also by prereperfusion injection (%IS/AR = 32.0 +/- 2.0% by 1.2 mg/kg SB). These results suggest that EPO and PC afford additive infarct size-limiting effects by additive phosphorylation of GSK-3beta at the time of reperfusion by Akt-dependent and -independent mechanisms.     

Reduced hexokinase II impairs muscle function 2 wk after ischemia-reperfusion through increased cell necrosis and fibrosis

We previously demonstrated that hexokinase (HK) II plays a key role in the pathophysiology of ischemia-reperfusion (I/R) injury of the heart (Smeele et al. Circ Res 108: 1165-1169, 2011; Wu et al. Circ Res 108: 60-69, 2011). However, it is unknown whether HKII also plays a key role in I/R injury and healing thereafter in skeletal muscle, and if so, through which mechanisms. We used male wild-type (WT) and heterozygous HKII knockout mice (HKII(+/-)) and performed in vivo unilateral skeletal muscle I/R, executed by 90 min hindlimb occlusion using orthodontic rubber bands followed by 1 h, 1 day, or 14 days reperfusion. The contralateral (CON) limb was used as internal control. No difference was observed in muscle glycogen turnover between genotypes at 1 h reperfusion. At 1 day reperfusion, the model resulted in 36% initial cell necrosis in WT gastrocnemius medialis (GM) muscle that was doubled (76% cell necrosis) in the HKII(+/-) mice. I/R-induced apoptosis (29%) was similar between genotypes. HKII reduction eliminated I/R-induced mitochondrial Bax translocation and oxidative stress at 1 day reperfusion. At 14 days recovery, the tetanic force deficit of the reperfused GM (relative to control GM) was 35% for WT, which was doubled (70%) in HKII(+/-) mice, mirroring the initial damage observed for these muscles. I/R increased muscle fatigue resistance equally in GM of both genotypes. The number of regenerating fibers in WT muscle (17%) was also approximately doubled in HKII(+/-) I/R muscle (44%), thus again mirroring the increased cell death in HKII(+/-) mice at day 1 and suggesting that HKII does not significantly affect muscle regeneration capacity. Reduced HKII was also associated with doubling of I/R-induced fibrosis. In conclusion, reduced muscle HKII protein content results in impaired muscle functionality during recovery from I/R. The impaired recovery seems to be mainly a result of a greater susceptibility of HKII(+/-) mice to the initial I/R-induced necrosis (not apoptosis), and not a HKII-related deficiency in muscle regeneration.     

Changes in excitation-contraction coupling in an isolated ventricular myocyte model of cardiac stunning

To investigate cardiac stunning, we recorded intracellular [Ca(2+)], contractions, and electrical activity in isolated guinea pig ventricular myocytes exposed to simulated ischemia and reperfusion. After equilibration, ischemia was simulated by exposing myocytes to hypoxia, acidosis, hyperkalemia, hypercapnia, lactate accumulation, and substrate deprivation for 30 min at 37 degrees C. Reperfusion was simulated by exposure to Tyrode solution. Field-stimulated myocytes exhibited stunning upon reperfusion. By 10 min of reperfusion, contraction amplitude decreased to 43.0 +/- 5.5% of preischemic values (n = 15, P < 0.05), although action potential configuration and sarcoplasmic reticulum Ca(2+) stores, assessed with caffeine, were normal. Diastolic [Ca(2+)] and Ca(2+) transients (fura 2) were also normal in stunned myocytes. In voltage-clamped cells, peak L-type Ca(2+) current was reduced to 47.4 +/- 4.5% of preischemic values at 10 min of reperfusion (n = 21, P < 0.05). Contractions elicited by Ca(2+)-induced Ca(2+) release and the voltage-sensitive release mechanism were both depressed in reperfusion. Our observations suggest that stunning is associated with reduced L-type Ca(2+) current but that alterations in Ca(2+) homeostasis and release are not directly responsible for stunning.     

RETRACTED ARTICLE: Effect of preischemic treatment with fenofibrate,a peroxisome proliferator-activated receptor-α ligand,on hepatic ischemia–reperfusion injury in rats

Liver ischemia/reperfusion (I/R) injury is a serious clinical problem. The reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-α) are important mediators in liver I/R injury. This study was designed to investigate the effect of preischemic treatment with fenofibrate (Peroxisome proliferator-activated receptor- α agonist) on the oxidative stress and inflammatory response to hepatic I/R injury in rats. Hepatic I/R was induced by clamping the blood supply of the left lateral and median lobes of the liver for 60 min, followed by reperfusion for 4 h. Each animal group was pretreated with a single dose of fenofibrate (50 mg/kg body weight) intraperitoneally 1 h before ischemia. At the end of reperfusion, blood samples and liver tissues were obtained to assess serum alanine aminotransferase (ALT), TNF-α, hepatic malondialdehyde (MDA) and superoxide dismutase activity (SOD). Liver specimens were obtained and processed for light and electron microscopic study. Hepatic I/R induced a significant elevation of serum ALT and TNF-α with significant elevation of hepatic MDA and reduction of SOD activity. Histopathological examination revealed hepatic inflammation, necrosis and apoptosis. Preischemic treatment with fenofibrate at a dose of 50 mg/kg significantly attenuated the biochemical and structural alterations of I/R-induced liver injury.     

Ischemia-reperfusion-induced calpain activation and SERCA2a degradation are attenuated by exercise training and calpain inhibition

The Ca2+-activated protease calpain has been shown to play a deleterious role in the heart during ischemia-reperfusion (I/R). We tested the hypothesis that exercise training would minimize I/R-induced calpain activation and provide cardioprotection against I/R-induced injury. Hearts from adult male rats were isolated in a working heart preparation, and myocardial injury was induced with 25 min of global ischemia followed by 45 min of reperfusion. In sedentary control rats, I/R significantly increased calpain activity and impaired cardiac performance (cardiac work during reperfusion = 24% of baseline). Compared with sedentary animals, exercise training prevented the I/R-induced rise in calpain activity and improved cardiac work (recovery = 80% of baseline). Similar to exercise, pharmacological inhibition of calpain activity resulted in comparable cardioprotection against I/R injury (recovery = 86% of baseline). The exercise-induced protection against I/R-induced calpain activation was not due to altered myocardial protein levels of calpain or calpastatin. However, exercise training was associated with increased myocardial antioxidant enzyme activity (Mn-SOD, catalase) and a reduction in oxidative stress. Importantly, exercise training also prevented the I/R-induced degradation of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a. These findings suggest that increases in endogenous antioxidants may diminish the free radical-mediated damage and/or degradation of Ca2+ handling proteins (such as SERCA2a) typically observed after I/R. In conclusion, these results support the concept that calpain activation is an important component of I/R-induced injury and that exercise training provides cardioprotection against I/R injury, at least in part, by attenuating I/R-induced calpain activation.     

Adenosine A1 receptor activation reduces myocardial reperfusion effects on intrinsic cardiac nervous system

The intrinsic cardiac nervous system is the final common integrator of regional cardiac function. The ischemic myocardium modifies this nervous system. We sought to determine the role that intrinsic cardiac neuronal P(1) purinergic receptors play in transducing myocardial ischemia and the subsequent reperfusion. The activity generated by ventricular neurons was recorded concomitant with cardiac hemodynamic variables in 44 anesthetized pigs. Regional ventricular ischemia was induced by briefly occluding (30 s) the ventral interventricular coronary artery distal to the arterial blood supply of identified ventricular neurons. Adenosine (100 microM) was administered to these neurons via their local arterial blood supply during or immediately after transient coronary artery occlusion. Occlusion was also performed following local administration of adenosine A(1) [8-cyclopentyl-1,3-dipropylxanthine (DPCPX)] or A(2) [3,7-dimethyl-1-propargylxanthine (DMPX)] receptor blocking agents. The activity generated by ventricular neurons was modified by transient coronary artery occlusion and the subsequent reperfusion (||Delta|| 112 +/- 14 and 168 +/- 34 impulses/min, respectively; P < 0.01 vs. preischemic states). Locally administered adenosine attenuated neuronal responses to reperfusion (-75%; P < 0.01 compared with normal reperfusion) but not ischemia. The neuronal stabilizing effects that adenosine elicited during reperfusion persisted in the presence of DMPX but not DPCPX. It is concluded that activation of neuronal adenosine A(1) receptors stabilizes the intrinsic cardiac nervous system during reperfusion.     

Role and mechanism of PKC in ischemic preconditioning of pig skeletal muscle against infarction

Protein kinase C (PKC) inhibitors, chelerythrine (Chel, 0.6 mg) and polymyxin B (Poly B, 1.0 mg), and PKC activators, phorbol 12-myristate 13-acetate (PMA, 0.05 mg) and 1-oleoyl-2-acetyl glycerol (OAG, 0.1 mg), were used as probes to investigate the role of PKC in mediation of ischemic preconditioning (IPC) of noncontracting pig latissimus dorsi (LD) muscles against infarction in vivo. These drugs were delivered to each LD muscle flap (8 x 12 cm) by 10 min of local intra-arterial infusion. It was observed that LD muscle flaps sustained 43 +/- 5% infarction when subjected to 4 h of global ischemia and 24 h of reperfusion. IPC with three cycles of 10 min ischemia-reperfusion reduced muscle infarction to 25 +/- 3% (P < 0.05). This anti-infarction effect of IPC was blocked by Chel (42 +/- 7%) and Poly B (37 +/- 2%) and mimicked by PMA (19 +/- 10%) and OAG (14 +/- 5%) treatments (P < 0.05), given 10 min before 4 h of ischemia. In addition, the ATP-sensitive K(+) (K(ATP)) channel antagonist sodium 5-hydroxydecanoate attenuated (P < 0.05) the anti-infarction effect of IPC (37 +/- 2%), PMA (44 +/- 17%), and OAG (46 +/- 9%). IPC, OAG, and Chel treatment alone did not affect mean arterial blood pressure or muscle blood flow assessed by 15-microm radioactive microspheres. Western blot analysis of muscle biopsies obtained before (baseline) and after IPC demonstrated seven cytosol-associated isoforms, with nPKCepsilon alone demonstrating progressive cytosol-to-membrane translocation within 10 min after the final ischemia period of IPC. Using differential fractionation, it was observed that nPKCepsilon translocated to a membrane compartment other than the sarcolemma and/or sarcoplasmic reticulum. Furthermore, IPC and preischemic OAG but not postischemic OAG treatment reduced (P < 0.05) muscle myeloperoxidase activity compared with time-matched ischemic controls during 16 h of reperfusion after 4 h of ischemia. Taken together, these observations indicate that PKC plays a central role in the anti-infarction effect of IPC in pig LD muscles, most likely through a PKC-K(ATP) channel-linked signal-transduction pathway.