Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs

Transient episodes of ischemic preconditioning (PC) render myocardium protected against subsequent lethal injury after ischemia and reperfusion. Recent studies indicate that application of short, repetitive ischemia only during the onset of reperfusion after the lethal ischemic event may obtain equivalent protection. We assessed whether such ischemic postconditioning (Postcon) is cardioprotective in pigs by limiting lethal injury. Pentobarbital sodium-anesthetized, open-chest pigs underwent 30 min of complete occlusion of the left anterior descending coronary artery and 3-h reflow. PC was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. Postcon was elicited by three cycles of 30-s reperfusion, followed by 30-s reocclusion, after the 30-min occlusion period and before the 3-h reflow. Infarct size (%area-at-risk using triphenyltetrazolium chloride macrochemistry; means +/- SE) after 30 min of ischemia was 26.5 +/- 5.2% (n = 7 hearts/treatment group). PC markedly limited myocardial infarct size (2.8 +/- 1.2%, n = 7 hearts/treatment group, P < 0.05 vs. controls). However, Postcon had no effect on infarct size (37.8 +/- 5.1%, n = 7 hearts/treatment group). Within the subendocardium, Postcon increased phosphorylation of Akt (74 +/- 12%) and ERK1/2 (56 +/- 10%) compared with control hearts subjected only to 30-min occlusion and 15-min reperfusion (P < or = 0.05), and these changes were not different from the response triggered by PC (n = 5 hearts/treatment group). Phosphorylation of downstream p70S6K was also equivalent in PC and Postcon groups. These data do not support the hypothesis that application of 30-s cycles of repetitive ischemia during reperfusion exerts a protective effect on pig hearts subjected to lethal ischemia, but this is not due to a failure to phosphorylate ERK and Akt during early reperfusion.     

Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43

Connexin 43 (Cx43) is localized at left ventricular (LV) gap junctions and in cardiomyocyte mitochondria. A genetically induced reduction of Cx43 as well as blockade of mitochondrial Cx43 import abolishes the infarct size (IS) reduction by ischemic preconditioning (IP). With progressing age, Cx43 content in ventricular and atrial tissue homogenates is reduced. We now investigated whether or not 1) the mitochondrial Cx43 content is reduced in aged mice hearts and 2) IS reduction by IP is lost in aged mice hearts in vivo. Confirming previous results, sarcolemmal Cx43 content was reduced in aged (>13 mo) compared with young (<3 mo) C57Bl/6 mice hearts, whereas the expression levels of protein kinase C epsilon and endothelial nitric oxide synthase remained unchanged. Also in mitochondria isolated from aged mice LV myocardium, Western blot analysis indicated a 40% decrease in Cx43 content compared with mitochondria isolated from young mice hearts. In young mice hearts, IP by one cycle of 10 min ischemia and 10 min reperfusion reduced IS (% of area at risk) following 30 min regional ischemia and 120 min reperfusion from 67.7 +/- 3.3 (n = 17) to 34.2 +/- 6.6 (n = 5, P < 0.05). In contrast, IP's cardioprotection was lost in aged mice hearts, since IS in nonpreconditioned (57.5 +/- 4.0, n = 10) and preconditioned hearts (65.4 +/- 6.3, n = 8, P = not significant) was not different. In conclusion, mitochondrial Cx43 content is decreased in aged mouse hearts. The reduced levels of Cx43 may contribute to the age-related loss of cardioprotection by IP.     

The delta-opioid receptor agonist DADLE at reperfusion protects the heart through activation of pro-survival kinases via EGF receptor transactivation

The specific delta-opioid receptor agonist [D-Ala(2)-D-Leu(5)]enkephalin (DADLE) protects against infarction in the heart when given before ischemia. In rabbit, this protection leads to phosphorylation of the pro-survival kinases Akt and extracellular signal-regulated kinase (ERK) and is dependent on transactivation of the epidermal growth factor receptor (EGFR). DADLE reportedly protects rat hearts at reperfusion. We therefore tested whether DADLE at reperfusion could protect isolated rabbit hearts subjected to 30 min of regional ischemia and 120 min of reperfusion and whether this protection is dependent on Akt, ERK, and EGFR. DADLE (40 nM) was infused for 1 h starting 5 min before reperfusion and reduced infarct size from 31.0 +/- 2.3% in the control group to 14.6 +/- 1.6% (P = 0.01). This protection was abolished by cotreatment of the metalloproteinase inhibitor (MPI) and the EGFR inhibitor AG1478. In contrast, 20 nM DADLE, although known to be protective before ischemia, failed to protect. Western blotting revealed that DADLE's protection was correlated to increase in phosphorylation of the kinases Akt and ERK1 and -2 in reperfused hearts (2.5 +/- 0.5, 1.6 +/- 0.2, and 2.3 +/- 0.7-fold of baseline levels, P < 0.05 vs. control). The DADLE-dependent increases in Akt and ERK1/2 phosphorylation were abolished by either MPI or AG1478, confirming a signaling through the EGFR pathway. Additionally, DADLE treatment increased phosphorylation of EGFR (1.4 +/- 0.2-fold, P = 0.03 vs. control). Thus the delta-opioid agonist DADLE protects rabbit hearts at reperfusion through activation of the pro-survival kinases Akt and ERK and is dependent on the transactivation of the EGFR.     

Cardioprotection mediated by sphingosine-1-phosphate and ganglioside GM-1 in wild-type and PKC epsilon knockout mouse hearts

Sphingosine-1-phosphate (S1P) protects neonatal rat cardiac myocytes from hypoxic damage through unknown signaling pathways. We tested the hypothesis that S1P-induced cardioprotection requires activation by the epsilon-isoform of protein kinase C (PKC epsilon) by subjecting hearts isolated from PKC epsilon knockout mice and wild-type mice to 20 min of global ischemia and 30 min of reperfusion. Pretreatment with a 2-min infusion of 10 nM S1P improved recovery of left ventricular developed pressure (LVDP) in both wild-type and PKC epsilon knockout hearts and reduced the rise in LV end-diastolic pressure (LVEDP) and creatine kinase (CK) release. Pretreatment for 2 min with 10 nM of the ganglioside GM-1 also improved recovery of LVDP and suppressed CK release in wild-type hearts but not in PKC epsilon knockout hearts. Importantly, GM-1 but not S1P, increased the proportion of PKC epsilon localized to particulate fractions. Our results suggest that GM-1, which enhances endogenous S1P production, reduces cardiac injury through PKC epsilon-dependent intracellular pathways. In contrast, extracellular S1P induces equivalent cardioprotection through PKC epsilon-independent signaling pathways.     

胰岛素保护缺血/再灌注心脏:PI3-K/Akt和JNKs信号通路间的交互作用

我们前期研究表明胰岛素可激活细胞内信号转导机制如磷脂酰肌醇3．激酶．蛋白激酶B．内皮型一氧化氮合酶．一氧化氮（P13-K-Akt-eNOS-NO）信号通路,减轻心肌缺血／再灌注（ischemia／reperfusion,I／R）损伤,改善缺血后心肌功能恢复。然而c-Jun氨基末端激酶（c-JunNH2-terminal kinase,JNK）信号通路在胰岛素保护I／R心肌中的作用尚不清楚,本研究旨在探讨JNK信号通路在胰岛素保护I／R心肌中的作用及其与P13．K／Akt信号通路间的相互关系。离体Sprague-Dawley大鼠心脏缺血30min后施行2h或4h的再灌注,缺血前用LY294002（15mmol／L）和SP600125（10mmol／L）灌注15min,分别阻断P13．K／Akt和磷酸化JNK（phosphorylated．JNK,p-JNK）活化,观测心脏功能、心肌梗死、细胞凋亡和蛋白磷酸化水平。与对照组相比,胰岛素再灌注2h后,心率、左心室发展压和左心室收缩／舒张最大速率均明显增加,梗死面积减少约16．1％[（28．9±2．0）％vs（45．0±4．0）％,n=6,P〈O．01],细胞凋亡指数从（27．6±113）％减少到（16．0±0．7）％（n=6,P〈O．01）,Akt的活性增加1．7倍（n=6,P〈0．05）,同时JNK活性增加1．5倍铆=6,P〈O．05）。用LY294002处理后,胰岛素对I／R心肌的保护作用消失;而用SP600125处理可增强胰岛素的保护作用,且可部分逆转LY294002的抑制作用。进一步观察发现SP600125减弱了Akt的磷酸化m=6,P〈0．05）。上述结果表明,在I／R心肌中,胰岛素可同时激活P13．K／Akt及JNK信号通路,且通过后者进一步增加Akt活化,从而减轻I／R损伤,改善心肌功能。这种P13．K／Akt与JNK信号通路交互机制对胰岛素保护I／R心肌有重要意义。     

Ischemic preconditioning protects by activating prosurvival kinases at reperfusion

Pharmacological activation of the prosurvival kinases Akt and ERK-1/2 at reperfusion, after a period of lethal ischemia, protects the heart against ischemia-reperfusion injury. We hypothesized that ischemic preconditioning (IPC) protects the heart by phosphorylating the prosurvival kinases Akt and ERK-1/2 at reperfusion. In isolated perfused Sprague-Dawley rat hearts subjected to 35 min of lethal ischemia, the phosphorylation states of Akt, ERK-1/2, and p70 S6 kinase (p70S6K) were determined after 15 min of reperfusion, and infarct size was measured after 120 min of reperfusion. IPC induced a biphasic response in Akt and ERK-1/2 phosphorylation during the preconditioning and reperfusion phases after the period of lethal ischemia. IPC induced a fourfold increase in Akt, ERK-1/2, and p70S6K phosphorylation at reperfusion and reduced the infarct risk-to-volume ratio (56.9 +/- 5.7 and 20.9 +/- 3.6% for control and IPC, respectively, P < 0.01). Inhibiting the IPC-induced phosphorylation of Akt, ERK-1/2, and p70S6K at reperfusion with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 or the MEK-1/2 inhibitor PD-98059 abrogated IPC-induced protection (46.3 +/- 5.8, 49.2 +/- 4.0, and 20.9 +/- 3.6% for IPC + LY-294002, IPC + PD-98059, and IPC, respectively, P < 0.01), demonstrating that the phosphorylation of these kinases at reperfusion is required for IPC-induced protection. In conclusion, we demonstrate that the reperfusion phase following sustained ischemia plays an essential role in mediating IPC-induced protection. Specifically, we demonstrate that IPC protects the heart by phosphorylating the prosurvival kinases Akt and ERK-1/2 at reperfusion.     

Inhibition of p38 MAPK and AMPK restores adenosine-induced cardioprotection in hearts stressed by antecedent ischemia by altering glucose utilization

p38 mitogen-activated protein kinase (MAPK) and 5'-AMP-activated protein kinase (AMPK) are activated by metabolic stresses and are implicated in the regulation of glucose utilization and ischemia-reperfusion (IR) injury. This study tested the hypothesis that inhibition of p38 MAPK restores the cardioprotective effects of adenosine in stressed hearts by preventing activation of AMPK and the uncoupling of glycolysis from glucose oxidation. Working rat hearts were perfused with Krebs solution (1.2 mM palmitate, 11 mM [(3)H/(14)C]glucose, and 100 mU/l insulin). Hearts were stressed by transient antecedent IR (2 x 10 min I/5 min R) before severe IR (30 min I/30 min R). Hearts were treated with vehicle, p38 MAPK inhibitor (SB-202190, 10 microM), adenosine (500 microM), or their combination before severe IR. After severe IR, the phosphorylation (arbitrary density units) of p38 MAPK and AMPK, rates of glucose metabolism (micromol x g dry wt(-1) x min(-1)), and recovery of left ventricular (LV) work (Joules) were similar in vehicle-, SB-202190- and adenosine-treated hearts. Treatment with SB-202190 + adenosine versus adenosine alone decreased p38 MAPK (0.03 +/- 0.01, n = 3 vs. 0.48 +/- 0.10, n = 3, P < 0.05) and AMPK (0.00 +/- 0.00, n = 3 vs. 0.26 +/- 0.08, n = 3 P < 0.05) phosphorylation. This was accompanied by attenuated rates of glycolysis (1.51 +/- 0.40, n = 7 vs. 3.95 +/- 0.65, n = 7, P < 0.05) and H(+) production (2.12 +/- 0.76, n = 7 vs. 6.96 +/- 1.48, n = 7, P < 0.05), and increased glycogen synthesis (1.91 +/- 0.25, n = 6 vs. 0.27 +/- 0.28, n = 6, P < 0.05) and improved recovery of LV work (0.81 +/- 0.08, n = 7 vs. 0.30 +/- 0.15, n = 8, P < 0.05). These data indicate that inhibition of p38 MAPK abolishes subsequent phosphorylation of AMPK and improves the coupling of glucose metabolism, thereby restoring adenosine-induced cardioprotection.     

Low-pressure reperfusion alters mitochondrial permeability transition

We hypothesized that low-pressure reperfusion may limit myocardial necrosis and attenuate postischemic contractile dysfunction by inhibiting mitochondrial permeability transition pore (mPTP) opening. Male Wistar rat hearts (n = 36) were perfused according to the Langendorff technique, exposed to 40 min of ischemia, and assigned to one of the following groups: 1) reperfusion with normal pressure (NP = 100 cmH(2)O) or 2) reperfusion with low pressure (LP = 70 cmH(2)O). Creatine kinase release and tetraphenyltetrazolium chloride staining were used to evaluate infarct size. Modifications of cardiac function were assessed by changes in coronary flow, heart rate (HR), left ventricular developed pressure (LVDP), the first derivate of the pressure curve (dP/dt), and the rate-pressure product (RPP = LVDP x HR). Mitochondria were isolated from the reperfused myocardium, and the Ca(2+)-induced mPTP opening was measured using a potentiometric approach. Lipid peroxidation was assessed by measuring malondialdehyde production. Infarct size was significantly reduced in the LP group, averaging 17 +/- 3 vs. 33 +/- 3% of the left ventricular weight in NP hearts. At the end of reperfusion, functional recovery was significantly improved in LP hearts, with RPP averaging 10,392 +/- 876 vs. 3,969 +/- 534 mmHg/min in NP hearts (P < 0.001). The Ca(2+) load required to induce mPTP opening averaged 232 +/- 10 and 128 +/- 16 microM in LP and NP hearts, respectively (P < 0.001). Myocardial malondialdehyde was significantly lower in LP than in NP hearts (P < 0.05). These results suggest that the protection afforded by low-pressure reperfusion involves an inhibition of the opening of the mPTP, possibly via reduction of reactive oxygen species production.     

Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning

Ischemic preconditioning (Pre-con) is an adaptive response triggered by a brief ischemia applied before a prolonged coronary occlusion. We tested the hypothesis that repetitive ischemia applied during early reperfusion, i.e., postconditioning (Post-con), is cardio-protective by attenuating reperfusion injury. In anesthetized open-chest dogs, the left anterior descending artery (LAD) was occluded for 60 min and reperfused for 3 h. In controls (n = 10), there was no intervention. In Pre-con (n = 9), the LAD was occluded for 5 min and reperfused for 10 min before the prolonged occlusion. In Post-con (n = 10), at the start of reperfusion, three cycles of 30-s reperfusion and 30-s LAD reocclusion preceded the 3 h of reperfusion. Infarct size was significantly less in the Pre-con (15 +/- 2%, P < 0.05) and Post-con (14 +/- 2%, P < 0.05) groups compared with controls (25 +/- 3%). Tissue edema (% water content) in the area at risk was comparably reduced in Pre-con (78.3 +/- 1.2, P < 0.05) and Post-con (79.7 +/- 0.6, P < 0.05) versus controls (81.5 +/- 0.4). Polymorphonuclear neutrophil (PMN) accumulation (myeloperoxidase activity, Deltaabsorbance.min-1.g tissue-1) in the area at risk myocardium was comparably reduced in Post-con (10.8 +/- 5.5, P < 0.05) and Pre-con (13.4 +/- 3.4, P < 0.05) versus controls (47.4 +/- 15.3). Basal endothelial function measured by PMN adherence to postischemic LAD endothelium (PMNs/mm2) was comparably attenuated by Post-con and Pre-con (15 +/- 0.6 and 12 +/- 0.6, P < 0.05) versus controls (37 +/- 1.5), consistent with reduced expression of P-selectin on coronary vascular endothelium in Post-con and Pre-con. Endothelial function assessed by the maximal vasodilator response of postischemic LAD to acetylcholine was significantly greater in Post-con (104 +/- 6%, P < 0.05) and Pre-con (109 +/- 5%, P < 0.05) versus controls (71 +/- 8%). Plasma malondialdehyde (microM/ml), a product of lipid peroxidation, was significantly less at 1 h of reperfusion in Post-con (2.2 +/- 0.2, P < 0.05) versus controls (3.2 +/- 0.3) associated with a decrease in superoxide levels revealed by dihydroethidium staining in the myocardial area at risk. These data suggest that Post-con is as effective as Pre-con in reducing infarct size and preserving endothelial function. Post-con may be clinically applicable in coronary interventions, coronary artery bypass surgery, organ transplantation, and peripheral revascularization where reperfusion injury is expressed.     

Ischemia induced activation of heat shock protein 27 kinases and casein kinase 2 in the preconditioned rabbit heart.

Protein kinase C (PKC), p38 MAP kinase, and mitogen-activated protein kinase-activated kinases 2 and 3 (MAPKAPK2 and MAPKAPK3) have been implicated in ischemic preconditioning (PC) of the heart to reduce damage following a myocardial infarct. This study examined whether extracellular signal-regulated kinase (Erk) 1, p70 ribosomal S6 kinase (p70 S6K), casein kinase 2 (CK2), and other hsp27 kinases are also activated by PC, and if they are required for protection in rabbit hearts. CK2 and hsp27 kinase activities declined during global ischemia in control hearts, whereas PC with 5 min ischemia and 10 min reperfusion increased their activities during global ischemia. Resource Q chromatography resolved two distinct peaks of hsp27 phosphotransferase activities; the first peak (at 0.36 M NaCl) appeared to correspond to the 55-kDa MAPKAPK2. Erk1 activity was elevated in both control and PC hearts after post-ischemic reperfusion, but no change was observed in p70 S6K activity. Infarct size (measured by triphenyltetrazolium staining) in isolated rabbit hearts subjected to 30 min regional ischemia and 2 h reperfusion was 31.0+/-2.6% of the risk zone in controls and was 10.3+/-2.2% in PC hearts (p<0.001). Neither the CK2 inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) nor the Mek1/2 inhibitor PD98059 infused during ischemia blocked protection by PC. The activation of CK2 and Erk1 in ischemic preconditioned hearts appear to be epiphenomena and not required for the reduction of infarction from myocardial ischemia.     

Protection against ventricular arrhythmias and cardiac death using adenosine and lidocaine during regional ischemia in the in vivo rat

Despite decades of research, there are few effective ways to treat ventricular fibrillation (VF), ventricular tachycardia (VT), or cardiac ischemia that show a significant survival benefit. Our aim was to investigate the combined therapeutic effect of two common antiarrhythmic compounds, adenosine and lidocaine (AL), on mortality, arrhythmia frequency and duration, and infarct size in the rat model of regional ischemia. Sprague-Dawley rats (n = 49) were anesthetized with pentobarbital sodium (60 mg.ml(-1).kg(-1) i.p.) and instrumented for regional coronary occlusion (30 min) and reperfusion (120 min). Heart rate, blood pressure, and a lead II electrocardiogram were recorded. Intravenous pretreatment began 5 min before ischemia and extended throughout ischemia, terminating at the start of reperfusion. After 120 min, hearts were removed for infarct size measurement. Mortality occurred in 58% of saline controls (n = 12), 50% of adenosine only (305 microg.kg(-1).min(-1), n = 8), 0% in lidocaine only (608 microg.kg(-1).min(-1), n = 8), and 0% in AL at any dose (152, 305, or 407 microg.kg(-1).min(-1) adenosine plus 608 microg.kg(-1).min(-1) lidocaine, n = 7, 8, and 6). VT occurred in 100% of saline controls (18 +/- 9 episodes), 50% of adenosine-only (11 +/- 7 episodes), 83% of lidocaine-only (23 +/- 11 episodes), 60% of low-dose AL (2 +/- 1 episodes, P < 0.05), 57% of mid-dose AL (2 +/- 1 episodes, P < 0.05), and 67% of high-dose AL rats (6 +/- 3 episodes). VF occurred in 75% of saline controls (4 +/- 3 episodes), 100% of adenosine-only-treated rats (3 +/- 2 episodes), and 33% lidocaine-only-treated rats (2 +/- 1 episodes) of the rats tested. There was no deaths and no VF in the low- and mid-dose AL-treated rats during ischemia, and only one high-dose AL-treated rat experienced VF (25.5 sec). Infarct size was lower in all AL-treated rats but only reached significance with the mid-dose treatment (saline controls 61 +/- 5% vs. 38 +/- 6%, P < 0.05). We conclude that a constant infusion of a solution containing AL virtually abolished severe arrhythmias and prevented cardiac death in an in vivo rat model of acute myocardial ischemia and reperfusion. AL combinational therapy may provide a primary prevention therapeutic window in ischemic and nonischemic regions of the heart.     

Acetaminophen in the post-ischemia reperfused myocardium

Acetaminophen was administered acutely at the onset of reperfusion after 20 min of low-flow, global myocardial ischemia in isolated, perfused guinea pig hearts (Langendorff) to evaluate its influence in the postischemia, reperfused myocardium. Similarly prepared hearts were treated with vehicle or with uric acid (another phenol for comparison). Functionally, acetaminophen-treated hearts (0.35 mM) achieved significantly greater recovery during reperfusion. For example, left ventricular developed pressures at 40 min reperfusion were 38 +/- 3, 27 +/- 3, and 20 +/- 2 in the presence of acetaminophen (P < 0.05, relative to the other two groups), vehicle, and uric acid, respectively. Coronary perfusion pressures and calculated coronary vascular resistances, in the acetaminophen-treated hearts, were significantly lower at the same time (e.g., coronary perfusion pressures in the three groups, respectively, were 40 +/- 2 [P < 0.05], 51 +/- 3, and 65 +/- 12 mm Hg). Under baseline, control conditions, creatine kinase ranged from 12-15 units/liter in the three groups. It increased to 35-40 units/liter (P < 0.05) during ischemia but was significantly reduced by acetaminophen during reperfusion (e.g., 5.3 +/- 0.8 units/liter at 40 min). Oxidant-mediated chemiluminescence in all three treatment groups during baseline conditions and ischemia was similar (i.e., approximately 1.5-2.0 min for peak luminescence to reach its half maximal value). It took significantly more time during reperfusion for the oxidation of luminol in the presence of acetaminophen (>20 min, P < 0.05) than in its absence (3-8 min in uric acid- and vehicle-treated hearts). These results suggest that administration of acetaminophen (0.35 mM), at the onset of reperfusion, provides anti-oxidant-mediated cardioprotection in the postischemia, reperfused myocardium.     

PR-39, a potent neutrophil inhibitor, attenuates myocardial ischemia-reperfusion injury in mice

We investigated the effects of PR-39, a recently discovered neutrophil inhibitor, in a murine model of myocardial ischemia-reperfusion injury. Mice were given an intravenous injection of vehicle (n = 12) or PR-39 (n = 9) and subjected to 30 min of coronary artery occlusion followed by 24 h of reperfusion. In addition, the effects of PR-39 on leukocyte rolling and adhesion were studied utilizing intravital microscopy of the rat mesentery. The area-at-risk per left ventricle was similar in vehicle- and PR-39-treated mice. However, myocardial infarct per risk area was significantly (P < 0.01) reduced in PR-39 treated hearts (21.0 +/- 3.8%) compared with vehicle (47.1 +/- 4.8%). Histological analysis of ischemic reperfused myocardium demonstrated a significant (P < 0.01) reduction in polymorphonuclear neutrophil (PMN) accumulation in PR-39-treated hearts (n = 6, 34.3 +/- 1.7 PMN/mm(2)) compared with vehicle-treated myocardium (n = 6, 59.7 +/- 3.1 PMN/mm(2)). In addition, PR-39 significantly (P < 0.05) attenuated leukocyte rolling and adherence in rat inflamed mesentery. These results indicate that PR-39 inhibits leukocyte recruitment into inflamed tissue and attenuated myocardial reperfusion injury in a murine model of myocardial ischemia-reperfusion.     

Apelin reduces myocardial reperfusion injury independently of PI3K/Akt and P70S6 kinase

Apelin, the endogenous ligand of the G protein-coupled APJ receptor, is a peptide mediator with emerging regulatory actions in the heart. The aim of the present studies was to explore potential roles of the apelin/APJ system in myocardial ischaemia/reperfusion injury. To determine the cardiac expression of apelin/APJ and potential regulation by acute ischaemic insult, Langendorff perfused rat hearts were subjected to regional ischaemia (left coronary artery occlusion, 35 min) or ischaemia followed by reperfusion (30 min). Apelin and APJ mRNA expression were then determined in ventricular myocardium by rt-PCR. Unlike APJ mRNA expression, which remained unchanged, apelin mRNA was upregulated 2.4 fold in ventricular myocardium from isolated rat hearts undergoing ischaemia alone, but returned back to control levels after 30 min reperfusion. We then proceeded to test the hypothesis that treatment with exogenous apelin is protective against ischaemia/reperfusion injury. Perfused hearts were subjected to 35 min left main coronary artery occlusion and 120 min reperfusion, after which infarct size was determined by tetrazolium staining. Exogenous Pyr(1)-apelin-13 (10(-8 )M) was perfused either from 5 min prior to 15 min after coronary occlusion, or from 5 min prior to 15 min after reperfusion. Whilst ineffective when used during ischaemia alone, apelin administered during reperfusion significantly reduced infarct size (47.6+/-2.6% of ischaemic risk zone compared to 62.6+/-2.8% in control, n=10 each, p<0.05) in hearts subject to temporary coronary occlusion followed by reperfusion. This protective effect was not abolished by co-administration of the PI3K inhibitor wortmannin (10(-7 )M, infarct size 49.8+/-4.1%, n=4) or the P70S6 kinase inhibitor rapamycin (10(-9 )M, 41.8+/-8.8%, n=4). In conclusion these results suggest that apelin may be a new and potentially important cardioprotective autacoid, upregulated rapidly after myocardial ischaemia and acting through an unknown pathway.     

The novel adipocytokine visfatin exerts direct cardioprotective effects

Visfatin is an adipocytokine capable of mimicking the glucose-lowering effects of insulin and activating the pro-survival kinases phosphatidylinositol-3-OH kinase (PI3K)-protein kinase B (Akt) and mitogen-activated protein kinase kinase 1 and 2 (MEK1/2)-extracellular signal-regulated kinase 1 and 2 (Erk 1/2). Experimental studies have demonstrated that the activation of these kinases confers cardioprotection through the inhibition of the mitochondrial permeability transition pore (mPTP). Whether visfatin is capable of exerting direct cardioprotective effects through these mechanisms is unknown and is the subject of the current study. Anaesthetized C57BL/6 male mice were subjected to in situ 30 min. of regional myocardial ischaemia and 120 min. of reperfusion. The administration of an intravenous bolus of visfatin (5 x 10(-6) micromol) at the time of myocardial reperfusion reduced the myocardial infarct size from 46.1+/-4.1% in control hearts to 27.3+/-4.0% (n>or= 6/group, P<0.05), an effect that was blocked by the PI3K inhibitor, wortmannin, and the MEK1/2 inhibitor, U0126 (48.8+/-5.5% and 45.9+/-8.4%, respectively, versus 27.3+/-4.0% with visfatin; n>or= 6/group, P<0.05). In murine ventricular cardiomyocytes subjected to 30 min. of hypoxia followed by 30 min. of reoxygenation, visfatin (100 ng/ml), administered at the time of reoxygenation, reduced the cell death from 65.2+/-4.6% in control to 49.2+/-3.7%(n>200 cells/group, P<0.05), an effect that was abrogated by wortmannin and U0126 (68.1+/-5.2% and 59.7+/-6.2%, respectively; n>200 cells/group, P>0.05). Finally, the treatment of murine ventricular cardiomyocytes with visfatin (100 ng/ml) delayed the opening of the mPTP induced by oxidative stress from 81.2+/-4 sec. in control to 120+/-7 sec. (n>20 cells/group, P<0.05) in a PI3K- and MEK1/2-dependent manner. We report that the adipocytokine, visfatin, is capable of reducing myocardial injury when administered at the time of myocardial reperfusion in both the in situ murine heart and the isolated murine cardiomyocytes. The mechanism appears to involve the PI3K and MEK1/2 pathways and the mPTP.     

Controlled reperfusion after hypothermic heart preservation inhibits mitochondrial permeability transition-pore opening and enhances functional recovery

We investigated whether low-pressure reperfusion may attenuate postischemic contractile dysfunction, limits necrosis and apoptosis after a prolonged hypothermic ischemia, and inhibits mitochondrial permeability transition-pore (MPTP) opening. Isolated rats hearts (n = 72) were exposed to 8 h of cold ischemia and assigned to the following groups: 1) reperfusion with low pressure (LP = 70 cmH(2)O) and 2) reperfusion with normal pressure (NP = 100 cmH(2)O). Cardiac function was assessed during reperfusion using the Langendorff model. Mitochondria were isolated, and the Ca(2+) resistance capacity (CRC) of the MPTP was determined. Malondialdehyde (MDA) production, caspase-3 activity, and cytochrome c were also assessed. We found that functional recovery was significantly improved in LP hearts with rate-pressure product averaging 30,380 +/- 1,757 vs. 18,000 +/- 1,599 mmHg/min in NP hearts (P < 0.01). Necrosis, measured by triphenyltetrazolium chloride staining and creatine kinase leakage, was significantly reduced in LP hearts (P < 0.01). The CRC was increased in LP heart mitochondria (P < 0.01). Caspase-3 activity, cytochrome c release, and MDA production were reduced in LP hearts (P < 0.001 and P < 0.01). This study demonstrated that low-pressure reperfusion after hypothermic heart ischemia improves postischemic contractile dysfunction and attenuates necrosis and apoptosis. This protection could be related to an inhibition of mitochondrial permeability transition.     

Chronically administered acetaminophen and the ischemia/reperfused myocardium

Male and female Hartley strain guinea pigs weighing 280 +/- 10 g were given acetaminophen-treated water ad libitum for 10 days. Sham-treated control animals were given similar quantities of untreated tap water (vehicle-treated control group). On Day 10, hearts were extracted, instrumented, and exposed to an ischemia (low-flow, 20 min)/reperfusion protocol. Our objective was to compare and contrast ventricular function, coronary circulation, and selected biochemical and histological indices in the two treatment groups. Left ventricular developed pressure in the early minutes of reperfusion was significantly greater in the presence of acetaminophen, e.g., at 1 min, 40 +/- 4 vs 21 +/- 3 mmHg (P < 0.05). Coronary perfusion pressure was significantly less from 3 to 40 min of reperfusion in the presence of acetaminophen. Creatine kinase release in vehicle-treated hearts rose from 42 +/- 14 (baseline) to 78 +/- 25 units/liter by the end of ischemia. Corresponding values in acetaminophen-treated hearts were 36 +/- 8 and 44 +/- 14 units/liter. Acetaminophen significantly (P < 0.05) attenuated release of creatine kinase. Chemiluminescence, an indicator of the in vitro production of peroxynitrite via the in vivo release of superoxide and nitric oxide, was also significantly attenuated by acetaminophen. Electron microscopy indicated a well-preserved myofibrillar ultrastructure in the postischemic myocardium of acetaminophen-treated hearts relative to vehicle-treated hearts (e.g., few signs of contraction bands, little or no evidence of swollen mitochondria, and well-defined light and dark bands in sarcomeres with acetaminophen; opposite with vehicle). We conclude that chronic administration of acetaminophen provides cardioprotection to the postischemic, reperfused rodent myocardium.     

Myocardial function defined by strain rate and strain during alterations in inotropic states and heart rate

For porcine myocardium, ultrasonic regional deformation parameters, systolic strain (epsilon(sys)) and peak systolic strain rate (SR(sys)), were compared with stroke volume (SV) and contractility [contractility index (CI)] measured as the ratio of end-systolic strain to end-systolic wall stress. Heart rate (HR) and contractility were varied by atrial pacing (AP = 120-180 beats/min, n = 7), incremental dobutamine infusion (DI = 2.5-20 microg. kg(-1). min(-1), n = 7), or continuous esmolol infusion (0.5 mg. kg(-1). min(-1)) + subsequent pacing (120-180 beats/min) (EI group, n = 6). Baseline SR(sys) and epsilon(sys) averaged 5.0 +/- 0.4 s(-1) and 60 +/- 4%. SR(sys) and CI increased linearly with DI (20 microg. kg(-1). min(-1); SR(sys) = 9.9 +/- 0.7 s(-1), P < 0.0001) and decreased with EI (SR(sys) = 3.4 +/- 0.1 s(-1), P < 0.01). During pacing, SR(sys) and CI remained unchanged in the AP and EI groups. During DI, epsilon(sys) and SV initially increased (5 microg. kg(-1). min(-1); epsilon(sys) = 77 +/- 6%, P < 0.01) and then progressively returned to baseline. During EI, SV and epsilon(sys) decreased (epsilon(sys) = 38 +/- 2%, P < 0.001). Pacing also decreased SV and epsilon(sys) in the AP (180 beats/min; epsilon(sys) = 36 +/- 2%, P < 0.001) and EI groups (180 beats/min; epsilon(sys) = 25 +/- 3%, P < 0.001). Thus, for normal myocardium, SR(sys) reflects regional contractile function (being relatively independent of HR), whereas epsilon(sys) reflects changes in SV.     

Protective role of protein kinase C epsilon activation in ischemia-reperfusion arrhythmia

PURPOSE: Ischemic heart disease carries an increased risk of malignant ventricular tachycardia (VT), fibrillation (VF), and sudden cardiac death. Protein kinase C (PKC) epsilon activation has been shown to improve the hemodynamics in hearts subjected to ischemia/reperfusion. However, very little is known about the role of epsilon PKC in reperfusion arrhythmias. Here we show that epsilon PKC activation is anti-arrhythmic and its inhibition is pro-arrhythmic. METHOD: Langendorff-perfused isolated hearts from epsilonPKC agonist (epsilonPKC activation), antagonist (epsilonPKC inhibition) transgenic (TG), and wild-type control mice were subjected to 30 min stabilization period, 10 min global ischemia, and 30 min reperfusion. Action potentials (APs) and calcium transients (CaiT) were recorded simultaneously at 37 degrees C using optical mapping techniques. The incidence of VT and VF was assessed during reperfusion. RESULTS: No VT/VF was seen in any group during the stabilization period in which hearts were perfused with Tyrode's solution. Upon reperfusion, 3 out of the 16 (19%) wild-type mice developed VT but no VF. In epsilonPKC antagonist group, in which epsilonPKC activity was downregulated, 10 out of 13 (76.9%) TG mice developed VT, of which six (46.2%) degenerated into sustained VF upon reperfusion. Interestingly, in epsilonPKC agonist mice, in which the activity of epsilonPKC was upregulated, no VF was observed and only 1 out of 12 mice showed only transient VT during reperfusion. During ischemia and reperfusion, CaiT decay was exceedingly slower in the antagonist mice compared to the other two groups. CONCLUSION: Moderate in vivo activation of epsilonPKC exerts beneficial antiarrhythmic effect vis-a-vis the lethal reperfusion arrhythmias. Abnormal CaiT decay may, in part, contribute to the high incidence of reperfusion arrhythmias in the antagonist mice. These findings have important implications for the development of PKC isozyme targeted therapeutics and subsequently for the treatment of ischemic heart diseases.