Ischemia-reperfusion leads to depletion of glutathione content and augmentation of malondialdehyde production in the rat heart from overproduction of oxidants: Can caffeic acid phenethyl ester (CAPE) protect the heart?

During restoration of blood flow of the ischemic heart induced by coronary occlusion, free radicals cause lipid peroxidation with myocardial injury. Lipid peroxidation end-products, such as malondialdehyde (MDA), have been used to assess oxygen free radical-mediated injury of the ischemic-reperfused (I/R) myocardium in rats. This experimental study assessed the preventive effect of caffeic acid phenthyl ester (CAPE), antioxidant, on I/R-induced lipid peroxidation in the rat heart. We are also interested in the role of CAPE on glutathione (GSH) levels, an antioxidant whose levels are influenced by oxidative stress. I/R leads to the depletion of GSH which is the major intracellular nonprotein sulphydryl and plays an important role in the maintenance of cellular proteins and lipid in their functional state and acts primarily to protect these important structures against the threat of oxidation. In addition, we also examined morphologic changes in the heart by using light microscopy. The left coronary artery was occluded for 30 min and then reperfused for 120 min more before the experiment was terminated. CAPE (50 M kg^–1) was administered 10 min prior to ischemia and during occlusion by infusion. At the end of the reperfusion period, rats were sacrificed, and the heart was quickly removed for biochemical determination and histopathological analysis. I/R was accompanied by a significant increase in MDA production and decrease in GSH content in the rat heart. Administration of CAPE reduced MDA production and prevented depletion of GSH content. These beneficial changes in these biochemical parameters were also associated with parallel changes in histopathological appearance. These findings imply that I/R plays a causal role in heart injury due to overproduction of oxygen radicals or insufficient antioxidant and CAPE exert cardioprotective effects probably by the radical scavenging and antioxidant activities.     

Soluble epoxide hydrolase inhibition and gene deletion are protective against myocardial ischemia-reperfusion injury in vivo

Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids. EETs are formed from arachidonic acid during myocardial ischemia and play a protective role against ischemic cell death. Deletion of sEH has been shown to be protective against myocardial ischemia in the isolated heart preparation. We tested the hypothesis that sEH inactivation by targeted gene deletion or pharmacological inhibition reduces infarct size (I) after regional myocardial ischemia-reperfusion injury in vivo. Male C57BL\6J wild-type or sEH knockout mice were subjected to 40 min of left coronary artery (LCA) occlusion and 2 h of reperfusion. Wild-type mice were injected intraperitoneally with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), a sEH inhibitor, 30 min before LCA occlusion or during ischemia 10 min before reperfusion. 14,15-EET, the main substrate for sEH, was administered intravenously 15 min before LCA occlusion or during ischemia 5 min before reperfusion. The EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE) was given intravenously 15 min before reperfusion. Area at risk (AAR) and I were assessed using fluorescent microspheres and triphenyltetrazolium chloride, and I was expressed as I/AAR. I was significantly reduced in animals treated with AUDA-BE or 14,15-EET, independent of the time of administration. The cardioprotective effect of AUDA-BE was abolished by the EET antagonist 14,15-EEZE. Immunohistochemistry revealed abundant sEH protein expression in left ventricular tissue. Strategies to increase 14,15-EET, including sEH inactivation, may represent a novel therapeutic approach for cardioprotection against myocardial ischemia-reperfusion injury.     

Nicorandil induces late preconditioning against myocardial infarction in conscious rabbits

Nicorandil has been shown to induce an infarct-limiting effect similar to that induced by the early phase of ischemic preconditioning (PC). The goals of this study were to determine whether nicorandil induces a delayed cardioprotection that is analogous to the late phase of ischemic PC and, if so, whether nicorandil-induced late PC is associated with upregulation of cardioprotective proteins. Chronically instrumented, conscious rabbits received vehicle (intravenous normal saline; control group, n = 10), nicorandil (100 microg/kg bolus + 30 microg x kg(-1) x min(-1) i.v. for 60 min; nicorandil group, n = 10), or ischemic PC (6 cycles of 4-min coronary occlusion/4-min reperfusion; PC group, n = 8). Twenty-four hours later, rabbits underwent a 30-min coronary occlusion, followed by 3 days of reperfusion. Myocardial infarct size was significantly reduced in rabbits pretreated with nicorandil (27.5 +/- 5.3% of the risk region) or with ischemia (30.3 +/- 4.2%) versus controls (59.1 +/- 4.7%, P < 0.05 vs. both). Furthermore, the expression of cyclooxygenase-2 (COX-2) and Bcl-2 was significantly elevated (+38% and +126%, respectively; P < 0.05) in myocardium of rabbits given nicorandil 24 h earlier versus controls. We conclude that nicorandil induces delayed cardioprotection against myocardial infarction similar to that afforded by the late phase of ischemic PC, possibly by upregulating COX-2 and Bcl-2.     

Platelet-activating factor induces cardioprotection in isolated rat heart akin to ischemic preconditioning: role of phosphoinositide 3-kinase and protein kinase C activation

Ischemic preconditioning (IP) is a cardioprotective mechanism against myocellular death and cardiac dysfunction resulting from reperfusion of the ischemic heart. At present, the precise list of mediators involved in IP and the pathways of their mechanisms of action are not completely known. The aim of the present study was to investigate the role of platelet-activating factor (PAF), a phospholipid mediator that is known to be released by the ischemic-reperfused heart, as a possible endogenous agent involved in IP. Experiments were performed on Langendorff-perfused rat hearts undergoing 30 min of ischemia followed by 2 h of reperfusion. Treatment with a low concentration of PAF (2 x 10(-11) M) before ischemia reduced the extension of infarct size and improved the recovery of left ventricular developed pressure during reperfusion. The cardioprotective effect of PAF was comparable to that observed in hearts in which IP was induced by three brief (3 min) periods of ischemia separated by 5-min reperfusion intervals. The PAF receptor antagonist WEB-2170 (1 x 10(-9) M) abrogated the cardioprotective effect induced by both PAF and IP. The protein kinase C (PKC) inhibitor chelerythrine (5 x 10(-6) M) or the phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 (5 x 10(-5) M) also reduced the cardioprotective effect of PAF. Western blot analysis revealed that following IP treatment or PAF infusion, the phosphorylation of PKC-epsilon and Akt (the downstream target of PI3K) was higher than that in control hearts. The present data indicate that exogenous applications of low quantities of PAF induce a cardioprotective effect through PI3K and PKC activation, similar to that afforded by IP. Moreover, the study suggests that endogenous release of PAF, induced by brief periods of ischemia and reperfusion, may participate to the triggering of the IP of the heart.     

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.     

Extent of damage in ischemic,nonreperfused, and reperfused myocardium of anesthetized rats

To investigate the localization of the earliest damage in ischemic and ischemic-reperfused myocardium, anesthetized rats were subjected to coronary occlusion for 15, 30, 45, or 90 min. One-half of the animals in each group had no reperfusion, whereas the other half was reperfused for 14 min. With the use of histological methods, preferentially in the periphery of the area at risk, localized zones were detected that lacked the hypoxia-specific increase in NADH fluorescence. The extent of these areas displaying injured tissue was found to be significantly smaller in the ischemic-nonreperfused hearts than in the ischemic-reperfused organs (15-min ischemia: 0.22 +/- 0.12% vs. 43.0 +/- 5.0%; 30-min ischemia: 5.7 +/- 2.7% vs. 64.6 +/- 2.9%; 45-min ischemia: 5.6 +/- 1.2% vs. 66.0 +/- 7.5%; 90-min ischemia: 39.3 +/- 5.5% vs. 86.7 +/- 1.8% of the area at risk). The results point to a localized initiation of the damage close to the surrounding oxygen-supplied tissue during ischemia and an expansion of this injury by intercellular actions into yet-intact areas upon reperfusion.     

Local Arginase Inhibition during Early Reperfusion Mediates Cardioprotection via Increased Nitric Oxide Production

Consumption of L-arginine contributes to reduced bioavailability of nitric oxide (NO) that is critical for the development of ischemia-reperfusion injury. The aim of the study was to determine myocardial arginase expression and activity in ischemic-reperfusion myocardium and whether local inhibition of arginase within the ischemic myocardium results in increased NO production and protection against myocardial ischemia-reperfusion. Anesthetized pigs were subjected to coronary artery occlusion for 40 min followed by 4 h reperfusion. The pigs were randomized to intracoronary infusion of vehicle (n = 7), the arginase inhibitor N-hydroxy-nor-L-arginine (nor-NOHA, 2 mg/min, n = 7), the combination of nor-NOHA and the NO synthase inhibitor N^G-monomethyl-L-arginine (L-NMMA, 0.35 mg/min, n = 6) into the jeopardized myocardial area or systemic intravenous infusion of nor-NOHA (2 mg/min, n = 5) at the end of ischemia and start of reperfusion. The infarct size of the vehicle group was 80±4% of the area at risk. Intracoronary nor-NOHA reduced infarct size to 46±5% (P<0.01). Co-administration of L-NMMA abrogated the cardioprotective effect mediated by nor-NOHA (infarct size 72±6%). Intravenous nor-NOHA did not reduce infarct size. Arginase I and II were expressed in cardiomyocytes, endothelial, smooth muscle and poylmorphonuclear cells. There was no difference in cytosolic arginase I or mitochondrial arginase II expression between ischemic-reperfused and non-ischemic myocardium. Arginase activity increased 2-fold in the ischemic-reperfused myocardium in comparison with non-ischemic myocardium. In conclusion, ischemia-reperfusion increases arginase activity without affecting cytosolic arginase I or mitochondrial arginase II expression. Local arginase inhibition during early reperfusion reduces infarct size via a mechanism that is dependent on increased bioavailability of NO.     

Leukocyte and endothelial cell adhesion molecules in a chronic murine model of myocardial reperfusion injury

Expression of endothelial and leukocyte cell adhesion molecules is a principal determinant of polymorphonuclear neutrophil (PMN) recruitment during inflammation. It has been demonstrated that pharmacological inhibition of these molecules can attenuate PMN influx and subsequent tissue injury. We determined the temporal expression of alpha-granule membrane protein-40 (P-selectin), endothelial leukocyte adhesion molecule 1 (E-selectin), and intercellular cell adhesion molecule 1 (ICAM-1) after coronary artery occlusion and up to 3 days of reperfusion. The expression of all of these cell adhesion molecules peaked around 24 h of reperfusion. We determined the extent to which these molecules contribute to PMN infiltration by utilizing mice deficient (-/-) in P-selectin, E-selectin, ICAM-1, and CD18. Each group underwent 30 min of in vivo, regional, left anterior descending (LAD) coronary artery ischemia and 24 h of reperfusion. PMN accumulation in the ischemic-reperfused (I/R) zone was assessed using histological techniques. Deficiencies of P-selectin, E-selectin, ICAM-1, or CD18 resulted in significant (P < 0.05) attenuation of PMN infiltration into the I/R myocardium (MI/R). In addition, P-selectin, E-selectin, ICAM-1, and CD18 -/- mice exhibited significantly (P < 0.05) smaller areas of necrosis after MI/R compared with wild-type mice. These data demonstrate that MI/R induces coronary vascular expression of P-selectin, E-selectin, and ICAM-1 in mice. Furthermore, genetic deficiency of P-selectin, E-selectin, ICAM-1, or CD18 attenuates PMN sequestration and myocardial injury after in vivo MI/R. We conclude that P-selectin, E-selectin, ICAM-1, and CD18 are involved in the pathogenesis of MI/R injury in mice.     

Effects of adenosine and acadesine on interstitial nucleosides and myocardial stunning in the pig.

5-Amino-4-imidazolecarboxamide riboside (AICAr) or acadesine has been proposed to exert cardioprotection by enhancing adenosine production in ischemic myocardium. However, there are conflicting reports on acadesine's effects in ischemic myocardium and few studies in which myocardial adenosine levels have been measured. The purpose of this study was to determine whether acadesine increases interstitial fluid adenosine levels and attenuates myocardial stunning or potentiates the effects of adenosine in the intact pig. In pentobarbital-anesthetized pigs, myocardial stunning was induced by 10 min left anterior descending coronary artery occlusion and 90 min reperfusion. Regional ventricular function was assessed by measuring systolic wall thickening, and interstitial nucleosides were estimated by cardiac microdialysis. Control hearts were compared with hearts treated with acadesine, adenosine, and adenosine plus acadesine. Adenosine pretreatment (100 microg x kg(-1) x min(-1), intracoronary) immediately prior to ischemia increased interstitial adenosine levels 9-fold and improved postischemic functional recovery from a control value of 17.6 +/- 4.1% to 43.6 +/- 3.4% of preischemic systolic wall thickening. In contrast, acadesine (20 mg/kg i.v. bolus 10 min prior to ischemia + 0.5 mg x kg (-1) x min(-1), i.v. infusion through 60 min reperfusion) had no effect on interstitial fluid adenosine levels or the recovery of regional function (21.5 +/- 5.9% recovery), nor were the functional effects of adenosine potentiated by acadesine. These findings indicate that acadesine does not enhance myocardial adenosine levels, attenuate myocardial stunning, or potentiate the cardioprotective effects of adenosine in the pig.     

Effect of ibuprofen and indomethacin on the O2 supply/consumption balance in ischemic rabbit myocardium

The purpose of this study was to determine if differences in the cardioprotective abilities of ibuprofen and indomethacin were due to their differing abilities to alter the O2 supply/consumption ratio in the ischemic myocardium. Experiments were done on 21 anesthetized open-chest rabbits. Regional flow (using radioactive microspheres), O2 extraction, O2 consumption, and O2 supply/consumption ratio were determined 1 hr after occlusion of the left anterior descending coronary artery in controls and animals given iv 10 mg/kg ibuprofen or iv 3 mg/kg indomethacin. Myocardial blood flow was depressed in the occluded region compared to the nonoccluded region after occlusion for all treatments. O2 extraction in the occluded region was elevated compared to the nonoccluded region for all treatments after occlusion. No differences in O2 consumption were noted between any treatment within the occluded or nonoccluded regions. The O2 supply/consumption ratio was lower in the occluded region compared to the nonoccluded region for all treatments. No differences in this ratio were noted between any treatment. Thus, the effects of indomethacin or ibuprofen on ischemia are not related to acute changes in myocardial O2 supply/consumption balance.     

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.     

Remote renal preconditioning-induced cardioprotection: a key role of hypoxia inducible factor-prolyl 4-hydroxylases

Remote preconditioning is a unique phenomenon in which brief episodes of ischemia and reperfusion to remote organ protect the target organ against sustained ischemia–reperfusion (I/R)-induced injury. Protective effects of remote renal preconditioning (RRPC) are well established in heart, but their mechanisms still remain to be elucidated. So, the present study was designed to investigate the possible role of oxygen-sensing hypoxia inducible factor-prolyl 4-hydroxylases (HIF-P4Hs) in RRPC-induced cardioprotection in rats. Remote renal preconditioning was performed by four episodes of 5 min renal artery occlusion and reperfusion. Isolated rat hearts were perfused on Langendorff apparatus and were subjected to global ischemia for 30 min followed by 120 min reperfusion. The levels of lactate dehydrogenase (LDH) and creatine kinase (CK) were measured in coronary effluent to assess the degree of myocardial injury. Extent of myocardial infarct size and coronary flow rate was also measured. Ethyl 3,4-dihydroxybenzoate (EDHB) and α-ketoglutarate (α-KG) were employed as HIF-P4Hs inhibitor and activator, respectively. Diethyldithiocarbamic acid (DDCA) was employed as NFkB inhibitor. Remote renal preconditioning prevented I/R-induced myocardial injury and produced cardioprotective effects. Pharmacological preconditioning with EDHB (100 mg kg⁻¹ i.p.) mimicked the cardioprotective effects of RRPC. However, α-KG (200 mg kg⁻¹ i.p.) and DDCA (150 mg kg⁻¹ i.p.) abolished cardioprotective effects of RRPC and EDHB. So, it may be concluded that inhibition of HIF-P4H has a key role in RRPC-induced cardioprotection. Further, remote preconditioning-induced HIF-P4H inhibition may have triggered a transduction pathway involving activation of NFkB.     

N-oleoyldopamine, a novel endogenous capsaicin-like lipid, protects the heart against ischemia-reperfusion injury via activation of TRPV1

N-oleoyldopamine (OLDA), a bioactive lipid originally found in the mammalian brain, is an endovanilloid that selectively activates the transient receptor potential vanilloid type 1 (TRPV1) channel. This study tests the hypothesis that OLDA protects the heart against ischemia and reperfusion (I/R) injury via activation of the TRPV1 in wild-type (WT) but not in gene-targeted TRPV1-null mutant (TRPV1(-/-)) mice. Hearts of WT or TRPV1(-/-) mice were Langendorffly perfused with OLDA (2 x 10(-9) M) in the presence or absence of CGRP8-37 (1 x 10(-6) M), a selective calcitonin gene-related peptide (CGRP) receptor antagonist; RP-67580 (1 x 10(-6) M), a selective neurokinin-1 receptor antagonist; chelerythrine (5 x 10(-6) M), a selective protein kinase C (PKC) antagonist; or tetrabutylammonium (TBA, 5 x 10(-4) M), a nonselective K(+) channel antagonist, followed by 35 min of global ischemia and 40 min of reperfusion (I/R). Left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), coronary flow (CF), and left ventricular peak positive dP/dt (+dP/dt) were evaluated after I/R. OLDA improved recovery of cardiac function after I/R in WT but not TRPV1(-/-) hearts by increasing LVDP, CF, and +dP/dt and by decreasing LVEDP. CGRP8-37, RP-67580, chelerythrine, or TBA abolished the protective effect of OLDA in WT hearts. Radioimmunoassay showed that the release of substance P (SP) and CGRP after OLDA treatment was higher in WT than in TRPV1(-/-) hearts, which was blocked by chelerythrine or TBA. Thus OLDA exerts a cardiac protective effect during I/R injury in WT hearts via CGRP and SP release, which is abolished by PKC or K(+) channel antagonists. The protective effect of OLDA is void in TRPV1(-/-) hearts, supporting the notion that TRPV1 mediates OLDA-induced protection against cardiac I/R injury.     

Ceramide-induced preconditioning involves reactive oxygen species

INTRODUCTION: Ceramide induces programmed cell death and it is thought to contribute to cardiac ischemia/reperfusion (I/R) injury. In contrast, we have demonstrated that administration of low doses of ceramide engenders cardiac preconditioning (PC). Ceramide is known to generate reactive oxygen species (ROS) in cells. Since mechanisms triggering the ceramide-induced cardioprotection remain unknown, we investigated the role of ROS in the genesis of this protective mechanism. METHODS: Using an isolated Langendorff-perfused rat heart model, four groups (n > or = 6 in each group) were considered: Control hearts underwent 30 min index regional ischemia and 120 min of reperfusion. In the ceramide group, hearts were preconditioned with c2-ceramide 1 microM for 7 min followed by 10 min washout prior to the I/R insult. In additional groups, MPG (1 mM), a synthetic antioxidant was given for 15 min alone or bracketing the ceramide perfusion. In each group, infarct size was determined at the end of the reperfusion period and superoxide dismutases (CuZnSOD and MnSOD) and catalase activities were evaluated. RESULTS: Ceramide preconditioning reduced the infarct/area at risk (I/AAR) ratio (8.3 +/- 1.1% for ceramide vs. 36.4 +/- 1.2% for control, p < 0.001). Perfusion with MPG abolished the preconditioning effect of ceramide (I/AAR ratio = 36.7 +/- 4.9%). Ceramide was also associated with a 29% and 38% increase in catalase and CuZnSOD activities, respectively, compared with control group. CONCLUSION: Production of reactive oxygen species following ceramide preconditioning of the ischemic-reperfused heart appears to play a role in the cardioprotective effect of ceramide.     

Effect of modulating cardiac A1 adenosine receptor expression on protection with ischemic preconditioning

Activation of A(1) adenosine receptors (A(1)ARs) may be a crucial step in protection against myocardial ischemia-reperfusion (I/R) injury; however, the use of pharmacological A(1)AR antagonists to inhibit myocardial protection has yielded inconclusive results. In the current study, we have used mice with genetically modified A(1)AR expression to define the role of A(1)AR in intrinsic protection and ischemic preconditioning (IPC) against I/R injury. Normal wild-type (WT) mice, knockout mice with deleted (A(1)KO(-/-)) or single-copy (A(1)KO(+/-)) A(1)AR, and transgenic mice (A(1)TG) with increased cardiac A(1)AR expression underwent 45 min of left anterior descending coronary artery occlusion, followed by 60 min of reperfusion. Subsets of each group were preconditioned with short durations of ischemia (3 cycles of 5 min of occlusion and 5 min of reperfusion) before index ischemia. Infarct size (IF) in WT, A(1)KO(+/-), and A(1)KO(-/-) mice was (in % of risk region) 58 +/- 3, 60 +/- 4, and 61 +/- 2, respectively, and was less in A(1)TG mice (39 +/- 4, P < 0.05). A strong correlation was observed between A(1)AR expression level and response to IPC. IF was significantly reduced by IPC in WT mice (35 +/- 3, P < 0.05 vs. WT), A(1)KO(+/-) + IPC (48 +/- 4, P < 0.05 vs. A(1)KO(+/-)), and A(1)TG + IPC mice (24 +/- 2, P < 0.05 vs. A(1)TG). However, IPC did not decrease IF in A(1)KO(-/-) + IPC mice (63 +/- 2). In addition, A(1)KO(-/-) hearts subjected to global I/R injury demonstrated diminished recovery of developed pressure and diastolic function compared with WT controls. These findings demonstrate that A(1)ARs are critical for protection from myocardial I/R injury and that cardioprotection with IPC is relative to the level of A(1)AR gene expression.     

Protein kinase C is involved in cardioprotective effects of ischemic preconditioning on infarct size and ventricular arrhythmia in rats in vivo

Protein kinase C (PKC) has been known to play an important role in ischemic preconditioning (IP). This study was designed to examine whether the translocation of PKC is associated with the cardioprotective effects of IP in vivo on infarct size and ventricular arrhythmias in a rat model.Using anesthetized rats, heart rate, systolic blood pressure, infarct size and ventricular arrhythmias during 45 min of coronary occlusion were measured. PKC activity was assayed in both the cytosolic and cell membrane fraction . Brief 3-min periods of ischemia followed by 10 min of reperfusion were used to precondition the myocardium. Calphostin C was used to inhibit PKC.Infarct size was significantly reduced by IP (68.1 (2.5)%, mean (S.E.) vs. 45.2 (3.4)%, p < 0.01). The reduction in infarct size by IP was abolished by pretreatment with calphostin C. The total number of ventricular premature complex (VPC) during 45 min of coronary occlusion was reduced by IP (1474 (169) beats/45 min vs. 256 (82) beats/45 min, p < 0.05). The reduction the total number of VPC induced by IP was abolished by the administration of calphostin C before the episode of brief ischemia. The same tendency was observed in the duration of ventricular tachycardia and the incidence of ventricular fibrillation. PKC activity in the cell membrane fraction transiently increased immediately after IP (100 vs. 142%, p < 0.01) and returned to baseline 15 min after IP. Pretreatment with calphostin C prevented the translocation of PKC.The translocation of PKC plays an important role in the cardioprotective effect of IP on infarct size and ventricular arrhythmias in anesthetized rats.     

Na(+)/H(+) exchange inhibition prevents endothelial dysfunction after I/R injury

Whereas inhibition of the Na(+)/H(+) exchanger (NHE) has been demonstrated to reduce myocardial infarct size in response to ischemia-reperfusion injury, the ability of NHE inhibition to preserve endothelial cell function has not been examined. This study examined whether NHE inhibition could preserve endothelial cell function after 90 min of regional ischemia and 180 min of reperfusion and compared this inhibition with ischemic preconditioning (IPC). In a canine model either IPC, produced by one 5-min coronary artery occlusion (1 x 5'), or the specific NHE-1 inhibitor eniporide (EMD-96785, 3.0 mg/kg) was administered 15 min before a 90-min coronary artery occlusion followed by 3 h of reperfusion. Infarct size (IS) was determined by 2,3,5-triphenyl tetrazolium chloride staining and expressed as a percentage of the area-at-risk (IS/AAR). Endothelial cell function was assessed by measurement of coronary blood flow in response to intracoronary acetylcholine infusion at the end of reperfusion. Whereas neither control nor IPC-treated animals exhibited a significant reduction in IS/AAR or preservation of endothelial cell function, animals treated with the NHE inhibitor eniporide showed a marked reduction in IS/AAR and a significantly preserved endothelial cell function (P < 0.05). Thus NHE-1 inhibition is more efficacious than IPC at reducing IS/AAR and at preserving endothelial cell function in dogs.     

Effect of ischemia and reperfusion without airway occlusion on vascular barrier function in the in vivo mouse lung.

Ischemia-reperfusion (I/R) lung injury causes increased vascular permeability and edema. We developed an in vivo murine model of I/R allowing measurement of pulmonary vascular barrier function without airway occlusion. The left pulmonary artery (PA) was occluded with an exteriorized, slipknotted suture in anesthetized C57BL/6J mice. The effect of ischemic time was determined by subjecting mice to 5, 10, or 30 min of left lung ischemia followed by 150 min of reperfusion. The effect of reperfusion time was determined by subjecting mice to 30 min of left lung ischemia followed by 30 or 150 min of reperfusion. Changes in pulmonary vascular barrier function were measured with the Evans blue dye (EBD) technique, dual-isotope radiolabeled albumin (RA), bronchoalveolar lavage (BAL) protein concentration, and wet weight-to-dry weight ratio (WW/DW). Increasing left lung ischemia with constant reperfusion time or increasing left lung reperfusion time after constant ischemic time resulted in significant increases in left lung EBD content at all times compared with both right lung values and sham surgery mice. The effects of left lung ischemia on lung EBD were corroborated by RA but the effects of increasing reperfusion time differed, suggesting binding of EBD to lung tissue. An increase in WW/DW was only detected after 30 min of reperfusion, suggesting edema clearance. BAL protein concentrations were unaffected. We conclude that short periods of I/R, without airway occlusion, increase pulmonary vascular permeability in the in vivo mouse, providing a useful model to study molecular mechanisms of I/R lung injury.