Efficacy of a growth hormone-releasing peptide mimetic in cardiac ischemia/reperfusion injury.

The cardioprotective efficacy of the pyrazolinone-piperidine dipeptide growth hormone secretagogue (GHS) CP-424,391 was studied in an in vivo rabbit model of ischemia and reperfusion. CP-424,391 was administered at 25 mg/kg p.o. x 7 days. Ischemia was induced by left coronary artery occlusion for 30 min, after which the heart was reperfused for 2 h. At the end of reperfusion, animals were euthanized and the infarct size was determined. The area at risk of infarct was not different between the control (45.8+/-3.7%, n=6) and CP-424,391-treated groups (36.9+/-4.3%, n=11). The infarct size of the control animals was 49.5+/-7.1% and was significantly (P<0.05) lower in the CP-424,391-treated group (infarct size=17.3+/-3.0). There was a trend, albeit not significant, for the left ventricular function to recover to a greater extent in CP-424,391-treated rabbits. Thus, the treatment of rabbits for 7 days with CP-424,391 was cardioprotective against ischemia/reperfusion injury.

In vivo myocardial infarct size reduction by a caspase inhibitor administered after the onset of ischemia.

The aim of this study was to determine the effect of different administration protocols on the cardioprotective efficacy of the non-selective, irreversible caspase inhibitors N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) and bocaspartyl-(OMe)-fluoromethylketone (BocD.fmk) in a rat in vivo ischemia and reperfusion paradigm. Hearts were made ischemic for 45 min and reperfused for 180 min. Under these conditions, it was determined that zVAD.fmk was cardioprotective when administered before or after the onset of ischemia, whereas BocD.fmk was efficacious only when administered before the onset of ischemia. This is the first report of in vivo cardioprotection by a caspase inhibitor when administered after the onset of ischemia.

In vivo models of myocardial ischemia and reperfusion injury: application to drug discovery and evaluation.

This review discusses the pharmacology of regional myocardial ischemia and reperfusion (I/R) injury and the utilization of in vivo animal models in the preclinical development of novel therapeutic compounds. The manuscript aims to provide an overview of a number of different cardioprotective strategies that have been successful from a preclinical perspective and to also present where possible results of clinical trials of the respective compounds. Myocardial ischemia reperfusion injury may be manifested as myocardial stunning, ventricular arrhythmias, coronary vascular dysfunction, or the development of a myocardial infarct. This review is principally concerned with preclinical studies related to reduction of infarct size. The pathophysiology of the reperfusion injury process is complex, including primarily cellular and humoral components of inflammation, as well as myocellular ionic and metabolic disturbances. This review will discuss strategies directed at oxygen-derived free radicals, neutrophils, adenosine, and the sodium-hydrogen exchanger (NHE). The results of preclinical cardioprotective studies are influenced by the paradigm used therefore methodological considerations will also be presented where appropriate.

Differential effect of a selective cyclooxygenase-2 inhibitor versus indomethacin on renal blood flow in conscious volume-depleted dogs.

Renal effects of a selective cyclooxygenase-2 (COX-2) inhibitor [MF-Tricyclic; 3-(3,4-difluorophenyl)-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone] were studied in control and volume-depleted conscious dogs. MF-Tricyclic was compared with the nonselective COX-1/COX-2 inhibitor indomethacin. Six instrumented male dogs were randomly selected to receive MF-Tricyclic or indomethacin at 10 mg/kg. Volume depletion was effected by a sodium-restricted diet (14 days) with administration of furosemide (7.5 mg/kg, i.v.) the day before the experiment. Indomethacin ablated systemic COX-1 activity (p < 0.05), whereas MF-Tricyclic did not affect this activity. Each compound achieved plasma concentrations in excess of their respective median inhibitory concentrations (IC50 values) against canine COX-2. In controls, neither compound affected mean arterial pressure (MAP), heart rate (HR), renal blood flow (RBF), fractional excretion (FE) Na+, or FE K+. In volume-depleted dogs, indomethacin reduced RBF (p < 0.05), whereas MF-Tricyclic did not affect this parameter. Indices of renal function in volume-depleted dogs were not affected. These data are consistent with the view that the effects of indomethacin on RBF are a consequence of inhibition of COX-1 activity. Furthermore, in these studies, short-term administration of a selective COX-2 inhibitor was without deleterious effects on renal function.

Effect of ramiprilat or captopril on myocardial infarct size: assessment in canine models of ischemia alone and ischemia with reperfusion.

Cardioprotective effects of the angiotensin-converting enzyme inhibitors captopril and ramiprilat were studied in two in vivo canine models of myocardial ischemic injury: 90 min of regional ischemia with 18 h reperfusion (protocol I) and 6 h of continuous ischemia without reperfusion (protocol II). In protocol I, neither ramiprilat (50 micrograms/kg) nor captopril (5 mg/kg + 0.25 mg/kg/h) reduced infarct size after 18 h of reperfusion (vs. controls). In protocol II, drugs were administered directly into both left anterior descending coronary artery and left circumflex branch. Compared to controls, continuous intracoronary administration of ramiprilat (40 ng/kg/min) or captopril (400 ng/kg/min) did not reduce infarct size. Thus neither captopril nor ramiprilat protected the heart from injury under conditions of ischemia with reperfusion or ischemia without reperfusion.

Co-localization of the cysteine protease caspase-3 with apoptotic myocytes after in vivo myocardial ischemia and reperfusion in the rat.

The aim of our study was to characterize the temporal relationship of apoptosis to regional myocardial ischemia and reperfusion and we aimed to determine the effect of ischemia and reperfusion on the distribution of the pro-apoptotic cysteine protease caspase-3 (CPP 32, apopain, Yama) in an in vivo rat model. Male Sprague-Dawley rats (250-400 g) were anesthetized with sodium pentobarbital (65 mg/kg, i.p.), the left external carotid artery was isolated to monitor arterial pressure and a left thoracotomy was performed. Regional myocardial ischemia was induced by occluding the left main coronary artery for 45 min. The heart was reperfused for 0, 60, 120 or 180 min. TUNEL staining of formalin-fixed, paraffin-embedded left ventricle, and DNA fragmentation analysis, showed that apoptosis occurred during 45 min of ischemia alone, but further developed during the 3-h reperfusion period. Immunohistochemical analysis of ischemic/reperfused left ventricle showed caspase-3 levels were substantially elevated and localized in the ischemic/reperfused region, and that caspase-3 co-localized to TUNEL positive myocytes. Therefore, regional myocardial ischemia serves as a stimulus for myocyte apoptosis, and this form of cell death progresses time-dependently after the onset of reperfusion. Our studies implicate caspase-3 to be involved in apoptotic cell death in ischemic/reperfused rat heart.

Cell death attenuation by 'Usurpin', a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD-95 (Fas, APO-1) receptor complex.

Apoptotic cell suicide initiated by ligation of CD95 (Fas/APO-1) occurs through recruitment, oligomerization and autocatalytic activation of the cysteine protease, caspase-8 (MACH, FLICE, Mch5). An endogenous mammalian regulator of this process, named Usurpin, has been identified (aliases for Usurpin include CASH, Casper, CLARP, FLAME-1, FLIP, I-FLICE and MRIT). This protein is ubiquitously expressed and exists as at least three isoforms arising by alternative mRNA splicing. The Usurpin gene is comprised of 13 exons and is clustered within approximately 200 Kb with the caspase-8 and -10 genes on human chromosome 2q33-34. The Usurpin polypeptide has features in common with pro-caspase-8 and -10, including tandem 'death effector domains' on the N-terminus of a large subunit/small subunit caspase-like domain, but it lacks key residues that are necessary for caspase proteolytic activity, including the His and Cys which form the catalytic substrates diad, and residues that stabilize the P1 aspartic acid in substrates. Retro-mutation of these residues to functional caspase counterparts failed to restore proteolytic activity, indicating that other determinants also ensure the absence of catalytic potential. Usurpin heterodimerized with pro-caspase-8 in vitro and precluded pro-caspase-8 recruitment by the FADD/MORT1 adapter protein. Cell death induced by CD95 (Fas/APO-1) ligation was attenuated in cells transfected with Usurpin. In vivo, a Usurpin deficit was found in cardiac infarcts where TUNEL-positive myocytes and active caspase-3 expression were prominent following ischemia/reperfusion injury. In contrast, abundant Usurpin expression (and a caspase-3 deficit) occurred in surrounding unaffected cardiac tissue, suggesting reciprocal regulation of these pro- and anti-apoptotic molecules in vivo. Usurpin thus appears to be an endogenous modulator of apoptosis sensitivity in mammalian cells, including the susceptibility of cardiac myocytes to apoptotic death following ischemia/ reperfusion injury.

Methods for studying experimental myocardial ischemic and reperfusion injury.

This review describes methodologies used to study experimental myocardial ischemic and reperfusion injury. Myocardial reperfusion injury may be manifest as myocardial stunning, ventricular arrhythmias, coronary vascular dysfunction, or the extension of the area of myocyte necrosis beyond that due to the ischemic insult alone. This review discusses methodology pertaining to the latter form of reperfusion injury. The pathophysiology of the reperfusion injury process is complex, including primarily cellular and humoral components of inflammation, as well as myocellular ionic and metabolic disturbances. Since the extent of injury may be influenced by methodological considerations this review aims to discuss the principle means of characterizing reperfusion injury in the experimental setting. The methods discussed are principally those related to in vivo research. Where appropriate, advantages, disadvantages, or alternate methods will be presented. Lastly, as understanding of the pathophysiology of reperfusion injury increases, newer techniques utilizing murine models, the study of apoptotic cell death, and the role of gender may be used more frequently and are thus briefly reviewed.

Heat stress protects the perfused rabbit heart from complement-mediated injury.

We determined if heat stress induction of heat shock protein (HSP) 70 modulates complement activation in an experimental model of xenograft rejection. Male New Zealand White rabbits were heat stressed (core body temperature to 42 degrees C for 15 min; n = 9). Control rabbits (n = 13) were not exposed to heat stress. Hearts were removed 18 h later and perfused by the Langendorff method. After equilibration, human plasma (source of human complement) was added to the perfusion medium. Hemodynamic variables recorded during perfusion with human plasma were improved in hearts from heat-stressed animals compared with control hearts. Assembly of the soluble membrane attack complex was reduced in the interstitial fluid effluent from the heat-stressed hearts. Electron microscopic evidence of ultrastructural changes was attenuated in the hearts from heat-stressed rabbits. Myocardial tissue from heat-stressed animals exhibited an increase in inducible HSP 70 that was virtually absent in the hearts of control rabbits. Previous whole body hyperthermia protects the rabbit heart against the detrimental effects of heterologous plasma, suggesting that heat-stress induction of HSP 70 limits the extent of complement activation by a discordant vascularized tissue (xenograft). Induction of heat stress proteins by the donor organ might be an important mechanism affecting the outcome of xenograft transplants.

5-hydroxydecanoate fails to attenuate ventricular fibrillation in a conscious canine model of sudden cardiac death.

The electrophysiologic and antifibrillatory properties of 5-hydroxydecanoate, a KATP channel antagonist, were studied in a conscious canine model of sudden cardiac death. After a surgically induced myocardial infarction, animals were subjected to programmed electrical stimulation to identify those at risk for sudden cardiac death. 5-Hydroxydecanoate was administered as a bolus (10 mg/kg i.v.) followed by an infusion, 10 mg/kg/h (group 1, n = 12) or 30 mg/kg bolus followed by an infusion, 30 mg/kg/h (group 2, n = 8) i.v., while vehicle treated animals received a 0.9% sodium chloride solution (group 3, n = 11). The administration of 5-hydroxydecanoate did not alter the ventricular effective refractory period or the QTc interval. Anterior wall myocardial infarcts, expressed as a percentage of the left ventricle, did not differ among groups. Infusions of 5-hydroxydecanoate did not confer significant protection from sudden cardiac death (death within 60 min of posterolateral ischemia) due to ventricular fibrillation: group 1, 50%; group 2, 38%; and group 3, 18%. The data demonstrate that a continuous infusion of 5-hydroxydecanoate (10 and 30 mg/kg/h, i.v.) does not provide protection from ischemia-induced ventricular fibrillation in the postinfarcted conscious canine.

Cardioprotective effects of heparin or N-acetylheparin in an in vivo model of myocardial ischaemic and reperfusion injury.

OBJECTIVE: The aim was to determine if either heparin or N-acetylheparin could reduce the extent of myocardial injury resulting from 90 min of coronary artery occlusion and 6 h of reperfusion in the anaesthetised dog. METHODS: Heparin or N-acetylheparin was given in three repeated intravenous doses of 2 mg.kg-1. Drug or vehicle (0.9% saline) was given 75 min after onset of ischaemia and 90 and 180 min after reperfusion. To ensure an equal degree of myocardial ischaemia induced by left circumflex coronary artery occlusion among the three groups of animals studied, only animals with ischaemic zone blood flow of < or = 0.16 ml.min-1.g-1 were included in the final analysis. RESULTS: Ischaemic zone blood flow was 0.068(SEM 0.0016) ml.min-1.g-1 in control animals (n = 13), 0.083(0.017) ml.min-1.g-1 in heparin treated animals (n = 10), and 0.094(0.010) ml.min-1.g-1 in N-acetylheparin treated animals (n = 10). Baseline haemodynamic variables did not differ among the three groups studied. Heparin treatment alone significantly increased bleeding time and activated partial thromboplastin time. Electrocardiographic ST segment elevation, an indicator of regional ischaemia at the onset of coronary occlusion, was not different among control, heparin, or N-acetylheparin groups. The area of the left ventricle at risk of infarct was 39.8(1.5)%, 38.6(0.7)%, and 37.3(2.0)% in control, heparin, and N-acetylheparin treated groups, respectively. Myocardial infarct size, as a percentage of area at risk, was 43.0(3.7)%, 30.7(3.9)%, and 24.5(3.7)% in control, heparin, and N-acetylheparin treated animals, respectively (P < 0.05, control v heparin and N-acetylheparin). CONCLUSIONS: The glycosaminoglycans, heparin or N-acetylheparin, can reduce the extent of myocardial injury associated with regional ischaemia and reperfusion in the canine heart. The mechanism of cytoprotection is unrelated to alterations in the coagulation cascade and may involve inhibition of complement activation in response to tissue injury.

MS-551 protects against ventricular fibrillation in a chronic canine model of sudden cardiac death.

We studied the electrophysiologic and antifibrillatory properties of MS-551 (1,3-dimethyl-6-((2-[N-hydroxy-ethyl)-3-(4-nitrophenyl) propylamino] ethylamino) 2,4(1H,3H) pyrimidinedione hydrochloride) in a conscious canine model of sudden cardiac death. Three to 5 days after surgically induced myocardial infarction (MI: 2-h occlusion of the left anterior descending coronary artery, LAD), animals were subjected to programmed electrical stimulation (PES) to identify those at risk for sudden cardiac death. MS-551 was administered (2.0, 3.0, or 4 x 2.0 mg/kg intravenously, i.v.). Vehicle-treated animals received 0.9% sodium chloride solution for injection. MS-551 (multiple-dose regimen) increased ventricular effective refractory period (VERP) from 112 +/- 4 to 137 +/- 4 ms (p < 0.05) as compared with vehicle treatment, which did not alter VERP (125 +/- 6 to 121 +/- 5 ms). MS-551 prolonged QTc interval from a predrug value of 293 +/- 8 to 333 +/- 18 ms postdrug. The size of surgically induced MI did not differ among groups: 2.0 mg/kg, 23 +/- 4%; 3.0 mg/kg, 28 +/- 2%; 4 x 2.0 mg/kg, 25 +/- 3%; and vehicle, 28 +/- 3% of the left ventricle. Single bolus administration of MS-551 (2.0 or 3.0 mg/kg i.v.) did not confer significant protection against sudden cardiac death. However, repeated administration of MS-551 protected against sudden cardiac death in 8 of 10 dogs as compared with 2 of 12 in the vehicle-treated group (p < 0.05). The data indicate that a multiple-dose regimen of MS-551 provides protection against ischemia-induced ventricular fibrillation (VF) in the postinfarcted heart. The mechanism by which MS-551 achieves its antifibrillatory effect most likely depends on its ability to prolong VERP of myocardium without altering ventricular conduction velocity.

Effect of ranolazine on infarct size in a canine model of regional myocardial ischemia/reperfusion.

We assessed ranolazine's potential to reduce myocardial injury resulting from 90-min occlusion and 18-h reperfusion of left circumflex coronary artery (LCX) in anesthetized dogs. Ranolazine, a putative antianginal agent, has exhibited positive results in a variety of experimental models associated with the ischemic myocardium. Previous studies demonstrated that ranolazine possesses a mechanism of action involving increases in the amount of active pyruvate dehydrogenase during ischemia, suggesting that the compound may act to promote glucose utilization. Ranolazine was administered as a bolus of 3.3 mg/kg, followed by a constant infusion of 7.2 mg/kg/h for 20 h. The loading dose was administered 30 min before LCX occlusion. Control animals received appropriate volumes of vehicles (loading and infusion). Hemodynamics were unchanged between ranolazine and vehicle groups. Three animals in each group were excluded because of ventricular fibrillation (VF). There was no difference in degree of ST segment change between control and ranolazine-treated groups at any time during LCX occlusion. The area at risk (AAR) of infarct was 40.1 +/- 1.7 and 38.9 +/- 1.3% in control-treated (n = 13) and randolazine-treated (n = 8) animals, respectively (p = 0.631). Myocardial infarct size (IS) was 31.7 +/- 5.2 and 36.6 +/- 8.5% in control and ranolazine-treated animals, respectively (p = 0.603). No significant changes were observed in plasma content of enzymatic markers at 0.5, 2.0, and 18.0 h of reperfusion. The results of this in vivo study indicate that ranolazine did not provide protection from injury to regionally ischemic and reperfused myocardium despite its reported antiischemic activity.

Radiological effect of a low level waste site on the environment.

Environmental surveillance at the fence line of a low-level radioactive waste disposal site on the Nevada Test Site includes sampling for air particulates, radioiodines, tritium in atmospheric moisture, and airborne transuranics, plus an array of thermoluminescent dosimeters for measurement of external gamma exposures. The results obtained from this surveillance are displayed and discussed. The calculated effluents and the resultant effective dose equivalents to workers and the general public are discussed. It is concluded that, in the 15 years since its establishment, this waste disposal site has led to no significant radiological exposure to workers, the general public, or the environment.

Inhibition of in vivo myocardial ischemic and reperfusion injury by a synthetic manganese-based superoxide dismutase mimetic.

We determined whether an organic superoxide dismutase mimetic could reduce myocardial injury resulting from a 90-min occlusion of the left circumflex coronary artery, followed by 18 hr of reperfusion in an anesthetized canine. The superoxide dismutase-mimetic studied (SC-52608) was a synthetic Mn-based macrocyclic compound. SC-52608 or the inactive analog SC-54385 was administered as four doses of 4 mg/kg i.v. Drug, inactive analog or vehicle was administered 30 and 15 min before ischemia and 15 min and immediately before reperfusion. To ensure parity of left circumflex coronary artery occlusion-induced ischemia, only animals with ischemic zone blood flow of less than 0.15 ml/min/g were included in the final analysis. Ischemic zone blood flow was 0.069 +/- 0.016 ml/min/g in control animals (n = 10), 0.072 +/- 0.010 ml/min/g in SC-52608-treated animals (n = 11) and 0.053 +/- 0.011 ml/min/g in SC-54385-treated (n = 9) animals. A transient hypotensive effect was observed upon SC-52608 administration. Hemodynamic parameters were otherwise unaffected by SC-52608 or SC-54385. The areas at risk of infarct were 39.6 +/- 1.9%, 38.7 +/- 1.1% and 39.4 +/- 1.1% in control, SC-52608-treated and SC-54385-treated animals, respectively. Myocardial infarct sizes (% of area at risk of infarct) were 44.2 +/- 5.6%, 25.7 +/- 4.3% and 35.1 +/- 4.9% in control, SC-52608-treated and SC-54385-treated animals, respectively (P < .05 control vs. SC-52608-treated). Therefore, the synthetic superoxide dismutase mimetic protected the regionally ischemic and reperfused myocardium from injury, implicating oxygen-derived radicals in the tissue-injury process.

Cardioprotective effects of ranolazine (RS-43285) in the isolated perfused rabbit heart.

OBJECTIVE: The aim was to examine the putative cardioprotective effects of the novel antianginal agent, ranolazine, using an isolated rabbit heart model of ischaemia and reperfusion. METHODS: Hearts from male New Zealand White rabbits were perfused in the Langendorff mode with a recirculating Krebs buffer at a constant flow of 20-25 ml.min-1. After equilibration, hearts were treated with ranolazine (10 or 20 microM) or vehicle control for 10 min before exposure to a 30 min period of global ischaemia and 60 min reperfusion; a normoxic control group was also studied. Haemodynamic variables (left ventricular pressure), myocardial creatine kinase, and potassium release were measured at baseline (preischaemic) and at selected points during reperfusion; tissue calcium and ATP content were also measured and electron microscopy was performed. RESULTS: Left ventricular developed pressure during reperfusion was improved (p < 0.05) in a concentration dependent manner by 10 and 20 microM ranolazine (the baseline value was unaffected) with the latter dose resulting in a return to preischaemic values. The release of creatine kinase and potassium was reduced in the ranolazine groups (p < 0.05). A 2.5-fold increase in tissue calcium content in vehicle treated hearts at the end of reperfusion (compared to normoxic time control) was reduced by 10 microM ranolazine (p < 0.05) and completely prevented by 20 microM ranolazine. Similarly, the decrease in tissue ATP was largely inhibited by ranolazine in a concentration dependent manner. Electron microscopy showed that 20 microM ranolazine prevented the occurrence of many indications of reperfusion injury observed in vehicle treated control hearts, for example, blurring of myofibrillar Z bands, derangement of myofibrillar architecture, disruption of mitochondrial cristae and matrices, and the appearance of electron-dense bodies within them. The deposition of lanthanum chloride, a marker of blood vessel integrity, is also modified in the ranolazine treated hearts. CONCLUSIONS: Ranolazine has impressive cardioprotective properties in an isolated rabbit heart model of ischaemia and reperfusion, suggesting that the drug warrants further research into its precise mechanism of action.

Antiarrhythmic agent, MS-551, protects against pinacidil + hypoxia-induced ventricular fibrillation in Langendorff-perfused rabbit isolated heart.

We studied the electrophysiologic and antifibrillatory effects of the class III agent MS-551 in a rabbit isolated heart model in which ventricular fibrillation (VF) occurs reproducibly under conditions of hypoxia/reoxygenation in the presence of the ATP-dependent potassium channel opener, pinacidil. Ten minutes after MS-551 or vehicle administration, addition of pinacidil (1.25 microM) to the buffer was followed by a 12-min hypoxic period and 40-min reoxygenation. At a low concentration of MS-551 (1.0 microM), VF occurred in 5 of 6 hearts, the same incidence as in the control group (5 of 6). In contrast 0 of 6 hearts treated with 15 microM MS-551 developed VF (p < 0.05 vs. vehicle). Ventricular effective refractory period (VERP) was determined in a separate group of isolated hearts (n = 13). Pinacidil alone, during normoxic perfusion, decreased VERP 48 +/- 11% (p < 0.05) 15 min after exposure. Five minutes of hypoxia alone also decreased VERP (57 +/- 8%, p < 0.05). Under normoxic conditions, MS-551 increased ERP 31 +/- 10% (p < 0.05 vs. baseline). VERP prolongation by MS-551 was reduced in the presence of pinacidil but remained 22 +/- 6% (p < 0.05) above baseline. The results suggest that VERP shortening owing to pinacidil-mediated ATP-dependent K+ channel opening is associated with development of VF in isolated heart. MS-551 attenuates the pinacidil-mediated decrease in VERP and prevents pinacidil+hypoxia-reoxygenation-induced VF. Because pinacidil and hypoxia open myocardial KATP channels, putatively decreasing VERP, MS-551 may exert its antifibrillatory effect through partial blockade of KATP channels.