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.

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.

The assessment of potential for QT interval prolongation with new pharmaceuticals: impact on drug development.

Few examinations of a single physiological variable can end the development of a putative new pharmaceutical. Prolongation of the electrocardiographic QT interval is one of these tests. Recognizing the removal of several approved and widely used medicines, worldwide regulatory authorities have raised a heightened awareness on the submission of data surrounding the ventricular repolarization process. This review will discuss the anatomy and physiology surrounding the generation of the electrocardiographic QT interval and the consequences of its alteration. In addition, relevant models of preclinical safety and general guidelines for clinical examination in this area are discussed along with the impact of incorporating these assays into the drug development process.

Localization of cyclooxygenase isozymes in cardiovascular tissues of dogs treated with naproxen.

Prostaglandins have diverse roles in the cardiovascular system mediating both physiologic and inflammatory responses. Two cyclooxygenase isoforms, cyclooxygenase-1 and cyclooxygenase-2, catalyze prostaglandin production. In many tissues and cell types studied, cyclooxygenase-1 is constitutively active whereas cyclooxygenase-2 expression is primarily responsible for prostaglandin production during inflammation. However, little information exists concerning which isoform is responsible for prostaglandin-mediated effects in the heart. We examined cyclooxygenase-1 and cyclooxygenase-2 expression in heart and vascular tissue of dogs using isoform-specific antibodies. In addition, tissues from dogs treated with naproxen (5-10mg/kg/day), an inhibitor of prostaglandin production were also examined. Cyclooxygenase-1 expression was evident in endothelial cells of the microvasculature of the heart, aorta and renal artery. Cyclooxygenase-1 expression was also found in fibrocytes of the tricuspid valve and in the chordae tendinae. Animals treated with naproxen exhibited a similar pattern and intensity of cyclooxygenase-1 staining. No cyclooxygenase-2 expression was evident in cardiac tissue. However, minimal cyclooxygenase-2 immunoreactivity was present in the vascular endothelial cells of small myocardial blood vessels located in several regions of the heart as well as in endothelial cells of the aorta. These data may expand our understanding of the effects of non-steroidal anti-inflammatory drugs on cardiac function.

Investigation of mechanism of drug-induced cardiac injury and torsades de pointes in cynomolgus monkeys.

BACKGROUND AND PURPOSE: Drug candidates must be thoroughly investigated for their potential cardiac side effects. During the course of routine toxicological assessment, the compound RO5657, a CCR5 antagonist, was discovered to have the rare liability of inducing torsades de pointes (polymorphic ventricular arrhythmia) in normal, healthy animals. Studies were conducted to determine the molecular mechanism of this arrhythmia. EXPERIMENTAL APPROACH: Toxicological effects of repeat dosing were assessed in naïve monkeys. Cardiovascular effects were determined in conscious telemetry-implanted monkeys (repeat dosing) and anaesthetized instrumented dogs (single doses). Mechanistic studies were performed in guinea-pig isolated hearts and in cells recombinantly expressing human cardiac channels. KEY RESULTS: In cynomolgus monkeys, RO5657 caused a low incidence of myocardial degeneration and a greater incidence of ECG abnormalities including prolonged QT/QTc intervals, QRS complex widening and supraventricular tachycardia. In telemetry-implanted monkeys, RO5657 induced arrhythmias, including torsades de pointes and in one instance, degeneration to fatal ventricular fibrillation. RO5657 also depressed both heart rate (HR) and blood pressure (BP), with no histological evidence of myocardial degeneration. In the anaesthetized dog and guinea-pig isolated heart studies, RO5657 induced similar cardiovascular effects. RO5657 also inhibited Kv11.1 and sodium channel currents. CONCLUSIONS AND IMPLICATIONS: The molecular mechanism of RO5657 is hypothesized to be due to inhibition of cardiac sodium and Kv11.1 potassium channels. These results indicate that RO5657 is arrhythymogenic due to decreased haemodynamic function (HR/BP), decreased conduction and inhibition of multiple cardiac channels, which precede and are probably the causative factors in the observed myocardial degeneration.

Effects of troglitazone and pioglitazone on cytokine-mediated endothelial cell proliferation in vitro.

We examined whether troglitazone and pioglitazone, antidiabetic thiazolidinediones, would directly induce endothelial cell proliferation or influence cytokine-driven proliferation in vitro. Monolayers of Balb/c mouse aortic endothelial cells were treated with troglitazone or pioglitazone in the absence of fetal bovine serum. Endothelial cells also were exposed to varying concentrations of basic fibroblast growth factor (bFGF) or insulin with or without either thiazolidinedione. After 48 h, 3-[4,5-dimethylthiozol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assays were performed to quantitate endothelial cell proliferation by using the various treatment regimens. The data demonstrate that the antidiabetic thiazolidinediones troglitazone and pioglitazone negligibly affect direct endothelial cell proliferation in vitro. Furthermore, troglitazone and pioglitazone significantly inhibit bFGF-induced endothelial cell mitogenesis, whereas only high concentrations of troglitazone affect insulin-mediated proliferation.

Selective inhibition of the alternative complement pathway by sCR1[desLHR-A] protects the rabbit isolated heart from human complement-mediated damage.

Evidence is presented that treatment with a selective inhibitor of the alternative complement pathway, sCR1[desLHR-A], protects the ex vivo perfused rabbit heart from human complement-mediated injury. Hearts from male New Zealand white rabbits were perfused in the Langendorff mode. After equilibration, normal human plasma was added to the perfusate as a source of complement. Concomitant with the addition of human plasma, vehicle (n = 13), soluble complement receptor type 1 (sCR1) (n = 10), or sCR1[desLHR-A], a truncated version of sCR1 that lacks the C4b binding region (n = 10) was included in the perfusate. Hemodynamic variables were obtained for all groups before (baseline) and after the addition of human plasma. Compared to vehicle-treated hearts, variables recorded during perfusion with human plasma including coronary perfusion pressure, left ventricular developed pressure, and left ventricular end diastolic pressure, along with a reduction of creatine kinase efflux, were improved in hearts perfused with either complement inhibitor. In addition, in vitro hemolysis assays were utilized to discriminate between the classical and alternative pathways. The addition of sCR1 to human serum prevented both the classical and alternative pathway-mediated hemolysis while sCR1[desLHR-A] prevented only the alternative pathway-mediated lysis. This study indicates that deletion of the C4b-binding site from sCR1 results in a new pharmacological moiety, sCR1[desLHR-A], that primarily inhibits the alternative pathway of human complement.

Effects of heparin and N-acetyl heparin on ischemia/reperfusion-induced alterations in myocardial function in the rabbit isolated heart.

Evidence is presented that heparin pretreatment produces protective effects on myocardial tissue distinct from its anticoagulant activity. The present study examines the ability of heparin sulfate and N-acetyl heparin (a derivative of heparin devoid of anticoagulant effects) to protect the heart from injury associated with global ischemia and reperfusion. Male New Zealand White rabbits were administered either heparin sulfate (n = 7, 300 U/kg i.v.), N-acetyl heparin (n = 6, 1.73 mg/kg i.v.), or vehicle (n = 6). Two hours after treatment, the hearts were removed, perfused on a Langendorff apparatus, and subjected to 30 minutes of global ischemia, followed by 45 minutes of reperfusion. During reperfusion, creatine kinase concentrations in the coronary sinus effluent were greater in hearts from vehicle-treated rabbits compared with hearts from N-acetyl heparin-treated and heparin-treated rabbits. Left ventricular end-diastolic pressure after 45 minutes of reperfusion in the vehicle-treated group was 64 +/- 15 mm Hg compared with 17 +/- 4 and 10 +/- 3 mm Hg in the heparin-pretreated and N-acetyl heparin-pretreated groups, respectively. Heparin, but not N-acetyl heparin, increased the activated partial thromboplastin time, consistent with its known anticoagulant action. Heparin and N-acetyl heparin inhibited complement-mediated erythrocyte lysis in a concentration-dependent manner. The glycosaminoglycans, in contrast to r-hirudin, reduced complement activation-induced injury in the rabbit isolated heart. The results demonstrate that heparin or N-acetyl heparin, administered to the intact rabbit, protects the isolated heart from subsequent myocardial dysfunction secondary to ischemia/reperfusion. The cardioprotective effects of heparin and N-acetyl heparin are independent of an antithrombin mechanism.

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.

Reviparin-sodium prevents complement-mediated myocardial injury in the isolated rabbit heart.

The cytoprotective action of reviparin-sodium (LU-47311: Clivarin), a low-molecular-weight heparin, was examined in an ex vivo model of complement-mediated myocardial injury. The effective concentration of reviparin was determined by using an in vitro rabbit erythrocyte-lysis assay using 4% normal human plasma. Reviparin (0.01-2.73 mg/ml) reduced erythrocyte lysis in a concentration-dependent manner. Subsequently, 0.91 mg/ml of reviparin was evaluated in an ex vivo rabbit isolated-heart model of human complement-mediated injury. Hearts perfused in the presence of 0.91 mg/ml of reviparin (n = 10) demonstrated significant preservation of ventricular function compared with vehicle-treated hearts (n = 10), as evidenced by coronary artery perfusion pressure, left ventricular developed pressure, and left ventricular end-diastolic pressure. A reduction in myocyte creatine kinase release was observed in reviparin-treated hearts compared with controls. Myocardial injury in vehicle-treated hearts was associated with an increased assembly of the membrane-attack complex, as determined by immunohistochemical localization of C5b-9 neoantigen. Reviparin decreased fluid-phase Bb formation detected in the lymphatic drainage of plasma-perfused hearts. The results of this study demonstrate that reviparin inhibits complement-mediated myocardial injury as assessed in an ex vivo experimental model of complement activation.

LU 51198, a highly sulfated, low-molecular-weight heparin derivative, prevents complement-mediated myocardial injury in the perfused rabbit heart.

Evidence is presented that treatment with a highly sulfated low-molecular-weight heparin fraction, LU 51198, protects the ex vivo perfused rabbit heart from human complement-mediated injury. Hearts from male New Zealand White rabbits were perfused under constant flow in the Langendorff mode. After equilibration, normal human plasma was added to the perfusate as a source of complement. Either control (n = 8) or LU 51198 (0.6 mg/ml; n = 7) was added to the perfusion medium 10 min before the addition of human plasma. Hemodynamic variables were obtained for both groups before treatment of human plasma. Hemodynamic variables were obtained for both groups before treatment (baseline), 10 min after treatment (zero) and after the addition of human plasma. Compared to control-treated hearts, variables recorded during perfusion with human plasma, including coronary perfusion pressure, left ventricular developed pressure, and left ventricular end-diastolic pressure, along with a reduction of creatine kinase and potassium efflux, were significantly improved in hearts treated with LU 51198 (P < .05). ELISA assays were used to analyze lymphatic effluent for the presence of iC3b, Bb and SC5b-9 proteins derived from the activation of human complement. The increased presence of the Bb fragment in the effluent obtained from LU 51198-perfused hearts suggests an accelerated dissociation of the convertases responsible for complement amplification, an observation that coincided with protection from complement-mediated damage in the presence of the glycosaminoglycan. The lysis of rabbit red blood cells upon exposure to human plasma was inhibited by LU 51198, which is evidence of the drug's ability to modulate complement reactivity. The results of this study indicate that a highly sulfated heparin fraction, LU 51198, can reduce tissue injury and preserve discordant organ function that otherwise would be compromised during activation of the human complement cascade.

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.

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.