Gregory S. Friedrichs

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.

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.

Thrombolytic effects of recombinant fibrolase or APSAC in a canine model of carotid artery thrombosis.

BACKGROUND Thrombolytic agents used clinically rely on the activation of plasminogen to plasmin. Plasmin possesses multiple actions including increasing thrombin activity and activation of platelets. Thus, after successful thrombolytic therapy, arterial hyperactivity and reocclusion may be the result of a predominant plasmin-induced thrombogenic action at the site of the residual thrombus. Fibrolase, a direct-acting fibrinolytic enzyme from southern copperhead snake venom, induces rapid clot lysis in vitro. Fibrolase does not rely on plasminogen activation or any other bloodborne components for activity and is not inhibited by any of the rapidly acting serine proteinase inhibitors in blood. METHODS AND RESULTS We investigated the efficacy of fibrolase to lyse an occlusive thrombus formed in the carotid artery of the anesthetized dog. Electrolytic injury was initiated in both the right and left carotid arteries. Thirty minutes after both arteries were occluded, each vessel was infused with either fibrolase (4 mg/kg over 5 minutes) or physiological saline (over 5 minutes). In two separate groups of dogs, anisoylated plasminogen streptokinase activator complex (APSAC) (0.1 U/kg) was infused into the occluded vessel. In the artery infused with fibrolase, five of five dogs exhibited patency within 6 +/- 1 minutes of the infusion (P < .05 versus vehicle-treated artery; Fishers exact test). In the contralateral carotid artery that received vehicle, the occlusion was maintained throughout the experimental protocol. APSAC alone lysed the thrombus in each vessel within 17 +/- 3 minutes. Five minutes after the end of fibrolase administration and in one of the groups administered APSAC, a glycoprotein (GP)IIb/IIIa antibody, 7E3 (0.8 mg/kg IV), was administered to prevent reocclusion of the patent artery. After 7E3 administration, the vessel treated with fibrolase remained patent in four of five dogs, and six of six APSAC-treated vessels were patent for the remainder of the observation period (2 hours). CONCLUSIONS These studies demonstrate that local administration of fibrolase lyses a carotid arterial thrombus rapidly without excessive hemorrhage or hemodynamic compromise. The enzyme in combination with antiplatelet therapy (7E3) offers a unique mechanism for clot dissolution and may prove useful as a clinically efficacious alternative to presently used thrombolytic agents or may act in a synergistic manner with plasminogen activators.

Protective Effects of Ranolazine on Ventricular Fibrillation Induced by Activation of the ATP-Dependent Potassium Channel in the Rabbit Heart.

Background: The authors studied the antifibrillatory effects of the adenosine-triphosphate (ATP)-sparing metabolic modulator ranolazine in a rabbit isolated heart model in which ventricular fibrillation occurs under conditions of hypoxia/reoxygenation in the presence of the ATP-dependent potassium channel opener pinacidil. Methods and Results: Ten minutes after ranolazine or vehicle administration, addition of pinacidil (1.25 μM) to the buffer was followed by a 12-minute hypoxic period and 40 minutes of reoxygenation. At a reduced concentration of ranolazine (10 μM), ventricular fibrillation occurred in 60% of the hearts. compared to 89% in the control group (P = NS). In contrast, only three of nine hearts (33%) treated with 20 μM ranolazine developed ventricular fibrillation (P <.05 vs vehicle). Hemodynamic parameters including coronary perfusion pressure, left ventricular developed pressure, and ±dP/dt were not affected by the presence of ranolazine in the perfusion medium. Ranolazine did not prevent or modify the negative inotropic or coronary vasodilator actions of pinacidil, suggesting a mechanism of action independent of potassium channel antagonism. Conclusions: Ranolazine significantly reduced the incidence of ventricular fibrillation in the hypoxic/reoxygenated heart exposed to the ATP-dependent potassium channel opener, pinacidil. The reported ability of ranolazine to prevent the decrease in cellular ATP during periods of a reduced oxygen supply may account for its observed antifibrillatory action. By maintaining intracellular ATP, ranolazine may modulate or prevent further opening of the ATP-dependent potassium channel in response to hypoxia and/or pinacidil.

Sphingosine modulates myocyte electrophysiology, induces negative inotropy, and decreases survival after myocardial ischemia

Contractility studies in isolated feline myocytes have demonstrated that sphingosine, a metabolite stimulated by tumor necrosis factor (TNF) binding, decreases intracellular calcium release and depresses inotropic activity. This study investigated the electrophysiologic effects of sphingosine in isolated cat myocytes as well as the cardiodynamic consequence of TNF, sphingosine, and its metabolic precursors in vivo. In cat myocytes, sphingosine markedly decreased action potential duration, lowered action potential plateau, and inhibited L-type calcium current (I Ca-L ). After administration of TNF, sphingomyelin, C2-ceramide, or sphingosine, only C2-ceramide and sphingosine depressed cardiac function in normal rats. Negative inotropic effects of C2-ceramide were attenuated by N-oleoylethanolamine (NOE), a ceramidase inhibitor that blocks sphingosine formation. Rats pretreated with NOE before undergoing 30 min of acute regional myocardial ischemia followed by 150 min of reperfusion exhibited improved survival. Most deaths could be attributed to acute pump failure accompanied by bradycardia. Myocardial infarct size and peak serum TNF were not different between NOE- and vehicle-treated groups (3,908 ± 1097 pg/ml and 3,027 ± 846 pg/ml, respectively). These results indicate that sphingosine exerts direct inhibitory effects on the action potential and I Ca-L in isolated feline myocytes, consistent with previously reported sphingosine activity on I Ca-L in isolated rat myocytes. The in vivo study suggests that reducing sphingosine production with N-oleoylethanolamine attenuates cardiodepression and can improve overall survival after ischemic injury. Clearly, agents that modulate sphingosine production limit cardiodepression and may provide a therapeutic benefit in clinical conditions of myocardial inflammatory injury.

Effects of tedisamil (KC-8857) on cardiac electrophysiology and ventricular fibrillation in the rabbit isolated heart.

1 The direct cardiac electrophysiological and antifibrillatory actions of tedisamil (KC‐8857) were studied in rabbit isolated hearts. 2 Tedisamil (1, 3, and 10 μm), prolonged the ventricular effective refractory period (VRP) from 120±18ms (baseline) to 155±19, 171±20, and 205±14 ms, respectively. Three groups of isolated hearts (n = 6 each) were used to test the antifibrillatory action of tedisamil. Hearts were perfused with 1.25 μm pinacidil, a KATP channel activator. Hearts were subjected to hypoxia for 12 min followed by 40 min of reoxygenation. Ventricular fibrillation (VF) developed during hypoxia and reoxygenation in both the control and 1 μm tedisamil‐treated groups (5/6 and 4/6, respectively). Tedisamil (3 μm) reduced the incidence of VF (0/6, P = 0.007 vs. control). 3 In a separate group of hearts, VF was initiated by electrical stimulation. The administration of 0.3 ml of 10 mM tedisamil, via the aortic cannula, terminated VF in all hearts, converting them to normal sinus rhythm. 4 Tedisamil (3 μm) reversed pinacidil‐induced negative inotropic effects in rabbit isolated atrial muscle which were equilibrated under normoxia, as well as in atrial muscle subjected to hypoxia and reoxygenation. 5 The results demonstrate a direct antifibrillatory action of tedisamil in vitro. The mechanism responsible for the observed effects may involve modulation by tedisamil of the cardiac ATP‐regulated potassium channel, in addition to its antagonism of IK and Ito.

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

Summary We studied the electrophysiologic and anti-fibrillatory properties of MS-551 (l,3-dimethyl-6-{(2-[N-hydroxy-ethyl)-3-(4-nitrophenyl) propylamino] ethy-lamino} 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 ± 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 ± 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.

Antifibrillatory Efficacy of Long-term Tedisamil Administration in a Postinfarcted Canine Model of Ischemic Ventricular Fibrillation

The electrophysiologic and antifibrillatory properties of tedisamil (KC-8857) were studied in vivo in a conscious canine model of sudden cardiac death. Male mongrel dogs were anesthetized, and surgical anterior myocardial infarction was induced by a 2-h occlusion, with reperfusion of the left anterior descending coronary artery. Three to five days after infarction, dogs were subjected to programmed electrical stimulation (PES) to identify those at risk for ischemia-induced ventricular fibrillation. Previous studies documented that dogs with a significant anterior-wall infarction develop ventricular tachycardia in response to PES and are at an increased risk for sudden cardiac death on imposition of a transient ischemic event in a region remote from the infarct-related artery. PES-inducible animals were randomized to either oral placebo or oral tedisamil treatment (3 mg/kg, b.i.d for 4 days, Group 1, n = 8). Control animals received empty gelatin capsules (Group 2, n = 8). The effective refractory period and QTc interval were unchanged after 3 days of oral placebo or tedisamil dosing. Arrhythmic activity after drug administration was not observed in dogs treated with tedisamil. PES induction of ventricular tachycardia was reduced significantly in the tedisamil-treated group (100% inducible before drug vs. 9% inducible after drug; p < 0.05). In the sudden-cardiac-death protocol, tedisamil reduced the incidence of lethal ischemic arrhythmias developing in response to acute posterolateral myocardial ischemia. Tedisamil-treated animals exhibited a 100% compared with a 25% survival rate in the control group (p < 0.05). Anterior-wall infarct size, expressed as a percentage of the left ventricle, did not differ between groups: Group 1 = 20 +/- 1%; Group 2 = 22 +/- 1%. Our findings suggest that tedisamil might be useful in the prevention of malignant ventricular arrhythmias in myocardial ischemic injury.

Orally Effective CVS-1123 Prevents Coronary Artery Thrombosis in the Conscious Dog

BACKGROUND We examined the oral efficacy of a direct thrombin inhibitor, CVS-1123 [(CH3CH2CH2)(2)-CH-CO-Asp (OCH3)-Pro-Arg-CHO; MW, 575]. The object was to determine whether thrombin inhibition could reduce the incidence of occlusive coronary artery thrombosis in response to arterial wall injury. METHODS AND RESULTS Arterial wall injury was induced in conscious dogs by a 150-muA anodal current applied to the intimal surface of the circumflex coronary artery 30 minutes after oral CVS-1123 (20 mg/kg every 8 hours for three doses; n = 11) or placebo containing diluent (n = 10). Dogs were monitored for 8 hours and at 24 hours. The coronary artery remained patent for 24 hours in 8 of 11 CVS-1123-treated dogs. All dogs (n = 10) in the placebo group developed a sustained, occlusive arterial thrombus. Two hours after the initial oral dose, the plasma CVS-1123 concentration was 13 +/- 1 microgram/mL, reaching a maximum of 15 +/- 1 micrograms/mL after the second dose and 4.4 +/- 0.5 micrograms/mL at 24 hours. Ex vivo platelet aggregation to gamma-thrombin was inhibited and activated partial thromboplastin time was increased after treatment with CVS-1123 (P < .05). CONCLUSIONS The direct thrombin inhibitor CVS-1123 is effective after oral administration in reducing the incidence of primary thrombus formation in an experimental model of arterial wall injury. Thrombin-specific inhibitors, such as CVS-1123, may be alternative antithrombotic agents in clinical settings in which heparin-associated thrombosis is a complicating factor or when long-term anticoagulation is required.

Tedisamil in a chronic canine model of atrial flutter

Tedisamil inhibits several cardiac potassium channels including Ito, Ikr, and the adenosine triphosphate (ATP)-sensitive potassium channel (I(KATP)), which may be important in the initiation and maintenance of atrial arrhythmias. We herein report the efficacy of tedisamil in terminating and protecting against the reinduction of atrial flutter (AFL) in a conscious canine model. Sustained AFL (> 15 min) was induced in eight of 10 mongrel dogs by programmed atrial stimulation (PAS) 2-41 days after producing a surgical barrier to conduction in the right atrium. At the time of surgery, an epicardial electrode was attached to the right atrial appendage for pacing and recording. Normal saline, 1 ml/kg, was infused after 15 min of AFL as placebo. Tedisamil (1.0 mg/kg) was given intravenously after 30 min of sustained AFL while recording surface ECGs and atrial electrograms. Conversion to sinus rhythm was achieved in 10 of 10 trials (eight dogs) in a mean time of 20.5 s (SD, +/- 11.8 s). Tedisamil had a negative chronotropic effect lasting > or =2 h and was protective against the reinduction of AFL. In five dogs, PAS was able to induce AFL in only two of seven trials 2 h after drug infusion. The corrected QT interval (QTc) was lengthened for the first 15 min after tedisamil administration (mean, +/- 39.3 ms; p < 0.05), but thereafter returned to baseline. The QRS interval was not altered by tedisamil. Saline alone, given after 15 min of sustained AFL, converted AFL in one of 11 trials (eight dogs) but did not alter the RR interval, QTc, or QRS interval compared with values measured during AFL. No significant adverse effects of tedisamil were observed. The results indicate that tedisamil is effective in interrupting and/or preventing reinduction of canine AFL, possibly by prolonging atrial refractoriness through inhibition of one or more potassium ion repolarizing currents in atrial muscle. Further studies are required to address the exact mechanism by which tedisamil exerts its antiarrhythmic effect.