Abstract
Background:
In postmyocardial infarction patients, transient episodes of ischemia are associated with a greater incidence of sudden cardiac death (SCD). Ventricular tachycardia and ventricular fibrillation (VF) are responsible for the majority of SCDs, but current pharmacological interventions for prevention of lethal ventricular arrhythmias are less than satisfactory. We investigated the efficacy of HBI-3000 (HBI), a novel antiarrhythmic agent, in preventing SCD in a conscious canine model.
Methods:
After 3 to 7 days of a surgically induced myocardial infarction (ie, 90-minute occlusion of the left anterior descending coronary artery followed by 30 minutes of reperfusion), conscious animals were administered vehicle (0.9% NaCl solution for injection) or HBI (15 mg/kg) intravenously. An occlusive thrombus at a site remote from the previous myocardial infarction was induced by electrolytic injury to the intimal surface of the left circumflex coronary artery.
Results:
Control animals developed premature ventricular complexes (PVCs) followed by ventricular tachycardia, which terminated in VF in 5 of the 8 dogs. HBI reduced the frequency of PVCs, and only 1 of the 9 HBI-treated animals developed VF (P < .05). In a separate group of postinfarcted animals, the electrical conversion threshold was assessed before and after the intravenous administration of HBI (5, 10, or 15 mg/kg) or flecainide (3 mg/kg), a class IC antiarrhythmic agent. The electrical conversion threshold was not altered by HBI, whereas the administration of flecainide increased the threshold (P < .01 vs baseline).
Conclusions:
The data indicate that HBI is an effective antiarrhythmic and antifibrillatory agent for the prevention of VF or sudden cardiac death.
Introduction
Sudden cardiac death (SCD) is described as death due to an unexpected cessation of cardiac activity and hemodynamic collapse in individuals with or without preexisting heart disease. 1,2 Recurrent, transient episodes of ischemia—either at rest or with exercise—are associated with a greater incidence of sudden death in patients with ischemic heart disease. 3 Electrocardiographic monitoring of ambulatory patients who experienced SCD show that ventricular tachycardia degenerating into ventricular fibrillation (VF) is the most common arrhythmic pattern and ultimate cause of death. 4 Clinical approaches designed to prevent SCD must include (1) the identification of patients possessing a vulnerable myocardial substrate for lethal arrhythmias and (2) the application of effective measures in preventing the development of VF in the presence of triggering events. Implantable cardioverter defibrillators (ICDs) have been demonstrated to reduce mortality in high-risk patients 5 but do not prevent recurrent ventricular arrhythmias; therefore, pharmacological interventions with direct antiarrhythmic effects are used as concomitant therapy to protect against SCD and reduce the frequency of appropriate or inappropriate shocks. 6
HBI-3000 (HBI) is a novel antiarrhythmic agent that is currently in clinical trial as a potential treatment for atrial fibrillation and ventricular arrhythmias. In human ventricular myocytes, HBI exerted blocking effects on sodium, potassium, and calcium ion channels. HBI did not induce early afterdepolarizations or other electrophysiological abnormalities. 7 The present study examined the efficacy of HBI in a canine model of SCD. Our animal model incorporates many of the etiologic features of sudden death and may constitute a relevant model to identify pharmacological agents that are anti- or proarrhythmic. 8 The goal of this investigation was to determine whether HBI could prevent the development of VF precipitated by an acute ischemic event superimposed on a previously infarcted myocardium in the conscious animal. Additional studies were performed to further evaluate the actions of HBI on the electrical conversion threshold (ECT) in the ischemic heart. We report that HBI provides protection against the development of VF in response to acute ischemia in conscious, postinfarcted dogs. The experimental drug candidate may be useful for the prevention of lethal ventricular arrhythmias in the setting of myocardial ischemic injury.
Materials and Methods
Guidelines for Animal Research
The procedures used in this study are in accordance with the guidelines of the University of Michigan Committee on the Use and Care of Animals. The American Association of Accreditation of Laboratory Animal Care accredits the University of Michigan, and the animal care use program conforms to the standards in The Guide for the Care and Use of Laboratory Animals (NIH Publ. No. 86-23). The Michigan Unit for Laboratory Animal Medicine provided all veterinary care.
Surgical Preparation
Purpose-bred beagles were anesthetized with an intravenous (iv) administration of sodium pentobarbital (30 mg/kg). The dogs were ventilated with room air with the use of a cuffed endotracheal tube and a Harvard respirator (Harvard Apparatus, Holliston, Massachusetts) adjusted to deliver a stroke volume of 30 mL/kg at 12 breaths/min.
A left thoracotomy was performed between the fourth and fifth ribs, the pericardium was opened, and the heart was suspended in a pericardial cradle. The left anterior descending (LAD) coronary artery was isolated at the apex of the left atrial appendage, and the left circumflex (LCX) coronary artery was isolated 1 cm from its origin. Left ventricular anterior wall ischemic injury was induced by a 90-minute occlusion of the LAD coronary artery using a loop of silicone rubber tubing passed through a polyethylene tube followed by gradual release (30 minutes) of the ligature to establish reperfusion of the vessel.
An intravascular electrode (Teflon-insulated, silver-coated, copper wire) was inserted into the lumen of the proximal LCX coronary artery and was sutured in place onto the surface of the heart. The electrode was implanted for the subsequent application of an anodal electrical current to the intimal surface of the coronary artery. The surgical incision was closed, dressings were applied to the wound site, and a protective harness was placed on the animal. Postoperative analgesia was maintained by local infiltration of the surgical wound site with 0.75% bupivacaine hydrochloride.
Sudden Cardiac Death in the Conscious Canine Model
Female, purpose-bred beagles weighing 11.2 ± 0.8 kg were assigned randomly to 1 of the 2 groups: group 1 (n = 8) was administered 0.9% NaCl solution for injection, while group 2 (n = 9) received the test compound HBI (15 mg/kg, iv). The surgically prepared animals were ambulatory within 6 to 8 hours and displayed frequent runs of monomorphic and polymorphic ventricular tachycardia, which subsided gradually over the course of 3 to 5 days. The animals were returned daily to the laboratory for monitoring of the lead II electrocardiogram (ECG) for 30 minutes while the animals were conscious and resting comfortably in a harness. The ECG was recorded on a Grass model 7 polygraph (Astro-Med, West Warwick, Rhode Island) interfaced to the data acquisition program Ponemah (Data Sciences International, St Paul, Minneapolis, Minnesota).
After 3 to 7 postoperative days, the ECG showed sinus rhythm with occasional premature ventricular complexes (PVCs) and the absence of ventricular tachycardia during the observation period. Baseline ECG recordings were monitored for 30 minutes while the postinfarcted animal rested in a supporting harness. The previously implanted wire electrode was connected to the positive pole (anode) of a dual-channel square wave generator (Grass S88 stimulator) and a Grass Constant Current Unit (model CCU1A). The cathode was placed at a remote, subcutaneous site. The animals were administered vehicle or HBI and then subjected to electrolytic injury of the intimal surface of the proximal LCX coronary artery by application of an anodal current (300 μA). When a current is applied through the electrode in the blood stream, which contains ions and water, electrolysis takes place. The toxic products of electrolysis (e.g. free radicals) errode the endothelium, leading to the spontaneous development of an occlusive arterial thrombus at the site of vessel injury. 9 The current was discontinued after 3 hours of posterolateral myocardial ischemia or until SCD. The surviving animals were returned to the animal care facility and were observed for an additional 21 hours.
Sudden cardiac death was defined as death due to a cardiac event occurring within 24 hours from the onset of myocardial ischemia as determined from the changes in the ST segment (depression and/or elevation) recorded from the lead II ECG. Upon the death of an animal, the heart was removed, and the LCX coronary artery was examined to confirm the presence of an occlusive thrombus or deep vessel wall injury. Heart sections were subjected to histochemical staining with triphenyltetrazolium chloride to confirm the presence of irreversible myocardial infarction in the area of distribution of the LAD coronary artery.
Antiarrhythmic Actions in the Postinfarcted Heart
In a separate study, the electrocardiograms (ECGs) of 4 conscious animals that had survived the SCD protocol were monitored. Each of the 4 animals had electrocardiographic evidence of a recent myocardial infarction as well as continuous runs of ventricular tachycardia and/or ectopic ventricular complexes during the initial 45-minute baseline monitoring period. The conscious animals were administered HBI intravenously in increasing doses—15 minutes apart—of 5, 10, and 15 mg/kg for a cumulative dose of 30 mg/kg. Quantifying the number of abnormal ventricular complexes during the entire observation period was used to assess the antiarrhythmic efficacy of HBI. The number of premature ventricular complexes was counted for 2 minutes, and the subsequent 2 minutes were not counted. The counting of PVCs during the 2-minute on–off intervals was repeated over the course of the protocol.
Hemodynamic Effects of HBI in the Normal Heart
Three animals were sedated intramuscularly (IM) with ketamine (15 mg/kg) and xylazine (2 mg/kg). 10 Arterial blood pressure was monitored by cannulating the left common carotid artery with a Millar MikroTip pressure catheter (Miller Instruments, Houston, Texas); the jugular vein was cannulated for the iv administration of HBI.
After the animals became ambulatory, they rested comfortably in a harness in a quiet environment. The HBI dosing regimen was administered as follows: 1, 3, 10, and 30 mg/kg, for a cumulative dose of 44 mg/kg. Each dose was administered over a period of 10 minutes followed by a subsequent dose 10 minutes later. Heart rate and blood pressure were monitored continuously.
Electrical Conversion Threshold in the Ischemic Heart
Purpose-bred beagles were anesthetized with sodium pentobarbital (30 mg/kg), intubated, and mechanically ventilated with room air at a tidal volume of 30 mL/kg. The right femoral artery was instrumented with a Millar MikroTip pressure catheter (Millar Instruments) to monitor arterial blood pressure. The heart was exposed via a left thoracotomy, the pericardium was opened, and the heart was suspended in a pericardial cradle. The LCX coronary artery was isolated and occluded 2 cm distal to its origin. The lead II ECG was monitored continuously to ensure that regional ischemia was maintained for 3 hours before starting induction of VF and determination of the electrical conversion threshold. VF was induced by applying the 2 poles of a 9-V NiCad battery to the apex of the heart. Sustained VF was confirmed by examination of the lead II ECG. Twenty seconds after the induction of VF, direct current (DC) countershock was applied to the heart with the use of a capacitor-discharge direct current defibrillator (Physio-Control, Model 640, Redmond, Washington). The current was increased in 5-Joule (J) increments every 20 seconds (5-30 J) until coordinated electrical activity was restored (ie, sinus rhythm). Animals that did not convert were assigned a value of 30 J. Conversion was defined as return to spontaneous rhythm and effective ventricular contractile activity. The lowest intensity countershock required to restore sinus rhythm was recorded as the ECT.
Three groups were studied to assess the effective dose of HBI on the ECT in the ischemic heart (5 mg/kg, n = 6; 10 mg/kg, n = 7; and 15 mg/kg, n = 9). A separate group of animals was treated with the class IC antiarrhythmic agent flecainide (3 mg/kg, n = 12).
Statistical Analysis
The data are expressed as mean ± standard error of the mean (SEM). The number of SCD incidences between vehicle and HBI-treated animals was analyzed by Fisher exact test. The ECT before and after drug administration was analyzed by paired t test. Repeated measures analysis of variance followed by Tukey post hoc test was performed to compare the average number of abnormal ventricular complexes and the hemodynamic effects of HBI. Data were considered statistically significant at P < .05. Statistical analyses were performed using Graph Pad Prism 5.0a (GraphPad Software, San Diego, California).
Results
HBI Attenuates Sudden Cardiac Death
Baseline ECGs showed normal sinus rhythm in each of the animals that had recovered from the surgical induction of myocardial infarction. Subsequent induction of electrolytic injury to the LCX coronary artery was accompanied by progressive changes in the lead II ECG. Of the 8 control animals, 5 developed premature ventricular complexes degenerating to ventricular tachycardia followed by VF or SCD (Figure 1A). Each of the 9 animals in the HBI-treated group developed ST depression, which is indicative of ischemia in the remote region of the left ventricle (Figure 1B). Of the 9 HBI-treated animals, 8 survived the SCD protocol. This was significantly different (P < .05) from the placebo-treated animals in which only 3 of the 8 dogs survived (Figure 2).
Typical pattern of electrocardiographic changes observed in a postinfarcted canine 3 days after acute myocardial infarction. A, In the control animal, the electrocardiogram recording indicated the development of premature ventricular complexes (4), degenerating to ventricular tachycardia (5), and then, finally, ventricular fibrillation (6) or sudden cardiac death. B, The animal treated with HBI-3000 (15 mg/kg, intravenous) did not result in ventricular fibrillation. ST segment depression (3) was indicative of a remote myocardial ischemia.
Survival curve of HBI-3000 (15 mg/kg, intravenous, white squares) and vehicle-treated animals (black circles) after the onset of posterolateral myocardial ischemia. Data shown are a 24-hour survival after electrolytic injury to the left circumflex coronary artery (LCX); *P < .05, Fisher exact test.
HBI is Antiarrhythmic
Figure 3 summarizes the antiarrhythmic actions of HBI in 4 animals that had survived the SCD protocol. Each of the animals exhibited premature ventricular complexes during the pretreatment phase of the experiment. The administration of HBI (5, 10, and 15 mg/kg) resulted in a restoration of sinus rhythm and a dose-dependent decrease in the frequency of the number of abnormal ventricular complexes.
Average number of ventricular complexes of the 4 animals that had survived the sudden cardiac death protocol. A dose-dependent reduction in the frequency of the abnormal ventricular complexes was observed. The cumulative dose of 30 mg/kg HBI was able to achieve near total suppression of the abnormal ventricular complexes with a return to sinus rhythm (P > .05, repeated measures analysis of variance) and no evidence of proarrhythmia.
Hemodynamic Effects of HBI
The data in Table 1 indicate that mean arterial blood pressure (MAP) decreases with increasing doses of HBI. The MAP was significantly lower after the administration of 30 mg/kg HBI compared to baseline values. It should be noted that the cumulative dose of 44 mg/kg is well beyond the effective therapeutic dose in the SCD protocol. The decrease in blood pressure was accompanied by an increase in heart rate, which was not statistically significant. Significant changes in electrocardiographic intervals were not observed.
Effect of HBI-3000 on Hemodynamics.a
Abbreviations: bpm, beats per minute; MAP, mean arterial blood pressure; SEM, standard error of the mean.
aValues are mean ± SEM.
b P < .05 versus pretreatment.
HBI Does Not Alter the Electrical Conversion Threshold
The effect of HBI on the electrical conversion threshold was compared with flecainide (Figure 4). The baseline ECT among all experimental groups was relatively the same (P > .05, 1-way analysis of variance). The ECT was not significantly altered after the administration of HBI. Although animals treated with high-dose HBI (15 mg/kg) demonstrated an increase in the ECT, the before and after drug administration data were not significantly different. In contrast, an iv administration of flecainide (3 mg/kg) increased the ECT (P < .01) compared to the respective baseline values.
The electrical conversion threshold before and after treatment with HBI-3000 (shaded bars) or flecainide (white bar, n = 12). The ECT was not altered significantly after treatment with HBI at 5 (black, n = 6), 10 (dark gray, n = 7), or 15 (light gray, n = 9) mg/kg. The administration of flecainide significantly increased the ECT after treatment (**P < .01, paired t test).
A limited study was repeated in the normal canine heart (data not shown). Flecainide-treated animals (n = 2) could not be converted by DC countershock over the range of 5 to 30 J. In contrast, HBI-treated animals (10 mg/kg, iv, n = 2) were converted at an average threshold of 7.5 ± 2.5 J. The before treatment threshold was 10 J and 7.5 ± 2.5 J for HBI and flecainide-treated animals, respectively.
Discussion
Sudden Cardiac Death Animal Model
Our laboratory developed a conscious canine model of anterior myocardial infarction that was susceptible to the development of VF upon induction of ischemia in a region remote from the previous infarction. Previous experimental studies 3,8 have provided sufficient evidence that the superimposition of a transient, ischemic event on a previously injured ventricular myocardium can lead to an electrical instability, culminating in VF. Occlusion and reperfusion of the LAD coronary artery renders the canine heart predisposed to electrical induction of ventricular arrhythmias. Within the infarcted region, the salvaged myocardial fibers may provide reentrant pathways causing sustained tachyarrhythmias. The addition of a second ischemic event due to the initiation of thrombosis in the LCX coronary artery causes a remote region in the heart to support a reentry pattern, which engages the initial damaged region, resulting in VF. 8
Our SCD model closely resembles the events that occur in humans with coronary heart disease. The experimental model, which can be conducted in the anesthetized as well as in the ambulatory canine, has been extensively used in our laboratory for the evaluation of agents that might be useful in preventing the development of SCD in high-risk patients who have survived a primary sudden cardiac arrest. 11 –13 In the present study, we demonstrated that HBI exhibits favorable antiarrhythmic and antifibrillatory actions in the postinfarcted canine heart. Of the 8 vehicle-treated animals, 5 developed VF, whereas 8 of the 9 HBI-treated animals survived the experimental protocol. The data demonstrate that HBI is effective in preventing the development of ventricular arrhythmias and VF in the early period following acute myocardial ischemia.
Electrophysiological Profile of HBI
HBI-3000 (sulcardine sulfate) is a novel antiarrhythmic agent that was well tolerated in phase 1 studies. HBI is a multi-ion channel blocker, and it inhibits—in order of efficacy—the late sodium current (INa-L), rapidly activating delayed rectifier potassium current (IKr),
The cardiac late sodium current (INa-L) is a therapeutic target for treating electrical and contractile dysfunction in myocardial ischemia and heart failure. 15 The INa-L current is enhanced in pathologic conditions and is recognized to be proarrhythmic by reducing repolarization reserve (ie, net outward current) and prolonging the action potential duration. 16,17 Selective inhibition of INa-L has been associated with improvement of electrical function in hearts made ischemic. 18 Furthermore, inhibition of INa-L has not been reported to have adverse consequences in normal or diseased hearts. Thus, the electrophysiological profile and properties of HBI should exert optimal antiarrhythmic effects, and this was demonstrated in our preclinical studies.
Antiarrhythmic Actions of HBI
HBI’s antiarrhythmic actions were studied by quantifying the number of abnormal ventricular complexes in the postinfarcted canine heart that is subjected to a remote ischemic event. The 90-minute occlusion of the anterior descending coronary artery followed by reperfusion results in an area of irreversibly injured myocytes dispersed among viable myocardial tissue. This pattern of tissue injury results in the development of repetitive episodes of polymorphic ventricular complexes in the conscious animal. The underlying mechanism for the genesis of the abnormal ventricular rhythm is the result of reentry. 19 In each of the animals, a cumulative dose of HBI 30 mg/kg suppressed abnormal complexes and restored sinus rhythm. The limited study size population warrants further studies to completely assess HBI’s antiarrhythmic potential.
Effect of HBI on the Electrical Conversion Threshold
Antiarrhythmic drugs have been reported to alter the electrical conversion threshold; therefore, an important consideration is whether the ICD will provide adequate energy for defibrillation during long-term follow-up and to what extent concomitant antiarrhythmic treatment will affect the energy level required to achieve successful defibrillation. 20 Here, HBI was administered intravenously in doses of 5, 10, or 15 mg/kg. Studies were conducted in which the energy required to terminate VF was determined in the normal canine heart as well as in the setting of acute myocardial infarction. Flecainide (3.0 mg/kg, iv), a class IC sodium channel blocker, was used as a comparator. Hernandez et al have previously demonstrated that the administration of flecainide significantly increases the defibrillation threshold in the anesthetized dog. 21 Class I antiarrhythmic drugs, including flecainide, are also known to increase all-cause mortality in patients with postmyocardial infarction 22 and are therefore not recommended in the prevention of SCD. 23 We observed a significant increase in the ECT only in the flecainide-treated animals. The high-dose HBI-treated group (15 mg/kg) also demonstrated an increase in ECT compared to baseline values, although not statistically significant. Future experimental or clinical studies with high-dose HBI should be used with caution.
Conclusion
There is a clinical need to develop safe and effective antiarrhythmic and antifibrillatory drugs that are capable of preventing unexpected death due to lethal ventricular arrhythmias. Consistent with previous studies 7,24 , the results of this investigation suggest that HBI has an ability to prevent the development of VF in response to an acute ischemic event at a site distant from an area of previous myocardial infarction. The antiarrhythmic actions of HBI make the experimental drug a potential candidate for continued studies in humans to determine its efficacy in treating atrial and ventricular arrhythmias and preventing secondary sudden cardiac death.
Footnotes
Acknowledgment
We thank HUYA Bioscience International, LLC, especially Dr Mireille Gillings for providing HBI-3000 and funding support.
Authors’ Notes
This research was conducted at the University of Michigan Medical School.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by HUYA Bioscience International, LLC and the Cardiovascular Research Fund of the University of Michigan.