Evgeny P. Anyukhovsky

Regional Differences in Electrophysiological Properties of Epicardium, Midmyocardium, and Endocardium In Vitro and In Vivo Correlations

BACKGROUND Microelectrode studies have described a population of cells within the midmyocardium (M cells) displaying a steep rate dependence of action potential duration (APD) and high Vmax compared with endocardial (Endo) and epicardial (Epi) cells. METHODS AND RESULTS We studied repolarization in different myocardial layers in vitro and in situ. In addition to confirming the results of earlier studies, we found that after abrupt lengthening of the cycle length (CL), APDs in M cells reached a new steady state faster than in Epi or Endo cells: the time to achieve 90% of the difference in APD (t90) was 13.3 +/- 0.7 minutes in Endo cells, 12.8 +/- 1.1 minutes in Epi cells, and 2.6 +/- 0.4 minutes in M cells (P < .05 compared with Epi or Endo) when CL changed from 400 to 1000 ms. In situ, we registered activation-recovery intervals (ARIs) in bipolar electrograms obtained from different myocardial layers in conditions of AV block and His-bundle pacing. At all CLs from 300 to 2000 ms, ARIs were equal in all myocardial layers from Epi to Endo cells. Steady-state ARIs coincided with APD of M cells registered in vitro in the physiological range of CL from 300 to 700 ms. When CL was changed from 300 to 1000 ms, the ARI followed the rapid time course typical of M cells (t90 = 2.6 +/- 0.5, 2.2 +/- 0.4, 2.5 +/- 0.4, 2.6 +/- 0.5, and 2.3 +/- 0.4 minutes for Epi; 3-, 5-, and 7-mm sub-Epi; and Endo cells, respectively). CONCLUSIONS In contrast to in vitro results, there is no significant difference in repolarization among myocardial layers in the intact normal canine heart.

Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates.

Background—We hypothesized that administration of the HCN2 gene to the left bundle-branch (LBB) system of intact dogs would provide pacemaker function in the physiological range of heart rates. Methods and Results—An adenoviral construct incorporating HCN2 and green fluorescent protein (GFP) as a marker was injected via catheter under fluoroscopic control into the posterior division of the LBB. Controls were injected with an adenoviral construct of GFP alone or saline. Animals were monitored electrocardiographically for up to 7 days after surgery, at which time they were anesthetized and subjected to vagal stimulation to permit emergence of escape pacemakers. Hearts were then removed and injection sites visually identified and removed for microelectrode study of action potentials, patch clamp studies of pacemaker current, and/or immunohistochemical studies of HCN2. For 48 hours postoperatively, 7 of 7 animals subjected to 24-hour ECG monitoring showed multiple ventricular premature depolarizations and/or ventricular tachycardia attributable to injection-induced injury. Thereafter, sinus rhythm prevailed. During vagal stimulation, HCN2-injected dogs showed rhythms originating from the left ventricle, the rate of which was significantly more rapid than in the controls. Excised posterior divisions of the LBB from HCN2-injected animals manifested automatic rates significantly greater than the controls. Isolated tissues showed immunohistochemical and biophysical evidence of overexpressed HCN2. Conclusions—A gene-therapy approach for induction of biological pacemaker activity within the LBB system provides ventricular escape rhythms that have physiologically acceptable rates. Long-term stability and feasibility of the approach remain to be tested.

Cellular electrophysiologic properties of old canine atria provide a substrate for arrhythmogenesis

OBJECTIVE The incidence of atrial fibrillation increases with age. We hypothesized that aging-associated changes in the atrial action potential (AP) and conduction velocity provide a substrate for abnormal conduction and arrhythmogenesis. METHODS We used microelectrode techniques to record AP from the endocardium of the right atrial wall of dogs aged 1-5 (adult) and >8 years (old). Conduction velocity was measured between two microelectrodes 3-10 mm apart. Histological study was carried out to assess fibrosis. RESULTS Whereas resting potential, AP amplitude and V(max) did not differ with age, the plateau was more negative and AP duration was longer in old tissue. The L-type calcium current (I(Ca,L)) agonist Bay K8644 (10(-8)-10(-6) mol/l) elevated the plateau and shortened APD more in old than in adult, such that AP contour in old atria approached that of adult. In contrast, the I(Ca,L) blocker nisoldipine (10(-8)-10(-5) mol/l) depressed the plateau in adult and had no effect in old. There was no difference between the two groups in conduction velocity of normal beats, whereas for early premature impulses, reduced conduction velocity and a wider time window manifesting slow conduction were detected in old in comparison to adult tissue. A twofold increase in the amount of fibrous tissue was detected in old atria. CONCLUSIONS Our data show significant differences in contour of AP in adult and old atria. The responses to Bay K8644 and nisoldipine suggest a decreased I(Ca,L) in old atrial tissue. The alterations in AP contour and increased fibrosis may be responsible for slower conduction of early premature beats in old atria. The age-related changes in conduction of premature beats are consistent with those observed in patients with paroxysmal atrial fibrillation and may contribute to the greater propensity to atrial fibrillation in the aged.

Evolution and resolution of long-term cardiac memory.

BACKGROUND Cardiac memory (CM) refers to T-wave changes induced by ventricular pacing or arrhythmia that accumulate in magnitude and duration with repeated episodes of abnormal activation. We report herein the kinetics of long-term CM and its association with the ventricular action potential. METHODS AND RESULTS Dogs were paced from the ventricles at rates of 110 to 120 bpm for approximately 3 weeks. CM characterized by gradual sinus rhythm T vector rotation toward the paced QRS vector evolved in all dogs regardless of pacing site (left ventricular [LV] anterior apex or base, posterior LV, or right ventricular free wall). Cardiac hemodynamics and myocardial flow (microsphere studies) were unaltered by the pacing. Recovery time for the memory T wave to return to control increased with duration of the previous pacing. The protein synthesis inhibitor cycloheximide markedly (P<.05) and reproducibly attenuated evolution of CM. When pacing was performed from the atrium, CM did not occur. Standard microelectrode techniques were used to study action potential from the LV free wall of control and CM dogs. CM was associated with increased action potential duration in epicardial and endocardial but not midmyocardial cells, significantly altering the transmyocardial gradient for repolarization. CONCLUSIONS CM is a dynamic process for which the final T vector is predicted by the paced QRS vector and which is associated with significant changes in epicardial and endocardial but not midmyocardial cell action potential duration, such that the transmural gradient of repolarization is altered. It is unaccompanied by evidence of altered hemodynamics or flow, requires a change in pathway of activation, and appears to require new protein synthesis.

Repolarization Gradients in the Canine Left Ventricle Before and After Induction of Short-Term Cardiac Memory

Background—Questions remain about the contributions of transmural versus apicobasal repolarization gradients to the configuration of the T wave in control settings and after the induction of short-term cardiac memory. Methods and Results—Short-term cardiac memory is seen as T-wave changes induced by altered ventricular activation that persists after restoration of sinus rhythm. We studied cardiac memory in anesthetized, open-chest dogs paced from the ventricle for 2 hours. Unipolar electrograms were recorded from as many as 98 epicardial and 144 intramural sites, and activation times and activation-recovery intervals (ARIs) were measured. In separate experiments, epicardial monophasic action potentials were recorded. We found no appreciable left ventricular intramural gradients in repolarization times (activation time+ARI) in either control conditions or after the induction of memory. In controls, there was a left ventricular apicobasal gradient, with the shortest repolarization times in anterobasal regions and longest repolarization times posteroapically. After induction of memory, repolarization times shortened uniformly throughout the ventricular wall. Monophasic action potential duration at 90% repolarization decreased by ≈10 ms after induction of memory. Conclusions—In the intact canine left ventricle at physiological rates, there is no transmural gradient in repolarization. Apicobasal gradients in repolarization time, with shortest repolarization times in anterobasal areas and longest repolarization times in posteroapical regions, are important in the genesis of the T wave. Repolarization times and monophasic action potentials at the 90% repolarization level shorten after the induction of memory. The deeper T wave in the ECG after induction of memory may be explained by the more rapid phase 3 of the action potential.

Effects of Quinidine on Repolarization in Canine Epicardium, Midmyocardium, and Endocardium: I. In Vitro Study

BACKGROUND In the companion article, we report a significant difference in quinidine effects on the action potential duration between surface (epicardial and endocardial) cells and midmyocardial cells (M cells) of canine left ventricle in vitro. This article considers two questions raised by the previous study: (1) Are the complex quinidine effects in vitro reflected in its actions on the heart in situ? (2) What are the cellular determinants of quinidine effects on QT interval in ECG? METHODS AND RESULTS We used plunge and surface electrodes to measure activation-recovery intervals (ARIs) of bipolar electrograms obtained from epicardium, endocardium, and midmyocardium (3, 5, and 9 mm from epicardium) of canine left ventricle in conditions of AV block and right ventricular pacing. Quinidine was infused continuously; its plasma level increased from 1.6+/-0.1 microg/mL at 30 minutes to 7.6+/-0.7 microg/mL at 180 minutes. At cycle lengths (CLs) from 300 to 1500 ms, there was no ARI gradient across the ventricular wall before and during quinidine infusion. At a CL of 300 ms, therapeutic concentrations of quinidine prolonged ARIs and QT intervals. At a CL of 1500 ms, ARIs were significantly prolonged at low quinidine concentrations. With an increase of quinidine concentration, this effect subsided and disappeared. CONCLUSIONS In situ, quinidine-induced prolongation of repolarization is uniform in all myocardial layers and follows the pattern observed in M cells in vitro. The ability of quinidine in therapeutic concentrations to prolong repolarization at rapid heart rates can contribute to its antiarrhythmic efficacy.

Epicardial border zone overexpression of skeletal muscle sodium channel, SkM1, normalizes activation, preserves conduction and suppresses ventricular arrhythmia: an in silico, in vivo, in vitro study

Background— In depolarized myocardial infarct epicardial border zones, the cardiac sodium channel (SCN5A) is largely inactivated, contributing to low action potential upstroke velocity (&OV0312; max), slow conduction, and reentry. We hypothesized that a fast inward current such as the skeletal muscle sodium channel (SkM1) operating more effectively at depolarized membrane potentials might restore fast conduction in epicardial border zones and be antiarrhythmic. Methods and Results— Computer simulations were done with a modified Hund-Rudy model. Canine myocardial infarcts were created by coronary ligation. Adenovirus expressing SkM1 and green fluorescent protein or green fluorescent protein alone (sham) was injected into epicardial border zones. After 5 to 7 days, dogs were studied with epicardial mapping, programmed premature stimulation in vivo, and cellular electrophysiology in vitro. Infarct size was determined, and tissues were immunostained for SkM1 and green fluorescent protein. In the computational model, modest SkM1 expression preserved fast conduction at potentials as positive as −60 mV; overexpression of SCN5A did not. In vivo epicardial border zone electrograms were broad and fragmented in shams (31.5±2.3 ms) and narrower in SkM1 (22.6±2.8 ms; P=0.03). Premature stimulation induced ventricular tachyarrhythmia/fibrillation >60 seconds in 6 of 8 shams versus 2 of 12 SkM1 (P=0.02). Microelectrode studies of epicardial border zones from SkM1 showed membrane potentials equal to that of shams and &OV0312; max greater than that of shams as membrane potential depolarized (P<0.01). Infarct sizes were similar (sham, 30±2.8%; SkM1, 30±2.6%; P=0.86). SkM1 expression in injected epicardium was confirmed immunohistochemically. Conclusions— SkM1 increases &OV0312; max of depolarized myocardium and reduces the incidence of inducible sustained ventricular tachyarrhythmia/fibrillation in canine infarcts. Gene therapy to normalize activation by increasing &OV0312; max at depolarized potentials may be a promising antiarrhythmic strategy.

An alpha-1-adrenergic receptor subtype is responsible for delayed afterdepolarizations and triggered activity during simulated ischemia and reperfusion of isolated canine Purkinje fibers.

BACKGROUND We used standard microelectrode techniques to study delayed afterdepolarizations and triggered activity induced by alpha 1-adrenergic stimulation in canine Purkinje fibers in the setting of simulated ischemia and reperfusion. METHODS AND RESULTS The ischemic environment included 10.8 mM [Ca2+]o, 10 mM [K+]o, 40-50 mm Hg PO2, 20 mM lactate (pH 6.7), and 1 x 10(-7) M phenylephrine. During ischemia, there was the variable occurrence of abnormal automaticity, early afterdepolarizations, and delayed afterdepolarizations. During reperfusion, 100% of preparations manifested delayed afterdepolarizations and 40% manifested triggered activity. Decreasing PO2 to less than 20 mm Hg markedly reduced the incidence of delayed afterdepolarizations and triggered activity, as did increasing PO2 to values of more than 90 mm Hg. WB 4101, an alpha 1-subtype-selective competitive blocker that antagonizes the effects of alpha 1-agonists to induce phosphoinositide metabolism and increase [Ca2+]i, significantly reduced the incidence of delayed afterdepolarizations and triggered activity. In contrast, the alpha 1-subtype-selective blocker chloroethylclonidine, an alkylating agent, had no effect on afterdepolarizations or triggered activity. CONCLUSIONS Our results indicate that a specific alpha 1-adrenergic pathway is involved in the induction of triggered activity in the setting of ischemia and reperfusion and suggest that interventions used to block this specific pathway have the potential to be antiarrhythmic. They also emphasize the importance of the magnitude of hypoxia in the expression of the arrhythmias.

Increased Cell-Cell Coupling Increases Infarct Size and Does not Decrease Incidence of Ventricular Tachycardia in Mice.

Increasing connexin43 (Cx43) gap junctional conductance as a means to improve cardiac conduction has been proposed as a novel antiarrhythmic modality. Yet, transmission of molecules via gap junctions may be associated with increased infarct size. To determine whether maintaining open gap junction channels impacts on infarct size and induction of ventricular tachycardia (VT) following coronary occlusion, we expressed the pH- and voltage-independent connexin isoform connexin32 (Cx32) in ventricle and confirmed Cx32 expression. Wild-type (WT) mice injected with adenovirus-Cx32 (Cx32inj) were examined following coronary occlusion to determine infarct size and inducibility of VT. There was an increased infarct size in Cx32inj hearts as compared to WT (WT 22.9 ± 4%; Cx32inj 44.3 ± 5%; p < 0.05). Programmed electrical stimulation showed no difference in VT inducibility in WT and Cx32inj mice (VT was reproducibly inducible in 55% of shams and 50% of Cx32inj mice (p > 0.05). Following coronary occlusion, improving cell–cell communication increased infarct size, and conferred no antiarrhythmic benefit.

Effects of Quinidine on Repolarization in Canine Epicardium, Midmyocardium, and Endocardium

BACKGROUND The antiarrhythmic action of quinidine is associated with a slowing of conduction and prolongation of repolarization. The latter effect has no consistent correlation with quinidine actions on action potential duration (APD) in isolated tissue experiments. To enhance our understanding of the mechanisms of quinidine action, we studied its effect on APD in canine epicardial, midmyocardial, and endocardial tissues. METHODS AND RESULTS Standard microelectrode techniques were used to study the effects of quinidine 2.5 to 20 micromol/L on APD in ventricular epicardial, endocardial, and transmural (M-cell) slabs at cycle lengths (CLs) from 300 to 4000 ms. Qualitatively different time courses of actions and concentration- and rate-dependent effects were seen in M cells compared with the others. In endocardium and epicardium, quinidine induced monotonic and concentration-dependent APD prolongation at all CLs. In contrast, the effects of quinidine in M cells varied from prolongation to shortening, depending on duration of superfusion, concentration, and CL. Experiments with E4031 and TTX suggested that in M cells, quinidine-induced APD lengthening was attributable to block of delayed rectifier potassium current and APD shortening was due to inhibition of TTX-sensitive steady-state sodium current. CONCLUSIONS In vitro, there is a significant difference of quinidine effects in M cells versus epicardial and endocardial cells that appears to reflect differences in the contributions of specific ion channels to the APD at the three sites. The differences may influence the actions of quinidine on repolarization of the heart in situ and determine both the proarrhythmic and antiarrhythmic actions of the drug.