Failure of Impulse Propagation in a Mathematically Simulated Ischemic Border Zone: Influence of Direction of Propagation and Cell-to-Cell Electrical Coupling

Propagation in a Modeled Ischemic Border Zone. Introduction: It is suggested that heterogeneous extracellular potassium concentration, cell-to-cell coupling, and geometric nonuniformities of the ischemic border zone contribute to the incidence of unidirectional block and subsequent development of lethal ventricular arrhythmias.
Method and Results: A discrete electrical network was used to model a single cardiac fiber with a [K⁺]_e gradient characteristic of an ischemic border zone. Directional differences in propagation were evaluated by creating discrete regions with increased gap junctional resistance within the [K⁺]_e gradient. Furthermore, the effect of homogeneity/heterogeneity of cell length on impulse propagation through the [K⁺]_e gradient in the presence of increased gap junctional resistance was evaluated. The results indicate that failure of impulse propagation occurs at the junction between partially uncoupled and normally coupled cells. Furthermore, propagation failure was more likely to occur as the impulse propagated from a region of high [K⁺]_e to low [K⁺]_e. Heterogeneity in cell length contributes to the variability in the occurrence of unidirectional and bidirectional block.
Conclusions: The onset of cellular uncoupling in an ischemic border zone may interact with the inherent [K⁺]_e gradient leading to unidirectional conduction block. This mechanism may be important for the generation of reentrant arrhythmias at the ischemic border zone.     

Alpha1-Adrenergic Receptor Modulation of Repolarization in Canine Purkinje Fibers

Alpha-Agonists and Repolarization. Introduction: Alpha-adrenergic receptor stimulation increases contractility and prolongs repolarization. These effects are modulated by α₁-adrenergic receptor-mediated inhibition of transsarcolemmal potassium currents.
Methods and Results: We used standard microelectrode techniques to study the actions of 4-aminopyridine (4-AP), which blocks the transient outward current, I_to, and WAY-123,398, which blocks the delayed rectifier, I_k, on canine Purkinje fiber action potential prolongation induced by phenylephrine. At a basic cycle length of 1 second, phenylephrine (0.1 to 10 μ) dose-dependently prolonged action potential duration at 90% repolarization (APD₉₀) from 331 ± 10 msec to 400 ± 12 msec (P < 0.05) at phenylephrine, 10 μ. Phenylephrine did not change phase 1 or plateau height. 4-AP (0.1 mM) decreased phase 1 magnitude, shifted plateau height to more positive potentials (from 0.1 ± 1.8 mV to 14.3 ± 1.1 mV [P < 0.05]), and shortened APD₉₀ from 318 ± 9 msec to 294 ± 8 msec (P < 0.05). 4-AP did not block phenylephrine effects on APD₉₀, which increased, at 10 μ phenylephrine, from 294 ± 8 msec to 342 ± 6 msec (P < 0.05). In contrast, WAY-123,398 (0.1 μ) prolonged APD₉₀ from 360 ± 6 msec to 452 ± 6 msec (P < 0.05), and had no effect on plateau height. In the presence of WAY-123,398, phenylephrine no longer increased APD_9o.
Conclusion: (1) Agents that block I_to shorten APD in Purkinje fibers; and (2) the α-agonist mediated increase of APD in canine Purkinje fibers can be explained by inhibition of I_k.     

I(Ks) block by HMR 1556 lowers ventricular defibrillation threshold and reverses the repolarization shortening by isoproterenol without rate-dependence in rabbits

Introduction: The slow delayed rectifier K⁺ current (I_Ks) contributes little to ventricular repolarization at rest. It is unclear whether I_Ks plays a role during ventricular fibrillation (VF) or ventricular repolarization at rapid rates during β-adrenergic stimulation.
Methods and Results: In an in vivo rabbit model, we evaluated the effects of HMR 1556 (1 mg Kg⁻¹+ 1 mg kg ⁻¹ hr ⁻¹ i.v.), a selective I_Ks blocker, on monophasic action potential duration at 90% repolarization (MAPD₉₀), ventricular effective refractory period (VERP), and defibrillation threshold (DFT). In perfused rabbit hearts, the effects of HMR 1556 (10 and 100 nM) in the presence of isoproterenol (5 nM) on MAPD₉₀ and VERP were studied at cycle lengths (CLs) 200–500 msec. In vivo , HMR 1556 prolonged MAPD₉₀ by 6 ± 1 msec at CL 200 msec (P < 0.01, n = 6), lowered DFT from 558 ± 46 V to 417 ± 31 V (P < 0.01), and decreased the coefficient of variation in the VF inter-beat deflection intervals from 8.9 ± 0.6% to 6.5 ± 0.4% (P < 0.05) compared with control. In perfused rabbit hearts, isoproterenol shortened MAPD₉₀ by 5 ± 1 msec at CL 200 msec and 11 ± 4 msec at CL 500 msec (P < 0.05, n = 7). This shortening was reversed by HMR 1556 (P < 0.05), and both effects were rate-independent.
Conclusion: I_Ks block increases VF temporal organization and lowers DFT, and I_Ks that is activated following β-adrenergic stimulation contributes to ventricular repolarization without rate dependence.     

Action Potential Duration, Rate of Stimulation, and Intracellular Sodium

In the first section of this short review the change of the cardiac action potential (APD) with the rate of stimulation under physiological conditions is described and mechanistically analyzed. A fast phase of adaptation is mainly caused by changes in gating characteristics of ionic currents, and rapid modulation of the Na⁺/Ca²⁺ exchanger. The slower phase is largely conditioned by incomplete recovery from inactivation of the late Na⁺ current (late I_Na) and changes in ion concentrations of [K⁺]_e, [Na⁺]_i, and [Ca²⁺]_i, which cause secondary changes in the permeation and the gating of ion channels and flux through transporters. In a second section, an analysis is presented of the rate dependence of APD in pathological conditions and its importance in the genesis of arrhythmias in hypertrophy, heart failure, congenital, and acquired LQT syndromes is summarized. The role of the late I_Na, Na⁺, and Ca²⁺ overload is emphasized. Special attention is given to the paradoxical transient lengthening of APD in LQT3 syndrome for the sudden increase in rate in this setting. The third section consists of a short commentary on Na⁺ and Ca²⁺ overload and drugs which block the late I_Na.     

Differential Effects of Cytochalasin D and 2, 3 Butanedione Monoxime on Isometric Twitch Force and Transmembrane Action Potential in Isolated Ventricular Muscle: Implications for Optical Measurements of Cardiac Repolarization

Cytochalasin D Induced Cardiac Contraction Failure. Introduction : 2,3-Butanedione monoxime (BDM) has been widely used to inhibit contraction during optical recordings of cardiac membrane voltage changes, even though it markedly abbreviates cardiac action potentials.
Methods and Results : We compared the effects of BDM and of the F-actin disrupter cytochalasin D (cyto D) on isometric twitch force and transmembrane action potentials in isolated canine right ventricular trabeculae superfused with Tyrode's solution (2 mmol/L CaCl₂, 37°C) and stimulated at 0.5 Hz. BDM at 10 mmol/L and cyto D at 80 μmol/L were equally effective in reducing peak isometric force to 10%± 3% (n = 6; mean ± SEM) and 8%± 1% (n = 8), respectively. Neither agent significantly altered resting tension. While 10 mmol/L BDM markedly shortened the action potential duration at 90% repolarization (APD₉₀) from 198 ± 7 msec to 146 ± 9 msec ( P < 0.001), 80 μmol/L cyto D had no significant effects on APD₉₀ or on any other action potential parameter. The effects of BDM on peak isometric force and APD were completely reversible after 15 minutes of washout, whereas in the cyto D group contractile force continued to be reduced (13%± 3%) and action potential characteristics did not show significant changes from control values after a 60-minute period of superfusion with cyto D-free Tyrode's solution.
Conclusion : We conclude that cyto D should be considered an alternative excitation-contraction uncoupler for optical mapping studies of cardiac repolarization.     

Potent Antiarrhythmic Effects of Chronic Amiodarone in Canine Pulmonary Vein Sleeve Preparations

Objectives: To examine the effects of chronic amiodarone on the electrophysiology of canine pulmonary vein (PV) sleeve preparations and left ventricular wedge preparation.
Background: Amiodarone is commonly used for the treatment of ventricular and supraventricular arrhythmias. Ectopic activity arising from the PV plays a prominent role in the development of atrial fibrillation (AF).
Methods: Standard microelectrode techniques were used to evaluate the electrophysiological characteristics of superfused PV sleeve (left superior or inferior) and arterially perfused left ventricular (LV) wedge preparations isolated from untreated and chronic amiodarone-treated dogs (amiodarone, 40 mg/kg daily for 6 weeks).
Results: In PV sleeves, chronic amiodarone (n = 6) induced a significant increase in action potential duration at 90% repolarization (APD₉₀) and a significant use-dependent reduction in V_max leading to 1:1 activation failure at long cycle lengths (basic cycle length of 124 ± 15 ms in control vs 420 ± 320 ms after chronic amiodarone [P < 0.01]). Diastolic threshold of excitation increased from 0.3 ± 0.2 to 1.8 ± 0.7 mA (P < 0.01). Delayed and late phase 3 early afterdepolarizations and triggered activity could be induced in PV sleeve preparations using acetylcholine (ACh, 1 μM), high calcium ([Ca²⁺]_o= 5.4 mM), isoproterenol (Iso, 1 μM), or their combination in 6 of 6 untreated PV sleeves, but in only 1 of 5 chronic amiodarone-treated PV sleeve preparations. V_max, conduction velocity, and 1:1 activation failure were much more affected in PV sleeves versus LV wedge preparations isolated from amiodarone-treated animals.
Conclusions: The results point to potent effects of chronic amiodarone to preferentially suppress arrhythmogenic substrates and triggers arising from the PV sleeves of the dog.     

Azimilide (NE-10064) Can Prolong or Shorten the Action Potential Duration in Canine Ventricular Myocytes:

Azimilide Effects on Membrane Voltage and Current. Introduction : Azimilide (NE-10064) has antiarrhythmic and antifibrillatory effects in canine models of ventricular arrhythmia. The goal of the present study was to examine the effects of azimilide on action potential and membrane currents of canine ventricular myocytes.
Methods and Results : Membrane voltage and current were recorded using the whole cell, patch clamp method. Azimilide at 1 /μM induced a consistent prolongation of action potential duration (APD): on average APD₉₀ was prolonged by 25% and 17% at stimulation rates of 0.33 and 1 Hz, respectively. Elevating the drug concentration to 5 μM induced APD prolongation in some cells but APD shortening in the others at 0.33 Hz, and a consistent APD shortening at 1 Hz. Azimilide suppressed the following currents (K_d in parenthesis): I_Kr (< 1 μM at -20 mV), I_Ks, (1.8 μM at +30 mV), L-type Ca current (17.8 μM at +10 mV), and Na current (19 μM at -40 mV). Azimilide was a weak blocker of the transient outward and inward rectifier currents (K_d≥ 50 μM at +50 and -140 mV, respectively). Azimilide blocked I_Kr, I_Ks, and I_Na in a use-dependent manner. Furthermore, azimilide reduced a slowly inactivating component of Na current that might be important for maintaining the action potential plateau in canine ventricular myocytes.
Conclusion : Azimilide has variable effects on APD in canine ventricular myocytes due to Us blocking effects on multiple currents with different potencies. Its Class III antiarrhythmic action is most likely seen at low concentrations (< 5 μM).     

Quantitative Reconstruction of Cardiac Electromechanics in Human Myocardium:

Introduction: Myocytes from normal and failing myocardium show significant differences in electromechanical behavior. Mathematical modeling of the behavior provides insights into the underlying physiologic and pathophysiologic mechanisms. Electromechanical models of cardiomyocytes exist for various species, but models of human myocytes are lacking.
Methods and Results: A mathematical model of electromechanics in normal and failing cardiac myocytes in humans was created by assembly and adaptation of parameters of an electrophysiologic model at the level of single cells and a force development model at the level of the sarcomere. The adaptation was performed using data from recent studies of ventricular myocytes and myocardium. The model was applied to quantitatively reconstruct measurement data from different experimental studies of normal and failing myocardium. Several simulations were performed to quantify the transmembrane voltage V_m , intracellular concentration of calcium[ Ca²⁺ ] _i , the [ Ca²⁺ ] _i –force relationship, and force transients. Furthermore, frequency dependencies and restitution of action voltage duration to 90% recovery APD₉₀, peak [ Ca²⁺ ] _i , duration to 50% force recovery FD₅₀, and peak force were determined.
Conclusion: The presented mathematical model was capable of quantitatively reconstructing data obtained from different studies of electrophysiology and force development in normal and failing myocardium of humans. In future work, the model can serve as a component for studying macroscopic mechanisms of excitation propagation, metabolism, and electromechanics in human myocardium. (J Cardiovasc Electrophysiol, Vol. 14, pp. S210-S218, October 2003, Suppl.)     

Electrophysiologic Features of Torsades de Pointes:

Torsades de Pointes in the Isolated Rabbit Heart. Introduction : The exact electrophysiologic mechanism of torsades de pointes (TdP) is under intense investigation. No isolated animal heart model of this particular arrhythmia exists.
Methods and Results : In isolated rabbit hearts, TdP was induced by means of bradyeardia in the presence of a high concentration of d-sotalol (10⁻⁴ M) and shortly after lowering the concentration of potassium and magnesium in the perfusate. Multiple simultaneous epicardial and endocardial monophasic action potentials (MAPs) and volume-conducted 12-lead ECGs were recorded. d-Sotalol prolonged repolarization and increased dispersion of ventricular repolarization compared to baseline recordings. With the onset of low potassium and magnesium concentrations, repolarization was further prolonged and dispersion of repolarization was further increased followed by the occurrence of early afterdepolariZations (EADs) in the majority of MAP recordings, i.e., at both endocardial and epicardial locations of both ventricles, upon increase of EAD amplitude, triggered arrhythmias with TdP of up to 42 heats ensued in 10 of 11 hearts studied. MAP duration at 90% repolarization (APD₉₀), dispersion of APD₉₀, and the incidence of EADs as well as dispersion of the QT interval and T wave area were significantly higher in heats triggering higemini, couplets, or runs of TdP.
Conclusion : TdP observed in this new isolated heart model was associated with markedly increased dispersion of ventricular repolarization and the occurrence of EADs in multiple locations of the heart. TdP is initiated when the amplitude of an EAD reaches threshold for initiation of the first beat of an episode.     

Measurements of Electrophysiological Effects of Components of Acute Ischemia in Langendorff-Perfused Rat Hearts Using Voltage-Sensitive Dye Mapping

Introduction: This study was carried out to evaluate optical mapping in the presence of cytochalasin-D as a method for measuring electrophysiological responses in general, and in particular the responses to acute ischemia in the Langendorff-perfused rat heart. Cytochalasin-D is commonly used to reduce contraction for the purpose of suppressing motion artifacts in voltage-sensitive dye recordings of cardiac membrane potential.
Methods and Results: Observations using optical mapping were complemented by recordings of the surface electrogram to provide information independent of the optical measurements. Perfusion of Langendorff-perfused rat hearts with 3 μM cytochalasin-D resulted in a 24% prolongation of the QT interval of surface electrograms indicating that cytochalasin-D prolongs the rat ventricular action potential. Individual components of the electrophysiological response to acute ischemia were globally induced as follows: (1) opening of K_ATP channels was induced by perfusion of 2 μM P-1075, (2) accumulation of extracellular K⁺ was simulated by increasing perfusate [K⁺] to 12 mM, and (3) acidosis was simulated by reducing perfusate pH to 6.5. The responses to these interventions could be reliably documented using optical recordings, as well as from surface electrograms. Whole-cell patch clamp measurements on isolated rat ventricular myocytes indicate that cytochalasin-D produces an approximately 2.5-fold increase in P-1075-induced I_K,ATP.
Conclusion: These results provide the necessary background information for interpreting electrophysiological measurements during acute ischemia in the presence of cytochalasin-D.     

Comparison of the Rate-Dependent Properties of the Class III Antiarrhythmic Agents Azimilide (NE-10064) and E-4031

Rate Dependence of Azimilide and E-4031. Introduction : Reverse rate-dependence, a lessening in Class III antiarrhythmic agent action potential duration (APD) prolongation as heart rate is increased, has been proposed to be related to an incomplete deactivution of the slow component (I_Ks) of the delayed rectifier K⁺ current (I_K). The rate-dependent properties of block of I_K by azimilide were compared to E-4031, which selectively blocks the rapid component (I_Kr) of I_k, in guinea pig ventricular muscle.
Methods and Results : Azimilide prolonged APD in isolated papillary muscles in a concentration-dependent manner and to a greater degree than E-4031. Both agents prolonged APD less at fast than slow rates, consistent with a similar reverse rate-dependent effect. Isolation of azimilide block of I_Ks by subtraction of APD during E-4031 plus azimilide from E-4031 alone revealed rate-independent prolongation of APD. In voltage clamp experiments on single ventricular myocytes, activation of I_ks was similar following 30 seconds of conditioning pulses of physiological duration (125 to 200 msec) with either a fast (cycle length 250 msec) or slow (cycle length 2000 msec) rate. The block of I_Ks by azimilide 3 μM was greater after a fast conditioning pulse train.
Conclusions : Selective block of I_ks prolongs APD in a rate-independent manner. In voltage clamped myocytes, no evidence of a rate-dependent accumulation of I_Ks was observed. These findings support a mechanism of reverse rate-dependent APD prolongation by Class III antiarrhythmic agents that block I_Kr independent of I_Ks.     

The Phosphodiesterase III Inhibitor Olprinone Decreases Sensitivity of Rat Kupffer Cells to Endotoxin

Background: Sensitivity of Kupffer cells to endotoxin [lipopolysaccharide (LPS)] and overproduction of tumor necrosis factor-α (TNF-α) are critical for progression of alcoholic liver injury. Therefore, suppression of TNF-α should prove useful for treatment of alcoholic liver injury. However, a transient increase of intracellular calcium ([Ca²⁺]_i) is required for LPS-induced TNF-α production by the macrophage cell line. The phosphodiesterase III inhibitor olprinone has been shown to suppress [Ca²⁺]_i level in vascular smooth muscle cells. Accordingly, the purpose of this study was to determine whether olprinone could prevent sensitization of Kupffer cells to endotoxin.
Methods: Kupffer cells were isolated by collagenase digestion and differential centrifugation. LPS was added to Kupffer cells 24 hr after incubation with or without olprinone (0.1 μmol/liter). After addition of LPS (10 μg/ml) to culture media, [Ca²⁺]_i was measured using a fluorescent indicator, fura-2.
Results: LPS increased [Ca²⁺]_i of Kupffer cells in control rats from basal levels (28 ± 4 nmol/liter) to 280 ± 14 nmol/liter. This increase was blunted by olprinone (91 ± 8 nmol/liter). Similarly, olprinone diminished the LPS (1 μg/ml)-induced TNF-α production by Kupffer cells by 30% (2220 ± 116 vs. 1386 ± 199 pg/ml; p < 0.05).
Conclusions: These results indicate that olprinone decreases sensitivity of Kupffer cells to endotoxin.     

Function, Regulation, Pharmacology, and Molecular Structure of ATP-Sensitive K+ Channels in the Cardiovascular System

K_ATP Channels in Cardiovascular System. ATP-sensitive K⁺ (K_ATP) channels are inhibited by intracellular ATP and activated by intracellular nucleoside diphosphates, and thus provide a link between cellular metabolism and excitability. K_ATP channels are widely distributed in various tissues and may be associated with diverse cellular functions. In the heart, the K_ATP channel appears to be activated during ischemic or hypoxic conditions and may be responsible for the increase of K⁺ efflux and shortening of the action potential duration. Therefore, opening of this channel may result in cardioprotective as well as proarrhythmic effects. In the vascular smooth muscle, the K_ATP channel is believed to mediate the relaxation of vascular tone. Thus, K_ATP channels play important regulatory roles in the cardiovascular system. Furthermore, K_ATP channels are the targets of two important classes of drugs, i.e., the antidiabetic sulfonylureas, which block the channels, and a series of vasorelaxants called "K⁺ channel openers," which tend to maintain the channels in an open conformation. Recently, the molecular structure of K_ATP channels has been clarified. The K_ATP channel in pancreatic β-cells is a complex composed of at least two subunits, a member of inwardly rectifying K⁺ channels and a sulfonylurea receptor. Subsequently, two additional homologs of the sulfonylurea receptor, which form cardiac and smooth muscle type K_ATP channels, respectively, have been reported. Further works are now in progress to understand the molecular mechanisms of K_ATP channel function.     

Do HbSS erythrocytes lose KCl in physiological conditions?

KCl cotransporter activity in sickle (HbSS) red blood cells (RBCs) was measured in cells suspended in 'simple' physiological saline, saline augmented with inorganic salts, and autologous plasma. Our results showed that the transporter was only functioning at 20% of the level of cells in saline when cells were resuspended in autologous plasma. Kinetic analysis of the data showed that plasma decreased both V _max and K _m for K⁺ of the transporter. The plasma factor(s) responsible was heat-stable and dialysable (i.e. size &60; 10 kD).
Adding magnesium, calcium, inorganic phosphate or bicarbonate to 'simple' saline to mimic the effect of plasma revealed that Mg²⁺ and Ca²⁺ had no significant effect at physiological concentrations. P_i was not effective at 1.1 m M  , but did inhibit significantly (42±2%) at 5.6 m M  . HCO⁻₃   had a major inhibitory effect on K⁺ influx when added to saline, and was identified as the principal candidate for the plasma effect.
We suggest bicarbonate may play a significant role in modifying KCl cotransport, and hence HbSS cell volume in vivo . It acts by altering the set point of the transporter via the signalling systems involved in its regulation.     

Reduced Inward Rectifying and Increased E-4031-Sensitive K+ Current Density in Arrhythmogenic Subendocardial Purkinje Myocytes from the Infarcted Heart

Outward Currents in Purkinje Cells from 48-Hour Infarcted Heart. Introduction : Subendocardial Purkinje myocytes from the 4K-hour infarcted heart (IZPCs) have reduced resting potentials, possibly due to altered inwardly rectifying K⁺ currents I_KI. Abnormal depolarization-activated outward K⁺ currents could contribute to long triangularly shaped action potentials of IZPCs.
Methods and Results : We used whole cell patch recordings to compare cesium-sensitive I_KI and 4-aminopyridine (4-AP)-resistant, noninactivating sustained I_K between normal Purkinje myocytes (NZPCs) and IZPCs. IZPCs showed decreased net membrane currents. Two IZPC groups were distinguished, based on 4-AP-resistant outward K⁺ currents. IZPC-I had isochronal I_KI current-voltage relations similar to NZPCs whereas IZPC-II showed significantly reduced I_KI and increased outward plateau currents. To study the sustained I_K in the presence of the Class III antiarrhythmic agent E-4031, a two-pulse protocol was used to inactivate transient outward currents, followed by step depolarizations. E-4031-sensitive currents were significantly greater in IZPCs at depolarized potentials (> 0 mV). Similar to NZPCs, IZPC E-4031 currents showed time dependence during depolarization, lack of rectification at positive steps, and voltage-dependent recovery from block.
Conclusion : Decreased I_KI may account for reduced resting potentials in IZPCs. E-4031-sensitive currents in NZPCs, unlike those in canine ventricular myocytes, are sensitive to 4-AP and are larger in IZPCs.     

Bradykinin is a mediator, but unlikely a trigger, of antiarrhythmic effects of ischemic preconditioning

Objective : Brief reversible ischemic episodes (ischemic preconditioning, IPC) protect the heart against arrhythmias during a subsequent prolonged low-flow ischemia. We have recently shown that this protection involves release of bradykinin, activation of bradykinin B₂ receptors followed by opening of sarcolemmal, but not mitochondrial ATP-sensitive K⁺ channels. The goal of this study was to clarify a trigger and/or mediator role of bradykinin in the antiarrhythmic effects of IPC during low-flow ischemia.
Methods : Isolated perfused rat hearts underwent 60 minutes of low-flow ischemia induced by reducing perfusion pressure followed by 60 minutes of reperfusion. Preconditioning was induced by 2 × 5 minutes episodes of zero-flow ischemia. In yet other groups, preconditioned or nonpreconditioned hearts were treated either with bradykinin (10 nmol/L) or with HOE 140 (bradykinin B₂ receptor antagonist, 100 nmol/L).
Results : IPC reduced the number of ventricular premature beats, as well as the incidence of ventricular tachycardia and of ventricular fibrillation during low-flow ischemia. In addition, this protection was abolished by HOE 140 given during low-flow ischemia. Pharmacological preconditioning using short bradykinin perfusion instead of IPC did not show antiarrhythmic effects. However, bradykinin administered during low-flow ischemia and reperfusion reduced the number of ventricular premature beats and the incidence of ventricular tachycardia and of ventricular fibrillation during low-flow ischemia.
Conclusion: Bradykinin is a mediator, but unlikely a trigger, of antiarrhythmic effects of IPC during low-flow ischemia.     

On the Mechanism of Inhibition of KATP Channels by Glibenclamide in Rat Ventricular Myocytes

Glibenclamide Block of K_ATP Channels. Introduction: The mechanism by which glibenclamide inhibits K_ATP channel activity has been examined in membrane patches from isolated rat ventricular cells. Methods and Results: Inside-out patches were exposed to zero, or low, [ATP] to activate K_ATP channels. Glibenclamide did not affect single channel conductance, but reversibly reduced channel open probability from either side of the membrane. Internal (cytoplasmic) glibenclamide inhibited with half-maximal inhibitory [glibenclamide] = 6 μM, Hill coefficient = 0.35. Complete channel inhibition was not observed, even at 300 μM [glibenclamide]. The response to step increases of internal [glibenclamide] could be resolved into two phases of channel inhibition (t_{1/2, fast}, < 1 sec, t_{1/2, slow}= 10.5 ± 0.9 sec, n = 8). Step decrease of [glibenclamide] caused a single resolvable phase of reactivation (t_1/2= 20.4 ± 0.7 sec, n = 16). Channel inhibition by internal glibenclamide could be relieved by ADP, but only in the presence of Mg²⁺.
Conclusion: Glibenclamide can inhibit K_ATP channels from either side of the membrane, with block from one side being competitive with block from the other. Internal MgADP antagonizes the blocking action of glibenclamide. Glibenclamide inhibition of cardiac K_ATP channels differs quantitatively and qualitatively from the inhibition of pancreatic K_ATP channels.     

Potentiating Effect of Acetylcholine on Stimulation by Isoproterenol of L-Type Ca2+ Current and Arrhythmogenic Triggered Activity in Guinea Pig Ventricular Myocytes

ACh Facilitation of Triggered Activity. Introduction : The objective of this study was to determine whether the effect of isoproterenol (Iso) to increase L-type Ca²⁺ current [I_ca(L)] and action potential duration (APD) was potentiated in ventricular myocytes following termination of an exposure of these cells to acetylcholine (ACh), and whether this potentiating effect of Ach could he arrhythmogenic.
Methods and Results : Transmembrane currents and potentials of guinea pig Isolated ventricular myocytes were measured using the whole cell, patch clamp technique. Stimulation of I_ca(L) and prolongation of APD caused by Iso (10 nmol/L) were attenuated in the presence of Ach (10 μ mol/L), but were transiently enhanced by 111%± 20% and 214%± 44%, respectively, following termination of a 2- to 4-minute exposure of myocytes to ACh. No changes were observed in the absence of Iso. Both the amplitude and incidence of isoinduced transient inward current, afterdepolarizations, and sustained triggered activity were greater immediately after termination of exposure to ACh than before application of ACh.
Conclusion : Stimulation by Iso of I_ca(L) is transiently enhanced in guinea pig ventricular myocytes following termination of exposure of these cells to ACh. The rebound increase of Iso-stimulated I_ca(L) is associated with an increase of APD and induction of arrhythmogenic triggered activity.     

Role of L-Type Calcium Channel Window Current in Generating Current-Induced Early Afterdepolarizations

Ionic Mechanism of EADs. Introduction: Early afterdepolarizations (EADs) can give rise to triggered activity and thereby produce cardiac arrhythmias. We used the whole-cell patch clamp technique to examine the relationship between L-type Ca²⁺ channel window current and the generation of EADs in single ventricular myocytes isolated from guinea pig hearts.
Methods and Results: With a high concentration of EGTA in the internal solution and Na⁺-containing physiologic external solution, EADs were induced in unclamped cells by injecting intracellular depolarizing current pulses. During voltage clamp protocols designed to simulate action potentials interrupted by EADs, we recorded an inward shift in total current up to 0.7 pA/pF over 400 msec at test steps in the range of the take-off potential for EADs. Cd^2t (0.2 mM) blocked most of the inward shift of current during the test steps and abolished EADs. When the same voltage clamp protocol was used following perfusion with an Na⁺-free, K⁺-free external solution, the Cd²⁺-sensitive inward currents recorded during the test steps were similar to those obtained in physiologic external solution. The overlapping range of potentials for partial activation of the d and f variables of L-type Ca²⁺ current ("window" region) measured in Na⁺-free, K⁺-free external solution was virtually the same as the voltage range of the Cd²⁺–sensitive inward currents.
Conclusion: Our experiments suggest that: (1) EADs can arise under conditions of high EGTA buffering of intraccllular [Ca²⁺]; and (2) under these conditions, L-type Ca²⁺ channel window current plays a major role in the initiation of EADs.