Canine Cardiac Purkinje Fibers Research Articles

Comprehensive in vitro pro-arrhythmic assays demonstrate that omecamtiv mecarbil has low pro-arrhythmic risk.

Omecamtiv mecarbil (OM) is a myosin activator (myotrope), developed as a potential therapeutic agent for heart failure with reduced ejection fraction. To characterize the potential pro‐arrhythmic risk of this novel sarcomere activator, we evaluated OM in a series of International Conference on Harmonization S7B core and follow‐up assays, including an in silico action potential (AP) model. OM was tested in: (i) hERG, Nav1.5 peak, and Cav1.2 channel assays; (ii) in silico computation in a human ventricular AP (hVAP) population model; (iii) AP recordings in canine cardiac Purkinje fibers (PF); and (iv) electrocardiography analysis in isolated rabbit hearts (IRHs). OM had low potency in the hERG (half‐maximal inhibitory concentration [IC50] = 125.5 µM) and Nav1.5 and Cav1.2 assays (IC50 > 300 µM). These potency values were used as inputs to investigate the occurrence of repolarization abnormalities (biomarkers of pro‐arrhythmia) in an hVAP model over a wide range of OM concentrations. The outcome of hVAP analysis indicated low pro‐arrhythmia risk at OM concentration up to 30 µM (100‐fold the effective free therapeutic plasma concentration). In the isolated canine PF assay, OM shortened AP duration (APD)60 and APD90 significantly from 3 to 30 µM. In perfused IRH, ventricular repolarization (corrected QT and corrected JT intervals) was decreased significantly at greater than or equal to 1 µM OM. In summary, the comprehensive proarrhythmic assessment in human and non‐rodent cardiac models provided data indicative that OM did not delay ventricular repolarization at therapeutic relevant concentrations, consistent with clinical findings.

An Inotropic Action Caused by Muscarinic Receptor Subtype 3 in Canine Cardiac Purkinje Fibers

Objective. The objective of this study was to investigate the inotropic mechanisms and the related muscarinic receptor subtype of acetylcholine (ACh) in canine cardiac Purkinje fibers. Materials and Methods. Isolated Purkinje fiber bundles were used for the measurement of contraction. The receptor subtype was determined using PCR and real-time PCR methods. Results. ACh evoked a biphasic response with a transient negative inotropic effect followed by a positive inotropic effect in a concentration-dependent manner. The biphasic inotropic actions of ACh were inhibited by the pretreatment with atropine. Caffeine inhibited the positive inotropic effect of ACh. ACh increased inositol-1,4,5-trisphosphate content in the Purkinje fibers, which was abolished by atropine. Muscarinic subtypes 2 (M2) and 3 (M3) mRNAs were detected in the canine Purkinje fibers albeit the amount of M3 mRNA was smaller than M2 mRNA. M1 mRNA was not detected. Conclusion. These results suggest that the positive inotropic action of ACh may be mediated by the activation of IP3 receptors through the stimulation of M3 receptors in the canine cardiac Purkinje fibers.

Properties and Molecular Composition of Mouse and Canine Cardiac Purkinje Fiber ATP-Sensitive K + Channels

ATP-sensitive potassium (KATP) channels consist of pore-forming (Kir6.1 and Kir6.2) and sulfonylurea receptor (SUR1, SUR2A or SUR2B) subunits. We compared the biophysical and pharmacological properties, as well as the molecular composition of KATP channels in cardiac Purkinje cells and ventricular myocytes. Mouse Purkinje cells and myocytes were enzymatically isolated from Cntn2-EGFP Purkinje cell reporter mice. KATP channels recorded from Purkinje cells (inside-out patch clamp configuration) were inhibited by ATP with an IC50 of 125±18µM (Hill coefficient of 2.6), which was significantly higher than that of the ventricular KATP channel (68±10µM). The slope conductance of the Purkinje cell KATP channel in symmetric 140 mM K+ was also lower (57±3pS versus 76±1pS in the ventricle). Kinetic analysis showed similar mean open (respectively 2.2±0.16 and 2.9±0.95ms) and closed times (respectively 0.4±0.02 and 0.4±0.02ms) in Purkinje cells and ventricular myocytes. The Purkinje fiber KATP channel was activated preferentially by tolbultamide (200µM) and less by levcromakalim (30µM), whereas the reverse pharmacological profile was observed for the ventricular KATP channel. Using real-time semi-quantitative RT-PCR, we detected expression of Kir6.1, Kir6.2, and SUR2 mRNA in both mouse cell types. Purkinje fibers expressed more Kir6.1 and less total SUR2 than ventricular myocytes. Conventional RT-PCR demonstrated increased SUR2B expression relative to SUR2A in Purkinje cells. Western blotting confirmed expression of Kir6.1, Kir6.2, SUR2A and SUR2B protein in both dog Purkinje fibers and ventricular tissues with Kir6.1 and SUR2B levels elevated in the Purkinje fiber. Consistently, our confocal microscopy imaging data demonstrated the presence of Kir6.1, Kir6.2, and SUR2B subunits on the Purkinje cell. In summary, KATP channels in cardiac Purkinje cells manifest unique channel properties and molecular composition. They may have a differential electrophysiological response (eg. conduction velocity and arrhythmia) during periods of ischemia.

Development of cardiomyopathy and atrial tachyarrhythmias associated with activating autoantibodies to beta-adrenergic and muscarinic receptors

A 71-year-old male with well-controlled hypertension developed atrial tachyarrhythmias in 2002 and a restrictive cardiomyopathy in 2006 to 2007. Sera from 1992, 2001, and 2006 to 2008 demonstrated activating autoantibodies against β-adrenergic (AAβAR) and M2 muscarinic receptors (AAM2R). These sera have been characterized for bioactivity using in vitro assays of cardiac contractility and automaticity using a canine cardiac Purkinje fiber assay as well as protein kinase assay activation in H9c2 cells. These assays demonstrated concurrent positive βAR and inhibitory M2R effects that were blocked by nadolol and atropine, respectively. In a canine pulmonary vein atrial sleeve preparation, sera diluted 1:100 produced atrial hyperpolarization that was blocked by atropine. Atrial tachyarrhythmias developed in 2002 in the presence of a persistent bradycardia. Serial echocardiograms demonstrated progressive diastolic dysfunction in the absence of cardiac hypertrophy between 2006 and 2007. A dual-chamber pacemaker was installed with combined βAR (nadolol) and M2<3R (oxybutynin) blockade, resulting in marked suppression of atrial ectopy and improved diastolic function. The estimated pulmonary artery pressure decreased and exercise tolerance returned. Blood pressure has remained normal with β-blockade. AAβAR and AAM2R prospectively influenced atrial and ventricular function in this patient, and specific receptor blockade was associated with improved cardiac function.

Nitroglycerin on contractile force in canine cardiac Purkinje fibers

The objective of this study was to investigate the effects of nitroglycerin (NG) on the contractile force in order to elucidate regulatory roles of cyclic guanosine monophosphate (cGMP) in the bundle of canine cardiac Purkinje fibers. NG suppressed the developed tension in canine Purkinje fibers immersed in normal Tyrode's solution. The contraction was also elicited in the depolarizing solution containing 30 mM K+ by adding isoproterenol (Iso) or tetraethylammonium (TEA). The suppression caused by NG was more marked in fibers in the depolarizing solution treated with TEA than in the depolarizing solution treated with Iso. 8-Bromo-cGMP and sodium nitroprusside had a slight inhibition on the contraction in the fibers. Methylene blue did not affect the decrease in contractile force induced by NG. The weaker inhibition caused by NG on the contraction in the presence of Iso might result from a possible increase in intracellular cAMP levels in the fibers.

Multiple agents potentiate alpha1-adrenoceptor-induced conduction depression in canine cardiac purkinje fibers.

Halothane more so than isoflurane potentiates an alpha1-adrenoceptor (alpha1-AR)-mediated action of epinephrine that abnormally slows conduction in Purkinje fibers and may facilitate reentrant arrhythmias. This adverse drug interaction was further evaluated by examining conduction responses to epinephrine in combination with thiopental and propofol, which "sensitize" or reduce the dose of epinephrine required to induce arrhythmias in the heart, and with etomidate, which does not, and responses to epinephrine with verapamil, lidocaine, and l-palmitoyl carnitine, a potential ischemic metabolite. Action potentials and conduction times were measured in vitro using two microelectrodes in groups of canine Purkinje fibers stimulated at 150 pulses/min. Conduction was evaluated each minute after exposure to 5 microm epinephrine (or phenylephrine) alone or with the test drugs. Changes in the rate of phase 0 depolarization (Vmax) and the electrotonic spread of intracellular current were measured during exposure to epinephrine with octanol to evaluate the role of inhibition of active and passive (intercellular coupling) membrane properties in the transient depression of conduction velocity. Lidocaine (20 microm) and octanol (0.2 mm) potentiated alpha1-AR-induced conduction depression like halothane (0.4 mm), with maximum depression at 3-5 min of agonist exposure, no decrease of Vmax, and little accentuation at a rapid (250 vs. 150 pulses/min) stimulation rate. Thiopental (95 microm), propofol (50 microm), and verapamil (2 microm) similarly potentiated epinephrine responses, whereas etomidate (10 microm) did not. Between groups, the decrease of velocity induced by epinephrine in the presence of (10 microm) l-palmitoyl carnitine (-18%) was significantly greater than that resulting from epinephrine alone (-6%; 0.05 </= P </= 0.10). Current injection experiments were consistent with marked transient inhibition of cell-to-cell coupling correlating with alpha1-AR conduction depression in fibers exposed to octanol. Anesthetic "sensitization" to the arrhythmogenic effects of catecholamines may be a special case of a more general phenomenon by which not only some anesthetics and antiarrhythmic drugs but also possible ischemic fatty acid metabolites potentiate conduction depression due to acute alpha1-AR-mediated cell-to-cell uncoupling.

Electrophysiological characterization of BRL-32872 in canine Purkinje fiber and ventricular muscle: Effect on early after-depolarizations and repolarization dispersion

Amongst the different pharmacological approaches to the treatment of cardiac arrhythmias, compounds with multiple electrophysiological activities appear to exhibit a reduced adverse effect profile. BRL-32872 ( N-(3,4-dimethoxyphenyl)- N-[3[[2-(3,4-dimethoxyphenyl) ethyl] propyl]-4-nitrobenzamide hydrochloride) is a typical example of an antiarrhythmic agent with combined K + and Ca 2+ blocking actions. In this study, we investigated the effects of BRL-32872 on early after-depolarizations and on dispersion of repolarization. Action potentials were recorded either in canine cardiac Purkinje fibers alone or in preparations containing both ventricular muscle and the attached Purkinje fibers. In Purkinje fibers, BRL-32872 (0.3–10 μM) induced a bell-shaped concentration-dependent increase in action potential duration. At 90% of repolarization, the action potential was prolonged at concentrations up to 1 μM and was shortened when the concentration of BRL-32872 was further increased. In all 17 experiments, BRL-32872 did not cause early after-depolarizations in Purkinje fibers. On the contrary, BRL-32872 (3 μM) systematically suppressed early after-depolarizations induced by clofilium (4-chloro- N, N-diethyl- N-heptylbenzenebutanaminium tosylate, 1 μM), a selective inhibitor of the delayed rectifier K + current. A similar effect was observed once with 1 μM BRL-32872, a concentration able to prolong Purkinje fiber action potentials. Simultaneous recording of action potentials in ventricular and Purkinje preparations showed that increasing concentrations of BRL-32872 (0.3–10 μM) induced a limited increase in the difference of repolarization time between the two tissues. The selective K + channel inhibitor E-4031 ( N-(4-(1-[2-(6-methyl-2-pyridyl) ethyl]-4-piperidyl)-carbonyl] phenyl) methanesulfonamide dihydrochloride dihydrate) exhibited a significant concentration-dependent increase in dispersion of repolarization. We conclude from the present results that the Ca 2+ blocking activity of BRL-32872 (i) prevents the occurrence of early after-depolarizations associated with action potential prolongation and (ii) limits an excessive increase in action potential duration heterogeneity. These electrophysiological features might represent the basis for antiarrhythmic compounds with reduced proarrhythmic profile.

Muscarinic Activation Causes Biphasic Inotropic Response and Decreases Cellular Na+ Activity in Canine Cardiac Purkinje Fibers

In this study, the effects of carbachol (CCh) on twitch tension, intracellular Na⁺ activity (aⁱ_Na), and action potential were simultaneously measured in canine cardiac Purkinje fibers in order to examine the regulation of inotropy through muscarinic receptors and its relation to aⁱ_Na. In fibers driven at 1 Hz, CCh (10 μM) initially and transiently decreased and then increased the twitch tension by 36 ± 8%. The action potential showed a significant elevation of the plateau and a significant shortening of the duration at 90% repolarization (APD₉₀), from 403 ± 7 to 389 ± 7 ms. The aⁱ_Na decreased from 7.4 ± 0.4 to 6.7 ± 0.3 mM (n = 23, p < 0.05). Atropine (1 μM) decreased the twitch tension by 21 ± 6% (n = 7, p < 0.05) without significant effects on the action potential and aⁱ_Na, and inhibited the effects of CCh. Cs⁺ (20 mM) increased the plateau height and APD₉₀, enhanced the twitch tension by 66 ± 24%, but decreased aⁱ_Na from 7.3 ± 0.3 to 6.3 ± 0.4 mM (n = 6, p < 0.05). In the presence of 20 mM Cs⁺, some fibers generated slow responses. The addition of 10 μM CCh further increased the twitch tension and APD₉₀, and decreased aⁱ_Na from 6.3 ± 0.4 to 5.3 ± 0.3 mM. Ouabain (0.3 μM) increased the twitch tension and aⁱ_Na, and inhibited the CCh-induced decrease of aⁱ_Na. In the presence of ouabain, 20 mM Cs⁺ depolarized the fiber and generated slow responses with a decreased aⁱ_Na. The addition of 10 μM CCh enhanced the slow action potential, and increased aⁱ_Na although there was a transient decrease during early exposure. These results suggest that activation of muscarinic receptors in canine Purkinje fibers results in an enhancement of the Na⁺-K⁺ pump activity and a biphasic inotropic response, probably via different receptor subtypes. The inhibitory effect, most likely through M₂ receptors, is associated with the activation of K⁺ channels. The stimulatory effect, on the other hand, is probably due to the action on the M₁ receptors, resulting in increases in Ca²⁺ currents.

Muscarinic activation causes biphasic inotropic response and decreases cellular Na+ activity in canine cardiac Purkinje fibers.

In this study, the effects of carbachol (CCh) on twitch tension, intracellular Na+ activity (aiNa), and action potential were simultaneously measured in canine cardiac Purkinje fibers in order to examine the regulation of inotropy through muscarinic receptors and its relation to aiNa. In fibers driven at 1 Hz, CCh (10 microM) initially and transiently decreased and then increased the twitch tension by 36 +/- 8%. The action potential showed a significant elevation of the plateau and a significant shortening of the duration at 90% repolarization (APD90), from 403 +/- 7 to 389 +/- 7 ms. The aiNa decreased from 7.4 +/- 0.4 to 6.7 +/- 0.3 mM (n = 23, p < 0.05). Atropine (1 microM) decreased the twitch tension by 21 +/- 6% (n = 7, p < 0.05) without significant effects on the action potential and aiNa, and inhibited the effects of CCh. Cs+ (20 mM) increased the plateau height and APD90, enhanced the twitch tension by 66 +/- 24%, but decreased aiNa from 7.3 +/- 0.3 to 6.3 +/- 0.4 mM (n = 6, p < 0.05). In the presence of 20 mM Cs+, some fibers generated slow responses. The addition of 10 microM CCh further increased the twitch tension and APD90, and decreased aiNa from 6.3 +/- 0.4 to 5.3 +/- 0.3 mM. Ouabain (0.3 microM) increased the twitch tension and aiNa, and inhibited the CCh-induced decrease of aiNa. In the presence of ouabain, 20 mM Cs+ depolarized the fiber and generated slow responses with a decreased aiNa. The addition of 10 microM CCh enhanced the slow action potential, and increased aiNa although there was a transient decrease during early exposure. These results suggest that activation of muscarinic receptors in canine Purkinje fibers results in an enhancement of the Na+-K+ pump activity and a biphasic inotropic response, probably via different receptor subtypes. The inhibitory effect, most likely through M2 receptors, is associated with the activation of K+ channels. The stimulatory effect, on the other hand, is probably due to the action on the M1 receptors, resulting in increases in Ca2+ currents.

The effects of halothane on abnormal automaticity in canine cardiac Purkinje fibers.

Abnormal automaticity is the spontaneous beating of cardiac cells with abnormally depolarized resting membrane potentials. The effects of halothane on cardiac arrhythmias caused by abnormal automaticity are controversial, with either antiarrhythmic effects or enhancement of abnormal automaticity reported by different authors. The goal of the present investigation was to clarify the effects of halothane on abnormal automaticity induced by superfusing excised canine Purkinje fibers (PF) with barium chloride. Intracellular microelectrodes recorded action potentials from fibers superfused with buffer solution in a tissue bath. Barium chloride 0.25 mM reduced maximal diastolic potential from -82.1 +/- 5.6 mV to -67.4 +/- 9.4 mV (mean +/- SD, P < 0.05). Fibers developed abnormal automatic rhythms at a rate of 47.1 +/- 5.9 bpm. Halothane, 0.5%-4%, was added to the superfusate. Halothane reduced the rate of firing in a dose-dependent manner, so that abnormal automaticity was abolished by 4% halothane and reduced by lesser concentrations. Serendipitously, during barium superfusion, two additional fibers developed early afterdepolarizations, a cause of triggered arrhythmias in patients with long Q-T syndrome. Halothane abolished early afterdepolarizations in each. In this model of barium toxicity in excised canine PF, halothane antagonized both abnormal automaticity and early afterdepolarizations. Life-threatening cardiac arrhythmias may occur during anesthesia. An arrhythmia called abnormal automaticity occurs after heart attacks and can be mimicked by adding barium to small segments of heart tissue. Halothane abolished abnormal automaticity in these tissues, which suggests that it or similar agents may benefit patients prone to developing such abnormal rhythms during surgery.

The Effects of Halothane on Abnormal Automaticity in Canine Cardiac Purkinje Fibers

Abnormal automaticity is the spontaneous beating of cardiac cells with abnormally depolarized resting membrane potentials.The effects of halothane on cardiac arrhythmias caused by abnormal automaticity are controversial, with either antiarrhythmic effects or enhancement of abnormal automaticity reported by different authors. The goal of the present investigation was to clarify the effects of halothane on abnormal automaticity induced by superfusing excised canine Purkinje fibers (PF) with barium chloride. Intracellular microelectrodes recorded action potentials from fibers superfused with buffer solution in a tissue bath. Barium chloride 0.25 mM reduced maximal diastolic potential from -82.1 +/- 5.6 mV to -67.4 +/- 9.4 mV (mean +/- SD, P < 0.05). Fibers developed abnormal automatic rhythms at a rate of 47.1 +/- 5.9 bpm. Halothane, 0.5%-4%, was added to the superfusate. Halothane reduced the rate of firing in a dose-dependent manner, so that abnormal automaticity was abolished by 4% halothane and reduced by lesser concentrations. Serendipitously, during barium superfusion, two additional fibers developed early afterdepolarizations, a cause of triggered arrhythmias in patients with long Q-T syndrome. Halothane abolished early afterdepolarizations in each. In this model of barium toxicity in excised canine PF, halothane antagonized both abnormal automaticity and early afterdepolarizations. Implications: Life-threatening cardiac arrhythmias may occur during anesthesia. An arrhythmia called abnormal automaticity occurs after heart attacks and can be mimicked by adding barium to small segments of heart tissue. Halothane abolished abnormal automaticity in these tissues, which suggests that it or similar agents may benefit patients prone to developing such abnormal rhythms during surgery. (Anesth Analg 1998;86:488-92)

Memory and complex dynamics in cardiac Purkinje fibers.

The contribution of cumulative changes in action potential duration (memory) to complex cellular electrophysiological behavior was investigated in canine cardiac Purkinje fibers. Complex behavior induced during constant pacing was caused by reciprocal interactions between the time to full repolarization (TFR), where TFR = response duration + latency, and the diastolic interval (DI). The relationship between TFR and the preceding DI during complex behavior differed from that obtained using a standard restitution protocol. In particular, higher-order periodicities and chaos were produced in fibers in which the restitution curve lacked the prerequisites for such behavior. To investigate whether shifts in the restitution curve might be expected during rapid pacing, the relationship between TFR of a test response (TFR(n + 1)) and the immediately preceding response (TFR(n)) was determined. For any fixed DI(n), reduction of TFR(n) from 240 to 130 ms was accompanied by a corresponding reduction of TFR(n + 1), whereas as TFR(n) was reduced further to 120 ms, TFR(n + 1) increased. Because of the dependence of TFR(n + 1) on TFR(n) (memory) and on the preceding DI(n) (restitution), the slope of the low-dimensional relationship between TFR(n + 1) and DI(n) at a constant pacing cycle length depended on the slopes of the restitution and memory functions. These results suggest that rapid accumulation and dissipation of memory may contribute importantly to complex electrical behavior in cardiac tissue.

Electrophysiological effects of EGIS-7229, a new antiarrhythmic agent, in isolated mammalian and human cardiac tissues.

The cellular electrophysiological effects of EGIS-7229 (5-chlor-4-[N-(3,4-dimethoxy-phenyl-ethyl)-amino-propylamino]-3 (2H)-pyridazinone fumarate), a novel antiarrhythmic agent, was studied using conventional microelectrode techniques in canine cardiac Purkinje fibers and papillary muscle preparations obtained from man, rabbits and guinea pigs. Low concentration of EGIS-7229 (3 mumol/l) selectively lengthened action potential duration (both APD50 and APD90) in all preparations. The effect of higher concentrations (30-100 mumol/l) of EGIS-7229 on action potential duration was variable depending on the preparation studied: in rabbit and human papillary muscles both APD50 and APD90 were lengthened, in canine Purkinje fibers APD90 was lengthened but APD50 was shortened, while in guinea pig papillary muscles both APD50 and APD90 were shortened by high concentrations of the drug. At these higher concentrations EGIS-7229 also decreased the maximum velocity of action potential upstroke (Vmax) and depressed the plateau of action potentials without affecting the resting membrane potential or action potential amplitude. Both reduction of Vmax and lengthening of APD were frequency dependent. The former effect was more prominent at higher pacing frequencies, while the latter was more pronounced at lower driving rates. In guinea pig papillary muscle, the time constant of recovery from Vmax-block was 719 +/- 33 ms (n = 18) and the rate of onset of the block was 1.81 +/- 0.06 AP-1 (n = 16) in the presence of 100 mumol/l EGIS-7229. EGIS-7229 had a complex action on refractoriness in guinea pig papillary muscles: ERP was lengthened at low concentrations (3 to 10 mumol/l), unchanged at 30 mumol/l and shortened at 100 mumol/l. The ratio of ERP/APD90, however, was significantly increased at concentrations higher than 3 mumol/l. In canine Purkinje fiber, when the delayed rectifier K current (IK) was blocked by d-sotalol (60 mumol/l) and APD was shortened back to its control value by additional application of nicorandil (15 mumol/l), APD was not affected by 3 mumol/l but was shortened by 30 mumol/l of EGIS-7229. 100 mumol/l EGIS-7229 shortened APD in guinea pig papillary muscle. This effect of EGIS-7229 was effectively prevented by nifedipine pretreatment (10 mumol/ l). In this preparation, EGIS-7229 also decreased the Vmax of the slow action potential, evoked in the presence of 20 mmol/l external K+ plus 0.5 mmol/l Ba2+. It is likely that EGIS-7229 at low concentrations blocks IK in human, canine, rabbit and guinea pig cardiac preparations, but at higher concentrations also inhibits Ca and Na currents. Therefore, EGIS-7229 appears to carry mixed class III, IV and IB antiarrhythmic properties.

P-479 - Ca2+-dependent modulation by caffeine of rate-force relation in canine cardiac Purkinje fibers

P-479 - Ca2+-dependent modulation by caffeine of rate-force relation in canine cardiac Purkinje fibers

Beta-adrenergic modulation of Na-K pump activity in young and adult canine cardiac Purkinje fibers.

We used standard microelectrode techniques to study the developmental changes and beta-adrenergic modulation of membrane potential and of Na-K pump activity in adult (> 1 yr of age) and neonatal (2-10 days) canine Purkinje fibers. Isoproterenol (10(-7) M) increased the rate of development and magnitude of pacing-induced hyperpolarization of adult fibers driven at a 1-s cycle length. This effect of isoproterenol was attenuated by treating dogs with pertussis toxin (PTX), (30 micrograms/kg). Other adult and neonatal fibers were superfused with a Tyrode solution containing Ba2+ 0.2 mM, Cs+ 2 mM, and 10(-6) M verapamil, thus leading to depolarization and cessation of spontaneous activity. The Na-K pump was studied by alternating solutions containing [K] at 0 mM (inhibiting the pump) and 4 mM (reactivating the pump). Although the kinetics of the Na-K pump appeared faster in neonatal fibers than in adult fibers, measurement of cell surface-to-volume ratio compensated for the difference. We therefore conclude that 1) the apparent age-related changes in Na-K pump activity in canine Purkinje fibers in fact reflect cell surface-to-volume ratio and, 2) the beta-adrenergic agonist-induced hyperpolarization in adults requires the presence of a PTX-sensitive G protein for its occurrence.

α2-Adrenergic receptor binding in canine Purkinje fibers

Autoradiograms were developed from free running strands of Purkinje fibers and from left ventricular myocardium of dog hearts exposed to [7- methoxy- 3H]prazosin or [ O- methyl- 3H]yohimbine in the presence or absence of excessive non-radioactive ligands. Both Purkinje fiber and ventricular myocardium showed high density, tissue-specific binding to [ 3H]prazosin. In contrast, high density, tissue-specific binding to [ 3H]yohimbine was present in Purkinje fibers but not in ventricular myocardium. Membrane fractions showed high affinity, saturable, and displaceable binding with [ 3H]yohimbine in preparations from canine cardiac Purkinje fibers but not those from canine cardiac ventricular myocardium. Scatchard analysis of the canine Purkinje membrane α 2-adrenergic receptor binding showed a B max of 54.9 fmol/mg protein with a K d of 6.25 nM. These results confirm the electrophysiological findings that post-junctional α 2-adrenergic receptors are present in Purkinje fibers.

Electrophysiological effects of dridocainide on isolated canine, guinea-pig and human cardiac tissues

The cellular electrophysiological effects of dridocainide (EGIS-3966), a novel class I antiarrhythmic agent, was studied using conventional microelectrode techniques in canine cardiac Purkinje fibres and papillary muscle preparations obtained from humans and guinea-pigs. In each preparation, dridocainide (0.6-2 mumol/l) decreased the maximum velocity of action potential upstroke (Vmax) in a frequency-dependent manner, although marked differences were observed in its effects in Purkinje fibre and ventricular muscle preparations. In canine Purkinje fibres, action potential duration measured at 50% and 90% of repolarization was decreased, while action potential duration measured at 10% of repolarization was increased by dridocainide. In addition, the plateau of the action potential was depressed by the drug. These changes in action potential configuration were not observed in guinea pig or human papillary muscles. The offset kinetics of the dridocainide-induced Vmax block were different in Purkinje fibres and in ventricular muscle: the slow time constant of recovery of Vmax was estimated to be 2.5 s in dog Purkinje fibre and 5-6 s in human and guinea-pig papillary muscle. In guinea-pig papillary muscle, the rate of onset of the Vmax block was 0.15 and 0.2 per action potential in the presence of 0.6 and 2 mumol/l dridocainide, respectively. Dridocainide also decreased the force of contraction in this preparation. On the basis of the present results, dridocainide appears to possess mixed class I.C and I.A properties, with I.C predominance in human and guinea-pig ventricular muscle. Present results also indicate that results of conventional classification of class I drugs may depend on the parameters chosen, as well as on the preparation selected.

In Vivo and In Vitro Electrophysiologic Effects of Terodiline on Dog Myocardium

Terodiline hydrochloride, widely prescribed for urinary incontinence, has been reported to cause bradycardia and torsades de pointes. In this study, we characterized the electrophysiologic effects of terodiline in dog cardiac tissues in vivo and in isolated canine cardiac Purkinje fibers. Terodiline (1 to 10 microM) resulted in dose-dependent reduction of action potential amplitude and maximal upstroke velocity (Vmax). The threshold for these effects was approximately 2 microM (0.6 mg/L), and the changes were cycle-length dependent. Terodiline (> or = 2 microM) also depressed the action potential plateau but did not significantly alter action potential duration at concentrations < or = 10 microM. In vivo studies demonstrated that high doses of terodiline (3 mg/kg) lengthened AH and HV intervals, slowed spontaneous sinus rate, prolonged ventricular refractoriness, and inhibited vagally induced slowing of the sinus node. Sympathetic effects on spontaneous sinus rate were unchanged. In both isolated canine Purkinje fibers and anesthetized dogs, terodiline did not evoke afterdepolarizations, repetitive firing, or ventricular tachyarrhythmias under normal or hypokalemic conditions. Our findings suggest that terodiline (> or = 1 to 2 microM) leads to blockade of sodium and calcium channels as well as muscarinic receptors in canine cardiac tissues.

Electrophysiological effects of alpha 2-adrenergic stimulation in canine cardiac Purkinje fibers

The effects of alpha 2-adrenergic stimulation on action potentials were measured in isolated canine Purkinje fibers. Action potential durations at 50 and 90% of repolarization (APD50 and APD90) were significantly prolonged by 0.25 microM l-norepinephrine + 0.5 microM dl-propranolol (NE+P) from baseline values of 166 +/- 7 and 249 +/- 9 (SE) ms (n = 7) to 174 +/- 7 and 265 +/- 9 ms, respectively (P < 0.05 for both). Selective alpha 2-blockade with 0.01 microM yohimbine (YO) reduced this prolongation by NE+P in APD50 and APD90 to 169 +/- 7 and 256 +/- 8 ms, respectively (P < 0.05 compared with NE+P). Additional selective alpha 1-blockade with 0.01 microM prazosin (PZ) completely blocked the effects of NE+P, returning APD50 and APD90 to 163 +/- 7 and 250 +/- 9 ms (not different from baseline). After incubation of isolated Purkinje fibers with pertussis toxin (1 microgram/ml), which reduced the availability of a 41-kDa membrane protein for ADP ribosylation by 70 +/- 7% (n = 4, P < 0.05), YO failed to reverse the prolongation in action potential durations brought on by NE+P, but the effects of PZ were intact. The effects of alpha 2-stimulation on beta-adrenergic-induced delayed afterdepolarizations (DADs) were studied by burst pacing of Purkinje fibers in Tyrode solution containing 7.5 mM Ca2+. The DADs induced in the presence of NE+PZ (beta- + alpha 2-stimulation) were significantly smaller in amplitude and required a shorter pacing cycle length to reach threshold than those induced in the presence of NE+PZ+YO (unopposed beta-adrenergic stimulation). Furthermore sustained triggered activity, seen in five of eight preparations under beta-stimulation, could no longer be elicited in the presence of beta- + alpha 2-stimulation. These results suggest that the postjunctional alpha 2-adrenergic receptors in canine Purkinje fibers are coupled to a pertussis toxin-sensitive G protein and that stimulation of these receptors leads to action potential prolongation and suppression of DADs induced by beta-adrenergic stimulation.

Effects of Hypothermia, Potassium, and Verapamil on the Action Potential Characteristics of Canine Cardiac Purkinje Fibers

Hypothermia may induce hypokalemia and increase intracellular Ca2+ by affecting serum K+ and Ca2+ fluxes across the cell membrane. These ionic alterations may significantly change the electrophysiologic characteristics of the cardiac action potential and may induce cardiac arrhythmias. The current study was undertaken to determine whether electrophysiologic changes in Purkinje fibers induced by hypothermia could be reversed by manipulating the extracellular K+ and transmembrane Ca2+ fluxes by Ca2+ channel blockade with verapamil. A conventional microelectrode method was used to determine the effects of hypothermia (32 +/- 0.5 degrees C and 28 +/- 0.5 degrees C) and various external K+ concentrations ([K+]o) (2.3, 3.8, and 6.8 mM) on maximum diastolic potential, maximum rate of phase 0 depolarization (Vmax), and action potential duration (APD) at 50% (APD50) and at 95% (APD95) repolarization in isolated canine cardiac Purkinje fibers. To evaluate the contribution of the slow inward Ca2+ current to action potential changes in hypothermia, the experiments were repeated in the presence of the Ca(2+)-channel antagonist verapamil (1 microM). Variations of [K+]o induced the expected shifts in maximum diastolic potential, and hypothermia (28 degrees C) induced moderate depolarization, but only when [K+]o was > or = 3.9 mM (P < 0.05). Hypothermia decreased Vmax at all [K+]o studied (P < 0.05). Regardless of the temperature, Vmax was not affected by verapamil when [K+]o was < or = 3.9 mM, but at 6.8 mM [K+]o in hypothermia Vmax was significantly lower in the presence of verapamil. Hypothermia increased both the APD50 and the APD95. The effects of verapamil on APD were temperature and [K+]o dependent; between 37 degrees C and 28 degrees C with 2.3 mM [K+]o in the superfusate, verapamil did not affect APD. At 28 degrees C in the presence of verapamil, the APD50 and APD95 decreased only if the [K+]o was > or = 3.9 mM. Verapamil and K+ supplementation in hypothermia may exert an antiarrhythmic effect, primarily by reducing the dispersion fo prolonged APD.