The antiarrhythmic actions of bisaramil and penticainide result from mixed cardiac ion channel blockade.

Decades of focus on selective ion channel blockade has been dismissed as an effective approach to antiarrhythmic drug development. In that context many older antiarrhythmic drugs lacking ion channel selectivity may serve as tools to explore mixed ion channel blockade producing antiarrhythmic activity. This study investigated the non-clinical electrophysiological and antiarrhythmic actions of bisaramil and penticainide using in vitro and in vivo methods. In isolated cardiac myocytes both drugs directly block sodium currents with IC50 values of 13µM (bisaramil) and 60µM (penticainide). Both drugs reduced heart rate but prolonged the P-R, QRS and Q-T intervals of the ECG (due to sodium and potassium channel blockade) in intact rats. They reduced cardiac conduction velocity in isolated rat hearts, increased the threshold currents for capture and fibrillation (indices of sodium channel blockade) and reduced the maximum following frequency as well as prolonged the effective refractory period (indices of potassium channel blockade) of electrically stimulated rat hearts. Both drugs reduced ventricular arrhythmias and eliminated mortality due to VF in ischemic rat hearts. The index of cardiac electrophysiological balance (iCEB) did not change significantly over the dose range evaluated; however, different drug effects resulted when changes in BP and HR were considered. While bisaramil is a more potent sodium channel blocker compared to penticainide, both produce a spectrum of activity against ventricular arrhythmias due to mixed cardiac ion channel blockade. Antiarrhythmic drugs exhibiting mixed ion channel blockade may serve as tools for development of safer mixed ion channel blocking antiarrhythmic drugs.

Pharmacological and toxicological activity of RSD921, a novel sodium channel blocker.

BACKGROUND: RSD921, the R,R enantiomer of the kappa (k) agonist PD117,302, lacks significant activity on opioid receptors. METHODS: The pharmacological and toxicological actions were studied with reference to cardiovascular, cardiac, antiarrhythmic, toxic and local anaesthetic activity. RESULTS: In rats, dogs and baboons, RSD921 dose-dependently reduced blood pressure and heart rate. In a manner consistent with sodium channel blockade it prolonged the PR and QRS intervals of the ECG. Furthermore, in rats and NHP, RSD921 increased the threshold currents for induction of extra-systoles and ventricular fibrillation (VFt), and prolonged effective refractory period (ERP). In rats, RSD921 was protective against arrhythmias induced by electrical stimulation and coronary artery occlusion. Application of RSD921 to voltage-clamped rat cardiac myocytes blocked sodium currents. RSD921 also blocked transient (ito) and sustained (IKsus) outward potassium currents, albeit with reduced potency relative to sodium current blockade. Sodium channel blockade due to RSD921 in myocytes and isolated hearts was enhanced under ischaemic conditions (low pH and high extracellular potassium concentration). When tested on the cardiac, neuronal and skeletal muscle forms of sodium channels expressed in Xenopus laevis oocytes, RSD921 produced equipotent tonic block of sodium currents, enhanced channel block at reduced pH (6.4) and marked use-dependent block of the cardiac isoform. RSD921 had limited but quantifiable effects in subacute toxicology studies in rats and dogs. Pharmacokinetic analyses were performed in baboons. Plasma concentrations producing cardiac actions in vivo after intravenous administration of RSD921 were similar to the concentrations effective in the in vitro assays utilized. CONCLUSIONS: RSD921 primarily blocks sodium currents, and possesses antiarrhythmic and local anaesthetic activity.

Spontaneous intrauterine growth restriction due to increased litter size in the guinea pig programmes postnatal growth, appetite and adult body composition.

Intrauterine growth restriction (IUGR) and subsequent neonatal catch-up growth are implicated in the programming of increased appetite, adiposity and cardiometabolic diseases. Guinea pigs provide an alternate small animal model to rodents to investigate mechanisms underlying prenatal programming, being relatively precocial at birth, with smaller litter sizes and undergoing neonatal catch-up growth after IUGR. The current study, therefore, investigated postnatal consequences of spontaneous IUGR due to varying litter size in this species. Size at birth, neonatal, juvenile (post-weaning, 30-60 days) and adolescent (60-90 days) growth, juvenile and adolescent food intake, and body composition of young adults (120 days) were measured in 158 male and female guinea pigs from litter sizes of one to five pups. Compared with singleton pups, birth weight of pups from litters of five was reduced by 38%. Other birth size measures were reduced to lesser degrees with head dimensions being relatively conserved. Pups from larger litters had faster fractional neonatal growth and faster absolute and fractional juvenile growth rates (P<0.005 for all). Relationships of post-weaning growth, feed intakes and adult body composition with size at birth and neonatal growth rate were sex specific, with neonatal growth rates strongly and positively correlated with adiposity in males only. In conclusion, spontaneous IUGR due to large litter sizes in the guinea pig causes many of the programmed sequelae of IUGR reported in other species, including human. This may therefore be a useful model to investigate the mechanisms underpinning perinatal programming of hyperphagia, obesity and longer-term metabolic consequences.

A progressive assessment strategy improves student learning and perceived course quality in undergraduate physiology.

In 2010, second-year physiology (n = 165) had a traditional single 3-h end-of-semester exam. To provide diagnostic feedback earlier, for students enrolled in 2011 (n = 128), we incorporated an in-class exam at 3 wk in addition to the final exam. Based on initial analysis and positive student comments, for the 2012 cohort (n = 148), we expanded this to incorporate four 1-h in-class exams every 3 wk plus a short final integrative exam. Average scores from exams and questionnaires (student evaluations of learning and teaching, 10 questions) were compared among 2010, 2011, and 2012 cohorts. We also compared scores in the practical component of the course, which had a constant format for all cohorts. Data are given as means ± SD; statistical analyses were done with unpaired two-way Students t-tests. From 2010 to 2012, there was a significant improvement in total exam scores (59.7 ± 15.8 vs. 68.6 ± 14.2, P < 0.001) but no significant change in total practical scores (72.3 ± 9.0 vs. 74.4 ± 10.2, P = 0.05), indicating that the rise in exam score was not due to higher academic abilities of the 2012 cohort. Overall mean student evaluation of learning and teaching responses (4.9 ± 0.4 vs. 5.3 ± 0.3, P = 0.015) and overall percent broad agreement (66.0 ± 8.0 vs. 79.2 ± 7.5, P = 0.003) indicated a significant improvement in student satisfaction. In conclusion, both learning outcome and perceived course quality were enhanced by the increased frequency of examinations, possibly by promoting consistent student study habits.

Amitriptyline pharmacologically preconditions rat hearts against cardiac ischemic-reperfusion injury.

BACKGROUND/OBJECTIVES: Amitriptyline (AMY) is a tricyclic anti-depressant that has recently been shown to have anti-inflammatory properties. We investigated whether AMY is cardioprotective against reperfusion injury in ex-vivo rat hearts. METHODS: Thirty adult Sprague-Dawley rat hearts were perfused ex-vivo in a Langendorff apparatus. All hearts except SHAM (n = 6, perfused for 110 min.) received 30 min no-flow ischemia followed by 40 min reperfusion (I-R). One group (n = 6) was untreated before I-R (non-preconditioned; NPC), another non-preconditioned group was perfused with 10 µM amitriptyline for 30 min before I-R (NPC-AMY, n = 6). One group was preconditioned with 3 × 5-minute periods of ischemia before I-R (PC, n = 6) and a fifth group was preconditioned in the presence of 10 µM amitriptyline (PC-AMY, n = 6). p38 phosphorylation and HMGB1 levels were quantified using Western blots. Data was analysed using multiway ANOVAs with Tukey HSD and linear regression models with Sobel mediator tests. RESULTS: NPC hearts recovered poorly (LVDP recovered to 26.5 ± 10.5% of pre-ischemic values, compared to PC hearts (82.8 ± 14.9%: P < 0.05)). PC-AMY (69.9 ± 6.16%) and NPC-AMY (90.3 ± 10.0%) groups both recovered well (P < 0.05). The Sobel mediator test suggested that p38 activity may be indirectly involved in the amitriptyline induced cardioprotection (P < 0.05). HMGB1 was lower in amitriptyline treated hearts compared to the non-preconditioned hearts (P < 0.05) but the multiway ANOVA test suggests that HMGB1 was not involved in amitriptyline induced protection. CONCLUSIONS: Amitriptyline at 10 µM protects hearts against ischemic-reperfusion injury which may be partially mediated through p38 phosphorylation.

Minimum Information about a Cardiac Electrophysiology Experiment (MICEE): standardised reporting for model reproducibility, interoperability, and data sharing.

Cardiac experimental electrophysiology is in need of a well-defined Minimum Information Standard for recording, annotating, and reporting experimental data. As a step towards establishing this, we present a draft standard, called Minimum Information about a Cardiac Electrophysiology Experiment (MICEE). The ultimate goal is to develop a useful tool for cardiac electrophysiologists which facilitates and improves dissemination of the minimum information necessary for reproduction of cardiac electrophysiology research, allowing for easier comparison and utilisation of findings by others. It is hoped that this will enhance the integration of individual results into experimental, computational, and conceptual models. In its present form, this draft is intended for assessment and development by the research community. We invite the reader to join this effort, and, if deemed productive, implement the Minimum Information about a Cardiac Electrophysiology Experiment standard in their own work.

Riluzole protects against cardiac ischaemia and reperfusion damage via block of the persistent sodium current.

BACKGROUND AND PURPOSE: Current strategies to ameliorate cardiac ischaemic and reperfusion damage, including block of the sodium-hydrogen exchanger, are therapeutically ineffective. Here we propose a different approach, block of the persistent sodium current (INaP). EXPERIMENTAL APPROACH: Left ventricular pressure was measured as an index of functional deficit in isolated, Langendorff perfused, hearts from adult rats, subjected to 30 min global ischaemia and reperfusion with vehicle only (control) or riluzole (1-10 microM) in the perfusate. Cell shortening and intracellular Ca2+ concentrations [Ca2+](i) were measured in adult rat isolated myocytes subjected to hypoxia and re-oxygenation. The block of transient and persistent sodium currents by concentrations of riluzole between 0.01 and 100 microM were assessed in rat isolated myocytes using patch clamp techniques. KEY RESULTS: In perfused hearts, riluzole produced a concentration-dependent cardioprotective action, with minor protection from 1 microM and produced rapid and almost complete recovery upon reperfusion from 3 and 10 microM. In isolated myocytes, riluzole at 3 and 10 microM greatly attenuated or prevented the hypoxia- and reperfusion-induced rise in [Ca2+](i) and the contractile deficit. In patch clamp experiments, riluzole blocked the persistent sodium current with an IC(50) of 2.7 microM, whereas the block of the transient sodium current was only apparent at concentrations above 30 microM. CONCLUSIONS AND IMPLICATIONS: Riluzole preferentially blocked INaP and was protective in cardiac ischaemia and reperfusion. Thus block of the persistent sodium current would be a viable method of ameliorating cardiac ischaemic and reperfusion damage.

The cardiac persistent sodium current: an appealing therapeutic target?

The sodium current in the heart is not a single current with a mono-exponential decay but rather a mixture of currents with different kinetics. It is not clear whether these arise from distinct populations of channels, or from modulation of a single population. A very slowly inactivating component, [(INa(P))] I(Na(P)) is usually about 1% of the size of the peak transient current [I(Na(T))], but is enhanced by hypoxia. It contributes to Na(+) loading and cellular damage in ischaemia and re-perfusion, and perhaps to ischaemic arrhythmias. Class I antiarrhythmic agents such as flecainide, lidocaine and mexiletine generally block I(NA(P)) more potently than block of I(Na(T)) and have been used clinically to treat LQT3 syndrome, which arises because mutations in SCN5A produce defective inactivation of the cardiac sodium channel. The same approach may be useful in some pathological situations, such as ischaemic arrhythmias or diastolic dysfunction, and newer agents are being developed with this goal. For example, ranolazine blocks I(Na(P)) about 10 times more potently than I(Na(T)) and has shown promise in the treatment of angina. Alternatively, the combination of I(Na(P)) block with K(+) channel block may provide protection from the induction of Torsades de Pointe when these agents are used to treat atrial arrhythmias (eg Vernakalant). In all of these scenarios, an understanding of the role of I(Na(P)) in cardiac pathophysiology, the mechanisms by which it may affect cardiac electrophysiology and the potential side effects of blocking I(Na(P)) in the heart and elsewhere will become increasingly important.

Gene expression of stretch-activated channels and mechanoelectric feedback in the heart.

1. Mechanoelectric feedback (MEF) in the heart is the process by which mechanical forces on the myocardium can change its electrical properties. Mechanoelectric feedback has been demonstrated in many animal models, ranging from isolated cells, through isolated hearts to whole animals. In humans, MEF has been demonstrated directly in both the atria and the ventricles. It seems likely that MEF provides either the trigger or the substrate for some types of clinically important arrhythmias. 2. Mechanoelectric feedback may arise because of the presence of stretch-sensitive (or mechano-sensitive) ion channels in the cell membrane of the cardiac myocytes. Two types have been demonstrated: (i) a non-specific cation channel (stretch-activated channel (SAC); conductance of approximately 25 pS); and (ii) a potassium channel with a conductance of approximately 100 pS. The gene coding for the SAC has not yet been identified. The gene for the potassium channel is likely to be TREK, a member of the tandem pore potassium channel gene family. We have recorded stretch-sensitive potassium channels in rat isolated myocytes that have the properties of TREK channels expressed in heterologous systems. 3. It has been shown that TREK mRNA is expressed heterogeneously in the rat ventricular wall, with 17-fold more expression in endocardial compared with epicardial cells. This difference is reflected in the TREK currents recorded from endocardial and epicardial cells using whole-cell patch-clamp techniques, although the difference in current density was less pronounced (approximately threefold). Consistent with this, we show here that when the ventricle is stretched by inflation of an intraventricular balloon in a Langendorff perfused rat isolated heart, action potential shortening was more pronounced in the endocardium (30% shortening at 40 mmHg) compared with that in the epicardium (10% shortening at the same pressure). 4. Computer models of the mechanics of the (pig) heart show pronounced spatial variations in strain in the myocardium with large transmural differences (in the left ventricle in particular) and also large differences between the base and apex of the ventricle. 5. The importance of MEF and the non-homogeneous gene expression and strain distribution for arrhythmias is discussed.

Heterogeneous expression of tandem-pore K+ channel genes in adult and embryonic rat heart quantified by real-time polymerase chain reaction.

1. Many members of the tandem-pore K+ channel gene family have been reported to be present in cardiac cells. However, the pattern of gene expression of these channels in the heart is a matter of some dispute. 2. Here, we used reverse transcription and real-time quantitative polymerase chain reaction to investigate the pattern of gene expression of nine members of the tandem-pore K+ channel genes in adult and embryonic rat heart. The genes (TWIK-1, TWIK-2, TASK-1, TASK-2, TASK-3, TREK-1, TREK-2, TRAAK and KCNK6) were quantified, relative to glyceraldehyde-3-phosphate dehydrogenase (GADPH), in all four chambers of adult rat hearts and in the ventricles of embryonic rat hearts. 3. All these genes were detected in at least one chamber of the heart, with a predominance of TWIK-2, TASK-1 and TREK-1 expression. The expression of TWIK-2 was higher in the right atrium than in other cardiac chambers, TASK-1 was expressed more in atria than in ventricles and TREK-1 was highly expressed in the right ventricle. 4. The expression levels of the three predominant genes in embryonic rat ventricle are much lower than their expression in adult rat ventricles. 5. The physiological implications of the differential gene expression of the tandem-pore K+ channels is discussed.

Suppression of calcium sparks in rat ventricular myocytes and direct inhibition of sheep cardiac RyR channels by EPA, DHA and oleic acid.

The anti-arrhythmic effects of long-chain polyunsaturated fatty acids (PUFAs) may be related to their ability to alter calcium handling in cardiac myocytes. We investigated the effect of eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA) on calcium sparks in rat cardiac myocytes and the effects of these PUFAs and the monounsaturated oleic acid on cardiac calcium release channels (RyRs). Visualization of subcellular calcium concentrations in single rat ventricular myocytes showed that intensity of calcium sparks was reduced in the presence of EPA and DHA (15 micro M). It was also found that calcium sparks decayed more quickly in the presence of EPA but not DHA. Sarcoplasmic vesicles containing RyRs were prepared from sheep hearts and RyR activity was determined by either [(3)H]ryanodine binding or by single-channel recording. Bilayers were formed from phosphatidylethanolamine and phosphatidylcholine dissolved in either n-decane or n-tetradecane. EPA inhibited [(3)H]ryanodine binding to RyRs in SR vesicles with K(I) = 40 micro M. Poly- and mono-unsaturated free fatty acids inhibited RyR activity in lipid bilayers. EPA (cytosolic or luminal) inhibited RyRs with K(I) =32 micro M and Hill coefficient, n(1) = 3.8. Inhibition was independent of the n-alkane solvent and whether RyRs were activated by ATP or Ca(2+). DHA and oleic acid also inhibited RyRs, suggesting that free fatty acids generally inhibit RyRs at micromolar concentrations.

Trek-like potassium channels in rat cardiac ventricular myocytes are activated by intracellular ATP.

Large (111 +/- 3.0 pS) K+ channels were recorded in membrane patches from adult rat ventricular myocytes using patch-clamp techniques. The channels were not blocked by 4-AP (5 mM), intracellular TEA (5 mM) or glybenclamide (100 mM). Applying stretch to the membrane (as pipette suction) increased channel open probability (Po) in both cell-attached and isolated patches (typically, Po approximately equals 0.005 with no pressure; approximately equals 0.328 with 90 cm H2O: Vm = 40 mV, pHi = 7.2). The channels were activated by a decrease in intracellular pH; decreasing pHi to 5.5 from 7.2 increased Po to 0.16 from approx. 0.005 (no suction, Vm held at 40 mV). These properties are consistent with those demonstrated for TREK-1, a member of the recently cloned tandem pore family. We confirmed, using RT-PCR, that TREK-1 is expressed in rat ventricle, suggesting that the channel being recorded is indeed TREK-1. However, we show also that the channels are activated by millimolar concentrations of intracellular ATP. At a pH of 6 with no ATP at the intracellular membrane face, Po was 0.048 +/-0.023, whereas Po increased to 0.22 +/- 0.1 with 1 mM ATP, and to 0.348 +/- 0.13 with 3 mM (n = 5; no membrane stretch applied). The rapid time course of the response and the fact that we see the effect in isolated patches appear to preclude phosphorylation. We conclude that intracellular ATP directly activates TREK-like channels, a property not previously described.

Subconductance states of the cardiac K(ATP) channel revealed by partial block with glybenclamide.

Currents carried by single ATP-sensitive potassium channels (K(ATP) channels) were recorded in membrane patches isolated from adult rat ventricular myocytes. Channel currents were blocked completely by ATP at millimolar concentrations and by glybenclamide at micromolar concentrations. However, at lower glybenclamide concentrations (1-1000 nM), a partial block, manifest as a subconductance state, was often seen. At concentrations of 100-300 nM the mean size of the subconductance state was 33+/-2.7 pS (175 mM potassium in the pipette; n=13). The size of the conductance substate varied slightly with the concentration of glybenclamide, (42 pS at 1 nM, 34 pS at 100 nM and 31 pS at 1 microM), while the open time of the subconductance state decreased with increasing glybenclamide concentration (n=4). ATP (4 mM) completely blocked both the main conductance state of the channel and the subconductance state induced by glybenclamide. Submaximal concentrations of ATP also appeared to induce subconductance states, but these could not be resolved into discrete conductance levels. The observation that subconductance states can be induced by low concentrations of glybenclamide may have implications for models of how the binding of glybenclamide is translated into closure of the Kir6.2 pore.

Interaction of lidocaine with the cardiac sodium channel: effects of low extracellular pH are consistent with an external blocking site.

Brief extracellular application of millimolar concentrations of lidocaine affected sodium currents recorded in isolated rat ventricular myocytes in two ways: 1) a reduction of the maximum current consistent with a channel blocking action, and 2) a negative shift in the voltage dependence of inactivation consistent with an interaction with the inactivated state of the channel. Both effects occurred very rapidly (<< 1 s). Decreasing extracellular pH to 6.4 increased the potency for channel block (EC50 1.8 +/- 0.2 mM at pH 7.4 and 0.8 +/- 0.1 mM at pH 6.5) and decreased the potency to shift inactivation (V(1/2) shift -42 mV by 1 mM lidocaine at pH 7.4 and -12.6 mV at pH 6.5). Channel block was slightly less at +90 mV compared to -40 mV at either pH (not statistically significant). The increase in potency for block at decreased extracellular pH, while intracellular pH is buffered, and the lack of voltage dependence of block, suggest that the charged form of lidocaine can block the channel by interacting with a site near the extracellular mouth, although alternative explanations are discussed.

Effects of dietary n-3 fatty acids on contractility, Na+ and K+ currents in a rat cardiomyocyte model of arrhythmia.

The n-3 polyunsaturated fatty acids (PUFAs) have been reported to prevent ventricular fibrillation in human clinical studies and in studies involving experimental animals and isolated cardiomyocytes. This study aimed to determine whether dietary n-3 PUFAs could prevent isoproterenol and free radical-induced arrhythmic (asynchronous) contractile activity in adult rat cardiomyocytes and whether whole-cell Na(+) and K(+) currents measured by patch-clamp techniques were affected. Dietary supplementation with fish oil for 3 weeks significantly increased the proportion of total n-3 PUFAs in ventricular membrane phospholipids compared with saturated fat supplementation (18.8 +/- 0.6% vs. 8.1 +/- 1.0%, respectively). Cardiomyocytes from the fish oil group were less susceptible to isoproterenol-induced asynchronous contractile activity than were those from the saturated fat group [EC(50) values: 892 +/- 130 nM, n = 6 and 347 +/- 91 nM, n = 6 (P < 0.05), respectively]. Fish oil supplementation also prolonged the time taken to develop asynchronous contractile activity induced by superoxide and hydrogen peroxide. The voltage dependence of inactivation of Na(+) currents were significantly altered (-73.5 +/- 1.2 mV, n = 5 vs. -76.7 +/- 0.7 mV, n = 5, P < 0.05, for saturated fat and fish oil treated groups, respectively). The voltage dependence of activation of Na(+) and K(+) currents was not significantly affected by the dietary fish oil treatment. These results demonstrate the antiarrhythmic effects of dietary fish oil in a cardiomyocyte model of arrhythmia.

Inhibition of cardiac sodium currents in adult rat myocytes by n-3 polyunsaturated fatty acids.

1. The acute effects of n-3 polyunsaturated fatty acids were determined on whole-cell sodium currents recorded in isolated adult rat ventricular myocytes using patch clamp techniques. 2. The n-3 polyunsaturated fatty acids docosahexaenoic acid (22:6, n-3), eicosapentaenoic acid (20:5, n-3) and alpha-linolenic acid (18:3, n-3) dose-dependently blocked the whole-cell sodium currents evoked by a voltage step to -30 mV from a holding potential of -90 mV with EC50 values of 6.0 +/- 1.2, 16.2 +/- 1.3 and 26.6 +/- 1.3 microM, respectively. 3. Docosahexaenoic acid, eicosapentaenoic acid and alpha-linolenic acid at 25 microM shifted the voltage dependence of activation of the sodium current to more positive potentials by 9.2 +/- 2.0, 10.1 +/- 1.1 and 8.3 +/- 0.9 mV, respectively, and shifted the voltage dependence of inactivation to more negative potentials by 22.3 +/- 0.9, 17.1 +/- 3.7 and 20.5 +/- 1.0 mV, respectively. In addition, the membrane fluidising agent benzyl alcohol (10 mM) shifted the voltage dependence of activation to more positive potentials by 7.8 +/- 2.5 mV and shifted the voltage dependence of inactivation to more negative potentials (by -24.6 +/- 3.6 mV). 4. Linoleic acid (18:2, n-6), oleic acid (18:1, n-9) and stearic acid (18:0) were either ineffective or much less potent at blocking the sodium current or changing the voltage dependence of the sodium current compared with the n-3 fatty acids tested. 5. Docosahexaenoic acid, eicosapentaenoic acid, alpha-linolenic acid and benzyl alcohol significantly increased sarcolemmal membrane fluidity as measured by fluorescence anisotropy (steady-state, rss, values of 0.199 +/- 0. 004, 0.204 +/- 0.006, 0.213 +/- 0.005 and 0.214 +/- 0.009, respectively, compared with 0.239 +/- 0.002 for control), whereas stearic, oleic and linoleic acids did not alter fluidity (the rss was not significantly different from control). 6. The potency of the n-3 fatty acids docosahexaenoic acid, eicosapentaenoic acid and alpha-linolenic acid to block cardiac sodium currents is correlated with their ability to produce an increase in membrane fluidity.

Variability of channel subconductance states of the cardiac sodium channel induced by protease.

Conductance and subconductance levels of voltage-activated sodium channels recorded using patch clamp techniques from isolated cardiac myocytes were accurately determined using signal processing techniques. From the tabulated amplitude distributions of the conductance levels, we inferred the most likely underlying distribution by applying the method of the kernel density estimate. When myocytes were prepared by dissociation of the heart with a solution containing collagenase as the only digestive enzyme, the fully open conductance level of the channel was 23 pS, with two prominent sublevels at 8 and 16 pS (280 mM sodium). When cells were dissociated in an identical manner but with solution containing added protease, the most frequent open levels of the channel were 9 and 15 pS. In these latter recordings, the channel also opened to 22 pS, but did so only rarely. The main conductance levels in cells dissociated with protease were essentially the same as the subconductance states in cells dissociated without protease. We infer that the conductance sublevels normally seen are, within experimental errors, 1/3 and 2/3 of the fully open level, and that the proteolytic enzyme modifies the channel such that it tends to open predominantly to the subconductance levels.

Sodium channel-blocking properties of spiradoline, a kappa receptor agonist, are responsible for its antiarrhythmic action in the rat.

Spiradoline (U-62,066E), a selective kappa (kappa) receptor agonist, was examined for actions on the cardiovascular system and on myocardial ionic currents in rats. We initially characterized cardiac, hemodynamic, and antiarrhythmic actions of spiradoline in isolated perfused rat hearts and pentobarbital-anesthetized rats. Electrophysiologic studies in isolated myocytes were used to elucidate the mechanism for changes observed in vivo in the ECG, as well as for antiarrhythmic actions against electrical and ischemia-induced arrhythmias. In isolated rat hearts, spiradoline reduced heart rate and cardiac contractility and increased the PR interval and QRS width of the ECG in a concentration-dependent manner. In anesthetized rats, spiradoline dose-dependently reduced blood pressure and heart rate and prolonged the PR interval and QRS width. At slightly higher doses, it increased the QaT interval of the ECG. RSh, an index of sodium channel blockade in the rat, also was dose-dependently increased. Electrical stimulation of the left ventricle suggested that spiradoline may exert its antiarrhythmic action by blockade of myocardial sodium currents. The electrophysiologic actions of spiradoline on sodium currents, the transient outward (i(to)) and sustained plateau potassium (ik(sus)) currents were studied in isolated cardiac rat myocytes by whole-cell patch-clamp techniques. Spiradoline (15-500 microM) reduced peak sodium current in a rapid, reversible, and concentration-dependent manner; it also increased the rate of decay of I(to) and reduced the amplitude of Ik(sus). At a concentration of 150 microM, spiradoline produced a 24 +/- 2 mV hyperpolarizing shift in sodium current inactivation kinetics but did not alter activation processes. Spiradoline showed both tonic and frequency-dependent components of sodium current block. Thus spiradoline produced its antiarrhythmic actions via sodium channel blockade in myocardial tissue, although higher doses also block potassium currents. This combined ion channel-blocking property may be of added clinical benefit in the setting of myocardial ischemia.

Pacemaking in the heart: the interplay of ionic currents.

1. There is still a degree of controversy about which currents drive pacemaking in the sinoatrial node or sinus venous. Early attempts to identify a single 'pacemaker current' in these tissues, based on voltage-clamp data, were largely unsuccessful, prompting the search for other mechanisms that may contribute to rhythmic activity. 2. Whole-cell patch-clamp recording from single cells isolated from the sinus venosus of the toad has shown that a voltage-dependent sodium current may play a role in pacemaking. This current has a transient component that contributes to the action potential upstroke and an inactivation-resistant component that contributes to the diastolic depolarization. The relative importance of this current in pacemaking is still controversial. 3. The development of computer models of pacemaking has contributed greatly to our understanding of how ionic currents can interact to produce rhythmic activity. Results are presented from one such model, 'Oxsoft Heart', to illustrate the different contributions of Ir and INa and to highlight the concept that pacemaking is driven by the integrated activity of many processes, rather than by any one current in particular. 4. Present models of pacemaking fail to accurately reproduce biological observations for certain situations. It is becoming clear that many processes contribute to pacemaking and have yet to be fully incorporated into models. Recent results regarding the role of intracellular calcium buffering and release and their implications, are discussed in this context. 5. The control of pacemaking by neurotransmitters is discussed. The limitations of single cell models in reproducing many of the complex responses to nerve stimulation of multicellular tissue, such as postinhibitory rebound, are discussed and possible improvements to models are suggested.

The effects of propofol on macroscopic and single channel sodium currents in rat ventricular myocytes.

1. The effects of the injectable anaesthetic agent propofol (di-isopropyl phenol) were examined on sodium currents and single sodium channels by use of patch-clamp techniques in ventricular myocytes isolated from rat hearts. 2. Propofol dose-dependently blocked the whole cell sodium currents evoked by a voltage step to -30 mV from a holding potential of -90 mV with an EC50 of 14.8+/-2.3 microM (mean+/-s.e.mean). 3. Propofol caused a substantial hyperpolarizing shift in the voltage-dependence of inactivation of sodium currents (168 microM (30 microg ml(-1)) propofol caused a -14 mV shift (P<0.01); 56 microM caused a -8 mV shift (P<0.05)). A smaller shift in the voltage-dependence of activation was produced (4 mV by 168 microM (not statistically significant)), but this was to more depolarized potentials. The maximal sodium conductance, as judged from the activation and inactivation curves, was reduced by 13% by 168 microM propofol (not statistically significant), but propofol did not affect the reversal potential of the current-voltage relationship. 4. The macroscopic rate of inactivation, as measured by the time constant of the exponential fall of current amplitude from the peak current, was also slowed by propofol, from a control time constant of 1.78+/-0.31 ms to 2.93+/-0.47 ms (mean+/-s.e.mean, n=8, P<0.05) by 168 microM propofol. Despite the increase in the time constant, the macroscopic inactivation remained well fitted by a single exponential. The macroscopic rate of activation was also slowed, but to a lesser degree (<10%, not statistically significant) by 168 microM propofol. 5. Propofol slowed the rate of recovery from inactivation of the sodium current, as measured by a two pulse protocol. Propofol (168 microM) increased the time constant of recovery, measured at -100 mV and room temperature, from a control value of 55+/-5.9 ms to 141+/-24.2 ms (mean+/-s.e.mean, n=8, P<0.01). Although the time constant was increased at all voltages measured, the intrinsic voltage-dependence of the rate of recovery was not changed. 6. Single channel recordings showed that the mean open time of single sodium channels was dramatically reduced by propofol (from 0.50+/-0.02 ms in control to 0.28+/-0.01 ms by 56 +/-M propofol and to 0.24+/-0.01 ms by 168 microM, both significantly different from control, P<0.01). Single channel conductance was not changed by either concentration of propofol.