Dietary Fish Oil and Atrial Fibrillation.Introduction: Dietary fish oil is thought to reduce sudden cardiac death by suppressing ventricular arrhythmias but little is known about its impact on atrial arrhythmias. We examined the effect of dietary fish oil on the rabbit model of stretch-induced vulnerability to atrial fibrillation (AF).Methods and Results: Six-week-old rabbits were fed standard rabbit pellets supplemented with 5% tuna fish oil (n = 6) or supplemented with 5% sunflower oil (n = 6) for 12 weeks. Six rabbits raised on the standard diet were used as controls. In Langendorff-perfused hearts intraatrial pressures were increased in a stepwise manner and rapid burst pacing applied to induce AF at increasing intraatrial pressures until AF was sustained (> 1 minute). Atrial refractory periods were recorded at each pressure. Increased atrial pressure resulted in a reduction in atrial refractory period and a propensity for induction of sustained AF. Higher pressures were needed to induce and sustain AF in the fish oil group compared with the sunflower oil and control groups. The stretch-induced drop in refractory period was also less marked in the fish oil group. Red blood cell, atrial, and ventricular omega-3 fatty acid levels were significantly higher in the fish oil group. The ratio of atrial n-6/n-3 polyunsaturated fatty acids was 13 +/- 0.9 with sunflower oil and 1.5 +/- 0.01 with fish oil (P < 0.001).Conclusions: Incorporation of dietary omega-3 fatty acids into atrial tissue reduces stretch-induced susceptibility to AF.

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.

Stretching the myocardium can have profound effects on its electrical activity, a process called mechanoelectric feedback. Mechanoelectric feedback can be powerful enough to trigger arrhythmias, and it is likely to be the underlying trigger for some clinically intractable arrhythmias. It seems likely that the response to stretch involves at least two types of mechanosensitive ion channels, a nonspecific cation channel, and a potassium channel (or channels). Currents carried by nonspecific cation channels have been well characterised in cardiac tissue, although the gene has not yet been identified. Very recently, a peptide toxin isolated from Grammastola spatulata has been shown to block this channel and to be an effective antiarrhythmic agent against stretch-induced atrial arrhythmias. Stretch-activated potassium currents and single channels have also been recorded in cardiac cells; in the case of these channels, the genes coding for them have recently been cloned and shown to be members of a new potassium channel gene family, the "tandem pore" channels. Although some nonspecific activators and blockers of these channels exist, there is at present no specific blocker, which limits investigation of the role of these channels in arrhythmogenesis. The potential for the development of blockers of both types of stretch-activated channels as therapeutic agents is discussed, Drug Dev. Res. 55:53-58, 2002. (C) 2002 Wiley-Liss, Inc.

Real-time reverse transcription (RT) PCR is currently the most sensitive method for the detection of low-abundance mRNAs. Two relative quantitative methods have been adopted: the standard curve method and the comparative C(T) method. The latter is used when the amplification efficiency of a reference gene is equal to that of the target gene; otherwise the standard curve method is applied. Based on the simulation of kinetic process of real-time PCR, we have developed a new method for quantitation and normalization of gene transcripts. In our method, the amplification efficiency for each individual reaction is calculated from the kinetic curve, and the initial amount of gene transcript is derived and normalized. Simulation demonstrated that our method is more accurate than the comparative C(T) method and would save more time than the relative standard curve method. We have used the new method to quantify gene expression levels of nine two-pore potassium channels. The relative levels of gene expression revealed by our quantitative method were broadly consistent with those estimated by routine RT-PCR, but the results also showed that amplification efficiencies varied from gene to gene and from sample to sample. Our method provides a simple and accurate approach to quantifying gene expression level with the advantages that neither construction of standard curve nor validation experiments are needed.

This study investigated the effects of dietary omega-3 polyunsaturated fatty acids on calcium handling mechanisms in cardiac myocytes, with the hypothesis that this effect underlies some of the antiarrhythmic properties of these compounds. Adult male Sprague Dawley rats had their standard chow supplemented with either lard (57% saturated and 40% monounsaturated fat), canola oil (60% monounsaturated, 33% polyunsaturated) or fish oil (78% polyunsaturated). Isolated cardiac atrial myocytes from these animals were loaded with fluo-3AM and examined with laser scanning confocal microscopy. The dietary interventions resulted in considerable changes in the membrane phospholipid composition of cardiac cell membranes, particularly the ratio of n-6 to n-3 (2.17 with lard supplement and 1.28 with fish oil supplement). Calcium sparks in myocytes from rats which received saturated fat were significantly more prolonged than those from rats which received fish oil. (Lard = 105.4 +/- 18.9 ms; Fish oil = 43.5 +/- 4.7 ms: mean +/- s.e.m). The results for canola oil were intermediate (56.4 +/- 9.0 ms). The prolongation of the sparks in rats fed lard was primarily due to a higher proportion of sparks with long plateaus and/or slowed kinetics in this group. The frequency of sparks was not significantly different in cells from any group. We conclude that calcium handling mechanisms in rat atrial myocytes are affected by inclusion of different fats in the diet, correlated with changes in the cell membrane phospholipid composition, and speculate that this may underlie some of the antiarrhythmic properties of these dietary compounds.

Real time RT-PCR is the most sensitive method for quantitation of gene expression levels. The accuracy can be dependent on the mathematical model on which the quantitative methods are based. The generally accepted mathematical model assumes that amplification efficiencies are equal at the exponential phase of the reactions for the same amplicon. However, no methods are available to test the assumptions regarding amplification efficiency before one starts the real time PCR quantitation. Here we further develop and test the validity of a new mathematical model which dynamically fits real time PCR data with good correlation (R(2)=0.9995+/-0.002, n=50). The method is capable of measuring cycle-by-cycle PCR amplification efficiencies and demonstrates that these change dynamically. Validation of the method revealed the intrinsic relationship between the initial amount of gene transcript and kinetic parameters. A new quantitative method is proposed which represents a simple but accurate quantitative method.

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 [EC50 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.

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.

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 mu M, respectively.3. Docosahexaenoic acid, eicosapentaenoic acid and alpha-linolenic acid at 25 mu M 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, r(ss), 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 r(ss) was not significantly different from control).6. The potency of the n-3 fatty acids docosahexaenoic acid, eicosapentaenoic acid and alpha-linolenic acid to Mock cardiac sodium currents is correlated with their ability to produce an increase in membrane fluidity.

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.

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.

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. 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 EC 50 of 14.8±2.3 μ M (mean±s.e.mean). Propofol caused a substantial hyperpolarizing shift in the voltage‐dependence of inactivation of sodium currents (168 μ M (30 μg ml −1 ) propofol caused a −14 mV shift ( P <0.01); 56 μ M caused a −8 mV shift ( P <0.05)). A smaller shift in the voltage‐dependence of activation was produced (4 mV by 168 μ M (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 μ M propofol (not statistically significant), but propofol did not affect the reversal potential of the current ‐ voltage relationship. 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 μ M 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 μ M propofol. Propofol slowed the rate of recovery from inactivation of the sodium current, as measured by a two pulse protocol. Propofol (168 μ M ) 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. 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 μ M propofol and to 0.24±0.01 ms by 168 μ M , both significantly different from control, P <0.01). Single channel conductance was not changed by either concentration of propofol.

1. The effect of propofol on cardiac whole-cell sodium currents and single sodium channels in rat isolated ventricular myocytes was examined using patch-clamp techniques. 2. Propofol caused a block of the whole-cell sodium current, the potency of block depending on the holding potential. When cells were held at -90 mV, the EC50 was 2.8 micrograms/mL. When cells were held more hyperpolarized (at -140 mV), the EC50 increased to 44.0 micrograms/mL. 3. Although the degree of block produced by the same concentration of propofol was different at different holding potentials, the time course of onset and recovery from block was the same. 4. The current/voltage relationship for the sodium current showed a pronounced block of peak current by propofol (40-50% block of the maximum current by 30 micrograms/mL propofol), with a minimal shift in the voltage dependence of activation and no shift in reversal potential. 5. The voltage dependence of the steady state inactivation curve was shifted to more hyperpolarized potentials by propofol (shift of 18 and 8 mV by 30 and 10 micrograms/mL propofol, respectively). 6. Single channel records showed that propofol caused a shortening of the mean channel open time (from a mean of 0.59 to 0.38 ms by 10 micrograms/mL propofol), but no change in the channel amplitude. 7. It is concluded that propofol produces a block of sodium currents in cardiac myocytes at concentrations that are comparable to those that may be attained during anaesthesia.

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.

1. The effects of bisaramil on sodium currents in rat isolated cardiac myocytes were examined by use of tight-seal, whole-cell patch clamp techniques. Bisaramil produced a concentration-dependent, readily reversible reduction in peak transient sodium current. When the sodium current was evoked at 3 s intervals the estimated ED50 for bisaramil was about 11 microM. 2. Bisaramil (16 microM) produced a shift in the inactivation curve to hyperpolarized potentials of about 10 mV, but produced no change in the voltage-dependence of activation. 3. The block of the sodium current by bisaramil showed a profound use-dependence. A concentration of 10 microM produced a considerable block of the current with repeated stimulation. The recovery from block was biphasic, showing fast and slow components which had time constants of about 40 ms and 5 s respectively. 4. Bisaramil produced little tonic block of the sodium current at concentrations of 100 microM; at 300 microM it produced tonic block of around 50%, with extreme use-dependence. 5. Bisaramil appeared not to interact primarily with the inactivated form of the channel, since lengthening the depolarizing pulses did not affect the degree of block produced.