Mechanisms underlying adaptation of action potential duration by pacing rate in rat myocytes

Heart rate is an essential determinant of cardiac performance. In rat ventricular myocytes, a sudden increase in rate yields to a prolongation of the action potential duration (APD). The mechanism underlying this prolongation is controversial: it has been proposed that the longer APD is due to either: (1) a decrease in K⁺ currents only or (2) an increase in Ca²⁺ current only. The aim of this study was to quantitatively investigate the contribution of Ca²⁺ and K⁺ currents in the adaptation of APD to pacing rate. Simulation using a mathematical model of ventricular rat cardiac cell model [Pandit, S.V., Clark, R.B., Giles, W.R., Demir, S.S., 2001. A mathematical model of action potential heterogeneity in adult rat left ventricular myocytes. Biophys. J. 81, 3029–3051] predicted a role in the prolongation of APD for K⁺ currents only. In patch clamp experiments, increasing the pacing rate leads to a significant increase in APD in both control and detubulated myocytes, although it was more marked in control than detubulated myocytes. Supporting the model prediction, we observed that increasing stimulation frequency leads to a decrease in K⁺ currents in voltage clamped rat ventricular myocytes (square and action potential waveforms), and to a similar extent in both cell types. We have also observed that frequency-dependent facilitation of Ca²⁺ current occurred in control cells but not in detubulated cells (square and action potential waveforms). From these experiments, we calculated that the relative contribution of Ca²⁺ and K⁺ currents to the longer APD following an increase in pacing rate is 65% and 35%, respectively. Therefore, in contrast to the model prediction, Ca²⁺ current has a significant role in the adaptation of APD to pacing rate. Finally, we have introduced a simplistic modification to the Pandit's model to account for the frequency-dependent facilitation of Ca²⁺ current.     

Ionic currents in cardiac myocytes of squid, Alloteuthis subulata

This paper provides the first study of voltage-sensitive membrane currents present in heart myocytes from cephalopods. Whole cell patch clamp recordings have revealed six different ionic currents in myocytes freshly dissociated from squid cardiac tissues (branchial and systemic hearts). Three types of outward potassium currents were identified: first, a transient outward voltage-activated A-current (I_A), blocked by 4-aminopyridine, and inactivated by holding the cells at a potential of −40 mV; second, an outward, voltage-activated, delayed rectifier current with a sustained time course (I_K); and third, an outward, calcium-dependent, potassium current (I_K(Ca)) sensitive to Co²⁺ and apamin, and with the characteristic N-shaped current voltage relationship. Three inward voltage-activated currents were also identified. First, a rapidly activating and inactivating, sodium current (I_Na), blocked by tetrodotoxin, inactivated at holding potentials more positive than −40 mV, and abolished when external sodium was replaced by choline. Second, an L-type calcium current (I_Ca,L) with a sustained time course, suppressed by nifedipine or Co²⁺, and enhanced by substituting Ca²⁺ for Ba²⁺ in the external medium. The third inward current was also carried by calcium ions, but could be distinguished from the L-type current by differences in its voltage dependence. It also had a more transient time course, was activated at more negative potentials, and resembled the previously described low-voltage-activated, T-type calcium current. Accepted: 24 September 1999     

Selenium-induced alterations in ionic currents of rat cardiomyocytes

In the present study, rats were treated with sodium selenite (5 micromol/kg body weight/day, ip) for 4 weeks and the parameters of contractile activity, action potential, L-type Ca2+-current (ICaL), as well as transient outward (Ito), inward rectifier (IK1), and steady state (Iss) K+-currents were investigated. Sodium selenite treatment increased rat blood glucose level and lowered plasma insulin level, significantly. This treatment also caused slightly prolongation in action potential with no significant effects on spontaneous contraction parameters and intracellular Ca2+ transients of the heart preparations. These effects were associated with marked alterations in the kinetics of both ICaL and Ito including a significant slowing in both inactivation time constants of ICaL and a significant shift to negative potential at half-inactivation of these channels without any change in the current density. Also, there was a significantly faster inactivation of Ito and no shift in half-inactivation of this channel without any change in its current density. Consequently, there was a approximately 50% increase in total charges carried by Ca2+ current and approximately 50% decrease in total charges carried by K+ currents of the treated rat cardiomyocytes. Additionally we observed a significant inhibition in IK1 density in treated rat cardiomyocytes. Oxidized glutathione level was significantly increased (70%) while the observed decrease in reduced glutathione was much less. Since a shift in redox state of regulatory proteins is related with cell dysfunction, selenium-induced increase in blood glucose and decrease in plasma insulin may correlate these alterations. These alterations, in the kinetics of the channels and in IK1 density, might lead to proarrhythmic effect of chronic selenium supplementation.     

Positive inotropic action of NMDA receptor antagonist (+)-MK801 in rat heart

(+)-MK801, a noncompetitive NMDA receptor antagonist, was reported to exhibit anticonvulsive and neuroprotective activities during the postischemic period. Intravenous administration of (+)-MK801 produced tachycardia in rats, but bradycardia in pigs. We examined the mechanical and electrophysiological effects of (+)-MK801 on rat cardiac tissues. (+)-MK801 dose-dependently increased (3–100 µM) twitch tension in rat atria and ventricular strips. The spontaneous beating rate in rat right atria, however, was dose-dependently decreased by (+)-MK801. The inotropic effect of (+)-MK801 was affected neither by ₁-antagonist (1 µM prazosin) nor by ₁-adrenoceptor antagonist (3 µM atenolol), but significantly by a transient outward K⁺ channel blocker (3 mM 4-aminopyridine). (+)-MK801 did not cause any significant change of intracellular cAMP content. Electrophysiological study in rat ventricular cells revealed that (+)-MK801 concentration-dependently prolonged the action potential duration with a concomitant decrease in the maximum rate of the action potential upstroke (V_max) and an increase in the recovery time constant of V_max. Voltage clamp study showed that (+)-MK801 (3 µM) reduced inward Na⁺ current (I_Na), along with a slowing of its recovery from inactivation and a slight negative shift of its voltage-dependent steady-state inactivation curves. At a much higher concentration (30 µM), (+)-MK801 slightly reduced the amplitude of L-type calcium inward current (I_Ca), although the voltage dependence of its steady-state inactivation was unaffected. For the potassium currents in rat ventricular cells, 3 µM of (+)-MK801 reduced the peak transient outward current (I_to), steady-state outward current (I_ss) and inward current through K₁ channels. The inhibition of I_to was associated with a prominent negative shift in the voltage dependence of its steady-state inactivation curve. The outward current through K₁ channels was unaffected. These results indicate that (+)-MK801 may be a strong I_Na and I_to blocker with some I_Ca blocking activity. The inhibition of I_to and other K⁺ efflux would prolong action potential duration, produce positive inotropic action and contribute to the negative chronotropic effect of (+)-MK801.     

Intracellular calcium and the relationship to contractility in an avian model of heart failure

Global contractile heart failure was induced in turkey poults by furazolidone feeding (700 ppm). Abnormal calcium regulation appears to be a key factor in the pathophysiology of heart failure, but the cellular mechanisms contributing to changes in calcium fluxes have not been clearly defined. Isolated ventricular myocytes from non-failing and failing hearts were therefore used to determine whether the whole heart and ventricular muscle contractile dysfunctions were realized at the single cell level. Whole cell current- and voltage-clamp techniques were used to evaluate action potential configurations and L-type calcium currents, respectively. Intracellular calcium transients were evaluated in isolated myocytes with fura-2 and in isolated left ventricular muscles using aequorin. Action potential durations were prolonged in failing myocytes, which correspond to slowed cytosolic calcium clearing. Calcium current-voltage relationships were normal in failing myocytes; preliminary evidence suggests that depressed transient outward potassium currents contribute to prolonged action potential durations. The number of calcium channels (as measured by radioligand binding) were also similar in non-failing and failing hearts. Isolated ventricular muscles from failing hearts had enhanced inotropic responses, in a dose-dependent fashion, to a calcium channel agonist (Bay K 8644). These data suggest that changes in intracellular calcium mobilization kinetics and longer calcium-myofilament interaction may be able to compensate for contractile failure. We conclude that the relationship between calcium current density and sarcoplasmic reticulum calcium release is a dynamic process that may be altered in the setting of heart failure at higher contraction rates. Accepted: 1 March 2000     

Protaglandins and the antiarrhythmic effect of preconditioning in the isolated rat heart

Our study evaluated the relationship between the endogenous production of prostacyclin and the antiarrhythmic effect of ischemic preconditioning against ischemic and reperfusion-induced tachyarrhythmia. Langendorff perfused rat hearts underwent 30 min regional ischemia with reperfusion. Preconditioning was induced by a single episode of 5 min ischemia and 15 min reperfusion. Prostaglandin 6-keto F₁ (a stable metabolite of prostacyclin) was determined in the coronary effluent.In the control group the incidence of tachyarrhythmia was 31 % during ischemia and 67% during reperfusion. Preconditioning did not affect ischemic arrhythmias but attenuated arrhythmias a reperfusion (8%, p < 0.01) and was associated with increased release of prostacyclin prior to reperfusion. Aspirin abolished the antiarrhythmic effect of preconditioning against reperfusion tachyarrhythmias. However, no relationship was found between suppression of prostacyclin production and the occurrence of arrhythmia in individual hearts.Thus, our findings suggest that metabolites of arachidonic acid via the cyclooxygenase pathway are involved in the protective effect of ischemic preconditioning against reperfusion-induced tachyarrhythmias. (Mol Cell Biochem 160/161: 249–255, 1996)     

Pharmacological evaluation of R(+)-pulegone on cardiac excitability: Role of potassium current blockage and control of action potential waveform

IntroductionR(+)-pulegone is a ketone monoterpene and it is the main constituent of essential oils in several plants. Previous studies provided some evidence that R(+)-pulegone may act on isolated cardiac myocytes. In this study, we evaluated in extended detail, the pharmacological effects of R(+)-pulegone on cardiac tissue.MethodsUsing in vivo measurements of rat cardiac electrocardiogram (ECG) and patch-clamp technique in isolated myocytes we determinate the influence of R(+)-pulegone on cardiac excitability.ResultsR(+)-pulegone delayed action potential repolarization (APR) in a concentration-dependent manner (EC₅₀ = 775.7 ± 1.48, 325.0 ± 1.30, 469.3 ± 1.91 μM at 10, 50 and 90% of APR respectively). In line with prolongation of APR R(+)-pulegone, in a concentration-dependent manner, blocked distinct potassium current components (transient outward potassium current (I_to), rapid delayed rectifier potassium current (I_Kr), inactivating steady state potassium current (I_ss) and inward rectifier potassium current (I_K1)) (EC₅₀ = 1441 ± 1.04; 605.0 ± 1.22, 818.7 ± 1.22; 1753 ± 1.09 μM for I_to, I_Kr, I_ss and I_K1, respectively). The inhibition occurred in a fast and reversible way, without changing the steady-state activation curve, but instead shifting to the left the steady-state inactivation curve (V_1/2 from −56.92 ± 0.35 to −67.52 ± 0.19 mV). In vivo infusion of 100 mg/kg R(+)-pulegone prolonged the QTc (∼40%) and PR (∼62%) interval along with reducing the heart rate by ∼26%.ConclusionTaken together, R(+)-pulegone prolongs the APR by inhibiting several cardiomyocyte K⁺ current components in a concentration-dependent manner. This occurs through a direct block by R(+)-pulegone of the channel pore, followed by a left shift on the steady state inactivation curve. Finally, R(+)-pulegone induced changes in some aspects of the ECG profile, which are in agreement with its effects on potassium channels of isolated cardiomyocytes.     

Changes in the calcium current among different transmural regions contributes to action potential heterogeneity in rat heart

To clarify the transmural heterogeneity of action potential (AP) time course, we examined the regulation of L-type Ca²⁺ current (I_Ca,L) by voltage and Ca²⁺-dependent mechanisms. Currents were recorded using patch clamp of single rat subepicardial (EPI) and subendocardial (ENDO) of left ventricular, right ventricular (RV) and septal (SEP) cardiomyocytes. Voltage clamp commands were derived from ENDO and EPI APs or rectangular voltage pulses.During rectangular pulses, peak I_Ca,L was significantly greater in EPI than in other cells. The inactivation of I_Ca,L by Ca²⁺-dependent mechanisms (suppressed by ryanodine and BAPTA) was present in all cells but greater in extent in ENDO and SEP cells. Activation and inactivation curves for all regions show subtle differences that are Ca²⁺ sensitive, with Ca²⁺ inactivation shifting the activation variables negative by ∼ 7 mV and inactivation variables positive by 2-7 mV (EPI being least, RV greatest). In AP-clamps, the peak I_Ca,L was significantly smaller in ENDO than in EPI cells, while the integrated current was significantly larger in ENDO than in EPI cells. The results are discussed with regard to the interplay of AP time course and net Ca²⁺ influx.     

Effects of streptozotocin-induced diabetes on action potentials in the sinoatrial node compared with other regions of the rat heart

In vivo biotelemetry studies have demonstrated that heart rate (HR) is progressively and rapidly reduced after administration of streptozotocin (STZ) and that the reduction in HR can be partially normalized with insulin replacement. Reductions in HR have also been reported in isolated perfused heart and superfused right atrial preparations suggesting that intrinsic defects in the heart are at least partly responsible for the bradycardia. The regional effects of STZ-induced diabetes mellitus (DM) on action potentials (APs) in the sinoatrial node (SAN), right and left atria and ventricles have been compared in the spontaneously beating Langendorff perfused rat heart 10–12 weeks after treatment. HR was significantly reduced in STZ-induced diabetic rat heart (174 ± 9 BPM) compared to controls (241 ± 12 BPM). The duration of AP repolarization at 50% and 70% from peak AP was significantly prolonged in SAN, right atrium and right ventricle from STZ-induced diabetic rat compared to age-matched controls. In the SAN AP duration (APD) at 50% and 70% were 51.7 ± 2.2 and 59.5 ± 2.3 ms in diabetic rat heart compared to 45.2 ± 1.7 and 50.0 ± 1.6 ms in controls, respectively. In contrast APD at 50% and 70% were not significantly altered in the left atrium and left ventricle. Regional defects in the expression and/or electrophysiology of SAN ion channels, and in particular those involved in AP repolarization, might underlie heart rhythm disturbances in the STZ-induced DM rat.     

Toxic concentrations of selenite shortens repolarization phase of action potential in rat papillary muscle

Selenium (Se) has long been recognized as both an essential mammalian nutrient and a hazardous element. Sodium selenite is commonly used as a dietary supplement for the treatment of Se deficiency. On the other hand, chronic Se toxicity has been demonstrated to affect the major organs, including the heart, in experimental animals. This study examines the effects of high concentrations of extracellular selenite (in the range of 0.001–1 mM) application into the recording bath on the electrical properties of rat papillary muscles. Conventional glass semifloating microelectrodes were used to record intracellular action potentials (APs) in isolated rat papillary muscles. The amplitude of APs and the resting membrane potential of papillary muscles were not changed following a 20-min selenite (1 mM) application compared to the first minute of its application. Freshly isolated ventricular myocytes by an enzymatic method were used to determine the selenite-induced alterations in Na⁺ currents. Na⁺ currents, measured at 22°C, by the whole-cell patch-clamp technique, decreased by 38±8% in the presence of 1 mM selenite for 5 min. These selenite-induced effects were not reversed, but are restored by dithiothreitol (1 mM). These results demonstrated that toxic concentrations of selenite induced a significant shortening in AP duration as a result of an increase in the rate of repolarization. Our findings also suggest that a decrease in Na⁺ currents, in addition to Ca²⁺ currents, may play a role in the shortening of AP duration in rat papillary muscles.     

Light-triggered action potentials in the liverwort Conocephalum conicum

The response to light of a liverwort, Conocephalum conicum L., measured as a change in the resting potential, consists of two stages. The first stage is a slight depolarization dependent on light intensity. This plays the role of a generator potential (GP) which induces the second stage - an action potential of the all-or-none character. Action potentials induced by light and by electrical stimuli have the same properties, i.e. identical time course, propagation velocity, and refractory periods. A summation occurs of sub threshold light stimuli and of light and electrical stimuli. The presence of 5⋅10-⁻⁶ M DCMU cancelled the light response and blocked - by inhibition of the electron transport chain - the mechanism leading to GP generation. However, this effect did not produce any change in the response to electrical stimuli.     

Stretch-grown axons retain the ability to transmit active electrical signals

Little is known about extensive nervous system growth after axons reach their targets. Indeed, postnatal animals continue to grow, suggesting that axons are stretched to accommodate the expanding body. We have previously shown that axons can sustain stretch-growth rates reaching 1 cm/day; however, it remained unknown whether the ability to transmit active signals was maintained. Here, stretch-growth did not alter sodium channel activation, inactivation, and recovery or potassium channel activation. In addition, neurons generated normal action potentials that propagated across stretch-grown axons. Surprisingly, Na and K channel density increased due to stretch-growth, which may represent a natural response to preserve the fidelity of neuronal signaling.     

Intracellular mechanisms of hypoxia-induced calcium increase in rat sensory neurons

Elevation of cytosolic level of Ca(2+) was measured by spatial screening of freshly isolated dorsal root ganglion neurons loaded with Fura-2AM after subjecting them to a moderate hypoxic solution (pO(2)=10-40 mmHg). Short exposure of neurons to hypoxia resulted in a reversible elevation of intracellular Ca(2+) to about 120% in the cell center and to 80% in the cell periphery. Such elevation could be almost completely eliminated by removal of Ca(2+) or Na(+) from external medium or application of nifedipine, an L-type calcium channel blocker. Remarkable antihypoxic efficiency (58%) was achieved by preapplication of mitochondrial protonophore CCCP. A conclusion is made that in sensory neurons the hypoxia-induced elevation of cytosolic Ca(2+) is induced by combined changes of function in three cell substructures: voltage-operated L-type Ca(2+) and Na(+) channels and Ca(2+) accumulation by mitochondria. Mitochondria are important for spatial difference in the hypoxia-induced Ca(2+) elevation due to their specific location in these neurons.     

内源性儿茶酚胺参与白细胞介素-2对大鼠离体心脏的作用

目的: 研究内源性儿茶酚胺是否参与白细胞介素-2(IL-2)的心脏作用.方法: 采用Langendorff离体心脏灌流模型,观察室性早搏次数、左室发展压(LVDP)、左室舒张末压(LVEDP)、心率(HR)和冠脉流量(CF)的变化.结果: ①50 U/ml IL-2增加离体心脏的室性早搏次数,增加LVDP、LVEDP、HR和CF.②利舍平(reserpine)或普萘洛尔(propranolol)预处理后,50 U/ml IL-2对离体心脏的作用消失;phentolamine预处理后,不改变50U/ml IL-2的离体心脏作用.③200 U/ml IL-2增加离体心脏的室性早搏次数,对LVDP、LVEDP、HR和CF无显著增加作用.④reserpine或propranolol预处理后,200 U/ml IL-2增加离体心脏的室性早搏次数作用不明显,LVDP、HR和CF降低、LVEDP增高.结论: 内源性儿茶酚胺介导了IL-2的致离体心脏心律失常、正性变时和变力作用,较高浓度的IL-2抑制离体心脏的功能.     

An eikonal-curvature equation for action potential propagation in myocardium

We derive an eikonal-curvature equation to describe the propagation of action potential wavefronts in myocardium. This equation is used to study the effects of fiber orientation on propagation in the myocardial wall. There are significant computational advantages to the use of an eikonal-curvature equation over a full ionic model of action potential spread. With this model, it is shown that the experimentally observed misalignment of spreading action potential ellipses from fiber orientation in level myocardial surfaces is adequately explained by the rotation of fiber orientation through the myocardial wall. Additionally, it is shown that apparently high propagation velocities on the epicardial and endocardial surfaces are the result of propagation into the midwall region and acceleration along midwall fibers before reemergence at an outer surface at a time preceding what could be accomplished with propagation along the surface alone.Research was supported in part by NSF Grant DMS-8801446     

The effects of static magnetic field on action potential propagation and excitation recovery in nerve

Calculations using the Hodgkin–Huxley and one-dimensional cable equations have been performed to determine the expected sensitivity of conduction and refractoriness to changes in the time constant of sodium channel deactivation at negative potentials, as reported experimentally by Rosen (Bioelectromagnetics 24 (2003) 517) when voltage-gated sodium channels are exposed to a 125 mT static magnetic field. The predicted changes in speed of conduction and refractory period are very small.     

Quantitative analysis of sodium and potassium activation delays in fresh axons of the squid: Loligo forbesi

Activation kinetics of the sodium and potassium conductances were re-examined in fresh axons of Loligo forbesi exhibiting very little if any potassium accumulation and a very small leak conductance, special attention being paid to the initial lag phase which precedes the turning-on of the conductances. The axons were kept intact and voltage-clamped at 2–3°C.In all cases, the rising phase of the currents could be fitted with very good accuracy using the Hodgkin-Huxley (1952) equations although, in most cases, the turning-on of the conductance did not coincide with the beginning of the depolarizing test pulse. The delay which separates the change in potential and the turning-on of current (the activation delay) was analyzed quantitatively for different prepulse and pulse potentials. The measured activation delay differed significantly from the delay predicted by the original HH equations. This difference (the non-HH delay) varied with prepulse and pulse potentials. For the potassium current, the relationship between the non-HH delay and pulse potential for a constant prepulse was bell shaped, the maximum value (0.7 ms for a prepulse to –80 mV) being reached for about 0 mV For this same current, the relationship between the non-HH delay and the prepulse potential for a constant pulse potential was sigmoidal, starting from a minimum value of around 0.5 ms at –100 mV and rising to 5 ms at –15 mV Essentially similar results were obtained for the sodium current although the non-HH delay was three to five times smaller and the dependency upon prepulse potential not significant. These results are in agreement with previous observations on squid axons and frog nodes of Ranvier and suggest that the opening of an ionic channel is preceded by a short but essential voltage-dependent conformational change of the channel protein. Offprint requests to: Y. Pichon     

人参皂甙Rd通过调节SNI大鼠背根神经节钠、钾电流抑制痛敏

目的：观察人参皂甙Rd（ginsenoside Rd）对大鼠坐骨神经分支选择性损伤（spared sciatic nerve injury,SNI）引起的痛敏的影响及其作用机制。方法：坐骨神经分支选择性损伤术后7天,观察腹腔注射不同浓度人参皂甙Rd后大鼠后足的机械性缩足反应阈值（paw withdrawl mechanical threshold,PWMT）的变化;在术后7天,急性分离并取出大鼠腰4和腰5段背根节,对整节DRG上的中小型神经元运用全细胞膜片钳技术进行记录。结果：坐骨神经分支选择性损伤术后7天,大鼠出现明显的机械性痛敏,腹腔注射5 mg/ml和10 mg/ml的人参皂甙Rd能剂量依赖性的翻转大鼠机械性痛敏;坐骨神经分支选择性损伤能明显地增大SNI大鼠DRG中小型神经元上的钠电流以及减小电压依赖性钾电流,而100μM人参皂甙Rd能有效翻转该钠、钾电流的变化。结论：人参皂甙Rd能有效地改善坐骨神经分支选择性损伤引起的机械性痛敏,其机制可能与人参皂甙Rd明显地调节SNI大鼠DRG中小型神经元上的电压依赖性钠、钾电流有关。     

The effect of n-alkanols on the stationary current voltage behavior and action potential of myelinated nerve

Stationary current voltage characteristics and the action potential of single myelinated nerve fibres were measured to examine the effect of $\text{n-}\text{alkanols}$ (methanol to octanol) on the electrophysiological function of the axon membrane. K⁺-depolarized membranes show alkanol-dependent shifts of $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$, the membrane transition voltage, whereas in veratridine-depolarized membranes such $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}\text{-}\text{shifts}$ are not observed. In the latter case, $\text{n-}\text{alkanols}$ reduce both the stationary Na⁺ current and the conductivity step between the high- and low-ohmic conductivity state of the membrane. Action potential amplitude, however, is less affected by the alkanols as is the stationary Na⁺ current. The results are compared with the alkanol-dependent changes of the thermotropic phase transition in phospholipid bilayers.