Abrupt rate accelerations or premature beats cause life-threatening arrhythmias in mice with long-QT3 syndrome.

Deletion of amino-acid residues 1505-1507 (KPQ) in the cardiac SCN5A Na(+) channel causes autosomal dominant prolongation of the electrocardiographic QT interval (long-QT syndrome type 3 or LQT3). Excessive prolongation of the action potential at low heart rates predisposes individuals with LQT3 to fatal arrhythmias, typically at rest or during sleep. Here we report that mice heterozygous for a knock-in KPQ-deletion (SCN5A(Delta/+)) show the essential LQT3 features and spontaneously develop life-threatening polymorphous ventricular arrhythmias. Unexpectedly, sudden accelerations in heart rate or premature beats caused lengthening of the action potential with early afterdepolarization and triggered arrhythmias in Scn5a(Delta/+) mice. Adrenergic agonists normalized the response to rate acceleration in vitro and suppressed arrhythmias upon premature stimulation in vivo. These results show the possible risk of sudden heart-rate accelerations. The Scn5a(Delta/+) mouse with its predisposition for pacing-induced arrhythmia might be useful for the development of new treatments for the LQT3 syndrome.

The effect of external pH on the delayed rectifying K+ current in cardiac ventricular myocytes.

The effects of extracellular pH (pHe) on the delayed rectifying K+ current iKr in rabbit ventricular myocytes were studied using the whole-cell-clamp technique. Since a variety of results have been reported on the effect of pH on expressed hERG channels, our aim was to investigate the effects of pH on iKr channels in their native environment. iKr is reduced by extracellular acidification and its deactivation is faster. Extracellular acidification results in a marked shift of the steady-state activation curve to more positive potentials, while alkalinization does not produce a significant shift. E1/2= - 11.3 mV, -20.2 mV, -21.4 mV at pHe 6.5, 7.4, 8.5 respectively; the slope factor is 6.2 mV, and is not affected by pHe. Deactivation of iKr is biexponential, with time constants of the order of 0.5 s and 10 s at -50 mV. Both time constants decrease with external acidification. Also the contribution of the fast component to the total amplitude becomes larger with acidification. Acidification also decreases the fully activated iKr current. Our experiments demonstrate that extracellular acidification reduces iKr by increasing the rate of deactivation, causing a shift of the voltage dependence of activation and producing a voltage-dependent block of the fully activated iKr current.

Downregulation of delayed rectifier K(+) currents in dogs with chronic complete atrioventricular block and acquired torsades de pointes.

BACKGROUND: Acquired QT prolongation enhances the susceptibility to torsades de pointes (TdP). Clinical and experimental studies indicate ventricular action potential prolongation, increased regional dispersion of repolarization, and early afterdepolarizations as underlying factors. We examined whether K(+)-current alterations contribute to these proarrhythmic responses in an animal model of TdP: the dog with chronic complete atrioventricular block (AVB) and biventricular hypertrophy. METHODS AND RESULTS: The whole-cell K(+) currents I(TO1), I(K1), I(Kr), and I(Ks) were recorded in left (LV) and right (RV) ventricular midmyocardial cells from dogs with 9+/-1 weeks of AVB and controls with sinus rhythm. I(TO1) density and kinetics and I(K1) outward current were not different between chronic AVB and control cells. I(Kr) had a similar voltage dependence of activation and time course of deactivation in chronic AVB and control. I(Kr) density was similar in LV myocytes but smaller in RV myocytes (-45%) of chronic AVB versus control. For I(Ks), voltage-dependence of activation and time course of deactivation were similar in chronic AVB and control. However, I(Ks) densities of LV (-50%) and RV (-55%) cells were significantly lower in chronic AVB than control. CONCLUSIONS: Significant downregulation of delayed rectifier K(+) current occurs in both ventricles of the dog with chronic AVB. Acquired TdP in this animal model with biventricular hypertrophy is thus related to intrinsic repolarization defects.

Cardiac ionic currents and acute ischemia: from channels to arrhythmias.

The aim of this review is to provide basic information on the electrophysiological changes during acute ischemia and reperfusion from the level of ion channels up to the level of multicellular preparations. After an introduction, section II provides a general description of the ion channels and electrogenic transporters present in the heart, more specifically in the plasma membrane, in intracellular organelles of the sarcoplasmic reticulum and mitochondria, and in the gap junctions. The description is restricted to activation and permeation characterisitics, while modulation is incorporated in section III. This section (ischemic syndromes) describes the biochemical (lipids, radicals, hormones, neurotransmitters, metabolites) and ion concentration changes, the mechanisms involved, and the effect on channels and cells. Section IV (electrical changes and arrhythmias) is subdivided in two parts, with first a description of the electrical changes at the cellular and multicellular level, followed by an analysis of arrhythmias during ischemia and reperfusion. The last short section suggests possible developments in the study of ischemia-related phenomena.

Repolarizing K+ currents ITO1 and IKs are larger in right than left canine ventricular midmyocardium.

BACKGROUND: The ventricular action potential exhibits regional heterogeneity in configuration and duration (APD). Across the left ventricular (LV) free wall, this is explained by differences in repolarizing K+ currents. However, the ionic basis of electrical nonuniformity in the right ventricle (RV) versus the LV is poorly investigated. We examined transient outward (ITO1), delayed (IKs and IKr), and inward rectifier K+ currents (IK1) in relation to action potential characteristics of RV and LV midmyocardial (M) cells of the same adult canine hearts. METHODS AND RESULTS: Single RV and LV M cells were used for microelectrode recordings and whole-cell voltage clamping. Action potentials showed deeper notches, shorter APDs at 50% and 95% of repolarization, and less prolongation on slowing of the pacing rate in RV than LV. ITO1 density was significantly larger in RV than LV, whereas steady-state inactivation and rate of recovery were similar. IKs tail currents, measured at -25 mV and insensitive to almokalant (2 micromol/L), were considerably larger in RV than LV. IKr, measured as almokalant-sensitive tail currents at -50 mV, and IK1 were not different in the 2 ventricles. CONCLUSIONS: Differences in K+ currents may well explain the interventricular heterogeneity of action potentials in M layers of the canine heart. These results contribute to a further phenotyping of the ventricular action potential under physiological conditions.

Antiarrhythmic drugs and cardiac ion channels: mechanisms of action.

In this review a description and an analysis are given of the interaction of antiarrhythmic drugs with their molecular target, i.e. ion channels and receptors. Our approach is based on the concept of vulnerable parameter, i.e. the electrophysiological property which plays a crucial role in the genesis of arrhythmias. To prevent or stop the arrhythmia a drug should modify the vulnerable parameter by its action on channel or receptor targets. In the first part, general aspects of the interaction between drugs channel molecules are considered. Drug binding depends on the state of the channel: rested, activated pre-open, activated open, or inactivated state. The change in channel behaviour with state is presented in the framework of the modulated-receptor hypothesis. Not only inhibition but also stimulation can be the result of drug binding. In the second part a detailed and systematic description and an analysis are given of the interaction of drugs with specific channels (Na+, Ca2+, K+, "pacemaker") and non-channel receptors. Emphasis is given to the type of state-dependent block involved (rested, activated and inactivated state block) and the change in channel kinetics. These properties vary and determine the voltage- and frequency-dependence of the change in ionic current. Finally, the question is asked as to whether the available drugs by their action on channels and receptors modify the vulnerable parameter in the desired way to stop or prevent arrhythmias.

Phenylephrine-induced stimulation of Na+/Ca2+ exchange in rat ventricular myocytes.

OBJECTIVE: The effect of an alpha-adrenergic agonist, phenylephrine, on the Na+/Ca2+ exchange current in rat ventricular myocytes was investigated. METHODS: The Na+/Ca2+ exchange current was measured at room temperature in rat ventricular myocytes as the whole-cell current induced by addition of extracellular Na+ and Ca2+, while blocking Na+ current by setting the holding potential at -30 mV, K+ currents by intracellular Cs+, TEA+ and by extracellular Ba2+, Ca2+ current by nifedipine and Na+ pump current by ouabain or by 0 extracellular K+. RESULTS: Under these experimental conditions, application of external Na+ and Ca2+ induced a current which was further increased by phenylephrine. Phenylephrine (80 microM) increased the current by up to 31.0 +/- 5.4% of control at all membrane potentials tested both below and above the reversal potential. The reversal potential (+21.0 +/- 3.2 mV), which corresponded with the theoretical reversal potential for the Na+/Ca2+ exchange current under our ionic conditions (+21.3 mV), was not changed by phenylephrine (+23.2 +/- 4.1 mV). Applying phenylephrine in the absence of Na+/Ca2+ exchange (0 Na+e, 0 Ca2+e) did not change the current. The effect was resistant to propranolol, a beta-adrenergic blocker, but prevented by prazosin, an alpha-receptor antagonist, by neomycin, an inhibitor of phospholipase C, and by chelerythrine, a selective inhibitor of protein kinase C. Phorbol 12-myristate 13-acetate failed to stimulate the current. The effect remained similar under conditions of high (HEPESi = 5 mM) and low (HEPESi = 0.5 mM) intracellular pH buffering. CONCLUSION: Our data indicate that phenylephrine stimulates the Na+/Ca2+ exchange, both in the forward and the reverse modes, probably via a protein kinase C-dependent pathway.

Effects of cetirizine on the delayed K+ currents in cardiac cells: comparison with terfenadine.

1. The aim of the present experiments was to analyse the effect of the H1-histamine antagonist, cetirizine, on the delayed K+ currents in cardiac cells and to compare its effects with another H1-histamine antagonist terfenadine, known to possess proarrhythmic effects. 2. Whole cell currents were measured by use of the single electrode patch-clamp technique in rabbit and guinea-pig myocytes. 3. The activation relationship for the IKr current in rabbit ventricular myocytes was depressed and its voltage-dependence shifted in the negative direction with a V1/2 value -13.4+/-2.4 mV under control conditions which changed to -19.1+/-1.9 mV (n=4) in the presence of 0.1 mM cetirizine. 4 In rabbit ventricular myocytes the IC50 for block of IKr was 108+/-8 microM (n=5); in guinea-pig ventricular myocytes this concentration of cetirizine reduced the rapidly activating component IKr to 49+/-4.5% (n=5), while the slowly activating IKs was less affected and only inhibited to 79+/-2.3% (n=5). 5 The block of IKr did not show use-dependence and the time course of the tail current was not changed, suggesting rested-state block or fast activated-state block and no rapid recovery on deactivation. No important difference was found in the activity of the two enantiomers of cetirizine. 6 Terfenadine in comparison was more potent in blocking IKr, the IC50 being 96+/-15 nM (n=6). 7 Based on the present results and information in the literature on binding, it was concluded that cetirizine is a relatively selective H1-histamine receptor antagonist, with minor effects on K+ currents. The IC50 concentration for IKr block in heart cells was 1.000 times higher than the concentrations needed to block H1 histamine receptors. The occurrence of cardiac arrhythmias due to K+ current blockade is therefore unlikely with this drug.

Modulation of transient outward current by extracellular protons and Cd2+ in rat and human ventricular myocytes.

1. The effects of extracellular acidosis and Cd2+ on the transient outward current (Ito) have been investigated in rat and human ventricular myocytes, using the whole-cell patch-clamp technique. 2. In rat myocytes, exposure to acidic extracellular solution (pH 6.0) shifted both steady-state activation and inactivation curves to more positive potentials, by 20.5 +/- 2.7 mV (mean +/- S.E.M.; n = 4) and 19.8 +/- 1.2 mV, respectively. Cd2+ also shifted the activation and inactivation curves in a positive direction in a concentration-dependent manner. 3. In human myocytes, the steady-state activation and inactivation curves were located at more positive potentials. The effect of Cd2+ was similar, but acidosis had less effect than in rat myocytes (e.g. pH 6.0 shifted activation by only 7.2 +/- 2.2 mV and inactivation by 13.7 +/- 0.5 mV; n = 4). 4. In both species, the effect of acidosis decreased with increasing concentrations of Cd2+ and vice versa, suggesting competition between H+ and Cd2+ for a common binding site. 5. The data indicate that acidosis and divalent cations influence Ito via a similar mechanism and act competitively in both rat and human myocytes, but that human cells are less sensitive to the effects of acidosis.

T-type Ca2+ current as a trigger for Ca2+ release from the sarcoplasmic reticulum in guinea-pig ventricular myocytes.

1. We have investigated whether Ca2+ entry through T-type Ca2+ channels participates in triggering Ca2+ release from the sarcoplasmic reticulum (SR) in single guinea-pig ventricular myocytes (whole-cell voltage clamp, K5fura-2 as [Ca2+]i indicator; all monovalent cations replaced by impermeant ions to record uncontaminated Ca2+ currents; T = 23 or 36 degrees C). 2. T-type Ca2+ currents were elicited from a holding potential of -90 mV during steps to -50 to -20 mV. For steps to -50 mV, very small [Ca2+]i transients could be recorded with high loading of the SR (peak Delta[Ca2+]i, 67 +/- 41 nM; n = 9). 3. For steps to -40, -30 and -20 mV, we compared the amplitude of Ca2+ release for a holding potential of -50 mV with L-type Ca2+ current only to Ca2+ release for a holding potential of -90 mV with both T- and L-type Ca2+ current. Significantly more Ca2+ release was observed with T-type current present, and both the T-type current and the additional Ca2+ release were suppressed by 50 microM NiCl2. 4. Ca2+ influx through T-type Ca2+ channels triggered less Ca2+ release than a comparable Ca2+ influx through L-type Ca2+ channels. 5. Rapid block of T-type Ca2+ current during the action potential (50 microM NiCl2 during steady-state stimulation at 1 or 2 Hz) did not immediately reduce Ca2+ release, although a small decrease was observed after longer application. 6. We conclude that T-type Ca2+ current can trigger Ca2+ release from the SR albeit less efficiently than L-type Ca2+ current. T-type current is most likely to provide only a small contribution to the trigger for Ca2+ release in normal conditions. These results support the hypothesis that L-type Ca2+ channels have a privileged role in excitation-contraction coupling.

Neurospecificity of phyto-bufadienolides is not related to differences in Na+/K+ pump inhibition.

The aim of the present study was to investigate the effects of neuro- (cumulative) and cardiotoxic (non-cumulative) bufadienolides originating from plants (phyto-bufadienolides) on the Na+/K+ pump current (Ip) in cardiac (rat and guinea pig) and dorsal root ganglion cells (guinea pig), and on Ca2+ currents in cardiomyocytes (guinea pig). All bufadienolides tested (non-cumulative drugs: thesiuside, tyledoside C; lanceotoxin B and tyledoside F for the neurotoxic group) were potent blockers of Ip at concentrations in the micro- and submicromolar range. K0.5 values for Ip inhibition in dorsal root ganglion neurones were slightly lower compared to cardiomyocytes, but the order of potency was similar in both cell types. Both classes of bufadienolides were equipotent in suppressing Ip, generated by high- and low-affinity pump isoforms. Phenomena related to pump inhibition, as hypercontracture and increase in T-type Ca2+ current in cardiomyocytes, were influenced to the same extent. Therefore, from these results, neurospecificity of some bufadienolides could not be explained by differences in Na+/K+ pump affinity.

Na(+)-Ca2+ exchange in rat dorsal root ganglion neurons.

The role of the Na(+)-Ca2+ exchanger was examined in isolated rat dorsal root ganglion (DRG) neurons. Neurons were dialyzed with the Ca2+ indicator Indo-1. Ca2+ transients were elicited by depolarizing the cells from -80 to 0 mV for 100 ms under voltage clamp conditions. In most cells (45 of 67), the decay of intracellular Ca2+ concentration ([Ca2+]i) could be fitted with a single exponential with a time constant of 2.43 s. In the remaining 22 cells, the decay of [Ca2+]i could be described with a double exponential with time constants of 0.76 and 11.84 s. In cells that displayed a biphasic [Ca2+]i relaxation, Na(+)-free medium caused resting [Ca2+]i to increase from 116 to 186 nM; the slow component of recovery to basal [Ca2+]i was nearly abolished in Na(+)-free medium or by application of 5 mM Ni2+. In 35 of 45 cells displaying a monophasic [Ca2+]i decay, omitting external Na+ increased the time constant of [Ca2+]i decay from 2.02 to 3.63 s. In the remaining 10 cells, Na(+)-free solution did not affect Ca2+ handling. The time constant of [Ca2+]i relaxation was voltage dependent. These findings demonstrate the important role of the Na(+)-Ca2+ exchanger in DRG neurons. Its presence was further confirmed both at the mRNA and the protein level.

Mechanism of L- and T-type Ca2+ channel blockade by flunarizine in ventricular myocytes of the guinea-pig.

Flunarizine is a substance known to block voltage-dependent Ca2+ channels in smooth muscle and neuronal cells. Reports on the effect on voltage-dependent cardiac Ca2+ channels are however sparse. Therefore, the mechanism of action of flunarizine on two types of voltage-dependent cardiac Ca2+ channels, the L- and T-type, in single ventricular myocytes of the guinea-pig was investigated using the whole-cell voltage clamp technique. Both channel types can be blocked by flunarizine in a time-, frequency-, voltage-, Ca(2+)-, and proton-dependent way. While the overall mechanism of action on cardiac myocytes is similar to the one reported for other cell types, we found that cardiomyocytes are less susceptible to block (Kd 3.3-11 mM). We also describe a complete analysis of the different components of block, together with evidence for open channel state block and drug-induced changes in channel gating. These findings provide new insights into the mechanism of action of flunarizine on voltage-dependent Ca2+ channels.

Na+ current and Ca2+ release from the sarcoplasmic reticulum during action potentials in guinea-pig ventricular myocytes.

1. Ca2+ release from the sarcoplasmic reticulum (SR) was examined in enzymatically isolated single guinea-pig ventricular myocytes by monitoring [Ca2+]i with fura-2 during whole-cell recording of action potentials at room temperature (23-25 degrees C). Modulation of Ca2+ release by the Na+ current (INa) was studied by manipulating Na+ influx through the Na+ channel. 2. For a comparable Ca2+ loading of the SR, brief hyperpolarizing currents applied at the peak of the action potential increased Ca2+ release, while depolarizing pulses had the opposite effect. Similar currents applied before the action potential did not affect Ca2+ release. 3. Application of tetrodotoxin (TTX; 60 microM) moderately reduced Ca2+ release from the SR, but this effect was delayed in comparison with the immediate block of INa. An early effect of TTX was to increase Ca2+ release. 4. Replacement of Na+ with Li did not reduce Ca2+ release, but led to a progressive increase in Ca2+ release, resulting in spontaneous activity. 5. Ca2+ channel blockers (CdCl2, 100 microM; nisoldipine, 20 microM; or nifedipine, 20 microM) drastically reduced Ca2+ release from the SR. 6. Voltage clamp experiments confirmed that TTX blocked INa and its associated [Ca2+]i transient during voltage steps from -90 to -50 mV. INa and its associated [Ca2+]i transient were equally suppressed following replacement of Na+ with N-methyl-D-glucamine (NMDG+), but the [Ca2+]i transient was not suppressed following replacement of Na+ with Li+. 7. The INa-associated transient was sensitive to Ca2+ channel blockers. During steps from -50 to 0 mV, it appeared that the dihydropyridine antagonists often did not provide full block of the calcium current (ICa). 8. During current clamp stimulation at 1 Hz in the presence of TTX (60 microM), the Ca2+ content of the SR was decreased, due to the changes in action potential configuration and to changes in [Na+]i. 9. Our experiments indicate that the Ca2+ entry coupled to Na+ influx via the Na+ channel does not contribute substantially to the trigger for Ca2+ release from the SR during action potentials (23-25 degrees C). However, INa modulates Ca2+ release by affecting the Ca2+ load of the SR.

The alpha-dendrotoxin footprint on a mammalian potassium channel.

alpha-Dendrotoxin, a 59-amino acid basic peptide from the venom of Dendroaspis angusticeps (green mamba snake), potently blocks some but not all voltage-dependent potassium channels. Here we have investigated the relative contribution of the individual alpha-subunits constituting functional Kv1.1 potassium channels to alpha-dentroxin binding. Three residues critical for alpha-dentrotoxin binding and located in the loop between domains S5 and S6 were mutated (A352P, E353S, and Y379H), and multimeric cDNAs were constructed encoding homo- and heterotetrameric channels composed of all possible ratios of wild-type and mutant alpha-subunits. Complete mutant channels were about 200-fold less sensitive for the alpha-dendrotoxin block than complete wild-type channels, which is attributable to a smaller association rate. Analysis of the bimolecular reaction between alpha-dendrotoxin and the different homo- and heteromeric channel constructs revealed that the association rate depends on the number of wild-type alpha-subunits in the functional channel. Furthermore, we observed a linear relationship between the number of wild-type alpha-subunits in functional channels and the free energy for alpha-dendrotoxin binding, providing evidence that all four alpha-subunits must interact with alpha-dendrotoxin to produce a high affinity binding site.

[Ca2+]i-dependent membrane currents in guinea-pig ventricular cells in the absence of Na/Ca exchange.

Transient inward currents (Iti) during oscillations of intracellular [Ca2+] ([Ca2+]i) in ventricular myocytes have been ascribed to Na/Ca exchange. We have investigated whether other Ca2+-dependent membrane currents contribute to Iti in single guinea-pig ventricular myocytes, by examining membrane currents during [Ca2+]i oscillations and during caffeine-induced Ca2+ release from the sarcoplasmic reticulum in the absence of Na+. Membrane currents were recorded during whole-cell voltage clamp and [Ca2+]i measured simultaneously with fura-2. In the absence of Na/Ca exchange, i.e., with Li+, Cs+ or N-methyl-D-glucamine (NMDG+) substituted for Na+, the cell could be loaded with Ca2+ by repetitive depolarizations to +10 mV, resulting in spontaneous [Ca2+]i oscillations. During these oscillations, no inward currents were seen, but instead spontaneous Ca2+ release was accompanied by a shift of the membrane current in the outward direction at potentials between -40 mV and +60 mV. This [Ca2+]i-dependent outward current shift was not abolished when NMDG+ was substituted for internal monovalent cations, nor was it sensitive to substitution of external Cl-. It was however, sensitive to the blockade of ICa by verapamil. These results suggest that the transient outward current shift observed during spontaneous Ca2+ release represents [Ca2+]i-dependent transient inhibition of ICa. Similarly, during the [Ca2+]i transients induced by brief caffeine (10 mM) applications, we could not detect membrane currents attributable to a Ca2+-activated nonselective cation channel, or to a Ca2+-activated Cl- channel; however, transient Ca2+-dependent inhibition of ICa was again observed. We conclude that neither the Ca2+-activated nonselective cation channel nor the Ca2+-activated Cl- channel contribute significantly to the membrane currents during spontaneous [Ca2+]i oscillations in guinea-pig ventricular myocytes. However, in the voltage range between -40 mV and +60 mV Ca2+-dependent transient inhibition of ICa will contribute to the oscillations of the membrane current.

Intracellular citrate induces regenerative calcium release from sarcoplasmic reticulum in guinea-pig atrial myocytes.

Ca2+ release from the sarcoplasmic reticulum was studied in voltage-clamped guinea-pig atrial myocytes. Cells were dialysed with a pipette solution containing the Ca2+ indicator 1- [2-amino-5-(6-carboxyindol-2-yl) phenoxy]-2-(2'-amino-5'-methylphenoxy) ethane-N,N,N',N'-tetraacetic acid] (Indo-1, 100 microM) and as main anion either chloride or the low-affinity Ca2+ buffer citrate. Intracellular Ca2+ transients (Cai transients) were elicited by depolarizations from a holding potential of -50 mV. In chloride-dialysed cells, Cai transients showed a bell-shaped dependence on the amplitude of the depolarizing pulse. In citrate-dialysed cells, membrane depolarizations were associated with a small rise in [Ca2+]i. These small changes in [Ca2+]i were either followed by a large Cai transient or failed to induce large changes in [Ca2+]i. The peak amplitude of the large Cai transient did not vary with the amplitude of the depolarizing pulse. These results demonstrate that in the presence of intracellular chloride, Ca2+ release in atrial cells is a graded process triggered by Ca2+ influx. Using citrate as the main intracellular anoin, Ca2+ release triggered by Ca2+ entry was no longer graded but occurred in a regenerative manner. The results are discussed in terms of two models in which citrate, affects the spatial distribution of [Ca2+]i or the loading state of the sarcoplasmic reticulum.