Pharmacological inhibition of the hERG potassium channel is modulated by extracellular but not intracellular acidosis.

INTRODUCTION: Human ether-à-go-go related gene (hERG) is responsible for channels that mediate the rapid delayed rectifier K(+) channel current (I(Kr) ), which participates in repolarization of the ventricles and is a target for some antiarrhythmic drugs. Acidosis occurs in the heart in some pathological situations and can modify the function and responses to drugs of ion channels. The aim of this study was to determine the effects of extracellular and intracellular acidosis on the potency of hERG channel current (I(hERG)) inhibition by the antiarrhythmic agents dofetilide, flecainide, and amiodarone at 37 °C. METHODS AND RESULTS: Whole-cell patch-clamp recordings of I(hERG) were made at 37 °C from hERG-expressing Human Embryonic Kidney (HEK293) cells. Half-maximal inhibitory concentration (IC(50)) values for I(hERG) tail inhibition at -40 mV following depolarizing commands to +20 mV were significantly higher at external pH 6.3 than at pH 7.4 for both flecainide and dofetilide, but not for amiodarone. Lowering pipette pH from 7.2 to 6.3 altered neither I(hERG) kinetics nor the extent of observed I(hERG) blockade by any of these drugs. CONCLUSION: Conditions leading to localized extracellular acidosis may facilitate heterogeneity of action of dofetilide and flecainide, but not amiodarone via modification of hERG channel blockade. Such effects depend on the external pH change rather than intracellular acidification.

Enhanced inhibitory effect of acidosis on hERG potassium channels that incorporate the hERG1b isoform.

Extracellular acidosis occurs in the heart during myocardial ischemia and can lead to dangerous arrhythmias. Potassium channels encoded by hERG (human ether-à-go-go-related gene) mediate the cardiac rapid delayed rectifier K+ current (IKr), and impaired hERG function can exacerbate arrhythmia risk. Nearly all electrophysiological investigations of hERG have centred on the hERG1a isoform, although native IKr channels may be comprised of hERG1a and hERG1b, which has a unique shorter N-terminus. This study has characterised for the first time the effects of extracellular acidosis (an extracellular pH decrease from 7.4 to 6.3) on hERG channels incorporating the hERG1b isoform. Acidosis inhibited hERG1b current amplitude to a significantly greater extent than that of hERG1a, with intermediate effects on coexpressed hERG1a/1b. IhERG tail deactivation was accelerated by acidosis for both isoforms. hERG1a/1b activation was positively voltage-shifted by acidosis, and the fully-activated current-voltage relation was reduced in amplitude and right-shifted (by ∼10 mV). Peak IhERG1a/1b during both ventricular and atrial action potentials was both suppressed and positively voltage-shifted by acidosis. Differential expression of hERG isoforms may contribute to regional differences in IKr in the heart. Therefore inhibitory effects of acidosis on IKr could also differ regionally, depending on the relative expression levels of hERG1a and 1b, thereby increasing dispersion of repolarization and arrhythmia risk.

Na/Ca exchange and Na/K-ATPase function are equally concentrated in transverse tubules of rat ventricular myocytes.

Formamide-induced detubulation of rat ventricular myocytes was used to investigate the functional distribution of the Na/Ca exchanger (NCX) and Na/K-ATPase between the t-tubules and external sarcolemma. Detubulation resulted in a 32% decrease in cell capacitance, whereas cell volume was unchanged. Thus, the surface-to-volume ratio was used to assess the success of detubulation. NCX current (I(NCX)) and Na/K pump current (I(pump)) were recorded using whole-cell patch clamp, as Cd-sensitive and K-activated currents, respectively. Both inward and outward I(NCX) density was significantly reduced by approximately 40% in detubulated cells. I(NCX) density at 0 mV decreased from 0.19 +/- 0.03 to 0.10 +/- 0.03 pA/pF upon detubulation. I(pump) density was also lower in detubulated myocytes over the range of voltages (-50 to +100 mV) and internal [Na] ([Na](i)) investigated (7-22 mM). At [Na](i) = 10 mM and -20 mV, I(pump) density was reduced by 39% in detubulated myocytes (0.28 +/- 0.02 vs. 0.17 +/- 0.03 pA/pF), but the apparent K(m) for [Na](i) was unchanged (16.9 +/- 0.4 vs. 17.0 +/- 0.3 mM). These results indicate that although thet-tubules represent only approximately 32% of the total sarcolemma, they contribute approximately 60% to the total I(NCX) and I(pump). Thus, the functional density of NCX and Na/K pump in the t-tubules is 3-3.5-fold higher than in the external sarcolemma.

Two pore residues mediate acidosis-induced enhancement of C-type inactivation of the Kv1.4 K(+) channel.

Acidosis inhibits current through the Kv1.4 K(+) channel, perhaps as a result of enhancement of C-type inactivation. The mechanism of action of acidosis on C-type inactivation has been studied. A mutant Kv1.4 channel that lacks N-type inactivation (fKv1.4 Delta2-146) was expressed in Xenopus oocytes, and currents were recorded using two-microelectrode voltage clamp. Acidosis increased fKv1.4 Delta2-146 C-type inactivation. Replacement of a pore histidine with cysteine (H508C) abolished the increase. Application of positively charged thiol-specific methanethiosulfonate to fKv1.4 Delta2-146 H508C increased C-type inactivation, mimicking the effect of acidosis. Replacement of a pore lysine with cysteine (K532C) abolished the acidosis-induced increase of C-type inactivation. A model of the Kv1.4 pore, based on the crystal structure of KcsA, shows that H508 and K532 lie close together. It is suggested that the acidosis-induced increase of C-type inactivation involves the charge on H508 and K532.

Na+-Ca2+ exchange activity is localized in the T-tubules of rat ventricular myocytes.

Detubulation of rat ventricular myocytes has been used to investigate the role of the t-tubules in Ca2+ cycling during excitation-contraction coupling in rat ventricular myocytes. Ca2+ was monitored using fluo-3 and confocal microscopy. In control myocytes, electrical stimulation caused a spatially uniform increase in intracellular [Ca2+] across the cell width. After detubulation, [Ca2+] rose initially at the cell periphery and then propagated into the center of the cell. Application of caffeine to control myocytes resulted in a rapid and uniform increase of intracellular [Ca2+]; the distribution and amplitude of this increase was the same in detubulated myocytes, although its decline was slower. On application of caffeine to control cells, there was a large, rapid, and transient rise in extracellular [Ca2+] as Ca2+ was extruded from the cell; this rise was significantly smaller in detubulated cells, and the remaining increase was blocked by the sarcolemmal Ca2+ ATPase inhibitor carboxyeosin. The treatment used to produce detubulation had no significant effect on Ca2+ efflux in atrial cells, which lack t-tubules. Detubulation of ventricular myocytes also resulted in loss of Na+-Ca2+ exchange current, although the density of the fast Na+ current was unaltered. It is concluded that Na+-Ca2+ exchange function, and hence Ca2+ efflux by this mechanism, is concentrated in the t-tubules, and that the concentration of Ca2+ flux pathways in the t-tubules is important in producing a uniform increase in intracellular Ca2+ on stimulation.

Effects of the protein kinase A inhibitor H-89 on Ca2+ regulation in isolated ferret ventricular myocytes.

We investigated the effects of a protein kinase A (PKA) inhibitor, H-89 {N-[2-(p-bromocinnamylamino)ethyl]-5-iso-quinolinesulphonamide}, on Ca2+ regulation in Fura-2-loaded ferret myocytes. H-89 (10 micromol/l) decreased the amplitude of the Fura-2 transient to 28. 2+/-4.3% (P<0.001) of control and prolonged its duration, characterized by a decrease in the rate of decline of Ca2+ to diastolic levels: t1/2 increased from 311+/-35 ms to 547+/-43 ms (P<0.001, n=7). Reduced Ca2+ uptake by the sarcoplasmic reticulum (SR) in the presence of H-89 was also indicated by a decrease in the SR Ca2+ content, as assessed with caffeine. The apparent slowing of the SR Ca2+-ATPase was not caused by changes in phosphorylation of phospholamban (PLB). However, Ca2+ uptake in microsomal vesicles prepared from canine hearts and fast-twitch rat skeletal muscle (which lacks PLB) was decreased by 34.1 and 46.8% (n=3), respectively, suggesting that H-89 has a direct inhibitory effect on the SR Ca2+-ATPase. In electrophysiological experiments, 5.0 micromol/l H-89 decreased the L-type Ca2+ current (ICa) by 39.5% (n=6) and slowed the upstroke of the action potential and, in some cases, caused loss of excitability without changes in the resting membrane potential. In summary, data show that [Ca2+ ]i regulation, and hence contraction, is sustained by PKA-mediated phosphorylation, even in the absence of beta-agonists. However, the use of H-89 as a tool to study the role of this signalling pathway is limited by the non-specific effects of H-89 on the SR Ca2+-ATPase.

Rate-dependent abbreviation of Ca2+ transient in rat heart is independent of phospholamban phosphorylation.

The mechanisms underlying the accelerated decline of the intracellular Ca2+ transient that occurs in cardiac muscle when stimulation rate is increased have been investigated in ventricular myocytes from rat hearts. Increasing stimulation rate from 0.1 to 0.5 and 1 Hz decreased the time taken for the Ca2+ transient to decline from its peak to 50% of its peak value in cells generating action potentials, when the duration of depolarization was held constant by voltage clamp, and when Na/Ca exchange was inhibited. The sarcoplasmic reticulum Ca2+ adenosinetriphosphatase inhibitor thapsigargin inhibited rate-dependent abbreviation of the Ca2+ transient. However, neither a chemical inhibitor of Ca(2+)-calmodulin-dependent protein kinase II (KN62) nor a peptide inhibitor of this enzyme (calmodulin-binding domain peptide) had a significant effect on rate-dependent abbreviation of the Ca2+ transient. Analysis of the phosphorylation of the regulatory sites Ser16 and Thr17 of phospholamban showed no significant change in phosphorylation with changes of stimulation rate. These data suggest that rate-dependent shortening of the Ca2+ transient is due predominantly to enhanced Ca2+ uptake by the sarcoplasmic reticulum without changes in phospholamban phosphorylation.

Sarcoplasmic reticulum Ca2+ content, L-type Ca2+ current and the Ca2+ transient in rat myocytes during beta-adrenergic stimulation.

1. The effect of beta-adrenergic stimulation on the relationship between the intracellular Ca2+ transient and the amplitude of the L-type Ca2+ current (ICa) has been investigated in ventricular myocytes isolated from rat hearts. Intracellular [Ca2+] was monitored using fura-2 during field stimulation and while membrane potential was controlled using voltage clamp techniques. 2. The increase in the amplitude, and the rate of decline, of the Ca2+ transient produced by isoprenaline (1.0 mumol l-1) was not significantly different in myocytes generating action potentials and in those voltage clamped with pulses of constant duration and amplitude. 3. Under control conditions, the current-voltage (I-V) relationship for ICa was bell shaped. The amplitude of the Ca2+ transient also showed a bell-shaped voltage dependence. In the presence of isoprenaline, the amplitude of both ICa and the Ca2+ transient was greater at all test potentials and the I-V relationship maintained its bell-shaped voltage dependence. However, the size of the Ca2+ transient was no longer graded with changes in the amplitude of ICa: a small ICa could now elicit a maximal Ca2+ transient. 4. Rapid application of caffeine (10 mmol l-1) was used to elicit Ca2+ release from the sarcoplasmic reticulum (SR). Isoprenaline increased the integral of the subsequent rise in cytoplasmic [Ca2+] to 175 +/- 13% of control. 5. Abbreviation of conditioning pulse duration in the presence of isoprenaline was used to reduce the amplitude of the Ca2+ transient to control levels. Under these conditions, the amplitude of the Ca2+ transient was again graded with the amplitude of ICa in the same way as under control conditions. 6. Nifedipine (2 mumol l-1) was also used to decrease Ca2+ transient amplitude in the presence of isoprenaline. In the presence of isoprenaline and nifedipine, the amplitude of the Ca2+ transient again showed a bell-shaped voltage dependence. 7. The SR Ca(2+)-ATPase inhibitor thapsigargin (2.5 mumol l-1) reduced the effect of isoprenaline on the amplitude of the Ca2+ transient. In the presence of thapsigargin, the size of the Ca2+ transient increased as ICa increased in response to isoprenaline. 8. These data suggest that the increase in the amplitude of the Ca2+ transient produced by beta-adrenergic stimulation in cardiac muscle is due to an increase in the gain of the SR Ca2+ release process, due principally to an increase in the Ca2+ content of the SR.

Effects of acidosis on phosphorylation of phospholamban and troponin I in rat cardiac muscle.

Acidosis inhibits Ca2+ transport by the sarcoplasmic reticulum of cardiac muscle and decreases the Ca2+ sensitivity of the contractile proteins, although the mechanisms underlying these changes are unclear. We have investigated the hypothesis that changes in the phosphorylation of the regulatory proteins phospholamban and troponin I might play a role in the acidosis-induced changes in the function of the sarcoplasmic reticulum and the myofilaments, respectively. Langendorff-perfused rat hearts were labeled with 32P and then perfused with either control (pH 7.4) or acid (pH 6.8) physiological salt solution, in both the absence and presence of isoproterenol. The incorporation of 32P into phospholamban and troponin I was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sarcoplasmic reticulum and myofibrillar proteins, followed by autoradiography and liquid scintillation counting. The data show that acidosis has no effect on the phosphorylation of phospholamban in the absence of isoproterenol but that, in the presence of isoproterenol, acidosis increased the phosphorylation of phospholamban. However, acidosis increased the phosphorylation of troponin I, in both the absence and the presence of isoproterenol. Acidosis did not alter the adenosine 3',5'-cyclic monophosphate content of the hearts but did inhibit type 1 phosphatase. These data show that acidosis can alter the phosphorylation of these two proteins and suggest that these changes underlie, in part the changes observed in cardiac muscle during acidosis.

The mechanism underlying the cardiotoxic effect of the toxin from the jellyfish Chironex fleckeri.

We have investigated the mechanisms underlying the cardiac effects of the toxin from the box jellyfish Chironex fleckeri. Papillary muscles isolated from the hearts of ferrets and ventricular myocytes isolated from the hearts of ferrets and rats were used. Force, intracellular [Ca2+], and membrane potential were monitored in the papillary muscles; contraction, intracellular [Ca2+], intracellular [Na+], and membrane currents were monitored in the isolated myocytes. Application of the toxin to these preparations resulted in a large increase in intracellular [Ca2+] and the adverse symptoms of Ca2+ overload (aftercontractions, spontaneous contractions, a decrease in developed force, and an increase in resting force). The response of papillary muscles to the toxin was not inhibited by blockers of Ca2+ or Na+ channels or by inhibitors of the sarcoplasmic reticulum, Na+/K+ ATPase, or Na+/H+ exchange. The response to the toxin was, however, blocked by prior exposure to a solution which contained no Na+ and by Ni2+. In the isolated myocytes, as well as an increase in intracellular [Ca2+], the toxin also caused an increase in intracellular [Na+] and the appearance of a current which was inward at negative potentials and reversed at about -10 mV. These data can be explained by the toxin increasing Na+ influx into the cell. The increase in intracellular [Na+] will then increase intracellular [Ca2+] via the Na+/Ca2+ exchange mechanism, thus producing the observed Ca2+ overload.

Ouabain enhances basal and stimulus-induced cytoplasmic calcium concentrations in platelets.

Incubation of platelet-rich plasma (PRP) with ouabain, an inhibitor of sodium/potassium ATPase (Na+/K+ ATPase), induced a significant rise in basal platelet intracellular calcium concentration [( Ca2+]i) when measured using fura 2. Ouabain induced an enhanced aggregation response to low doses of collagen in both PRP and washed platelets loaded with aequorin. In aequorin loaded platelets this enhanced aggregation response was associated with an enhanced rise in [Ca2+]i such that the relationship between [Ca2+]i and aggregation was unchanged. As inhibition of plasma membrane Na+/K+ ATPase would lead to a raised intracellular sodium ion concentration [( Na+]i) the results suggest that in the platelet, [Na+]i can modulate [Ca2+]i and hence influence the response of platelets to stimuli such as collagen.

Myocardial contractile function during ischemia and hypoxia.

There is good evidence that elevated [Ca2+]i, produced by an influx of Ca2+ in exchange for Na+, is the underlying pathology in reperfusion or reoxygenation damage. Further measurements of [Na+]i and [Ca2+]i during ischemia and reperfusion, coupled with information about metabolic levels, are needed to confirm or refute this hypothesis. Contributions to cell damage by other mechanisms, e.g., oxygen free radicals, certainly cannot yet be excluded.

A nuclear magnetic resonance study of metabolism in the ferret heart during hypoxia and inhibition of glycolysis.

31P nuclear magnetic resonance was used to measure the relative concentrations of phosphorus-containing metabolites in Langendorff-perfused ferret hearts. Intracellular concentrations of inorganic phosphate ([Pi]i), phosphocreatine ([PCr]i), ATP ([ATP]i) and H+ (pHi) were monitored under control conditions and while oxidative phosphorylation and/or glycolysis were prevented. Mechanical performance was assessed by recording the pressure developed in a balloon placed in the left ventricle. Oxidative phosphorylation was prevented either by replacement of O2 with N2 or by addition of cyanide. When the rate of oxidative phosphorylation was reduced by either method, developed pressure fell to a stable level of about 35% of control after 5 min. The pHi (control value 6.98) first increased to a peak of 7.07 after 2 min but then decreased to give a stable acidosis (pH 6.85). [PCr]i decreased rapidly to about 15% of the control value after 5 min whereas [ATP]i declined very slowly, reaching about 90% of the control value after 10 min. Reduction in the rate of glycolysis was achieved either (i) by removal of external glucose and depletion of glycogen stores by a long (1-2 h) period of stimulation or (ii) by removal of glucose and application of 2-deoxyglucose (1 mM) for 30-60 min. These procedures had only a small effect on pressure development, [ATP]i, [PCr]i and pHi. Measurements of lactate production showed that these procedures reduced the rate of glycolysis by a factor of about 10. When oxidative phosphorylation was prevented during periods when the rate of glycolysis was reduced, developed pressure fell to less than 5% of control after 5 min and there was a subsequent increase in resting pressure (hypoxic contracture). pHi (control value 7.03) first increased to a peak of 7.12 and then declined to about pH 7.00, but there was no subsequent acidosis. [PCr]i fell rapidly to about 10% of control after about 5 min while [ATP]i declined to about half of its control value over 10 min. It is concluded that (i) when oxidative phosphorylation alone is prevented, the changes in pHi can account for a substantial part of the changes in developed pressure. The increase in [Pi]i probably also contributes to the decline of developed pressure. (ii) When oxidative phosphorylation was prevented under conditions in which the rate of glycolysis was also reduced, the more pronounced decline in developed pressure which occurs within 5 min cannot be accounted for by pHi changes and is probably not explained by the rise in [Pi]i or by the moderate fall of [ATP]i.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of intracellular [Ca2+] and [H+] in contractile failure of the hypoxic heart.

When oxidative metabolism is inhibited in heart muscle, developed tension often increases slightly before decreasing below control. We have examined the possible mechanisms underlying these changes in developed tension in two series of experiments. In the first series of experiments, the photoprotein aequorin was used to monitor intracellular free [Ca2+] [( Ca2+]i) in papillary muscles during inhibition of oxidative phosphorylation, using either cyanide or hypoxia. The observed changes of developed tension were independent of changes in [Ca2+]i. It was therefore possible that these changes of tension were due to changes of intracellular pH (pHi). We tested this idea in a second series of experiments, using 31P nuclear magnetic resonance to monitor pHi, [ATP], and phosphocreatine concentration [( PCr]) in Langendorff-perfused ferret hearts. During the application of cyanide, pHi increased transiently before decreasing to below control. [PCr] decreased throughout this period, but [ATP] did not change. It is concluded that the observed changes of pHi could account for most of the observed changes of developed tension. It is suggested that the initial increase of pHi is due to PCr breakdown and the subsequent decrease of pHi to accelerated anaerobic glycolysis.

Measurements of intracellular calcium concentration in heart muscle: the effects of inotropic interventions and hypoxia.

The use of aequorin as an intracellular calcium indicator in ventricular muscle is described. If the increase of intracellular calcium concentration associated with each contraction (the calcium transient) is measured during inotropic interventions, it is possible to distinguish two classes of inotropic intervention. One class leads to changes in the calcium transient which parallel the changes in tension. The second class leads to changes in the calcium transient and tension which are different in magnitude or direction. In this latter class, changes in the sensitivity of the contractile proteins to calcium are occurring and represent an important part of the inotropic mechanism. When oxidative phosphorylation is inhibited in an isolated mammalian papillary muscle, tension declines but the amplitude of the calcium transients is unaffected. Intracellular acidosis caused by lactate production associated with the increased rate of glycolysis is the probable mechanism of this decline in tension. When both oxidative phosphorylation and glycolysis are inhibited, both the calcium transient and the developed tension decline rapidly to zero. This profound contractile failure may be a consequence of a decline in the free energy of hydrolysis of ATP so that the sarcoplasmic reticulum can no longer accumulate calcium. Hypoxic contractures occur in the absence of significant increases in resting [Ca2+]i and are probably due to rigor produced by the low [ATP].

The relationship between hypoxic pulmonary vasoconstriction and arterial oxygen tension in the intact dog.

The relationship between the magnitude of hypoxic pulmonary vasoconstriction (h.p.v.) and arterial oxygen tension (Pa,O2) has been studied in intact anaesthetized dogs. Radioactive 133Xe was infused continuously into the inferior vena cava. A tracheal divider made it possible to vary the inspired gas composition to each lung independently. With constant ventilation the 133Xe in the mixed expired gas from each lung was proportional to the blood flow to that lung. Unilateral ventilation of the left lung with 7% oxygen produced a diversion of blood flow away from the lung and a reduction in Pa,O2. Repeated hypoxic stimuli produced a progressively greater reduction in the blood flow to the hypoxic lung and a progressive increase in Pa,O2. Administration of a beta-adrenergic agonist, dobutamine hydrochloride, during ventilation of the left lung with 7% oxygen, resulted in an increased blood flow to the left lung and a further decrease in Pa,O2. Addition of CO2 to the inspired gas resulted in an increased diversion of blood flow away from the hypoxic lung but a decrease in Pa,O2. Respiratory alkalosis induced by over-ventilation decreased the hypoxic vasoconstriction and increased Pa,O2 slightly. However acid-base changes induced by infusion of 1 N-lactic acid or 8.4% NaHCO3 had no significant effects on the magnitude of the hypoxic vasoconstriction, or on Pa,O2, during hypoxic ventilation of the left lung. The magnitude of hypoxic vasoconstriction and Pa,O2 in the experiments described in 3, 4 and 5 above were positively correlated (r = 0.885), showing that the vasoconstriction may help to maintain Pa,O2. It is suggested that the effects of CO2 on h.p.v. and Pa,O2 may be explained largely by the changes in alveolar oxygen pressure (PA,O2) which are secondary to changes in PA,CO2.