T-wave morphology can distinguish healthy controls from LQTS patients.

Long QT syndrome (LQTS) is an inherited disorder associated with prolongation of the QT/QTc interval on the surface electrocardiogram (ECG) and a markedly increased risk of sudden cardiac death due to cardiac arrhythmias. Up to 25% of genotype-positive LQTS patients have QT/QTc intervals in the normal range. These patients are, however, still at increased risk of life-threatening events compared to their genotype-negative siblings. Previous studies have shown that analysis of T-wave morphology may enhance discrimination between control and LQTS patients. In this study we tested the hypothesis that automated analysis of T-wave morphology from Holter ECG recordings could distinguish between control and LQTS patients with QTc values in the range 400-450 ms. Holter ECGs were obtained from the Telemetric and Holter ECG Warehouse (THEW) database. Frequency binned averaged ECG waveforms were obtained and extracted T-waves were fitted with a combination of 3 sigmoid functions (upslope, downslope and switch) or two 9th order polynomial functions (upslope and downslope). Neural network classifiers, based on parameters obtained from the sigmoid or polynomial fits to the 1 Hz and 1.3 Hz ECG waveforms, were able to achieve up to 92% discrimination between control and LQTS patients and 88% discrimination between LQTS1 and LQTS2 patients. When we analysed a subgroup of subjects with normal QT intervals (400-450 ms, 67 controls and 61 LQTS), T-wave morphology based parameters enabled 90% discrimination between control and LQTS patients, compared to only 71% when the groups were classified based on QTc alone. In summary, our Holter ECG analysis algorithms demonstrate the feasibility of using automated analysis of T-wave morphology to distinguish LQTS patients, even those with normal QTc, from healthy controls.

The Romano-Ward syndrome--1964-2014: 50 years of progress.

This year marks the 50th anniversary of publication in the then Journal of the Irish Medical Association of the seminal work by Irish paediatrician Professor Conor Ward entitled 'A new familial Cardiac Syndrome in Children'. The condition soon became known by the eponym Romano-Ward Syndrome and is now recognised as the congenital Long QT Syndrome. Here we review the major developments in the field over the past fifty years, with special mention of the important contributions made by Irish researches.

The S631A mutation causes a mechanistic switch in the block of hERG channels by CnErg1.

We have studied the interaction of CnErg1, a member of the gamma-KTX subfamily of scorpion toxins with the inactivation-deficient S631A hERG channel. In the background of this mutation, we observed a mechanistic switch from turret block, characteristic of the action of gamma-KTXs on Kv11-type channels, to pore plugging, characteristic of alpha-KTX block of Kv1-type channels. We suggest this reflects destabilization of the outer pore (turret region) of hERG allowing access of the toxin molecule to directly plug the conduction pathway.

Mechanism of block of the hERG K+ channel by the scorpion toxin CnErg1.

The scorpion toxin CnErg1 binds to human ether-a-go-go related gene (hERG) K(+) channels with a 1:1 stoichiometry and high affinity. However, in contrast to other scorpion toxin-ion channel interactions, the inhibition of macroscopic hERG currents by high concentrations of CnErg1 is incomplete. In this study, we have probed the molecular basis for this incomplete inhibition. High concentrations of CnErg1 had only modest effects on hERG gating that could not account for the incomplete block. Furthermore, the residual current in the presence of 1 microM CnErg1 had normal single channel conductance. Analysis of the kinetics of CnErg1 interaction with hERG indicated that CnErg1 binding is not diffusion-limited. A bimolecular binding scheme that incorporates an initial encounter complex and permits normal ion conduction was able to completely reproduce both the kinetics and steady-state level of CnErg1-hERG binding. This scheme provides a simple kinetic explanation for incomplete block; that is, relatively fast backward compared to forward rate constants for the interconversion of the toxin-channel encounter complex and the blocked toxin-channel complex. We have also examined the temperature-dependence of CnErg1 binding to hERG. The dissociation constant, K(d), for CnErg1 increases from 7.3 nM at 22 degrees C to 64 nM at 37 degrees C (i.e., the affinity decreases as temperature increases) and the proportion of binding events that lead to channel blockade decreases from 70% to 40% over the same temperature range. These temperature-dependent effects on CnErg1 binding correlate with a temperature-dependent decrease in the stability of the putative CnErg1 binding site, the amphipathic alpha-helix in the outer pore domain of hERG, assayed using circular dichroism spectropolarimetry. Collectively, our data provides a plausible kinetic explanation for incomplete blockade of hERG by CnErg1 that is consistent with the proposed highly dynamic conformation of the outer pore domain of hERG.

Effect of S5P alpha-helix charge mutants on inactivation of hERG K+ channels.

The ether-à-go-go (EAG) family of voltage-gated K(+) channels contains three subfamilies, EAG, ether-à-go-go related (ERG) and ether-à-go-go like (ELK). The human ether-à-go-go related gene (hERG) K(+) channel has been of significant interest because loss of function in the hERG channel is associated with a markedly increased risk of cardiac arrhythmias. The hERG channel has unusual kinetics with slow activation and deactivation but very rapid and voltage-dependent inactivation. The outer pore region of the hERG K(+) channel is predicted to be different from that of other members of the voltage-gated K(+) channel family. HERG has a much longer linker between the fifth transmembrane domain (SS) and the pore helix (S5P linker) compared to other families of voltage-gated K(+) channels (43 amino acids compared to 14-23 amino acids). Further, the S5P linker contains an amphipathic alpha-helix that in hERG channels probably interacts with the mouth of the pore to modulate inactivation. The human EAG and rat ELK2 channels (hEAG and rELK2) show reduced or no inactivation in comparison to hERG channels, yet both channels are predicted to contain a similarly long S5P linker to that of hERG. In this study, we have constructed a series of chimaeric channels consisting of the S1-S6 of hERG but with the S5P alpha-helical region of either hEAG or rELK2, and one consisting of the S1-S6 of rELK2 but with the S5P alpha-helical region of hERG to investigate the role of the S5P linker in inactivation. Our studies show that charged residues on the alpha-helix of the S5P linker contribute significantly to the differences in inactivation characteristics of the EAG family channels. Further, individually mutating each of the hydrophilic residues on the S5P alpha-helix of hERG to a charged residue had significant effects on the voltage dependence of inactivation and the two residues with the greatest affect when mutated to a lysine, N588 and Q592, both lie on the same face of the S5P alpha -helix. We suggest that inactivation of hERG involves the interaction of this face of the S5P alpha-helix with a charged residue on the remainder of the outer pore domain of the channel.

Cortisol influences the ontogeny of both alpha- and beta-subunits of the cardiac sodium channel in fetal sheep.

During development, the heart has to adapt to changes in shape, size and, at birth, to significant changes in arterial pressure. The orderly contraction of the heart is dependent on the coordinated expression of ion channels at appropriate densities in individual cardiac myocytes. The present study demonstrated that the expression of the alpha-subunit of the cardiac sodium channel, SCN5a, was high at mid gestation but then decreased until 10 days before birth before increasing again. Whereas the beta-subunit, SCN1b, gradually increased in expression towards partum, there was no detectable expression of SCN3b at any gestational time point. Fetal adrenalectomy prior to the normal prepartum surge in cortisol caused a reduction in expression of SCN1b and a 7.0 kb transcript of SCN5a, but not the major 8.5 kb transcript. Conversely, cortisol infusion into immature fetuses precociously increased expression levels of SCN1b and the SCN5a 7.0 kb transcript. The results show that cortisol regulates cardiac SCN gene expression in fetal sheep during late gestation. These findings could have implications for the aetiology of sudden infant death syndrome and for the intrauterine programming of adult cardiovascular disease.

The sodium channel beta-subunit SCN3b modulates the kinetics of SCN5a and is expressed heterogeneously in sheep heart.

1. Cardiac sodium channels are composed of a pore-forming alpha-subunit, SCN5a, and one or more auxiliary beta-subunits. The aim of this study was to investigate the role of the recently discovered member of the beta-subunit family, SCN3b, in the heart. 2. Northern blot and Western blot studies show that SCN3b is highly expressed in the ventricles and Purkinje fibres but not in atrial tissue. This is in contrast to the uniform expression of SCN1b throughout the heart. 3. Co-expression of SCN3b with the cardiac-specific alpha-subunit SCN5a in Xenopus oocytes resulted in a threefold increase in the level of functional sodium channel expression, similar to that observed when SCN1b was co-expressed with SCN5a. These results suggest that both SCN1b and SCN3b improve the efficiency with which the mature channel is targeted to the plasma membrane. 4. When measured in cell-attached oocyte macropatches, SCN3b caused a significant depolarising shift in the half-voltage of steady-state inactivation compared to SCN5a alone or SCN5a + SCN1b. The half-voltage of steady-state activation was not significantly different between SCN5a alone and SCN5a + SCN3b or SCN5a + SCN1b. 5. The rates of inactivation for SCN5a co-expressed with either subunit were not significantly different from that for SCN5a alone. However, recovery from inactivation at -90 mV was significantly faster for SCN5a + SCN1b compared to SCN5a + SCN3b, and both were significantly faster than SCN5a alone. 6. Thus, SCN1b and SCN3b have distinctive effects on the kinetics of activation and inactivation, which, in combination with the different patterns of expression of SCN3b and SCN1b, could have important consequences for the integrated electrical activity of the intact heart.

Effects of premature stimulation on HERG K(+) channels.

1. The unusual kinetics of human ether-à-go-go-related gene (HERG) K(+) channels are consistent with a role in the suppression of arrhythmias initiated by premature beats. Action potential clamp protocols were used to investigate the effect of premature stimulation on HERG K(+) channels, transfected in Chinese hamster ovary cells, at 37 degrees C. 2. HERG K(+) channel currents peaked during the terminal repolarization phase of normally paced action potential waveforms. However, the magnitude of the current and the time point at which conductance was maximal depended on the type of action potential waveform used (epicardial, endocardial, Purkinje fibre or atrial). 3. HERG K(+) channel currents recorded during premature action potentials consisted of an early transient outward current followed by a sustained outward current. The magnitude of the transient current component showed a biphasic dependence on the coupling interval between the normally paced and premature action potentials and was maximal at a coupling interval equivalent to 90 % repolarization (APD(90)) for ventricular action potentials. The largest transient current response occurred at shorter coupling intervals for Purkinje fibre (APD(90) - 20 ms) and atrial (APD(90) - 30 ms) action potentials. 4. The magnitude of the sustained current response following premature stimulation was similar to that recorded during the first action potential for ventricular action potential waveforms. However, for Purkinje and atrial action potentials the sustained current response was significantly larger during the premature action potential than during the normally paced action potential. 5. A Markov model that included three closed states, one open and one inactivated state with transitions permitted between the pre-open closed state and the inactivated state, successfully reproduced our results for the effects of premature stimuli, both during square pulse and action potential clamp waveforms. 6. These properties of HERG K(+) channels may help to suppress arrhythmias initiated by early afterdepolarizations and premature beats in the ventricles, Purkinje fibres or atria.

Normal conduction of surface action potentials in detubulated amphibian skeletal muscle fibres.

1. The influence of the transverse (T) tubules on surface action potential conduction was investigated by comparing electrophysiological and confocal microscopic assessments of tubular changes in osmotically shocked and control fibres from frog sartorius muscle. 2. The membrane-impermeant fluorescent dye, di-8-ANEPPs spread readily from the bathing extracellular solution into the tubular membranes in control, intact fibres. Prior exposure of muscles to a hypertonic glycerol-Ringer solution, its replacement by an isotonic Ca(2+)-Mg(2+) Ringer solution and cooling sharply reduced such access. In contrast, dye application in the course of this osmotic shock procedure stained the large tubular vacuoles hitherto associated with successful muscle detubulation. 3. Conduction velocities in intact, control fibres (1.91 +/- 0.048 m s(-1), mean +/- S.E.M., n = 32 fibres) agreed with earlier values reported at room temperature (18-21 degrees C) and were unaffected by prior episodes of steady cooling to 8-10 degrees C (1.91 +/- 0.043 m s(-1), n = 30). 4. Cooling to 11.5 degrees C reduced these velocities (1.47 +/- 0.081 m s(-1), n = 25) but action potential waveforms still included early overshoots and the delayed after-depolarizations associated with tubular electrical activity. 5. In contrast, action potentials from cooled, superficial fibres in osmotically shocked muscles lacked after-depolarization phases implying tubular detachment. Their mean conduction velocities (1.62 +/- 0.169 m s(-1), n = 25) were not significantly altered from values obtained in untreated controls or in intact fibres in muscle similarly treated with glycerol, in direct contrast to earlier results. 6. Cooling produced similar reductions in maximum rates of voltage change dV/dt in action potentials from all fibre groups with lower rates of change shown by detubulated fibres. 7. Use of an antibody to a conserved epitope of the alpha-subunit of voltage-gated sodium channels suggested a concentration of sodium channels close to the mouths of the T tubules. 8. These electrophysiological and anatomical findings are consistent with a partial independence of electrical events in the transverse tubules from those responsible for the rapid conduction of surface regenerative activity. 9. The findings are discussed in terms of a partial separation of the electrical activity propagated over the surface membrane, from the initiation of propagated activity within the T tubules, by the triggering of the sodium channels clustered selectively around the mouths of the T tubules.

HERG K+ channels: friend and foe.

The K+ channel encoded by the human ether-à-go-go related gene (HERG) is one of many ion channels that are crucial for normal action potential repolarization in cardiac myocytes. HERG encodes the pore-forming subunit of the rapid component of the delayed rectifier K+ channel, I(K(Vr)). HERG K+ channels are of considerable pharmaceutical interest as possible therapeutic targets for anti-arrhythmic agents and as the molecular target responsible for the cardiac toxicity of a wide range of pharmaceutical agents. Recent studies of the molecular basis of the promiscuity of HERG K+ channel drug binding has not only started to shed light on this tricky pharmaceutical problem but has also provided further insights into the structure and function of HERG K+ channels.

The effect of Mg2+ on cardiac muscle function: Is CaATP the substrate for priming myofibril cross-bridge formation and Ca2+ reuptake by the sarcoplasmic reticulum?

Kinetics are established for the activation of the myofibril from the relaxed state [Smith, Dixon, Kirschenlohr, Grace, Metcalfe and Vandenberg (2000) Biochem. J. 346, 393-402]. These require two troponin Ca2+-binding sites, one for each myosin head, to act as a single unit in initial cross-bridge formation. This defines the first, or activating, ATPase reaction, as distinct from the further activity of the enzyme that continues when a cross-bridge to actin is already established. The pairing of myosin heads to act as one unit suggests a possible alternating mechanism for muscle action. A large positive inotropic (contraction-intensifying) effect of loading the Mg2+ chelator citrate, via its acetoxymethyl ester, into the heart has confirmed the competitive inhibition of the Ca2+ activation by Mg2+, previously seen in vitro. In the absence of a recognized second Ca2+ binding site on the myofibril, with appropriate binding properties, the bound ATP is proposed as the second activating Ca2+-binding site. As ATP, free or bound to protein, can bind either Mg2+ or Ca2+, this leads to competitive inhibition by Mg2+. Published physico-chemical studies on skeletal muscle have shown that CaATP is potentially a more effective substrate than MgATP for cross-bridge formation. The above considerations allow calculation of the observed variation of fractional activation by Ca2+ as a function of [Mg2+] and in turn reveal simple Michaelis-Menten kinetics for the activation of the ATPase by sub-millimolar [Mg2+]. Furthermore the ability of bound ATP to bind either cation, and the much better promotion of cross-bridge formation by CaATP binding, give rise to the observed variation of the Hill coefficient for Ca2+ activation with altered [Mg2+]. The inclusion of CaADP within the initiating cross-bridge and replacement by MgADP during the second cycle is consistent with the observed fall in the rate of the myofibril ATPase that occurs after two phosphates are released. The similarity of the kinetics of the cardiac sarcoplasmic reticulum ATPase to those of the myofibril, in particular the positive co-operativity of both Mg2+ inhibition and Ca2+ activation, leads to the conclusion that this ATPase also has an initiation step that utilizes CaATP. The first-order activation by sub-millimolar [Mg2+], similar to that of the myofibril, may be explained by Mg2+ involvement in the phosphate-release step of the ATPase. The inhibition of both the myofibril and sarcoplasmic reticulum Ca2+ transporting ATPases by Mg2+ offers an explanation for the specific requirement for phosphocreatine (PCr) for full activity of both enzymes in situ and its effect on their apparent affinities for ATP. This explanation is based on the slow diffusion of Mg2+ within the myofibril and on the contrast of PCr with both ATP and phosphoenolpyruvate, in that PCr does not bind Mg2+ under physiological conditions, whereas both the other two bind it more tightly than the products of their hydrolysis do. The switch to supply of energy by diffusion of MgATP into the myofibril when depletion of PCr raises [ATP]/[PCr] greatly, e.g. during anoxia, results in a local [Mg2+] increase, which inhibits the ATPase. It is possible that mechanisms similar to those described above occur in skeletal muscle but the Ca2+ co-operativity involved would be masked by the presence of two Ca2+ binding sites on each troponin.

Loss of the normal epicardial to endocardial gradient of cftr mRNA expression in the hypertrophied rabbit left ventricle.

The electrical instability of hypertrophied and failing hearts is caused by delayed repolarisation, which is thought to be due in part to altered levels and/or patterns of expression of ion channel genes. The aim of this study was to investigate changes in the levels and pattern of cystic fibrosis transmembrane conductance regulator (cftr) mRNA expression in a combined pressure and volume overload model of heart failure in the rabbit, using in situ mRNA hybridisation. There was a decrease in cftr mRNA expression, primarily due to a decrease in epicardial expression and, hence, loss of the normal epicardial to endocardial gradient of cftr mRNA expression in the rabbit left ventricle. In contrast there was an increase in atrial natriuretic factor (anf) mRNA expression in the hypertrophied hearts with preferential reexpression in subendocardial regions. The patterns of both cftr and anf mRNA expression in the hypertrophied hearts were similar to those seen in embryonic hearts. This suggests that the reversion to an embryonic pattern of gene expression in cardiac hypertrophy applies to ion channel genes. The loss of the normal transmural gradient of repolarising ion channels is likely to contribute to instability of repolarisation in the hypertrophied heart and hence increased risk of cardiac arrhythmias in patients with heart failure.

The effect of heptanol on the electrical and contractile function of the isolated, perfused rabbit heart.

Changes in cardiac gap junction expression, such as those following myocardial infarction and produced in connexin knockout mice, are associated with a predisposition to arrhythmias. The present experiments investigated the effects of heptanol, a reversible gap junction inhibitor, on isolated Langendorff-perfused rabbit hearts. The introduction and withdrawal of heptanol inhibited both pressure generation and electrical conduction. These effects were completely reversible. Possible mechanisms for these findings were investigated through measurement of the concentration dependence of heptanol's effects upon conduction velocity and repolarization duration. Low concentrations of heptanol (less than 0.3 mM) caused small but significant increases in the delay between the stimulus (delivered to the basal septum) artefact and local activation of the left ventricle, as measured from bipolar electrogram (BEG) recordings. There was a steep increase in the latency between stimulus and left-ventricular activation at concentrations of heptanol above 0.3 mM. These findings are explicable by earlier reports of heptanol actions on gap junctions in vitro and modelling studies of the effects of reduced gap junction conductance on conduction velocity. Heptanol decreased repolarization duration, measured from the activation recovery interval (ARI) of BEGs, and monophasic action potential duration at 70% repolarization (MAPD70). Heptanol also reduced left-ventricular developed pressure (LVDP), and the maximum rates of contraction and relaxation of the left ventricle; these effects were concentration dependent and reversible. However, changes in ARIs, LVDP and the maximum rates of change of pressure lacked the steep response to 0.3-1.0 mM heptanol shown by the latency. These other effects are therefore likely to be mediated by cellular targets other than gap junctions. Perfusion of hearts with heptanol was also associated with a high incidence of arrhythmias. During premature stimulation protocols arrhythmias could be induced in hearts perfused with 0.1-0.3 mM heptanol but not at higher concentrations. This suggests that there is a critical range of slowed conduction that permits the development of re-entrant arrhythmias in the normal heart, although the effects of heptanol on repolarization duration may also contribute to its pro-arrhythmic activity.

Developmental regulation of the gradient of cftr expression in the rabbit heart.

Gradients of ion channels across the left ventricular free wall of the heart have been found for a number of repolarizing ion channels. Amongst these are the cAMP-activated chloride channels encoded by cftr. In this report, we show that the epicardial (higher) to endocardial (lower) gradient of cftr mRNA found in adult rabbit hearts is not present in embryonic hearts. The gradient starts to develop shortly after birth, and over a period of 5-6 weeks increases to the levels found in the adult. This is the first report of the developmental regulation of any cardiac ion channel mRNA gradient.

Estimation of systolic and diastolic free intracellular Ca2+ by titration of Ca2+ buffering in the ferret heart.

Spectroscopic Ca(2+)-indicators are thought to report values of free intracellular Ca(2+) concentration ([Ca(2+)](i)) that may differ from unperturbed values because they add to the buffering capacity of the tissue. To check this for the heart we have synthesized a new (19)F-labelled NMR Ca(2+) indicator, 1, 2-bis-[2-bis(carboxymethyl)amino-4,5-difluorophenoxy]ethane ('4, 5FBAPTA'), with a low affinity (K(d) 2950 nM). The new indicator and four previously described (19)F-NMR Ca(2+) indicators 1,2-bis-[2-(1 - carboxyethyl)(carboxymethyl)amino - 5 - fluorophenoxy]ethane ('DiMe-5FBAPTA'), 1, 2-bis-[2-(1-carboxyethyl)(carboxymethyl)amino-4-fluorophenoxy]ethane ('DiMe-4FBAPTA'), 1, 2-bis-[2-bis(carboxymethyl)amino-5-fluorophenoxy]ethane ('5FBAPTA') and 1, 2-bis-[2-bis(carboxymethyl)amino-5-fluoro-4-methylphenoxy]ethane ('MFBAPTA'), with dissociation constants for Ca(2+) ranging from 46 to 537 nM, have been used to measure [Ca(2+)](i), over the range from less than 100 nM to more than 3 microM, in Langendorff-perfused ferret hearts (30 degrees C, pH 7.4, paced at 1.0 Hz) by (19)F-NMR spectroscopy. Loading hearts with indicators resulted in buffering of the Ca(2+) transient. The measured end-diastolic and peak-systolic [Ca(2+)](i) were both positively correlated with indicator K(d). The positive correlations between indicator K(d) and the measured end-diastolic and peak-systolic [Ca(2+)](i) were used to estimate the unperturbed end-diastolic and peak-systolic [Ca(2+)](i) by extrapolation to K(d)=0 (diastolic) and to K(d)=infinity (systolic) respectively. The extrapolated values in the intact beating heart were 161 nM for end-diastolic [Ca(2+)](i) and 2650 nM for peak-systolic [Ca(2+)](i), which agree well with values determined from single cells and muscle strips.

Ca2+ buffering in the heart: Ca2+ binding to and activation of cardiac myofibrils.

The measurement of cardiac Ca(2+) transients using spectroscopic Ca(2+) indicators is significantly affected by the buffering properties of the indicators. The aim of the present study was to construct a model of cardiac Ca(2+) buffering that satisfied the kinetic constraints imposed by the maximum attainable rates of cardiac contraction and relaxation on the Ca(2+) dissociation rate constants and which would account for the observed effects of (19)F-NMR indicators on the cardiac Ca(2+) transient in the Langendorff-perfused ferret heart. It is generally assumed that the Ca(2+) dependency of myofibril activation in cardiac myocytes is mediated by a single Ca(2+)-binding site on troponin C. A model based on 1:1 Ca(2+) binding to the myofilaments, however, was unable to reproduce our experimental data, but a model in which we assumed ATP-dependent co-operative Ca(2+) binding to the myofilaments was able to reproduce these data. This model was used to calculate the concentration and dissociation constant of the ATP-independent myofilament Ca(2+) binding, giving 58 and 2.0 microM respectively. In addition to reproducing our experimental data on the concentration of free Ca(2+) ions in the cytoplasm ([Ca(2+)](i)), the resulting Ca(2+) and ATP affinities given by fitting of the model also provided good predictions of the Ca(2+) dependence of the myofibrillar ATPase activity measured under in vitro conditions. Solutions to the model also indicate that the Ca(2+) mobilized during each beat remains unchanged in the presence of the additional buffering load from Ca(2+) indicators. The new model was used to estimate the extent of perturbation of the Ca(2+) transient caused by different concentrations of indicators. As little as 10 microM of a Ca(2+) indicator with a dissociation constant of 200 nM will cause a 20% reduction in peak-systolic [Ca(2+)](i) and 30 microM will cause approx. 50% reduction in the peak-systolic [Ca(2+)](i) in a heart paced at 1.0 Hz.

Molecular and functional distributions of chloride conductances in rabbit ventricle.

The regulation of cardiac electrical activity is critically dependent on the distribution of ion channels in the heart. For most ion channels, however, the patterns of distribution and what regulates these patterns are not well characterized. The most likely candidates for the genes that encode the cAMP- and swelling-activated chloride conductances in the heart are an alternatively spliced variant of CFTR and ClC-3, respectively. In this study we have 1) measured the density of CFTR and ClC-3 mRNA levels across the left ventricular free wall (LVFW) of the rabbit heart using in situ hybridization and 2) measured the corresponding current density of cAMP- and swelling-activated chloride channels in myocytes isolated from subepicardial, midmyocardial, and subendocardial regions of the LVFW. There was a highly significant gradient in the whole cell slope conductance of cAMP-activated chloride currents; normalized slope conductance at 0 mV was 15.7 +/- 1.8 pS/pF (n = 9) in subepicardial myocytes, 7.8 +/- 1.5 pS/pF (n = 11) in midmyocardial myocytes, and 4.9 +/- 1.1 pS/pF (n = 9) in subendocardial myocytes. The level of CFTR mRNA was closely correlated with the density of cAMP-activated chloride conductances in different regions of the heart, with the level of CFTR mRNA being three times higher in the subepicardium than in the subendocardium. The whole cell slope conductance of swelling-activated chloride channel activity, measured 3-5 min after the commencement of cell swelling, was higher in myocytes isolated from the subepicardium than in myocytes isolated from the midmyocardium or subendocardium. In contrast, there was a uniform expression of ClC-3 mRNA across the LVFW of the rabbit heart. These results suggest that the control of gene expression is an important contributor in regulating the distribution of cAMP-activated chloride channels in the rabbit heart but that it may be less important for the swelling-activated chloride channels.

Action potential shortening through the putative beta4-adrenoceptor in ferret ventricle: comparison with beta1- and beta2-adrenoceptor-mediated effects.

The electrophysiological responses to (-)-CGP 12177 ((-)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one), an agonist for the putative beta4-adrenoceptor, were investigated on isolated perfused ferret hearts paced at 100 min(-1) and compared to those of (-)-noradrenaline and (-)-adrenaline, mediated through beta1- and beta2-adrenoceptors respectively. The three agonists decreased ventricular monophasic action potential duration but prolonged the action potential plateau; beta3-adrenoceptor-selective agonists had no effect. (-)-CGP 12177 was the most potent, but (-)-noradrenaline the most efficacious; both agonists caused ventricular extra-systoles. Because only (-)-noradrenaline but not (-)-CGP 12177 elicited shortening of the refractory period, the mechanism of arrhythmias mediated through beta1- and putative beta4-adrenoceptors may be different.

Changes in ventricular repolarization during acidosis and low-flow ischemia.

Myocardial ischemia, primarily a metabolic insult, is also defined by altered cardiac mechanical and electrical activity. We have investigated the metabolic contributions to the electrophysiological changes during low-flow ischemia (7.5% of the control flow) using 31P NMR spectroscopy to monitor metabolic parameters, suction electrodes to study epicardial monophasic action potentials, and 86Rb as a tracer for K+-equivalent efflux during low-flow ischemia in the Langendorff-perfused ferret heart. Shortening of the action potential duration at 90% repolarization (APD90) was most marked between 1 and 5 min after induction of ischemia, at which time it shortened from 261 +/- 4 to 213 +/- 8 ms. The period of marked APD90 shortening was accompanied by a fivefold increase in the rate of 86Rb efflux, both of which were inhibited by the ATP-sensitive K+ (KATP)-channel blockers glibenclamide and 5-hydroxydecanoate (5-HD), as well as by a significant fall in intracellular pH (pHi) from 7.14 +/- 0.02 to 6.83 +/- 0.03 but no change in intracellular ATP concentration ([ATP]i). We therefore investigated whether a fall in pHi could be the metabolic change responsible for modulating cardiac KATP channel activity in the intact heart during ischemia. Both metabolic (30 mM lactate added to extracellular solution) and respiratory (PCO2 increased to 15%) acidosis caused an initial lengthening of APD90 to 112 +/- 1.5 and 113 +/- 0.9%, respectively, followed by shortening during continued acidosis to 106 +/- 1.2 and 106 +/- 1.4%, respectively. The shortening of APD90 during continued acidosis was inhibited by glibenclamide, consistent with acidosis causing activation of KATP channels at normal [ATP]i. The similar responses to metabolic (induced by adding either l- or d-lactate) and respiratory acidosis suggest that lactate has no independent metabolic effect on action potential repolarization.