Mathematical model of an adult human atrial cell: the role of K+ currents in repolarization.

We have developed a mathematical model of the human atria myocyte based on averaged voltage-clamp data recorded from isolated single myocytes. Our model consists of a Hodgkin-Huxley-type equivalent circuit for the sarcolemma, coupled with a fluid compartment model, which accounts for changes in ionic concentrations in the cytoplasm as well as in the sarcoplasmic reticulum. This formulation can reconstruct action potential data that are representative of recordings from a majority of human atrial cells in our laboratory and therefore provides a biophysically based account of the underlying ionic currents. This work is based in part on a previous model of the rabbit atrial myocyte published by our group and was motivated by differences in some of the repolarizing currents between human and rabbit atrium. We have therefore given particular attention to the sustained outward K+ current (I[sus]), which putatively has a prominent role in determining the duration of the human atrial action potential. Our results demonstrate that the action potential shape during the peak and plateau phases is determined primarily by transient outward K+ current, I(sus) and L-type Ca2+ current (I[Ca,L]) and that the role of I(sus) in the human atrial action potential can be modulated by the baseline sizes of I(Ca,L), I(sus) and the rapid delayed rectifier K+ current. As a result, our simulations suggest that the functional role of I(sus) can depend on the physiological/disease state of the cell.

Electrophysiological analyses of threshold conditions and rate-dependent failure of excitation in single myocytes from rabbit ventricle.

The changes in transmembrane ionic currents that underlie normal excitability and rate-dependent failure were studied in single cells from rabbit ventricle by using whole cell voltage clamp methods. When trains of brief (1 to 2 ms) stimuli are applied at strengths very close to the threshold for excitation, a number of different patterns of action potential entrainment and failure are observed. In an individual cell, a characteristic pattern of entrainment or failure can be maintained for a relatively long time, allowing both a detailed description and a quantitative investigation of the ionic basis for this phenomenon. Three hypotheses for rate-dependent failure of excitation in rabbit ventricle were examined. The first is that following relatively high rates of stimulation, the intracellular calcium ion concentration increases and, secondarily, a background inwardly rectifying potassium ion current (IK1) decreases, thereby lowering the excitation threshold. The second hypothesis is that residual activation of the delayed rectifier potassium ion current (IK) causes the stimulus to become subthreshold as the rate of stimulation increases. The third hypothesis is that small changes in the time and voltage dependence of the inactivation and reactivation of the sodium ion current (INa) result in less net inward ion current for a given waveform of depolarization, and the cell therefore becomes inexcitable (eg, to every second stimulus). The calcium ion hypothesis was tested by buffering changes in intracellular calcium ion concentrations with BAPTA. The results strongly suggest that changes in intracellular calcium ion concentrations do not contribute significantly to the observed patterns of failure of excitation. The delayed rectifier hypothesis was evaluated using the class III antiarrhythmic drug dofetilide, which selectively blocks a large fraction of the IK current in rabbit ventricle. Dofetilide slightly decreased the stimulus threshold, suggesting that residual activation of the rapidly activated outward conductance of potassium ions is an important variable under some conditions. The INa hypothesis was tested by altering the size of INa with tetrodotoxin (TTX). After TTX application, the threshold for excitation increased significantly, and rate-dependent failure and entrainment were no longer observed until the stimulus strength was increased. These results show that INa is an essential variable underlying normal excitation and entrainment in rabbit ventricle. This is plausible because INa is 20 to 50 times larger than either the maximum outward current due to IK1 or the fully activated IK in this tissue.

Electrotonic modulation of electrical activity in rabbit atrioventricular node myocytes.

Electrotonic effects of electrically coupling atrioventricular (AV) nodal cells to each other and to real and passive models of atrial and ventricular cells were studied using a technique that does not require functional gap junctions. Membrane potential was measured in each cell using suction pipettes. Mutual entrainment of two spontaneously firing AV nodal cells was achieved with a junctional resistance (Rj) of 500 M omega, which corresponds to only 39 junctional channels, assuming a single-channel conductance of 50 pS. Coupling of AV nodal and atrial cells at Rj of 50 M omega caused hyperpolarization of the nodal cell, decreasing its action potential duration and either slowing or blocking diastolic depolarization in the AV node myocyte. Opposite changes occurred in the atrial action potential. When AV nodal and ventricular cells were coupled at Rj of 50 M omega, nodal diastolic potential was markedly hyperpolarized and diastolic depolarization was completely blocked with little change in ventricular diastolic potential. However, coupling did elicit marked changes in the action potential duration of both cells, with prolongation in the nodal cell and shortening in the ventricular cell. Nodal maximum upstroke velocity was increased by both atrial and ventricular coupling, as expected from the hyperpolarization that occurred. With an Rj of 50 M omega, spontaneous firing was blocked in all single AV nodal pacemaker cells during coupling to a real or passive model of an atrial or ventricular cell. These results demonstrate that action potential formation and waveform in a single AV nodal cell is significantly affected by electrical coupling to other myocytes.

Modification of gap junction conductance by divalent cations and protons in neonatal rat heart cells.

Myocytes were isolated from neonatal rat hearts and grown in culture dishes. Pairs of cells were selected to study the effect of divalent cations and protons on the conductance of gap junctions, gj. The experimental approach involved the dual voltage-clamp method and cell dialysis via patch pipette, i.e. gj was monitored while the cytosolic level of Ca2+, Mg2+, Sr2+, Ba2+ or H+ was modified in one of the cells. A dose-dependent decrease in gj developed when pCa of the pipette solution was lowered (range: pCa = 7.7-2.42, equivalent to a [Ca2+] of 20 nM-3.8 mM). The gj/pCa-relationship revealed a Hill coefficient n of 0.87 and a half-maximal concentration pKCa of 3.5. Pretreatment with 3 mM NiCl2 and 1 micron ryanodine to minimize the removal of cytosolic Ca2+ did not significantly affect the response to gj. Similarly, gj was decreased in a dose-dependent fashion when pHi in the pipette solution was lowered (range: pH = 7.2-5.0, corresponding to a [H+] of 63 nM-10 microns). The gj/pH-relationship yielded an n of 0.92 and a pKH of 5.85. Pretreatment with 1 mM amiloride to minimize the extrusion of protons enhanced the effects of pH on gj. Simultaneous alterations in pCa and pH demonstrated an additive type of action of Ca2+ and H+ on gj. This is consistent with the existence of two types of sensors which contribute separately to the functional state of gj. No significant decrease in gj was detectable when the pipette solution contained Mg2+ or Ba2+ (up to 5 mM). Partial uncoupling was observed with pipette solution containing 5 mM Sr2+. We conclude that gj of neonatal and adult cardiomyocytes exhibit different ionic sensitivities. This discrepancy may reflect differences in connexin expression and/or molecular intermediates involved in regulating gj.

Outward currents underlying repolarization in human atrial myocytes.

OBJECTIVE: The goals of this study were to identify the types of outward potassium (K+) currents that are activated at membrane potentials corresponding to the plateau of the action potential in human atrial myocytes, and to compare their properties with published data describing the K+ channels which have been cloned from a human cDNA library. METHODS: Specimens of right atrial appendages were obtained from patients undergoing cardiac surgery. Single myocytes were isolated enzymatically and whole cell voltage- and current-clamp recording techniques were applied. RESULTS: The outward K+ current in most cells consisted of transient and sustained (non-inactivating) components. 4-Aminopyridine (4-AP, 50 microM) broadened the action potential and increased the plateau height by blocking a Ca(2+)-independent transient outward K+ current (I(t)). The transient and the pedestal components could also be separated by using two pulse voltage-clamp protocols to inactivate them: the transient component was inactivated completely by 400 ms depolarizing pre-pulses (-80 to 0 mV). In contrast, the inactivation of the pedestal component was not complete even when very long (2500 ms) pre-pulses were applied. The time-course of inactivation of the K+ currents in most cells could be described mathematically by the sum of two exponential functions. The faster of the two processes underlying inactivation was voltage-independent for membrane voltages between +10 and +40 mV. The dependence of the recovery kinetics (reactivation) of I(t) on [K+]o was also studied. When [K+]o was reduced from 5.4 to 1.0 mM, reactivation slowed significantly. In a small fraction of atrial cells, a slowly activating delayed rectifier current was also identified. CONCLUSIONS: These results provide additional information concerning the ionic mechanism(s) for early and late repolarization, and they allow findings from electrophysiologically viable human atrial cells to be related to recent information regarding the molecular biology of potassium currents in human heart.

Short-term diabetes alters K+ currents in rat ventricular myocytes.

The electrophysiological properties of single ventricular myocytes from control rats and from rats made diabetic by streptozotocin (STZ) injection (100 mg/kg body weight) have been investigated using whole-cell voltage-clamp measurements. Our major goal was to define the effects of diabetes on rate-dependent changes in action potential duration and the underlying outward K+ currents. As early as 4 to 6 days after STZ treatment, significant elevation of plasma glucose levels occurs, and the action potential duration increases. In both control and diabetic rats, when the stimulation rate is increased, the action potential is prolonged, but this lengthening is considerably more pronounced in myocytes from diabetic rats. In ventricular myocytes from diabetic rats, the Ca(2+)-independent transient outward K+ current (I(t)) is reduced in amplitude, and its reactivation kinetics are slowed. These changes result in a smaller I(t) at physiological heart rates. The steady-state outward K+ current (IK) also exhibits rate-dependent attenuation, and this phenomenon is more pronounced in cells from diabetic rats. These STZ-induced changes in I(t) and IK also develop when a lower dose (55 mg/kg) of STZ is used and measurements are made after 7 weeks of treatment. These electrophysiological effects are not related to the hypothyroid conditions that accompany the diabetic state, since they cannot be reversed by replacement of the hormone L-triiodothyronine to physiological levels. Direct effects of STZ could be ruled out, since preceding the STZ injection with a bolus injection of 3-O-methylglucose, which prevents development of hyperglycemia, prevents the electrophysiological changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen peroxide induced changes in membrane potentials in guinea pig ventricular muscle: permissive role of iron.

STUDY OBJECTIVE: It has been proposed that oxygen free radicals trigger reperfusion arrhythmias. The mechanism of these arrhythmias is not clear. Thus, the effect of H2O2 on cellular action potentials was examined. DESIGN: Trabecular muscles were superfused either with H2O2 alone or with H2O2 in combination either with H2O2 alone or with H2O2 in combination with iron ions or an iron chelating agent or various scavengers of oxygen free radicals. The effect of reduction of the superfusate calcium from 1.8 to 0.2 mmol.litre-1 on H2O2 induced changes was also studied. EXPERIMENTAL MATERIAL: Thin trabecular muscles isolated from the hearts of guinea pigs (200-300 g) of either sex were used. MEASUREMENTS AND MAIN RESULTS: H2O2 (0.6 mmol.litre-1) caused a reproducible sequence of changes consisting of an initial increase in plateau height and in action potential duration, followed after 12-14 min by rapid action potential shortening accompanied by resting membrane depolarisation, reduction in action potential amplitude and dV/dt max, and by occasional appearance of late afterdepolarisations, leading finally to loss of excitability. This sequence of changes was: (1) accelerated by higher concentrations of H2O2, FeCl3 (0.1 mmol.litre-1), and FeCl2 (0.1 mmol.litre-1); (2) prevented by dimethylthiourea (10 mmol.litre-1) and desferrioxamine (2 mmol.litre-1); (3) not influenced by superoxide dismutase (150 units.ml-1), mannitol (5-50 mmol.litre-1) or PBN (50 mumols.litre-1); and (4) not prevented by a reduction of the superfusate calcium. CONCLUSIONS: The electrophysiological alterations induced by H2O2 are caused by a hydroxyl radical formed intracellularly in the iron catalysed Fenton reaction.

Effects of in vitro generated oxygen free radicals on transmembrane potentials in ventricular cardiac muscle.

The effects of in vitro generated oxygen free radicals on transmembrane potentials in guinea pig papillary muscles were investigated. Superfusion of the muscle with the oxygen free radical generating medium (1 mM xanthine + 10 U/l xanthine oxidase) for 40 min altered significantly neither the resting nor the action potentials characteristics. We propose that electrophysiological changes leading to the reperfusion-induced arrhythmias may be secondary to an inhomogenous change in the coronary myocardial perfusion produced by the radicals.