SUMMARY1. Slow inward tail currents attributable to electrogenic sodium-calcium exchange can be recorded by imposing hyperpolarizing voltage clamp pulses during the normal action potential of isolated guinea-pig ventricular cells. The hyperpolarizations return the membrane to the resting potential (between -65 and -88 m V) allowing an inward current to be recorded. This current usually has peak amplitude when repolarization is imposed during the first 50 ms after the action potential upstroke, but becomes negligible once the final phase of repolarization is reached. The envelope of peak current tail amplitudes strongly resembles that of the intracellular calcium transient recorded in other studies.2. Repetitive stimulation producing normal action potentials at a frequency of 2 Hz progressively augments the tail current recorded immediately after the stimulus train. Conversely, if each action potential is prematurely terminated at 01 Hz, repetitive stimulation produces a tail current much smaller than the control value. The control amplitude of inward current is only maintained if interrupted action potentials are separated by at least one full 'repriming' action potential. These effects mimic those on cell contraction (Arlock & Wohlfart, 1986) and suggest that progressive changes in tail current are controlled by variations in the amplitude and time course of the intracellular calcium transient.3. When intracellular calcium is buffered sufficiently to abolish contraction, the tail current is abolished. Substitution of calcium with strontium greatly reduces the tail current.4. The inward tail current can also be recorded at more positive membrane potentials using standard voltage clamp pulse protocols. In this way it was found that temperature has a large effect on the tail current, which can change from net inward at 22°C to net outward at 37 'C. The largest inward currents are usually recorded at about 30 'C. It is shown that this effect is attributable predominantly to the temperature sensitivity of activation of the delayed potassium current, iK, whose decay can then mask the slow tail current at high temperatures.5. Studies of the relationship between the tail current and the membrane calcium current, iCa, have been performed using a method of drug application which is capable of perturbing ica in a very rapid and highly reversible manner. Partial block of iCa with cadmium does not initially alter the size of the associated inward current T. M. EGAN AND OTHERS tail. When iCa is increased by applying isoprenaline, the percentage augmentation of the associated tail current is much greater but occurs more slowly. Similarly, the tail current recovers to its initial value more slowly than does ica.6. These results are interpreted to indicate that the sodium-calcium exchange current flows during the time course of the cardiac action potential and that its amplitude is more closely related to intracellular calcium release than to the membrane calcium current per se. Calculation of the exchange current flowing durin...

Abstract-A tetrodotoxin-sensitive persistent sodium current, I pNa , was found in guinea pig ventricular myocytes by whole-cell patch clamping. This current was characterized in cells derived from the basal left ventricular subendocardium, midmyocardium, and subepicardium. Key Words: persistent sodium current Ⅲ guinea pig ventricle Ⅲ regional differences F irst indications for a slowly inactivating, persistent sodium current in cardiac cells came from studies on Purkinje fibers of dogs and rabbits.1,2 This was followed by the discovery of slowly inactivating sodium channels in ventricular myocytes of rats. Subsequently, a small late sodium current was described in guinea pig ventricular myocytes using single-channel and whole-cell patch clamping. 4 This study also suggested a significant effect of the late sodium current on action potential duration in ventricular cells: application of 60 mol/L tetrodotoxin (TTX) reversibly shortened action potential duration at 95% repolarization (ADP 95 ) by about 10%. Kiyosue et al 4 speculated that this was because of block of slowly inactivating sodium channels.Additional studies investigated the changes in the I-V relation of a slowly inactivating, TTX-sensitive sodium current (I pNa ) in rat ventricular myocytes in the absence and presence of hypoxia. 5,6 This persistent sodium current increased during hypoxia, and, thus, it was suggested that it could be involved in the development of early after depolarizations (EADs) and arrhythmias during hypoxic states. Most recently, Maltsev et al 7 showed that a persistent sodium current (which they called the late sodium current) is present in ventricular myocytes from human midmyocardium in normal donor hearts and heart failure patients. Interestingly, they found a similar (15% to 20%) reduction in action potential duration (APD) after application of 1.5 mol/L TTX, as previously described 4 in guinea pigs. They also showed that TTX abolished EADs in myocytes isolated from heart failure patients. Computer modeling studies fit these data surprisingly well. It has been demonstrated 8 that EADs can be induced in a cell model by increasing I pNa ; this mechanism also requires that the inactivation curve of the fast sodium current is shifted in the depolarizing direction, as has been observed in an SCN5A missense mutation. 9 An increase in late sodium current is also present in some well-known genetic predispositions to arrhythmias, for example, one type of the long-QT syndrome.

SUMMARY1. We studied the effects of low temperature on the action potentials and membrane currents of guinea-pig ventricular myocytes, using a tight-seal whole-cell clamp technique.2. The action potential duration at 95 % repolarization was prolonged from 146+ 33 ms (mean+ S.D., n = 6) at 33-34°C (control temperature) to 314+ 83 ms at 24-25°C (low temperature).3. In whole-cell clamp experiments, low temperature decreased the calcium current (ICa), the delayed rectifier potassium current (IK)' and the inwardly rectifying potassium current (IK.) with 'apparent' Qlo (temperature coefficient) values of 2-3 + 06 for ICa' 4-4 + 1-2 for 'K tail current and 1-5 + 03 for IK. (n = 7).4. The effect of low temperature onIK was further studied in the presence of 06 ,(M nicardipine to block ICa. The decay phase of the IK tail consisted of two exponential components. The fast but not the slow component was highly sensitive to the temperature change with an apparent Q1o of 4-5.5. We found that a component of time-independent current is also sensitive to the temperature. The current had a linear I-V relationship and remained almost unchanged after inhibition of Na+-K+ pump in K+-free external solution.6. Using our mathematical model of the ventricular action potential (a modification from the DiFrancesco-Noble model), we simulated the action potential at low temperature by modifying some of the membrane currents, namely IK' IK1, ICa and a component of background current. It was shown that simultaneous changes in these currents could reproduce approximately 75 % of the action prolongation induced by low temperature.

SUMMARY1. A component of inward current has been identified in isolated guinea-pig ventricular cells that is closely correlated with the contraction of the cell and not with the rapidly activated calcium current. This is a delayed current most clearly seen as a current 'tail' after 50-200 ms depolarizing pulses. At 22°C the delayed current has a maximum amplitude of -05 nA at -40 mV (consistently 10-20 %O of the peak amplitude of the calcium current) and decays with a half time of -150 ms.2. Paired-pulse protocols show that at pulse intervals (300-400 ms) at which the calcium current is nearly fully reprimed, the delayed component is very small. It recovers over a time course of several seconds, as does the contraction. Adrenaline speeds the decay of the delayed current (-50°/) and similarly accelerates cell relaxation. Adrenaline also shortens the recovery time of both the contraction and the delayed current. 3. During long trains of repetitive pulses, the delayed current amplitude follows that of the contraction 'staircase'. The half-time of the decay of the current 'tail' also matches that of contraction and suggests that both may reflect the time course of the underlying intracellular calcium transient.4. The half-time of decay of the delayed current is only moderately voltage dependent over the potential range -80 to 0 mV. The amplitude of the delayed current normally reaches a minimum around -20 mV and increases at more negative potentials.5. The voltage dependence and kinetics of decay of the current show that it should flow and decay largely during the action potential plateau and repolarization rather than during diastole.6. Diffusion of high concentrations of EGTA into cells abolishes the delayed current and cell contraction. Under these conditions the fast calcium current is increased and its inactivation delayed.7. When calcium is replaced by strontium, the delayed current amplitude is greatly reduced even though the contraction is larger and slower.8. The results are consistent with the hypothesis that the delayed inward current is activated by the intracellular calcium transient. It may be carried by the sodium-calcium exchange process and/or by calcium-activated non-specific channels (especiallv when internal calcium is elevated by reduction of external sodium).9. In the presence of 1 ,tM-ryanodine, the calcium current is greatly reduced, whereas the delayed current is not significantly altered.

SUMMARY1. The arrhythmogenic transient inward current, iTI, and contractions were recorded in isolated guinea-pig ventricular myocytes, after exposure to strophanthidin or low external K+ (0-5 mM), using a single-microelectrode voltage-clamp technique and an optical measure of contraction.2. The inward current, iTI, and after-contraction occurred on repolarization after a depolarizing pre-pulse. Longer pre-pulses to more positive potentials increased the size and reduced the latency of iTI* Oscillatory currents and contractions also occurred during pulses to positive potentials.3. The voltage dependence of iTI was studied by repolarizing to different potentials after a constant depolarizing pulse. Inward currents preceded after-contractions at all potentials. The iTI was maximal at about -50 mV, diminishing in magnitude at more negative and positive potentials. It remained inward at potentials up to +47 mV. The contraction exhibited a similar voltage dependence. The currentvoltage relation varied in the same cell with longer exposure to glycosides. Thus, the voltage dependence of iTI reflected not only that of an underlying ionic mechanism but also the effects of potential on intracellular Ca2" oscillations which trigger iTI' 4. Uniformity of internal Ca2+ transients was achieved by clamping to different potentials at the peak of an inward current. The iTI remained inward at positive potentials. An inward tail current, seen on repolarizing during iTI at the end of a depolarizing pre-pulse, progressively increased at negative potentials. This voltage dependence may be close to that of the Ca2+-activated inward current responsible for 1TI 5. Replacement of Na+ by Li+ initially increased the magnitude of iTI, but further exposure abolished the inward current, while the after-contractions continued to increase. The potential dependence of iTI was not affected by exposure to zero Na+. Replacement of Ca2+ by Sr2+ also abolished iTI and the after-contraction, but the main effect was to slow their occurrence.6. The voltage dependence of the Ca2+-activated inward current in guinea-pig ventricular myocytes leads us to favour electrogenic Na-Ca exchange current as a major component of iTI, under our experimental conditions.