Rapid onset of lysophosphatidylcholine-induced modification of whole cell cardiac sodium current kinetics.

Lysophosphatidylcholine (LPC), an ischemic metabolite implicated in arrhythmogenesis, has been shown to modulate aspects of Na+ channel gating, but its effects on steady-state availability (h infinity), recovery from inactivation, and the timing of onset and possible reversibility, have not been characterized. We studied Na current (INa) by the whole-cell patch clamp technique on isolated rat ventricular myocytes at 22 degrees C with reduced Na+ (45 mM out, 5 mM in) from a holding potential of -150 mV. Changes in the electrophysiological parameters were measured after LPC 10 microM was added to the bath and compared to time controls (TC) taken from the time of seal formation. LPC decreased peak current for a test potential to -30 mV by about 20%. The peak current voltage relationship shifted in a positive direction by about 5 mV after LPC as compared to a small 2 mV negative shift in TC cells. LPC shifted the steady-state availability curve in the hyperpolarizing direction by about 6 mV. LPC perfusion caused a slowing of the decay of INa, and also a slowing of recovery from inactivation. Onset of the effects occurred within 6 min after adding LPC to the bath and were statistically significant with respect to TC cells between 12 and 16 min. In three cells, some of the effects on INa were either arrested or partially reversed by washout and cell survival was less than 20 min if LPC was not removed from the bath. These LPC induced changes in INa would tend to slow conduction and increase refractoriness, effects also seen in acutely ischemic myocardium. We therefore conclude that LPC action on INa may potentiate the arrhythmogenic substrate and that the onset of these changes are sufficiently rapid to play a role in the electrical instability of acute ischemia.

Slowly recovering cardiac sodium current in rat ventricular myocytes: effects of conditioning duration and recovery potential.

INTRODUCTION: Recovery of the Na channel from inactivation is essential to the normal conduction and refractoriness of the myocardium. In addition to fast recovery, occurring within several milliseconds at hyperpolarized potentials, a component of the current exhibits slow recovery occurring over hundreds of milliseconds. Long conditioning depolarizations potentiate slow recovery. METHODS AND RESULTS: This study was designed to test conditioning durations (tc) between 0.25 and 4 seconds (s) as to whether recovery was slowed by an effect on the fast (tau f) and slow (tau s) time constants of recovery, the relative amplitude of the slow component (As), or both. We studied Na channel recovery at -150 mV from inactivation using whole cell voltage clamp of rat ventricular cells at 23 degrees C using a two-pulse recovery protocol. Longer conditioning durations dramatically increased A2 (from 12% for tc = 500 msec to 37% for tc = 4000 msec, P < 0.01). Neither tau f (6 vs 5 msec) nor tau s (115 vs 140 msec) were significantly affected. In a second set of experiments, the recovery potential was depolarized to a potential at which the sodium current was 70% available (approximately equal to - 105 mV). This recovery potential had no significant effect on A2, but both tau f and tau s were significantly slower (e.g., at tc = 2 s, tau s = 147 msec and As = 28% at Vr = - 150 mV, and tau s = 456 msec and As = 29% at Vr approximately equal to - 105 mV). In addition, a 1- to 2-msec lag in the onset of recovery was prominent at the depolarized recovery potentials. CONCLUSIONS: Our results support a model for slow recovery where conditioning duration determines entry into an inactivated state from which Na channels recover slowly, and recovery potential determines the rate of recovery from this state. A kinetic scheme with at least three inactivated states is proposed. These results also have implications for cardiac excitability under conditions, such as ischemia, where membranes are depolarized.

Cytoskeleton modulates gating of voltage-dependent sodium channel in heart.

To investigate the role of the cytoskeleton in cardiac Na+ channel gating, the action of cytochalasin D (Cyto-D), an agent that interferes with actin polymerization, was studied by whole cell voltage clamp and cell-attached and inside-out patches from rat and rabbit ventricular cardiac myocytes. Cyto-D (20-40 microM) reduced whole cell peak Na+ current by 20% within 12 min and slowed current decay without affecting steady-state voltage-dependent availability or recovery from inactivation. Brief treatments (< 10-15 min) of cell-attached patches by Cyto-D (20 microM) in the bath induced short bursts of Na+ channel openings and prolonged decays of ensemble-averaged currents. Bursting of the Na+ channel was more pronounced when the cell suspension was pretreated with Cyto-D (20 microM) for 1 h before seal formation. Application of Cyto-D on the cytoplasmic side of inside-out patches resulted in more dramatic gating changes. Peak open probability was reduced by > 50% within 20 min, and long bursts of openings occurred. Washout of Cyto-D did not restore ensemble-averaged current amplitude, but burst duration decreased toward control values. Cyto-D also induced an additional slower component to open and closed times. These results suggest that Cyto-D, through effects on cytoskeleton, induced cardiac Na+ channels to enter a mode characterized by a lower peak open probability but a greater persistent activity as if the inactivation rate was slowed. The cytoskeleton, in addition to localizing integral membrane proteins, apparently also plays a role in regulating specific detailed functions of integral membrane proteins such as the gating of Na+ channels.