Faculty Bibliography

We have previously shown that whole-cell L-type Ca2+ current that was stimulated through beta-adrenergic receptors was negatively modulated by alpha 1-adrenergic activation. In the present study, we investigated the kinetic basis of this modulation at the single-channel level in adult rat ventricular myocytes using Ba2+ as the charge carrier. Unitary current sweeps were evoked by 300-ms depolarizing pulses to 0 mV, from a holding potential of -50 mV at 0.5 Hz. During control conditions, the ensemble-averaged current amplitude was 0.18 +/- 0.01 pA (n = 7). To achieve beta-adrenergic stimulation (beta effect), cells were superfused with norepinephrine (10 mumol/L) in the presence of prazosin (10 mumol/L), an alpha 1-adrenergic blocker. beta-adrenergic stimulation enhanced ensemble-averaged current (from 0.18 +/- 0.01 to 0.75 +/- 0.04 pA, P < .05, n = 7), increased the open-time constants, and decreased the closed-time constants. To activate alpha 1-receptors while maintaining the beta-adrenergic stimulation, cells were superfused with norepinephrine alone (alpha 1 + beta effects). alpha 1-adrenergic activation reduced ensemble-averaged current (from 0.75 +/- 0.04 to 0.41 +/- 0.03 pA, P < .05, n = 7), decreased open-time constants, and increased closed-time constants. alpha 1-adrenergic activation also inhibited ensemble-averaged currents stimulated by a low concentration (10 mumol/L) of 8-bromo-cAMP but not by (-)Bay K 8644 (1 mumol/L). Calphostin C (1 mumol/L), a specific inhibitor of protein kinase C, attenuated alpha 1-adrenergic inhibition on beta-adrenergic-stimulated unitary currents. We conclude that alpha 1-adrenergic activation exerts an inhibitory effect on beta-adrenergic-stimulated unitary Ba2+ current at the single-channel level. The shortening of the open-time and the lengthening of the closed-time constants and the increase in blank sweeps may explain the inhibition of the Ca(2+)-channel activity and the reduction in whole-cell Ca2+ current previously reported. This inhibition is in part mediated through the protein kinase C pathway.

INTRODUCTION/BACKGROUND:Cocaine has been shown to have broad cardiovascular effects that could be life threatening. Most of the reported electrophysiologic effects of cocaine have been studied in normal but not infarcted myocardium. METHODS AND RESULTS/RESULTS:Using microelectrode techniques, we investigated the electrophysiologic effects of cocaine on endocardial canine Purkinje fibers that survived 1 day of myocardial infarction. In quiescent infarcted preparations, stimulated trains were followed by subthreshold delayed afterdepolarizations (DADs), in the presence of propranolol (1 microM). Cocaine (10 microM) decreased the amplitude of DADs from 6.1 +/- 1.8 mV to 3.0 +/- 1.3 mV (P < 0.05, n = 6). When stimulated preparations (n = 23) showing no triggered activity during control (+propranolol) were superfused with a low concentration of caffeine (1 mM) or high extracellular Ca2+ (8.1 mM), triggered activity was induced. Subsequent cocaine (10 microM) superfusion prevented the induction of caffeine- and high Ca(2+)-induced triggered activity. Cocaine's effects were reversible upon washout. In preparations that showed triggered activity during control conditions (+propranolol), the mean cycle length of triggered activity was 755 +/- 45 msec. Cocaine (10 microM) superfusion lengthened the cycle length to 1030 +/- 141 msec and terminated triggered activity with a subthreshold DAD (n = 12). In addition, cocaine and ryanodine (10 microM) suppressed triggered activity in a similar manner when tested in the same preparations (n = 4). During control conditions, cocaine did not cause any significant change on the rate of rise of action potential upstroke (from 55.6 +/- 24.3 to 54.5 +/- 28.6 V/sec, n = 8) and maximum diastolic potential (from -58.4 +/- 4.3 to -56.6 +/- 6.5 mV, n = 8). In the absence of propranolol, 50 microM but not 10 microM cocaine induced early afterdepolarizations in 62% of the preparations exhibiting triggered activity during control conditions. CONCLUSION/CONCLUSIONS:The results suggest that cocaine modulates DADs and triggered activity in infarcted endocardial fibers via direct inhibition of cyclic release of Ca2+ from sarcoplasmic reticulum (SR) independently from a local anesthetic or sympathomimetic effect. This SR inhibition could account for the myocardial depressant effect of cocaine. However, while cocaine suppressed DADs, its induction of EADs can precipitate malignant ventricular arrhythmias in the setting of cocaine overdose and infarction.

Ketanserin (KT), an antihypertensive agent, has been shown to prolong action potential duration (APD) and QT interval and to induce torsade de pointes in some patients. We previously suggested that the prolongation of APD could be due to KT inhibition of the fast component of the delayed rectifier current (IKr) in guinea-pig myocytes. However, in other tissue such as human atrium, Purkinje fibers, epicardial cells, and rat ventricular myocytes, the transient outward potassium current (Ito) is one of the major repolarizing currents. We investigated the possibility that KT could also increase APD by blocking Ito. Action potentials and membrane currents were recorded from rat ventricular myocytes known to have a large Ito by using whole-cell patch-clamp techniques. We found that KT (50 mumol/L) significantly prolonged APD at 50% repolarization by 218% (P < .05) and APD at 90% repolarization by 256% (P < .05) with no significant effect on other action potential parameters. Time-dependent Ito and sustained current (ISus) were measured in the presence of 400 nmol/L nisoldipine during depolarizing pulses to 40 mV from a holding potential of -100 mV every 10 seconds. KT resulted in a concentration- and time-dependent inhibition of charge area of Ito evaluated by integration with an EC50 of 8.3 mumol/L. The inhibitory effect of KT (10 mumol/L) was seen at voltages from 0 to 80 mV without any shift of the current-voltage relation of peak Ito. KT did not significantly change activation, inactivation, and reactivating curves of Ito. Kinetic analysis of Ito showed a biexponential fit of inactivation in 80.5% of total tracings studied at voltages between -30 and 80 mV (n = 149, R = .99 +/- .01). The inhibitory effect of KT was more prominent on charge areas of the slow component (Qs) than the fast component (Qf) of Ito (Qf = 33.2 +/- 6.2 s.pA and Qs = 235.5 +/- 7.4 s.pA for the control condition; 12.4 +/- 4.3 and 59.6 +/- 17 s.pA for KT at 40 mV; n = 4). The binding association (k) and dissociation (l) constants at 40 mV were 9.0 +/- 0.9x10(6) M-1.s-1 and 86.6 +/- 0.3 s-1, respectively. KT also blocked ISus in a dose-dependent manner with an EC50 of 11.2 mumol/L and had no significant effect on both the inward rectifier potassium current and the L-type calcium current.(ABSTRACT TRUNCATED AT 400 WORDS)

INTRODUCTION/BACKGROUND:Early afterdepolarizations (EADs) are among the mechanisms proposed to underlie ventricular arrhythmias. Sea anemone toxin, ATXII, known to delay Na inactivation and to induce plateau level voltage oscillations, was used to study the formation of EADs. METHODS AND RESULTS/RESULTS:Action potential and membrane currents were studied in rat ventricular myocytes using whole cell current and voltage clamp techniques. Phase plane trajectories were generated by plotting membrane potential (V) versus the first time derivative of membrane potential (dV/dt). Under current clamp conditions, ATXII (40 nM) consistently prolonged the action potential and induced EADs. The EADs developed at a plateau voltage between -10 and -40 mV. Calcium channel blockers, verapamil 10 microM and cobalt 4 mM, and the sarcoplasmic reticulum modulator, ryanodine (1 microM), did not antagonize ATXII effects on the action potential and EADs. However, Na channel blockers, tetrodotoxin 0.3 microM and lidocaine 40 microM, and rapid stimulation consistently shortened the prolonged action potential and suppressed EADs. Under voltage clamp conditions in the presence of ATXII, a slowly decaying inward current followed the fast inward current during depolarizing pulses. Membrane currents flowing at or later than 100 msec after the test pulse were analyzed. The control isochronal current-voltage (I-V) curves showed no late inward currents. In the presence of ATXII, all the isochronal I-V curves showed an inward current that was more prominent between -40 and 0 mV. The ATXII-induced current at the 100-msec isochrone activated at a potential of approximately -60 mV, peaked at about -20 mV, and reversed at +40 mV consistent with the Na current I-V curve. The isochronal I-V curves obtained after lidocaine superfusion resembled those of the control. The phase plane trajectory of the action potential obtained with ATXII showed an oscillatory behavior corresponding to the EAD range of potential; within this voltage range, the isochronal I-V curves were shown to cross the abscissa three times instead of once. CONCLUSION/CONCLUSIONS:These results suggest that, in this experimental model, neither sarcolemmal L-type Ca current nor sarcoplasmic reticulum Ca release plays a significant role in the genesis of ATXII-induced EADs. EADs are generated by a voltage-dependent balance between a markedly prolonged Na inward current and K outward currents within the voltage plateau range of the action potential but not by Ca current reactivation and inactivation.

OBJECTIVES/OBJECTIVE:This study was designed to determine the effects of glucose-insulin infusion on ischemia-induced changes in extracellular potassium ([K+]o) accumulation and the associated electrophysiologic abnormalities in the canine heart. BACKGROUND:Although glucose-insulin-potassium infusion has been shown to limit myocardial injury in acute ischemia, its effect on ischemia-induced electrophysiologic alterations has not been investigated. METHODS:Recordings of [K+]o and local electrograms from the normal, border and ischemic zones were obtained during serial (10-min) left anterior descending coronary artery occlusions in the control state and after infusion of glucose-insulin (eight dogs), glucose alone (six dogs) or insulin alone (eight dogs). RESULTS:Glucose-insulin infusion caused significant reduction in the rise of [K+]o during the entire period of ischemia in both ischemic and border zones associated with significant improvement in the degree of intramyocardial conduction delay. At 10 min of ischemia, [K+]o was reduced from a mean control level of 15.9 +/- 3.7 to 10.1 +/- 4.3 mmol/liter (p < 0.005) in the ischemic zone and from 6.8 +/- 1.9 to 5.5 +/- 1.1 mmol/liter (p < 0.05) in the border zone. The electrogram duration was shortened from a mean control value of 102 +/- 13 to 78 +/- 12 ms in the ischemic zone and from 79.2 +/- 7.8 to 58.1 +/- 6.6 ms in the border zone (p < 0.005). Glucose alone caused significant reduction in [K+]o during the initial 6 min of ischemia, only in the ischemic zone. Conversely, insulin caused no changes in [K+]o accumulation during ischemia. Neither glucose nor insulin alone had any effect on ischemia-induced intramyocardial conduction delay. CONCLUSIONS:The present study demonstrated that the combination of glucose and insulin is essential for the salutary effect of reducing [K+]o accumulation during ischemia and improving the associated intramyocardial conduction delay. It could be postulated that glucose in the presence of insulin increases the glycolytic flux, thereby providing adequate adenosine triphosphate for suppressing the cardiac adenosine triphosphate-sensitive potassium ion channels. The latter are, at least partially, responsible for the [K+]o rise in the early phase of ischemia. This study highlights the antiarrhythmic potential of interventions that modulate the metabolic consequences of ischemia.

We investigated the mechanism by which alpha 1-adrenergic activation regulates basal and stimulated whole cell L-type Ca current (ICa) in rat ventricular myocytes using the physiological neurotransmitter, norepinephrine (NE, 10 microM). Stimulation of alpha 1-adrenoceptors, achieved by NE + 10 microM esmolol (a beta-receptor antagonist), had no significant effect on basal ICa. alpha 1-adrenergic activation had a marked inhibitory effect on ICa elevated by beta activation (NE + 1 microM) prazosin, an alpha 1-receptor antagonist) or activation of adenylyl cyclase by forskolin (25 microM); the inhibitory effect was reversible upon washout. However, alpha 1-adrenergic stimulation had no significant effect on ICa previously increased by intracellular application of cAMP (25 microM). The inhibitory effect seen on ICa elevated by NE showed no significant shift of either I-V or inactivation curves. It is unlikely that the inhibitory effect of alpha 1-adrenergic stimulation on NE or forskolin-elevated ICa is mediated through activation of Ca-dependent protein kinase C or changes in intracellular free Ca (pCa = 8.5, EGTA 5 mM) or cAMP-dependent phosphodiesterase. We conclude that alpha 1-adrenergic inhibition of beta-adrenergic stimulated-ICa is probably mediated through an as yet unknown G-protein. This inhibitory effect could serve as a regulatory feedback mechanism in physiological and pathophysiological settings.