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Background: Recent clinical and experimental data suggest that testosterone may protect males against the deleterious effects of repolarization-prolonging drugs. This study tests the hypothesis that 5alpha-dihydrotestosterone (DHT) protects normal females against drug-induced excessive prolongation of repolarization., Methods and Results: We used microelectrode techniques to study isolated preparations of rabbit ventricular endocardium from age-matched normal control female rabbits and female rabbits treated with DHT for 4 weeks. Serum 17beta-estradiol levels were identical in the control and DHT-treated animals, whereas DHT levels were high (equaling those in normal males) only in the DHT-treated animals. Basal action potential duration to 90% repolarization (APD90) was significantly shorter in DHT-treated (155+/-7.4 ms, n=32) than control females (178+/-6.7 ms, n=29; P<0.05) at cycle length=1000 ms. The increase in APD90 induced by 10(-8) mol/L dofetilide at cycle length=1000 ms was significantly less in DHT-treated females than normal females (DeltaAPD90=8+/-7 and 29+/-5 ms, respectively, P<0.05). At 10(-6) mol/L dofetilide, the incidence of early afterdepolarizations was 28% in DHT-treated and 55% in normal female rabbits (P<0.05)., Conclusions: Elevating DHT levels diminishes the effects of dofetilide to increase APD and induce early afterdepolarizations in females. Moreover, treatment of females with DHT results in prolongation of APD and an incidence of early afterdepolarization equal to values previously reported by us for dofetilide-treated normal males. That serum levels of 17beta-estradiol were the same in DHT-treated and untreated females suggests that estradiol is not involved in the response to dofetilide. Thus, these data suggest that DHT and perhaps other androgenic hormones may protect normal females against the risk of dofetilide-induced arrhythmia.

Background: Regional function in stunned myocardium is usually thought to be more depressed in the endocardium than the epicardium. This has been attributed to the greater loss of blood flow at the endocardium during ischemia., Methods and Results: We measured transmural distributions of 3D systolic strains relative to local myofiber axes in open-chest anesthetized dogs before 15 minutes of left anterior descending coronary artery occlusion and during 2 hours of reperfusion. During ischemia, regional myocardial blood flow was reduced 84% at the endocardium and 32% at the epicardium (P<0.005, n=7), but changes in end-systolic fiber length from baseline were transmurally uniform. Relative to baseline, radial segments in stunned tissue were significantly thinner at the endocardium than the epicardium at end systole (24+/-5% versus 16+/-3%; P<0.05, n=8), consistent with previous reports. Unlike radial and cross-fiber segments, however, the increase of end-systolic fiber lengths in stunned myocardium had no significant transmural gradient (23+/-8% epicardium versus 21+/-4% endocardium). We also observed significant 3D diastolic dysfunction in the ischemic-reperfused region transmurally., Conclusions: Myocardial ischemia/reperfusion in the dog results in a significant transmural gradient of dysfunction between epicardial and endocardial layers in radial and cross-fiber segments, but not for fiber segments, despite a gradient in blood flow reduction during ischemia. Perhaps systolic fiber dysfunction rather than the degree of perfusion deficit during the preceding ischemic period may be the main determinant of myocardial dysfunction during reperfusion.

Background: Mechanisms for longer rate-corrected QT intervals and higher incidences of drug-induced torsade de pointes in women than in men are incompletely defined, although gonadal steroids are assumed to be important determinants of these differences., Methods and Results: We used microelectrode techniques to study isolated rabbit right ventricular endocardium from control male and female and castrated male (ORCH) and female (OVX) rabbits. Action potential duration to 30% repolarization (APD(30)) was significantly shorter in male than female and in ORCH than OVX at a cycle length of 500 ms. The I(Ks) blocker chromanol 293B had no effect on APD in males or females. The I(Kr) blocker dofetilide prolonged APD in female and ORCH more than in male and OVX. At 10(-)(6) mol/L dofetilide (cycle length=1 second), the incidence of early afterdepolarizations was: female, 67%; ORCH, 56%; male, 40%; and OVX, 28%. Serum 17beta-estradiol levels were unrelated to the effects of dofetilide, but as testosterone levels increased, the dofetilide effect to increase APD diminished, as did early afterdepolarization incidence., Conclusions: Sex-related differences in basal right ventricular endocardial AP configuration persist in castrated rabbits, suggesting that extragonadal factors contribute to the differences in ventricular repolarization. In this model, drugs that block I(Kr) but not I(Ks) prolong repolarization in a way that suggests that protection from excess prolongation in males is attributable to testosterone, whereas the risk of excess prolongation of repolarization in females is related to sex-determined factors in addition to estrogen.

Background: The antiarrhythmic action of quinidine is associated with a slowing of conduction and prolongation of repolarization. The latter effect has no consistent correlation with quinidine actions on action potential duration (APD) in isolated tissue experiments. To enhance our understanding of the mechanisms of quinidine action, we studied its effect on APD in canine epicardial, midmyocardial, and endocardial tissues., Methods and Results: Standard microelectrode techniques were used to study the effects of quinidine 2.5 to 20 micromol/L on APD in ventricular epicardial, endocardial, and transmural (M-cell) slabs at cycle lengths (CLs) from 300 to 4000 ms. Qualitatively different time courses of actions and concentration- and rate-dependent effects were seen in M cells compared with the others. In endocardium and epicardium, quinidine induced monotonic and concentration-dependent APD prolongation at all CLs. In contrast, the effects of quinidine in M cells varied from prolongation to shortening, depending on duration of superfusion, concentration, and CL. Experiments with E4031 and TTX suggested that in M cells, quinidine-induced APD lengthening was attributable to block of delayed rectifier potassium current and APD shortening was due to inhibition of TTX-sensitive steady-state sodium current., Conclusions: In vitro, there is a significant difference of quinidine effects in M cells versus epicardial and endocardial cells that appears to reflect differences in the contributions of specific ion channels to the APD at the three sites. The differences may influence the actions of quinidine on repolarization of the heart in situ and determine both the proarrhythmic and antiarrhythmic actions of the drug.

Background: In the companion article, we report a significant difference in quinidine effects on the action potential duration between surface (epicardial and endocardial) cells and midmyocardial cells (M cells) of canine left ventricle in vitro. This article considers two questions raised by the previous study: (1) Are the complex quinidine effects in vitro reflected in its actions on the heart in situ? (2) What are the cellular determinants of quinidine effects on QT interval in ECG?, Methods and Results: We used plunge and surface electrodes to measure activation-recovery intervals (ARIs) of bipolar electrograms obtained from epicardium, endocardium, and midmyocardium (3, 5, and 9 mm from epicardium) of canine left ventricle in conditions of AV block and right ventricular pacing. Quinidine was infused continuously; its plasma level increased from 1.6+/-0.1 microg/mL at 30 minutes to 7.6+/-0.7 microg/mL at 180 minutes. At cycle lengths (CLs) from 300 to 1500 ms, there was no ARI gradient across the ventricular wall before and during quinidine infusion. At a CL of 300 ms, therapeutic concentrations of quinidine prolonged ARIs and QT intervals. At a CL of 1500 ms, ARIs were significantly prolonged at low quinidine concentrations. With an increase of quinidine concentration, this effect subsided and disappeared., Conclusions: In situ, quinidine-induced prolongation of repolarization is uniform in all myocardial layers and follows the pattern observed in M cells in vitro. The ability of quinidine in therapeutic concentrations to prolong repolarization at rapid heart rates can contribute to its antiarrhythmic efficacy.

Background: Because of the marked difference in the incidence and severity of cardiovascular diseases between men and premenopausal women, several groups have studied the effect of sex steroids, particularly estrogen, on vascular endothelial prostacyclin (PGI2) release. No previous studies have addressed the effect of estrogen on endocardial endothelial cells (EECs), which are involved in the modulation of the myocardium and potentially in downstream pulmonary and systemic vascular tone. Furthermore, all previous studies of estrogen effects on cultured endothelial cell function have used cells grown under standard static cell culture conditions, thereby ignoring the contribution of flow, the ubiquitous environmental endothelial stimulus., Methods and Results: The effect of 17 beta-estradiol pretreatment (100 ng/mL, 72 hours) on cultured sheep EEC PGI2 release in response to multiple physiologically relevant stimuli was studied. EECs were grown in six-well plates (static conditions) or on microcarrier beads and perfused at a constant flow with normoxic (PO2 = 150 mm Hg, PCO2 = 35 mm Hg) or hypoxic (PO2 = 35 mm Hg, PCO2 = 35 mm Hg) Krebs solution. The stable metabolite of PGI2, 6-keto-PGF1 alpha, was determined in samples from both static and perfusion experiments by direct radioimmunoassay. 17 beta-Estradiol pretreatment did not alter basal or stimulated (arachidonic acid, 1 mumol/L, 10 mumol/L; A23187, 10 mumol/L; and bradykinin, 1 mumol/L) PGI2 release in static conditions. Untreated and acutely treated (100 ng/mL added to perfusate) EECs responded to flow with a time-dependent increase in PGI2 release that plateaued between 60 and 100 minutes. In contrast, 17 beta-estradiol-pretreated, perfused EECs did not increase PGI2 release over time. During perfusion, acute hypoxia increased PGI2 release: 140 +/- 65 (normoxia) to 296 +/- 113 pg (hypoxia) 6-keto-PGF1 alpha/mg per minute. 17 beta-Estradiol inhibited hypoxia-induced PGI2 release: 296 +/- 113 pg (untreated EECs, hypoxia) versus 159 +/- 60 pg (17 beta-estradiol pretreated, hypoxia) 6-keto-PGF1 alpha/mg per minute., Conclusions: This study demonstrates for the first time an inhibitory effect of 17 beta-estradiol on flow- and acute hypoxia-induced increase in PGI2 release from perfused EECs in the absence of any effect on pharmacologically stimulated PGI2 release from static cultures. These effects of 17 beta-estradiol may explain in part the well-recognized gender and estrogen effects in cardiovascular diseases and highlight the importance of flow in studies of endothelial cell function.

Background: Pinacidil is known to augment a time-independent outward current in cardiac tissues by activating the ATP-regulated potassium channels. Activation of this current, IK-ATP, is thought to be responsible for increased potassium permeability in ischemia. The contribution of IK-ATP activation to arrhythmogenesis and the role of activation of this current in suppression of arrhythmias are areas of great interest and debate. Because electrical depression attending myocardial ischemia is more accentuated in ventricular epicardium than in endocardium, we endeavored to contrast the effects of pinacidil-induced IK-ATP activation on the electrophysiology of canine ventricular epicardium and endocardium., Methods and Results: Standard microelectrode techniques were used. Pinacidil (1 to 5 mumol/L) produced a marked dispersion of repolarization and refractoriness in isolated canine ventricular epicardium as well as between epicardium and endocardium. In endocardium, pinacidil abbreviated action potential duration (APD90) and refractoriness by 8.0 +/- 2.3%. In epicardium, the effects of pinacidil were nonhomogeneous. At some sites, pinacidil induced an all-or-none repolarization at the end of phase 1 of the action potential, resulting in 55.5 +/- 8.7% abbreviation of APD90 and refractoriness. Adjacent to these were sites at which the dome was maintained with only minor changes in APD and refractoriness. Extrasystolic activity displaying features of reentry was observed in isolated sheets of epicardium (63.2%) after exposure to pinacidil (1 to 5 mumol/L) but never in its absence. Dispersion of repolarization and ectopic activity was most readily induced in epicardium by a slowing of the stimulation rate in the presence of pinacidil. Electrical homogeneity was restored and arrhythmias abolished after washout of pinacidil or addition of either a transient outward current blocker, 4-aminopyridine, or a blocker of the ATP-regulated potassium channels, glybenclamide., Conclusions: Our data suggest that the activation of IK-ATP can produce a marked dispersion of repolarization and refractoriness in epicardium as well as between epicardium and endocardium, leading to the development of extrasystolic activity via a mechanism that we have called phase 2 reentry. The available data also suggest that blockade of the transient outward current and/or the ATP-regulated potassium channels may be useful antiarrhythmic interventions under ischemic or "ATP depleted" conditions.

Background: We recently reported that sodium channel block can produce opposite effects on action potential duration (APD) and refractoriness in epicardial versus endocardial tissues of the canine ventricle. In addition, strong sodium channel current inhibition was found to cause loss of the action potential dome in epicardium but not endocardium, thus inducing a marked dispersion of repolarization and refractoriness between epicardium and endocardium as well as among neighboring epicardial sites. The marked heterogeneity that evolves under these conditions provides a substrate for the development of arrhythmias. Flecainide was found to induce extrasystolic activity more readily than other sodium blockers. The present study contrasts the electrophysiological actions of flecainide in canine ventricular epicardium and endocardium and examines the characteristics of flecainide-induced arrhythmias in epicardial sheets of canine ventricle., Methods and Results: Standard microelectrode techniques were used. Flecainide (10-20 microM) produced either prolongation or marked abbreviation of APD in epicardium but only minor changes in the APD of endocardium. Marked abbreviation of APD in epicardium was due to loss of the action potential dome (plateau phase). Arrhythmias displaying characteristics of reentry could be readily induced in flecainide-treated preparations either by increasing the stimulation rate or by introduction of extrastimuli. Flecainide-induced slowing of conduction, more accentuated at the faster stimulation rates, appeared to act synergistically with the drug-induced dispersion of repolarization to generate reentry in these relatively small sheets of epicardium. 4-Aminopyridine, a transient outward current (Ito) blocker, reversed the flecainide-induced marked abbreviation of APD in epicardium and abolished reentrant activity in all cases. Flecainide failed to induce reentry in preparations pretreated with 4-aminopyridine., Conclusions: Our data suggest that the presence of a prominent Ito in epicardium contributes the development of marked electrical heterogeneity in the ventricle after exposure to flecainide. Flecainide-induced dispersion of repolarization, especially when accompanied by prominent conduction delays, results in extrasystolic activity via a mechanism that we have termed "phase 2 reentry." Our results also suggest a role for Ito blockers in the treatment of reentrant arrhythmias.

We developed methods for identifying beta-adrenergic receptors in human right ventricular endomyocardial biopsy tissue with the radioligand (-)[125I]iodocyanopindolol (ICYP). Specific ICYP binding in a crude, high-yield membrane preparation derived from endomyocardial biopsy tissue was high (specificity greater than 90%), of high affinity (KD around 20 pM), saturable and stereospecific for the (-) vs the (+) isomer of isoproterenol. Subjects with mild-moderate and severe biventricular dysfunction had respective decreases in beta-adrenergic receptor density of 38.2% and 57.7% when normalization methods were averaged, with no significant differences in ICYP dissociation constant. A subgroup of subjects was subdivided by left ventricular ejection fraction (LVEF) into those with mild cardiac dysfunction (LVEF less than 0.50 greater than 0.40) and severe heart failure (LVEF less than 0.20) and given graded sequential infusions of dobutamine and calcium gluconate. Those with severe cardiac dysfunction had marked impairment of the dobutamine dP/dt and stroke work index response, whereas these responses to calcium did not differ in the two groups. These data indicate that in the intact human heart endomyocardial biopsy may be used for direct analysis of beta-adrenergic receptors, heart failure-associated myocardial beta-adrenergic down-regulation begins with mild-moderate ventricular dysfunction, reduction in myocardial beta-receptor density is related to degree of heart failure, and beta-receptor down-regulation is associated with pharmacologically specific impairment of the beta-agonist-mediated contractile response.