The incidence rates of long QT syndrome (LQTS) and drug-induced torsades de pointes (TDP) are higher in women than men. Although gonadal steroids are assumed to play an important role in the gender-based differences in cardiac electrophysiological properties, the underlying mechanisms of the gender-based differences are not fully understood. In particular I Kr, which comprises the repolarization phase of the action potential, has not been well understood in its modulation by sex hormones. To assess this, we examined the effects of the female sex hormone β-estradiol on the human ether-a-go-go-related gene (hERG)-encoded potassium current stably expressed in human embryonic kidney-293 (HEK) cells. We demonstrated that hERG currents were inhibited by β-estradiol maximally to 62%of control with an IC50 of 1.3 μM and a Hill coefficient of 0.87, which might account for the sex-related differences in LQTS. We also examined whether estrogen modulated drug-induced blocking effects on hERG currents or not. With simultaneous application of 10 μM erythromycin, which is known to block hERG currents but not in low doses, the blocking effects of β-estradiol on hERG currents were enhanced. Namely, hERG currents were inhibited maximally to 45.8%of control with an IC50 of 59 nM (P < 0.02) by β-estradiol with 10 μM erythromycin. We conclude here that a significant block of hERG currents by β-estradiol may account for the sex-related differences in LQTS and the synergic effects of β-estradiol and erythromycin indicate a higher risk of drug-induced TDP in women than men.

Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from total ICa activated from a holding potential (Vh) of -80 mV. L-type current (IL) was activated by depolarizing steps from Vh -30 or -40 mV. The difference current (IT) was obtained by subtracting IL from ICa. IT could also be distinguished pharmacologically from IL in these cells. IT was selectively blocked by 40-160 .mu.M Ni2+, whereas IL was suppressed by 1 .mu.M D600 or 2 .mu.M nifedipine. The Ni2+-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those of IT and IL defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells, IT and IL could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nontheless, in most NS cells, ICa was partly blocked by Ni2+ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separate IT and IL by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis.In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V1/2) of the Ni2+-resistant current IL averaged -18 mV. In contrast, V1/2 of the Ni2+-sensitive IT was -60 mV in 14-day cells, -52 mV in 7-day S cells, and -43 mV in 7-day NS cells. The half-activation potential was near -2 mV for IL at both ages, but that of IT was -38 mV in 14-day and -29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components of ICa is a negative shift with embryonic age in the activation and inactivation relationships of IT along the voltage axis.

T-type calcium channels (IT channels) were studied in cell-attached patch electrode recordings from the ventricular cell membrane of 14-day embryonic chick heart. All experiments were performed in the absence of Ca2+ with Na+ (120 mM) as the charge carrier. IT channels were distinguished from L-type calcium channels (IL) by their more negative activation and inactivation potential ranges; their smaller unitary slope conductance (26 pS), and their insensitivity to isoproterenol or D600. Inactivation kinetics were voltage dependent. The time constant of inactivation was 37 msec when the membrane potential was depolarized 40 mV from rest (R + 40 mV), and 20 msec at R + 60 mV. The frequency histogram of channel open times (.tau.o) was fit by a single-exponential curve while that of closed times (.tau.c) was bi-exponential. .tau.o was the same at R + 40 mV and R + 60 mV whereas .tau.c was shortened at R + 60 mV. The open-state probability (Po) increased with depolarization: 0.35 at R + 40 mV, 0.8 at R + 60 mV and 0.88 at R + 80 mV. This increase in Po at depolarized potentials could be accounted for by the decrease in .tau.c.

To clarify ionic mechanisms underlying successive changes in action potential repolarization upon sudden increase in driving rate or initiation of rapid drive after a rest, membrane potentials and currents were recorded from isolated rabbit ventricular myocytes using the suction-pipette whole-cell clamp method. When 20 action potentials were elicited with a stimulus frequency of 2.0 Hz after a rest period of 20 s, the plateau and action potential duration showed complex changes in successive beats, whereas the plateau and action potential duration showed complex changes in successive beats, whereas they were nearly constant with stimulation at 0.1 Hz. There were only weak correlations between changes in action potential parameters and preceding diastolic intervals. The changes were prominent in the first 10 beats but subsided gradually thereafter, attaining nearly steady configurations of action potentials. When depolarizing pulses were applied at a fast rate, under the voltage clamp, the amplitudes of the initial inward current in the presence of tetrodotoxin changed greatly depending on the pulse numbers and diastolic intervals, whereas the delayed outward K+ current changed little. Variations of the initial inward current in successive pulses were caused by different degrees of activation and recovery from inactivation in the Ca2+ current, the Ca2+ -sensitive and -insensitive transient outward current. While inhibition of either one or two current components decreased the action potential alterations, blocking the three components completely abolished them. These results indicate that alteractions of the Ca2+ -sensitive and -insensitive transient outward current together with the Ca2+ current contribute to the action potential alteractions after initiation of rapid drive or an increase in driving rates.

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. We have demonstrated spontaneous (Ca2+)i oscillations in hMSCs without agonist stimulation, which result primarily from release of Ca2+ from intracellular stores via InsP3 receptors. In this study, we further investigated functions and contributions of Ca2+ transporters on plasma membrane to generate (Ca2+)i oscillations. In confocal Ca2+ imaging experiments, spontaneous (Ca2+)i oscillations were observed in 193 of 280 hMSCs. The oscillations did not sustain in the Ca2+ free solution and were completely blocked by the application of 0.1 mM La3+. When plasma membrane Ca2+ pumps (PMCAs) were blocked with blockers, carboxyeosin or caloxin, (Ca2+)i oscillations were inhibited. Application of Ni2+ or KBR7943 to block Na+-Ca2+ exchanger (NCX) also inhibited (Ca2+)i oscillations. Using RT-PCR, mRNAs were detected for PMCA type IV and NCX, but not PMCA type II. In the patch clamp experiments, Ca2+ activated outward K+ currents (IKCa) with a conductance of 170+-21.6 pS could be recorded. The amplitudes of IKCa and membrane potential (Vm) periodically fluctuated liked to (Ca2+)i oscillations. These results suggest that in undifferentiated hMSCs both Ca2+ entry through plasma membrane and Ca2+ extrusion via PMCAs and NCXs play important roles for (Ca2+)i oscillations, which modulate the activities of IKCa to produce the fluctuation of Vm.

Osmotic pressure and ion movements in and out of the vitreous space after open-sky vitrectomy were studied in the in vivo eyecup of rabbits. The osmotic pressure of several hypo-osmotic solutions rose over a period of time by the penetration of ions from the surrounding tissue; increases in the concentration of sodium, chloride, potassium, and calcium were documented. Appearance and disappearance of ions were independent of the total osmolarity and depended on the concentration gradient for each ion.

Action potentials and membrane currents were recorded from isolated single ventricular cells from rabbit hearts using the suction pipette whole-cell clamp method. Action potentials elicited after short diastolic intervals of less than 2 seconds showed an increase and prolongation of the plateau compared to those elicited after a 10-second rest period. The recovery of the tetrodotoxin-insensitive secondary inward current revealed a transient increase at short diastolic intervals above the level of full recovery (after 10 seconds). The increased secondary inward current recovery, however, was voltage-dependent, and the period of its increase did not cover the entire diastolic intervals of the action potential overshoots, suggesting the contribution of another ionic current to the changes in potential. During depolarizing voltage steps, from + to -20 mV, a rapid activating and then inactivating outward current was elicited, which overlapped the calcium current. This outward current exhibited time- and voltage-dependent properties similar to those of the transient outward current in Purkinje and other cardiac preparations. The recovery of the transient outward current was slow, achieving only 75% of its full level at 2 seconds, whereas the same level of calcium current recovery was achieved at 200 milliseconds. The application of 4-aminopyridine suppressed most of the transient outward current, and the rest of the current was abolished by caffeine or Co2+. The 4-aminopyridine sensitive transient outward current exhibited slow recovery kinetics compared to those of the other or calcium current, and its inhibition caused elimination of the augmented plateau during electrical restitution. The application of verapamil or Co2+ for inhibition of secondary inward current also abolished the action potential overshoot. These results indicate that an increase and prolongation of the plateau at short diastolic intervals are produced by the slower recovery from inactivation in the 4-aminopyridine-sensitive transient outward current than that in the calcium current.

BACKGROUND Homer, known as a scaffolding protein that regulates postsynapse signaling in neurons, has been poorly explored in cardiac research. We show the fundamental properties of Homer 1 in mouse heart in association with cardiac ryanodine receptor (RyR), a binding protein of Homer 1. METHODS AND RESULTS Immunohistochemistry of adult mouse heart with Homer 1 antibody showed striated staining on Z-bands both in atria and ventricles. The interactions between Homer 1 and RyR were confirmed by co-immunoprecipitation assays. Immunostaining of adult isolated cardiomyocytes showed partial co-localization of both proteins. In neonatal primary cultures, targeting of Homer 1 preceded that of RyR in their Z-band arrangement. CONCLUSIONS Homer 1 binds to RyR in adult mouse heart and precedes RyR in Z-band arrangement in the early postnatal period.