K(+) currents responsible for repolarization in mouse ventricle and their modulation by FK-506 and rapamycin.

Modulation of mouse ventricular action potentials and K(+) currents was examined using the whole cell patch-clamp technique. The composite mouse ventricular K(+) current (consisted of an outward transient followed by a slowly decaying sustained component. Use of the K(+) channel blockers tetraethylammonium and 4-aminopyridine and a transgenic mouse model revealed three pharmacologically and kinetically distinct currents: I(to), which contributed to the transient component; I(K), which contributed to the sustained component; and a slowly activating current (I(slow)), which contributed to both components. The immunosuppressant FK-506 increased action potential duration at 90% repolarization by 66.7% by decreasing the sustained component (-48% at +60 mV) and prolonging recovery from inactivation (by 26% at 200 ms) of the transient component. These effects were isolated to I(K) and I(to), respectively. Rapamycin had strikingly similar effects on these currents. Both FK-506 and rapamycin are known to target the immunophilin FKBP12. Thus we conclude that FKBP12 modulates specific mouse K(+) channels, and thus the mouse ventricular action potential, by interacting directly with K(+) channel proteins or with other associated regulatory proteins.

Independent inhibition of calcineurin and K+ currents by the immunosuppressant FK-506 in rat ventricle.

FK-506 increases the cytosolic Ca2+ concentration transient in rat ventricular myocytes by prolonging the action potential through inhibition of the K+ currents Ito and IK [J. Physiol. (Lond.) 501: 509-516, 1997]. Physiological and biochemical techniques were used in parallel to examine the electrophysiological mechanisms and the role of calcineurin inhibition in these effects. FK-506 prolonged the recovery of Ito from inactivation. Thus Ito inhibition was frequency dependent, with no decrease at 0.2 Hz (recorded at +50 mV from -70 mV) but a 40% decrease at 2.0 Hz. In contrast, inhibition of IK ( approximately 60%) was time and voltage independent. At 25 microM, FK-506 (by 65%) and cyclosporin A (by 57%) inhibited calcineurin activity in myocyte extracts. However, only FK-506 increased the cytosolic Ca2+ concentration transient in field-stimulated myocytes. Furthermore, FK-506 was still active on K+ currents when cells were dialyzed with 10 mM EGTA. These results demonstrate that calcineurin inhibition is not responsible for the functional effects of FK-506 in heart and suggest that IK and Ito are modulated by FK-506-binding proteins or directly by FK-506.

Effect of the immunosupressant FK506 on excitation-contraction coupling and outward K+ currents in rat ventricular myocytes.

1. We examined the effects of the immunosupressant drug FK506 on excitation-contraction coupling in isolated rat ventricular myocytes. [Ca2+]i transients were recorded using the intracellular Ca2+ indicators fluo-3 and indo-1 while action potentials (APs) or membrane currents were recorded using patch-type microelectrodes in the whole cell mode. 2. FK506 (25 microM) rapidly and reversibly increased the magnitude of the [Ca2+]i transient in intact cells without changing resting [Ca2+]i or the kinetics of the [Ca2+]i transient, a finding consistent with previous reports that investigated the actions of FK506 on the sarcoplasmic reticulum Ca2+ release channel. 3. The 36% increase in the [Ca2+]i transient produced by FK506 was accompanied by a 293% increase in AP duration (by 293%). Importantly, the addition of FK506 had no effect on the [Ca2+]i transient when the depolarizing duration was controlled in voltage clamp experiments. The increased AP duration could be explained by a marked inward shift in the net membrane current that was observed in these experiments. 4. The net inward current change was not directly responsible for a change in Ca2+ influx, since no change in L-type Ca2+ current (ICa) was observed. Instead, FK506 inhibited both the transient outward K+ current (Ito) and the delayed rectifier K+ current (IK). 5. We conclude that FK506 increases the [Ca2+]i transient during normal contractions by an indirect action: it prolongs the action potential. This action does not appear to depend on the established action of FK506 on the ryanodine receptor. Instead, the inhibition of outward K+ currents prolongs the AP which secondarily increases Ca2+ influx and/or decreases Ca2+ efflux.

Initial contractile response of isolated rat heart cells to halothane, enflurane, and isoflurane.

BACKGROUND: In several beating cardiac muscle preparations, a short-lived increase in twitch tension or amplitude has been observed when they were exposed abruptly to solutions containing halothane or enflurane. As exposure to the anesthetics was continued, the expected negative inotropic effect became evident after the short-lived increase in twitch. No such increase in twitch has been reported during exposure to isoflurane. It has been hypothesized that this short-lived increase in twitch is caused by an enhancement of calcium release from the sarcoplasmic reticulum, but other mechanisms have not been excluded. METHODS: Freshly isolated, single rat ventricular cells were stimulated to beat at room temperature and abruptly exposed to solutions containing halothane (0.25-0.64 mM), enflurane (0.69-1 mM), or isoflurane (0.31-0.54 mM). During these exposures, twitch amplitude was measured and intracellular calcium concentration was followed using the calcium-sensitive dye indo-1. In some experiments, the whole-cell patch-clamp technique was used to measure membrane current. In addition, in several cells the sarcoplasmic reticulum calcium content was assessed through the response to brief pulses of caffeine. RESULTS: Both the twitch amplitude and the intracellular calcium transient were increased temporarily in cells abruptly exposed to halothane or enflurane. No such behavior was found with isoflurane. After continued exposure to all three agents, both the twitch amplitude and the calcium transient were less than control. During the beats exhibiting an increase in twitch, no alteration in the relation between cell length (twitch amplitude) and the intracellular calcium transient was found compared with control conditions. In addition, the temporary increase in twitch amplitude occurred in cells contracting under voltage-clamp control when halothane was introduced, and it was not associated with any increase in the calcium current. The sarcoplasmic reticulum calcium content at the time of the halothane-induced increase in twitch also was not increased. CONCLUSIONS: The short-lived increase in twitch after abrupt exposure to halothane or enflurane is related to increased intracellular calcium during the beat and not to any changes in myofilament sensitivity to calcium. Because these results eliminate most alternative explanations for this phenomenon, the authors conclude that halothane, and probably also enflurane, increases the fraction of calcium released from the sarcoplasmic reticulum with each heart beat. Isoflurane appears to lack this action.

Dynamic modulation of excitation-contraction coupling by protein phosphatases in rat ventricular myocytes.

1. The effects of the serine/threonine protein phosphatases (PP) type 1 and 2A on L-type Ca2+ current (ICa) and the intracellular [Ca2+]i transient were examined in rat ventricular myocytes. ICa was measured under voltage clamp using patch-type microelectrodes in the whole-cell mode with the cells in a steady state of sarcoplasmic reticulum (SR) Ca2+ loading. [Ca2+]i transients were measured simultaneously using the fluorescent Ca2+ indicator indo-1 (50 microM) which was added to the pipette filling solution along with PP-1 or PP-2A (4 units ml-1). 2. PP-1 had no effect on the ICa-V relationship but decreased the [Ca2+]i-voltage relationship (by 43% at 0 mV). PP-2A decreased both ICa-V (by 26% at 0 mV) and the [Ca2+]i transient-voltage (by 65% at 0 mV). Excitation-contraction coupling gain, defined as (delta [Ca2+]i/ICa), was decreased to 43% of control by PP-1 and to 29% of control by PP-2A at-28 mV. 3. Diastolic [Ca2+]i (i.e.[Ca2+]i measured immediately before each voltage clamp pulse) was not altered by PP-1 or PP-2A and neither phosphatase changed steady-state SR Ca2+ content, as measured with caffeine. 4. We conclude that the reduced [Ca2+]i transient following the application of PP-1 was due to reduced SR Ca2+ release channel activity. The effects of PP-2A, while more broadly based, were still consistent with a decrease in SR Ca2+ release channel activity. 5. Our experiments, combined with recent experiments by others, suggest that the basal state of contractility in heart is dynamically regulated by dephosphorylation and phosphorylation of the SR Ca2+ release channel.

Alternative splicing of the Na(+)-Ca2+ exchanger gene, NCX1.

We describe an analysis of the NCX1 gene and show that various tissues express different alternatively spliced forms of the gene. Alternative splicing has been confirmed by the genomic analysis of the Na(+)-Ca2+ exchanger gene. We also describe the Drosophila Na(+)-Ca2+ exchanger as having many of the same structural characteristics of the mammalian exchangers and this locus as possibly undergoing alternative splicing in the same region that has been described in the NCX1 gene. The general structure of the exchangers is similar to that of the alpha-subunit of the (Na(+)+ K+)-A Pase. Finally, sequence comparison of the various molecules demonstrates that structural characteristics of these molecules are more strongly conserved than the primary sequence of these products.

Repletion of sarcoplasmic reticulum Ca after ryanodine in rat ventricular myocytes.

The ryanodine (R)-induced loss of sarcoplasmic reticulum (SR) Ca2+ and the abilities of the SR to accumulate Ca2+ and participate in contractile activation after R were studied in rat ventricular myocytes. Indo 1 fluorescence (IF) indexed cytosolic Ca2+, and caffeine assayed SR Ca2+. Before R, there was a negative staircase, and the SR accumulated Ca2+ at rest. During stimulation (0.5 Hz), R decreased IF and contraction, converting the negative staircase to positive. When R was pulsed onto resting cells, IF increased and cells shortened, subsequently behaving as if stimulated in R. After R, there was no caffeine-releasable Ca2+ at rest, and little accumulated during 0.5-Hz stimulation. At high rates, caffeine-releasable Ca2+ and diastolic IF increased. In isoproterenol and R, IF transients and contractions recovered at 0.5 Hz with a marked positive staircase and little diastolic IF increase. Within 10 beats, SR Ca2+ accumulated to pre-R levels. R eliminated the positive inotropic effect of paired-pulse stimulation, but isoproterenol temporarily restored it. Twitch contractions in thapsigargin, an SR Ca2+ pump blocker, and isoproterenol were slow compared with control or R + isoproterenol. R leaks SR Ca2+ into the cytosol. SR Ca2+ can be repleted in R by high-rate stimulation or by low-rate stimulation with a beta-adrenergic agonist. SR Ca2+ release in R can be temporarily restored if Ca2+ influx and SR Ca2+ pumping are increased enough to overcome the SR Ca2+ leak.

Cytosolic calcium and myofilaments in single rat cardiac myocytes achieve a dynamic equilibrium during twitch relaxation.

1. Single isolated rat cardiac myocytes were loaded with either the pentapotassium salt form or the acetoxymethyl ester (AM) form of the calcium-sensitive fluorescent probe, Indo-1. The relationship of the Indo-1 fluorescence transient, an index of the change in cytosolic calcium [Ca2+]i concentration, to the simultaneously measured cell length during the electrically stimulated twitch originating from slack length at 23 degrees C was evaluated. It was demonstrated that even if the Ca2+ dissociation rate from Indo-1 was assumed to be as slow as 10 s-1, the descending limb ('relaxation phase') of the Indo-1 fluorescence transient induced by excitation under these conditions is in equilibrium with the [Ca2+]i transient. Additionally, the extent of Indo-1 loading employed did not substantially alter the twitch characteristics. 2. A unique relationship between the fluorescence transient and cell length was observed during relaxation of contractions that varied in amplitude. This was manifest as a common trajectory in the cell length vs. [Ca2+]i phase-plane diagrams beginning at the time of cell relengthening. The common trajectory could also be demonstrated in Indo-1 AM-loaded cells. The Indo-1 fluorescence-length relation defined by this common trajectory is steeper than that described by the relation of peak contraction amplitude and peak fluorescence during the twitch contractions. 3. The trajectory of the [Ca2+]i-length relation elicited via an abrupt, rapid, brief (200 ms) pulse of caffeine directly onto the cell surface or by 'tetanization' of cells in the presence of ryanodine is identical to the common [Ca2+]i-length trajectory formed by electrically stimulated contractions of different magnitudes. As the [Ca2+]i and length transients induced by caffeine application or during tetanization in the presence of ryanodine develop with a much slower time course than those elicited by electrical stimulation, the common trajectory is not fortuitous, i.e. it cannot be attributed to equivalent rate-limiting steps for the decrease of [Ca2+]i and cell relengthening. 4. The [Ca2+]i-length relation defined by the common trajectory shifts appropriately in response to perturbations that have previously been demonstrated to alter the steady-state myofilament Ca2+ sensitivity in skinned cardiac fibres. Specifically, the trajectory shifts leftward in response to an acute increase in pH or following the addition of novel myofilament calcium-sensitizing thiadiazinone derivatives; a rightward shift occurs in response to an acute reduction in pH or following the addition of butanedione monoxime.(ABSTRACT TRUNCATED AT 400 WORDS)

The cytosolic calcium transient modulates the action potential of rat ventricular myocytes.

1. The modulation of the action potential by the cytosolic Ca2+ (Cai2+) transient was studied in single isolated rat ventricular myocytes loaded with the acetoxymethyl ester form of the Ca(2+)-sensitive fluorescent dye Indo-1. Stimulation following rest and exposure to ryanodine were used to change the amount of Ca2+ released from the sarcoplasmic reticulum and thus the size of the Cai2+ transient. The Cai2+ transient was measured as the change, upon stimulation, in the ratio of Indo-1 fluorescence at 410 nm to that at 490 nm (410/490) and action potentials or membrane currents were recorded using patch-type microelectrodes. 2. When stimulation was initiated following rest, the magnitude of the Cai2+ transient decreased in a beat-dependent manner until a steady state was reached. The negative staircase in the Cai2+ transient was accompanied by a similar beat-dependent decrease in the duration of the action potential, manifested primarily as a gradual loss of the action potential plateau (approximately -45 mV). A slow terminal phase of repolarization of a few millivolts in amplitude was found to parallel the terminal decay of the Cai2+ transient. 3. The terminal portion of phase-plane loops of membrane potential (Vm) vs. Indo-1 ratio from all of the beats of a stimulus train followed a common linear trajectory even though the individual beats differed markedly in the duration and amplitude of the action potential and Cai2+ transient. 4. When the stimulation dependence of the Cai2+ transient was titrated away with submaximal exposure to ryanodine, the stimulation-dependent changes in the action potential plateau and terminal phase of repolarization were also eliminated. The same effect was noted in cells which, fortuitously, did not show a staircase in the Cai2+ transient following a period of rest. 5. When action potentials were triggered immediately following spontaneous release of Ca2+ from the sarcoplasmic reticulum, which results in a small depolarization at the resting potential, phase-plane loops (Vm vs. Indo-1 ratio) of the spontaneous events followed the same linear trajectory as the terminal phase of repolarization in the loops of the stimulated beats. 6. Following repolarization from brief voltage clamp pulses (to minimize time and voltage-dependent currents associated with depolarization), an inward current was observed that rose and fell in phase with the Cai2+ transient. This current was present at -70 mV, near the resting potential, and at -40 mV, a potential relevant to the plateau of the action potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Rest decay of calcium transients and contractility in feline ventricular myocytes.

To define the respective contributions of sarcoplasmic reticulum Ca2+ release and action potential-dependent Ca2+ influx to the activator Ca2+, rest decay of the cytosolic free Ca2+ (Ca2+i) transient and contractility was measured in feline ventricular myocytes loaded with the Ca2(+)-sensitive fluorescent dye indo-1. Myocytes were stimulated to a steady state and then rested for periods ranging from 2 to 120 s. Rest decay was assayed with both resumption of stimulation and rapid extracellular applications of caffeine. The protocol was then repeated after exposure to ryanodine. Ryanodine changed the Ca2+i transients during steady-state stimulation from large, rapidly rising transients starting near the resting Ca2+i level to small, slowly rising Ca2+i transients superimposed on an elevated diastolic Ca2+i. Under control conditions the stimulation- and caffeine-induced Ca2+i transients exhibited slow monotonic rest decay with similar time constants of decay (180 and 202 s, respectively, from a representative cell). After ryanodine, the rest decay of stimulation-induced Ca2+i transients was seemingly no different. However, no Ca2+i transient could be elicited by caffeine after 15 s of rest. These results suggest that 1) under normal conditions the primary source of activator Ca2+ is the sarcoplasmic reticulum, and 2) the rest decay of contractility seen in feline ventricular myocytes results from time-dependent loss of Ca2+ from this organelle.

Voltage and beat dependence of Ca2+ transient in feline ventricular myocytes.

The positive contractile staircase after a period of rest is attributable to a positive staircase in the magnitude of the Ca2+ transient. The present study used voltage-clamp techniques and the fluorescent Ca2+ indicator, indo-1, to examine the effects of membrane potential, the duration of depolarization, and the slow inward Ca2+ current (Isi) in the regulation of the magnitude of the steady-state Ca2+ transient and the development of the steady state during the positive staircase. In the steady state, the Ca2+ transient was greatest at +10 mV, the potential at which Isi was also the greatest. However, the Isi-voltage relationship was much more bell-shaped than the Ca2+ transient-voltage relationship. The magnitude and duration of the steady-state Ca2+ transient was not affected by pulse durations as short as 25 ms. However, prolonged voltage pulses were essential to maintain the steady state. The development of the positive staircase was very voltage dependent. After a rest period, a positive staircase was seen when voltage-clamp drives were done to +30 mV but not when done to -10 mV, potentials that elicit Isi of comparable magnitude. These results support the idea that the early peak of Isi can act as a trigger for release of Ca2+ from the sarcoplasmic reticulum. However, sarcoplasmic reticulum Ca2+ loading is dependent on prolonged depolarization and may be mediated through Na+-Ca2+ exchange.

A comparison of cytosolic free Ca2+ in resting feline and rat ventricular myocytes.

The Ca2+-sensitive dye quin-2 was used to measure the cytosolic free Ca2+ (Cai2+) in suspensions of ventricular myocytes isolated from cat and rat ventricles. Following an isolation procedure that was similar for both species, the cells were loaded with quin-2 AM (25 microM) for 30 min at 37 degrees C. After two washes to remove extracellular dye, the cells were resuspended for fluorescence measurements. Extracellular Ca2+ was 2.0 mM. Resting Cai2+ in the rat (121 +/- 11 nM) was found to be significantly higher than in the cat (57 +/- 4 nM). These results are discussed in terms of known differences in excitation-contraction coupling between these two species.