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Am J Physiol Heart Circ Physiol 275: H2041-H2052, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 6, H2041-H2052, December 1998

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

W. H. duBell1, S. T. Gaa1, W. J. Lederer2, and T. B. Rogers1

1 Department of Biochemistry and Molecular Biology, School of Medicine, and 2 Departments of Molecular Biology and Biophysics and Physiology, Medical Biotechnology Center and School of Medicine, University of Maryland, Baltimore, Maryland 21201

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
References

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 (~60%) was time and voltage independent. At 25 µM, 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.

immunophilins; transient outward current; potassium current; excitation-contraction coupling

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
References

FK-506-BINDING PROTEINS (FKBPs) are the intracellular receptors for the immunosuppressant drug FK-506 (33). The functions of FKBPs are largely unknown. However, two modes of action have been identified for one such protein, FKBP12: 1) inhibition of the Ca2+- and calmodulin-activated protein phosphatase calcineurin and 2) regulation of intracellular Ca2+ release channel properties. It is well known that the immunosuppressant actions of FK-506 result from the ability of the FK-506-FKBP12 complex to bind to and inhibit calcineurin in T lymphocytes (23, 24, 28, 33). FKBP12 is also an integral component of intracellular Ca2+ release channels, as first noted in the Ca2+ channel complex found in the sarcoplasmic reticulum of skeletal muscle (20, 31). Removal of FKBP12 causes changes in the kinetic and conductance properties of this channel (20, 31). Interesting experiments have also revealed that FKBP12 is associated with the inositol 1,4,5-trisphosphate receptor, a related intracellular Ca2+ release channel, where it may also play a modulatory role (10, 11).

Recent evidence also suggests that FKBPs are broadly relevant to cardiac function. FKBP12 and the related isoform FKBP12.6, the products of separate genes, are expressed in adult canine cardiac myocytes (23, 32). Timmerman et al. (32) reported that, despite making up only a small fraction of the total FKBP in heart cells, FKBP12.6 is the only isoform associated with the cardiac ryanodine receptor (32), a role subserved in skeletal muscle by FKBP12 (20, 31). The functional role of FKBP12.6 has not been resolved, inasmuch as its removal from the cardiac ryanodine receptor complex has been reported to have no effect on the isolated channels (32) or to induce the appearance of subconductance states in, and increase the open probability of, these channels (21, 34). Consistent with these latter reports, FK-506 has also been shown to change the properties of Ca2+ sparks (26, 34), spatially resolved Ca2+ release events thought to reflect some of the in situ properties of the cardiac sarcoplasmic reticulum Ca2+ release channel (12).

Recent studies also implicate FKBPs in the process of cardiac myocyte development and growth. Clinically, FK-506 can provoke congestive heart failure in pediatric transplant patients, in whom the drug is used to prevent rejection of transplanted organs (4), although the mechanism of this action remains unknown. In a more recent report, lethal ultrastructural and functional defects appeared in the developing hearts of FKBP12 knockout mice (29). The native ryanodine receptors isolated from cardiac and skeletal muscle in these mice exhibited prolonged openings and subconductance states (29). This result suggests, at least in developing heart, that FKBP12 imparts some of the characteristics of cardiac ryanodine receptor behavior. However, regardless of the questions raised by these reports, it is clear that FKBPs have critically important functions in mammalian heart cells.

We recently identified specific K+ channels as possible sites for FKBP regulation in heart (15). FK-506 decreased the K+ currents Ito and IK, resulting in action potential prolongation that increased the magnitude of the cytosolic Ca2+ concentration ([Ca2+]i) transient through altered cellular Ca2+ balance (15). This is of great potential importance, because K+ currents are critical in determining the shape and duration of the cardiac action potential, and their modulation can have important consequences for cardiac function through alterations in contractility and rhythm (5). In the present study the macroscopic electrophysiological mechanisms of the effects of FK-506 on Ito and IK were examined. In addition, physiological and biochemical techniques were used in parallel to test whether one model of FKBP action, the inhibition of calcineurin, is involved in mediating the effects of FK-506 on cardiac K+ currents.

    METHODS
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Abstract
Introduction
Methods
Results
References

Cardiac Myocyte Preparation

Hearts were removed from adult Sprague-Dawley rats (250-275 g) after deep anesthesia had been induced by pentobarbital sodium (30 mg/kg ip; Abbott Laboratories, N. Chicago, IL). The aorta was quickly cannulated for Langendorff perfusion, and ventricular myocytes were isolated, as previously described (14), by perfusion of the coronary arteries with a buffer containing 50 µM Ca2+, collagenase (type II; Worthington Biochemical, Lakewood, NJ), and protease (type XIV). The isolated cells were suspended, at room temperature, in HEPES-buffered DMEM with 10% FCS. Unless otherwise indicated, the reagents used for the cell isolation and other procedures were obtained from Sigma Chemical (St. Louis, MO).

Measurement of Phosphatase Activity

Tissue preparation. Immediately after isolation, suspensions of rat ventricular myocytes were centrifuged at 1,600 rpm for 30 s. The supernatant was removed, and the cells were resuspended and centrifuged in 3 ml of 0.9% NaCl with 1 mM MgCl2. The resulting pellet was resuspended in lysing buffer [50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mM EDTA, 0.5 mM dithiothreitol, 5 mg/ml leupeptin, 5 mg/ml aprotinin], then homogenized using a Teflon-glass rod. The homogenate was centrifuged at 5°C in a Beckman Airfuge (rotor A-10, 30 psi, 178,000 g, 95,000 rpm) for 10 min. The supernatant, containing the cytosolic fraction of the extract, was removed and stored on ice for subsequent use in the calcineurin assay. Calcineurin activity was normalized to protein content, as determined using the assay of Bradford (7), with BSA as a standard.

Calcineurin assay. The assay was based on an established protocol (19) that measures the ability of calcineurin to dephosphorylate a synthetic phosphopeptide, [Ala97]-RII-(81---99). The designation refers to a peptide encompassing amino acids 81-99 of the RII subunit of cAMP-dependent protein kinase in which the amino acid at position 97 has been changed to alanine. The peptide corresponds to the phosphorylation site on this protein (6, 19). The calcineurin pseudosubstrate (synthesized by the University of Maryland Biopolymer Core Facility) was labeled with [32P]Pi using [gamma -32P]ATP (NEN Life Science Products, Boston, MA) and the catalytic subunit of protein kinase A (PKA), as described by Hubbard and Klee (19). Briefly, 30 µCi of [gamma -32P]ATP (1 mM) and 45 µl of 3.3 mM [Ala97]-RII-(81---99) were added to 0.8 ml of reaction buffer [40 mM Tris (pH 6.8), 0.4 mM EGTA-Tris, 0.8 mM EDTA-Tris, 4 mM MgCl2, 0.1 mg/ml BSA]. The reaction was started with the addition of 4 µg of PKA. After 30 min at 30°C, an additional 2 µg of PKA were added, and incubation was continued for an additional 30 min. Separation of the labeled peptide was carried out on a disposable C18 column (Waters, Milford, MA) prewashed with 3 ml of 30% acetonitrile in 0.1% trifluoroacetic acid (TFA) and 5 ml of 0.1% TFA. The column containing the phosphorylated peptide was washed with 0.1% TFA until the effluent radioactivity was <1,000 cpm/µl. The peptide was then eluted with successive 0.5-ml additions of 30% acetonitrile in 0.1% TFA. The pooled radiolabeled fraction was evaporated in a Speed-Vac concentrator and stored at -70°C.

Calcineurin activity was measured by incubation of cardiac cell extracts (25 µg protein, see above) in 60 µl of reaction buffer {20 mM Tris (pH 8), 100 mM NaCl, 6 mM MgCl2, 0.5 mM dithiothreitol, 0.1 mg/ml BSA, 5 µM [32P]Pi-labeled [Ala97]-RII-(81---99)}. The released [32P]Pi was isolated and quantified as described by Hubbard and Klee (19). The specific (Ca2+ dependent) activity was defined as the amount of [32P]Pi released in the reaction minus the radioactivity released in a parallel reaction carried out in the presence of 5 mM EGTA. The assay was linear with respect to time (1-25 min) and amount of tissue (1-25 µg protein). Other experiments revealed that okadaic acid (100 nM; Upstate Biotechnology, Lake Placid, NY), an inhibitor of the protein phosphatases PP-1 and PP-2A, decreased the total amount of phosphatase activity without decreasing the Ca2+-dependent (calcineurin) signal. The Ca2+-dependent signal represented >90% of the total phosphatase activity measured in this assay, and all measurements of calcineurin activity reported in this study were made in the presence of 100 nM okadaic acid. In some experiments a calcineurin autoinhibitory peptide (CIP; Biomol, Plymouth Meeting, PA), derived from the autoinhibitory sequence of the alpha -subunit of calcineurin (18), was included in the reaction mixture. In some others the calmodulin inhibitor R-2457 (17) was used. All assays were performed within 3 h of cell isolation.

Measurement of [Ca2+]i Transients and Membrane K+ Currents

[Ca2+]i transients were recorded, as previously described (15), from myocytes loaded with the fluorescent Ca2+ indicator fluo 3. Fluorescence emission (F) was measured at 510-610 nm with an excitation wavelength of 488 nm. The [Ca2+]i transients are reported as relative fluorescence, or F/F0, where F is the fluorescence emission at each point and F0 is the resting fluorescence at the start of each recording. Cells were loaded with fluo 3 (Molecular Probes, Eugene, OR) by a 15-min incubation with 5 µM fluo 3-AM, prepared from a 442 mM stock in DMSO and 20% (wt/wt) Pluronic F-127 (Molecular Probes). After resuspension in buffer without fluo 3, cells were placed in a bath on the fluorescence microscope and superfused (0.5 ml/min, 30°C) with extracellular buffer consisting of (in mM) 137 NaCl, 5 KCl, 20 HEPES, 15 D-glucose, 1.3 MgSO4, 1 NaH2PO4, and 2 CaCl2, with pH adjusted to 7.4 with KOH. The cells were field stimulated (model S48, Grass, Warwick, RI) at 2.0 Hz by 3-ms pulses with a magnitude 1.5× threshold.

The electrophysiological experiments were carried out on a Nikon Diaphot 300 inverted microscope mounted on a vibration isolation table (Technical Manufacturing, Peabody, MA). The voltage-clamp amplifier was an Axopatch 200A with a CV202A head stage (Axon Instruments, Burlingame, CA). The experiments were conducted under computer control (pClamp software, Axon Instruments). All the voltage-clamp recordings were made using patch-type microelectrodes (0.7-2.0 MOmega , TW150F, WPI, Sarasota, FL) in the whole cell mode. The pipette filling solution consisted of (in mM) 130 KCl, 15 HEPES, 1 MgCl2, and 5 MgATP, with pH adjusted to 7.2 with KOH. For the experiments that required very low [Ca2+]i, the pipette solution included 10 mM EGTA, and the concentration of KCl was reduced to 115 mM to compensate for the additional KOH required to obtain pH 7.2. The extracellular buffer was the same as that described above, except CaCl2 concentration was reduced to 0.25 mM. Tetrodotoxin (10 µM; Calbiochem, La Jolla, CA) and nifedipine (5 µM) were included to block Na+ and L-type Ca2+ currents, respectively. Membrane current recordings were low-pass filtered at 1 kHz, digitized at 5 kHz, and stored on a computer for subsequent analysis. Ito magnitude was calculated as the difference between the fast peak of outward current and the steady-state current at the end of the pulse. IK magnitude was calculated as the difference between the holding current and the steady-state current at the end of the voltage-clamp pulse. All currents were normalized for differences in cell size by measuring cell capacitance, and the average data are reported as current densities (pA/pF). Cell capacitance was calculated by integrating the uncompensated capacity transients elicited by 10-ms hyperpolarizing pulses from -70 to -80 mV immediately after whole cell access was established. Stock solutions (in ethanol; Pharmco Products, Brookfield, CT) of FK-506 (25 mM; gift of Fujisawa, Melrose Park, IL), cyclosporin A (20 mM; Calbiochem), and nifedipine (20 mM) were used to add these agents to the experimental buffers. In all cases the concentration of vehicle was the same in control and experimental solutions and never exceeded 0.125%. All the functional experiments ([Ca2+]i transient measurements and electrophysiological experiments) were performed at 30°C on acutely dissociated cells within 8 h of isolation. The [Ca2+]i transient measurements reported were recorded in 6 cells from a total of 3 preparations, and the electrophysiological experiments were carried out on a total of 24 cells from 10 cardiac myocyte preparations. The cells used for electrophysiological experiments were usually subjected to more than one experimental protocol, with all protocols performed before and after addition of FK-506. Values are means ± SE, with statistical significance (P < 0.05) assessed using Student's paired t-test.

    RESULTS
Top
Abstract
Introduction
Methods
Results
References

It was recently shown that the immunosuppressant FK-506 increased the magnitude of the [Ca2+]i transient by inhibiting the outward K+ currents IK and Ito in rat ventricular myocytes, rather than by acting on the ryanodine receptor (15). By reducing K+ currents, FK-506 prolonged the action potential, which secondarily increased Ca2+ influx and/or decreased Ca2+ efflux, thereby increasing the [Ca2+]i transient. In the present study we examined 1) the macroscopic electrophysiological mechanisms of the effects of FK-506 on Ito and IK and 2) the role of calcineurin inhibition in these effects.

Effects of FK-506 on the Current-Voltage Relationship of IK and Ito

Because of the distinctly different kinetics of Ito and IK in rat ventricle (3), each current component can be resolved from the net current recorded in the absence of K+ channel blockers but in the presence of nifedipine (to block L-type Ca2+ current) and tetrodotoxin (to block Na+ current). Figure 1A shows superimposed membrane currents recorded during voltage-clamp pulses to several test potentials from a holding potential of -70 mV. Figure 1B shows the method for measuring Ito and IK from the composite current waveform. Ito, a rapidly activating current that inactivates completely within ~200 ms (3), is measured as the difference between the early peak of outward current and the steady-state current remaining at the end of the voltage-clamp pulse. IK, which activates less rapidly but inactivates very little over the course of a 300-ms voltage-clamp pulse (3), is calculated as the difference between the steady-state current at the end of the pulse and the holding current.


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  		Fig. 1.   Effects of FK-506 on voltage dependence of K+ currents (Ito and IK). A: schematic of voltage protocol (top) and actual superimposed outward currents (bottom) recorded in control conditions from a holding potential of -70 mV to test potentials of -40, -20, 0, +20, +40, and +60 mV. B: method used for resolving Ito (difference between peak outward current and steady-state current at end of pulse) and IK (difference between steady-state current and holding current) from same recorded current. Holding potential was -70 mV, and current was recorded during a test pulse to +60 mV. C: effect of 25 µM FK-506 on current-voltage relationship of IK, calculated as described in B. Experiments were done from a holding potential of -70 mV. Test pulses (300 ms) ranged from -50 to +60 mV and were imposed every 3 s until protocol was completed. Average magnitude of IK (n = 10 from 6 cardiac myocyte preparations) is plotted against test potential before and after 25 µM FK-506. D: effect of 25 µM FK-506 on current-voltage relationship of Ito. * Statistically significant difference. Ito was measured from same current recordings used for C, and details are as described in C. E: effect of 5 µM FK-506 on current-voltage relationship of IK calculated as described in B (n = 4 from 3 cardiac myocyte preparations). * Statistically significant difference.

The effects of FK-506 (25 µM) on the current-voltage (I-V) relationship of IK and Ito were examined using this method. Each cell was depolarized at 3-s intervals, from a holding potential of -70 mV, to potentials ranging from -50 to +60 mV. This ensured measurement of each current through the range of potentials that encompassed the threshold for activation of the current and the maximum overshoot of the rat ventricular action potential (15). The protocol was then repeated on the same cell after addition of 25 µM FK-506. IK and Ito were resolved from the resulting composite currents as described above. FK-506 produced a marked, statistically significant (P < 0.001) decrease in the magnitude of IK, ranging from 41% at -30 mV to 63% at +60 mV, at all potentials tested (Fig. 1C). In contrast, the decrease in Ito produced by 25 µM FK-506 (Fig. 1D) was small (~10%) and was statistically significant (P < 0.05) only between -10 and +60 mV, inclusive (Fig. 1D). This is in contrast to the large effect of 25 µM FK-506 on Ito reported previously (15). The reasons for this discrepancy are examined below. Identical protocols were also used to examine the effects of 5 µM FK-506 on Ito and IK (n = 4). This concentration has been shown to produce a positive inotropic effect in rat ventricular myocytes (26) (data not shown). Not surprisingly, given the small effect of 25 µM FK-506 on Ito under identical conditions (Fig. 1D), 5 µM FK-506 had no effect on the I-V relationship of Ito (data not shown). However, 5 µM FK-506 inhibited IK (Fig. 1E). The difference reached significance at -10 mV [2.75 ± 0.32 pA/pF (control) vs. 2.50 ± 0.32 pA/pF (FK-506), P = 0.001], and between +20 and +60 mV, IK was inhibited ~30% by 5 µM FK-506 [+60 mV: 10.1 ± 1.15 (control) vs. 7.01 ± 0.77 pA/pF (FK-506), P = 0.006]. To ensure a maximal effect on Ito and IK, all subsequent electrophysiological studies were carried out at 25 µM FK-506.

Effect of FK-506 on Ito Steady-State Inactivation

To investigate the mechanism of the effect of FK-506 on Ito, the voltage dependencies of steady-state inactivation of Ito were compared before and after FK-506. A change in this parameter will affect the number of ion channels available for activation at a given membrane potential. Figure 2A shows a schematic of the voltage-clamp protocol used for these experiments. A 300-ms test pulse to +50 mV was imposed after the cell was held for 1.2 s at potentials ranging from -100 to -2.5 mV. The protocol was then repeated after application of FK-506. Figure 2B shows the family of currents recorded during test pulses from a single cell before and after FK-506. To best illustrate Ito, only the current recorded during the first 40 ms of each test pulse is shown, and the offset introduced by IK has been removed by subtracting the value of the steady-state current at the end of each pulse. There was no effect of FK-506 on the maximal magnitude of Ito [recorded from a holding potential of -100 mV: 30.9 ± 5 (control) vs. 30.3 ± 3 pA/pF (FK-506), P = 0.858]. To analyze the effects of FK-506 on Ito steady-state inactivation, the average normalized Ito magnitude (ratio of test to maximum Ito) was plotted against test potential (Fig. 2C), and the resulting curves were analyzed by fitting them with a Boltzmann equation (see legend for Fig. 2). Before FK-506, Ito exhibited voltage dependence of steady-state inactivation comparable to that described by others in rat ventricular myocytes in the absence of inorganic Ca2+ channel blockers (1). Although FK-506 had no effect on maximum Ito, the normalized magnitude of Ito after FK-506 was significantly reduced at test potentials between -92.5 mV [0.984 ± 0.01 (control) vs. 0.940 ± 0.01 (FK-506), P = 0.002] and -62.5 mV [0.714 ± 0.06 (control) vs. 0.602 ± 0.08 (FK-506), P = 0.043]. Although this could account for the small change in Ito produced by FK-506 in Fig. 1, it is inconsistent with the large decrease in Ito evoked by FK-506 in a previous study (15).


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  		Fig. 2.   Effects of 25 µM FK-506 on voltage dependence of steady-state inactivation of Ito. A: schematic of voltage protocol. Cells were held at -70 mV. A voltage ramp (400 ms) to new test potential (-100 to -2.5 mV) was imposed, and cell was held at test potential for 1,200 ms until a 300-ms test pulse to +50 mV was imposed. Interval between each episode was 8 s. B: superimposed currents (Ito) recorded during each test pulse before (top) and after (bottom) FK-506. To adequately display Ito, only first 40 ms of test pulse are displayed, and offset introduced by IK has been removed by subtracting steady-state current at end of test pulse. C: voltage dependence of steady-state inactivation of Ito. Average normalized Ito magnitude (ratio of test to maximum Ito) is plotted against test potential before and after FK-506. Data were fit to Boltzmann equation as follows: I = Imax /1 + exp(Vm - V0.5)/k, where I is recorded current, Imax is maximum current, Vm is membrane potential, V0.5 is potential where inactivation is 50%, and k is slope factor. Imax was 30.9 ± 5 and 30.3 ± 3.0 pA/pF in control and FK-506, V0.5 was -58 and -59.5 mV in control and FK-506, and k was 5.47 and 6.49 in control and FK-506, respectively. Data represent average of 5 cells from 3 cardiac myocyte preparations. * Statistically significant difference.

Effect of FK-506 on Recovery From Inactivation of Ito

Another important determinant of the magnitude of a membrane current is its rate of recovery from inactivation. Slower recovery kinetics translate into decreased current under conditions of repeated stimulation if the stimulus interval is shorter than the time required for full recovery from inactivation. Thus a paired-pulse voltage-clamp protocol was used to test the hypothesis that FK-506 prolonged the recovery from inactivation of Ito. A schematic of the protocol is shown in Fig. 3A. The holding potential was -70 mV. Pairs of 200-ms voltage-clamp pulses to +50 mV were imposed. A rest period ranging from 20 to 200 ms, increasing in 20-ms increments, separated each pulse. The interval between each episode of pulse pairs was 10 s. After exposure to FK-506, the entire protocol was repeated. Figure 3B shows representative currents from an experiment of this type. In both cases the currents recorded during the first pulse of each pair are nearly identical. In fact, the magnitude of I0 did not change over the course of the entire experiment. The average value of I0 in the control condition was 28 ± 1.5 pA/pF, whereas the value with FK-506 was 26.0 ± 1.0 pA/pF, indicating no rundown of Ito over the course of the experiment. Ito recorded during the test pulses gradually increases as the test interval is increased. Furthermore, the recovery of Ito appears to be markedly slower in the presence of FK-506. This is confirmed in the summary data shown in Fig. 3C. It is clear that there is less current at each test interval in the presence of FK-506, such that Ito recovery is >90% complete at 200 ms in the absence of FK-506 and only 79% complete in its presence (P < 0.05). This demonstrates that FK-506 markedly slows the recovery of Ito from inactivation.


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  		Fig. 3.   Effects of 25 µM FK-506 on recovery from inactivation of Ito. A: schematic of voltage-clamp protocol. Holding potential was -70 mV. Pairs of 200-ms voltage-clamp pulses to +50 mV were imposed. A rest period ranging from 20 to 200 ms, increasing in 20-ms increments, separated each pulse. Interval between each episode of pulse pairs was 10 s. B: superimposed current recordings in absence and presence of FK-506. C: average effect of FK-506 on recovery from inactivation of Ito. Magnitude of Ito recorded with 2nd pulse (Itest) was normalized to magnitude of Ito recorded with 1st pulse (I0), and resulting value is plotted against rest interval (n = 5 cells from 3 cardiac myocyte preparations). * Statistically significant difference.

Because of the finite kinetics of Ito recovery (Fig. 3C), the steady-state magnitude of the current should exhibit frequency dependence. Furthermore, because FK-506 slows the recovery of Ito from inactivation, it should exaggerate the effect of stimulation rate on the steady-state magnitude of Ito. This hypothesis was tested by imposing trains of voltage-clamp pulses (-70 to +50 mV, 200-ms duration) at stimulation rates ranging from 0.2 to 2.0 Hz. A rest period of 1 min was imposed between trains to allow full recovery of Ito. After exposure to FK-506 the entire protocol was repeated. The average magnitude of beat 1 Ito was 33.6 ± 3.1 and 34.9 ± 3.0 pA/pF in the control condition and with FK-506, respectively (P = 0.315), again indicating the lack of rundown of Ito over time. Figure 4A shows the superimposed current records from trains of 2.0, 1.0, 0.5, and 0.2 Hz in the absence and presence of FK-506. All these records were obtained from the same cell and are representative of the summary data (Fig. 4, B and C). Under control conditions the change from beat 1 to beat 10 is statistically significant only at 1.0 Hz (-7.2%, P = 0.033) and 2.0 Hz (-11.2%, P = 0.04). However, after FK-506 application, there is a marked attenuation in Ito at the higher frequencies (Fig. 4C), such that the change from beat 1 to beat 10 is statistically significant at 0.5 Hz (-13.7%, P = 0.019), 1.0 Hz (-24.2%, P = 0.011), and 2.0 Hz (-40.2%, P = 0.008). Thus, although FK-506 does not inhibit the magnitude of the first pulse, it does bring about a marked decline in Ito during a train of stimuli, with the extent of the decrease becoming greater as the stimulation frequency is increased. These data provide additional confirmation that FK-506 slows the rate of Ito recovery from inactivation. Furthermore, in concert with the effect of FK-506 on the voltage dependence of Ito steady-state inactivation, these data explain the discrepancy between the small effect of FK-506 on the I-V relationship of Ito reported in the present study (~10% decrease, Fig. 1) and the large effect (~30% decrease) reported previously (15). In the present study, Ito was measured from a holding potential of -70 mV, with 3 s between each test pulse. In contrast, the previous study was carried out from a holding potential of -60 mV with 2 s between each test pulse. The results shown in Figs. 2-4 demonstrate that both of these differences in the I-V protocol would accentuate the inhibitory effect of FK-506 on Ito.


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  		Fig. 4.   Effect of 25 µM FK-506 on frequency dependence of Ito magnitude. Trains of 10 200-ms voltage-clamp pulses (-70 to +50 mV) were imposed at frequencies ranging from 2 to 0.2 Hz after a 1-min rest period. A: superimposed current recordings from 10 pulse voltage-clamp trains at (from left to right) 2.0, 1.0, 0.5, and 0.2 Hz in absence and presence of FK-506. To emphasize effect on peak of Ito, only 1st 50 ms of each pulse are shown. B: beat dependence of Ito magnitude at stimulation rates of 2.0, 1.0, 0.5, and 0.2 Hz. Magnitude of Ito from each beat was normalized to Ito recorded in 1st beat of pulse train, and average (n = 4 cells from 2 cardiac myocyte preparations) is plotted against pulse number. C: beat dependence of Ito magnitude from same 4 cells in B and at same stimulation rates after exposure to 25 µM FK-506.

Effects of FK-506 on IK

The electrophysiological mechanism of the inhibition of IK by FK-506 was also examined. Figure 5 shows the effects of FK-506 on the voltage dependence of steady-state inactivation of IK measured from the same composite currents used to analyze the effects of FK-506 on the steady-state inactivation of Ito (Fig. 2B). Figure 5A shows the family of currents recorded from a single cell before and after FK-506 application. The vertical scale is adjusted to best illustrate the effect of FK-506 on IK, and as a result, Ito is completely off-scale in the control recording and is truncated in the recording made in the presence of FK-506. In FK-506 there is nearly complete suppression of IK regardless of the holding potential. The relationship between average normalized IK magnitude and holding potential is displayed in Fig. 5B. The shape of the control relationship is complex. The major effect of FK-506 was to decrease maximum IK by 66%, from 14.7 ± 1.8 to 4.94 ± 1.1 pA/pF (n = 5, P < 0.001). There was little additional voltage-dependent inactivation of IK at more depolarized holding potentials. Thus FK-506 inhibits IK through a voltage-independent mechanism that, unlike the effect on Ito, does not allow recovery to the pre-FK-506 value. Consequently, the effects of FK-506 on the recovery from inactivation of IK were not investigated. These results demonstrate that the electrophysiological mechanisms of the effects of FK-506 on Ito and IK are quite distinct.


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  		Fig. 5.   Effects of 25 µM FK-506 on voltage dependence of steady-state inactivation of IK. A: superimposed membrane currents recorded during each test pulse by using protocol shown in Fig. 2A before (left) and after (right) FK-506. Records are shown at same horizontal and vertical gain and are truncated for better visualization of IK. B: voltage dependence of steady-state inactivation of IK. Magnitude of each IK recording, before and after FK-506, was normalized to current recorded during pulse from a holding potential of -100 mV in absence of FK-506 (maximum IK). Average normalized IK magnitude (ratio of test to maximum IK) is plotted vs. test potential before and after FK-506 (n = 5 cells from 3 cardiac myocyte preparations).

Calcineurin Activity in Isolated Cardiac Myocytes

The results described in Figs. 1-5 reveal that FK-506 has profound effects on Ito and IK in rat heart cells. The molecular mechanisms by which FK-506 acts to alter K+ currents are unknown. Because FK-506 is known to inhibit calcineurin (23, 24, 28), it seemed likely that inhibition of this phosphatase may underlie or contribute to the action of FK-506 on cardiac Ito and/or IK as well. The ability of immunosuppressants to inhibit calcineurin in heart was measured directly in cardiac myocyte extracts. Calcineurin activity was assayed by quantitating the release of [32P]Pi from [32P]Pi-labeled [Ala97]-RII-(81---99), an established calcineurin substrate (6, 19). The results of these experiments are presented in Fig. 6A, which shows the amount of [32P]Pi released from the pseudosubstrate peptide, 6.8 ± 2.6 fmol Pi · mg protein-1 · min-1, in the presence of 100 µM Ca2+ and 150 nM calmodulin, which should provide the optimal condition for calcineurin activation (22). The phosphatase activity is inhibited (P < 0.05) ~62%, to 2.54 ± 1.06 fmol Pi · mg protein-1 · min-1, by the addition of 500 nM R-24571, a potent calmodulin inhibitor (17, 25). The phosphatase activity was inhibited (P < 0.05) by ~90%, to 0.718 ± 0.27 fmol Pi · mg protein-1 · min-1, when Ca2+ was chelated by addition of 5 mM EGTA to the reaction mixture. The phosphatase activity is also inhibited by a peptide derived from the autoinhibitory sequence of the alpha -subunit of calcineurin (i.e., CIP; Fig. 6B) (18). This profile of Ca2+ and calmodulin dependence and sensitivity to CIP confirms the presence of calcineurin in cardiac myocytes, as previously demonstrated by others (6, 25, 30).


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  		Fig. 6.   Calcineurin activity in crude extracts of isolated adult rat ventricular myocytes measured as liberation of [32P]Pi from [32P]Pi-labeled [Ala97]-RII-(81---99) pseudosubstrate peptide. A: effect of various interventions on phosphatase activity. Activity ([32P]Pi counts/min) was measured in presence of 100 µM Ca2+ and 150 nM exogenous calmodulin, Ca2+ and calmodulin with 500 nM R-24571 (a potent calmodulin antagonist), and Ca2+ and calmodulin with 5 mM EGTA. Experiments were performed in parallel, and each data point is average of 5 measurements obtained from same 5 cardiac myocyte preparations. B: dose-dependent inhibition of calcineurin activity by FK-506, cyclosporin A, and calcineurin autoinhibitory peptide (CIP). Data are plotted as percentage of calcineurin activity remaining after addition of indicated concentration of agent. Data points are averages of several (n = 4-8) independent measurements.

In T lymphocytes the inhibition of calcineurin by immunosuppressants results from an obligatory binding step that activates an immunophilin: FKBP12 for FK-506 (24) and cyclophilin for cyclosporin A (24). Furthermore, efficient calcineurin inhibition depends on the relative stoichiometry of these binding proteins compared with calcineurin (23). Thus it was important to know whether FK-506 inhibited calcineurin in the myocyte extracts under the conditions in which it was employed in the K+ current studies described above. Figure 6B shows that FK-506 produced dose-dependent inhibition of calcineurin activity, with ~50% inhibition observed at 1 µM FK-506 and ~75% inhibition at 100 µM FK-506. Cyclosporin A (Fig. 6B) also inhibited calcineurin activity in a dose-dependent manner, up to 25 µM. Concentrations greater than this could not be tested because of the low solubility of cyclosporin A. These experiments reveal that there is a sufficient quantity of FKBP12 and cyclophilin in rat ventricular myocytes to mediate the inhibition of calcineurin activity by immunosuppressants under the conditions used in our studies.

Effects of FK-506 and Cyclosporin A on the [Ca2+]i Transient

Figure 6B demonstrates that FK-506 and cyclosporin A inhibit cardiac calcineurin. If the functional effects of FK-506 are due to calcineurin inhibition, then cyclosporin A should also alter cardiac cell function. The effects of both immunosuppressants on the [Ca2+]i transient were measured and compared in field-stimulated rat ventricular myocytes loaded with the Ca2+-sensitive fluorescent indicator fluo 3. A stimulus rate of 2 Hz was chosen to provide optimal inhibition of Ito and IK by FK-506 (Figs. 2-5 and 7). FK-506 (10 µM) increased the magnitude of the steady-state field-stimulated [Ca2+]i transient (Fig. 7, A and B), with little or no effect on its time course. This is demonstrated by the normalized, superimposed [Ca2+]i transients shown in Fig. 7C. In distinct contrast, the same concentration of cyclosporin A had no measurable effect on [Ca2+]i transient magnitude or time course (n = 3, Fig. 7, D-F). Because FK-506 and cyclosporin A inhibit calcineurin activity in cardiac myocyte extracts in a similar manner (Fig. 6), the results from Fig. 7 suggest that the inotropic effect of FK-506, and thus its ability to modulate K+ currents, is dissociated from its calcineurin-inhibitory action.


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  		Fig. 7.   Effects of 10 µM FK-506 and 10 µM cyclosporin A on field-stimulated cytosolic Ca2+ concentration ([Ca2+]i) transients in single isolated rat ventricular myocytes. A: steady-state [Ca2+]i transients in control and after 10 min in FK-506. B: superimposed [Ca2+]i transients, displayed with an expanded time base, from recordings in A. Each waveform is average of 10 [Ca2+]i transients in A. C: averaged [Ca2+]i transients from B normalized to maximum systolic and minimum diastolic values of F/F0 (where F is fluorescence emission and F0 is resting fluorescence) in each waveform. D-F: identical series recorded before and after exposure of a fluo 3-loaded rat ventricular myocyte to 10 µM cyclosporin A.

Effects of FK-506 on IK and Ito in Low-[Ca2+] Filling Solution

To further test this hypothesis, the effects of FK-506 on K+ currents were measured under conditions in which intracellular calcineurin activation was blocked. The experiments shown in Figs. 2-5 were repeated in a separate group of cells in which 10 mM EGTA was included in the pipette filling solution. The calculated free [Ca2+]i of this solution is <50 nM, and biochemical studies showed nearly complete inhibition of calcineurin activity under these conditions (Fig. 6A). Furthermore, influx of Ca2+ was minimized, as in Figs. 2-5, to prevent localized increases in Ca2+ concentration that would allow localized activation of calcineurin. This was accomplished by using zero-Na+ filling solution (to prevent reverse-mode Na/Ca exchange), by maintaining a low extracellular Ca2+ concentration (0.25 mM), and by including 5 µM nifedipine in the extracellular solution. Importantly, the native properties of Ito and IK were unaffected under these conditions. The control plots in Fig. 8, A, B, and E (no FK-506) are nearly identical to those recorded in the absence of [Ca2+]i buffering. Furthermore, the effects of FK-506 on the steady-state inactivation of Ito (Fig. 8A) and IK (Fig. 8E), the recovery from inactivation of Ito (Fig. 8B), and the stimulation rate dependence of Ito magnitude (Fig. 8D) were not altered by prior elimination of calcineurin activity. Because the effects of FK-506 on Ito and IK are not mitigated in any way by this filling solution, these results provide convincing and compelling evidence that inhibition of calcineurin is not the mechanism by which FK-506 modulates Ito or IK. Furthermore, these results provide novel insight into the mechanisms of K+ channel regulation in heart.


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  		Fig. 8.   Effects of FK-506 on parameters of IK and Ito recorded with 10 mM EGTA in pipette filling solution. A: voltage dependence of steady-state inactivation of Ito. Average normalized Ito magnitude is plotted against test potential before and after FK-506 (n = 5 cells from 2 preparations of cardiac myocytes). Dashed lines are from plots, without error bars, of data shown in Fig. 2C (without EGTA). B: average effect of FK-506 on recovery from inactivation of Ito. Normalized magnitude of Ito is plotted against rest interval (n = 4 cells from 2 cardiac myocyte preparations). Dashed lines are from Fig. 3C (without EGTA). C: average stimulation dependence of Ito magnitude (n = 5) at stimulation rates of 2.0, 1.0, 0.5, and 0.2 Hz. D: average stimulation dependence of Ito magnitude from same 5 cells (from 2 cardiac myocyte preparations) and at same stimulation rates after exposure to 25 µM FK-506. E: voltage dependence of steady-state inactivation of IK. Average normalized IK magnitude is plotted against test potential before and after FK-506 (n = 5 cells from 2 cardiac myocyte preparations). Dashed lines are from Fig. 5B (without EGTA).

    DISCUSION

FK-506 and Cardiac K+ Currents

Intracellular Ca2+ release channels (8, 9, 11, 21, 31, 34) and the protein phosphatase calcineurin (23, 24, 28, 33) are known targets of FK-506 action. It has recently been shown that FK-506 can also inhibit the K+ currents Ito and IK in rat ventricular myocytes (15). The initial goal of the present report was to characterize the electrophysiological mechanisms underlying the inhibition of both of these currents by FK-506. An unexpected finding was that these mechanisms were quite distinct. IK inhibition resulted from a time- and voltage-independent reduction in the probability of opening and/or single channel conductance. In contrast, the main effect of FK-506 on Ito was to prolong its recovery from inactivation. This latter result explains why the extent of Ito inhibition in FK-506 depends on the stimulation rate.

Because it is well known that the immunosuppressant action of FK-506 is mediated by the protein phosphatase calcineurin (33), it seemed likely that its action on cardiac K+ channels could also depend on the inhibition of this phosphatase. A series of biochemical experiments confirmed the presence of calcineurin in heart cells, as seen by others (17, 25, 30), and demonstrated that the cardiac enzyme was indeed inhibited by the immunosuppressants FK-506 and cyclosporin A. These results revealed pools of FKBP12 and cyclophilin that could be exploited in functional studies to examine the mechanism of FK-506 action in heart. Although both FK-506 and cyclosporin A inhibited calcineurin, only FK-506 produced a positive inotropic response, an effect shown previously to result from the inhibition of K+ currents (15). Furthermore, the inhibitory actions of FK-506 on Ito and IK were not altered when it was applied to cells in which [Ca2+]i was held at very low levels by dialyzing the cytosol with a high concentration of a chemical buffer (EGTA) and eliminating possible pathways for Ca2+ influx. Although there may be differentially regulated pools of calcineurin within these cells, this protocol minimizes the possibility that sarcolemmal K+ channels were accessible to any pool of active calcineurin. Thus the functional effects of FK-506 in rat ventricular myocytes, both the increase in inotropic state and the inhibition of K+ currents, are independent of its ability to inhibit calcineurin.

Calcineurin in Heart

The role of calcineurin in heart is unknown. Our results suggest that calcineurin is not involved in determining the magnitude of the steady-state cardiac contraction in the absence of neurohumoral stimulation, since cyclosporin A had no inotropic effect and we could not identify any functional effects of FK-506 that could be attributed to inhibition of this phosphatase. However, the biochemical evidence (6, 25, 30) (Fig. 6) makes it clear that calcineurin is present in these cells. One possible short-term role for cardiac calcineurin is the reversal of kinase-dependent phosphorylations that result in or from an increase in average [Ca2+]i. Recent evidence suggests a more long-term role for calcineurin, implicating the phosphatase in the development of cardiac hypertrophy (27). FK-506 and cyclosporin A blocked the hypertrophy response to phenylephrine and ANG II in cultured neonatal rat heart cells (27). Transgenic mice expressing 3-68 copies of a gene for an activated (Ca2+-independent) form of calcineurin developed cardiac hypertrophy and heart failure (27). This hypertrophy could be prevented with cyclosporin A (27). However, the extrapolation from these results to the role of calcineurin in promoting cardiac hypertrophy in response to disease remains unclear.

FKBP12 in Heart

It is becoming clear that FKBP12 has a broad array of intracellular functions relevant to the heart. This is emphasized by a recent report showing that FKBP12 knockout mice present with lethal birth defects localized to the heart, including dilated cardiomyopathies and septal defects (29). A most remarkable facet of that study was that the single channel properties of the cardiac muscle ryanodine receptors, as measured in lipid bilayer membranes, were altered in these mice, despite the fact FKBP12.6 was still present (29). Previously, FKBP12.6, and not FKBP12, had been identified as the exclusive FKBP isoform interacting with the adult canine cardiac ryanodine receptor, despite the much higher concentration of FKBP12 in the cytosol (20). The discrepancy may result from developmental differences in cardiac ryanodine receptor expression. Surprisingly, the morphology of the skeletal muscle in FKBP12 knockout mice was normal (29). An intriguing possibility, based on the results of the present study, is that the pathology is restricted to the heart because of altered cardiac K+ channel function and the resultant disruption of Ca2+ homeostasis. Regardless of the explanation, it is clear that normal cardiac tissue development is dependent on FKBP12 expression, revealing a complex and important role for this immunophilin in mammalian heart.

FK-506 and Intracellular Signaling

Because there is little information on the signaling pathways that regulate IK and Ito, the results with FK-506 provide an opportunity for new insight into their modulation. It has been shown that alpha 1-adrenergic stimulation inhibits IK and Ito (2, 16). Furthermore, similar to FK-506 (15), alpha 1-adrenergic stimulation produces a positive inotropic effect only during action potential stimulation, and not under voltage-clamp stimulation (16). It is interesting to speculate that FK-506 and alpha 1-adrenergic agonists share a common signaling pathway that involves FKBP12 and an "endogenous FK-506." Clearly, further research is required to clarify the role of FKBPs in the inhibition of K+ currents by FK-506. However, regardless of the specific signaling pathways involved, these results suggest that the precise and coordinated modulation of K+ currents can be effectively exploited as an inotropic mechanism in mammalian heart.

    ACKNOWLEDGEMENTS

We thank Fujisawa USA (Melrose Park, IL) for generously supplying the FK-506 used in our experiments.

    FOOTNOTES

This work is supported in part by National Institutes of Health Grants HL-27867 and AG-14637.

Portions of this work have appeared in abstract form (13).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: T. B. Rogers, Dept. of Biochemistry and Molecular Biology, 108 North Greene St., Baltimore, MD 21201.

Received 24 April 1998; accepted in final form 5 August 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
References

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Am J Physiol Heart Circ Physiol 275(6):H2041-H2052
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