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(Circulation Research. 1996;79:1110-1121.)
© 1996 American Heart Association, Inc.

Articles

Effects of FK-506 on Contraction and Ca²⁺ Transients in Rat Cardiac Myocytes

Eileen McCall, Li Li, Hiroshi Satoh, Thomas R. Shannon, Lothar A. Blatter, Donald M. Bers

the Department of Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Ill.

Correspondence to Donald M. Bers, PhD, Department of Physiology, Loyola University Chicago, Stritch School of Medicine, 2160 S First Ave, Maywood, IL 60153. E-mail dbers@luc.edu.

	Abstract

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FK-506 binding protein (FKBP) has been reported to be closely associated with the ryanodine receptor in skeletal and cardiac muscle and to modulate sarcoplasmic reticulum (SR) Ca²⁺ release channel gating in isolated channels. FK-506 can inhibit the activity of FKBP, thereby reversing its effects on SR Ca²⁺ release. We investigated the function of FKBP during normal contractions and Ca²⁺ transients in intact rat ventricular myocytes loaded with fluorescent Ca²⁺ indicators. FK-506 significantly increased steady state twitch Ca²⁺ transients and contraction amplitudes even under conditions in which the SR Ca²⁺ load and Ca²⁺ current were unaltered, suggesting that FK-506 increases the fraction of SR Ca²⁺ released during excitation-contraction (E-C) coupling. Action potentials were somewhat prolonged, consistent with the larger Ca²⁺ transients causing greater inward Na⁺-Ca²⁺ exchange current. FK-506 did not affect SR Ca²⁺ uptake but modestly decreased Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange in intact cells (although no effect on Na⁺-Ca²⁺ exchange was seen in sarcolemmal vesicles). In most cells, FK-506 caused an increase in SR Ca²⁺ content during steady state stimulation, as assessed by caffeine-induced contractures. This was probably due to the inhibition of Ca²⁺ efflux via Na⁺-Ca²⁺ exchange. FK-506 also accelerated the rest decay of SR Ca²⁺ content and increased the frequency of resting Ca²⁺ sparks about fourfold. The increase in frequency of these basic Ca²⁺ release events was not associated with changes in the amplitude or duration of the Ca²⁺ sparks. We conclude that FK-506 increases the fraction of SR Ca²⁺ released during normal twitches and enhances the rate of SR Ca²⁺ release during rest. FK-506 also inhibits Na⁺-Ca²⁺ exchange, although this effect may be indirect. These effects are consistent with an important SR-stabilizing effect of FKBP in intact rat ventricular myocytes.

Key Words: FK-506 • cardiac muscle • sarcolemmal Ca²⁺ channel • sarcoplasmic reticulum • ryanodine receptor

	Introduction

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The immunosuppressant agent FK-506 has been shown to bind to a number of homologous immunophilin proteins known as FKBPs in a variety of organisms, ranging from yeast to humans. Immunosuppression seems to be due to FK-506/FKBP inhibition of the Ca²⁺-dependent phosphatase calcineurin. This, in turn, prevents the calcineurin-dependent activation of interleukin-2 transcription and T-cell proliferation.¹

Numerous studies have demonstrated the important function of the RyR or SR Ca²⁺ release channel in E-C coupling in a variety of muscle types.² A 12-kD FKBP copurifies stoichiometrically with and coimmunoprecipitates with the skeletal muscle RyR.^{3 4} In addition FK-506 causes dissociation of FKBP from the skeletal RyR.³ A homologous 12.6-kD FKBP is found with the RyR in cardiac muscle,^{5 6} and FKBP has also been reported to be associated with the inositol tris-phosphate receptor from rat cerebellum and modifies its function.⁷ Furthermore, other FKBPs have been reported, such as FKBP-13, which is found in the endoplasmic reticulum of canine pancreatic cells.⁸

Several recent studies have shown that FKBP modulates SR Ca²⁺ release channel gating of both skeletal and cardiac RyRs incorporated into lipid bilayers.^{9 10 11 12} Brillantes et al⁹ found that FKBP stabilized rabbit skeletal muscle RyR channel openings. That is, the channels did not open as often (with reduced overall open probability), but when they opened, they were much more likely to reach the full conductance level. FK-506 reversed the effects of FKBP, causing more frequent openings and the appearance of different subconductance states. Kaftan et al¹² found that rapamycin (which binds to FKBP-like FK-506) also increases the overall open probability of the cardiac RyR but again decreases single-channel conductance. FKBP has an enzymatic cis-trans peptidyl prolyl isomerase activity, which is inhibited by both FK-506 and rapamycin,¹³ but this activity is not essential for immunosuppression.¹⁴ It is not yet clear whether this activity is essential for the effects of FKBP on the RyR.

Although the bilayer studies above provide valuable insight into the molecular action of FKBP and FK-506, little is known about how Ca²⁺ transients in intact heart cells are affected. The aims of the present study were to evaluate the effects of FK-506 on cellular Ca²⁺ regulation in intact ventricular myocytes during E-C coupling and rest. Several experimental techniques were used, including measurements of cell contractility, [Ca²⁺]_i, voltage-clamp currents, and resting Ca²⁺ spark characteristics. These Ca²⁺ sparks are basic units of SR Ca²⁺ release flux during both rest and E-C coupling measured with an LSCM.^{15 16 17} These stereotypical Ca²⁺ sparks may result from either a single SR Ca²⁺ release channel or a small cluster working as a functional unit.

The results suggest that in intact rat ventricular myocytes FK-506 increases SR Ca²⁺ release during both rest and E-C coupling. It may also decrease the ability of Na⁺-Ca²⁺ exchange to extrude Ca²⁺ from the cell. Portions of the present study have been previously presented in abstract form.^{18 19}

	Materials and Methods

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Myocyte Isolation
Isolation of rat ventricular myocytes was carried out as previously described.^{20 21} Briefly, hearts were excised from adult male Sprague-Dawley rats (300 g) under sodium pentobarbital anesthesia (140 mg/kg IP), mounted on a Langendorff perfusion apparatus, and perfused with nominally Ca²⁺-free HEPES-buffered Tyrode's solution for 5 minutes at 37°C. The heart was then perfused with the same solution containing 1 mg/mL collagenase (type B, Boehringer Mannheim) and 0.16 mg/mL pronase (Boehringer Mannheim) until it became flaccid (10 to 30 minutes), after which the digested tissue was separated and filtered. The resultant cell suspension was rinsed several times, with progressive increases in [Ca²⁺] to 1 mmol/L. Before experimental use, the myocytes were plated into Plexiglas superfusion chambers, with the glass bottoms of the chambers treated with laminin (GIBCO) to increase cell adhesion.

Measurement of Cell Shortening and Rest Behavior
Myocyte shortening was measured as previously described.²¹ Myocytes were superfused with Tyrode's solution at 22°C and 5 mL/min flow rate. Shortening was measured using a video–edge detection system (Crescent Electronics). Cells were transilluminated by a red light source, to avoid interference with indo 1 epifluorescence measurements (see below). Cells were equilibrated at 22°C for 20 minutes before experimental use. All experimental protocols were carried out at room temperature. After equilibration, the cells were stimulated at 0.5 Hz via platinum field electrodes. The amplitude of contractions elicited by SS stimulation (0.5 Hz) was measured during superfusion with Ca²⁺-containing Tyrode's solution, in the absence and presence of 5 µmol/L FK-506. This concentration was chosen because it produced a marked effect in the absence of cell toxicity (ie, at higher FK-506 concentration, the cells tended to contract spontaneously and did not relax fully). The myocytes were allowed to equilibrate with FK-506 for 10 minutes, by which time the SS twitch contraction amplitude had reached a new stable level.

Measurement of Intracellular Ca²⁺
Ratiometric Measurement
[Ca²⁺]_i and contractility were measured simultaneously during these experiments. The fluorescent Ca²⁺ indicator indo 1-AM (10 µmol/L, Molecular Probes Inc) was loaded as previously described,²² ie, at 15- to 20-minute incubation at room temperature, followed by washing with Ca²⁺-containing Tyrode's solution for at least 30 minutes. The indicator was excited at 365±5 nm, and fluorescence emitted was measured at 405±5 and 485±5 nm. The fluorescence ratio (405/485) and [Ca²⁺]_i were calculated after subtracting the background fluorescence.²²

Analysis of Ca²⁺ Sparks With LSCM
Isolated rat myocytes were loaded with fluo 3-AM (10 µmol/L, Molecular Probes) for 30 minutes at room temperature. Fluo 3 fluorescence imaging was performed on an LSCM (LSM-410, Carl Zeiss) equipped with an argon ion laser (model 2014 series, 25 mW, Uniphase) coupled to an inverted microscope (Axiovert 100, Carl Zeiss) with a Zeiss x40 oil immersion Plan-Neofluor objective (numerical aperture, 1.3). Fluo 3 fluorescence was excited at the 488-nm laser line, and emitted fluorescence was collected through a 515-nm long-pass emission filter. The line-scan mode was used for the quantitative analysis of Ca²⁺ sparks. A single myocyte was scanned repetitively (250 Hz) along a line parallel to the longitudinal axis, avoiding nuclei. The pixel size was 0.05 µm², and with a z resolution of 1 µm, the volume of each pixel (voxel) was 0.05 µm³. Data were analyzed on a Macintosh computer with IDL image-processing software (Research Systems).

Fluo 3 fluorescence was transformed to [Ca²⁺]_i by a "pseudo-ratio" method.^{15 16} Briefly, [Ca²⁺]_i was derived from the following equation: [Ca²⁺]_i=K_d(F/F₀)/{(K_d/[Ca²⁺]_irest)+1-(F/F₀)}, where K_d is the dissociation constant for fluo 3 (1.1 µmol/L²³ ), F is the fluorescence intensity, F₀ is the intensity at rest (mean of lowest 50 pixels at each point) and was assumed to be 150 nmol/L, and [Ca²⁺]_irest is [Ca²⁺]_i at rest.²⁴

Rat myocytes were field-stimulated at 0.5 Hz. After the Ca²⁺ transients had reached SS, stimulation was stopped, and the spark frequency was analyzed during a 20-s period of rest (10 line-scan images). The criteria for inclusion as a Ca²⁺ spark was that the peak amplitude of the Ca²⁺ transient exceeded 60 nmol/L (averaging five adjacent pixels) and that the duration of the half amplitude exceeded 8 ms. The number of sparks counted along each line-scan image was normalized spatially and temporally as the spark frequency (pL^-1·s^-1), where 1 spark per line-scan image corresponds to 20 pL^-1·s^-1.

Assessment of SR Ca²⁺ Load: Caffeine Contractures
The rapid application of a caffeine-containing solution induces a Ca²⁺ transient and contracture in cardiac myocytes, and the amplitude can be used as an index of SR Ca²⁺ content.^{21 25 26} In some experiments, 10 mmol/L caffeine was added to 0Na,0Ca so that Na⁺-Ca²⁺ exchange was also blocked. This solution was introduced into the chamber via a quick-switching device, similar to that described by Bassani et al.²¹ The stimulation and perfusion protocols used were similar to those described above, except that a rapid pulse of caffeine was applied to the myocyte at the end of a 2- to 180-s rest interval in order to measure SS and postrest SR Ca²⁺ loads. Caffeine application was continued for 10 s, by which time the caffeine contracture had begun to decline. The tip was then washed to remove any residual caffeine solution, and the cell perfused with Tyrode's solution for 2 minutes before resumption of stimulation. In some experiments, 10 mmol/L caffeine was also added directly to a normal Tyrode's solution containing Na⁺, such that Ca²⁺ extrusion and [Ca²⁺]_i decline were attributable primarily to Na⁺-Ca²⁺ exchange.²¹

Preparation and Assay of Cardiac Homogenates and Microsomes
Homogenized rat ventricular muscle, microsomes, and sarcolemmal vesicles were used to measure Na⁺,K⁺-ATPase, K⁺-stimulated pNPPase, and Na⁺-Ca²⁺ exchange activity. Rat ventricle was minced and homogenized with a Polytron in 5 mL of solution containing 250 mmol/L sucrose and 20 mmol/L MOPS-Tris (pH 7.4). Microsomes were prepared by centrifugation of the homogenate at 10 000g for 20 minutes at 4°C, after which the supernatant was collected and centrifuged at 160 000g for 40 minutes. Pellets were resuspended in a 140 mmol/L KCl and 40 mmol/L HEPES (pH 7.2) for use in the K⁺-stimulated pNPPase and Na⁺,K⁺-ATPase assays. Pellets for preparation of sarcolemmal vesicles for Na⁺-Ca²⁺ exchange were suspended in 250 mmol/L sucrose and 20 mmol/L MOPS-Tris (pH 7.4), layered over a 20% sucrose gradient, and centrifuged at 120 000g for 60 minutes at 4°C. Membranes were collected from the top and pelleted at 160 000g for 60 minutes. Pellets were resuspended in 140 mmol/L NaCl and 20 mmol/L MOPS-Tris (pH 7.4), quick-frozen in liquid N₂, and stored at -70°C. Total protein was measured by the method of Lowry et al.²⁷

Ouabain-sensitive K⁺-stimulated pNPPase activity was measured in microsomes (50 µg/mL) incubated in the presence of (mmol/L) p-nitrophenylphosphate 5, MgCl₂ 5, EDTA 1, KCl 20, and Tris 50 (pH 7.4) for 20 minutes at 37°C. Assays were performed in the presence and absence of 5 mmol/L ouabain. NaOH (0.667N) was added to the incubates, and the liberated p-nitrophenol was measured as absorbance at 410 nm compared with a standard curve.

Ouabain-sensitive Na⁺,K⁺-ATPase activity was measured with 4 µg/mL protein incubated with or without 5 mmol/L ouabain in 100 µL of a solution containing 50 µg/mL saponin and (mmol/L) Na₂ATP 3.5, NaCl 100, KCl 10, NaN₃ 10, MgCl₂ 1.87, EGTA 10, and PIPES 50 (pH 7.4). The reaction was allowed to proceed for 4.5 minutes at 37°C until it was stopped by the addition of 2 mL of 25 mmol/L sodium molybdate in 1 mol/L ammonium sulfate, and then malachite green (3 mg/100 mL) was incubated with the reaction mixture for 20 minutes on ice. Phosphate was measured as absorbance at 650 nm compared with a standard curve.

Na⁺-dependent ⁴⁵Ca uptake was measured in sarcolemmal vesicles essentially as described by Slaughter et al.²⁸ Briefly, sarcolemmal vesicles equilibrated with 140 mmol/L NaCl and 20 mmol/L MOPS-Tris (pH 7.4) were rapidly diluted 50-fold into 140 mmol/L KCl (or 140 mmol/L NaCl for controls), 12 µmol/L ⁴⁵CaCl₂, and 20 mmol/L MOPS-Tris (pH 7.4). Uptake was stopped by the addition of 4 mL of ice-cold 1 mmol/L EGTA, 200 mmol/L KCl, and 10 mmol/L MOPS-Tris (pH 7.4). Vesicles were collected by filtration onto glass fiber filters (Whatman GF/C). The ⁴⁵Ca on the filters was determined by liquid scintillation spectroscopy.

I_Ca and Action Potential Measurement
I_Ca was measured in rat ventricular myocytes using the perforated-patch technique,²⁹ allowing the cell to retain its usual intracellular environment and limiting I_Ca rundown. An Axopatch-200 amplifier (Axon Instruments) was used with patch electrodes of 1- to 2-M resistance (glass type TW150-6, World Precision Instruments). Electrodes were backfilled with 240 µg/mL amphotericin B in an intracellular solution containing (mmol/L) CsCl 55, cesium methanesulfonic acid 70, NaCl 8, MgCl₂ 1, HEPES 10, and EGTA 0.1, with the pH adjusted to 7.35 with CsOH at 22°C. Once a gigaseal was established, the patch was allowed to perforate for 15 to 30 minutes, after which stable whole-cell recordings with series resistances of 5 to 10 M could be made.

Myocytes were initially superfused with Tyrode's solution. To achieve SS I_Ca conditions, cells were held at -70 mV and then depolarized to 0 mV for 200 ms at 0.5 Hz for five pulses. The test pulse was initiated 2 s after the start of the last conditioning pulse. The cell was first depolarized by a 150-ms voltage ramp to -45 mV, in order to inactivate Na⁺ current, held at -45 mV for 100 ms, and then further depolarized for 200 ms to test potentials between -30 and +40 mV. I_Ca magnitude was measured as the difference between peak current and the residual current at the end of the pulse. Between test pulses, the cell was held at -70 mV for 25 s. The series was also repeated after 10-minute equilibration in Tyrode's solution containing 5 µmol/L FK-506. For comparison, this protocol was also carried out using the "ruptured-patch" technique with similar results.

Action potentials were recorded in current-clamp mode using the perforated–patch clamp technique. The bath solution was normal Tyrode's solution, and the pipette solution was selected to mimic the intracellular milieu and contained (mmol/L) KCl 30, potassium aspartate 110, NaCl 2.5, amphotericin B 200 µg/mL, and HEPES 5, adjusted to pH 7.2 with KOH.

Reagents and Solutions
Unless otherwise stated, experimental reagents used were of analytical grade and supplied by Sigma Chemical Co. FK-506 was a generous gift from Fujisawa USA Inc. A 10 mmol/L stock solution was made up in ethanol and stored at -20°C (final ethanol concentration, <0.05%). In pilot experiments, this concentration of ethanol had no effect on the mechanisms under study. Aliquots of this solution were added to the perfusate immediately before use. The normal Tyrode's solutions contained (mmol/L) NaCl 140, KCl 6, MgCl₂ 1, CaCl₂ 1, glucose 10, and HEPES 5, with the pH adjusted to 7.4 with NaOH at 22°C. Ca²⁺-free solution was the same, except that CaCl₂ was omitted. When both Ca²⁺ and Na⁺ were omitted (0Na,0Ca), 10 mmol/L EGTA was included, and Li⁺ replaced Na⁺.

	Results

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SS Twitches and SR Ca²⁺ Load
Effects on Contractions and Ca²⁺ Transients
Fig 1A shows original recordings of Ca²⁺ transients during SS stimulation of twitches at 0.5 Hz and during CafCs (in 0Na,0Ca) induced 2 s after the cessation of stimulation. From this experiment, it is evident that FK-506 (5 µmol/L) can increase the amplitude of the SS twitch Ca²⁺ transients, without a change of SR Ca²⁺ load (based on the unaltered amplitude of the Ca²⁺ transient induced by caffeine). The contractions corresponding to these Ca²⁺ transients showed the same characteristics.

View larger version (27K): [in this window] [in a new window]   Figure 1. FK-506 (FK) and SS [Ca2+]i and contractions. A, Original recordings of Ca2+ transients from SS twitches and CafCs obtained in a representative fluo 3–loaded rat myocyte under control conditions (left, a) and with 5 µmol/L FK (right, b). B, Pooled data of mean normalized peak SS and CafC [Ca2+]i (left) and contraction (right) amplitudes obtained under control conditions (Ctl) and with 5 µmol/L FK. Data shown are from six cells (mean±SEM, *P<.01). For Ctl, the mean SS CafC amplitude (25.7±2.6 µm) was about four times that of the mean SS twitch contraction (5.7±1.4 µm). The amplitudes of Ca2+ transient for the SS twitch and CafC were 597±101 and 1181±105 nmol/L, respectively.

On the average, cells showed a small increase in SS SR Ca²⁺ content with FK-506 (see below). To assess the effect of FK-506 on twitch amplitude at constant SR Ca²⁺ load, a subset of experiments (six of eight cells) was analyzed in which the caffeine-induced Ca²⁺ transient was not altered by FK-506 (100.4±6.3% of control). These results are shown in Fig 1B. In these experiments, FK-506 did not change the amplitude of the CafCs. The diastolic [Ca²⁺]_i between twitch contractions was also not significantly different in the presence of FK-506 (24.8±2.3 versus 25.7±2.3 arbitrary fluorescence units, n=13).

The observation that FK-506 could increase the twitch Ca²⁺ transients and contractions to 161% to 179% of control at a constant SR Ca²⁺ content in Fig 1 could be explained by an FK-506–induced increase in the fraction of SR Ca²⁺ released during the SS twitch. The fact that neither [Ca²⁺]_i nor contraction was changed by FK-506 during CafC suggests that myofilament Ca²⁺ sensitivity was not altered by FK-506 (otherwise contraction would be changed even if [Ca²⁺]_i was the same). Since the CafCs were unaltered in this series of experiments, we can also conclude that the inotropic effect of FK-506 does not require any change in SR Ca²⁺ content.

FK-506 increased SS twitch contractions and Ca²⁺ transients in every series of experiments, including cells that were not loaded with Ca²⁺ indicator, those loaded with the indicator indo 1, and other groups of cells studied using fluo 3. However, in most experimental groups other than that shown in Fig 1, FK-506 also moderately increased the SS SR Ca²⁺ content (as assessed by caffeine-induced contraction or Ca²⁺ transient amplitude). Even when the SR Ca²⁺ load was increased, the CafC change was smaller than for the twitch. For example, in a group of cells not loaded with fluorescent indicator, twitch contraction was increased by 56±16%, whereas CafCs were only increased by 31±6% (n=14). Similar results were seen in fluo 3–loaded cells (Fig 7B) and indo 1–loaded cells (not shown). These observations do not prevent clear conclusions from Fig 1 for the situation in which the SR Ca²⁺ load was unaltered by FK-506, but they do raise the possibility of a second effect of FK-506, which is either more variable or smaller in impact (see "FK-506 Can Increase SS SR Ca²⁺ Content").

View larger version (27K): [in this window] [in a new window]   Figure 7. Effects of FK-506 (FK) on Na+-Ca2+ exchange (Na/CaX) and Ca2+ transient during CafC. A, Peak Na+-Ca2+ exchange current (INa/CaX) during rapid and sustained application of 10 mmol/L caffeine (Caff) as in Fig 6, bottom left (n=4, *P=.02). Ctl indicates control conditions. B and C, Amplitude of Caff-induced Ca2+ transient (B) and time constant () of [Ca2+]i decline (C) using pooled results from intact cells as in Fig 5 and voltage-clamped cells as in Fig 6, top right (n=13, **P<.05). D, Effect of 5 µmol/L FK on the ratio of peak INa/CaX to the amplitude of the Ca2+ transient from four experiments like that shown in Fig 6, left panels. Both values in FK were normalized to the control values before taking the ratio. E, Effect of FK on Na/CaX activity in isolated sarcolemmal (SL) vesicles measured under controlled ionic conditions (n=3, P=NS; mean 45Ca uptake in control was 22.1 nmol/mg per minute).

The observation in Fig 1 of 50% to 100% larger twitch Ca²⁺ transients and contractions at constant SR Ca²⁺ load implies that in the presence of FK-506 there is either more Ca²⁺ being supplied or a dramatically slowed transport of Ca²⁺ out of the cytosol. Increased Ca²⁺ supply could be due to either increased I_Ca or a larger fractional Ca²⁺ release from the SR. Slowed extrusion of Ca²⁺ from the cytosol could be due to depressed SR Ca²⁺ pump or Na⁺-Ca²⁺ exchange activity. If such Ca²⁺ removal were dramatically slowed, a somewhat higher peak [Ca²⁺]_i would be expected.²² These possibilities are evaluated below.

Effect on SR Ca²⁺ Transport From the Cytosol
Fig 2 shows the characteristic effect of FK-506 to increase SS twitch Ca²⁺ transient amplitude (peak [Ca²⁺]_i, 580±80 nmol/L [control] versus 999±142 nmol/L [FK-506]; n=9; P<.001). However, FK-506 had no effect on the time constant of [Ca²⁺]_i decline (252±18 ms [control] versus 254±21 ms [FK-506], n=9, P=NS). In rat ventricle, the time course of [Ca²⁺]_i decline during the twitch is strongly dominated by the SR Ca²⁺-ATPase pump.²² We conclude that the increased twitch Ca²⁺ transient in Fig 1 is not attributable to a slowing of Ca²⁺ transport from the cytosol, particularly by the SR Ca²⁺-ATPase. It must be due to increased Ca²⁺ supply.

View larger version (19K): [in this window] [in a new window]   Figure 2. FK-506 (FK) and SR Ca2+ uptake during relaxation. A, Representative SS (0.5-Hz) twitch Ca2+ transients in a fluo 3–loaded cell in the absence and presence of 5 µmol/L FK. B, Mean peak [Ca2+]i and time constant () values under control conditions (Ctl) and with 5 µmol/L FK (n=9, *P=.001).

Careful inspection of Fig 2A also shows that FK-506 prolonged the time to peak [Ca²⁺]_i (69±5 ms [control] versus 103±11 ms [FK-506]). Although this could, in principle, be caused by slowed Ca²⁺ transport out of the cytosol, based on the foregoing considerations, it seems more plausible that this is due to either prolonged or larger Ca²⁺ release (or influx).

Effects on Ca²⁺ Supply
If FK-506 increased I_Ca significantly, it could increase the twitch Ca²⁺ transient in two ways, even at constant SR Ca²⁺ load: (1) More Ca²⁺ influx could directly contribute to the Ca²⁺ transient. (2) It would also cause a greater amount of SR Ca²⁺ release. Fig 3 shows SS I_Ca and current-voltage relationships obtained in the presence and absence of FK-506 using the perforated–patch clamp technique in five representative indo 1–loaded cells. There was no apparent effect of FK-506 on either the amplitude, kinetics, or voltage dependence of I_Ca.

View larger version (11K): [in this window] [in a new window]   Figure 3. FK-506 does not alter ICa. A, SS ICa resulting from a 200-ms step to 0 mV from a holding potential of -45 mV in the absence (Ctl) or presence of 5 µmol/L FK-506 in a representative myocyte. B, Mean conditioned current-voltage relationships obtained under Ctl conditions and with FK-506. Data are shown as mean±SEM (n=5). Vtest indicates test voltage.

A similar lack of effect of FK-506 on I_Ca was observed using the ruptured-patch technique, in which cells were dialyzed with a pipette solution including 10 mmol/L EGTA to prevent contraction (data not shown). From these experiments, we infer that increased sarcolemmal I_Ca can be ruled out as a possible route for increased Ca²⁺ supply to the cytosol during the twitch in the presence of FK-506. Therefore, we conclude that the increased Ca²⁺ transient amplitude in FK-506 in Fig 1 was due to an increased fraction of SR Ca²⁺ released during the twitch.

Effect of FK-506 on Action Potential Characteristics
Fig 4A shows a typical example of action potentials measured in current-clamp mode. The main effect of FK-506 on the action potential was a modest prolongation of the action potential. The action potential duration at 50% repolarization was slightly, but not significantly, increased (19.7±4.6 ms [control] versus 23.1±4.6 ms [FK-506], n=8). Although this could be related to changes in Ca²⁺ influx, the lack of effect on I_Ca makes this seem unlikely. It seems more plausible that the elevated plateau is secondary to the increased Ca²⁺ transient and greater Ca²⁺ extrusion via an inward, depolarizing Na⁺-Ca²⁺ exchange current.^{30 31}

View larger version (18K): [in this window] [in a new window]   Figure 4. FK-506 (FK) and SS action potentials. A, Action potentials recorded in current-clamp mode using perforated–patch clamp technique with stimulation by a small inward current injection applied at 0.5 Hz. B, Pooled results of the action potential duration at the point of 50% repolarization (APD50) under control conditions (Ctl) and with FK (n=8, P=NS).

Effect on Na⁺-Ca²⁺ Exchange
Although the time constant of [Ca²⁺]_i decline during the SS twitch was not altered (Fig 2), this primarily reflects the action of the SR Ca²⁺-ATPase rather than the Na⁺-Ca²⁺ exchange. Thus, a modest change in Na⁺-Ca²⁺ exchange might not be detectable in that protocol. However, since Na⁺-Ca²⁺ exchange is the main means of cellular Ca²⁺ extrusion, such changes could be of functional importance. To evaluate changes in Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange, caffeine was rapidly and continuously applied in normal Tyrode's solution to cause release of SR Ca²⁺ and prevent reuptake. In the presence of normal extracellular Na⁺, the rate of [Ca²⁺]_i decline during this CafC is mainly attributable to Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange.²²

Fig 5 shows representative recordings of caffeine-induced Ca²⁺ transients 2 s after a SS twitch in an intact field-stimulated cell (±5 µmol/L FK-506). Although there was cell-to-cell variation, the mean rate of [Ca²⁺]_i decline attributable to Na⁺-Ca²⁺ exchange was slower with FK-506 (=1.55±0.10 s [control] versus 2.13±0.09 s [FK-506], n=5, P<.01). Thus, it appears that FK-506 can decrease the ability of Na⁺-Ca²⁺ exchange to extrude Ca²⁺ from the cell by 25%.

View larger version (20K): [in this window] [in a new window]   Figure 5. FK-506 (FK) and Na+-Ca2+ exchange. A, Representative SS caffeine-induced Ca2+ transients in the presence of 140 mmol/L NaCl in a fluo 3–loaded cell in the absence and presence of 5 µmol/L FK. indicates the time constant of [Ca2+]i decline. B, The value for five experiments under control conditions (Ctl) and with FK (*P=.01).

We also performed this type of experiment in cells that were loaded with indo 1 and voltage-clamped, allowing the direct measurement of the Na⁺-Ca²⁺ exchange current during the SR Ca²⁺ release induced by caffeine. Fig 6 shows Ca²⁺ transients and Na⁺-Ca²⁺ exchange currents during a CafC (±5 µmol/L FK-506). The transient inward current measured under these conditions is carried entirely by Na⁺-Ca²⁺ exchange, as demonstrated by Delbridge et al.³² Fig 7A shows that on the average the peak Na⁺-Ca²⁺ exchange current was slightly smaller in the presence of FK-506 (0.97±0.14 pA/pF [control] versus 0.81±0.15 pA/pF [FK-506], P=.02, n=4). This decrease in Na⁺-Ca²⁺ exchange current occurred despite an increase in the amplitude of the Ca²⁺ transients during CafC in the presence of FK-506 in this experimental series (by 88±25%, P<.01, n=4). Fig 6C shows the [Ca²⁺]_i dependence of Na⁺-Ca²⁺ exchange current during the declining phases of the Ca²⁺ transients in Fig 6A. It can be appreciated that at any given [Ca²⁺]_i the Na⁺-Ca²⁺ exchange current is smaller in the presence of FK-506. Fig 7D shows that FK-506 decreased the ratio of the Na⁺-Ca²⁺ exchange current to peak [Ca²⁺]_i (a crude method to account for the effect of the larger Ca²⁺ transient on Na⁺-Ca²⁺ exchange). Again, these results indicate that Ca²⁺ extrusion by Na⁺-Ca²⁺ exchange is inhibited by FK-506.

View larger version (19K): [in this window] [in a new window]   Figure 6. Effects of FK-506 on Na+-Ca2+ exchange current and [Ca2+]i. Left, Ca2+ transients (top left) and Na+-Ca2+ exchange current (INa/CaX, bottom left) at -70 mV induced by rapid application of 10 mmol/L caffeine (Caff) after a series of five conditioning voltage-clamp pulses from -70 to 0 mV. Ctl indicates control conditions. Right, [Ca2+]i dependence of INa/CaX during the declining phase of the Ca2+ transients shown in the left panels. Note that in this cell the SR Ca2+ content is higher after FK-506, but the peak INa/CaX is reduced.

In Fig 7, panels B and C show pooled Ca²⁺ transient data from the CafC experiments performed in field-stimulated cells (as in Fig 5) together with those performed under voltage-clamp conditions (as in Fig 6). Although the Ca²⁺ transient amplitude was more strongly increased in the voltage-clamped cells, the group as a whole showed significant increases in both [Ca²⁺]_i and the time constant of [Ca²⁺]_i decline during caffeine application.

It is not clear whether this effect of FK-506 is directly on the Na⁺-Ca²⁺ exchange. One possibility is that the effect on the Na⁺-Ca²⁺ exchange is indirect and secondary to the effects on [Na⁺]_i or the Na⁺,K⁺-ATPase. For example, elevated [Na⁺]_i would decrease Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange. In an attempt to clarify this issue, we measured Na⁺-Ca²⁺ exchange in sarcolemmal vesicles and enzymatic activities of the Na⁺,K⁺-ATPase in homogenates and microsomes. Fig 7E shows that FK-506 did not alter the Na⁺-Ca²⁺ exchange activity in the isolated sarcolemma, where ionic conditions were controlled (Na⁺-dependent ⁴⁵Ca uptake was 102.8±1.2% of control). In homogenates, there was also no significant inhibition of Na⁺,K⁺-ATPase (104±6% of control) or the alternative enzyme assay for that pump (K⁺-stimulated pNPPase, 103±5% of control). These results suggest that the effect of FK-506 is not directly on either the Na⁺-Ca²⁺ exchange or the Na⁺,K⁺-ATPase. The effect may be secondary to effects on some other cellular constituent.^{33 34} The depressed Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange may also explain the increase in SS SR Ca²⁺ load observed in most cells (see "FK-506 Can Increase SS SR Ca²⁺ Content").

Effect of FK-506 on Rest Behavior
The results above indicate that FK-506 increases the fractional SR Ca²⁺ release during E-C coupling at SS twitches in isolated rat ventricular myocytes. FK-506 may also have effects on the resting SR Ca²⁺ content and release flux.

Effect of FK-506 on Rest Decay
Fig 8 shows the effect of rest on SR Ca²⁺ content in the absence and presence of FK-506, based on the amplitude of CafC-induced Ca²⁺ transients obtained after 2- to 180-s rest intervals. The data have been normalized to the SS CafC-induced Ca²⁺ transient amplitude (ie, after a 2-s rest). Under control conditions, the SR Ca²⁺ content remained relatively constant over the 3-minute rest period. FK-506 significantly decreased the SR Ca²⁺ content at all rest intervals tested. This is consistent with FK-506 increasing SR Ca²⁺ leak during rest, by increasing the overall open probability of SR Ca²⁺ release channels, thereby accelerating net Ca²⁺ loss from the SR.

View larger version (15K): [in this window] [in a new window]   Figure 8. FK-506 accelerates rest decay of SR Ca2+ content. The graph shows mean Ca2+ transient amplitudes induced by caffeine application after rest intervals of 2 to 180 s under control conditions () and with 5 µmol/L FK-506 () in indo 1–loaded rat ventricular myocytes. Data are normalized to the CafC after a 2-s rest and are shown as mean±SEM (n=5 to 11, *P<.05).

Effects of FK-506 on Resting Ca²⁺ Sparks
Ca²⁺ sparks are believed to represent elementary events of local SR Ca²⁺ release during both rest and E-C coupling in heart (whether due to a single SR Ca²⁺ release channel or a cooperative cluster).^{15 16 17} Thus, if FK-506 alters the resting Ca²⁺ leak from the SR, it may alter the properties of the spontaneous Ca²⁺ sparks measured with confocal microscopy.

Fig 9 shows line-scan images of Ca²⁺ sparks in a rat myocyte in the absence (panel A) and presence (panel B) of 10 µmol/L FK-506. Under control conditions, spark frequency was smallest during the initial few seconds after the last twitch and increased gradually toward a plateau over 10 to 20 s (see also Fig 10A). After the addition of FK-506, the spark frequency dramatically increased at all time intervals measured. Figure 10B shows that the overall spark frequency was increased about fourfold by FK-506 (mean values were 19.1±2.9 pL^-1·s^-1 [control] and 74.7±4.7 pL^-1·s^-1 [FK-506]; P<.01 by paired t test; n=10 cells).

View larger version (112K): [in this window] [in a new window]   Figure 9. Effect of FK-506 on spontaneous Ca2+ sparks in a single rat ventricular myocyte. Line-scan images of Ca2+ sparks are shown during rest in the absence (A) and presence (B) of 10 µmol/L FK-506 in a rat cell. [Ca2+]o was 2 mmol/L. The line-scan images indicate Ca2+ sparks 2 s after the last SS twitch Ca2+ transient (left), which is not shown, and from the 10th resting image after 20 s of rest (right). Before the rest, the cell was stimulated at 0.5 Hz. The number of spark events that reached threshold were as follows: for the left and right images, 2 and 7, respectively, in control, 17 and 19, respectively, in FK-506. Line recordings of [Ca2+]i shown under the line-scan images were taken from the spots indicated as 1, 2, or 3 along the line. The dashed lines indicate [Ca2+]i at 100 nmol/L.

View larger version (20K): [in this window] [in a new window]   Figure 10. Effect of FK-506 (FK) on rest-dependent changes of spark frequency. A, Time courses of mean spark frequencies normalized temporally and spatially (pL-1·s-1) in control conditions (Ctl, ) and with FK (). The broken lines show simple linear regressions. B, Mean spark frequencies during the 20 s of rest calculated from panel A. Data are mean±SEM from 10 paired experiments (*P<.05).

We also examined the effects of FK-506 on Ca²⁺ sparks with a higher SR loading state (eg, with 4 mmol/L Ca²⁺ Tyrode's solution, not shown). In this case, the control resting spark frequency was higher, such that FK-506 increased the spark frequency by a more moderate extent. However, the incidence of propagating Ca²⁺ waves was raised significantly by FK-506, from 0.91 to 3.18 min^-1 (n=6).

Characteristics of Individual Ca²⁺ Sparks
The extent of Ca²⁺ release from the SR is dependent not only on the spark frequency but also on the amount of Ca²⁺ released during an individual spark. Thus, the amount of Ca²⁺ released at a single Ca²⁺ spark is expected to depend on the amplitude and duration of the event. Since bilayer results with purified RyRs have shown channel opening to subconductance states in the presence of FK-506,⁹ this analysis seems particularly germane.

Fig 11 shows the distributions of the peak amplitudes (panel A) and the durations of half amplitude (panel B) of individual sparks. Both parameters followed approximately gaussian distributions and were not altered significantly by FK-506. The mean amplitudes and durations were 215±5 nmol/L and 27.4±1.1 ms in control (n=144) and 211±6 nmol/L and 26.5±1.2 ms in the presence of FK-506 (n=128, not significant). These findings imply that FK-506 does not modulate the individual characteristics of Ca²⁺ sparks. This is consistent with either the FK-506–induced increase in SR Ca²⁺ load being small or the effects on SR Ca²⁺ load being separate from the effects on SR Ca²⁺ release (otherwise, Ca²⁺ spark amplitude might be expected to be higher).

View larger version (27K): [in this window] [in a new window]   Figure 11. Effects of FK-506 on individual spark characteristics. Histograms show the distribution of spark amplitudes (A) and durations of the half amplitudes (B) in rat myocytes. Numbers of Ca2+ sparks were plotted as a function of the peak amplitude of [Ca2+]i and the duration of the half amplitudes in control conditions (open bar, n=144) and with FK-506 (stippled bar, n=128). Both the amplitudes and durations roughly followed gaussian distributions in both groups, and there were no significant differences in the mean peak amplitudes (215 vs 211 nmol/L) and the mean durations (27.4 vs 26.5 ms).

Fig 12 shows another manifestation of the increased resting spark frequency in the presence of FK-506. Here, the extracellular solution was rapidly changed to 0Na,0Ca (with EGTA) immediately after the last twitch to prevent both Ca²⁺ influx and Na⁺-Ca²⁺ exchange. During 20 s of rest, [Ca²⁺]_i gradually increased by 13.7±5.0 nmol/L in control, whereas it increased by 31.1±5.6 nmol/L in the presence of FK-506. Since Ca²⁺ could not have entered from the extracellular space, the elevated [Ca²⁺]_i is from an intracellular compartment, presumably the SR. Thus, the FK-506–dependent increase in Ca²⁺ spark frequency (and SR Ca²⁺ leak) is sufficient to elevate diastolic [Ca²⁺]_i, at least when Ca²⁺ extrusion by Na⁺-Ca²⁺ exchange is prevented. Indeed, the increased resting [Ca²⁺]_i levels with FK-506 may be a macroscopic manifestation of increase in the resting Ca²⁺ spark frequency.

View larger version (16K): [in this window] [in a new window]   Figure 12. Effect of FK-506 on resting [Ca2+]i in 0Na,0Ca. A, The superfusate was switched to 0Na,0Ca immediately after the last stimulated twitch contraction (in the absence or presence of FK-506). The rise in [Ca2+]i must be from intracellular stores. The [Ca2+]i displayed is the mean for the entire scanned line. B, Pooled results from five cells.

The FK-506–induced acceleration of rest decay of SR Ca²⁺ content (Fig 8) and the increased resting spark frequency (of unaltered amplitude and duration, Figs 9 through 11) are consistent with an FK-506–induced increase in the resting open probability of SR Ca²⁺ release channels in intact ventricular myocytes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we have attempted to provide an initial characterization of the effects of FK-506 on Ca²⁺ regulation in intact rat ventricular myocytes. Since FKBP and FK-506 have dramatic effects on the RyR in biochemical studies and when the isolated SR Ca²⁺ release channel is incorporated into lipid bilayers,^{4 5 10 11} it is important to understand their effects on the RyR in its natural cellular environment. In studying the intact cell, we also have the ability to detect whether FK-506 appears to affect other Ca²⁺ regulatory systems in addition to the SR Ca²⁺ release channel.

FK-506 Increases SS Twitches and SR Ca²⁺ Release
The increase in the amplitude of SS twitches could in principle be due to (1) increased myofilament Ca²⁺ sensitivity, (2) an increased amount of activating Ca²⁺ supplied to the myofilaments, or (3) a decreased rate of Ca²⁺ transport from the cytosol. An overall effect of FK-506 on myofilament Ca²⁺ sensitivity can be largely excluded, since the CafC in the key experiments summarized in Fig 1B were not altered in either contraction or Ca²⁺ transient amplitude. If FK-506 had increased myofilament Ca²⁺ sensitivity, one would expect an increased contraction for the same amplitude Ca²⁺ transient.

For a reduced rate of Ca²⁺ removal from the cytosol to explain the increase in Ca²⁺ transient and contraction, the effect would have to be dramatic. Indeed, Bassani et al²² found that even complete inhibition of the SR Ca²⁺ pump (the main means of Ca²⁺ transport from the cytosol) increased the amplitude of the twitch Ca²⁺ transient by only 25%. Furthermore, they found that this small increase in peak [Ca²⁺]_i was associated with a ninefold slowing of the time constant of twitch [Ca²⁺]_i decline in rat ventricular myocytes.²² Fig 2 shows that with FK-506 there was almost no change in the time constant of [Ca²⁺]_i decline during the twitch. Although some acceleration in [Ca²⁺]_i decline could be intrinsically due to the larger Ca²⁺ transient amplitude in FK-506 per se,³⁵ thereby masking a modest depression of Ca²⁺ removal from the cytosol, there is clearly no dramatic slowing of [Ca²⁺]_i decline. Since the SR Ca²⁺ pump is so dominant over the Na⁺-Ca²⁺ exchange in rat ventricular myocytes, this experiment might not reveal effects of FK-506 on Na⁺-Ca²⁺ exchange (see below). Bassani et al²² also showed that even complete block of the Na⁺-Ca²⁺ exchange did not affect the amplitude of the twitch Ca²⁺ transient in rat ventricular myocytes. Thus, the FK-506–induced increase in twitch Ca²⁺ transient cannot be attributed to decreased Ca²⁺ removal from the cytosol.

The main ways Ca²⁺ enters the cytosol during a twitch are across the sarcolemma (as I_Ca or Na⁺-Ca²⁺ exchange) and from the SR via the SR Ca²⁺ release channel. Measurements of I_Ca (Fig 3) show no apparent direct effect of FK-506 on this ionic current. The absolute amount of Ca²⁺ influx via Na⁺-Ca²⁺ exchange is probably small compared with that via I_Ca, although it has been suggested that Ca²⁺ entry via this system may be able to trigger SR Ca²⁺ release.^{2 36 37 38} Our measurements suggest that Ca²⁺ efflux via Na⁺-Ca²⁺ exchange is somewhat reduced by FK-506 (Figs 5 through 7). An allosteric inhibition of Na⁺-Ca²⁺ exchange would not be expected to allow more Ca²⁺ influx via this system during E-C coupling. On the other hand, the apparent effect of FK-506 on Na⁺-Ca²⁺ exchange could be due to a shift of the thermodynamic balance (ie, raising [Na⁺]_i). This would make Ca²⁺ entry via Na⁺-Ca²⁺ exchange more favorable during the early part of the action potential. Such enhanced Ca²⁺ influx via the Na⁺-Ca²⁺ exchange could contribute to the enhanced E-C coupling observed with FK-506.^{36 37 38} Such a shift would also tend to favor increased SR Ca²⁺ load, but increases in twitch Ca²⁺ transients were observed even in the absence of increased SR Ca²⁺ (Fig 1). Thus, it seems that increased Ca²⁺ influx across the sarcolemma is unlikely to explain the increased SS twitch Ca²⁺ transients observed with FK-506.

An increase in the SR Ca²⁺ content with the same Ca²⁺ release trigger and fraction of SR Ca²⁺ released could explain the elevated twitch and Ca²⁺ transient observed with FK-506. An increase in SR Ca²⁺ load can also increase the fraction of SR Ca²⁺ release, even with the same Ca²⁺ release trigger.³⁹ This effect may contribute to the increase in Ca²⁺ transient in experiments in which both SR Ca²⁺ load and twitch Ca²⁺ transients were enhanced. However, this cannot explain the 50% to 100% increase in twitch Ca²⁺ transients and contractions (observed in Fig 1) in which there was no change in SR Ca²⁺ content. Thus, although changes in SR Ca²⁺ load may contribute to alteration observed in some cells, it cannot explain the enhanced SS twitches in Fig 1.

Finally, there could be a higher fraction of SR Ca²⁺ released during the twitch in the presence of FK-506. Since the SR is the main source of activating Ca²⁺ and because FKBP is known to bind to and alter properties of the isolated SR Ca²⁺ release channel or RyR, this is both a logical and a parsimonious possibility. An FK-506–induced increase in the fraction of SR Ca²⁺ release at a twitch is entirely consistent with our results. That is, for a given amount of SR Ca²⁺ (Fig 1B) and trigger I_Ca, a greater fraction of SR Ca²⁺ released during the twitch could easily explain the increase in twitch Ca²⁺ transient. This would also be consistent with the results from RyRs in artificial bilayers,^{9 10 11 12} where FK-506 (or rapamycin) increased overall channel open probability under SS conditions with respect to [Ca²⁺]. Thus, we conclude that FK-506 increases the amount of SR Ca²⁺ released for a given SR Ca²⁺ content and Ca²⁺ influx trigger.

FK-506 Increases Resting SR Ca²⁺ Release
In addition to its effect to increase SR Ca²⁺ release during E-C coupling, FK-506 also appears to increase SR Ca²⁺ release during rest. This would be the simplest interpretation of both the acceleration of resting loss of global SR Ca²⁺ content (Fig 8) and the increased frequency of Ca²⁺ sparks observed during rest in rat ventricular myocytes (Figs 9 and 10). The accelerated rest decay of SR Ca²⁺ content with FK-506 could, in principle, also be due to increased Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange or decreased SR Ca²⁺-ATPase activity.⁴⁰ However, since the results in the present study indicate a reduced Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange with FK-506 (Figs 5 through 7) and unaltered Ca²⁺ transport by the SR Ca²⁺-ATPase (Fig 2), this possibility seems unlikely.

The increase in resting Ca²⁺ sparks and net SR Ca²⁺ loss observed in the present study are consistent with results from previous studies involving skeletal or cardiac RyRs incorporated into lipid bilayers.^{9 10 12} These studies have suggested that FKBP normally stabilizes the RyR, decreasing its overall open probability, and that FK-506 or rapamycin can reverse this effect, making it more likely to open (in our case, during either rest or E-C coupling). They have also reported that either removing FKBP from the RyR or treating the native channel with FK-506 or rapamycin causes the appearance of subconductance states of the release channel with frequent transitions to one of four different levels.

We did not detect any change in either the duration or amplitude of Ca²⁺ sparks in the presence of FK-506. Indeed, there was no appreciable difference in the shapes of the histograms in Fig 11, as might have been expected for the single-channel behavior reported in bilayers. The lack of alteration in individual spark characteristics does not refute the presence of subconductance states in intact cells but instead raises the issue of whether Ca²⁺ sparks in cardiac myocytes are due to release from a single channel or from a functional cluster of individual Ca²⁺ release channels.^{15 41 42} If the Ca²⁺ sparks do result from a group of release channels that are somehow cooperative, then our ability to extrapolate from the duration and amplitude of the Ca²⁺ sparks to the behavior of single channels in bilayers is limited. It is also not clear whether the elevated spark frequency is due to an increase in the sensitivity of the release channel to activating Ca²⁺ or some other effect.

Our results are in contrast to those recently reported by Xiao et al,⁴³ who found that 10 µmol/L FK-506 had no effect on contraction in isolated mouse cardiac myocytes, whereas 50 µmol/L caused an increase in the duration of spontaneous Ca²⁺ sparks, from 32 to 93 ms, without altering the frequency of observed Ca²⁺ sparks. They concluded that FKBP is involved in the process that terminates spark production. The reasons behind the differing results are not known. We did not routinely use FK-506 concentration as high as 50 µmol/L, but in some pilot experiments, we found that concentrations higher than 10 µmol/L caused spontaneous activity in cells, frequent macrosparks, and propagating Ca²⁺ waves.

FK-506 Can Increase SS SR Ca²⁺ Content
FK-506 caused an increase in the SR Ca²⁺ content in most cells that were stimulated at 0.5 Hz (aside from the subset of valuable results in Fig 1B). Given the preceding discussion, this is not due to a reduced fraction of SR Ca²⁺ released at a twitch or to a greatly stimulated SR Ca²⁺ pump. It also seems unlikely that the increase in SR Ca²⁺ load is due to greater Ca²⁺ influx via either I_Ca or Na⁺-Ca²⁺ exchange at each contraction. A more plausible explanation has to do with the reduced ability of Na⁺-Ca²⁺ exchange to extrude Ca²⁺ in the presence of FK-506 (eg, during CafC, Figs 5 through 7).

A simple increased fraction of SR Ca²⁺ release at a twitch (with all other things being equal) would be expected to increase Ca²⁺ extrusion by Na⁺-Ca²⁺ exchange and thus reduce SS SR Ca²⁺ load. However, we found that FK-506 also inhibited the Na⁺-Ca²⁺ exchange and also note that any inhibition of Na⁺-Ca²⁺ exchange (by itself) will tend to increase cellular Ca²⁺ load. Thus, since we always observed that SS SR Ca²⁺ load was either increased or unchanged, we conclude that this inhibition of Na⁺-Ca²⁺ exchange is sufficient to prevent the anticipated "unloading" of the SR (and could also explain the increases in SR Ca²⁺ load). We already know that in rat ventricular myocytes the Na⁺-Ca²⁺ exchange is a weak competitor with the SR Ca²⁺ pump.²² Thus, with inhibition of the Na⁺-Ca²⁺ exchange, an even larger fraction of the activating Ca²⁺ would be taken up by the SR. Even though the diastolic SR Ca²⁺ leak is increased, the net amount of leak is still small during the brief diastolic interval at 0.5-Hz stimulation, and the SR may sequester most of the net cellular Ca²⁺ gain.

It may also be noted that in cells in which the SS SR Ca²⁺ content increased, the degree of increase in the twitch contraction and Ca²⁺ transient was even greater. Quantitative conclusions are hampered by the possible nonlinearities in the [Ca²⁺]_i dependence of total cytosolic Ca²⁺. Nevertheless, it seems probable that both increased SR Ca²⁺ content and increased fractional SR Ca²⁺ release contribute to the inotropic effect in these cells.

It is not clear why the apparent increase in SR Ca²⁺ content with FK-506 was a somewhat heterogeneous observation. It is possible that there is some variation in the degree of inhibition of Na⁺-Ca²⁺ exchange. That is, in some cells, the slowed Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange may only just offset the anticipated SR "unloading effect" of the increased fractional SR Ca²⁺ release discussed above. However, in most cells, the Na⁺-Ca²⁺ exchange inhibition may be even stronger, resulting in a net increase in SR Ca²⁺ content. Furthermore, even for a given modest decrease in the rate of Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange, some cells might still be able to extrude the same amount of Ca²⁺ during a cardiac cycle but simply take longer to accomplish it. Thus, the inhibition of Na⁺-Ca²⁺ exchange and the typical increase in SS SR Ca²⁺ load are probably related.

FK-506 and Inhibition of Na⁺-Ca²⁺ Exchange
The inhibitory effect of FK-506 on Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange could be due to a direct effect of the agent on the Na⁺-Ca²⁺ exchanger or it could be secondary to other changes that limit the ability of the Na⁺-Ca²⁺ exchange to extrude Ca²⁺. Since FK-506 was found to reduce Na⁺,K⁺-ATPase activity via inhibition of the phosphatase calcineurin in renal cells,^{33 34} even a slight elevation of [Na⁺]_i could significantly alter the ability of the Na⁺-Ca²⁺ exchanger to extrude Ca²⁺. Indeed, since FK-506 did not directly inhibit either the Na⁺,K⁺-ATPase or the Na⁺-Ca²⁺ exchange measured in homogenate or vesicle preparations, the cellular environment may be important in the inhibitory effect. Thus, these results point to an indirect effect on one of these systems as a possible mediator of the decrease in Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange in the cellular studies. The nature of this effect will require further study.

Note that the slowing of Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange observed in the present study would not help to explain the accelerated rest decay of SR Ca²⁺ content (ie, slower Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange would be expected to slow rest decay of SR Ca²⁺). This must mean that the increase in resting SR Ca²⁺ release via FK-506 is dominant over the degree of Na⁺-Ca²⁺ exchange inhibition with respect to rest decay. That is, even a moderately reduced Na⁺-Ca²⁺ exchange may extrude more Ca²⁺ per unit time because of a greater increase in SR Ca²⁺ leak during rest. Given the 300% increase in Ca²⁺ spark frequency and the mean 25% decrease in Ca²⁺ extrusion via Na⁺-Ca²⁺ exchange, this general interpretation seems plausible.

Conclusions
FK-506 consistently increased the amplitude of SS twitches and Ca²⁺ transients and accelerated the resting loss of SR Ca²⁺. These two effects may be attributed to increased opening of the SR Ca²⁺ release channel during E-C coupling as well as rest. In most cells, there was also an increase in SS SR Ca²⁺ load and a slowed decline of [Ca²⁺]_i during CafCs. Both of these effects might be due to a decrease in the ability of Na⁺-Ca²⁺ exchange to extrude Ca²⁺ from the cytosol. The effect of FK-506 on Na⁺-Ca²⁺ exchange seems likely to be indirect, since neither Na⁺-Ca²⁺ exchange nor Na⁺,K⁺-ATPase appears to be directly affected by FK-506 in isolated systems.

From a more integrated physiological perspective, FK-506 produces a positive inotropic effect on rat ventricular muscle, apparently without altering myofilament Ca²⁺ sensitivity, I_Ca, or the SR Ca²⁺-ATPase. This positive inotropic effect is probably due to an increase in the fraction of SR Ca²⁺ released during a normal twitch. However, the increase in resting Ca²⁺ sparks (and waves) and the increase in SS SR Ca²⁺ load sometimes observed could both be proarrhythmogenic. That is, the elevated SR Ca²⁺ loading might contribute to Ca²⁺ overload and triggered arrhythmias, whereas the increased spark frequency (and appearance of waves) could predispose the myocardium to propagation of local Ca²⁺ release events. Although there is little clinical evidence of cardiac toxicity of FK-506, there has been a report of hypertrophic cardiomyopathy in pediatric transplant patients.⁴⁴

	Selected Abbreviations and Acronyms

 

0Na,0Ca = Na+- and Ca2+-free solution CafC = caffeine-induced contracture E-C = excitation-contraction FKBP = FK-506 binding protein ICa = Ca2+ current LSCM = laser scanning confocal microscope pNPPase = para-nitrophenylphosphatase RyR = ryanodine receptor SR = sarcoplasmic reticulum SS = steady state

	Acknowledgments

 
This study was supported by grants from the US Public Health Service (HL-30077 and HL-51941), American Heart Association, and Schweppe Foundation. Dr Blatter is an Established Investigator of the American Heart Association. The authors wish to thank Christina Zakavec Hovance for her careful work in isolating cardiac myocytes and performing biochemical assays. We would also like to thank Melanie Robinson for help with the biochemical assays and J.L. Puglisi and Dr Kenneth S. Ginsburg for help with the electrophysiological experiments.

	Footnotes

 
Previously presented in part in abstract form (Biophys J. 1995;68:A176; J Mol Cell Cardiol. 1996;28:A131).

Received June 24, 1996; accepted September 17, 1996.

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