INTRODUCTION

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Since the discovery of Ca²⁺ transport into energized mitochondria about 30 years ago, numerous studies have been conducted on its kinetics and regulation in mitochondria isolated from various tissues (for review, see Gunter and Pfeiffer (1)). It is now generally accepted that mitochondria act as a sink during cytosolic Ca²⁺ overload in diseased or damaged cells. However, the physiological role of Ca²⁺ transport into mitochondria in intact cells remains unresolved. In recent years, evidence has been accumulating that Ca²⁺ transport into mitochondria occurs in stimulated cells. For instance, it has been shown that mitochondria accumulate Ca²⁺ from an intracellular microdomain of high Ca²⁺ upon stimulation of cloned HeLa cells that express the Ca²⁺-sensitive photoprotein aequorin in mitochondria (2). Ca²⁺ uptake into mitochondria in situ has also been observed in stimulated pancreatic cells (3, 4), liver cells (5), neuronal cells (6-9), and adrenal chromaffin cells (10, 11). These studies indicate that mitochondria accumulate Ca²⁺ when the cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) increases in stimulated cells. In cardiac muscle cells, mitochondria appear to accumulate only about 1% of the [Ca²⁺]_c during relaxation of a single twitch (12). This fraction is not enough to significantly affect [Ca²⁺]_c and cell contractility. However, increase in mitochondrial matrix free Ca²⁺ concentration ([Ca²⁺]_m) has been observed when myocytes were stimulated to increase [Ca²⁺]_c (13-18). An increase in mitochondrial total Ca content with increased workload has also been observed in isolated cardiac myocytes by x-ray electron probe microanalysis (19). Increase in [Ca²⁺]_m in vivo in isolated heart preparations with increased workload has also been observed (20). The significance of Ca²⁺ uptake into mitochondria and increase in [Ca²⁺]_m in cardiac and other cell types, however, has not been elucidated. It has been suggested that the increase in [Ca²⁺]_m may stimulate Ca²⁺-sensitive matrix dehydrogenases, and thus augment the rate of ATP synthesis to meet the heightened energy demand (21-26). Verification of this hypothesis requires demonstration of Ca²⁺ uptake into mitochondria and the consequent increase in matrix NAD(P)H and ATP synthesis in stimulated cardiac myocytes. The conclusions of the studies referred to above are based on presumed localization of Ca²⁺-sensitive dyes in mitochondria and their localization, or use of nonspecific inhibitors to block Ca²⁺ uptake into mitochondria. Moreover, the electron probe microanalysis approach in isolated hearts failed to detect any significant increase in mitochondrial total calcium content with isoproterenol stimulation, and yet an increase in pyruvate dehydrogenase activity was observed (27). A specific inhibitor of Ca²⁺ uptake into mitochondria will be helpful to elucidate the role of Ca²⁺ uptake into mitochondria in cardiac muscle and to clarify certain controversies.

Ruthenium red (RR)¹ has been shown to inhibit Ca²⁺ uptake into isolated mitochondria (28, 29). However, it was also shown to inhibit Ca²⁺ release from SR (30). It was found to produce both positive or negative inotropic effects in isolated rat hearts depending on its concentration in the perfusion solution (31). These effects of RR were attributed to its ability to inhibit SR Ca²⁺ release or sarcolemmal Na⁺/Ca²⁺ exchange. The inhibition of Ca²⁺ uptake into mitochondria however was shown to be due to a contaminant in the commercial preparations of RR (32). Recently, an oxygen-bridged dimeric ruthenium amine complex, which absorbs light at 360 nm (named Ru360), has been reported to inhibit Ca²⁺-stimulated respiration in isolated liver mitochondria (33, 34). This compound would be an extremely useful tool for elucidating the role or the contribution of Ca²⁺ uptake into mitochondria in vivo in isolated hearts or in situ in isolated cardiomyocytes if it penetrates the cell membrane but does not affect other cellular Ca²⁺ transport processes or cardiac cell contractility. To date, the specificity of Ru360 in inhibiting Ca²⁺ uptake into mitochondria in vitro or in intact cells has not been determined.

The objectives of this study are to determine the: 1) potency, effectiveness, binding, and mechanism of action of Ru360 on Ca²⁺ uptake into mitochondria in vitro; 2) effects of Ru360 on processes involved in cardiac contraction, such as SR Ca²⁺ uptake and release, SL Na⁺/Ca²⁺ exchange, L-type Ca²⁺ current, and myofibrilar actomyosin ATPase; 3) effects of Ru360 on [Ca²⁺]_c transient and cell shortening; and 4) uptake of Ru360 into intact cardiac myocytes and inhibition of Ca²⁺ uptake into mitochondria in situ. The results demonstrate that Ru360 binds to mitochondria with high affinity and specifically blocks Ca²⁺ uptake into mitochondria in vitro and in situ in intact myocytes without affecting other cellular processes involved in cardiac contraction.

	EXPERIMENTAL PROCEDURES

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Preparation of Ru360 and ¹⁰³Ru360-- A procedure similar to that described by Ying et al. (33) was used to synthesize Ru360 starting with RuCl₃ (Sigma). The purified and crystallized preparation was reddish-brown and exhibited _max at 360 nm in distilled water and virtually no trace of light absorbance at 533 nm, unlike RR (K & K Co./ICN product) which exhibited _max at 533 nm and a trace of light absorbance at 360 nm. ¹⁰³Ru360 was synthesized from ¹⁰³RuCl₃ by a procedure similar to that described by Ying et al. (33).

Determination of the Chemical Structure of Ru360-- The molecular structure and crystal packing were determined by x-ray crystallography. A platelike crystal (approximately 0.4 × 0.2 × 0.1 mm) was mounted onto the tip of a glass fiber with epoxy resin. Intensity data at low (233 K) and room temperature (293 K) were collected in a Siemens molecular analytical research tool (SMART, v4.05) CCD diffractometer on Mo K radiation (analytical x-ray instruments, Siemens, Madison, WI). The data frames were processed, and appropriate corrections for decay and Lorentz polarization effects were applied using Siemens area detector INTegration routine (SAINT, v4.05). Semiempirical absorption and beam corrections were applied using Siemens area detector ABSorption correction routine (SADABS, by G. M. Sheldrick, University of Goettengen, Germany). The structure was solved by a combination of direct method of crystal analysis (SHELXTL, v5.03, by G. M. Sheldrick) and the difference Fourier technique, and refined by full-matrix least squares on F². Non-hydrogen atoms were refined with anisotropic displacement parameters with the exception of C #l, which was refined isotropically. The formate atoms (O #2, C #l, O #3) were held at the atomic positions where first located during the subsequent refinement. C #l showed large thermal motion, which when left to refine, led eventually to an unstable refinement. The positions of H-atom were calculated and treated with a standard riding model. The largest residual electron density was located near the disordered formate ligand.

Isolation of Mitochondria from Rat Heart-- Mitochondria were isolated from rat cardiac ventricles by the method of Matlib et al. (35). Male Wistar rats weighing about 300 g were anesthetized with 30 mg of nembutal/kg. The chest cavity was surgically opened, the heart excised, and immediately placed in ice-cold saline solution. After rinsing, the atria and aorta were cut off, and the ventricles immersed in an ice-cold medium containing 180 mM KCl, 10 mM EGTA, and 0.5% bovine serum albumin, pH 7.2. All subsequent steps were carried out at 0-4 °C. The tissue was weighed, minced with scissors, and homogenized with a glass-Teflon tissue homogenizer (Thomas Scientific, size C) according to procedure B (35). Mitochondria were isolated by differential centrifugation (35). The first crude mitochondrial pellet was resuspended in a medium containing 180 mM KCl and 10 mM EGTA, pH 7.4. The last wash was carried out in a medium containing 180 mM KCl and 0.05 mM EGTA, pH 7.2. The final pellet was suspended in this medium at 40-50 mg protein/ml. Protein concentration was determined by the Lowry et al. (36) method using bovine serum albumin to construct a linear standard curve.

Measurements of Ca²⁺ Uptake and Na⁺-induced Ca²⁺ Release from Mitochondria Isolated from Rat Heart-- The rate of Ca²⁺ uptake into isolated mitochondria and the rate of Na⁺-induced Ca²⁺ release at 37 °C were determined spectrophotometrically using arsenazo III (37). The assay medium (3 ml) contained 120 mM KCl, 10 mM MOPS-KOH buffer (pH 7.2), 5 mM pyruvate, 5 mM malate, 2 mM potassium phosphate buffer (pH 7.2), 50 µM arsenazo III, and 1 mg of protein. Ru360 or RR, at the desired final concentration, was added 1 min before Ca²⁺ uptake was initiated with the addition of 50 nmol of CaCl₂. To measure the rate of Na⁺-induced Ca²⁺ release, the mitochondria were allowed to accumulate added Ca²⁺ (50 nmol of CaCl₂) as described above. When the uptake of Ca²⁺ was completed, Ru360 or RR was added. One minute after the addition of Ru360 or RR, Na⁺-induced Ca²⁺ release was initiated by adding 10 mM NaCl.

Binding of ¹⁰³Ru360 to Isolated Mitochondria-- Isolated heart mitochondria (7 mg of protein) were incubated at 22 °C in 35 ml of medium containing 120 mM KCl, 10 mM MOPS-KOH buffer (pH 7.2), and 0.1-1 nM ¹⁰³Ru360. After 30 min of incubation, the samples were centrifuged at 20,000 × g for 10 min, and the supernatant was discarded. The surface of the pellet was gently rinsed twice with the above medium. The pellet was resuspended in a small volume, transferred into a test tube, and counted in a gamma counter. Nonspecific binding was determined by incubating the samples containing 0.1-1 nM ¹⁰³Ru360 in the presence of 10 µM unlabeled Ru360. Specific binding was determined by subtracting the nonspecific binding from the total binding.

Measurement of Ca²⁺ Release from SR in Situ in Lysed Cardiomyocytes-- The rate of Ca²⁺ uptake into SR in situ in digitonin-permeabilized rat cardiomyocytes in the presence and absence of Ru360 or RR (K & K/ICN Product) was determined at 37 °C in a medium containing 100 mM NaCl, 11 mM glucose, 20 mM BES, 0.2 mM EGTA, 19 µM rotenone, 10 µM oligomycin, 1 mM dithiothreitol, 10 mM phosphocreatine, 0.2 units/ml creatine phosphokinase, 10 mM Mg-ATP, and ⁴⁵CaCl₂ to maintain a free Ca²⁺ concentration of 1 µM at pH 7.2 (38). The increase in the rate of Ca²⁺ uptake in the presence of RR was used as an indication of inhibition of Ca²⁺ release from SR as established previously (38).

Measurement of Ca²⁺ Uptake into Isolated SR Vesicles-- SR vesicles from rat hearts were isolated according to a previously described procedure (39). The excised hearts were rinsed with ice-cold saline, immediately frozen in liquid nitrogen, and stored at 70 °C. The frozen hearts were powdered with stainless steel mortar and pestle in liquid nitrogen, and the powder was suspended in a solution containing 300 mM sucrose and 20 mM Tris-maleate, pH 7.0. The suspension was homogenized (20 strokes at about 100 rpm) in a glass Potter Elvehjelm fitted with a Teflon piston. The homogenate was centrifuged at 1000 × g for 15 min and the supernatant was saved. The pellet was resuspended in the same solution, homogenized, and centrifuged at 1000 × g for 15 min, and the subsequent supernatant was combined with the saved supernatant. The combined supernatant was centrifuged at 10,000 × g for 20 min and was filtered through four layers of cheesecloth. KCl was added to a final concentration of 0.6 M. The solution was centrifuged at 100,000 × g for 1 h, and the pellet was resuspended in same volume of 300 mM sucrose and 20 mM Tris-maleate buffer (pH 7.0). The suspension was centrifuged at 100,000 × g for 1 h, and the pellet was resuspended in a small volume of 20 mM Tris-maleate, 300 mM sucrose, and 100 mM KCl, pH 7.0.

Measurement of Na⁺-Ca²⁺ Exchange Activity of Isolated SL Vesicles-- SL vesicles from rat heart were isolated according to a procedure described previously (41), with the exception that the supernatant solution, after the first 8000 × g centrifugation and sedimentation of mitochondria, was used for the isolation of sarcolemmal vesicles. Ca²⁺ uptake into 150 mM Na⁺-loaded SL vesicles at 10 µM free Ca²⁺ was determined using ⁴⁵CaCl₂ as a tracer and Millipore filtration technique according to a previously described procedure (41).

Measurement of Actomyosin Ca²⁺-ATPase Activity of Isolated Myofibrils-- Myofibrils from rat heart were isolated by homogenization and centrifugation according to a previously described procedure (42). Ca²⁺-stimulated actomyosin ATPase activity in the presence and absence of Ru360 was determined according to a previously described procedure (43), in a solution containing 60 mM KCl, 30 mM imidazole, 7.5 mM MgCl₂, 5 mM sodium ATP, 2.4 µM thapsigargin to inhibit SR Ca²⁺-ATPase, 5 mM NaN₃ to inhibit mitochondrial ATPase, 1 mM EGTA and CaCl₂ to yield 10 µM free Ca²⁺ at pH 7.0.

Measurement of Voltage-dependent Ca²⁺ Channel Current in Heart Cells-- Cardiac myocytes from adult rat heart were isolated as described previously (44). The isolated cells were placed into a small chamber (1.4 ml which contained the external test solution) on the stage of an inverted microscope. The cells were constantly perfused with the external test solution at rate of 1.8 ml/min. The external test solution contained in mM: 150 tetraethylammonium chloride, 1.8 CaCl₂, 0.5 MgCl₂, 3,4-aminopyridine, 3 Hepes, 5.5 glucose, pH 7.4 adjusted with HCl. Voltage-clamp recordings were performed in whole-cell configuration of the patch-clamp method by using patch-clamp amplifier (Axopatch-1D, Axon Instruments, Foster City, CA) and fire-polished borosilicate glass pipettes (World Precision Instruments, Sarasota, FL) with resistance of 2-6 M when filled with pipette solution containing in mM: 110 CsOH, 20 CsCl, 110 L-glutamic acid, 3 MgCl₂, 5 disodium ATP, 5 disodium creatine phosphate, 10 EGTA, 5 Hepes, pH 7.2 adjusted with CsOH. The I_Ca(L) were elicited from a holding potential of 40 mV to test potential of +10 mV for 300 ms every 15 s in cells untreated or pretreated with 10 µM Ru360 at 22-25 °C.

Measurement of ¹⁰³Ru360 Uptake into Myocytes Isolated from Rat Heart-- Myocytes were isolated from rat hearts as described previously (38). The myocyte preparations used in this study were about 85% rod-shaped and Ca²⁺ tolerant. To determine the uptake of ¹⁰³Ru360, 2 × 10⁵ cells were suspended in 1 ml of Joklick's medium (Life Technologies, Inc.) containing 1 µM ¹⁰³Ru360 and incubated at 37 °C. At 1, 3, 5, 10, 15, 20, and 30 min after the addition of ¹⁰³Ru360, the cells in Microfuge tubes were sedimented by centrifugation at 12,000 × g for 15 s and washed twice with 1 ml of Joklick's medium without Ru360. The pellet was counted in a gamma counter. The amount (picomoles) of Ru360 taken up at each time point was calculated from the specific activity of the ¹⁰³Ru360.

Measurement of Cytosolic Ca²⁺ Transients and Shortening of Isolated Single Cardiac Myocytes-- Myocytes from rat heart were isolated according to Wimsatt et al. (38) and suspended in Krebs-Henseleit medium containing 1 mM CaCl₂ and 25 mM Hepes buffer (pH 7.4). The cells were loaded with indo-1 by incubating cells in 3 µM indo-1/AM for 20 min at 37 °C. The cells were washed and further incubated at room temperature for 30 min to complete the conversion of indo-1 ester to its free acid. The cells were allowed to attach on the surface of a glass coverslip in a plastic chamber (0.3-ml volume). The cells were continuously perfused at a rate of 0.5 ml/min with buffered Krebs-Henseleit solution containing 1 mM CaCl₂ at 22 °C in the absence or presence of 10 µM Ru360. They were field-stimulated at 0.2 Hz (pulses of 4-ms duration) with platinum wires attached to a Grass S9 stimulator. Cell shortening was recorded with a video-edge detection system (Crescent Electronics, Salt Lake City, UT). Cytosolic indo-1 fluorescence emission ratio of 405 and 485 nm with 365 nm excitation was recorded with a P. T. I. Deltascan Photometer (Photon Technology International, Monmouth, NJ) attached to a Nikon Diaphot-200 inverted microscope.

Measurement of Ca²⁺ Uptake into Mitochondria in Situ in Isolated Intact Cardiac Myocytes-- Ventricular myocytes were isolated from adult ferret hearts (45). The effect of Ru360 was determined under voltage-clamped conditions in which the cytosol was progressively loaded with Ca²⁺ via the SL Na⁺/Ca²⁺ exchanger as described previously (46). Standard whole-cell recording techniques and Axopatch-1B amplifier (Axon Instruments) were used for electrophysiological recordings. The holding potential was 40 mV. To evoke Ca²⁺ influx into the cell, depolarization pulses to +110 mV were applied, which allows Ca²⁺ entry via the Na⁺/Ca²⁺ exchanger (46). Signals for whole-cell current, cell contraction, and indo-1 fluorescence signals at two wavelengths (400 and 500 nm) were simultaneously recorded using pClamp 6 (Axon Instruments) and an IBM-PC compatible computer at a sampling rate of 125-500 Hz. Intracellular Ca²⁺ signals were detected as described previously (46). The cells were loaded with indo-1 by incubation in Tyrode solution containing 5 µM indo-1/AM for 40-45 min at 37 °C. This condition allows loading of the cells with 0.5-1.0 mM indo-1, approximately 75% of which was found to be in mitochondria (46). The indo-1 fluorescence was converted into intracellular free Ca²⁺ concentration ([Ca²⁺]_i), representing both [Ca²⁺]_c and [Ca²⁺]_m, by two different methods. In the first method, [Ca²⁺]_i was calculated from the indo-1 fluorescence ratio according to Grynkiewicz et al. (47)

(Eq. 1)

(Eq. 2)

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

Chemical Structure-- The chemical structure of the compound is presented in Fig. 1. The compound we synthesized has two ruthenium amine-formate nuclei bridged with an oxygen atom. It has three positive charges. Each ruthenium atom is positively charged with the remaining charge delocalized between the Ru-O-Ru bridge. The deduced chemical formula is C₂H₂₆N₈O₅Ru₂Cl₃ and the calculated molecular weight is 550.5. A solution of 18 µM Ru360 in distilled water exhibited maximum light absorbance at 360 nm, with no detectable absorbance at 533 nm indicating that the preparation is free from ruthenium red contamination (not shown). The x-ray crystallographic data were identical to Ru360 reported previously (34). Thus, the light absorbance, the chemical structure derived from crystallograpy, and the molecular weight data led to the conclusion that the compound is Ru360 reported by Ying et al. (33) and Emerson et al. (34).

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Chemical structure of Ru360 as revealed by x-ray crystallography. The compound is oxygen-bridged dinuclear amine complex. The compound has three Cl1 ions.

Effect of Ru360 and RR on Ca²⁺ Uptake and Na⁺-induced Ca²⁺ Release from Mitochondria in Vitro-- Ru360 has been shown to inhibit Ca²⁺-stimulated respiration of isolated rat liver mitochondria (33). However, its effect on Ca²⁺ uptake into isolated heart mitochondria has not been studied. Therefore, we conducted experiments to determine whether Ru360 was effective in inhibiting Ca²⁺ uptake into heart mitochondria, and whether it was more potent than RR. Increasing concentration of Ru360 progressively inhibited the rate of Ca²⁺ uptake into isolated rat heart mitochondria (Fig. 2A). The concentration of Ru360 which inhibited 50% (IC₅₀) of the control rate was 0.184 nM or 0.55 pmol/mg mitochondrial protein. RR also inhibited the rate of Ca²⁺ uptake into mitochondria with increasing concentration (Fig. 2B). However, the IC₅₀ was 6.85 nM or 20 pmol/mg of protein. Ru360 produced no effect on Na⁺-induced Ca²⁺ release from mitochondria (Fig. 2C). RR also produced no effect on the rate of Na⁺-induced Ca²⁺ release from mitochondria (not shown). These data indicate that Ru360 is about 40 times more potent than RR in inhibiting Ca²⁺ uptake into heart mitochondria in vitro. The lack of effect on Na⁺-induced Ca²⁺ release process indicates that Ru360 is a selective inhibitor of Ca²⁺ uptake into mitochondria in vitro.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   The inhibition of the rate of Ca2+ uptake into isolated rat heart mitochondria (Mito) by Ru360 (A) and RR (B). The data in A were collected from 10 separate mitochondrial preparations and with three separate Ru360 preparations. The vertical bars represent S.E. of the mean (n = 10). The data in B were collected from six mitochondrial preparations. Each data point is the mean ± S.E. (n = 6). The concentration required to inhibit 50% of the rate of untreated mitochondria (IC50) is presented. The data (mean ± S.E., n = 4) on the effect of Ru360 on the rate of Na+-induced Ca2+ release from mitochondria are also presented (C). The equation (Y = Vmax (1  (X/(X + IC50)))), where X = [Ru360] or [RR], was used to fit the curves in A and B.

Binding of ¹⁰³Ru360 to Isolated Mitochondria-- To determine whether binding of Ru360 to membrane causes inhibition of Ca²⁺ uptake into mitochondria, binding of ¹⁰³Ru-labeled Ru360 and its potency to inhibit Ca²⁺ uptake were studied. ¹⁰³Ru360 binds to isolated mitochondria in a saturable manner (Fig. 3). The total binding at all concentrations of ¹⁰³Ru360 was 4-5% of that in the medium, indicating that the ligand concentration was not a limiting factor. The nonspecific binding was about 30% of the total binding and increased linearly with increasing ¹⁰³Ru360 concentration (not shown). The maximum binding (B_max) was found to be 80 fmol/mg of protein with a dissociation constant (K_d) of 0.34 nM. The K_d is comparable to the functional IC₅₀ measured in Fig. 2A. These data indicate that Ru360 binds to a specific site in mitochondrial membrane, and that binding to this site is most likely responsible for inhibition of Ca²⁺ uptake into mitochondria.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Specific binding of 103Ru360 to isolated rat heart mitochondria as a function of 103Ru360 concentration. Nonspecific binding in the presence of unlabeled 10 µM Ru360 was approximately 30% of the total binding and linear with increasing concentrations of 103Ru360. The specific binding curve was fitted with the equation Bmax/(1 + Kd/[Ru360]).

Effect of Ru360 and RR on Ca²⁺ Release from SR in Situ-- The rate of Ca²⁺ uptake into SR is increased due to the inhibition of ryanodine-sensitive Ca²⁺-induced Ca²⁺ release channel by RR (38). As expected, RR increased Ca²⁺ uptake into SR in digitonin-lysed myocytes by about 10-fold (Fig. 4A). On the other hand, Ru360 produced very little effect even at concentration as high as 30 µM (Fig. 4A). These data indicate that, unlike RR, Ru360 does not inhibit ryanodine-sensitive Ca²⁺-induced Ca²⁺ release channel in the SR.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effects of Ru360 and RR on Ca2+ release from SR in situ in isolated and digitonin-lysed rat cardiac myocytes (A), and on Ca2+ uptake into isolated SR vesicles (B). The rate of Ca2+ uptake into SR was determined at 1 µM free Ca2+ concentration in the absence and presence of RR or Ru360. RR increases the rate of Ca2+ uptake by inhibiting the release process. Each data point represents mean ± S.E. of 4 separate myocytes preparations. The rate of Ca2+ uptake into isolated SR vesicles as a function of free Ca2+ in the absence or presence of 10 µM Ru360 was determined in triplicate. The data were fitted with a nonlinear curve fitting program. The horizontal bars represent S.E. of the mean of triplicate determinations. Represented are four similar experiments each with a different membrane preparation. The curves in B were fitted with the equation Y = {(Vmax  Vmin)/(1 + EC50/[Ca2+])n} + Vmin, where Vmax and Vmin are maximum and minimum rate, respectively, and n is the slope of the curve.

Effect of Ru360 on Ca²⁺ Uptake into Isolated SR Vesicles-- It could be argued that the lack of effect of Ru360 on Ca²⁺ release from SR measured in lysed cardiomyocytes was due to its inhibitory effect on the Ca²⁺ uptake process. Therefore, the effect of Ru360 on the rate of Ca²⁺ uptake of isolated SR vesicles was determined. The [Ca²⁺] for half of the maximum rate of Ca²⁺ uptake (EC₅₀) was 0.20 ± 0.02 µM Ca²⁺, and the maximum rate (V_max) was 63.5 ± 4.4 nmol/min/mg in untreated control vesicles (Fig. 4B). Ru360 (10 µM) exerted no effect on either the EC₅₀ (0.20 ± 0.01 µM) or the maximum rate (58.9 ± 8.0 nmol/min/mg) of Ca²⁺ uptake into isolated SR vesicles (n = 4). The data indicate that Ru360 does not inhibit Ca²⁺ uptake into SR. Therefore, the lack of effect of Ru360 on ryanodine-sensitive Ca²⁺-induced Ca²⁺ release from SR of lysed myocytes in Fig. 4A was not due to inhibition of the Ca²⁺ uptake process.

Effect of Ru360 on Sarcolemmal Na⁺/Ca²⁺ Exchanger-- It has been suggested that RR interferes with sarcolemmal Na⁺/Ca²⁺ exchange activity in isolated heart preparations (31). Therefore, the effect of Ru360 on Ca²⁺ uptake into Na⁺-loaded sarcolemmal vesicles was determined. Ru360 (10 µM) produced no effect on Ca²⁺ uptake into Na⁺-loaded sarcolemmal vesicles (Fig. 5A). However, dichlorobenzamil, which is known to inhibit sarcolemmal Na⁺/Ca²⁺ exchanger, inhibited Ca²⁺ uptake into Na⁺-loaded sarcolemmal vesicles (not shown).

View larger version (39K): [in this window] [in a new window]   Fig. 5.   The effect of Ru360 on Na+/Ca2+ exchange activity in sarcolemmal vesicles (A), and on Ca2+-stimulated actomyosin ATPase activity in myofibrils (B), isolated from rat heart. The rate of Ca2+ uptake into 150 mM Na+-loaded SL vesicles was determined in the absence or presence of 10 µM Ru360. The data in A represent mean ± S.E. of three experiments. The rate of thapsigargin-insensitive Ca2+-stimulated ATPase activity in the presence and absence of 10 µM Ru360 is presented. The data in B represent the mean ± S.E. of three experiments.

Effect of Ru360 on Myofibrilar Actomyosin-ATPase-- Ca²⁺-stimulated actomyosin ATPase plays a crucial part in cardiac muscle contraction. Therefore, we examined the effect of Ru360 on Ca²⁺-stimulated actomyosin ATPase activity in myofibrils isolated from rat hearts. Ru360 at 10 µM produced no effect on Ca²⁺-stimulated actomyosin ATPase activity in isolated myofibrils (Fig. 5B).

Effect of Ru360 on Voltage-dependent L-type Ca²⁺ Channel Current-- It has been suggested that RR inhibits voltage-dependent Ca²⁺ channel current in leukemia cells (48). To determine whether Ru360 produces a similar effect in cardiac myocytes, we examined its effects on voltage-dependent L-type Ca²⁺ channel current (I_Ca(L)) in whole-cell voltage-clamped single cardiac myocytes. Ru360 at 10 µM produced no significant effect on the voltage-dependent Ca²⁺ channel current amplitude or kinetics (Fig. 6).

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Effect of Ru360 on voltage-dependent L-type Ca2+ channel current in whole-cell patch-clamped single myocytes. The holding potential was 40 mV and the test potential was +10 mV. The amplitude and kinetics of the Ca2+ current was determined 1 min (a) and 3-6 min (b) after 10 µM Ru360 treatment, and after 5 min washout (c). Inset, Mean ± S.E. of data from 13 different cells.

¹⁰³Ru360 Uptake into Isolated Cardiomyocytes-- To be effective in inhibiting Ca²⁺ uptake into mitochondria in situ in isolated cardiomyocytes, Ru360 must enter the cells. Therefore, we determined whether Ru360 enters and accumulates into myocytes in a time-dependent manner. In this study, predominantly (80-90%) rod-shaped isolated cardiomyocytes were incubated at 37 °C in Joklick's medium containing 1 µM ¹⁰³Ru360, and its accumulation was determined after sedimentation of cells by centrifugation and removal of the free ¹⁰³Ru360 remaining in the supernatant solution. ¹⁰³Ru360 was taken up by myocytes with time of incubation in a biphasic manner (Fig. 7). The initial very rapid phase (_f = 3.6 s) probably reflects Ru360 binding to the outer surface of the cell. The amount bound at the surface (7.5 pmol of ¹⁰³Ru360/10⁶ cells) would correspond to ~250 nmol/liter cell volume (assuming a 30-pl cell volume) (49) or 1500 molecules/µm² of cell surface (assuming 1 µF/cm² membrane and 7 pF/pl) (49). The slow phase is almost 500 times slower (_s = 28 min) and probably reflects Ru360 gradually entering the cell. After 30 min the slow phase would be ~3 pmol/10⁶ cells or ~100 nmol/liter of cell volume.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Uptake of 103Ru360 into isolated myocytes from rat heart. The time-dependent uptake from 1 µM 103Ru360 is presented. The data represent the mean ± S.E. of six experiments. Uptake = Ampf (1  exp(t/f)) + Amps (1  exp(t/s)). f, fast; s, slow.

Effect on Contraction and Cytosolic Free Ca²⁺ Transients of Isolated Single Cardiac Myocytes-- Indo-1 loaded single myocytes perfused with Krebs-Henseleit-(20 mM) Hepes buffer (pH 7.4) and stimulated at 0.2 Hz exhibits normal contraction and [Ca²⁺]_c transients before treatment with Ru360. Perfusion of the cells with 10 µM Ru360 up to 30 min did not produce any significant effect on contraction or [Ca²⁺]_c transients (Fig. 8). We have also studied the effects of 30 and 50 µM Ru360. Only 50 µM Ru360 significantly decreased the amplitude of [Ca²⁺]_c and cell shortening (not shown). The results indicate that Ru360 does not affect normal [Ca²⁺]_c transients and shortening of isolated cardiac myocytes at concentrations that inhibit Ca²⁺ uptake into mitochondria.

View larger version (50K): [in this window] [in a new window]   Fig. 8.   Effect of Ru360 on contraction and cytosolic free Ca2+ transients in single rat ventricular myocytes. The upper trace represents cell length, and the lower trace represents indo-1 fluorescence (405/485) ratio in a single myocyte stimulated at 0.2 Hz immediately before and after perfusion with 10 µM Ru360 up to 30 min. The data were recorded at 3, 10, 20, and 30 min after the initiation of Ru360 perfusion. The cell was stimulated continuously in the period between recordings. Inset numbers are mean of data from 10 beats ± S.E. of three different cells (100% = predrug values).

Inhibition of Ca²⁺ Uptake into Mitochondria in Situ in Isolated Single Cardiomyocytes-- To determine whether Ru360 can block mitochondrial Ca²⁺ uptake in intact cells, we applied combined whole-cell patch-clamp and indo-1 fluorescence measurements in single ferret ventricular myocytes. The cells were preloaded with indo-1 under conditions in which about 75% of the indo-1 was trapped inside mitochondria and about 25% was in the cytosol (46). Cells were progressively loaded with Ca²⁺ via the sarcolemmal Na⁺/Ca²⁺ exchanger with repeated strong depolarizations of +110 mV. A pure cytosolic Ca²⁺ signal can be derived from the contraction L, while the indo-1 signal (F₄₀₀/F₅₀₀ ratio) is a mixture of cytosolic and mitochondrial free Ca²⁺ concentrations (46). Comparison of the normalized L and the F₄₀₀/F₅₀₀ ratio show that a difference between L and F₄₀₀/F₅₀₀ ratio developed as the [Ca²⁺]_i gradually increased (Fig. 9A). We have shown previously (46) that this difference between L and F₄₀₀/F₅₀₀ ratio can be blocked by Ru360 dialyzed into the cell through the patch-pipette. In the present study, incubation of the cell with externally applied 10 µM Ru360 in normal Tyrode solution for 30 min resulted in complete blockade of both the sustained increase in [Ca²⁺]_i ratio and the development of the kinetic difference between L and F₄₀₀/F₅₀₀ ratio (Fig. 9B). Without Ru360, the [Ca²⁺]_i signal (mixed mitochondria and cytosolic signal) increases far above the purely cytosolic [Ca²⁺]_CL signal, but this is reversed with 30 min of exposure to 10 µM Ru360 (Fig. 9B, lower panels). This is also consistent with blockade of mitochondrial Ca²⁺ uptake by Ru360. That is, when [Ca²⁺]_m is high the mixed indo-1 signal overestimates [Ca²⁺]_c and when [Ca²⁺]_m is very low (with Ru360) the mixed [Ca²⁺] signal underestimates [Ca²⁺]_c. These results indicate that Ru360 enters the cell and blocks Ca²⁺ uptake into mitochondria in intact cardiac myocytes.

View larger version (23K): [in this window] [in a new window]   Fig. 9.   Inhibition of Ca2+ uptake into mitochondria in situ in single voltage-clamped ferret ventricular myocytes by Ru360. [Ca]CL represents cytosolic [Ca2+] estimated from cell contraction L. [Ca]i represents intracellular [Ca2+] estimated from indo-1 fluorescence in both cytosol (~25%) and mitochondria (~75%). Cytosolic and mitochondrial Ca2+ signals are represented by contraction (L and [Ca]CL) and indo-1 fluorescence (F400/F500 and [Ca2+]i) signals, respectively. To assess mitochondrial calcium uptake, the upper traces of F400/F500 ratios and L were normalized and scaled so that the kinetic difference between the two traces reflects Ca2+ uptake into mitochondria (46). The arrow indicates the difference between time course of the normalized F400/F500 and L signals (assumed to reflect Ca2+ uptake into mitochondria). Panel A represents experiments without Ru360. During the first pulse, the purely cytosolic signal (L) exactly overlaps on the F400/F500 signal (first arrow) indicating no Ca2+ uptake into mitochondria. When the basal [Ca2+]i was increased from 0.2 to 0.5 µM after 15 additional depolarization pulses, a dramatic difference between kinetics of L and indo-1 signal was observed (second arrow). At pulses 17 and 18, the cytosolic [Ca2+], as indicated by L, declined quickly to basal level, while F400/F500 ratio remained elevated indicating Ca2+ uptake into mitochondria and increase in [Ca2+]m. Panel B represents results of another patch-clamped cell in identical conditions to that in panel A except that 10 µM Ru360 was added to the bathing solution. Note the differences between L and F400/F500 ratio during decay phase is completely eliminated by 10 µM Ru360, indicating inhibition of Ca2+ uptake into mitochondria.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

The results presented here demonstrate that Ru360 is more potent and specific than RR in inhibiting Ca²⁺ uptake into heart mitochondria. The lack of effect of Ru360 on Na⁺-induced Ca²⁺ release process of mitochondria indicates that it selectively inhibits the Ca²⁺ uptake process. The concentrations at which Ru360 binds to mitochondria also inhibited Ca²⁺ uptake. The results suggest that binding of Ru360 to a high affinity site in the mitochondrial membrane causes inhibition of Ca²⁺ uptake into mitochondria. The number of binding sites in the mitochondrial membranes appear small (80 fmol/mg of protein). Assuming 40 mg of mitochondrial protein/ml cell volume, this corresponds to 3-4 nM Ru360 binding sites in the cell. Furthermore, the 120 nmol of Ca²⁺/mg/min uptake rate blocked by Ru360 would correspond to ~25,000 calcium ions/s/Ru360 binding site. This rate of Ca²⁺ influx would be consistent with an ion channel rather than other transport mechanisms. However, whether the Ru360 binding site is located in the Ca²⁺ uptake uniporter protein or in a regulatory protein molecule associated with the uniporter cannot be determined until the uniporter protein or the Ru360 binding site is identified.

Ruthenium red has been shown not only to inhibit Ca²⁺ uptake into mitochondria but also SR ryanodine-sensitive Ca²⁺ release channel (30), sarcolemmal voltage-dependent Ca²⁺ channel (48), and the sarcolemmal Na⁺-Ca²⁺ exchanger (31). These effects of RR preclude its usefulness as a specific inhibitor of Ca²⁺ uptake into mitochondria in intact cells or organs. Despite these effects, RR has been used and continues to be used in isolated cells and isolated hearts. Although the results obtained from these studies were often consistent with inhibition of Ca²⁺ uptake into mitochondria, the contributions of inhibition of Ca²⁺ release from SR, voltage-dependent Ca²⁺ channel, or sarcolemmal Na⁺/Ca²⁺ exchanger could not be completely excluded. Results presented here demonstrate that Ru360 does not produce any of these effects. These characteristics of Ru360 distinguish it from RR and establish it as a more potent and specific inhibitor of mitochondrial Ca²⁺ uptake.

Since Ru360 specifically inhibits Ca²⁺ uptake into mitochondria, it could be a very useful tool for estimating the contribution of mitochondrial Ca²⁺ transport in the regulation of [Ca²⁺]_m and [Ca²⁺]_c. Moreover, it will also be useful to 1) demonstrate cause-effect relationship of Ca²⁺ uptake into mitochondria, 2) estimate contribution of mitochondrial Ca²⁺ uptake into cell function in normal or diseased states, 3) eliminate mitochondrial Ca²⁺ transport when experimental condition dictates, 4) identify hitherto unknown role of mitochondrial Ca²⁺ uptake, 5) elucidate the mechanism of Ca²⁺ uptake into mitochondria, and 6) identify molecular nature of the mitochondrial Ca²⁺ uptake uniporter.

An ideal inhibitor should specifically bind to mitochondria with high affinity in the same concentration range as the inhibition of Ca²⁺ uptake, permeate the cell membrane, and specifically inhibit Ca²⁺ uptake into mitochondria in situ in intact cells. Results presented here demonstrate that ¹⁰³Ru360 is indeed taken up by myocardial cells and is accumulated in the cytosol in a biphasic manner. The very rapid phase of ¹⁰³Ru360 uptake may be due to binding to the cell surface while the slow phase could be due to intracellular accumulation. The relatively slow uptake could be due to the positive charge and/or low lipid solubility of the compound. After 30 min, the slow uptake may amount to ~100 nM (see "Results"). This is still 500 times the IC₅₀ shown in Fig. 2A and 25 times the number of mitochondrial Ru360 binding sites in the cell. Thus the quantity of cellular uptake of 1 µM Ru360 in 30 min is certainly more than sufficient to explain the functional blockade of mitochondrial Ca²⁺ uptake observed with 10 µM Ru360 in patch-clamped myocytes (Fig. 9). The results also indicate that micromolar extracellular concentrations of Ru360 and prolonged incubation are required for accumulation in cytosol at concentrations sufficient for the inhibition of Ca²⁺ uptake into mitochondria in intact cardiac myocytes. Nevertheless, the results presented here also show that micromolar concentrations of Ru360 produce no effect on contractility of single cardiac myocytes. Therefore, Ru360 can be used at low micromolar concentrations to inhibit Ca²⁺ uptake into mitochondria without affecting cellular processes involved in cardiac contractility.

Ca²⁺ uptake into mitochondria in a functioning single cardiac myocyte has been controversial. Some investigators have demonstrated Ca²⁺ uptake into mitochondria in stimulated cells (13-20), while others failed to observe any increase even though Ca²⁺-sensitive matrix pyruvate dehydrogenase activity was increased (27). The disparity in the results could be due to differences in the state of the cells, the workload imposed, and on the experimental approach. Ru360 will be useful in the resolution of this controversy by verifying that Ca²⁺ uptake into mitochondria is indeed the consequence of the specific interventions. To demonstrate the utility of Ru360 in the inhibition of Ca²⁺ uptake into mitochondria in situ in single cardiomyocytes, we have employed a procedure of simultaneous measurements of cell shortening by edge-detection, [Ca²⁺]_c from changes in cell length during systole, and [Ca²⁺]_m from indo-1 fluorescence in whole cell patch-clamped single cardiac myocytes (46). In this procedure, strong stimulation of the cell caused a sustained increase in intracellular indo-1 fluorescence the kinetics of which was dissociated from cell contraction and relaxation reflecting normal [Ca²⁺]_c transients. We attributed the increase in indo-1 fluorescence and slowed kinetics to Ca²⁺ uptake into mitochondria and resultant increase in [Ca²⁺]_m. This also matches our previous report of the same effect of Ru360 delivered intracellularly through the patch pipette (46). The present experiments demonstrate that Ru360 enters intact functioning myocytes and inhibits Ca²⁺ uptake into mitochondria in situ without affecting cell contractility. However, it required up to 30 min incubation of the cells with 10 µM Ru360 to observe complete block of Ca²⁺ uptake into mitochondria. The outcome of this experiment further confirms our observation that incubation of intact cardiac myocytes for a period of time with Ru360 is required for sufficient intracellular accumulation for the inhibition of Ca²⁺ uptake into mitochondria in situ. Furthermore, this observation is in quantitative agreement with the slow phase of ¹⁰³Ru360 uptake by isolated myocytes.

In summary, we demonstrate in this study that Ru360 inhibits Ca²⁺ uptake without affecting Na⁺-induced Ca²⁺ release from mitochondria. We observed no effect of Ru360 on Ca²⁺ uptake or release from SR, Na⁺/Ca²⁺ exchange activity of SL, voltage-dependent L-type Ca²⁺ channel activity of cell membrane, or Ca²⁺-stimulated actomyosin-ATPase activity of myofibrils. Also, we observed no effect of Ru360 on [Ca²⁺]_c and shortening transients in field-stimulated single cardiomyocytes. We conclude that Ru360 is a novel specific inhibitor of mitochondrial Ca²⁺ uptake. Furthermore, it is a valuable tool in studying Ca²⁺ regulation of mitochondria, both in isolation and in intact cells.