INTRODUCTION

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CYCLOSPORIN A (CsA) is a powerful immunosuppressant that is widely used to prevent organ rejection and to treat certain autoimmune diseases (30). The immunosuppressive properties of this agent have been extensively studied (17). The generally accepted view is that CsA is inactive by itself. However, on binding to cyclophilin A (CyPA), the major intracellular CsA binding protein, it forms a CsA-CyPA complex and inhibits the phosphatase activity of calcineurin (CaN), a key regulatory component of the T-cell activation pathway (15, 17, 27). Inhibition of CaN results in reduced interleukin-2 transcription and, finally, in suppression of T-lymphocyte activation (34).

Despite the effectiveness of CsA as an immunosuppressant, the clinical application of CsA is limited by various toxic side effects of this agent, such as nephrotoxicity and cardiotoxicity (30). One of the causes for nephrotoxicity is an angiotensin II-stimulated rise in intracellular free Ca²⁺ (31). Although the causes of cardiotoxicity are not fully understood, it is possible that changes in intracellular Ca²⁺ concentrations (3, 24) could be a common cause for CsA-induced side effects. Studies using cyclosporin analogs and metabolites have suggested that the effect of CsA on intracellular Ca²⁺ concentrations may be independent of the immunosuppressive activities of CsA (16, 28).

During excitation-contraction coupling, a membrane action potential allows extracellular Ca²⁺ to enter the cardiac muscle cell and intracellular Ca²⁺ to be released from the Ca²⁺-sequestering organelle, the sarcoplasmic reticulum (SR). This transient increase in cytoplasmic free Ca²⁺ concentration triggers muscle contraction (11, 28). Ca²⁺ release from the SR is mediated by Ca²⁺-release channels (CRC) located at the junction of terminal cisternae and transverse tubular membranes (9). Therefore, the Ca²⁺-release process mediated by CRC plays the major role in muscle contraction (1). A physiological association of CaN to CRC-FK506 binding protein (FKBP12) receptor complexes has been reported (7). The associated CaN could modulate the gating modes of CRC in a complex with FKBP. However, CaN may not be the regulator of CRC in the presence of FK506 or rapamycin, because these immunosuppressant drugs could dissociate CaN as well as FKBP from CRC (7).

Detailed effects of CsA treatment on mechanical alterations of heart have been reported from studies using rat models (3, 24). The reported mechanical alterations of the heart (3, 24) include reduction of peak systolic pressure, increased sensitivity of myocardium to external Ca²⁺, enhanced occurrence of spontaneous contractions (3), and reduced maximal stress development. From the physiological study of skinned cardiac trabeculae, Banijamali et al. (3) postulated that the altered mechanical properties in the CsA-treated rat hearts could be associated with alterations of the CRC functions.

The present study was performed by using [³H]ryanodine binding and oxalate-supported Ca²⁺ uptake to test the hypothesis that CsA-mediated mechanical alterations of heart muscles (3, 24) are caused by molecular changes in the CRC complex. Western blot analysis was also performed to examine the expression level of CaN. The results of this study indicate that chronic CsA treatment leads to cardiac muscle-specific quantitative as well as qualitative alterations of CRC, which could cause the mechanical alterations found in CsA-treated rat hearts (3, 24). The putative underlying molecular mechanisms responsible for the altered CRC functions are discussed.

	MATERIALS AND METHODS

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Materials. [³H]ryanodine and ⁴⁵CaCl₂ were obtained from NEN. Caffeine, ruthenium red, rapamycin, cremophor EL, potassium oxalate, and anti-CaN antibody were purchased from Sigma. CsA in cremophor (50 mg/ml) was obtained from Sandoz Pharmaceutical (Basel, Switzerland). All other chemicals used were of analytic grade.

Treatment of animals with of CsA. Male Sprague-Dawley rats weighing 280-360 g were divided into two groups. The CsA group was treated with CsA (3.75, 7.5, or 15 mg · kg body wt¹ · day¹) dissolved in cremophor for 1-3 wk by subcutaneous injection into the back of the neck, as described previously (24). The control group was treated with the same volume of cremophor. Handling and care of animals were guided by the institutional animal care committee.

Whole homogenate preparation. Rats were anesthetized with pentobarbital sodium (50 mg/kg body wt), and then hearts were removed from rats and immersed in ice-cold 0.9% NaCl. After atrial tissues, visible fat, and connective tissues were removed, a part of the left ventricular tissue was homogenized twice for 20 s in 20 mM MOPS (pH 7.4), 1 M KCl, 1 µM leupeptin, 1 µM pepstatin, 1 µM aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, and 10 µg/mg trypsin inhibitor by using a Polytron PT 10 probe (Brinkmann) at a speed setting of 5 (21). Whole homogenates of the slow- and fast-twitch skeletal muscles were made by using soleus for slow-twitch red (type I), the deep region of the vastus lateralis for fast-twitch red (type IIA), and the superficial region of the vastus lateralis for fast-twitch white (type IIB) (13).

Ryanodine binding. Equilibrium ryanodine binding to whole homogenates was performed by incubation of 2.5 mg of whole homogenate in 250 µl of reaction mixture containing 1 M KCl, 20 mM MOPS (pH 7.4), 20 nM [³H]ryanodine (54.7 Ci/mmol), 1 mM EGTA, and 0.98 mM CaCl₂ for 2 h at 37°C (21, 33). Direct effects of CsA on the ryanodine binding were examined at 5-20 µM CsA solubilized in DMSO. Ca²⁺-activated ryanodine binding was measured in either of two reaction solutions, one containing 1 M KCl, 20 mM MOPS (pH 7.4), 1 mM EGTA, 20 nM [³H]ryanodine (54.7 Ci/mmol), and varied amounts of CaCl₂ to produce free Ca²⁺ concentrations of 0.02-3.16 µM, and the other containing 0.15 M KCl, 20 mM MOPS (pH 6.8), 1 mM EGTA, 15 nM [³H]ryanodine (54.7 Ci/mmol), and varied amounts of CaCl₂ to produce 0.03-10 µM free Ca²⁺ (10). Caffeine-activated ryanodine binding was measured in the caffeine concentration range of 0.25-20 mM, and ruthenium red-inhibited ryanodine binding was measured in the ruthenium red concentration range of 0.1-20 µM. Percent increase in ryanodine binding activated by caffeine was calculated from the difference between the binding at each caffeine concentration and the binding in the absence of caffeine. Percent inhibition by ruthenium red was calculated from the difference between the binding at each ruthenium red concentration and the binding in the absence of ruthenium red. One hundred microliters of polyethylene glycol (PEG) solution (30% PEG, 1 mM EDTA, and 50 mM Tris, pH 7.4) was added to each vial, and incubation was continued for 5 min at room temperature (8). Precipitated protein was sedimented for 5 min at 14,000 rpm in an Eppendorf microcentrifuge, and the pellets were rinsed twice with 0.4 ml of the relevant ryanodine binding buffer without radioactive ryanodine. The pellets were then solubilized in 100 µl of Soluene 350 (Packard) at 70°C for 30 min, and the solution was counted in 4 ml of Picofluor (Packard) by liquid scintillation (21). For nonspecific binding, 100-fold nonlabeled ryanodine (Calbiochem) was included.

Oxalate-supported Ca²⁺ uptake. Whole homogenates of rat hearts or the superficial region of the vastus lateralis (type IIB) were preincubated for 4 min at 37°C in 100 mM KCl, 20 mM MOPS (pH 6.8), and 10 mM NaN₃, with or without 500 µM ryanodine (13). The uptake reaction was begun by rapid sequential addition of 5 mM MgATP, 10 mM potassium oxalate, and 0.2 mM ⁴⁵CaCl₂. A Gelman prefilter and a Millipore filter (0.45 µm) were used together to facilitate filtration. The rate of Ca²⁺ uptake was calculated from the linear regression of Ca²⁺ uptake at 1, 2, 4, and 6 min (32).

Western blot analysis. Cardiac whole homogenate samples in SDS sample buffer (25) were run on SDS-PAGE gel, and the proteins on the gel were electrophoretically transferred to nitrocellulose (NC) paper (35). The transferred proteins on NC paper were incubated with blocking solution containing 5% bovine serum albumin and 0.1% Tween 20 in Tris-buffered saline (TBS) for 2 h at room temperature. After blocking, the membrane was treated with anti-CaN primary antibody for 4 h at room temperature. After the membrane was washed several times in TBS with 0.1% Tween 20, the membrane was incubated with alkaline phosphatase-conjugated goat anti-mouse IgG secondary antibody for 1.5 h. The immunoreactive proteins were developed with nitro blue tetrazolium. Band intensities on the NC paper were quantified by densitometry scanning.

Miscellaneous. Protein concentrations of whole homogenates were determined by the method of Bradford (5). All data are presented as means ± SE. Statistical significance was evaluated with the Student's unpaired t-test or ANOVA. P values <0.05 were considered significant.

	RESULTS

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Characteristics of CsA-treated rats. Treatment of rats with CsA (15 mg · kg body wt¹ · day¹) led to a 20% reduction of body weight and a 23% reduction of heart weight. Therefore, the ratios of heart weight to body weight were similar between the two groups (control: 3.02 ± 0.10 × 10³; CsA: 2.98 ± 0.21 10³). However, the 23% reduction in body weight could result from major metabolic changes.

Equilibrium ryanodine binding to whole homogenates of rat cardiac and skeletal muscles. To examine whether CsA treatment alters the characteristics of CRC in the SR of rat cardiac and skeletal muscles, ryanodine binding to whole homogenates was measured at various [³H]ryanodine concentrations using cardiac and skeletal muscles of control and CsA-treated (for 3 wk) rats (Fig. 1). As the [³H]ryanodine concentration was increased from 0 to 50 nM, ryanodine binding to whole homogenates was rapidly activated and saturated at 20-50 nM. Kinetic parameters [maximal binding (B_max) and dissociation constant (K_d)] of ryanodine binding (Table 1) were calculated by iterative computer fitting using the equation Y = B_max × [X/(K_d + X)]. In the heart, the densities of CRC, as determined by B_max of ryanodine to the receptor, was significantly lower in CsA-treated than in control animals (0.37 ± 0.04 vs. 0.52 ± 0.02 pmol/mg protein). The K_d of ryanodine to CRC was significantly higher in CsA-treated rats (4.55 ± 0.56 vs. 2.47 ± 0.36 nM, P < 0.05), indicating that the affinity to ryanodine is lower in CsA-treated rats. However, B_max and K_d of ryanodine binding to whole homogenates of slow- and fast-twitch skeletal muscles were not significantly altered by CsA treatment. Scatchard analyses of ryanodine binding showed similar B_max and K_d values for both control and CsA groups (data not shown). It is also interesting to note that B_max and K_d are similar in control heart and control fast-twitch red or fast-twitch white muscle, whereas in CsA-treated heart these parameters are similar to slow-twitch skeletal muscles (Table 1).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Equilibrium ryanodine binding to whole homogenates of control () and cyclosporin A (CsA)-treated () rat hearts was measured at 2.5-50 nM [3H]ryanodine by incubation of 2.5 mg of whole homogenates in 250 µl of reaction mixture [1 M KCl, 20 mM MOPS (pH 7.4), 1 mM EGTA, 0.98 mM CaCl2, and varied amounts of [3H]ryanodine (54.7 Ci/mmol)] for 2 h at 37°C, as described in MATERIALS AND METHODS. Values are means ± SE for 4 pairs of animals. [Ryanodine], ryanodine concentration.

Time and dose-dependent alterations of ryanodine binding to whole homogenates of rat hearts by CsA. The altered kinetics of ryanodine binding to the CsA-treated heart were further studied by varying the period of treatment or the amounts of injecting CsA (Figs. 2 and 3). Figure 2 depicts the time-dependent alterations of ryanodine binding parameters. One week of treatment did not yield any significant changes in both K_d and B_max of ryanodine binding. Two weeks of treatment, however, decreased B_max significantly without significant change in K_d. Figure 3 shows dose-dependent changes of ryanodine binding parameters. At 3.75 mg CsA · kg body wt¹ · day¹, there was no significant alteration of the kinetic parameters, whereas at 7.5 mg CsA · kg body wt¹ · day¹, B_max was significantly altered, without change in K_d.

View larger version (24K): [in this window] [in a new window]   View larger version (21K): [in this window] [in a new window]   Fig. 2.   Time-dependent CsA effects on ryanodine binding to whole homogenates of rat hearts. CsA group (hatched bars) was treated with CsA dissolved in cremophor (15 mg · kg body wt1 · day1) for 1, 2, or 3 wk. Control group (open bars) was treated with same volume of cremophor. Equilibrium ryanodine binding to whole homogenates of control and CsA-treated rat hearts was measured at 2.5-50 nM [3H]ryanodine, and binding parameters [maximal ryanodine binding (Bmax; A) and dissociation constant (Kd) of ryanodine (B)] were calculated as described in Fig. 1 and MATERIALS AND METHODS. Values are means ± SE for 4 pairs of animals. * Statistically significant difference (P < 0.05, unpaired Student's t-test) between control and CsA-treated rats.

View larger version (22K): [in this window] [in a new window]   View larger version (20K): [in this window] [in a new window]   Fig. 3.   Dose-dependent CsA effects on ryanodine binding to whole homogenates of rat hearts. CsA group was treated with 3 doses of CsA dissolved in cremophor (3.75, 7, or 15 mg · kg body wt1 · day1 indicated by hatched, cross hatched, or solid bars, respectively) for 3 wk. Control group (open bars) was treated with same volume of cremophor. Equilibrium ryanodine binding to whole homogenates of control and CsA-treated rat hearts was measured at 2.5-50 nM [3H]ryanodine, and the binding parameters Bmax (A) and Kd (B) were calculated, as described in Fig. 1 and MATERIALS AND METHODS. Values are means ± SE for 4 pairs of animals. * Statistically significant difference (P < 0.05, unpaired Student's t-test) between control and CsA-treated rats.

Ca²⁺-concentration dependence of ryanodine binding to whole homogenates of CsA-treated rat hearts. To examine the Ca²⁺-sensitivity of ryanodine binding to control and CsA-treated rat hearts, [³H]ryanodine binding was measured at various Ca²⁺ concentrations by using the usual facilitated ryanodine binding condition (pH 7.4 and 1 M KCl) (Fig. 2A) (32) or the more physiological condition (pH 6.8 and 0.15 M KCl) (Fig. 2B) (26). The binding parameters of Ca²⁺-activated ryanodine binding were calculated by iterative computer fitting using sigmoid curves in semilogarithmic scale. In the facilitated ryanodine binding condition (32), the Ca²⁺ concentration for half-maximal activation of ryanodine binding (EC₅₀) (control: 0.09 ± 0.01; CsA: 0.10 ± 0.01 µM) and cooperativity of Ca²⁺-activated ryanodine binding expressed as Hill coefficient (control: 2.14 ± 0.72; CsA: 2.75 ± 0.90) were similar between control and CsA-treated rat hearts (Fig. 4A). In the more physiological binding conditions (26), the EC₅₀ (control: 0.059 ± 0.008; CsA: 0.066 ± 0.006 µM) and Hill coefficient (control: 1.58 ± 0.22; CsA: 2.06 ± 0.20) were also similar between control and CsA-treated rat hearts (Fig. 4B).

View larger version (10K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   Fig. 4.   Ca2+ dependence of ryanodine binding to whole homogenates of control () and CsA-treated () rat hearts was measured in facilitated ryanodine binding solution [containing 1 M KCl, 20 mM MOPS (pH 7.4), 1 mM EGTA, 20 nM [3H]ryanodine (54.7 Ci/mmol), and varied amounts of CaCl2] (32) to produce 0.02-3.16 µM free Ca2+ (A) or in a more physiological ryanodine binding solution [containing 0.15 M KCl, 20 mM MOPS (pH 6.8), 1 mM EGTA, 15 nM [3H]ryanodine (54.7 Ci/mmol), and varied amounts of CaCl2] (26) to produce 0.03-10 µM free Ca2+ (B), as described in MATERIALS AND METHODS. Values are means ± SE for 4 pairs of animals. [Ca2+], Ca2+ concentration.

Caffeine-activated ryanodine binding to whole homogenates of CsA-treated rat hearts. To investigate the effect of CsA treatment on agonist sensitivity of cardiac CRC, ryanodine binding with increasing caffeine concentrations (0.25-20 mM) was measured at 0.03 µM Ca²⁺ (Fig. 5). EC₅₀ of caffeine for activation of ryanodine binding in CsA-treated animals was significantly lower than in the controls (3.00 ± 0.10 vs. 3.56 ± 0.17 mM, n = 4, P < 0.05), suggesting that caffeine sensitivity was increased by CsA. The calculated Hill coefficient was similar between the control and CsA groups (1.27 ± 0.15 vs. 1.30 ± 0.11) (Fig. 5, inset).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Caffeine-activated ryanodine binding to whole homogenates of control () and CsA-treated () rat hearts was measured in caffeine concentration ([caffeine]) range of 0.25-20 mM, as described in MATERIALS AND METHODS. Actual binding levels (means ± SE) without caffeine were 0.19 ± 0.02 (control) and 0.11 ± 0.01 pmol/mg protein (CsA). Corresponding values in presence of 20 mM caffeine were 0.43 ± 0.03 (control) and 0.25 ± 0.03 pmol/mg protein (CsA). Values are means ± SE for 4 pairs of animals. Inset: Hill plots of caffeine-activated ryanodine binding to control and CsA-treated rat hearts.

Ruthenium red-inhibited ryanodine binding to whole homogenates of CsA-treated rat hearts. Ruthenium red is an effective CRC blocker in both cardiac and skeletal muscles (2, 22). To examine the effects of ruthenium red on CRC of control and CsA-treated rat hearts, ryanodine binding in the presence of ruthenium red (0.1-20 µM) was determined at 3.16 µM Ca²⁺ (Fig. 6). The concentration of ruthenium red yielding half-maximal inhibition of ryanodine binding (IC₅₀) was significantly higher in CsA-treated rat hearts than in control hearts (5.36 ± 0.70 vs. 2.88 ± 0.50 µM, P < 0.05), suggesting that the mechanisms responsible for ruthenium red inhibition are altered by CsA treatment. The calculated Hill coefficient was similar between the control and CsA groups (1.35 ± 0.09 vs. 1.35 ± 0.20) (Fig. 6, inset).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Ruthenium red-inhibited ryanodine binding to control () and CsA-treated () rat hearts was measured in ruthenium red concentration ([ruthenium red]) range of 0.1-20 µM, as described in MATERIALS AND METHODS. Percent inhibition of ryanodine binding by ruthenium red was calculated from difference between binding at each ruthenium red concentration and binding in absence of ruthenium red. Actual binding levels (means ± SE) without ruthenium red were 0.52 ± 0.01 (control) and 0.34 ± 0.02 pmol/mg protein (CsA). Corresponding values in presence of 20 µM ruthenium red were 0.25 ± 0.01 (control) and 0.16 ± 0.01 pmol/mg protein (CsA). Values are means ± SE for 4 pairs of animals. Inset: Hill plots of ruthenium red-inhibited ryanodine binding to control and CsA-treated rat hearts.

Oxalate-supported Ca²⁺ uptake in presence or absence of ryanodine. To investigate the function of CRC and Ca²⁺-ATPase in CsA-treated rat hearts, oxalate-supported Ca²⁺ uptake was measured in whole homogenates in the presence or absence of 500 µM ryanodine (Fig. 7). The rates of Ca²⁺ uptake were calculated by linear regression. The difference in Ca²⁺ uptake between control and CsA-treated rat hearts in the presence of 500 µM ryanodine was negligible (control: 17.52 ± 0.61; CsA: 16.70 ± 0.54 nmol · mg protein¹ · min¹), indicating that Ca²⁺-ATPase activity was not altered by CsA treatment (Fig. 7A). Ryanodine-sensitive Ca²⁺ uptake, defined as the difference between the Ca²⁺ uptake in the presence of 500 µM ryanodine and the Ca²⁺ uptake in the absence of ryanodine, is a function of CRC, because the high concentration of ryanodine irreversibly blocks CRC (12). Ryanodine-sensitive Ca²⁺ uptake in CsA-treated animals was significantly lower than in the control animals (control: 8.89 ± 0.60; CsA: 5.83 ± 0.52 nmol · mg protein¹ · min¹, P < 0.05) (Fig. 7A), further indicating that CsA-treated rat hearts contained a lower density of functional CRC. However, in skeletal muscle, the ryanodine-sensitive Ca²⁺ uptake was similar between control and CsA groups (4.98 vs. 4.89 nmol · mg protein¹ · min¹) (Fig. 7B), suggesting that the density of functional CRC was not altered by CsA treatment in the skeletal muscle.

View larger version (14K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   Fig. 7.   Oxalate-supported Ca2+ uptake into sarcoplasmic reticulum (SR) in whole homogenates of control (circles) and CsA-treated (triangles) rat hearts (A) or fast-twitch white muscles (B) was measured in a bath containing 10 mg tissue/ml, 100 mM KCl, 20 mM MOPS (pH 6.8), and 5 mM NaN3, with (filled symbols) or without (open symbols) 500 µM ryanodine, 5 mM MgCl2, 5 mM ATP, 10 mM potassium oxalate, and 200 µM 45CaCl2 at 37°C. A: uptake rates with 500 µM ryanodine were 17.52 ± 0.61 (control) and 16.70 ± 0.54 nmol · mg protein1 · min1 (CsA), and those without 500 µM ryanodine were 8.63 ± 0.60 (control) and 10.87 ± 0.40 nmol · mg protein1 · min1 (CsA) (P < 0.05). B: uptake rates with 500 µM ryanodine were 23.60 ± 2.02 (control) and 26.00 ± 2.44 nmol · mg protein1 · min1 (CsA), and those without 500 µM ryanodine were 18.62 ± 1.42 (control) and 21.11 ± 0.57 nmol · mg protein1 · min1 (CsA). Values are means ± SE for 4 pairs of animals.

Western blot analysis. In light of the evidence that CsA forms a CsA-CyPA complex and consequently inhibits the phosphatase activity of CaN (27), we tested the hypothesis that the chronic treatment of rats with CsA leads to a compensatory upregulation of CaN in the heart by examining the expression level of CaN. The catalytic subunit of CaN band (relative molecular mass 59 kDa) in 120 µg of whole homogenate was hardly detected by Coomassie blue staining alone (Fig. 8A). However, the CaN band was clearly seen by Western blot analysis (Fig. 8B). The CaN bands for the CsA group were significantly denser than those for the control (1.89 ± 0.07 vs. 1.00 ± 0.04 arbitrary units, n = 3, P < 0.05), indicating that CaN in the heart was upregulated by the chronic treatment with CsA.

View larger version (40K): [in this window] [in a new window]   Fig. 8.   Western blot analysis of calcineurin (CaN) in whole homogenates of control and CsA-treated rat hearts. Whole homogenates (120 µg) of control and 3-wk CsA-treated rat hearts were run on 10% SDS-PAGE gel, and proteins on gel were transferred to nitrocellulose (NC) paper. NC paper was subjected to Western blot analysis using anti-CaN antibody and alkaline phosphatase-conjugated secondary antibodies. A: Coomassie blue-stained gel for control (lanes 1 and 2) and CsA-treated (lanes 3 and 4) rat hearts. B: Western blots of same gel in A for control (lanes 1 and 2) and CsA groups (lanes 3 and 4).

Effects of rapamycin on ryanodine binding to whole homogenates of control and CsA-treated rat hearts. Cameron et al. (7) showed evidence that CaN is an important regulator of inositol 1,4,5-trisphosphate (IP₃) receptors and CRC. The effects of rapamycin on ryanodine binding to cardiac whole homogenates were examined to investigate whether the ryanodine binding properties altered by CsA are associated with the upregulation of CaN (Fig. 8). Rapamycin at the concentration range of 5-20 µM was added to the ryanodine binding medium and incubated for 2 h. With an increase in the concentration of rapamycin, both B_max and K_d became similar to the control (Table 3). At the incubation with 20 µM rapamycin, the statistically significant differences in B_max and K_d of ryanodine binding between the two groups (Table 1) was no longer present (B_max: 0.47 ± 0.03 vs. 0.48 ± 0.02 pmol/mg protein; K_d: 2.95 ± 0.38 vs. 3.43 ± 0.17), suggesting that the ryanodine binding properties altered by CsA are associated with the upregulation of CaN. On the other hand, in the control group, both B_max and K_d of ryanodine binding were not significantly affected by addition of 5-20 µM rapamycin.

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

This study reports that chronic CsA treatment results in characteristic alterations of CRC functions in the SR of cardiac muscle (Figs. 1-7 and Table 1), which could explain the reported alterations of contractile properties in CsA-treated rat heart (3, 23). Chronic CsA treatment was accompanied by decreased B_max of ryanodine binding to CRC (Fig. 1 and Table 1) and decreased ryanodine-sensitive Ca²⁺ uptake (Fig. 7). CsA treatment was also associated with increased sensitivity of the CRC to the Ca²⁺-release agonist caffeine (Fig. 5) and with decreased affinity or sensitivity to the Ca²⁺- release blockers ryanodine and ruthenium red (Figs. 1 and 6, Table 1). Total Ca²⁺ uptake in the presence of 500 µM ryanodine (Fig. 7) was similar between control and CsA-treated animal groups, indicating that, in contrast to cardiac CRC, Ca²⁺-ATPase activity was not altered by CsA treatment. This result is consistent with the report by Banijamali et al. (3) that the rate of decay of extrasystolic potentiation is similar in control and CsA-treated rat cardiac trabeculae.

To examine whether chronic CsA-treatment could also affect skeletal muscle CRC, equilibrium ryanodine binding was also examined by using three types of rat skeletal muscles (slow-twitch red, fast-twitch red, and fast twitch-white muscles) (13) (Table 1). B_max and K_d of ryanodine binding are similar in skeletal muscles of control and CsA-treated rats (Table 1), suggesting that CsA-mediated alteration of CRC properties is tissue specific. Ryanodine-sensitive Ca²⁺ uptake in the fast-twitch skeletal muscle was not altered by CsA (Fig. 7B), suggesting further that skeletal CRC is not altered by CsA. B_max and K_d of ryanodine binding are similar in heart and fast-twitch red skeletal muscles (Tables 1). On the other hand, B_max of ryanodine binding in slow-twitch red muscle was 3.5- to 4.3-fold smaller than in the fast-twitch red and fast-twitch white muscle, respectively (Table 1). It is interesting to note that the similar differences of Ca²⁺ uptake capacity (3.9- to 6.8-fold) and the B_max of ryanodine binding (3.5- to 4.3-fold) were shown previously in slow- and fast-twitch rat skeletal muscles (23). The affinity of ryanodine binding (K_d) to slow-twitch muscle was 1.9- to 2.6-fold lower than that of the fast-twitch muscle types (Table 1). A similar difference in K_d between slow- and fast-twitch skeletal muscles was previously reported in rabbits (26). To our knowledge, this is the first report that directly compares ryanodine binding to whole homogenates between different muscle types of skeletal and cardiac muscles in rats.

The use of whole homogenates for ryanodine binding and oxalate-supported Ca²⁺ uptake enabled us to deduce all of the density of CRC (32) and the Ca²⁺ pump (12) in CsA-treated rat hearts. B_max of ryanodine binding to CRC was significantly decreased in CsA-treated rats (Table 1), similar to the effects of pressure overload-induced left ventricular hypertrophy (21). However, because the hearts were not hypertrophied by CsA treatment, the decreased B_max of ryanodine binding to CRC in CsA-treated animals was not caused by hypertrophy of the heart. Furthermore, in hypertrophied hearts the Ca²⁺ pump is downregulated (21), whereas its expression was not altered in CsA-treated rats (Fig. 7).

CsA treatment of rats has been reported to cause decreased left ventricular systolic pressure and decreased maximal stress development in the presence of Ca²⁺ (3, 24). The effects could directly be associated with the decreased B_max of ryanodine binding to CRC (Fig. 1 and Table 1), resulting in a reduction of Ca²⁺ release from the SR and a subsequent reduction of contractile properties (24). The sensitivity of cardiac CRC to caffeine, the well-known Ca²⁺-release agonist (22), is increased in CsA-treated animals (Fig. 5) and may be related with the reported higher sensitivity of the myocardium to external Ca²⁺ and the increased occurrence of spontaneous contractions (3). However, lack of significant changes in Ca²⁺ sensitivity in CsA-treated animals (Fig. 4) suggests that the higher sensitivity of the myocardium to external Ca²⁺ may directly be related to the activity of L-type Ca²⁺ channel (3). The affinity of CRC to ryanodine and its sensitivity to ruthenium red were changed in CsA-treated animals (Figs. 1 and 6, Table 1). Because ruthenium red and ryanodine may share the same binding site (14), it is tempting to speculate that the mechanism associated with the ryanodine/ruthenium red binding is altered in the CsA-treated animal heart.

Figures 2 and 3 and Table 2 indicate that the characteristic alterations of CRC are not directly caused by CsA. Analysis of the time- (Fig. 2) and dose-dependent (Fig. 3) changes of B_max and K_d shows that B_max and K_d are not altered at the same time, suggesting that the underlying molecular mechanisms causing the changes of B_max and K_d are different. These alterations of CRC properties by CsA may thus provide an excellent molecular tool to study structure and function of CRC.

The underlying molecular mechanisms for the modification of the characteristics of CRC by CsA are not yet resolved. The time-dependent modification of the CRC (Fig. 2) and lack of direct effect of CsA (Table 2) suggest that the immunosuppressive properties of this agent (see introduction) may not directly cause the modification (16, 34). Recent evidence has suggested that the tetrameric structures of CRC are stabilized by the channel-associated FKBP, the cytosolic receptor for the immunosuppressant drugs FK506 and rapamycin, and that the drugs inhibit the prolyl isomerase activity of FKBP and can dissociate FKBP from CRC (4, 6, 19, 29). Cameron et al. (7) showed evidence that CaN, which has protein phosphatase activity, is anchored by FKBP-CRC or FKBP-IP₃ complex and that CaN anchored to the IP₃ receptor via FKBP12 regulates the phosphorylation state of the receptor, resulting in a dynamic Ca²⁺-sensitive regulation of IP₃-mediated Ca²⁺ fluxes. Our recent immunoprecipitation data also show that CaN is tightly bound to rat cardiac CRC in the presence of 100 µM Ca²⁺, but not in the absence of Ca²⁺, and that there is more CaN found in the cardiac SR of CsA-treated animals than in controls (A. Bandyopadhyay and D. H. Kim, unpublished data).

Our data showing the CsA-mediated upregulation of CaN (Fig. 8) and the rapamycin-mediated recovery of the altered ryanodine binding parameters to the control level (Table 3) suggest that the increased level of CaN expression by CsA could decrease the phosphorylation state of CRC and, hence, could lead to the characteristic alterations of ryanodine binding.