INTRODUCTION

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PROLONGED EXPOSURE to microgravity during a spaceflight results in various physiological alterations in humans, including skeletal musculature atrophy, volume shifts, osteopenia, and hematologic alterations associated with weightlessness (22). Regarding cardiovascular parameters, weightlessness is known to induce a cephalic blood shift, increasing the volume load on the heart (hypovolemia), likely due to activation of cardiopulmonary mechanoreceptors and cardiovascular deconditioning on return to earth (tachycardia and orthostatic hypotension) associated with an altered baroreceptor reflex.

Hindlimb suspension in rats is a widely used animal model to simulate the effects of weightlessness. Hindlimb suspension has been shown to induce atrophy of the hindlimb musculature, a chronic elevated volume load on the heart, and a reduction in plasma volume (3). Whether heart rate and blood pressure are elevated (18) or not (3, 9) during tail suspension is still controversial. Regarding the molecular mechanisms underlying excitation-contraction coupling alterations, 14-day unloaded soleus muscles exhibit a marked increase in the expression of the fast sarcoplasmic reticulum (SR) Ca²⁺ pump and of both dihydropyridine and ryanodine receptors (RyRs) associated with shorter twitch contractions (21). Such adaptative alterations regarding excitation-contraction coupling and Ca²⁺ homeostasis after simulated weightlessness are poorly documented in cardiac cells. Recently, in tail-suspended rats, a small (15%) decrease in force contraction of skinned cardiac myocytes has been reported at a submaximal Ca²⁺ concentration ([Ca²⁺]), with no change in maximum tension (7).

In the present study, we investigated the effects of a 14-day hindlimb suspension on [³H]ryanodine binding to rat cardiac microsomes and on twitch cytosolic [Ca²⁺] ([Ca²⁺]_i) transients, SR Ca²⁺ content, and voltage-dependent Ca²⁺ channels in isolated ventricular myocytes. We show that long-term hindlimb suspension specifically increases both electrically evoked twitch [Ca²⁺]_i transient amplitude and [³H]ryanodine affinity for cardiac RyRs.

	MATERIALS AND METHODS

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Hindlimb suspension. Forty-seven male Wistar rats were housed in controlled conditions of temperature (20 ± 1°C) and were submitted to a 12:12-h light-dark cycle, with unlimited access to food and water. Weightlessness simulation was accomplished on 25 rats according to the Morey tail-suspension model (17) as previously described (15, 20). Briefly, a plastic hole-drilled disk was attached with adhesive tape to the tail and connected to a pulley. The animals were able to move freely in the cage by grasping a metal grid on the floor with their forelimbs. The hindlimbs were maintained above the cage floor and were nonweight bearing. The level of the head-down tilt position was ~30-40°. Hindlimb-suspended animals were maintained in this position for 14 days. Eight rats from this group were submitted to a recovery period of 4 days. Twenty-two control rats in individual cages were treated similarly except for the hindlimb suspension. This protocol was approved by the ad hoc committee of the Centre National des Etudes Spatiales and was in accordance with the guidelines on the care and use of animals required by the International Physiological Society.

Cardiac microsomes. Fourteen control rats and fourteen 14-day hindlimb-suspended rats (four of the latter were submitted to a 4-day recovery period) were killed by cervical dislocation, and the whole heart was rapidly excised. The ventricular myocardium was dissected and drained free of blood in ice-cold 0.9% NaCl, weighed, and immediately frozen at 80°C until membrane preparation. The cardiac microsomal fraction was isolated by differential centrifugation at 4°C. The ventricles were minced into small pieces and homogenized in ice-cold sucrose-HEPES buffer (0.3 M sucrose, 40 mM HEPES, 1 mM iodoacetamide, and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.4) with a Polytron (3 bursts of 15 s each at setting 10). The homogenate was centrifuged at 1,000 g for 10 min and 10,000 g for 20 min. The resulting supernatant was filtered through cheesecloth, and KCl was added to a final concentration of 0.5 M. The mixture was stirred for 30 min and centrifuged at 100,000 g for 20 min in a Beckman 45 Ti rotor. The pellets were resuspended in ice-cold 25 mM HEPES (pH 7.4) and homogenized with a glass-Teflon homogenizer. Protein concentration was determined according to Bradford (2), with the Bio-Rad kit with -globulin as a standard.

[³H]ryanodine binding assay. [³H]ryanodine binding was carried out as previously described (16). For saturation experiments and modulation by Ca²⁺, the incubation medium contained 1 M KCl, 25 mM HEPES (pH 7.4 at 37°C), 1 mM EGTA and CaCl₂ to set the desired free [Ca²⁺] (10 µM free [Ca²⁺] in saturation studies). After a 3-h incubation at 37°C, aliquots were filtered through Whatman GF/C glass fiber filters and washed three times with 8 ml of ice-cold 0.1 M Tris (pH 7.4 at 4°C). The filters were placed into scintillation vials containing 4 ml of liquid scintillation cocktail, and the retained radioactivity was measured in a Packard 1500 Tri-Carb counter. The specific binding was defined as the difference between binding in the absence (total binding) and presence (nonspecific binding) of 10 µM unlabeled ryanodine. Nonspecific binding accounted for <5% of total binding at 2 nM [³H]ryanodine.

Cell preparation, [Ca²⁺]_i measurements, and Ca²⁺ currents. Single ventricular cardiac myocytes were prepared from 8 control rats and 11 suspended rats (4 of these were submitted to a 4-day recovery period) as previously reported (4). Briefly, the hearts were retrogradely perfused with a Ca²⁺-free solution containing (in mM) 120 NaCl, 5 KCl, 1 MgCl₂, 5 NaHCO₃, 10 glucose, and 20 HEPES (pH 7.3 at 37°C) equilibrated with 95% O₂-5% CO₂. After a washing out, the perfusion was switched to the same solution containing 80 U/ml of collagenase and 0.04 mg/ml of Pronase E. When the heart became flaccid (after ~40 min), the ventricular tissue was dispersed, and the cell suspension was rinsed several times, with a gradual increase in [Ca²⁺] up to 1 mM. Then, the cells were plated on glass coverslips treated with laminin and loaded with 10 µM indo 1-AM for 15 min (1). The cells were washed with a physiological solution for at least 30 min and allowed to cleave the dye to the active indo 1 compound. Indo 1-AM-loaded cells were mounted in a perfusion chamber and placed on the stage of an inverted microscope. The emission field was restricted to a single cell. [Ca²⁺]_i was estimated from the 405- to 480-nm fluorescence ratio (15). The minimum and maximum fluorescence (R_min and R_max, respectively) values were determined in vivo (11) in cells from control and suspended rats. The solution for R_min measurement contained (in mM) 60 NaCl, 50 KCl, 1 MgCl₂, 5 HEPES, 5 EGTA, 10 glucose, 0.02 ionomycin, and 50 2,3-butanedione monoxime to inhibit cell contraction, pH 7.4 with NaOH. The solution for R_max measurement was the same except that EGTA was replaced with 2 mM CaCl₂. The cells were electrically stimulated at 0.5 Hz with 5-ms voltage pulses (1.5 times threshold) delivered through a pair of platinum electrodes placed on either side of the experimental chamber. Voltage-clamp and Ca²⁺-current recordings were made with a standard patch-clamp technique with a List EPC7 patch-clamp amplifier (Darmstadt-Eberstadt, Germany). The whole cell recording mode was performed with patch pipettes of 2- to 4-M resistance. Membrane potential and current records were stored and analyzed with an IBM-PC computer (pCLAMP, Axon Instruments, Foster City, CA). Cell capacitance was recorded in each cell tested by imposing 10-mV hyperpolarizing steps from the holding potential (40 mV) and analyzing the amplitude and time course of the recorded currents. Ca²⁺-current density is expressed as peak current amplitude per capacitance unit (in pA/pF).

	RESULTS

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Effects of hindlimb suspension on [Ca²⁺]_i transients in ventricular myocytes. After the 14 days of hindlimb unloading, no significant difference was observed between the body weight of the control rats (315 ± 18 g; n = 22) and that of the suspended rats (305 ± 15 g; n = 17). As previously reported (10, 21), there was a significant decrease in soleus muscle mass between groups. The mean soleus wet weight in control rats was 144.5 ± 4.8 mg (n = 22) compared with 68.5 ± 2.3 mg in suspended rats (n = 17; P < 0.05). In contrast, the cardiac ventricle wet weight was not significantly different in the control and suspended rats (control rats: 0.73 ± 0.04 g, n = 22; suspended rats: 0.75 ± 0.03 g, n = 17; P > 0.05). Isolated cardiac myocytes were submitted to electrical stimulations to evoke either a single-twitch [Ca²⁺]_i transient after a rest period of 5 min or repetitive [Ca²⁺]_i transients during a steady-state stimulation at 0.5 Hz. It is noteworthy that the amplitude of the single-twitch [Ca²⁺]_i was similar to that of the first twitch in the steady-state stimulation period during which a negative staircase effect was observed (Fig. 1A). The amplitude of the caffeine (10 mM)-induced [Ca²⁺]_i transient was used to assess the SR Ca²⁺ content (19). Caffeine was applied 10 s after the steady-state stimulation or after a rest period of 5 min. The R_min and R_max values were not different in control rats (0.414 ± 0.006 and 1.071 ± 0.046, respectively; n = 8) and suspended rats (0.424 ± 0.012 and 0.926 ± 0.058, respectively; n = 7; P > 0.05). As shown in Fig. 1, hindlimb suspension induced a significant increase in twitch [Ca²⁺]_i transients evoked by electrical stimulations. Both the single postrest [Ca²⁺]_i transient and the first [Ca²⁺]_i transient of the steady-state stimulation were similarly stimulated (Figs. 1B and 2A). Moreover, the amplitude of the last [Ca²⁺]_i transients during the steady-state stimulation period was also increased (Figs. 1B and 2A). In contrast, the caffeine-induced Ca²⁺ responses obtained after a rest period or 10 s after a steady-state stimulation period were not significantly modified by the suspension (Fig. 2B). In addition, the basal [Ca²⁺]_i level remained unchanged after hindlimb suspension (fluorescence ratios: control rats, 0.510 ± 0.004, n = 8; suspended rats, 0.530 ± 0.009, n = 7; P > 0.05). Four days after the rats were removed from suspension, the amplitude of the electrically evoked twitch [Ca²⁺]_i transients was significantly decreased (0.075 ± 0.012; n = 4), although a complete recovery was not obtained.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Electrically evoked twitch cytosolic Ca2+ concentration ([Ca2+]i) transients and caffeine (Caf; 10 mM)-induced Ca2+ responses in ventricular myocytes from control (A) and suspended (14 days; B) rats. Twitch [Ca2+]i transient was evoked by electrical stimulation after a 5-min rest period (PRT), and Caf-induced [Ca2+]i transient was evoked after a steady-state stimulation (SS) at 0.5 Hz. [Ca2+]i is expressed by indo 1 405- to 480-nm fluorescence ratio (405/480).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Twitch [Ca2+]i transients (A) and Caf-induced responses (B) in control (solid bars) and suspended (open bars) rats. Amplitudes of twitch Ca2+ responses were measured after PRT or as last response during steady-state stimulation (SST). Amplitudes of Caf (10 mM)-induced Ca2+ responses were measured after a 5-min rest period (PRCaf) and 10 s after steady-state stimulation (SSCaf). Data are means ± SE; nos. in parentheses, no. of rats. For each rat, results obtained from 8-12 cells were pooled. * Significantly different from respective control condition, P < 0.05.

Effects of hindlimb suspension on Ca²⁺ current. To determine whether the increased twitch [Ca²⁺]_i transients depended on Ca²⁺-channel stimulation, Ca²⁺ currents were measured during depolarizing pulses from 40 to +10 mV in cardiac myocytes from control and suspended rats. At a holding potential of 40 mV, the Na⁺ current is largely inactivated, and only L-type Ca²⁺ channels are present in these cells as previously reported (23). In addition, these currents were completely blocked by either 1 mM Cd²⁺ or 1 µM oxodipine. As shown in Fig. 3A, inward Ca²⁺ currents with a similar amplitude and time course were obtained in control and suspended rats. In averaged data, both the cell capacitance and current density were not significantly affected by a 14-day suspension (Fig. 3B). As illustrated by the current-voltage relationships in Fig. 4A, the threshold potential, the potential corresponding to peak current, and the apparent reversal potential were not different in control and suspended rats. The voltage-dependent inactivation was examined with the two-pulse protocol. Steady-state inactivation induced during a conditioning pulse of 20-s duration and variable amplitude was estimated by the reduction in peak current associated with a test depolarization to +10 mV. The decrease in the test current was taken as an index of inactivation of the Ca²⁺ current. The amplitude of the test current was normalized to its value at the most negative prepulse and was plotted against the prepulse potential value. The experimental points obtained from control and suspended rats were fitted by a Boltzmann equation, with a half-maximal inactivation at 35 mV and a slope factor of 5.6. The value for half-maximal inactivation is similar to that recently reported in rat ventricular myocytes (23).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   L-type Ca2+ currents in control and suspended rats. A: Ca2+ currents elicited from a holding potential of 40 to +10 mV in cardiac myocytes from a control (a) and a suspended (b) rat. B and C: pooled data of cell capacitance and current density measurements, respectively, in control (solid bars) and suspended (open bars) rats. Data are means ± SE; nos. in parentheses, no. of rats. For each rat, measurements were obtained from 3 to 5 cells.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Current-voltage relationships and steady-state inactivation curves for L-type Ca2+ current in control () and suspended () rats. A: Ca2+ currents elicited from a holding potential of 40 mV at a stimulation frequency of 0.05 Hz. B: steady-state inactivation curve obtained with 2-pulse protocol for L-type Ca2+ current from a holding potential of 60 mV (in presence of 1 µM TTX). Data were fit by a curve of form 1/[1 + exp(Vm  Vh)/k], where Vh is potential at which one-half of current is inactivated, Vm is membrane potential, and k is slope factor. Ca2+ current is expressed as a fraction of maximal current (I/Imax). Data are means ± SE; n = 8 control and 7 suspended rats. For each rat, measurements were obtained from 3 to 4 cells.

Effect of hindlimb suspension on [³H]ryanodine binding to cardiac microsomes. Both control and suspended rats exhibited high-affinity [³H]ryanodine binding in a one-site fashion (Fig. 5A) for the [³H]ryanodine concentration range tested. The maximal binding capacity (B_max; calculated from 4 experiments in control and suspended rats) remained essentially the same after 14 days of hindlimb suspension (4.29 ± 0.14 pmol/mg protein in suspended rats vs. 4.07 ± 0.16 pmol/mg protein in control rats; P > 0.05). The dissociation constant was lowered from 2.05 ± 0.02 nM in control rats (4 experiments) to 1.07 ± 0.01 nM in suspended rats (4 experiments), i.e., a twofold increase in affinity (P < 0.05; Fig. 5B). Four days after the rats were removed from suspension, the dissociation constant of [³H]ryanodine binding was significantly increased (1.65 ± 0.04 nM; n = 4), although a complete recovery was not obtained.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   [3H]ryanodine binding to cardiac microsomes from control and suspended rats. A: saturation experiment carried out on cardiac microsomes (0.4-0.8 mg/ml protein) from suspended rats with increasing concentrations of [3H]ryanodine for 3 h at 37°C showing total binding (), specific binding (), and nonspecific binding () defined with 10 µM unlabeled ryanodine. Triplicate estimates were used for each point; similar estimates were obtained from 4 separate experiments. B, bound; F, free. B: Scatchard analysis of specific binding in control () and suspended () rats. Typical experiments are shown (control rats: dissociation constant 2.01 nM, maximal binding capacity 4.27 pmol/mg protein, Hill coefficient 0.99; suspended rats: dissociation constant 1.03 nM, maximal binding capacity 4.33 pmol/mg protein, Hill coefficient 0.96). Incubation medium contained 1 M KCl and 10 µM free [Ca2+]. Inset: specific [3H]ryanodine binding for control () and suspended () rats.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Ca2+-dependent activation of [3H]ryanodine binding. A: cardiac microsomes from control () and suspended () rats were incubated in a medium containing 1 M KCl and 1 nM [3H]ryanodine in presence of increasing concentrations of free [Ca2+]. Each point is mean ± SE of 4 experiments. Nonspecific binding was measured in presence of 10 µM unlabeled ryanodine. B: normalized curves obtained by plotting specific binding/maximal (max) binding vs. [Ca2+] in control and suspended rats.

	DISCUSSION

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Dihydropyridine receptor/L-type Ca²⁺ channel and RyR/Ca²⁺-release channel are the key components of excitation-contraction coupling in cardiac myocytes, but alterations of these components on simulated weightlessness have not been documented. In the present study, we showed that long-term hindlimb suspension increased twitch [Ca²⁺]_i transients in single cardiac myocytes without affecting L-type Ca²⁺-current density and SR Ca²⁺ content. These effects are associated with an increase in affinity of [³H]ryanodine binding to cardiac microsomes without modifications of B_max, suggesting that hindlimb suspension may affect the activity rather than the expression level of RyRs.

The increase in amplitude of the [Ca²⁺]_i transient in suspended rats could arise from any of the following steps involved in excitation-contraction coupling: 1) an increase in Ca²⁺ current that supplies the trigger for RyR activation, 2) an increase in the sensitivity of the RyR to Ca²⁺, 3) a modulation of RyR activity by endogenous effectors, 4) a change in the number of RyRs, and 5) an increase in the amount of releasable Ca²⁺ from the SR. The fact that both the ventricular weight and cell capacitance of cardiac myocytes remained unchanged in suspended rats indicates the absence of cell surface expansion and cellular hypertrophy after a 14-day hindlimb suspension. In rat ventricular myocytes, we do not see any evidence for the existence of T-type Ca²⁺ channels as previously reported (23). Therefore, we may assume that the Ca²⁺ current measured from a holding potential of 40 mV is only due to activation of L-type Ca²⁺ channels. After hindlimb suspension, the L-type Ca²⁺-current density was not significantly affected. The gating properties of the L-type Ca²⁺ channels revealed by the current-voltage relationship and the steady-state inactivation remained unchanged after suspension, indicating that the change in [Ca²⁺]_i transient was not due to an increase in L-type Ca²⁺ current. The results also show that the amount of releasable SR Ca²⁺ evoked by caffeine was not modified by hindlimb suspension, suggesting that no change in the ability of the SR to store Ca²⁺ is involved in the reduction of the [Ca²⁺]_i transient after hindlimb suspension. Thus these results indicate that the increase in the twitch [Ca²⁺]_i transient could be the result of an increased Ca²⁺ release from the SR rather than a change in the Ca²⁺-current density or in the SR Ca²⁺ content per se.

Therefore, analysis of the number and properties of RyRs was performed by studying [³H]ryanodine binding to cardiac microsomes from control and suspended rats. As previously reported (5, 14), [³H]ryanodine binding is a sensitive method for assessing RyR activity. We found a stimulation of [³H]ryanodine binding in cardiac microsomes from suspended rats compared with that in control rats. This stimulatory effect was the result of an increase in [³H]ryanodine affinity without alteration of B_max, suggesting that hindlimb suspension might alter Ca²⁺-release channel activity. Similar modifications of [³H]ryanodine binding, i.e., an increase in affinity without modification of the B_max value have been obtained by increasing [Ca²⁺] (12) and after addition of exogenous phosphatases (13). Modulation of [³H]ryanodine binding was examined in the [Ca²⁺] range (0.1-10 µM) where Ca²⁺ bound to high-affinity activation sites on the RyRs (12). Our results indicate that cardiac RyRs of suspended rats exhibited a Ca²⁺-dependent increase in [³H]ryanodine binding, with a maximal value reaching 1.6-fold the increase in specific [³H]ryanodine binding to control RyRs. However, normalizing the curves to maximal [³H]ryanodine binding revealed that there was no change in the Ca²⁺ sensitivity of [³H]ryanodine binding in control and suspended rats. These results suggest that the increase in twitch [Ca²⁺]_i transients of suspended rats is not due to an increased Ca²⁺ affinity of the Ca²⁺ activation sites on the RyRs.

Activity of Ca²⁺-release channels and [³H]ryanodine binding to cardiac microsomes have been reported to be modulated by ions, lipid derivatives, nucleotides, cADP-ribose, endogenous polyamines, Ca²⁺-binding proteins, and phosphorylation (24), suggesting that hindlimb suspension may alter any of these cellular targets in cardiac myocytes. Recently, it has been reported that, in rat ventricular myocytes, dephosphorylation of RyRs by phosphatases PP-1 and PP-2A produces a decrease in the peak of electrically evoked [Ca²⁺]_i transients (6), suggesting that L-type Ca²⁺ current activates RyRs that are normally phosphorylated. In ferret cardiac myocytes, an increase in efficacy of Ca²⁺-induced Ca²⁺ release by Ca²⁺/calmodulin-dependent protein kinase II has been also proposed (11). In agreement with these data, it can be postulated that hindlimb suspension may increase the phosphorylation state of RyRs, leading to a better efficacy of Ca²⁺-induced Ca²⁺ release in cardiac myocytes from suspended rats. However, biochemical and functional experiments on the effects of diverse protein kinases and protein phosphatases on RyR activity in control and suspended rats are needed to understand the effects of the phosphorylation-dephosphorylation mechanism on Ca²⁺-induced Ca²⁺ release.

In conclusion, our results show that hindlimb suspension increases the efficacy of Ca²⁺-induced Ca²⁺ release in cardiac myocytes by a mechanism that modulates the intrinsic properties of RyRs rather than the density of these receptors.