Introduction

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The release of Ca²⁺ by the sarcoplasmic reticulum is of critical importance to excitation-contraction coupling, and altered intracellular Ca²⁺ homeostasis has been implicated in heart disease (Sordahl et al., 1973; Baudet et al., 1992). The sarcoplasmic reticulum Ca²⁺ channel (or ryanodine receptor), a protein with a large cytoplasmic domain showing high affinity for ryanodine, is the main mechanism implicated in the release of Ca²⁺ from the sarcoplasmic reticulum in cardiac muscle (Ogawa, 1994; Sitsapesan et al., 1995; Franzini-Armstrong and Protasi, 1997). The ryanodine Ca²⁺-release channel is activated by Ca²⁺ and by a large number of chemically diverse substances such as caffeine, halothane, and ryanodine (Laï et al., 1988; Rousseau et al., 1987, 1988; Rousseau and Meissner, 1989; Sitsapesan et al., 1995). Caffeine, which is used to study intracellular Ca²⁺ homeostasis in striated muscles, can determine the susceptibility of patients to malignant hyperthermia by the in vitro contracture test (Herrmann-Frank et al., 1996b). Effect of caffeine has been tested into planar lipid bilayers (Rousseau et al., 1988) and has shown that this substance that acts on the sarcoplasmic reticulum ryanodine Ca²⁺-release channel also increases the number and duration of open events without changing the conductance of the channel. However, caffeine has been found to exert various side effects. In particular, caffeine increases the Ca²⁺ sensitivity of cardiac and skeletal contractile proteins and inhibits phosphodiesterases (Butcher and Sutherland, 1962; Wendt and Stephenson, 1983).

Chlorocresols, which are preservatives often added to commercial preparations of succinylcholine, recently have been shown to be strong stimulators of the Ca²⁺-release channel in skeletal muscle and cerebellum (Zorzato et al., 1993). In heavy sarcoplasmic reticulum vesicles from rabbit skeletal back muscles, 4-chloro-m-cresol (4-CmC)-stimulated Ca²⁺ activated [³H]ryanodine binding with a half-maximal activation of about 100 µM, which suggests that it could be a potent tool in differentiating the Ca²⁺ release mechanism between normal muscles and those susceptible to malignant hyperthermia (Herrmann-Frank et al., 1996b).

In biopsies from muscle susceptible to malignant hyperthermia, 4-CmC evoked a caffeine-like contracture and has a concentration threshold lower than that in normal muscle (Herrmann-Frank et al., 1996b). Previous experiments have indicated that ferret heart is a good model for investigating excitation-contraction-coupling mechanisms (Huchet et al., 1992). Moreover, caffeine is a common tool for inducing contractile responses in intact and saponin-skinned cardiac fibers. For example, caffeine elicited a large transient contracture in isolated trabeculae from ferret heart by releasing Ca²⁺ from intracellular stores (Chapman and Léoty, 1976b).

The purpose of this study was to determine whether 4-CmC could generate caffeine-like responses in ferret cardiac muscle. The concentration dependence of 4-CmC-mediated release of Ca²⁺ from the sarcoplasmic reticulum was studied in intact cardiac trabeculae and saponin-skinned fibers in which the sarcoplasmic reticulum was loaded with Ca²⁺. In both preparations, the effect of 4-CmC on sarcoplasmic reticulum Ca²⁺ content was estimated by analysis of caffeine contracture after application of chlorocresol. In addition, the effects of 4-CmC on maximal Ca²⁺-activated tension and the Ca²⁺ sensitivity of myofibrils were analyzed by using Triton-skinned cardiac fibers. The results show that 4-CmC generates a contractile response in saponin-skinned but not intact fibers. The sarcoplasmic reticulum is implicated in 4-CmC response; more precisely, in Ca²⁺ release via the ryanodine receptor. Moreover, 4-CmC, like caffeine, has effects on maximal Ca²⁺-activated tension and the Ca²⁺ sensitivity of myofibrils.

	Materials and Methods

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All procedures in this study were performed in accordance with the stipulations of the Helsinki Declarations for the care and use of laboratory animals. Adult male ferrets were anaesthetized heavily by an ether vapor flow. After respiratory arrest, the heart was removed quickly and placed at room temperature in a physiological solution (see composition below).

Experiments in Intact Trabeculae. For contractile experiments, free-running trabeculae (50-250 µm in diameter; 5-8 mm in length) were isolated from the right ventricle. The cardiac preparation was placed on a coverslip in a drop of physiological solution, transferred to the experimental chamber, and mounted as described by Chapman and Léoty (1976a). Briefly, both ends of the muscle were snared carefully by fine platinum wire loops, one fixed to the experimental dish and the other to the tip of a transducer (KD 2300 displacement measuring system; Kaman, Colorado Springs, CO). The preparation was perfused continuously with physiological solution at 20 ml/min, and the system of perfusion described by Chapman and Léoty (1976a) allows a rapid change of the bathing solution in 0.2 s. A 10-mM caffeine-transient contracture was produced and the fiber was stretched until caffeine contracture amplitude was maximal. During the experiment, no significant change was observed in the characteristics of the transient contracture. The preparation then was perfused with 4-CmC solution at different concentrations for 2 min before application of the caffeine solution (2.5, 5, or 10 mM). The three concentrations of caffeine (2.5, 5, and 10 mM) used in the present experiments do not give identical responses. Moreover, the maximal response was obtained with 10 mM caffeine. Indeed, as shown by Baudet et al. (1992), the application of different concentrations of caffeine (0.1-10 mM) in ferret heart ventricular fibers evoked a transient contracture, whose strength showed a clear dependence on the drug concentration.

Chemically Skinned Ventricular Fibers. Short, cut bundles (150-300 µm in diameter; 2.0-2.5 mm in length) from papillary muscles of adult ferret heart were dissected and placed in a relaxing solution of pCa 9.0 (pCa = log[Ca²⁺]). Bundles were treated for 30 min in pCa 9.0 solution containing 50 µg/ml saponin (Endo and Iino, 1980). This treatment disrupts the sarcolemma but does not affect the ability of the sarcoplasmic reticulum to accumulate and release Ca²⁺. The preservation of sarcoplasmic reticulum function is indicated by the ability of caffeine to evoke contractures (Endo and Kitazawa, 1979). For Triton-skinned fibers, preparations were placed for 1 h in a relaxing solution (pCa 9.0) containing 1% (v/v) Triton X-100. After this skinning procedure, the fibers were transferred in a relaxing solution (pCa 9.0) that did not contain Triton X-100 before being mounted in the experimental chamber. This treatment permeabilizes the sarcolemma and the sarcoplasmic reticulum without affecting the biochemical and structural properties of the myofibrils, thereby allowing measurement of the Ca²⁺ sensitivity of contractile proteins and maximal Ca²⁺-activated tension. The saponin- or Triton-skinned bundles were transferred and mounted in an experimental system, as described by Huchet and Léoty (1993). This system allowed measurements of the tension developed by the preparation immersed in 2.5-ml tubes (Nalge Nunc Int., Roskilde, Denmark). These tubes were placed on a rotative plate fixed on a disc placed on a magnetic stirrer (Rotamag 10; Prolabo, Paris, France), and the solutions were mixed continuously with stir bars. Fibers were mounted between two stainless steel tubes. One end of a fiber was snared in a loop of fine hair pulled into a tube glued to a fixed rod that was part of the transducer (KD 2300; 0.5 unshielded; Kaman). The other end of the preparation was snared similarly to a tube glued to a rod that formed the arm of the transducer. The diameter and length of the skinned muscle fibers were measured under a binocular microscope. The preparation was adjusted to slack length and then stretched step by step until the tension developed at pCa 4.5 became maximal. Maximal tension (T_max) generally was reached when resting length was increased by 20%. All experiments were performed at 22°C.

Ca²⁺ Uptake and Release in Sarcoplasmic Reticulum of Saponin-Skinned Cardiac Muscle Fibers. For the experiments on saponin-skinned fibers, the preparations were immersed successively in five different solutions. This protocol allows the loading of the sarcoplasmic reticulum with Ca²⁺ and then the release of Ca²⁺ from sarcoplasmic reticulum through application of caffeine, which generates a transient contracture (Su and Hasselbach, 1984). The ionic composition of these solutions and the variations in EGTA, Ca²⁺, and Mg²⁺ concentrations are indicated below. The saponin-skinned preparation was placed first in solution 1 (pCa 9.0, 10 mM EGTA, 1 mM Mg²⁺, 25 mM caffeine), which depleted the sarcoplasmic reticulum of Ca²⁺. Solution 2 (pCa 9.0, 10 mM EGTA, 1 mM Mg²⁺) was used to wash out caffeine. Solution 3 (pCa 6.5, 10 mM EGTA, 1 mM Mg²⁺) was a sarcoplasmic reticulum Ca²⁺-loading solution obtained by mixing pCa 9.0, 10 mM EGTA, and 1 mM Mg²⁺ with pCa 4.5, 10 mM EGTA, and 1 mM Mg²⁺ in appropriate proportions, and has the same composition in ATP (3.16 mM) and in all other components as pCa 9.0 and pCa 4.5. Solution 4 (pCa 7.0 or 6.5, 0.1 mM EGTA, 0.1 mM Mg²⁺) was used to wash out solution 3 and to prepare the fiber for the next solution. Solution 5 (pCa 7.0 or 6.5, 0.1 mM EGTA, 0.1 mM Mg²⁺) was similar to solution 4, but contained different caffeine concentrations (2.5, 5, or 10 mM) added to induce Ca²⁺ release from the sarcoplasmic reticulum. Saponin-skinned fibers were incubated for 2 min in each solution, except in solution 5, for which incubation time was based on contracture duration. Each preparation was run sequentially through load-release cycles. At the beginning of the experiment, two cycles were performed with caffeine. 4-CmC-induced release of Ca²⁺ from the sarcoplasmic reticulum was investigated by using different concentrations of chlorocresol (0.1, 0.5, 1, or 2 mM) in solution 5 instead of caffeine. Immediately after application of 4-CmC, a fiber was immersed in 10 mM caffeine solution (solution 5) to estimate sarcoplasmic reticulum Ca²⁺ content. The reversibility of the effects of 4-CmC was tested by a control cycle without 4-CmC. For each contracture (caffeine or 4-CmC), amplitude (mN/mm²), time to peak (s), and half-relaxation time (s) were measured.

Triton X-100-Skinned Cardiac Muscle Fibers. Tension-pCa relationships (pCa = log[Ca²⁺]) were obtained by exposing Triton-skinned fibers sequentially to solutions of decreasing pCa. These intermediate solutions were obtained by mixing pCa 9.0 and pCa 4.5 solutions in appropriate quantities. At the beginning of each experiment, a full set of solutions containing different concentrations of Ca²⁺ was prepared, and each Ca²⁺ concentration was duplicated, one serving as the control and the other containing 4-CmC (0.05, 0.1, 0.5, 1, or 2 mM). Isometric tension was recorded continuously on chart paper (Linear Bioblock 1200; Linear Instruments, Reno, NV), and baseline tension was established at the steady state measured in a relaxing solution (pCa 9.0). Data for relative tensions were fitted by using a modified Hill equation (Huchet and Léoty, 1993):
The Hill coefficient, nH, and the pCa for half-maximal activation, pCa₅₀ = log₁₀(K/nH), were calculated for each experiment by using linear regression analysis. K corresponds to the Ca²⁺ concentration (M) that induced half-maximal activation: K = 10^pCa₅₀. The Hill coefficient for each fiber was calculated as the slope of the fitted straight lines. Resting tension was that for pCa 9.0, and T_max tension was obtained in pCa 4.5. pCa₅₀ expressed the apparent Ca²⁺ sensitivity of contractile proteins, and nH indicated the cooperativity (Ashley et al., 1991).

Skinned Fiber Solutions. The composition of the solutions, i.e., the Ca²⁺ concentration, was calculated by using the computer program of Godt and Nosek (1986). The basic solutions (pCa 9.0, 4.5, 6.5, and 7.0) used contained: 10 mM (pCa 9.0 and 4.5) or 0.1 mM EGTA (pCa 7.0 or 6.5), 30 mM imidazole, 30.6 mM Na⁺, 1 mM (pCa 9.0 and 4.5) or 0.1 mM Mg²⁺ (pCa 7.0 or 6.5), 3.16 mM Mg-ATP, 12 mM phosphocreatine, and 0.3 mM dithiothreitol. Ionic strength was adjusted to 160 mM with KCl, and the pH was adjusted to 7.1 with HCl or KOH. In saponin-skinned fiber experiments, solutions also contained phosphocreatine kinase (17.5 UI/ml) and sodium azide (1 mM). EGTA and phosphocreatine were obtained from Sigma Chemical Co. (St. Louis, MO), and 4-CmC was purchased from Fluka (Neu Ulm, Germany) and prepared as a stock solution (0.25 M) in dimethyl sulfoxide.

Physiological Solutions. The control physiological solution contained: 140 mM NaCl, 6 mM KCl, 3 mM CaCl₂, 5 mM glucose, and 5 mM HEPES. The pH was adjusted to 7.35 with Tris base.

Fitting of Inhibition Curves. For the test of inhibition of caffeine contracture, the percentage of decrease of response amplitude was estimated as compared with caffeine contracture in control conditions. The points obtained at various concentrations of 4-CmC were fitted by a sigmoid equation. IC₅₀ was the 4-CmC concentration producing half-maximal inhibition of caffeine contracture amplitude, and n was the slope of the linear section of the curves.

Statistical Analysis. All values are expressed as means ± S.E.M. Student's unpaired t test was used to compare the different parameters among groups. Statistical significance was reached when P  .05.

	Results

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Effects of 4-CmC on Resting Tension and Caffeine Contracture in Intact Trabeculae from Ferret Heart. The application of different concentrations of 4-CmC (0.1-2 mM) to quiescent isolated trabeculae produced no detectable change in resting tension (Fig. 1), possibly because of a lack of effect on the Ca²⁺-release mechanism of sarcoplasmic reticulum. To test this possibility, caffeine contractures were elicited after application of 4-CmC on intact cardiac trabeculae. Three concentrations of caffeine were chosen (2.5, 5, and 10 mM) to see whether the possible effect of 4-CmC was related to the concentration of caffeine. Figure 1 shows an example of the transient responses elicited by 2.5 mM caffeine in control conditions (first trace) and then after application of different concentrations of 4-CmC for 2 min. Treatment of trabeculae with 4-CmC reduced the amplitude of caffeine contracture and accelerated the relaxation phase of caffeine contracture. These results strongly suggest that 4-CmC decreases sarcoplasmic reticulum Ca²⁺ content in a dose-dependent manner in intact trabeculae without causing a rise in tension. The decrease in the amplitude of caffeine contracture (2.5, 5, or 10 mM) was plotted as a function of 4-CmC concentrations. The fitted curves obtained (Fig. 2) were similar for all three concentrations of caffeine. In the presence of 4-CmC, whatever caffeine concentrations were tested (2.5, 5, or 10 mM), the IC₅₀ was not significantly different, i.e., (P  .05): 0.27 ± 0.01 (n = 5), 0.29 ± 0.04 (n = 6), and 0.29 ± 0.03 (n = 5) mM 4-CmC for 2.5, 5, and 10 mM caffeine, respectively. Moreover, the slopes of the caffeine inhibition curves (n) were not significantly different, i.e., (P  .05): 1.60 ± 0.17 (n = 5), 1.67 ± 0.11 (n = 6), and 1.96 ± 0.14 (n = 5) for 2.5, 5, and 10 mM caffeine, respectively. The results showed that the IC₅₀ was similar for the three concentrations of caffeine tested. To analyze the effect of 4-CmC at the sarcoplasmic reticulum level, experiments then were performed on saponin-skinned fibers in which functional sarcoplasmic reticulum was preserved and sarcolemma was disrupted.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Effect of 4-CmC on caffeine contracture in ferret intact trabeculae. Arrows indicate the application of a 2.5-mM caffeine solution; the first trace is the control response to 2.5 mM caffeine. In the following run, the same fiber was exposed to a range of 4-CmC concentrations, i.e., 0.1 (a), 0.2 (b), 0.4 (c), 0.5 (d), and 1 (e) mM, followed immediately by application of 2.5 mM caffeine. Temperature is 22°C.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Inhibition curves of caffeine contracture for different concentrations of 4-CmC on ferret intact trabeculae. Data are shown as the percentage of inhibition of 2.5 (; n = 5), 5 (; n = 6), and 10 (; n = 5) mM caffeine contracture amplitudes for each concentration of 4-CmC, as compared with the control. Points are connected by a curve fitting a sigmoid. The slope of the curves (n) and the IC50 are indicated for each caffeine concentration in text. Vertical bars represent ±S.E.M.

Effects of 4-CmC on Sarcoplasmic Reticulum Ca²⁺-Release in Saponin-Skinned Cardiac Fibers of Adult Ferret: Comparison with Caffeine. In saponin-skinned fibers in which functional sarcoplasmic reticulum has been preserved, caffeine induces a transient contracture that indicates the presence of a definite amount of Ca²⁺ released by the sarcoplasmic reticulum. In our experiment, the caffeine in the Ca²⁺ releasing solution (solution 5) was replaced with 4-CmC at different concentrations (0.1, 0.5, 1, and 2 mM). 4-CmC induced a transient contracture in a dose-dependent manner (Fig. 3), yielding the dose-response curve plotted in Fig. 4A. The concentration threshold for 4-CmC (0.1 mM) was similar to that obtained with caffeine. At pCa 7.0, 4-CmC concentrations greater than 2 mM have been tested. Five and 10 mM 4-CmC induced smaller contractures than that obtained for 2 mM. For example, 2 mM 4-CmC contracture amplitude represented 28.0 ± 4.0% of T_max (n = 5) whereas 5 and 10 mM 4-CmC induced contractile responses of 27.0 ± 2.0 and 17.0 ± 3.5% of T_max (n = 4), respectively. According to the concentrations of 4-CmC tested, the maximal response was obtained for 2 mM 4-CmC. On the contrary, maximal amplitude of caffeine contracture was obtained for a higher concentration: 5 mM (Fig. 4B). The kinetics of 4-CmC contractures (for 0.1, 0.5, and 1 mM), i.e., time to peak and half-relaxation time, was faster than for caffeine contractures but not significantly different. For example, the half-relaxation time of the contracture induced by 1 mM 4-CmC or caffeine was 14.5 ± 2.9 s (n = 5) and 22.7 ± 7.0 s (n = 6), respectively. However, 4-CmC contractures at a given concentration were always smaller than those of caffeine. For 1 mM 4-CmC and caffeine, the relative amplitude of the contracture (amplitude/T_max control) was 0.14 ± 0.04 (n = 5) and 0.38 ± 0.07 (n = 5), respectively (Fig. 4). This could be explained if less Ca²⁺ was released by 4-CmC than by caffeine.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Effect of 4-CmC on caffeine contracture in ferret saponin-skinned cardiac fibers. Arrows indicate the application of a 5-mM caffeine solution; the first trace is the control response to 5 mM caffeine. In the following run, the same fiber was exposed to a range of 4-CmC concentrations, i.e., 0.1 (a), 0.2 (b), 0.4 (c), 0.5 (d), and 1 (e) mM, followed immediately by application of 5 mM caffeine. Temperature is 22°C.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   4-CmC (A; n = 5) and caffeine (B; n = 5) dose-response curves plotted for pCa 7.0 () and pCa 6.5 () on ferret cardiac saponin-skinned fibers. Contracture amplitudes are related to the control maximal-activated tension. In A, "a" represents the difference when the amplitudes of the 4-CmC contractures were compared with the maximal response obtained at 2 mM 4-CmC for each concentration of calcium 107 and 106.5 M. In B, "a" represents the difference in the amplitudes of the caffeine contractures obtained at 107 and 106.5 M calcium.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Inhibition curves of caffeine contracture for different concentrations of 4-CmC in ferret cardiac saponin-skinned fibers. Data are the percentage of inhibition of 2.5 (; n = 5), 5 (; n = 5), and 10 mM (; n = 7) caffeine contractures for each concentration of 4-CmC as compared with the control. Points were fitted by using a sigmoid equation, which yielded the slope of the curve (n) and the IC50. Vertical bars represent ±S.E.M. (see values in text).

View larger version (12K): [in this window] [in a new window]   Fig. 6.   4-CmC contracture (1 mM) after ryanodine treatment (100 µM) in cardiac saponin-skinned fibers at pCa 7.0. The experiments were conducted as indicated: cycles 1, 2, 3, and then 4. The cycle consists of the saponin-skinned fibers protocol that leads to a contracture. The first two traces correspond to the contractures obtained with 10 mM caffeine (1) and 1 mM 4-CmC (2) in control conditions, i.e., before ryanodine treatment. The last traces (3 and 4) were obtained after ryanodine treatment. Trace 3 represents the tension developed in the presence of 10 mM caffeine plus 100 µM ryanodine after three running challenges. Trace 4 is the 1-mM 4-CmC response after ryanodine treatment.

Effects of Ca²⁺ Concentration on Ca²⁺ Release Induced by Caffeine and 4-CmC. It has been shown that Ca²⁺ plays the major role in regulating cardiac sarcoplasmic reticulum Ca²⁺-release channel activity (Xu et al., 1996). Moreover, it has been shown that Ca²⁺ shifts dose-response curves to lower 4-CmC concentrations in sarcoplasmic reticulum vesicles of skeletal muscle when pCa 7 and pCa 6 are used (Herrmann-Frank et al., 1996a). Nevertheless, Györke et al. (1994) demonstrated that the Ca²⁺ sensitivity and Ca²⁺ activation rate of single cardiac and skeletal sarcoplasmic reticulum Ca²⁺ channels are similar when reconstituted in artificial planar bilayers, but in the cell there may be other factors that regulate calcium-induced calcium release. In this context, we tested the effects of cytoplasmic Ca²⁺ concentration on Ca²⁺ release induced by 4-CmC (0.1, 0.5, 1, and 2 mM) and compared the results with those obtained at pCa 7.0. The same experiments as those reported previously and carried out at pCa 7.0 (10⁷ M of intracellular Ca²⁺) were realized by using a modified intracellular Ca²⁺ concentration of 10^6.5 M (pCa 6.5). Up to 1 mM, 4-CmC induced similar responses for the two pCa tested (Fig. 4A). In the presence of 1 mM 4-CmC, contracture amplitude was 2.9 ± 0.5 mN/mm² (n = 5) and 2.0 ± 0.7 mN/mm² (n = 5) at pCa 7.0 and 6.5, respectively.

7

6.5

Effects of 4-CmC on Properties of Contractile Proteins. The maximal Ca²⁺-activated tension and apparent Ca²⁺ sensitivity of contractile proteins were analyzed in the absence and presence of different concentrations of 4-CmC (0.05, 0.1, 0.5, 1, and 2 mM) in cardiac Triton-skinned fibers (Fig. 7). In heart fibers, a significant decrease in maximal Ca²⁺-activated tension was observed in the presence of 4-CmC (Table 1), i.e., 16.9 ± 3.3% for 0.1 mM 4-CmC (n = 5) as compared with control fibers. This effect was enhanced with increasing concentrations. For each concentration of 4-CmC, the dose-response curves obtained with saponin-skinned fibers were recalculated by reporting the amplitude of 4-CmC contracture (mN/mm²) to the T_max developed in the presence of 4-CmC (Fig. 9). The results showed that up to 2 mM, 4-CmC contractures were lower than caffeine but not significantly different.

View larger version (6K): [in this window] [in a new window]   Fig. 7.   Effect of 4-CmC on Ca2+-activated tension and maximal Ca2+-activated tension (pCa 4.5) in cardiac Triton-skinned fibers. Representative traces of Triton-skinned fibers were obtained from cardiac muscle. Tension was induced by soaking fibers in solutions of decreasing pCa not containing (af) or containing (a'f') 2 mM 4-CmC. Values for pCa were: a, a' = 6.5; b, b' = 6.25; c, c' = 6; d, d' = 5.875; e, e' = 5.75; f, f' = 4.5. Then, fiber was returned in pCa 9.0, not containing (g) or containing (g') 2 mM 4-CmC.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Effects of various concentrations of 4-CmC on the myofibrillar Ca2+ sensitivity of ventricle Triton-skinned fibers from cardiac muscle. Isometric tension-pCa (log [Ca2+]) relationships were determined in cardiac fibers in the absence () or presence of 1 () and 2 () mM 4-CmC. Force is expressed as the percentage of Tmax at pCa 4.5 for each concentration of 4-CmC tested. Curves were fitted by the modified Hill equation (see text). Temperature is 22°C.

	Discussion

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Our results show that 4-CmC (0.1, 0.5, 1, and 2 mM) applied to saponin-skinned fibers induced a variation of tension related to Ca²⁺ release from the sarcoplasmic reticulum mediated mainly by the ryanodine receptor.

4-CmC recently has been described as a specific, potent activator of ryanodine receptor in skeletal muscle cells, and could replace caffeine as a useful pharmacological tool for the detection of malignant hyperthermia (Herrmann-Frank et al., 1996b). Moreover, 4-CmC can be used at concentrations 10-fold lower than those of caffeine. Caffeine is known to induce contractile responses in a dose-dependent manner in cardiac muscle (Chapman and Léoty, 1976b), whereas 4-CmC failed to produce a contractile response on intact fibers in the present study. In intact fibers, the absence of responses resulting from the application of 4-CmC could be due to a slow diffusion of 4-CmC across the sarcolemma, and then the calcium would not be released rapidly enough to result in a contracture. Such a possibility was not supported by the experiments conducted in skeletal muscle, where 4-CmC produces a contractile response (Herrmann-Frank et al., 1996a). Another possibility would be a lack of Ca²⁺ release from the sarcoplasmic reticulum. However, the decrease of caffeine contracture in a dose-dependent manner after treatment with 4-CmC indicated that sarcoplasmic reticulum Ca²⁺ content was reduced by 4-CmC. Moreover, in saponin-skinned fibers in which the sarcolemma was disrupted and the sarcoplasmic reticulum was preserved, 4-CmC induced a contractile response. It has been shown previously that 4-CmC had no effect on the sarcoplasmic reticulum Ca²⁺-ATPase in PC12 cell vesicles (Zorzato et al., 1993) and in intact skeletal muscle (Westerblad et al., 1998). Then it could be proposed that the absence of 4-CmC contracture was related to a Ca²⁺ extrusion by sarcolemmal Ca²⁺ mechanisms. Particularly, it has been demonstrated that in ferret heart the relaxation of caffeine contracture was mainly a result of sarcolemmal Ca²⁺-ATPase and Na⁺/Ca²⁺ exchanger (Bassani et al., 1995). A possible stimulation of these mechanisms by 4-CmC would support further the absence of 4-CmC contracture in intact ferret heart. In saponin-skinned fibers, the disappearance of 4-CmC contracture after application of 100 µM ryanodine associated with caffeine and the activation of ryanodine receptor by 4-CmC confirm that 4-CmC releases Ca²⁺ from the sarcoplasmic reticulum, probably by direct activation of the ryanodine receptor. 4-CmC, which is considered to be a specific activator of the skeletal ryanodine receptor (Herrmann-Frank et al., 1996a), also appears to be an activator of the cardiac ryanodine receptor. Our results indicate that inhibition of caffeine contracture by 4-CmC was not dependent on the concentration of caffeine used in intact and saponin-skinned fibers; this suggests that the potential site of 4-CmC action on the ryanodine receptor is different from that of caffeine. These results are consistent with those of Herrmann-Frank et al. (1996a), who suggested that 4-CmC acts on a different binding site than caffeine on sarcoplasmic reticulum vesicles. Moreover, their experiments indicated that 4-CmC activated the isolated channel more strongly when applied from luminal binding sites. These authors have shown that 4-CmC acts preferentially on the trans face of sarcoplasmic reticulum vesicles of skeletal muscle whereas caffeine acts mainly on the cis face as reported by Sitsapesan et al. (1995).

One reason for using 4-CmC on saponin-skinned fibers of adult ferret would be to activate Ca²⁺-release channels without producing side effects on contractile proteins. The use of Triton-skinned fibers made it possible to determine the effects of 4-CmC on the myofibrillar Ca²⁺ responsiveness of mammalian cardiac muscle. At concentrations above 0.5 mM, 4-CmC increased the Ca²⁺ sensitivity of the contractile apparatus. With 1 mM 4-CmC, the increase was significant for the pCa₅₀ control, showing a direct effect on sensitivity to the Ca²⁺ of contractile proteins. This effect could not be attributed to any action on ryanodine Ca²⁺-release channels in sarcoplasmic reticulum because the skinned fibers were treated with 1% Triton X-100 to solubilize sarcoplasmic reticulum membranes. Moreover, because highly buffered EGTA-active solutions were used, any calcium release from remaining active sarcoplasmic reticulum would have been buffered by this solution.

Our study showed a clear decrease in maximal activated force in the presence of 4-CmC, which was dose-dependent and particularly apparent. This may explain why the contractile responses produced with 4-CmC, as compared with caffeine, were less in saponin-skinned fibers and absent in intact fibers. It is likely that 4-CmC affects the biochemical states of cross-bridges during the working cycle, causing a reduction in their number and/or generating force.

However, the tension-pCa curve was shifted to lower concentrations in heart muscle. Because pCa-tension curves generally are assumed to reflect the calcium-binding properties of Troponin C, the effect of 4-CmC may have been due to a direct action on contractile proteins, more particularly, Troponin C. Further research is needed to determine how 4-CmC might affect myofilaments. In terms of our results, it is likely that 4-CmC at concentrations above 0.5 mM exerts a dose-dependent sensitizing effect on the contractile apparatus and, from 0.1 mM, decreases maximal Ca²⁺-activated tension for cardiac fibers. This could explain why 4-CmC on saponin-skinned fibers could induce lower contractures than with caffeine. Indeed, when using up to 10 mM caffeine, no variation in the T_max was observed. In contrast to the curves plotted in Fig. 4, the amplitude of the 4-CmC contractures were larger when effects of 4-CmC on the T_max were taken into account (Fig. 9). The results showed that with up to 2 mM, 4-CmC contractures were not significantly different from caffeine.

View larger version (17K): [in this window] [in a new window]   Fig. 9.   4-CmC dose-response curves (n = 5) plotted for pCa 7.0 () and pCa 6.5 () on ferret cardiac saponin-skinned fibers. Contracture amplitudes are related to the maximal-activated tension in the presence of 4-CmC. a, the difference when the amplitudes of the 4-CmC contractures were compared with the maximal response obtained at 2 mM 4-CmC for the each concentration of calcium, 107 and 106.5 M.

Interestingly, the effect of 4-CmC on myofibrillar responsiveness is reminiscent of that of caffeine in ferret and rat cardiac muscle (Wendt and Stephenson, 1983; Baudet and Ventura-Clapier, 1990). 4-CmC and caffeine both increase the Ca²⁺ sensitivity of the contractile apparatus. For example, 2 mM 4-CmC increases Ca²⁺ sensitivity from 6.02 ± 0.03 (n = 10) to 6.13 ± 0.02 (n = 8), whereas a 5-mM concentration of caffeine increases it from 5.80 ± 0.03 (n = 14) to 5.94 ± 0.01 (n = 14), as shown by Baudet and Ventura-Clapier (1990). Consequently, the contractile apparatus is more sensitive to 4-CmC.

In summary, 4-CmC induced tension variations in Ca²⁺-loaded fibers due to Ca²⁺ release from the sarcoplasmic reticulum in association with an effect on the contractile machinery. Experiments with caffeine and ryanodine suggested that 4-CmC releases Ca²⁺ by acting on the ryanodine receptor, which is as sensitive to 4-CmC as caffeine. With 4-CmC, as with caffeine, care must be taken in interpreting results. Further investigations should consider whether 4-CmC has any effect on the Na⁺/Ca²⁺ exchanger or Ca²⁺-ATPase of the sarcolemma.