Introduction

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Molecular and functional differences between fast-twitch [extensor digitorum longus (edl)] and slow-twitch (soleus) skeletal muscles have been well documented in recent years in relation to contractile proteins (Danielli-Betto et al., 1990), pumping mechanisms of the sarcoplasmic reticulum (Brandl et al., 1986), intracellular proteins, or calcium-release mechanisms from the sarcoplasmic reticulum (Jorgensen and Jones, 1986; Damiani and Margreth, 1994; Delbono and Meissner, 1996). In mammalian skeletal muscle, the main source of Ca²⁺ is the sarcoplasmic reticulum, from which Ca²⁺ is released mainly through the ryanodine receptor RyR1 (Takeshima et al., 1989; Ogawa, 1994; Franzini-Armstrong and Protasi, 1997). The mechanisms of Ca²⁺ release in fast- and slow-twitch fibers have been analyzed in sarcoplasmic reticulum vesicles (Lee et al., 1991) and in intact (Delbono and Meissner, 1996) and skinned (Salviati and Volpe, 1988) fibers through the use of different drugs and calcium-release modulators of ryanodine receptor (caffeine, Mg²⁺, Ca²⁺, ryanodine, doxorubicin). Although caffeine has been widely used as a Ca²⁺-releasing agent in intact and skinned fibers (Rousseau et al., 1988; Salviati and Volpe, 1988; Fryer and Neering, 1989; Su and Chang, 1995; Pagala and Taylor, 1998), it is known to exert various side effects, particularly inhibition of phosphodiesterases and increase in Ca²⁺ sensitivity of cardiac and skeletal contractile proteins (Butcher and Sutherland, 1962; Wendt and Stephenson, 1983).

Chlorocresols, especially 4-chloro-m-cresol (4-CmC), have recently been reported to be strong stimulators of ryanodine receptors in cerebellum, intact skeletal muscle, and cardiac skinned fibers (Zorzato et al., 1993; Herrmann-Frank et al., 1996a,b; Westerblad et al., 1998; Choisy et al., 1999). In particular, 4-CmC stimulated Ca²⁺-activated [³H]ryanodine binding on heavy sarcoplasmic reticulum vesicles from rabbit back muscles, producing a half-maximal activation at about 100 µM (Herrmann-Frank et al., 1996a,b). Moreover, 4-CmC increased the affinity of [³H]ryanodine binding on sarcoplasmic reticulum vesicles from malignant hyperthermia-susceptible muscle compared with normal muscle. Consequently, it has been proposed that 4-CmC could replace caffeine in the test for muscle susceptibility to malignant hyperthermia (Herrmann-Frank et al., 1996a,b). Furthermore, 4-CmC induced a caffeine-like transient contracture in intact fibers at concentrations 10 times less than that with caffeine (Herrmann-Frank et al., 1996a,b). Thus, the results in the literature indicate that slow- and fast-twitch muscles have different sensitivities to caffeine (Salviati and Volpe, 1988) and that 4-CmC is a more sensitive tool than caffeine (Herrmann-Frank et al., 1996a,b). In this context, the aim of this work was to compare the effect of 4-CmC on edl and soleus muscles and to investigate whether 4-CmC induces a more specific contractile response than caffeine. The experiments were conducted on saponin-skinned fibers (in which the sarcoplasmic reticulum and contractile apparatus were functional and the sarcolemma disrupted) of edl and soleus rat muscles. The effect of 4-CmC on the Ca²⁺ content of sarcoplasmic reticulum was estimated by analysis of caffeine contracture after the application of different concentrations of chlorocresol. The cytosolic Ca²⁺ concentration dependence of the 4-CmC response was also investigated. Moreover, the effects of 4-CmC on the contractile apparatus of edl and soleus Triton X-100-skinned fibers were tested.

	Materials and Methods

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All procedures in this study were performed according to a university committee and to the stipulations of the Helsinki Declarations for the care and use of laboratory animals.

Adult male rats were heavily anesthetized by an ether vapor flow. After respiratory arrest, the heart was quickly excised, and fast- and slow-twitch skeletal muscles (edl and soleus) were removed and placed at room temperature in a physiological solution that contained 140 mM NaCl, 6 mM KCl, 3 mM CaCl₂, 5 mM glucose, and 5 mM HEPES. The pH was adjusted to 7.4 with Tris-base. All experiments were conducted on chemically skinned preparations of hindlimb muscles.

Chemically Skinned Skeletal Fibers. Small bundles (100- to 250-µm diameter and 1.5-2.5 mm in length) of soleus and edl muscles were dissected and placed in a relaxing solution of pCa 9.0 (pCa = log₁₀[Ca²⁺]), of a composition that is reported in Table 1, for subsequent chemical skinned treatments (saponin or Triton X-100). Saponin 50 µg/ml (Endo and Iino, 1980) was prepared in a pCa 9.0 solution in which the preparations were immersed for 30 min under constant stirring. This treatment disrupts the sarcolemma but does not affect the ability of the sarcoplasmic reticulum to accumulate and release Ca²⁺. The preservation of the sarcoplasmic reticulum function is indicated by the capacity of caffeine to induce contractures (Endo and Kitazawa, 1978).

Ca²⁺ Load-Release Cycle in Sarcoplasmic Reticulum of Saponin-Skinned Skeletal Muscle Fibers. A single fiber was successively immersed in five different solutions (Table 1). This protocol makes it possible to load the sarcoplasmic reticulum with Ca²⁺ and then release it by applying caffeine (Su and Hasselbach, 1984). EGTA, Mg²⁺, Ca²⁺, and caffeine concentrations varied with the solutions. Solution 1 (pCa 9.0), consisting of 10 mM EGTA, 1 mM Mg²⁺, and 25 mM caffeine, was used to deplete the sarcoplasmic reticulum of Ca²⁺. Solution 2 was a caffeine-free washing solution and was similar to solution 1. Solution 3 (pCa 7.0), consisting of 10 mM EGTA and 1 mM Mg²⁺, allowed Ca²⁺ loading of the sarcoplasmic reticulum and was obtained by mixing pCa 9.0 and pCa 4.5, (10 mM EGTA, 1 mM Mg²⁺), in appropriate proportions. Solution 4 (pCa 7.0 or 7.5), consisting of 0.4 mM EGTA and 0.1 mM Mg²⁺, was used to wash out solution 3 and to prepare the fiber for the next solution. Solution 5 was similar to solution 4 but contained different concentrations of caffeine (0.1-25 mM) or 4-CmC (2.5 µM to 2 mM) added to induce transient contracture. At the beginning of the experiments, two or three challenges were performed with caffeine (10 mM). The experimental protocol consisted of a test cycle of 4-CmC contracture using different 4-CmC concentrations (2.5 µM to 2 mM) added to solution 5 in the place of caffeine. Immediately after the application of 4-CmC, the fiber was immersed in a 10 mM (or 2.5 mM) caffeine solution to estimate the decrease of sarcoplasmic reticulum Ca²⁺ content. Skinned fibers were incubated for 2 min in all solutions except solution 5, in which fiber was immersed until the end of contracture. The experiments were conducted in slow- and fast-twitch skeletal muscles. For caffeine or 4-CmC response, contracture amplitude (mN/mm²), time to peak (s), and time of half-relaxation (s) were measured. Contracture amplitudes were related to the maximal tension developed in the presence of 4-CmC (or caffeine), and the dose-response curves were fitted for each fiber.

Triton X-100-Skinned Skeletal Muscles Fibers. Tension-pCa relationships were obtained by exposing Triton-skinned fibers of slow- and fast-twitch skeletal muscles sequentially to solutions of decreasing pCa. The intermediate solutions were obtained by mixing pCa 9.0 and pCa 4.5 (10 mM EGTA, 1 mM Mg²⁺) solutions (Table 1) in appropriate quantities. Solutions containing different concentrations of Ca²⁺ were prepared. For each concentration, one solution served as a control and the other contained 4-CmC (0.01, 0.5, 1, or 2 mM). Isometric tension was recorded, as for saponin-skinned fibers. Baseline tension was established at the steady state measured in the relaxing solution pCa 9.0.

Skinned Fiber Solutions. The composition of the solutions (i.e., the Ca²⁺ concentrations used for saponin or Triton X-100 protocols) was calculated using the computer program of Godt and Nosek (1986). The composition of basic solutions (pCa 9.0, 4.5) was reported in Table 1. For each solution, ionic strength was adjusted to 160 mM with KCl and pH was adjusted to 7.1 with HCl or KOH. In saponin-skinned fiber experiments, solutions also contained phosphocreatine kinase (17.5 I.U./ml) and sodium azide (1 mM).

Analysis of Fitted Curves for Saponin-Skinned Fibers. For each experimented fiber, 4-CmC (or caffeine) contracture amplitudes were related to the maximal tension developed in the presence of 4-CmC (or caffeine), and results were fitted using a modified Hill equation (Huchet and Léoty, 1993), which gave an EC₅₀ and a Hill coefficient. Mean values calculated for these two parameters were used to plot the dose-response curves on which mean values of each concentration of 4-CmC (or caffeine) were reported.

Statistical Analysis. All values are expressed as mean ± S.E.M. Student's unpaired t test was used to compare the different parameters between edl and soleus. Statistical significance was reached when P < .05.

	Results

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4-CmC Effects on Sarcoplasmic Reticulum Ca²⁺-Release Mechanisms and Comparison with Caffeine. Different 4-CmC concentrations (2.5 µM to 2 mM) were added to the Ca²⁺-release solution (pCa 7.5). After an identical loading procedure in saponin-skinned fibers of edl and soleus muscles, the application of 4-CmC produced a caffeine-like transient contracture in a dose-dependent manner (Fig. 1, A and B). In edl fibers, a contracture threshold was found at 10 µM 4-CmC, and in soleus fibers, a threshold was found at larger concentrations of 25 to 50 µM (Fig. 2A). The maximal amplitude of 4-CmC response (Table 2) was also obtained at lower concentrations in edl (0.5 mM) than in soleus (2 mM). The 4-CmC-EC₅₀ (i.e., the 4-CmC concentration providing 50% of the maximal 4-CmC contracture) was significantly lower in edl (70 ± 10 µM, n = 9) than in soleus (180 ± 40 µM, n = 8; P < .05). The results for different concentrations of 4-CmC (0.5, 1 and 2 mM) showed that the time to peak (s) of the edl contracture was significantly shorter than for soleus (Table 3). At 1 mM 4-CmC, the time to peak of edl was 38% shorter than that observed for soleus. The time of half-relaxation of 4-CmC contracture was different for edl and soleus. Indeed, an increase in the 4-CmC concentration induced a decrease of the time of half-relaxation for soleus but increased it for edl (Table 3).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   4-CmC contractures in soleus (A) and edl (B) saponin-skinned fibers at pCa 7.5. The first trace corresponds to the application of 10 mM caffeine, and the last four traces represent responses obtained for 0.05, 0.1, 0.5, and 1 mM 4-CmC. The "squared" variation in tension occurring before the contracture is due to the change of solutions. Experiments were performed at 22°C.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   4-CmC dose-response curves for saponin-skinned fibers at pCa 7.5 (A) and pCa 7.0 (B). Amplitudes of the contractures obtained for soleus (n = 8, A; n = 6, B) and edl (n = 9, A; n = 8, B) are expressed as a percentage of maximal response. Curves were fitted using the modified Hill equation.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Caffeine dose-response curves for saponin-skinned fibers at pCa 7.5 (A) and pCa 7.0 (B). Amplitudes of the contractures obtained for soleus (n = 5, A; n = 3, B) and edl (n = 6, A; n = 7, B) are expressed as a percentage of maximal response. Curves were fitted using the modified Hill equation.

Effect of 4-CmC on Sarcoplasmic Reticulum Ca²⁺ Content. Caffeine contracture is commonly used to study sarcoplasmic reticulum Ca²⁺ content. In an attempt to determine whether 4-CmC affected sarcoplasmic reticulum Ca²⁺ content, caffeine contractures were elicited after application of different concentrations of 4-CmC to saponin-skinned slow- and fast-twitch muscles. Experiments conducted at pCa 7.5 consisted in applying different concentrations of 4-CmC (0.01-2 mM) for 1 min followed by the application of caffeine. To assess the effects of 4-CmC, 2.5 mM caffeine was selected as the concentration producing approximately 50% of maximal contracture and 10 mM as that producing maximal response.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Inhibition curves of caffeine contracture for different concentrations of 4-CmC in soleus (A) and edl (B) saponin-skinned fibers at pCa 7.5. Data are the percentages of inhibition of 2.5 (n = 5, A; n = 6, B) and 10 (n = 5, A; n = 10, B) mM caffeine contractures for each concentration of 4-CmC compared with the control. Points were fitted using a sigmoid equation, which yielded the slope of the curve (n) and the IC50. Vertical bars represent ±S.E.M.

Action of 4-CmC and Caffeine on Ryanodine Receptor. It is well known that the sarcoplasmic reticulum Ca²⁺-release channel is inhibited by concentrations of ryanodine 10 µM only if this receptor is activated (Alderson and Feher, 1987). Caffeine is an activator of the ryanodine receptors. Caffeine (10 mM) was associated with ryanodine (100 µM), and after three challenges, the caffeine contracture disappeared (Fig. 5, traces 1 and 2). In these conditions, to see whether 4-CmC was acting as caffeine, we applied 4-CmC (1 mM) with ryanodine (100 µM): after three challenges, 4-CmC contracture was totally suppressed (Fig. 5, traces 3 and 4). Then, because 4-CmC and caffeine associated with ryanodine had the same effect, it suggested that 4-CmC was acting on the same Ca²⁺-release mechanism as caffeine (i.e., the ryanodine receptor).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   4-CmC (1 mM) and caffeine contracture (10 mM) after ryanodine treatment (100 µM) in slow-twitch skeletal saponin-skinned fibers at pCa 7.5. Caffeine experiments (1 and 2) were conducted on one fiber, and 4-CmC experiments (3 and 4) were conducted on another fiber. Traces 1 and 3 correspond to the contractures obtained respectively with 10 mM caffeine (1) and 1 mM 4-CmC (3), in control conditions, i.e., before ryanodine treatment. Traces 2 and 4 represent respectively the tension developed in the presence of 10 mM caffeine associated with 100 µM ryanodine and in the presence of 1 mM 4-CmC associated with 100 µM ryanodine, after three running challenges. Tension remaining for trace 2 is due to an effect on contractile apparatus.

Cytosolic Ca²⁺ Modulation of Ca²⁺ Release Induced by 4-CmC. It has been shown that the effectiveness of caffeine in increasing the rate of Ca²⁺ release is dependent on the free Ca²⁺ concentration in the release medium (Rousseau et al., 1988); to further compare the two substances, the effect of an increase in free Ca²⁺ concentration on the Ca²⁺-release rate by caffeine and 4-CmC was tested. In saponin-skinned fibers, the influence of cytosolic (cis) Ca²⁺ concentrations (31 and 100 nM) on 4-CmC contractile responses was tested in both types of fibers. 4-CmC (2.5 µM to 2 mM) was applied to the Ca²⁺-release solution at pCa 7.0 (100 nM Ca²⁺), and contractile responses were compared with those obtained at pCa 7.5 (31 nM Ca²⁺). 4-CmC dose-responses curves plotted for pCa 7.0 showed that the threshold concentrations in both muscles (2.5-5 µM, n = 8 for edl; 10-50 µM, n = 6 for soleus) and the EC₅₀ values were shifted to lower values than those observed at pCa 7.5 (Fig. 2, A and B). Thus, edl saponin-skinned fibers showed a 7- to 10-fold lower 4-CmC-EC₅₀ when sarcoplasmic reticulum Ca²⁺-release channels were activated by 4-CmC in the presence of 100 nM Ca²⁺. For example, in edl, the EC₅₀ values were significantly different (P < .05) at pCa 7.5 and pCa 7.0: 70 ± 10 µM (n = 6) and 10 ± 2 µM (n = 8) of 4-CmC, respectively. Moreover, 4-CmC-EC₅₀ found at pCa 7.0 was significantly different between edl and soleus. An increase in intracellular Ca²⁺ induced a more reduced shift in the 4-CmC dose-response curves of soleus than edl. The EC₅₀ value was significantly potentiated (P < .05) more than twice as much when cis Ca²⁺ was increased by 69 nM in soleus muscle [i.e., 70 ± 10 µM (n = 6) compared with 180 ± 40 µM, n = 8, at pCa 7.5].

Effects of 4-CmC on Properties of Contractile Proteins. Maximal Ca²⁺-activated tension (T_max) and apparent Ca²⁺ sensitivity of contractile proteins (pCa₅₀) were analyzed in the absence and presence of different concentrations of 4-CmC (0.01, 0.5, 1, or 2 mM) in skeletal Triton X-100-skinned fibers. Tension in soleus and edl muscle fibers was measured in control conditions and in the presence of 4-CmC (Fig. 6). The relationships between steady-state Ca²⁺-activated tension and free Ca²⁺ concentrations in the presence and absence of 1 and 2 mM 4-CmC are illustrated in Fig. 7. In both edl and soleus fibers, the capacity of contractile proteins to develop force when maximally activated by Ca²⁺ (pCa 4.5) was reduced in the presence of increasing concentrations of 4-CmC (Table 4). The decrease in maximal tension was more pronounced in edl than in soleus (Table 4). With 2 mM 4-CmC, T_max was decreased by 59.4% in edl fibers (n = 9) compared with only 28.4% in soleus fibers (n = 12). The effect of large concentrations of 4-CmC (1 and 2 mM) on maximal Ca²⁺-activated tension was not reversible.

View larger version (10K): [in this window] [in a new window]   Fig. 6.   Effect of 2 mM 4-CmC on Ca2+-activated tension and maximal Ca2+-activated tension (pCa 4.5) of soleus (A) and edl (B). Tension was induced by soaking fibers in a solution of decreasing pCa not containing (a-g, soleus; a-h, edl) or containing (a'-g', soleus; a'-h', edl) 2 mM 4-CmC. Values for pCa were a = 7, b = 6.5, c = 6.25, d = 6, e = 5.875, f = 4.5, g = 9 in soleus and a = 7, b = 6.5, c = 6.25, d = 6.125, e = 6.0, f = 5.875, g = 4.5, h = 9 in edl.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Effects of various concentrations of 4-CmC on myofibrils Ca2+ sensitivity of slow- and fast-twitch Triton-skinned fibers from skeletal muscle. Tension-pCa curves were obtained with soleus (A) and edl (B) fibers. Isometric tension-pCa (log10[Ca2+]) relationships were determined in twitch fibers in the absence (n = 12, A; n = 10, B) or presence of 1 (n = 8, A; n = 10, B) or 2 (n = 12, A; n = 9, B) mM 4-CmC. Force is expressed as the percentage of maximal tension at pCa 4.5 for each concentration of 4-CmC tested. Curves were fitted using the modified Hill equation. Temperature was 22°C.

	Discussion

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This study shows that 4-CmC induces caffeine-like transient contractures in a dose-dependent manner in saponin-skinned fibers isolated from fast- and slow-twitch skeletal muscles of the rat and that the muscles show differences in sensitivity (Fig. 1, A and B). Previous studies have reported that 4-CmC is a potent activator of skeletal (Herrmann-Frank et al., 1996a,b; Westerblad et al., 1998) and cardiac (Choisy et al., 1999) ryanodine receptors. Unlike caffeine, which induces contractures with millimolar concentrations (Salviati and Volpe, 1988; Herrmann-Frank et al., 1996a,b), 4-CmC proved efficient with micromolar concentrations. This result is consistent with that of Herrmann-Frank et al. (1996b), who found a threshold activity for 75 µM 4-CmC on intact human skeletal fibers isolated from malignant hyperthermia nonsusceptible muscle. Moreover, other authors have shown that 4-CmC was efficient at micromolar concentrations on PC12 cells (Zorzato et al., 1993), intact mouse skeletal muscle (Westerblad et al., 1998), and frog skeletal fibers (Struk and Melzer, 1999). The difference in sensitivity to 4-CmC and caffeine of skeletal muscles could be explained by the presence of distinct site or sites of action of these two substances on the ryanodine receptor. Indeed, it has been reported that caffeine acts preferentially on the cytosolic side of the ryanodine receptor, whereas 4-CmC is more potent in activating the ryanodine receptor when applied on the luminal side (Herrmann-Frank et al., 1996a).

The decrease in caffeine contractures (2.5 and 10 mM) induced by 4-CmC suggests that 4-CmC releases Ca²⁺ from the sarcoplasmic reticulum. Furthermore, caffeine and 4-CmC contractures were totally abolished when ryanodine (100 µM) was associated with each of these substances, which would indicate that 4-CmC and caffeine activate the same Ca²⁺-release mechanism (i.e., the ryanodine receptor).

In saponin-skinned fibers, edl showed greater sensitivity (a lower threshold and EC₅₀) than soleus for 4-CmC, which was probably not due to an inhibitory action on the sarcoplasmic reticulum Ca²⁺ pump. Indeed, Zorzato et al. (1993) concluded that at 1 mM, chlorocresol did not involve inhibition of sarcoplasmic reticulum Ca²⁺ pump on longitudinal sarcoplasmic reticulum vesicles. Moreover, Westerblad et al. (1998) showed that on intact structure, 0.1 mM 4-CmC had no inhibitory effect on the Ca²⁺ ATPase of sarcoplasmic reticulum. Under our conditions of experiments used for saponin-skinned fibers, it is difficult to answer to this question.

Our results also showed that 4-CmC inhibited the caffeine contractures in edl and soleus muscles with a different sensitivity. The difference in 4-CmC sensitivity between fast- and slow-twitch skeletal muscles may also have resulted from the presence of various isoforms of ryanodine receptor (RyR1 and/or RyR3) in these two types of muscle (Conti et al., 1996) and/or the difference in ryanodine receptor gating (Shin et al., 1996). Different reports have shown that Ca²⁺-release kinetic is faster in intact fibers (Delbono and Meissner, 1996), in sarcoplasmic reticulum vesicles (Lee et al., 1991), and in skinned fibers (Salviati and Volpe, 1988) of edl than of soleus muscle. Our results indicated that the time to peak of 4-CmC contractures was shorter for edl than for soleus (Table 3).

The increase in cytosolic Ca²⁺ (31-100 nM, i.e., pCa 7.5-7.0) shifted the dose-response curves to lower 4-CmC concentrations in saponin-skinned fibers. These results are similar to those reported by Herrmann-Frank et al. (1996a) for sarcoplasmic reticulum vesicles of skeletal muscle, in which a decrease in cytosolic Ca²⁺ (900-100 nM) shifted the dose-response curve to higher 4-CmC concentrations. Moreover, 4-CmC used to detect pathological muscular structure (malignant hyperthermia), in which the resting myoplasmic Ca²⁺ concentration is increased, is described to make the ryanodine receptor more sensitive to [³H]ryanodine binding (Herrmann-Frank et al., 1996b). In our study, edl exhibited greater sensitivity than soleus to 4-CmC. This effect was more marked for higher than lower cytosolic Ca²⁺ activity, whereas under similar conditions the caffeine sensitivity of skeletal muscles was less affected. This difference between caffeine and 4-CmC could be explained by a 4-CmC binding site, presumably located on the luminal side of the ryanodine receptor and close to the potential high-affinity Ca²⁺ intrareticular binding site as suggested by Herrmann-Frank et al. (1996a).

Thus, 4-CmC appears to be a more useful pharmacological tool than caffeine in discriminating between the contractile responses of edl and soleus, especially if the side effects on contractile proteins can be reduced.

Triton X-100-skinned fibers were used to determine the effect of 4-CmC on the myofibrillar responsiveness of mammalian skeletal muscle. These results show that in both edl and soleus muscles, 4-CmC decreased maximal-activated tension in a dose-dependent manner, particularly in edl at concentrations of 0.5 mM. It could be proposed that 4-CmC affects the biochemical states of crossbridges during the working cycle. The tension-pCa curves were shifted to lower Ca²⁺ concentrations in soleus and to higher concentrations in edl. As pCa-tension curves are assumed to reflect the Ca²⁺-binding properties of troponin C (TnC), the effect of 4-CmC could be due to its direct action on contractile proteins, and more particularly on TnC. Indeed, striated muscles contain two isoforms derived from a single copy gene: TnC-fast (TnC-f) expressed in fast skeletal muscle and TnC-slow (TnC-s) in slow muscle (Wilkinson, 1980). A major difference between the two TnC isoforms concerns the Ca²⁺ binding loops. Our investigations indicate that the Hill coefficient in edl was significantly decreased by the application of 2 mM 4-CmC but only slightly modified in soleus fibers. Accordingly, one possible explanation for changes in the Ca²⁺ sensitivity of contractile proteins is that Ca²⁺ binding loops were affected by 4-CmC. Further research is required to determine in which way 4-CmC affects myofilaments. Interestingly, the effect of 4-CmC on myofibrillar responsiveness is reminiscent of that of caffeine in skeletal muscles (Wendt and Stephenson, 1983).

In conclusion, these results indicating that micromolar 4-CmC concentrations release Ca²⁺ via activation of the sarcoplasmic reticulum ryanodine receptor in mammalian skeletal muscle strongly support the findings of Herrmann-Frank et al. (1996a,b). The difference in sensitivity to cytosolic Ca²⁺ activity between caffeine and 4-CmC could be of importance to studies of muscular pathology resulting in part from increased intracellular Ca²⁺. Moreover, because edl is more sensitive than soleus to 4-CmC, this substance may be a better tool than caffeine in discriminating between edl and soleus contractile responses.