Sarcoplasmic Reticulum Ca Release by 4-Chloro-m-Cresol (4-CmC) in Intact and Chemically Skinned Ferret Cardiac Ventricular Fibers

The purpose of this study was to determine whether 4-chlorom-cresol (4-CmC) could generate caffeine-like responses in ferret cardiac muscle. The concentration dependence of 4-CmC-mediated release of Ca from the sarcoplasmic reticulum was studied in intact cardiac trabeculae and saponinskinned fibers in which the sarcoplasmic reticulum was loaded with Ca. In intact and saponin-skinned preparations isolated from right ventricle, the effect of 4-CmC on sarcoplasmic reticulum Ca content was estimated by analysis of caffeine contracture after application of chlorocresol. In addition, the effects of 4-CmC on maximal Ca-activated tension and the Ca sensitivity of myofibrils were analyzed by using Tritonskinned 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 the 4-CmC response; more precisely, in Ca release via the ryanodine receptor. Moreover, 4-CmC, like caffeine, has effects on maximal Ca-activated tension and the Ca sensitivity of myofibrils. The release of Ca by the sarcoplasmic reticulum is of critical importance to excitation-contraction coupling, and altered intracellular Ca homeostasis has been implicated in heart disease (Sordahl et al., 1973; Baudet et al., 1992). The sarcoplasmic reticulum Ca 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 Ca from the sarcoplasmic reticulum in cardiac muscle (Ogawa, 1994; Sitsapesan et al., 1995; Franzini-Armstrong and Protasi, 1997). The ryanodine Carelease channel is activated by Ca and by a large number of chemically diverse substances such as caffeine, halothane, and ryanodine (Laı̈ et al., 1988; Rousseau et al., 1987, 1988; Rousseau and Meissner, 1989; Sitsapesan et al., 1995). Caffeine, which is used to study intracellular Ca 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 Ca-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 Ca 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 Ca-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 Ca activated [H]ryanodine binding with a half-maximal activation of about 100 mM, which suggests that it could be a potent tool in differentiating the Ca 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 Ca from intracellular stores (Chapman and Léoty, 1976b). The purpose of this study was to determine whether Received for publication November 12, 1998. ABBREVIATIONS: 4-CmC, 4-chloro-m-cresol; Tmax, maximal tension. 0022-3565/99/2902-0578$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 290, No. 2 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 290:578–586, 1999 578 at A PE T Jornals on Jne 4, 2017 jpet.asjournals.org D ow nladed from 4-CmC could generate caffeine-like responses in ferret cardiac muscle. The concentration dependence of 4-CmC-mediated release of Ca from the sarcoplasmic reticulum was studied in intact cardiac trabeculae and saponin-skinned fibers in which the sarcoplasmic reticulum was loaded with Ca. In both preparations, the effect of 4-CmC on sarcoplasmic reticulum Ca content was estimated by analysis of caffeine contracture after application of chlorocresol. In addition, the effects of 4-CmC on maximal Ca-activated tension and the Ca 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 Ca release via the ryanodine receptor. Moreover, 4-CmC, like caffeine, has effects on maximal Ca-activated tension and the Ca sensitivity of myofibrils. Materials and Methods 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 mm 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. In normal Ringer’s solution, the concentration of caffeine that produced 50% of the maximal contracture was 3.71 mM in intact cardiac trabeculae. Consequently, 2.5and 5-mM caffeine concentrations were chosen as values close to the concentration that produced 50% of the maximal contracture and that induced responses that amplitude allowed to estimate the inhibitory effect of 4-CmC. Furthermore, a concentration of 10 mM caffeine also was tested to study the effect of 4-CmC on maximal responses. The same concentrations of caffeine were used in saponin-skinned fibers to have a satisfactory estimate of the 4-CmC effects on caffeine contractures and also to compare the results with those obtained in intact preparations. The variation in the amplitude (mN/mm) of caffeine contracture was normalized to the amplitude of the caffeine contracture before application of 4-CmC (0.1, 0.2, 0.3, 0.4, 0.5, 1, and 2 mM). All experiments were performed at 20°C. Chemically Skinned Ventricular Fibers. Short, cut bundles (150–300 mm 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 5 2log[Ca]). Bundles were treated for 30 min in pCa 9.0 solution containing 50 mg/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 Ca. 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 Ca sensitivity of contractile proteins and maximal Ca-activated tension. The saponinor 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 (Tmax) generally was reached when resting length was increased by 20%. All experiments were performed at 22°C. Ca 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 Ca and then the release of Ca 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, Ca, and Mg concentrations are indicated below. The saponinskinned preparation was placed first in solution 1 (pCa 9.0, 10 mM EGTA, 1 mM Mg, 25 mM caffeine), which depleted the sarcoplasmic reticulum of Ca. Solution 2 (pCa 9.0, 10 mM EGTA, 1 mM Mg) was used to wash out caffeine. Solution 3 (pCa 6.5, 10 mM EGTA, 1 mM Mg) was a sarcoplasmic reticulum Ca-loading solution obtained by mixing pCa 9.0, 10 mM EGTA, and 1 mM Mg with pCa 4.5, 10 mM EGTA, and 1 mM Mg 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 Mg) 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 Mg) was similar to solution 4, but contained different caffeine concentrations (2.5, 5, or 10 mM) added to induce Ca release from the sarcoplasmic reticulum. Saponinskinned 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 Ca 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 sarcoplas1999 4-CmC and Ca Release in Ferret Cardiac Fibers 579 at A PE T Jornals on Jne 4, 2017 jpet.asjournals.org D ow nladed from mic reticulum Ca 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/mm), time to peak (s), and half-relaxation time (s) were measured. Triton X-100-Skinned Cardiac Muscle Fibers. Tension-pCa relationships (pCa 5 2log[Ca]) were obtained by exposing Tritonskinned 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 Ca was prepared, and each Ca 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): Relative tension 5 T/Tmax 5 @Ca #nH/~K! 1 @Ca#. The Hill coefficient, nH, and the pCa for half-maximal activation, pCa50 5 2log10(K/nH), were calculated for each experiment by using linear regression analysis. K corresponds to the Ca concentration (M) that induced half-maximal activation: K 5 1050. 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 Tmax tension was obtained in pCa 4.5. pCa50 expressed the apparent Ca 21 sensitivity of contractile proteins, and nH indicated the cooperativity (Ashley et al., 1991). Skinned Fiber Solutions. The composition of the solutions, i.e., the Ca 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 Mg (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 6 S.E.M. Student’s unpaired t test was used to compare the different parameters among groups. Statistical significance was reached when P #