Anesthesiology 2001; 94:468 –74 © 2001 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Effects of Dantrolene on Rat Diaphragm Muscle during Postnatal Maturation Gilles Orliaguet, M.D.,* Olivier Langeron, M.D.,† Catherine Coirault, M.D., Ph.D.,‡ Sylvia Fratea, M.D.,§ Pierre Coriat, M.D., Bruno Riou, M.D., Ph.D.# Background: Dantrolene is the only known effective treat- ment for malignant hyperthermia. However, its effects on dia- phragm muscle during postnatal maturation remain unknown. Methods: The effects of dantrolene (10 8 to 10 4 M) were investigated in vitro on diaphragm muscle strips in adult rats and in postnatal rats aged 3, 10, and 17 days, and compared with those of ryanodine (10 8 to 10 6 M). The authors studied contraction and relaxation under isotonic and isometric con- ditions (29°C, Krebs-Henseleit solution, tetanic stimulation at 50 Hz). Data are mean  SD. Results: During postnatal maturation, the authors observed a progressive increase in active force developed per cross- sectional area (from 34  25 to 69  32 mN/mm 2 ; P < 0.05) and maximum shortening velocity (from 2.9  0.5 to 4.9  1.4 L max /s; P < 0.05). Dantrolene induced a negative inotropic effect in diaphragm muscles in isotonic and isometric condi- tions in all groups, but this effect was significantly less marked in the 3-day-old rats compared with older rats. Dantrolene did not induce significant lusitropic effects during postnatal matu- ration. Developmental changes in the pharmacologic response to dantrolene were more rapid than those of ryanodine. Conclusion: Dantrolene induced less pronounced negative inotropic effects on the diaphragm in neonatal rats as com- pared with adult rats. Our study suggests that developmental changes in the pharmacologic response to dantrolene are more rapid than those of ryanodine. IN skeletal and diaphragmatic muscles, postnatal matu- ration is associated with important ultrastructural changes, including changes in fiber type, distribution, and size 1 ; biochemical differentiation, including elimina- tion of embryologic and neonatal myosin isoforms 2,3 ; changes in metabolic capacity 4,5 ; and progressive devel- opment of the sarcoplasmic reticulum (SR). 5 These changes result in an improved diaphragm contractility. 6 However, the precise mechanisms by which maturation induces changes in the contractile performance of dia- phragm muscle remain a matter of debate. Dantrolene, the only known effective treatment for malignant hyperthermia, 6 is a postsynaptic skeletal mus- cle relaxant that inhibits calcium release during excita- tion– contraction coupling 7,8 and reduces the myoplas- mic free calcium concentration in a dose-dependent manner. 9 The molecular basis of the action of dantrolene remains not completely understood but is generally pre- sumed to involve either direct or indirect inhibitory effects on ryanodine receptor (RyR) Ca 2+ channels of the SR. 10,11 The effects of dantrolene on muscle during postnatal maturation remain unknown. In adults, dan- trolene induces a major negative inotropic effect on skeletal and diaphragmatic muscles. 12 During postnatal maturation, some quantitative and qualitative changes occur in the biochemical composition of the SR, espe- cially changes regarding RyR3, 13 an isoform of RyR that is predominantly expressed in the diaphragm. There- fore, the extent of the inotropic effect of dantrolene on diaphragmatic muscle during postnatal development could be related to the state of maturation of different element in the contractile system, particularly that of RyR. We therefore conducted an in vitro study on the effects of dantrolene on rat diaphragm muscle during postnatal maturation. We tested the hypothesis that mat- uration may modify the effects of dantrolene on dia- phragm muscles. Materials and Methods Animals and Study Design Care of the animals conformed to the recommenda- tions of the Helsinski Declaration, and the study was performed in accordance with the regulations of the official edict of the French Ministry of Agriculture. After birth, rat pups were kept in cages with their mother. Adult rats received rat chow and water ad libitum. A 12-h light– dark cycle was provided. Experiments were performed on Wistar rats aged 3 days (n = 18), 10 days (n = 18), 17 days (n = 20), and 10 –12 weeks (adult, n = 21). After brief anesthesia with ether, a median laparotomy was performed, and a muscle strip from the ventral costal diaphragm was carefully dissected from the mus- cle in situ while the ribs and the central tendon were left intact, as previously reported. 14,15 With this procedure, diaphragmatic fibers were parallel and of approximately equal length. 15 This diaphragm strip was vertically sus- * Assistant Professor, Department of Anesthesiology, Centre Hospitalo-Univer- sitaire Necker-Enfants Malades. † Assistant Professor, § Staff Anesthesiologist,  Professor and Chairman, # Director of the Laboratory of Experimental Anesthe- siology and Professor, Department of Anesthesiology, Centre Hospitalo-Univer- sitaire Pitié-Salpêtrière. ‡ Chargé de Recherche, Institut National de la Santé et de la Recherche Médicale Unité 451, Laboratoire d’Optique Applique ´e-Ecole Nationale Supe ´rieure des Techniques-Ecole Polytechnique, and Service de Physi- ologie Cardiovasculaire et Respiratoire, Centre Hospitalo-Universitaire Kremlin-Bicêtre. Received from the Laboratoire d’Anesthésiologie, Département d’Anesthésie- Réanimation, Centre Hospitalo-Universitaire Pitié-Salpétriêre, Université Pierre et Marie Curie, Paris, France; Département d’Anesthésie-Réanimation, Centre Hos- pitalo-Universitaire Necker-Enfants Malades, Paris, France; Institut National de la Santé et de la Recherche Médicale Unité 451, Laboratoire d’Optique Applique ´e- Ecole Nationale Supe ´rieure des Techniques-Ecole Polytechnique, Palaiseau, France; and Service de Physiologie Cardiovasculaire et Respiratoire, Centre Hos- pitalo-Universitaire Kremlin-Bicêtre, Le Kremlin-Bice ˆtre, France. Submitted for publication January 14, 1999. Accepted for publication October 11, 2000. Supported by grants from the Société Française d’Anesthésie et de Réanimation and the Association Française contre la Myopathie, Paris, France. Address reprint requests to Dr. Orliaguet: Département d’Anesthésie-Réanima- tion, Groupe Hospitalier Necker Enfants Malades, 149 rue de Sèvres, 75743 Paris Cedex 15, France. Address electronic mail to: gorlia@club-internet.fr. Individual article reprints may be purchased through the Journal Web site, www.anesthesiology.org. Anesthesiology, V 94, No 3, Mar 2001 468