Dantrolene normalizes serum creatine kinase in MDX mice.

The dystrophy of a mutant mouse (MDX) is allelic to Duchenne muscular dystrophy (DMD), with a shared lack of the subsarcolemmal protein, dystrophin. lZ4 The mouse’s dystrophin deficiency has such mild (subclinical) consequences that other factors must explain the pathophysiology of dystrophin-deficient dystrophies. Impaired calcium regulation occurs in MDX’ and DMD,8 and may be a secondary, perhaps crucial step in muscle destruction. ’ For this reason we chose to investigate dantrolene’s influence on the expression of a known marker of the dystrophin-deficient phenotype, (in man,’ mouse,‘ and dog”): elevated serum creatine kinase (CK). Four groups of mice were studied: mdx dystrophic mice treated with dantrolene (30 mg/kg/day, n = 1 1 ) or placebo solution (n = 17) and C57/BL10 normal mice treated with dantrolene suspension (30 mg/kg/day, n = 25) or placebo solution (n = 23). The dantrolene suspension (3.0 mg/mL suspended in 0.5% methyl-cellulose solution) and placebo solution (0.5% methyl-cellulose) were administered by gavage (0.01 mugram body weight) once daily after a 2-hour fast, beginning at age 14-17 days. The mice were sacrificed at ages 17-44 days to measure CK and limb weight. Animal care and handling was in accordance with NIH guidelines. CK assays were based on the methods of Oliver’ and Rosalki‘ (Creatine Kinase Kit #45-UV, and Creatine Kinase Control #CPK-I, Sigma Diagnostics, St. Louis, MO). Blood samples were drawn by cardiac puncture under ketamine anesthesia (0.1 mg/g) approximately 4 hours after gavage. Blood samples for measuring dantrolene by high-pressure liquid chromatography, were drawn from 12 control mice 1 , 8, and 24 hours after dosing. Hindlimb dorsiflexors and plantarflexors with attached tibia and fibula were weighed to the nearest milligram (Sartorius, model 2434) within 20 minutes of exsanguination. The limb was disjointed with scalpel, and the biceps femoris, seminiembranosis, and semitendinosis trimmed with sharp scissors. Chronic administration of dantrolene by gavage markedly lowered the serum CK levels in mdx mice @I < . 0001, Wilcoxon Rank Sums) but left levels in C57/ BLlO control mice unaffected (Figure 1). No deaths occurred in either the treated or placebo mdx groups. Three control animals died of aspiration pneumonias (two placebo, one dantrolene). Dantrolene treatments left both mdx and C57/BL10 mice noticably weaker for 2-3 hours in the first 3 days of treatment but not thereafter. All four groups showed comparable weight gains (leg and body) over the study period and similar muscle shape and color. Serum dantrolene concentration was 10.8 pg/mL one hour, 1.0 pg/ml 8 hours, and undetectable 24 hours after dosing. The reduction of CK in the mdx mouse via chronic administration of dantrolene has both therapeutic and pathophysiologic implications for MDX and by its allelic relationship, for DMD. CK, a myoplasmic enzyme, leaks into the blood by at least two routes’: muscle fiber necrosis that releases the myoplasmic contents, or altered sarcolemmai permeability without such necrosis. Greater serum CK from greater sarcoplasmic permeability may reflect the subsarcolemmal location of dystrophin and, perhaps, its function. ’,‘ Dantrolene acts on the t-tubule, inhibiting the sarcoplasmic reticulum’s calcium release. ” The dramatically lower CK in the dantrolene-treated MDX implicates defective calcium homeostasis in the pathophysiology of MDX and suggests a potential therapeutic effect. Our data tend to rule out two other explanations of the lowered CK levels: reduced muscle activity and enhanced CK excretion, both of which can lower serum levels without improving the course of muscle diseases. After some weakness in the first 3 days of dantrolene treatment, all mice were fully active. Further, leg weight was slightly higher in the dantrolene-treated group; inactivity would be expected to induce atrophy. Any dantrolene-induced enhanced excretion CK should have reduced CK levels in dantrolene-treated C57/BL10 mice; this did not occur. Despite these interesting findings, we do not yet recommend dantrolene as a therapy for DMD: (1) A reduction in CK does not prove a significant therapeutic