Elevation of extracellular osmolarity improves signs of myotonia congenita in vitro: a preclinical animal study.

KEY POINTS: During myotonia congenita, reduced chloride (Cl- ) conductance results in impaired muscle relaxation and increased muscle stiffness after forceful voluntary contraction. Repetitive contraction of myotonic muscle decreases or even abolishes myotonic muscle stiffness, a phenomenon called 'warm up'. Pharmacological inhibition of low Cl- channels by anthracene-9-carboxylic acid in muscle from mice and ADR ('arrested development of righting response') muscle from mice showed a relaxation deficit under physiological conditions compared to wild-type muscle. At increased osmolarity up to 400 mosmol L-1 , the relaxation deficit of myotonic muscle almost reached that of control muscle. These effects were mediated by the cation and anion cotransporter, NKCC1, and anti-myotonic effects of hypertonicity were at least partly antagonized by the application of bumetanide. ABSTRACT: Low chloride-conductance myotonia is caused by mutations in the skeletal muscle chloride (Cl- ) channel gene type 1 (CLCN1). Reduced Cl- conductance of the mutated channels results in impaired muscle relaxation and increased muscle stiffness after forceful voluntary contraction. Exercise decreases muscle stiffness, a phenomena called 'warm up'. To gain further insight into the patho-mechanism of impaired muscle stiffness and the warm-up phenomenon, we characterized the effects of increased osmolarity on myotonic function. Functional force and membrane potential measurements were performed on muscle specimens of ADR ('arrested development of righting response') mice (an animal model for low gCl- conductance myotonia) and pharmacologically-induced myotonia. Specimens were exposed to solutions of increasing osmolarity at the same time as force and membrane potentials were monitored. In the second set of experiments, ADR muscle and pharmacologically-induced myotonic muscle were exposed to an antagonist of NKCC1. Upon osmotic stress, ADR muscle was depolarized to a lesser extent than control wild-type muscle. High osmolarity diminished myotonia and facilitated the warm-up phenomenon as depicted by a faster muscle relaxation time (T90/10 ). Osmotic stress primarily resulted in the activation of the NKCC1. The inhibition of NKCC1 with bumetanide prevented the depolarization and reversed the anti-myotonic effect of high osmolarity. Increased osmolarity decreased signs of myotonia and facilitated the warm-up phenomenon in different in vitro models of myotonia. Activation of NKCC1 activity promotes warm-up and reduces the number of contractions required to achieve normal relaxation kinetics.