Low chloride conductance myotonia - in vitro investigations on muscle stiffness and the warm-up phenomenon

Abstract

Mutations in the chloride channel are causative for low chloride conductance (gCl-) myotonia. The phenotype shows muscle stiffness. By unknown mechanisms, this type of myotonia shows a reduction of stiffness after exercise, the so-called warm-up phenomenon. This study investigated myotonic stiffness and the warm-up phenomenon in vitro. Mechanographic registrations and resting membrane potential measurements were performed. Muscle samples from the animal model for low gCl-myotonia (ADR-mice) as well as pharmacologically induced myotonia were studied. The [K+]o and osmolarity were varied and pharmacological substances with potential clinical benefit were tested. Strong contraction triggers K+ accumulation in the small T-tubular compartment which is capable to initiate myotonic bursts, thereby prolonging muscle relaxation (myotonic stiffness). Further increase in K+ and the osmolarity led to a sustained membrane depolarization in a concentration-dependent manner. At 7 mM [K+]o the myotonic burst rate was suppressed more than 90 %. Repetitive contractions facilitated the warm-up phenomenon reflecting an enhanced stabilization of the membrane excitability. The big conductance calcium activated potassium channel (BK) agonist NS1608 led to a more severe relaxation deficit, but also to a more rapid onset of warm-up. Blocking the BK channel with paxilline prevented the onset of myotonia. Increasing gK+ via activation of the voltage gated potassium channels by retigabine enhanced the warm-up phenomenon. The sodium potassium chloride cotransporter type 1 inhibitor bumetanide prevented the depolarization and reversed the antimyotonic effect of high osmolarity. Blocking voltage gated Na+ channels with mexiletine and flecainide dose-dependently facilitated the warm-up phenomenon in ADR muscle. The findings have an important impact on dietary counseling and the use of drugs influencing gK+ and gNa+ to improve management and treatment of myotonic stiffness in low gCl-myotonia.