Enhanced excitation-coupled calcium entry in myotubes expressing malignant hyperthermia mutation R163C is attenuated by dantrolene

Abstract

Dantrolene is the drug of choice for the treatment of malignant hyperthermia (MH) and is also useful for treatment of spasticity or muscle spasms associated with several clinical conditions. The current study examines the mechanisms of dantrolene's action on skeletal muscle and shows that one of dantrolene's mechanisms of action is to block excitation-coupled calcium entry (ECCE) in both adult mouse flexor digitorum brevis fibers and primary myotubes. A second important new finding is that myotubes isolated from mice heterozygous and homozygous for the ryanodine receptor type 1 R163C MH susceptibility mutation show significantly enhanced ECCE rates that could be restored to those measured in wild-type cells after exposure to clinical concentrations of dantrolene. We propose that this gain of ECCE function is an important etiological component of MH susceptibility and possibly contributes to the fulminant MH episode. The inhibitory potency of dantrolene on ECCE found in wild-type and MH-susceptible muscle is consistent with the drug's clinical potency for reversing the MH syndrome and is incomplete as predicted by its efficacy as a muscle relaxant.

Fig. 1

Dantrolene depresses Ca²⁺ transient amplitude in skeletal myotubes. A, representative EC coupling response from WT myotubes triggered by repetitive pulse trains of 30 Hz (1-ms pulse duration) before (Ctrl), 10 min after perfusion of 10 μM dantrolene (Dan), and 10 min after initiating washout of the drug (Wash). The records shown were taken from continuous measurements of a single myotube. B, expanded time scale showing the initial rate of intracellular Ca²⁺ rise attributed to SR Ca²⁺ release during the first 100 ms of EC coupling elicited by a 30-Hz pulse train before (Ctrl) and 10 min after (Dan) application of dantrolene (Dan = 10 μM). The initial rate of Ca²⁺ rise was limited by the affinity of Ca²⁺ binding to Fluo-4. C, mean and S.E. of normalized Ca²⁺ transient amplitude in n = 15 cells before (Ctrl) and 10 min after application of 10 μM dantrolene (Dan) and after 10 min of washout period (Wash). Measurements of intracellular Ca²⁺ were acquired at 100 Hz using photometry of individual myotubes loaded with Fluo-4 as described under Materials and Methods.

Fig. 2

Dantrolene does not attenuate gating properties of reconstituted RyR1 channels at either 25 or 35°C. A, SDS-PAGE gel showing typical purity of RyR1 preparation used in BLM reconstitutions. B, recording and analysis of purified RyR1-FKBP12 single-channel activity were made as described in detail under Materials and Methods. The specific compositions of the cytoplasmic (cis) and lumenal (trans) solutions are indicated above each representative trace. The upper three traces show representative segments of recordings obtained from a channel at 25°C lasting >20 min after the addition of dantrolene to the cis/trans solutions. This entire experiment was performed on two independent bilayer experiments. The remaining traces (traces 4 - 8) were obtained from an independent BLM experiment that incorporated two channels equilibrated in cis/trans buffers at 35°C. Results from this experimental protocol were replicated in n = 4 independent experiments with the same results. P_o and mean open/closed dwell time values (τ_o/τ_c) were calculated using Clampfit 9.0 and are denoted above each representative trace. For the two-channel's mean open time values, only level one (τ_o1) was presented, whereas P_o represents the open probability of the two channels. The broken line with “c” indicates the 0 current level.

Fig. 3

Dantrolene does not interfere with SOCE. A, SOCE was measured under extreme conditions of long-term store depletion. Fluo-4-loaded myotubes were challenged with 200 nM thapsigargin (TG), an irreversible blocker of SR Ca²⁺ pump in the presence (Dan) or absence (Ctrl) of 10 μM dantrolene. At the end of TG treatment (≥10 min; depletion phase not shown), ~90% of the cells failed to respond to electrical stimulation and caffeine (data not shown). After depletion, the external solution was changed to one containing 2 mM Ca²⁺, and the rate of SOCE was monitored. B, summarized data of experiments shown in A for n = 24 (Ctrl) and n = 25 treated (Dan).

Fig. 4

Dantrolene inhibits Mn²⁺ entry triggered by electrical stimulation of primary myotubes. A, primary WT myotubes exhibited enhanced Mn²⁺ entry (500 μM in a nominally Ca²⁺-free external solution) in response to a 20-Hz train of electrical pulses (ES) monitored by the rate of quench of Fura-2 fluorescence excited at the isosbestic wavelength (trace “Ctrl”). Pretreatment of myotubes for 10 min with 10 μM dantrolene (Dan) reduced the initial rate of Mn²⁺ entry by 72%. B, summarized data of experiments shown in A for n = 18 Ctrl and n = 24 Dan cells. *, p < 0.001.

Fig. 5

Dantrolene inhibits ECCE and Mn²⁺ entry in a dose-dependent manner. A, pretreatment of WT myotubes with 500 μM ryanodine for 30 min locked RyR1 in an inactive conformation unresponsive to caffeine (data not shown). Despite the lack of any response to caffeine in cells loaded with Fluo-4, depolarization of ryanodinetreated myotubes triggered a large ECCE that persisted for the duration of ES indicated by the representative trace labeled “0”. Dantrolene (2-50 μM) inhibited ECCE in ryanodine-treated WT cells in a dose-dependent manner with an IC₅₀ value of 4.2 μM (inset). B, Mn²⁺ entry rate in Fura-2 loaded WT myotubes (not blocked with ryanodine) was also inhibited dose-dependently by dantrolene; n = 12 cells in A and n = 12-20 cells in B.

Fig. 6

Adult skeletal muscle fibers exhibit ECCE that is inhibited by dantrolene. A, WT adult FDB myofibers were assayed with the Mn²⁺ quench assay for the presence of ECCE. Fura-2-loaded myofibers were excited at 360 nm, and emission was collected at 510 nM. Bath perfusion of normal mouse Ringer was followed by perfusion with Mn²⁺-containing Ringer solution (bar labeled Mn²⁺) and then Mn²⁺ Ringer solution containing 40 mM K⁺ (bar labeled KCl). Dishes of myofibers were randomized to control treatment (no drug; Ctrl), dantrolene treatment (+Dan), ryanodine treatment (250 μM, ~1 h; Ry), or ryanodine treatment followed by a 10-min application of dantrolene (10 μM; Ry + Dan). B, summarized data from 23 and 19 fibers for Ry and Ry + Dan treatments, respectively. C, summarized data from nine fibers for Ctrl and Dan. The rates of Mn²⁺ quench of Fura-2 during depolarization with K⁺ were normalized to the quench rate before depolarization of each fiber. Analysis of variance revealed that mean rates were significantly enhanced after depolarization compared with before depolarization for each treatment group (*, p < 0.05).

Fig. 7

Myotubes expressing R163C-RyR1 have an exaggerated ECCE. A, rates of Mn²⁺ entry were measured in Fura-2-loaded myotubes prepared from WT mice and mice HET or HOM for MH susceptibility mutation R163C-RyR1. The rates of Mn²⁺ quench were measured in an external buffer containing 500 μMMn²⁺ + 300 μMCa²⁺ (bar labeled Mn²⁺) before and after delivery of a 20-Hz ES. B, summarized data of mean rate of Mn²⁺ quench for n = 50 WT, n = 69 HET, and n = 69 HOM myotubes. Rates of electrically evoked Mn²⁺ entry for WT was significantly lower than either HET or HOM (*, p < 0.0001).

Fig. 8

Dantrolene attenuates ECCE in WT, HET, and HOM R163CRyR1-expressing myotubes. Myotubes were challenged with electrical pulse trains and monitored for Mn²⁺ entry as described in Fig. 7. Another group of cells was treated with 10 μM dantrolene for 10 min. Data represent the mean rate calculated from responses of n = 20 (WT), 31 (HET), and 43 (HOM) cells. *, p < 0.001 compared with corresponding nontreated genotype.