Dantrolene requires Mg2+ to arrest malignant hyperthermia

Abstract

Malignant hyperthermia (MH) is a clinical syndrome of skeletal muscle that presents as a hypermetabolic response to volatile anesthetic gases, where susceptible persons may develop lethally high body temperatures. Genetic predisposition mainly arises from mutations on the skeletal muscle ryanodine receptor (RyR). Dantrolene is administered to alleviate MH symptoms, but its mechanism of action and its influence on the Ca²⁺ transients elicited by MH triggers are unknown. Here, we show that Ca²⁺ release in the absence of Mg²⁺ is unaffected by the presence of dantrolene but that dantrolene becomes increasingly effective as cytoplasmic-free [Mg²⁺] (free [Mg²⁺]_cyto) passes mM levels. Furthermore, we found in human muscle susceptible to MH that dantrolene was ineffective at reducing halothane-induced repetitive Ca²⁺ waves in the presence of resting levels of free [Mg²⁺]_cyto (1 mM). However, an increase of free [Mg²⁺]_cyto to 1.5 mM could increase the period between Ca²⁺ waves. These results reconcile previous contradictory reports in muscle fibers and isolated RyRs, where Mg²⁺ is present or absent, respectively, and define the mechanism of action of dantrolene is to increase the Mg²⁺ affinity of the RyR (or "stabilize" the resting state of the channel) and suggest that the accumulation of the metabolite Mg²⁺ from MgATP hydrolysis is required to make dantrolene administration effective in arresting an MH episode.

Keywords: dantrolene; magnesium; malignant hyperthermia; ryanodine receptor; skeletal muscle fiber.

Fig. 1.

Ca²⁺ transients evoked by the removal of Mg²⁺ are not inhibited by dantrolene. Selected images of cytoplasmic rhod-2 fluorescence during continuous xyt recordings (at 0.8 s·frame⁻¹) in rat skinned fibers acquired while applying Ca²⁺ release inducing 0 Mg²⁺ solution either in the absence (A) or presence of dantrolene (B) and 1 mM EGTA and 300 nM Ca²⁺ (Table S1). (Scale bar: 50 μm.) (C) Spatially averaged values of normalized fluorescence intensity (F/F₀) versus elapsed time from the full experiment that is represented in A and B. Applied free [Mg²⁺]_cyto is given at the top, and exchange of solutions is indicated by pale blue vertical bars. A 2-min time interval was allowed for after each low Mg²⁺ transient to recover SR Ca²⁺. The applied free [Ca²⁺]_cyto was kept constant throughout the recording. The presence of 50 µM dantrolene is indicated by the horizontal bar. Mean normalized peak amplitude values (F/F₀) (D) and FDHM (E) in the absence and presence of dantrolene (n = 5 fibers). A paired Student’s t test revealed no significant difference (P > 0.05) in both D and E.

Fig. S1.

Ca²⁺ transients evoked by the removal of Mg²⁺ are not inhibited by dantrolene in the presence of exogenous calmodulin. (A) Spatially averaged profile of xyt recordings of Ca²⁺ transients in rat skinned fibers evoked by the nominal removal of Mg²⁺ either in the absence (first Ca²⁺ release) or presence of dantrolene and exogenous calmodulin (second Ca²⁺ release) and the presence of 1 mM EGTA and 300 nM Ca²⁺ (see Table S1). Applied free [Mg²⁺]_cyto is given at the top. A 2-min time interval was allowed for after each low Mg²⁺ transient to recover SR Ca²⁺ levels in a solution with 1 mM free Mg²⁺ and 300 nM Ca²⁺. The presence of 50 µM dantrolene and 100 nM calmodulin is indicated by the horizontal bar. Mean normalized peak amplitude values (F/F₀) (B) and FDHM (C) in the absence and presence of dantrolene (n = 5 fibers). A paired Student’s t test revealed no significant difference (P > 0.05) in both B and C.

Fig. 2.

Inhibition of electrically evoked Ca²⁺ transients by dantrolene is dependent on the free [Mg²⁺]_cyto. (A) Original recordings in rat skinned fibers as obtained by confocal line scans parallel to the fiber long axis (Top), with corresponding line-averaged and normalized rhod-2 fluorescence signals (F/F₀, Bottom). Cytosolic Ca²⁺ transients were elicited by electrical field stimulation at 1 Hz in the absence (Left) or presence (Right) of 50 µM dantrolene at free [Mg²⁺]_cyto of 0.4 (Top), 1 (Middle), or 3 mM (Bottom). All solutions contained 1 mM EGTA and 100 nM free Ca²⁺ (Table S1). Note that rhod-2 fluorescence was recorded at 2 ms·line⁻¹. (B) Effect of dantrolene on Ca²⁺ transient peak amplitudes for the [Mg²⁺]_cyto as shown in A. Best fit of the data to a Hill equation in the absence and presence of 50 µM dantrolene yielded IC₅₀ values of 2.95 ± 1.42 and 1.73 ± 1.63 mM, respectively. Curves are significantly different from each other (Extrasum of square F test). The red data points represent the Ca²⁺ transient amplitudes in the presence of 100 nM exogenous calmodulin (CaM) in the presence of dantrolene. Note that these points are not different from the responses in the presence of dantrolene with only the endogenous calmodulin. (C) % inhibition of Ca²⁺ transient by dantrolene at indicated [Mg²⁺]_cyto. (D) Concentration-dependent inhibition of electrically evoked Ca²⁺ transients by dantrolene at 3 mM free [Mg²⁺]_cyto. Best fit Hill curve had an IC₅₀ value of 0.41 ± 2.61 µM. All data are derived from 5 to 18 fibers and presented as mean ± SEM.

Fig. 3.

Halothane-induced Ca²⁺ waves in human MHS fibers require increases in free [Mg²⁺]_cyto to be affected by dantrolene. (A) Spatially averaged cytoplasmic rhod-2 fluorescence in a fiber from subject A during changes in the internal bathing solution from one containing 1 mM free [Mg²⁺]_cyto to 0 Mg²⁺ followed by reintroduction of 1 mM free [Mg²⁺]_cyto and subsequent exposure to 1 mM halothane. Note that halothane induced repetitive Ca²⁺ waves in the presence of 1 mM free [Mg²⁺]_cyto and that data were acquired at 0.8 s·frame⁻¹. [Ca²⁺] was 100 nM in all solutions. (B) Examples of MHS fibers from subject A exposed to 1 mM halothane and 1, 2, and 3 mM free [Mg²⁺]_cyto in the presence or absence of 50 µM dantrolene. Frequency of halothane-induced Ca²⁺ waves in the presence of 1–3 mM free [Mg²⁺]_cyto and the presence and absence of dantrolene in fibers isolated from the biopsies of subject A (C) and subject B (D). Data in C and D were fit with a Hill equation. IC₅₀ values amounted to 3.2 ± 1.05 and 1.6 ± 1.06 mM (C) and 2.1 ± 1.05 and 1.6 ± 1.06 mM (D) in the presence of halothane and halothane plus dantrolene, respectively. Curves in the presence and absence of 5 (D) and 50 µM dantrolene (C) were significantly different from each other (extra sum of squares F test). All data points are derived from four to five fibers and presented as mean ± SEM.

Fig. S2.

The effect of low caffeine on human muscle fiber from subject A. (A) Spatially averaged profile of cytoplasmic rhod-2 fluorescence of human skinned fiber during the reduction of [Mg²⁺]_cyto from 1 to nominally 0, and the exposure to 3 mM caffeine and 1 mM Mg²⁺. The fiber is in the constant presence of 0.1 mM EGTA and 100 nM Ca²⁺. Note that regenerative Ca²⁺ waves were observed through the period in the presence of 3 mM caffeine. (B) Summary of the number of fibers from the biopsy used responding with regenerative Ca²⁺ waves to 1 mM halothane or 3 mM caffeine in the presence of 1 mM Mg²⁺. The numbers in the bars represent the number of fibers. The response to low concentrations of halothane and caffeine are consistent with the muscle being MH-susceptible (20).

Fig. S3.

Halothane-induced Ca²⁺ waves in MHS fibers from subject B. Examples of MHS fibers from subject B exposed to 1 mM halothane and 1.5, 2, and 3 mM Mg²⁺ in the presence or absence of 5 µM dantrolene.