Ca2+ influx via the Na+/Ca2+ exchanger is enhanced in malignant hyperthermia skeletal muscle

Abstract

Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca(2+) dysregulation. NCX is a bidirectional transporter that effluxes (forward mode) or influxes (reverse mode) Ca(2+) depending on cellular activity. Resting intracellular calcium ([Ca(2+)]r) and sodium ([Na(+)]r) concentrations are elevated in MH susceptible (MHS) swine and murine muscles compared with their normal (MHN) counterparts, although the contribution of NCX is unclear. Lowering [Na(+)]e elevates [Ca(2+)]r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca(2+) or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced [Ca(2+)]r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of [Ca(2+)]r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces [Ca(2+)]r in MHS muscle fibers and decreases the amplitude of [Ca(2+)]r rise triggered by halothane, but had no effect on [Ca(2+)]r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca(2+) transient elicited by high [K(+)]e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced [Ca(2+)]r and the Ca(2+) transient area induced by high [K(+)]e. These results demonstrate a functional NCX3 in skeletal muscle whose activity is enhanced in MHS. Moreover reverse mode NCX3 contributes to the Ca(2+) transients associated with K(+)-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.

Keywords: Calcium Imaging; Calcium Signaling; KB-R7943; Malignant Hyperthermia; Skeletal Muscle; Sodium-Calcium Exchange; YM-244769.

FIGURE 1.

Effect of Na⁺ withdrawal on [Ca²⁺]_r in swine MHN and MHS muscle fibers. Solutions were prepared replacing NaCl with NMG-HCl. Fibers were incubated 5 min and measurements were assessed by double-barreled selective microelectrodes. Data are expressed as mean ± S.E., n = 25 fibers/group, ***, p < 0.001; one-way analysis of variance and Tukey's t test.

FIGURE 2.

Effect extracellular Ca²⁺ removal on Na⁺ depletion-dependent [Ca²⁺]_r increases. Swine MHN and MHS muscle fibers were incubated for 5 min in Ca²⁺-free solution, Na⁺-free solution, or Na⁺/Ca²⁺-free solution. [Ca²⁺]_r was studied with double-barreled selective microelectrodes. Data are expressed as mean ± S.E., n = 15–30 fibers/group, ***, p < 0.001; one-way analysis of variance and Tukey's t test.

FIGURE 3.

T-tubule disruption modifies [Ca²⁺]_r increases induced by Na⁺ depletion in swine MHN and MHS muscle fibers. Cells were incubated in 350 mm glycerol solution for 1 h and then returned to swine physiological solution. Ca²⁺ concentrations were determined with double-barreled selective microelectrodes. Data are expressed as mean ± S.E., n = 15–30 fibers/group, **, p < 0.01; ***, p < 0.001; one-way analysis of variance and Tukey's t test.

FIGURE 4.

NCX inhibition diminished Na⁺ depletion-induced [Ca²⁺]_r increases in swine MHN and MHS muscle fibers. Fibers were incubated with the NCX blocker KB-R7943 (10 μm) for 5 min and Ca²⁺ concentrations were determined by selective microelectrodes. Data are expressed as mean ± S.E., n = 10–30 fibers/group; *, p < 0.05; **, p < 0.01; ***, p < 0.001; one-way analysis of variance and Tukey's t test.

FIGURE 5.

NCX reverse mode blockade modifies halothane-induced [Ca²⁺]_r increases in MHS murine muscle fibers. Mice were anesthetized with ketamine and xylazine. KB-R7943 (10 μm) or YM-244769 (1 μm) was added to the perfusion solution directly on vastus lateralis muscle. Halothane (1.5%) was administered using a vaporizer connected to the ventilator. Ca²⁺ concentrations were determined directly into vastus lateralis muscle fibers using double-barreled selective microelectrodes. Inset shows [Ca²⁺]_r in MHN murine muscles. Data are expressed as mean ± S.E., n = 19–40 fibers/group; *, p < 0.05; ***, p < 0.001; one-way analysis of variance and Tukey's t test.

FIGURE 6.

NCX reverse mode blockade modifies halothane-induced [Na⁺] increases in MHS murine muscle fibers. Mice were anesthetized with ketamine and xylazine. KB-R7943 (10 μm) or YM-244769 (1 μm) was added to the perfusion solution directly on vastus lateralis muscle. Halothane (1.5%) was administered using a vaporizer connected to the ventilator. Na⁺ concentrations were determined directly into vastus lateralis muscle fibers using double-barreled selective microelectrodes. Inset shows [Na⁺]_r in MHN murine muscles. Data are expressed as mean ± S.E., n = 10–25 fibers/group; ***, p < 0.001; one-way analysis of variance and Tukey's t test.

FIGURE 7.

YM-244769 reduces the Ca²⁺ response elicited by high potassium depolarization in adult MHS muscle fibers. FDB fibers were loaded with Fluo-4 AM (10 μm) for 30 min at room temperature and then exposed to 60 mm extracellular potassium in the presence or absence of YM-244769. A, representatives Fluo-4 fluorescence traces in response to extracellular potassium. Amplitude (B) and area under curve (C) of the obtained Ca²⁺ transients. D, normalized Ca²⁺ transients in both MHN and MHS in the presence or absence of YM-244769. The decay of the Ca²⁺ signals were fitted to a single exponential curve and the quantification of the decay constant is shown in E. The effect of YM-244769 on SR Ca²⁺ loading was assessed in MHS FDB fibers and myotubes with either caffeine or ionomycin treatment in Ca²⁺-free media. The area under the curve of the Ca²⁺ transient evoked by caffeine (20 mm) in FDB muscle fibers (F) or ionomycin (5 μm) in myotubes (G) was calculated under each experimental condition. Data are expressed as mean ± S.E., n are indicated in the graph bars. ns, no significant; *, p < 0.05; ***, p < 0.001.

FIGURE 8.

NCX3 expression in MHN and MHS skeletal muscle cells. Quadriceps muscles were dissected from 3–5-month-old mice and cultured differentiated myotubes from MHN and MHS mice were homogenized and NCX3 protein levels were assessed by Western blot and normalized by GAPDH levels. Data are expressed as mean ± S.E., n are indicated in the graph bars. ns, no significant difference; unpaired t test.

FIGURE 9.

NCX3 knockdown reduced [Ca²⁺]_r and the calcium response elicited by high potassium depolarization in MHS myotubes. Myoblasts isolated from MHN and MHS were differentiated for 1 day and then transfected with either scramble- or NCX3-siRNA for 48 h. A, NCX3 expression levels in transfected myotubes assessed by Western blot at 48 h. B, transfection efficiency was assessed by siRNA FITC fluorescence at 24 h. FITC and Bright Field overlay is shown on the right panel. C, [Ca²⁺]_r measurements in both MHN and MHS myotubes after 48 h of transfection. Dashed horizontal lines represent the [Ca²⁺]_r levels in both MHN and MHS untransfected myotubes. D, representative Ca²⁺ responses elicited by high potassium depolarization in transfected MHN and MHS myotubes after 48 h. Amplitude (E) and area under the curve (F) quantification of the recorded Ca²⁺ signals. Data are expressed as mean ± S.E., n are indicated in the graph bars. *, p < 0.05; **, p < 0.01; ***, p < 0.001.