Nonspecific sarcolemmal cation channels are critical for the pathogenesis of malignant hyperthermia

Abstract

Malignant hyperthermia (MH) susceptibility has been attributed to a leaky sarcoplasmic reticulum (SR) caused by missense mutations in RYR1 or CACNA1S, and the MH crisis has been attributed solely to massive self-sustaining release of Ca²⁺ from SR stores elicited by triggering agents. Here, we show in muscle cells from MH-RyR1^R163C knock-in mice that increased passive SR Ca²⁺ leak causes an enlarged basal influx of sarcolemmal Ca²⁺ that results in chronically elevated myoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) at rest. We discovered that Gd⁺³ and GsMTx-4 were more effective than BTP2 or expression of the dominant-negative Orai1^E190Q in reducing both Ca²⁺ entry and [Ca²⁺]_i, implicating a non-STIM1/Orai1 SOCE pathway in resetting resting [Ca²⁺]_i. Indeed, two nonselective cationic channels, TRPC3 and TRPC6, are overexpressed, and [Na]_i is chronically elevated in MH-RyR1^R163C muscle cells. [Ca²⁺]_i and [Na⁺]_i are persistently elevated in vivo and further increased by halothane in MH-RyR1^R163C/WT muscle. These increases are markedly attenuated by local perfusion of Gd⁺³ or GsMTx-4 and completely suppressed by dantrolene. These results contribute a new paradigm for understanding MH pathophysiology by demonstrating that nonselective sarcolemmal cation channel activity plays a critical role in causing myoplasmic Ca²⁺ and Na⁺ overload both at rest and during the MH crisis.—Eltit, J. M., Ding, X., Pessah, I. N., Allen, P. D., Lopez, J. R. Nonspecific sarcolemmal cation channels are critical for the pathogenesis of malignant hyperthermia.

Malignant hyperthermia (MH) is an autosomal dominant hypermetabolic condition triggered by volatile anesthetics, such as halothane, and depolarizing neuromuscular blockers, such as succinylcholine (1). The MH crisis is characterized by hypermetabolism, hypercapnia, hyperthermia, tachycardia, hypoxemia, muscle rigidity, and respiratory and metabolic acidosis. If left untreated, the mortality is >70%, but improved understanding, better monitoring, and availability of the skeletal muscle relaxant dantrolene has reduced the mortality to <8% (1, 2).

Molecular genetic studies have established the type 1 ryanodine receptor (RYR1) gene encoding the skeletal muscle sarcoplasmic reticulum (SR) Ca²⁺ release channel as the primary locus for MH susceptibility (3–6). More than 180 RYR1 mutations have been associated with MH and/or central core disease (6). Another locus implicated for susceptibility is CACNA1S, encoding the pore-forming subunit of the skeletal muscle L-type voltage-dependent Ca²⁺ channel (CaV1.1; refs. 7–9). The molecular mechanisms by which both RyR1 and CaV1.1 mutations confer MH susceptibility are unknown. A common characteristic of muscle expressing MH-RyR1 and MH-Ca_V1.1 mutations is an increased resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) in susceptible patients (10) and experimental models (11–14) compared to wild-type (WT) muscle. Exposure to triggering agents (i.e., halothane at clinically relevant concentrations) accentuates this difference causing [Ca²⁺]_i to rise severalfold in MH muscle, whereas exposure to the same concentration of halothane is without effect in WT muscle (13, 15, 16). Until now, the mechanism for the MH crisis has been attributed solely to massive self-sustaining release of Ca²⁺ from the SR on exposure to a triggering agent. Counter to this view, our results introduce a new paradigm that implicates nonspecific sarcolemmal cation entry channels as both the predominant source of acutely elevated Ca²⁺ during fulminant MH and a significant contributor to chronically elevated [Ca²⁺]_i in quiescent MH-susceptible muscles. Furthermore, prevention of nonspecific sarcolemmal cation entry represents one of the pharmacological mechanisms by which dantrolene can prevent or abort the MH crisis.

MATERIALS AND METHODS

Myotube culture

Primary myoblasts were generated from the hindlimb and forelimb muscles of neonatal homozygous F2 R163C C57BL6 (MH-RyR1^R163C) mice and their WT littermates (17, 18). Myoblasts were differentiated into myotubes by withdrawal of growth factors, as described previously (19). Animals were cared for and euthanized according to the standards set by the Harvard Medical School Institutional Animal Care and Use Committee.

Mn²⁺ quench

Differentiated WT and MH-RyR1^R163C primary myotubes were loaded with 5 μM Fura-2-AM (Invitrogen, Eugene, OR, USA) for 30 min at 37°C. The myotubes were then washed 3 times with mammalian Ringer solution (see Solutions) and transferred to the stage of a Nikon TE2000 inverted microscope (Nikon, Tokyo, Japan) and illuminated at the isosbestic wavelength for Fura-2 (360 nm). Fluorescence emission was captured at 512 nm from regions of interest within each myotube from 3–10 individual cells at 5 frames/s using a ×40, 1.3-NA objective. To measure the specific rate of dye quench by Mn²⁺ entry, the Ringer solution under constant perfusion was switched to Mn²⁺-containing solution (see Solutions) at 23°C. The basal signal in Ringer solution and in Mn²⁺-containing solution was fitted to a linear regression (y=a+bx) independently. The specific rate of Fura-2 quenching induced by Mn²⁺ entry was calculated subtracting the basal slope to the slope during the Mn²⁺ solution application and expressed as fluorescence arbitrary units (f.a.u.) per second.

Ca²⁺-selective microelectrodes

Double-barreled Ca²⁺-selective microelectrodes were prepared as described previously (12, 20). Pulled microelectrodes were backfilled first with the neutral carrier ETH 129 (21193; Fluka-Sigma-Aldrich, Buchs, Switzerland) and then with pCa 7 solution. Each Ca²⁺-selective microelectrode was individually calibrated as described previously (21), and only those with a linear relationship between pCa 3 and 7 (nernstian response, 29.5 and 30.5 mV/pCa unit at 23°C and 37°C, respectively) were used experimentally. To better mimic the intracellular ionic conditions, all calibration solutions were supplemented with 1 mM Mg²⁺ and 8 mM Na⁺. The 0.75-mm OD barrel was backfilled with 3 M KCl just before its use. Interference from Na⁺ and Mg²⁺ on the calibration of these electrodes was negligible (e.g., 20 mM Na⁺ and 3 mM Mg²⁺ did not alter the response at pCa7). After making measurements of [Ca²⁺]_i, all electrodes were recalibrated, and if the two calibration curves did not agree within 3 mV, data from that microelectrode were discarded. Before starting the studies, we determined by direct calibration that the Ca²⁺ sensitivity of Ca²⁺ microelectrodes was not affected between pCa 6 and 7 by any of the drugs used in the present study.

Na⁺-selective microelectrodes

Double-barreled Na⁺-selective microelectrodes were prepared in a manner similar to preparation of Ca²⁺-selective electrodes described above. The electrodes were backfilled first with the Na⁺-sensitive ion cocktail (Sodium Ionophore I Cocktail A; Fluka Sigma-Aldrich) based on the neutral ligand ETH-227 and 24 h later with a solution containing 8 mM NaCl. Microelectrodes were calibrated 24 h later in solutions containing different [Na⁺] and 1 mM MgCl₂. The microelectrodes gave virtually nernstian responses at free [Na⁺] between 100 and 10 mM. However, at concentrations between 10 and 1 mM [Na⁺], the electrodes had a subnernstian response (40–45 mV), but their response was stable and of a sufficient amplitude to be able to measure intracellular Na⁺ concentration ([Na⁺]_i). After making measurements of resting [Na⁺]_i, all electrodes were recalibrated, and if the two calibration curves did not agree within 3 mV, data from that microelectrode were discarded.

Ca²⁺ and Na⁺ microelectrode recordings

Microelectrode recordings were performed as described previously (12). Single myotubes or vastus lateralis muscle fibers were impaled either with double-barreled Ca²⁺- or double-barreled Na⁺-selective microelectrodes, and their potentials were recorded via high-impedance amplifier (WPI Duo 773 electrometer; WPI, Sarasota, FL, USA). The potential from the 3 M KCl microelectrode (V_m) was subtracted electronically from either the potential of the Ca²⁺ electrode (V_CaE), or the Na⁺ electrode (V_NaE) to produce a differential Ca²⁺-specific potential (V_Ca) or Na⁺-specific potential (V_Na) that represents the [Ca²⁺]_i or [Na⁺]_i, respectively. V_m, V_Ca, and V_Na were filtered (30–50 kHz) to improve the signal-to-noise ratio and stored in a computer for further analysis.

Permanent overexpression of Orai1^WT and Orai1^E190Q

The Orai1^WT and Orai1^E190Q cDNAs were cloned into a retroviral expression vector with a separate eGFP expression cassette (plasmids 12199 and 21662, respectively; Addgene, Cambridge, MA, USA). These plasmids were kindly provided by Dr. Anjana Rao (Harvard Medical School and Immune Disease Institute, Boston, MA, USA). We verified that the correct sequence of both inserts and retroviral particles was packaged in HEK 293 helper cells, and myoblasts were infected at an MOI of 5, allowed to recover for 12 h, and then selected with puromycin (0.5 μg/ml) for 1 wk. After the selection period, all remaining cells on the plate showed expression of the EGFP marker. Uninfected cells did not survive to the selection protocol.

Western blot analysis

The determination of the expression levels of transient receptor potential cation (TRPC) proteins TRPC1, TRPC3, and TRPC6 was done as described previously (22).

SR loading capacity

Differentiated WT and MH-RyR1^R163C primary myotubes were loaded with 5 μM Fluo-4-AM for 20 min at 37°C. The myotubes were placed on the stage of an epifluorescence microscope (Nikon TE2000) coupled to a digital acquisition system (Stanford Photonics, Stanford, CA, USA). The filter set used was excitation, 480/30 nm, T495LP dichroic and emission, 535/40 nm. The emission signal was acquired at a frequency of 30 frames/s. The amount of SR Ca²⁺ was estimated by taking the combined area under the curve of 3 sequential exposures to 20 mM caffeine in Ca²⁺-free medium to prevent Ca²⁺ entry to get the total area under the curve to assess the total amount of Ca²⁺ released. To estimate the total intracellular Ca²⁺ store, Fluo-4-AM-loaded myotubes were exposed to the Ca²⁺ ionophore 4Br-A23187 in Ca²⁺-free medium. The area of the released Ca²⁺ was computed for each myotube type studied.

[Ca²⁺]_i and [Na⁺]_i determination in mice in vivo

The measurements were done as described previously (23) with the following modifications. Heterozygous F12 C57BL6 (MH-RyR1^R163C/WT) knock-in mice and their WT littermates were anesthetized (100 mg/kg ketamine and 5 mg/kg xylazine), intubated and ventilated with air using a mouse ventilator. A rectal temperature probe was placed, to measure core temperature and to provide a feedback signal to the heating pad to keep the mice euthermic. A small incision was made in the skin in the upper lateral part of both hind legs, the vastus lateralis muscle was identified, and its aponeurosis was partially removed. The superficial fibers were exposed and perfused with mammalian Ringer. The determination of [Ca²⁺]_i or [Na⁺]_i was carried out in both legs simultaneously using ion-selective microelectrodes described above (Supplemental Fig. S2). The exposed fibers were perfused with Ringer solution (left hind leg, control) or Ringer with the tested drug (right hind leg, test drug) locally. The ventilator was connected to a halothane vaporizer that was switched from 0 to 1.5 vol% in air to administer halothane. Several muscle fibers were impaled to measure ion concentrations for each condition in each leg (control condition, after test-drug application and after halothane exposure).

Solutions

Mn²⁺-containing solution (quench solution) had the following composition (in mM): 140 NaCl, 5 KCl, 0.5 MnCl₂, 5.5 glucose, and 10 HEPES, pH 7.4. The mammalian Ringer solution used in this study had the following composition (in mM): 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 5 glucose, and 10 HEPES, pH 7.4. The Ca²⁺-free solution had the following composition (in mM): 140 NaCl, 5 KCl, 2 MgCl₂, 1 EGTA, 5 glucose, and 10 HEPES, pH 7.4. N-{4-[3,5-bis-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2; CAS 223499-30-7), Gd³⁺, 1-oleoyl-2-acetyl-sn-glycerol (OAG; CAS 86390-77-4), GsMTx-4, and dantrolene solutions were made by adding the desired concentration of the reagent to the physiological solution.

Statistics

All values are expressed as means ± se. Statistical analysis was performed using 1-way ANOVA and Tukey's t test for multiple measurements to determine significance (P<0.05).

RESULTS

Basal store-operated calcium entry (SOCE) is higher in resting homozygous MH-RyR1^R163C myotubes

We employed Mn²⁺ quench of Fura-2 fluorescence to quantify unidirectional divalent cation flux into intact cultured skeletal muscle cells at rest. This technique allows the estimation of Ca²⁺ permeation from the extracellular space into the intact cell (24). Mn²⁺-quench experiments show that resting entry is 77% greater in MH-RyR1^R163C myotubes than in WT cells (−1.30±0.07 f.a.u./s, n=160, and −0.74±0.05 f.a.u./s, n=107, respectively; P<0.001; Fig. 1). The Ca²⁺ release-activated Ca²⁺ current (CRAC) and TRPC channel blocker BTP2 (25) decreased Mn²⁺ entry 53% in WT cells and by 70% in MH-RyR1^R163C cells. The stretch-activated channel blocker Gd³⁺ (26) at a concentration of 1 μM caused a 57% reduction in Mn²⁺ quench in MH-RyR1^R163C cells but had no effect in WT cells. Increasing the concentration of Gd³⁺ to 25 μM reduced Mn²⁺ quench by 74% in WT cells and by 94%, almost completely blocking quench, in MH-RyR1^R163C cells (Fig. 1).

Figure 1.

Resting sarcolemmal divalent cation permeability is greater in MH-RyR1^R163C than WT cultured muscle cells. Sarcolemmal permeability to Ca²⁺ was estimated using the rate with which external Mn²⁺ quenches cytoplasmic Fura-2 fluorescence, as described in Materials and Methods. A) Change in the slope of the Fura-2 fluorescence (f.a.u./s) after switching to Mn²⁺ containing external solution (Mn²⁺) accounts for the rate of Mn²⁺ entry into the cells; traces showing the Mn²⁺ entry in quiescent WT and MH-RyR1^R163C cells are depicted. B) Rates of Fura-2 fluorescence quenching in the presence of BTP2 (5 μM), Gd³⁺ (1 μM), and Gd³⁺ (25 μM) were computed and plotted. Means ± se (n≥50 myotubes) for each condition are shown. **P < 0.01, ***P < 0.001 vs. control; ^&&&P < 0.001 vs. corresponding WT.

Elevated [Ca²⁺]_i in homozygous MH-RyR1^R163C myotubes is partially normalized by modulation of Orai1

Resting membrane potentials and [Ca²⁺]_i were measured in quiescent WT and MH-RyR1^R163C myotubes with double-barreled ion-specific microelectrodes. [Ca²⁺]_i was 2.8-fold higher in MH-RyR1^R163C cells than that observed in WT cells (334±2 nM, n=25, vs. 120±1 nM; n=25, respectively; P<0.001; Fig. 2). There were no differences in membrane potential between WT and MH-RyR1^R163C cells.

Figure 2.

Gd³⁺ and GsMTx-4 are more effective than BTP2 or overexpression of Orai1^E190Q in reducing [Ca²⁺]_i at rest in MH-RyR1^R163C cultured muscle cells. [Ca²⁺]_i at rest was measured in cells exposed to BTP2 (5 μM), stably transduced to overexpress Orai1^WT or Orai1^E190Q, exposed to Gd³⁺ (1 and 25 μM) or exposed to GsMTx-4 (5 μM). Means ± se (n≥10 myotubes) for each condition are shown. ***P < 0.001 vs. control; ^&&&P < 0.001 vs. corresponding WT.

Incubation of WT and MH-RyR1^R163C cells with BTP2 for 5 min reduced [Ca²⁺]_i in both groups, but the effect was greater in MH-RyR1^R163C (40% decrement) than in WT (17% decrement, Fig. 2). A similar reduction in [Ca²⁺]_i was observed when the dominant-negative form of Orai1^E190Q was overexpressed in MH-RyR1^R163C and WT cells. Overexpression of Orai1^WT did not affect [Ca²⁺]_i in either group (Fig. 2). Gd³⁺ (1 μM) decreased [Ca²⁺]_i by 40% in MH-RyR1^R163C myotubes but, as was the case for Mn²⁺ quench experiments above, had no effect on WT cells. On the other hand, 25 μM Gd³⁺ decreased [Ca²⁺]_i in both WT and MH myotubes, with the decrement being significantly greater in MH-RyR1^R163C myotubes (27 vs. 63%, respectively). GsMTx-4 (5 μM), a cationic hydrophobic polypeptide that blocks mechanosensitive (stretch-activated) ion channels that conduct Ca²⁺ and Na⁺ (27–29), also decreased [Ca²⁺]_i in both WT and MH-RyR1^R163C myotubes (20 and 58%, respectively; Fig. 2).

Differential expression of TRPC1, TRPC3, and TRPC6 in WT and homozygous MH-RyR1^R163C myotubes

Myotubes were exposed to OAG, a membrane-permeable diacylglycerol analog that is a known activator of TRPC3/6 channels (30, 31). After 5 min of OAG (30 μM) application, [Ca²⁺]_i increased by only 32% (from 122±1.4 nM, n=20, to 161±3.7 nM, n=10; P<0.001) in WT myotubes, whereas it increased 64% (from 329±2.6 nM, n=20, to 541±10.6 nM, n=10; P<0.001) in MH-RyR1^R163C cells. The effect of OAG could be completely blocked with addition of either 25 μM Gd³⁺ or 5 μM GsMTx-4 to the assay medium of either genotype (Fig. 3A). Western blot analysis showed that TRPC1 was significantly decreased in MH-RyR1^R163C myotubes compared to WT (0.5±0.06-fold, n=4), whereas the expression of both TRPC3 and TRPC6 was significantly increased (2.6±0.8-fold, n=4, and 2.4±0.3-fold, n=4, respectively) compared to WT (Fig. 3B).

Figure 3.

Expression of TRPC1, TRPC3, and TRPC6 in WT and MH-RyR1^R163C cultured muscle cells differ. A) [Ca²⁺]_i was measured in WT and MH-RyR1^R163C cultured muscle cells. Effect of 5-min exposure to OAG (30 μM, DAG analog) whether alone or in the presence of Gd³⁺ (25 μM) or GsMTx-4 (5 μM) is plotted. Means ± se (n≥10 myotubes) for each condition are shown. B) Representative Western blot analysis shows the expression of TRPC1, TRPC3, and TRPC6 in WT and MH-RyR1^R163C cells; GAPDH was also analyzed as loading control. Densitometric analysis of 4 independent Western blots for each TRPC protein is shown and expressed relative to the WT expression. Means ± se for each TRPC protein are shown. C) [Na⁺]_i was measured in WT and MH-RyR1^R163C cultured muscle cells. Effects of Orai1^WT or Orai1^E190Q overexpression and pretreatment with GsMTx-4 (5 μM) are shown. Means ± se (n≥10 myotubes) for each condition are shown. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.

Resting [Na⁺]_i is increased in homozygous MH-RyR1^R163C myotubes

Because the permeability of TRPCs is not Ca²⁺ specific, their overactivity could also possibly allow significant Na⁺ entry, which could increase resting [Na⁺]_i. In this regard, [Na⁺]_i was 2-fold higher in MH-RyR1^R163C myotubes than in WT (15.7±0.20 mM, n=23, vs. 7.9±0.17 mM, n=26, respectively; P<0.001; Fig. 3C). Overexpression of Orai1^WT did not change [Na⁺]_i in either WT or MH cells. Unexpectedly, the expression of the dominant-negative Orai1^E190Q, which is unable to conduct calcium current, caused a 12-13% decrease in [Na⁺]_i in both WT cells and MH cells (Fig. 3C). Application of GsMTx-4 (5 μM), which blocks Na⁺ current through stretch-activated channels (28), significantly reduced [Na⁺]_i in both groups of cells but caused a 36% decrease in MH and only an 18% decrease in WT cells (Fig. 3C).

Blocking sarcolemmal Ca²⁺ and Na⁺ entry attenuates halothane-induced increases in [Ca²⁺]_i and [Na⁺]_i in heterozygous MH-RyR1^R163C/WT mice in vivo

[Ca²⁺]_i or [Na⁺]_i was measured simultaneously in the right and left vastus lateralis muscles in heterozygous MH-RyR1^R163C/WT and WT mice before and after exposure to 1.5% halothane vapor in their inspired gas. Representative experiments for in vivo measurements of [Ca²⁺]_i for each treatment used are shown in Fig. 4. Mean changes in [Ca²⁺]_i or [Na⁺]_i before and after halothane for each treatment are summarized in Fig. 5.

Figure 4.

Determination of the intracellular Ca²⁺ concentrations in the vastus lateralis muscle in vivo. [Ca²⁺]_i was monitored using Ca²⁺-selective microelectrodes in the vastus lateralis muscle. Measurements were done in the right and left thigh simultaneously both in MH-RyR1^R163C/WT and WT mice. Exposed fibers were perfused with Ringer solution alone (left hind leg, control, solid line) or Ringer solution plus the tested drug (right hind leg, test drug, shaded line). Prior to the experiment, the animals were anesthetized using ketamine, intubated, and actively ventilated. The ventilator was connected to a halothane dispenser, which was switched from 0 to 1.5% where indicated. Drugs used in the experiments were dantrolene (40 μM), BTP2 (5 μM), Gd³⁺ (25 μM), or GsMTx-4 (5 μM). Each graph represents consecutive measurements done in a representative single mouse.

Figure 5.

Ca²⁺ and Na⁺ concentrations before and during halothane exposure in the vastus lateralis muscle in vivo. Intracellular Ca²⁺ determinations (A) or Na⁺ determinations (B) in vastus lateralis muscle were done using ion-selective microelectrodes and performed as described in Materials and Methods. The intracellular Ca²⁺ and Na⁺ concentrations in the absence (shaded columns) or in the presence (solid columns) of 1.5% halothane are shown. Experiments were done in the presence of BTP2 (5 μM), GsMTx-4 (5 μM), Gd³⁺ (25 μM), or dantrolene (40 μM). Means ± se for each condition are shown. Measurements were done in ≥5 mice/group***P < 0.001 vs. corresponding control treatment group; ^&P < 0.05, ^&&&P < 0.001 vs. corresponding WT group.

In MH-RyR1^R163C/WT mice, [Ca²⁺]_i measured in vivo was 328 ± 1.2 nM (n=76) and increased during halothane administration to 1240 ± 4.9 nM (n=76). In WT mice, [Ca²⁺]_i was 120 ± 0.3 nM (n=60) and did not increase with halothane exposure. Local pretreatment of MH-RyR1^R163C/WT muscle fibers with 5 μM BTP2 decreased [Ca²⁺]_i in MH-RyR1^R163C/WT mice to 209 ± 2.0 nM (n=13), and although it was able to attenuate the increase in [Ca²⁺]_i during halothane exposure to 583 ± 4.1 nM (n=22), it was unable to completely abrogate the response. Local pretreatment of MH-RyR1^R163C/WT muscle fibers with either GsMTx-4 or Gd³⁺ (5 and 25 μM, respectively) decreased [Ca²⁺]_i to 149 ± 2.1 nM (n=9) and 138 ± 1.9 nM (n=23), respectively, and both decreased the rise in [Ca²⁺]_i during halothane exposure by 81% (325±8.2 nM, n=23, and 303.7±3.70 nM, n=25, respectively) compared to the contralateral muscle pretreated with vehicle alone (Figs. 4 and 5). Local treatment of WT vastus lateralis fibers with any of the three drugs caused a small decrement in [Ca²⁺]_i similar to their influence on WT myotubes. Exposure to halothane had no effect on [Ca²⁺]_i in WT muscle with or without drug pretreatment. Local pretreatment of muscle fibers with 40 μM dantrolene decreased [Ca²⁺]_i to similar levels in both WT and MH-RyR1^R163C/WT muscle (84±0.8 nM, n=13, and 87±0.9 nM, n=11, respectively). Furthermore, the increase in [Ca²⁺]_i seen during halothane exposure was completely ablated in dantrolene-pretreated MH-RyR1^R163C/WT muscles.

To test the hypothesis that the increase in [Ca²⁺]_i during exposure to halothane during the MH crisis could be the result of an increased sarcolemmal Ca²⁺ entry through a nonspecific sarcolemmal cation channel, as was suggested from our results in MH-susceptible myotubes at rest, we measured [Na⁺]_i in vivo under the same conditions as above for [Ca²⁺]_i (Fig. 5B). Similar to our data from myotubes, [Na⁺]_i was elevated at rest in vastus lateralis of MH-RyR1^R163C/WT mice compared to WT (15.7±0.16 vs. 8.2±0.06 mM, n=38). [Na⁺]_i rose by 57% during halothane exposure (24.7±0.25 mM, n=51) in MH-RyR1^R163C/WT fibers but remained unchanged in WT. Pretreatment with either BTP2, GsMTx-4 (5 μM), or 25 μM Gd³⁺ significantly lowered [Na⁺]_i in MH-RyR1^R163C/WT muscle fibers (P<0.001 vs. untreated) but did not restore it to WT levels. Furthermore, all three blockers reduced (by 62, 66, and 72%, respectively; P<0.001 vs. untreated) but could not ablate the elevation in [Na⁺]_i seen in the contralateral leg during exposure to halothane. Similar to what we observed in measuring [Ca²⁺]_i, dantrolene pretreatment reduced [Na⁺]_i to WT levels in MH-RyR1^R163C/WT fibers at rest and prevented any increase in [Na⁺]_i in the treated leg during exposure to halothane.

DISCUSSION

Skeletal muscles in individuals and animals that are susceptible to MH have altered Ca²⁺ homeostasis at rest (10, 11). The most striking alteration is the increase of [Ca²⁺]_i and [Na⁺]_i both at rest and during a fulminant MH episode compared to the normal muscle (Figs. 2 and 4). Since RyR1 mutations have been described as the most common cause of this syndrome, there has been speculation that a persistent leak of Ca²⁺ ions from the SR should fully account for the increased [Ca²⁺]_i. This hypothesis was supported by several studies that described an increased passive SR Ca²⁺leak in muscle cells and heterologous cells expressing RyR1s with MH mutations (12, 32, 33).

In the present study, we provide experimental evidence that a mutation in RyR1 that enhances SR Ca²⁺ leak (12) in MH susceptible muscles is closely associated with enhanced sarcolemmal Ca²⁺ and Na⁺ permeability, thereby satisfying the boundary theorem, which established that steady-state (long-term) changes in Ca²⁺ set point must be driven by fluxes in the plasma membrane (22, 34–37). Mn²⁺ quench experiments showed that MH-RyR1^R163C muscle cells at rest have ∼80% more sarcolemmal cation permeability than WT, most likely a consequence of accentuated Ca²⁺ leak through RyR1^R163C that chronically partially depletes SR stores at rest (Supplemental Fig. S1), which, in turn, activates a type of SOCE, as demonstrated by an increase in sarcolemmal Mn²⁺ permeability. It is important to note that unlike the SOCE activated by complete depletion of SR stores with no possibility of reuptake, as is the case with thapsigargin treatment, the resting Ca²⁺ entry shows a very slow rate compared to those measured as a consequence of complete SR store depletion (22, 38). Thus, although resting rates of entry are significantly higher in MH-RyR1^R163C muscle cells than those observed with WT, they are not sufficient to cause a plateau in the quench of Fura-2 even after 90 s of Mn²⁺ exposure. To determine which channels were responsible for this increase, we used both pharmacological and molecular blockade of the classical Stim1/Orai1 (CRAC/SOCE) pathway. We found that BTP2 decreases Mn²⁺ permeability and [Ca²⁺]_i and does so to a greater extent in MH-RyR1^R163C than in WT cells. Furthermore, overexpression of the dominant-negative form of Orai1^E190Q yielded similar results. These observations support the hypothesis that the classical stromal interaction molecule 1 (STIM1)/Orai1 SOCE pathway is constitutively more active at rest in MH cells. However, although BTP2 exposure and Orai1^E190Q overexpression reduced [Ca²⁺]_i in MH-RyR1^R163C myotubes, they failed to normalize [Ca²⁺]_i to WT levels (Fig. 2). On the other hand, Gd³⁺ had a stronger effect in decreasing Mn²⁺ permeability, and both Gd³⁺ and GsMTx-4 lowered [Ca²⁺]_i in MH-RyR1^R163C myotubes to levels nearly identical to untreated WT cells (∼120 nM; Fig. 2). This, coupled with the fact that [Na⁺]_i is also elevated in MH-RyR1^R163C myotubes, strongly suggests that in addition to the classical SOCE pathway, other Ca²⁺ entry pathways must also be involved. Moreover, these two agents block the increase in [Ca²⁺]_i seen in untreated cells after acute exposure to the TRPC3/6 activator OAG (Fig. 3). Accordingly, expression of TRPC6 and the structurally similar TRPC3 is increased in MH-RyR1^R163C myotubes compared to WT. The fact that resting sarcolemmal cation influx is much greater in MH-RyR1^R163C than in WT myotubes implies an increased conductance of multiple Ca²⁺ entry pathways as a consequence of partial SR Ca²⁺ depletion (Supplemental Fig. S1). One long-term consequence of elevated RyR1-mediated SR Ca²⁺ leak is the chronic and partial depletion of SR stores, which enhances sarcolemmal Ca²⁺ and Na⁺ entry in MH-susceptible muscle cells; the latter is primarily responsible for resetting the [Ca²⁺]_i and [Na]_i at rest in MH muscles compared to their WT counterparts (Fig. 6). An important consequence of chronically elevated [Ca²⁺]_i is that it is likely to promote elevated production of mitochondrial ROS and metabolic adaptations observed in MH-susceptible muscle in the absence of triggering agents (39–41).

Figure 6.

Scheme showing the differences in sarcolemmal cation flow and intracellular resting [Ca²⁺] and [Na⁺] and its effects on mitochondrial ROS production between WT and heterozygous MH-RyR1^R163C/WT muscles. Proposed sites for dantrolene's action are indicated with the numbers 1–4.

In addition to an elevated [Ca²⁺]_i, we found that [Na]_i is also higher in MH-RyR1^R163C than in WT muscle cells. Previous measurements of resting [Na⁺]_i in mammalian muscle vary between 7 and 13 mM, depending on the technique used for its determination (42, 43). Thus, our resting [Na⁺]_i in WT muscle cells (7.9±0.17 mM) is within the published range, and for MH-RyR1^R163C/WT, the [Na]_i is slightly higher that the value (11.3±1 mM) that we found previously in intact muscle fibers isolated from MH-susceptible swine (Poland China-Pietrain) using the same technique (44). This increase in resting [Na⁺]_i in MH-RyR1^R163C muscle cells could arise either by increased influx or decreased efflux of Na⁺. The fact that the Na⁺ overload measured in vivo in heterozygous MH-RyR1^R163C/WT muscles is substantially attenuated by local application of Gd³⁺ and GsMTx-4 strongly suggests that the elevation is mediated by an increase in influx rather than a decrease in Na⁺ efflux (Fig. 5). On the basis of our observation that the expression of TRPC1 is down-regulated and that TRPC3 and TRPC6 are up-regulated in MH-RyR1^R163C muscles, the most likely mechanism for this increased influx is an increased conductance through the nonselective TRPC3/6 cation entry channels (Fig. 6 scheme). However, despite the fact that Orai1 has previously been described as absolutely Ca²⁺ selective (45), based on the fact that overexpression of the dominant-negative Orai1^E190Q caused a modest reduction of [Na⁺]_i in both WT and MH-RyR1^R163C muscle cells in addition to its effect on [Ca²⁺]_i, it is possible that Orai1 can allow a small amount of Na⁺ conductance that previously went undetected. Alternatively, overexpression of the mutated Orai1 channel in this environment could have altered either the expression of or conductance of associated TRPC channels (46–48), which then was indirectly responsible for this small decrease in Na⁺ conductance.

The levels of [Ca²⁺]_i at rest measured in vivo were similar to those measured in myotubes (compare Figs. 2 and 5), with MH-RyR1^R163C/WT muscles having almost 3-fold higher resting [Ca²⁺]_i than WT, and resting V_m was identical in WT and MH-RyR^R163C/WT mice. (see Table 1). When the animals were exposed to halothane, there was a small (∼4 mV) change in V_m, and [Ca²⁺]_i increased by 4-fold in MH-RyR^R163C/WT mice, while there was no change in either parameter in WT mice. In addition to the increase in [Ca²⁺]_i, resting [Na⁺]_i also increased during halothane exposure in MH-RyR1^R163C/WT muscles, suggesting that nonspecific sarcolemmal cationic channels are activated during the MH crisis. Pretreatment by local application of BTP2, GsMTx-4, and Gd³⁺ directly to the muscle reduced both the subsequent response to halothane-induced elevation of [Ca²⁺]_i by 59, 81, and 81%, respectively, and of [Na⁺]_i by 62, 66, and 72%, respectively. In the case of GsMTx-4 and Gd³⁺, the increase in [Ca²⁺]_i measured in the presence of halothane is not different than the resting [Ca²⁺]_i measured in untreated MH muscle (∼320 nM, Figs. 4 and 5). This inhibition of the halothane-induced increase in [Ca²⁺]_i by Ca²⁺ entry blockers further supports the hypothesis that sarcolemmal Ca²⁺ entry and not release from SR stores plays the critical role in maintaining the new steady-state [Ca²⁺]_i that has been observed during an MH crisis (15, 49). This suggestion is especially reinforced in the case of Gd³⁺ (25 μM), a highly charged membrane impermeant ion, which has previously been shown not to affect electrically induced RyR1-mediated Ca²⁺ release, as inferred through force production (43). We cannot rule out the possibility that the Ca²⁺ influx that takes place during halothane exposure may activate RyR1 through a Ca²⁺-induced-Ca²⁺release (CICR) mechanism, as previously suggested by others (50, 51). In principle, this possibility would be unlikely, since Mg²⁺ inhibition and the repression by CaV1.1 keeps the RyR1 silent at rest and tightly controlled by membrane voltage (CaV1.1 control; refs. 20, 52–54). However, it has been suggested that MH mutations affect the regulation of RyR1 by Mg²⁺ and Ca²⁺ (55, 56) and alter Ca_V1.1-mediated RyR1 repression (13), making enhanced CICR a possibility, providing a further means of store depletion, which would then be followed by increased Ca²⁺ entry via SOCE.

Table 1.

Resting membrane potential determinations in vivo (mV)

Pretreatment	Treatment
	No drug	BTP2	Gd3+	GsMTx-4	Dantrolene
Control
WT	−82 ± 1.4 (n=121)	−83 ± 1.1 (n=13)	−82 ± 1.3 (n=13)	−82 ± 1.7 (n=9)	−82 ± 1.2 (n=14)
R163C-MHS	−82 ± 1.3 (n=143)	−83 ± 1 (n=13)	−82 ± 1.4 (n=23)	−82 ± 1.7 (n=9)	−82 ± 1.4 (n=11)
Halothane
WT	−82 ± 1.2 (n=84)	−83 ± 1.2 (n=22)	−82 ± 1.5 (n=26)	−82 ± 1.4 (n=23)	−82 ± 1.5 (n=21)
R163C-MHS	−78 ± 1 (n=90)*	−78 ± 1.1 (n=22)*	−77 ± 1.3 (n=25)*	−78 ± 1 (n=23)*	−81 ± 1 (n=21)

Resting membrane potentials values are expressed as means ± sd; n represents the number of measurements carried out in muscle fibers from 18 WT and 24 R163C-MHS mice.

^*P < 0.05 vs. R163C-MHS without halothane (1-way ANOVA, Kruskal-Wallis test, Dunn's multiple-comparison test).

Of particular interest is the action of dantrolene in skeletal muscle. Although dantrolene's primary pharmacological mechanism of action has been related to the inhibition of Ca²⁺ release (57–60), it attenuates both RyR1-mediated Ca²⁺ leak (Fig. 6, target 1; ref. 12) and excitation-coupled Ca²⁺ entry (ECCE) in MH-susceptible myotubes during active stimulation (ref. 38 and Fig. 6, target 2). The mechanisms responsible for how dantrolene modulates cation influx through sarcolemmal pathways not directly mediated by bidirectional signaling between CaV1.1 and RyR1 have been less clear. Remarkably, in addition to lowering resting [Ca²⁺]_i (49, 61) and preventing the elevation induced by exposure to halothane, as previously reported (15, 62), dantrolene pretreatment also completely prevents the increase in [Na⁺]_i caused by halothane exposure in MH-RyR1^R163C/WT muscles. This finding supports the previously suggested possibility that one or more of dantrolene's targets reside in the plasma membrane (38, 63) and strongly suggests that the identity of this target is a nonspecific cation entry channel, likely TRPC3, TRPC6, or both (Fig. 6, target 4).

However, because the effect of dantrolene in lowering resting [Ca²⁺]_i is significantly more profound than that of the pure sarcolemmal Ca²⁺ entry blockers, either the pharmacological agents used are incomplete blockers of their intended sarcolemmal targets or there are additional targets of dantrolene likely to be related to blocking SR Ca²⁺ leak, such as the leak state of RyR1 (Fig. 6, target 1). Thus, we cannot discount the alternative possibility that the suppression of the halothane-induced increase in [Na⁺]_i and [Ca²⁺]_i by dantrolene is secondary to a direct action on RyR1-mediated Ca²⁺ leak. This could restore Ca²⁺ store filling, thereby attenuating Ca²⁺ permeation through the SOCE pathways. (Fig. 6, targets 3 and 4).

In summary, these results open a new paradigm in the understanding of the MH pathology, demonstrating that nonselective sarcolemmal cation permeability, separate from the classic STIM/Orai pathway, is activated by SR depletion and plays a critical role in the causing cytosolic Ca²⁺ and Na⁺ overload both at rest and during the MH crisis. In addition, they suggest the possibility that these channels are critical targets for dantrolene's therapeutic effects.