Transient receptor potential cation channels and calcium dyshomeostasis in a mouse model relevant to Malignant Hyperthermia

Abstract

Background:

Until recently, the mechanism for the malignant hyperthermia crisis has been attributed solely to sustained massive Ca²⁺ release from the sarcoplasmic reticulum on exposure to triggering agents. We now test the hypothesis that transient receptor potential cation (TRPC) channels are important contributors to the Ca²⁺ dyshomeostasis in a mouse model relevant to malignant hyperthermia.

Methods:

We examined the mechanisms responsible for Ca²⁺ dyshomeostasis in RYR1-pG2435R mouse muscles and muscle cells using calcium and sodium ion selective microelectrodes, manganese quench of Fura2 fluorescence, and western blots.

Results:

RYR1-pG2435R mouse muscle cells have chronically elevated intracellular resting calcium and sodium and rate of manganese quench (homozygous>heterozygous) compared to wildtype. After exposure to 1-oleoyl-2-acetyl-sn-glycerol, a TRPC3/6 activator, increases in intracellular resting calcium/sodium were significantly greater in RYR1-pG2435R muscles (from 153±11nM/10±0.5mM to 304±45nM/14.2±0.7mM in heterozygotes, p<0.001, from 251±25nM/13.9±0.5mM to 534±64nM/20.9±1.5mM in homozygotes p<0.001 compared to 123±3nM/8±0.1mM to 196±27nM/9.4±0.7mM in wildtype). These increases were inhibited both by simply removing extracellular Ca²⁺ or by exposure to either a nonspecific (gadolinium) or a newly available more specific pharmacologic agent (SAR7334) to block TRPC6 and TRPC3 mediated cation influx into cells. Furthermore, local pretreatment with SAR7334 partially decreased the elevation of intracellular resting calcium that is seen in RYR1-p.G2435R muscles during exposure to halothane. Western blot analysis showed that expression of TRPC3 and TRPC6 were significantly increased in RYR1-p.G2435R muscles in a gene dose-dependent manner, supporting their being a primary molecular basis for increased sarcolemmal cation influx.

Conclusions:

Muscle cells in knock-in mice expressing the RYR1-p.G2435R mutation are hypersensitive to TRPC3/6 activators. This hypersensitivity can be negated with pharmacologic agents that block TRPC3/6 activity. This reinforces our working hypothesis that TRPC channels play a critical role in causing intracellular calcium and sodium overload in malignant hyperthermia susceptible muscle, both at rest and during the malignant hyperthermia crisis.

Introduction.-

Malignant hyperthermia is an autosomal dominant hypermetabolic condition triggered by volatile anesthetics and depolarizing neuromuscular blockers¹. The malignant hyperthermia crisis is characterized by hypercapnia, tachycardia, hypoxemia, muscle rigidity, hypermetabolism, respiratory and metabolic acidosis and hyperthermia. Human malignant hyperthermia susceptible individuals appear to for the most part remain subclinical until challenged with pharmacologic triggering agents^1–3. Once the syndrome is triggered, if left untreated, the mortality of a fulminant malignant hyperthermia episode is >70%, but availability of dantrolene has reduced the mortality to <8%^1,2. Molecular genetic studies have established that the type 1 ryanodine receptor gene (RYR1) encoding the skeletal muscle sarcoplasmic reticulum Ca²⁺ release channel as the primary locus for malignant hyperthermia^3–6. More than 200 RYR1 mutations found throughout the gene have been associated with malignant hyperthermia⁶. To date, one spontaneously occurring porcine model (p.R615C) and 4 murine knock-in models expressing the murine equivalent of human (p.R163C⁷, p.Y522S⁸, p.G2434R⁹ and p.T4826I¹⁰) RYR1 mutations have been studied in detail. All 5 models exhibit fulminant anesthetic-triggered malignant hyperthermia episodes and heat intolerance with varying gene dose relationships. The RYR1-p.G2435R variant which is the focus of this study is a model for the most frequent variant associated with malignant hyperthermia in the United Kingdom and North America. In addition, this is the most common RYR1 variant to be associated with familial genotype-phenotype discordance¹¹. It is associated with a relatively weak in vitro contracture test phenotype and is less likely to be associated with an elevated serum creatine kinase compared with T4826I and R163C^12,13.

The exact molecular mechanisms by which RYR1 mutations confer malignant hyperthermia susceptibility are unknown. A common characteristic of muscle expressing malignant hyperthermia-RyR1 mutations compared to non-susceptible muscle is an increased resting intracellular calcium concentration in humans¹⁴ and animal models^15–18. We have shown that exposure to halothane or isoflurane at clinically relevant concentrations causes intracellular calcium concentration to rise several fold in malignant hyperthermia muscle in experimental animal models, whereas exposure to the same concentrations of halothane or isoflurane had no effect in non-susceptible muscle^17,19,20.

Until recently, the mechanism for the malignant hyperthermia crisis has been attributed solely to massive self-sustaining release of Ca²⁺ from the sarcoplasmic reticulum on exposure to triggering agents. Counter to this view, we have introduced a new paradigm that implicates nonspecific sarcolemmal cation entry channels as both the predominant source of acute elevations in intracellular calcium concentration and intracellular sodium concentration during fulminant malignant hyperthermia and are significant contributors to chronically elevated intracellular calcium concentration and intracellular sodium concentration in quiescent malignant hyperthermia-susceptible muscles^21,22.

The aim of the current study was to use newly available more specific pharmacologic agents and physiologic studies to probe transient receptor potential cation channel (TRPC) function allowing us to refine their importance in initiating and supporting the malignant hyperthermia crisis in response to triggering agents, and with these new data test the hypothesis that TRPCs play a major role in the Ca²⁺ dyshomeostasis that we recently described in a RyR1-p.G2435R knock-in murine model of malignant hyperthermia⁹ similar to our previous observations in the RyR1-p.R163C mouse^21,22.

Material and Methods.-

Animals

Animals used in this study were housed in pathogen-free conditions with free access to food, water and 12-hour light-and-dark cycles. All experiments were undertaken with either UK Home Office, University of California at Davis IACUC or Mount Sinai Hospital IACUC approval. For single fiber and in vivo studies, a mixture of male and female mice between 12–16 weeks were used and for myotube studies, myoblasts were isolated from 10–12 day old neonatal mice. All experiments were terminal. For in vivo experiments the malignant hyperthermia animals were either allowed to succumb to isoflurane anesthesia when this was used, otherwise they and all wildtype animals were euthanized by cervical dislocation. For muscle collection all animals were euthanized by cervical dislocation immediately prior to dissection. Because homozygous animals were viable, animals were obtained and allocated to homozygous and heterozygous experimental groups by selective breeding of homozygous females to either homozygous or wildtype C57BL/6J males. Animals were used as available in the breeding colony when they reached the appropriate age. Studies on wildtype mice were done on 3–5-month-old C57BL/6J animals purchased from Charles River Laboratories (Wilmington MA) or the St. James’s animal facility (Leeds, UK) as needed.

Experimental Preparation

Experiments were conducted at room temperature (~23°C): (i) in vitro using flexor digitorum brevis muscle fibers from 3–5-month-old C57BL/6J (wildtype), heterozygous RYR1pG2435R and homozygous RYR1pG2435R mice. Single fibers were obtained by enzymatic digestion as described previously²¹. (ii) in vivo, in vastus lateralis fibers in wildtype and RYR1-p.G2435R mice anesthetized with ketamine/xylazine (100/5 mg/kg body weight) as described previously²². (iii) in vitro using myotubes differentiated from myoblasts from C57BL/6 (wildtype), RYR1-p.G2435R heterozygous, and RyR1-p.G2435R homozygous mice as described in detail previously⁹.

Preparation of Calcium and sodium selective microelectrodes

Double-barreled calcium and sodium selective microelectrodes were prepared as described previously^14,22. Each ion-selective microelectrode was individually calibrated before and after the determination of intracellular calcium concentration and intracellular Na⁺ concentration as described before²². Only those Ca²⁺ selective microelectrodes 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. The Na⁺ selective microelectrodes gave virtually Nernstian responses at free sodium concentrations between 100 and 10 mM. However, although at concentrations between 10 and 1 mM Na⁺, the electrodes had a sub-Nernstian response (40–45 mV), their response was of a sufficient amplitude to be able to measure intracellular sodium concentration. The sensitivity and response of the calcium and sodium selective microelectrodes were not directly affected by any of the drugs used in the present study.

Calcium and sodium determinations in muscle fibers

Microelectrode recordings were performed as described previously²². Single isolated adult flexor digitorum brevis muscle fibers (in vitro) or vastus lateralis muscle fibers (in vivo) from wildtype and RYR1-p.G2435R heterozygous and homozygous mice were impaled with either double-barreled Ca²⁺ or double-barreled Na⁺ -selective microelectrodes, and their potentials were recorded via a high-impedance amplifier (WPI Duo 773 electrometer; WPI, Sarasota, FL, USA). The potential from the 3 M potassium chloride microelectrode was subtracted electronically from either the potential of the calcium electrode or the sodium electrode to produce a differential calcium-specific potential or sodium-specific potential that represents the intracellular calcium concentration or intracellular sodium concentration, respectively. All electrode potentials were filtered (30–50 kHz) to improve the signal-to-noise ratio and stored in a computer for further analysis. Data from any given electrode impalement were accepted if the 3 M potassium chloride microelectrode potential was greater than or equal to −80mV, and if it was possible to make a stable recording of both the 3 M potassium chloride microelectrode potential and either the calcium-specific potential or sodium-specific potential for 60 seconds. These criteria resulted in rejection of data from 20% of in vivo impalements and 30–35% of in vitro impalements. There was no genotype effect on rejection rate.

Measurements of manganese quench of Fura2 in myotubes and isolated single fibers.

When the Ca²⁺ indicator Fura2 is exposed to manganese its fluorescence at its isobestic emission wavelength is quenched. Because TRPC, Orai1, 2, 3 and slow voltage gated Ca²⁺ channels are permeable to manganese as well as Ca²⁺, the rate of manganese quench can be used to determine the rate of Ca²⁺ entry into a cell.

We used manganese quench to assess sarcolemmal divalent cation entry in myotubes or isolated single adult muscle fibers loaded with 5 μM Fura2-AM for 30 mins at 37°C. The solution containing the Fura2-AM was washed off and the cells maintained for 25 mins at room temperature to allow de-esterification and equilibration of the intracellular Fura-2. The cells were observed through a 40× 1.3 NA objective on a Nikon Eclipse T2000 epifluorescence microscope using an excitation wave-length of 360+/−5 nm (the isosbestic wavelength of Fura-2 where the fluorescence of the dye is independent of the Ca²⁺ free versus Ca²⁺ bound state), and the emission signal measured at 510+/−40 nm. Images were captured with 2×2 binning at a rate of 5 fps using an intensified 12-bit digital intensified charge-coupled device (ORCA-ER) and IPLab software. Regions of interest in individual cells were analyzed using Image J software (NHLBI), with the data exported to GraphPad Prism 7. Prism was used to fit linear regression models (y=mx+c) for the basal signal in imaging buffer, and then independently in manganese-containing solutions with and without treatment drugs. The specific rate of Fura-2 quenching induced by manganese entry was calculated by subtracting the basal slope of decline of fluorescence over time from the slope during the application of manganese buffer (i.e. net slope) and is expressed as arbitrary fluorescence units per second. The net slope was calculated in the absence and presence of treatment drugs. Cells with a positive net slope following the addition of manganese buffer were excluded from analysis because this indicated a technical problem with the measurement. For the same reason the slope of the quench signal was normalized to zero when the net slope was positive following treatment with a cation channel antagonist.

Western blot analysis of TRPC3 and TRPC6 expression.-

Gastrocnemius muscles from all genotypes were dissected, minced, and homogenized in modified radioimmunoassay precipitation assay (RIPA) buffer. Total protein concentration was determined using the bicinchoninic acid (BCA) method (Thermo-Scientific, MA, USA). Samples of whole gastrocnemius homogenate were prepared as described by Altamirano et al. [18] and incubated overnight at 4°C with primary antibodies: rabbit anti -TRPC3, dilution 1:2500 (ab51560; Abcam, MA, USA), rabbit anti -TRPC6, dilution 1:2500 (ab62461, Abcam, MA, USA), human anti-actin, dilution of 1:5000 (SC8432; Santa Cruz, CA, USA). All of these antibodies have been validated previously by different research groups and our laboratory. The resolved bands were detected with a Storm 860 Imaging System (GE Bio-Sciences, NJ, USA). Protein levels were quantified using myImageAnalysis software (Thermo-Fisher Scientific, MA, USA) and normalized to b-actin.

Solutions

The mammalian Ringer solution used for experiments using muscle fibers had the following composition (in mM): 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 5 glucose, and 10 HEPES, pH 7.4. The imaging buffer used for the experiments with myotubes contained (in mM): 133 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 5.5 glucose, and 10 HEPES, pH 7.4. In the manganese buffer the CaCl₂ and MgCl₂ were substituted with 0.5 mM MnCl₂. 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. 1-oleoyl-2-acetyl-sn-glycerol, gadolinium, SAR7334 and hyperforin solutions were made by adding the desired concentration of the reagent to the physiological or manganese containing solutions.

Statistics

Neither randomization nor blinding of the investigator performing the test were used for these studies. No specific power calculation was conducted prior to experimentation but the rationale for the sample sizes used was based on our previous studies using this and other animal models of malignant hyperthermia with the same and different pharmacologic interventions. All values of intracellular ion concentrations are expressed as mean±SD, with N representing the number of mice used for the in vivo experiments, and n representing the number of myotubes or muscle fibers in which measurements were carried out. Statistical analysis for studies done on muscle fibers both in vitro and in vivo was performed using 1-way between genotypes ANOVA with Tukey’s post-test for multiple measurements to determine significance. Results of western blots were analyzed using Student’s unpaired independent t-test. Normal distribution of the data was verified by histogram analysis. For studies of manganese quench in myotubes, the results were not normally distributed, and as a result are expressed as the median and interquartile range. These results were analyzed using a Kruskal-Wallis test with Dunn’s multiple comparison to determine significance. p<0.05 was accepted as the minimum confidence level for significance for all comparisons but the P values obtained for each comparison are reported. With the exception of the myotube manganese quench studies, where positive quench data were excluded or normalized to zero as described above, and muscle fiber data where the criteria for a successful measurement was not met (based on either a depolarized membrane potential or lack of stable electrode recording for 60 seconds), no outlying data were removed from the data collected prior to or after analysis. All statistical analyses were performed using GraphPad Prism 7 software.

Results.-

Resting intracellular calcium and sodium concentrations

Malignant hyperthermia is characterized by an intracellular Ca²⁺ and Na⁺ dyshomeostasis^14,19,22,23. Consequently, intracellular calcium concentration or intracellular sodium concentration was measured in quiescent single muscle fibers isolated from wildtype, RYR1-p.G2435R heterozygous, and RYR1-p.G2435R homozygous cells using double-barreled ion-specific microelectrodes. intracellular calcium concentration was 120±2 nM (n=8) in wildtype, while it was 156±11 nM (n=11) in RYR1pG2435R heterozygous, and 275±15 nM (n=10) in homozygous (p <0.001 for both) (Figure 1A). Likewise, intracellular sodium concentration was more elevated in RYR1pG2435R heterozygous, and homozygous fibers than that observed in wildtype fibers. intracellular sodium concentration was 8±0.1 mM (n=8) in wildtype fibers, while it was 10±1 mM (n=9) in RYR1-p.G2435R heterozygotes and 14±0.6 mM (n=11) in RyR1-p.G2435R homozygotes respectively (p <0.001 for both) (Figure 1A). The resting membrane potentials were not different among genotypes (-82±1.2 mV (n=29) in resting wildtype muscle fibers, −82±1.3mV (n=31) in RYR1-p.G2435R heterozygous muscle fibers and -81±2.3 mV (n=34) in RYR1-p.G2435R homozygous muscle fibers).

Figure 1. Elevated intracellular Ca²⁺ and Na⁺ concentrations in RYR1-pG2435R malignant hyperthermia muscle cells.

Intracellular Ca²⁺ or Na⁺ concentration was measured in quiescent flexor digitorum brevis fibers isolated from Wild type, RYR1-pG2435R heterozygous, and RYR1-pG2435R homozygous mice (Fig. 1A) and in vivo from the vastus lateralis fibers in anesthetized Wild type, RYR1-pG2435R heterozygous and RYR1-pG2435R homozygous mice (Fig. 1B) using double-barreled ion-specific microelectrodes. In vitro: n_mice= 3/experimental condition, n_cell=15–18/genotype for intracellular Ca²⁺ concentration measurements; n_mice= 3/experimental condition, n_cell =15–18/genotype for intracellular Na⁺ concentration measurements.

In vivo: n_mice= 4/genotype, n_cell=13–16/experimental condition for intracellular Ca²⁺ concentration measurements; n_mice= 5/experimental condition, n_cell = 8–11/ genotype for intracellular Na⁺ concentration measurements. Values are expressed as means ± S.D. for each condition. One-way ANOVA with Tukey’s post-test, *p≤0.05.

As previously reported similar differences for intracellular calcium concentration and intracellular sodium concentration were obtained in the in-vivo measurements carried out on the vastus lateralis from wildtype and malignant hyperthermia mice⁹. In a new data set carried out for this study intracellular calcium concentration in wildtype was 122±3 nM (n=15) compared to 144±9 nM (n=14) and 259±31 nM (n=18) in RYR1-p.G2435R heterozygous and RYR1-p.G2435R homozygous muscles respectively (Figure 1B). intracellular sodium concentration was 8±0.1 mM (n=16) in wildtype compared to 9.5±1 mM (n=16) and 12.9±1.5 mM (n=13) in RYR1-p.G2435R heterozygous and RYR1-p.G2435R homozygous muscles respectively (p<0.001 compared to wildtype) (Figure 1B).

Taken together, the results demonstrate that there is a comparable intracellular calcium and sodium overload in both isolated RYR1-p.G2435R muscle cells and intact muscle fibers compared to wildtype and that resting intracellular ion recordings under in vitro conditions recapitulate those found in vivo.

1-oleoyl-2-acetyl-sn-glycerol induces an elevation of intracellular calcium and sodium concentrations

To directly investigate the effect of diacylglycerol on intracellular calcium and intracellular sodium concentrations single muscle fibers were exposed to 1-oleoyl-2-acetyl-sn-glycerol, a membrane-permeable diacylglycerol analog which has been shown to activate transient receptor potential cation channel subfamily C, member 3 (TRPC3) and member 6 (TRPC6) channels²⁴. Incubation of muscle fibers in 1-oleoyl-2-acetyl-sn-glycerol (100 μM) for 5 minutes produced an elevation of intracellular calcium concentration in all genotypes. In wildtype intracellular calcium concentration was increased by 60% (from 123±3 nM (n=11) to 196±27 nM (n=13) p<0.001), in RyR1-p.G2435R heterozygous fibers it was enhanced by 99% (from 153±11 nM (n=12) to 304±45 nM (n=11) p<0.001) and in RYR1-p.G2435R homozygous fibers it was elevated by 113% (from 251±25 nM (n=12) to 534±64 nM (n=13) p<0.001) (Figure 2A).

Figure 2. 1-oleoyl-2-acetyl-sn-glycerol provokes elevation of intracellular Ca²⁺ and Na⁺ concentrations.

Exposure of quiescent flexor digitorum brevis fibers to 1-oleoyl-2-acetyl-sn-glycerol 100 μM induced an elevation of intracellular Ca²⁺ (Fig. 2A) and Na⁺ concentration (Fig. 2B) that was greater in malignant hyperthermia (homozygous > heterozygous) than Wild type muscle fibers. Over the horizontal axis are indicated the experimental conditions. For intracellular Ca²⁺ concentration measurements: n_mice= 3/experimental condition, n_cell= 11–13/genotype. For intracellular Na⁺ concentration measurements: n_mice= 3/experimental condition, n_cell= 9–10/genotype. Values are expressed as means ± S.D. for each condition. One-way ANOVA with Tukey’s post-test, *p≤0.05.

Since the permeability of TRPCs is cation specific but not Ca²⁺ specific, their activation could also increase sodium entry and intracellular sodium concentration. Application of 1-oleoyl-2-acetyl-sn-glycerol (100μM) significantly elevated intracellular sodium concentration in all genotypes but its effect was greater in RYR1-p.G2435R muscle cells than in wildtype (Figure 2B). In wildtype incubation with 1-oleoyl-2-acetyl-sn-glycerol elevated intracellular sodium concentration by 18% (from 8±0.1 mM, n=10 to 9.4±0.7 mM, n=9, p<0.001) in RYR1-p.G2435R heterozygous by 42% (from 10±0.5 mM, n=10 to 14.2±0.7 mM, n=9, p<0.001) and in RYR1-p.G2435R homozygous by 50% (from 13.9±0.5 mM, n=9 to 20.9±1.5 mM, n=10, p<0.001).

These elevations of intracellular calcium and sodium concentrations induced by 1-oleoyl-2-acetyl-sn-glycerol were not affected by the voltage gated Ca²⁺ channel inhibitor nifedipine (10 μM) nor associated with changes in resting membrane potential in either wildtype or RYR1-p.G2435R muscle fibers (data not shown).

Removal of extracellular Ca²⁺ prevented increases in intracellular calcium concentration induced by 1-oleoyl-2-acetyl-sn-glycerol.

In a different set of experiments, we explored the contribution of the Ca²⁺ influx on intracellular calcium concentration elevation elicited by 1-oleoyl-2-acetyl-sn-glycerol. Single wildtype and RYR1-p.G2435R muscle fibers were incubated for 5 min in Ca²⁺-free solution prior to 1-oleoyl-2-acetyl-sn-glycerol (100μM) treatment. This exposure significantly reduced intracellular calcium concentration in all genotypes but had a greater effect in RYR1-p.G2435R than wildtype (Figure 3). In wildtype fibers Ca²⁺-free solution reduced intracellular calcium concentration from 123±4 nM (n=10) to 98±6 nM (n=12, p<0.001) while in RYR1-p.G2435R heterozygous fibers it was reduced from 161±26 nM (n=14) to 136±6 nM (n=12, p<0.001) and in RYR1-p.G2435R homozygous fibers it decreased from to 256±27 nM (n=12) to 176±17 nM (n=14, p<0.001). In addition, the observed rise in intracellular calcium concentration elicited by 1-oleoyl-2-acetyl-sn-glycerol was completely inhibited in both wildtype and RYR1-p.G2435R muscle fibers in the absence of extracellular Ca²⁺ (Figure 3).

Figure 3. Removal of extracellular Ca²⁺ prevents the 1-oleoyl-2-acetyl-sn-glycerol-induced increase in intracellular Ca²⁺ concentration.

Removal of extracellular Ca²⁺ lowered the intracellular Ca²⁺ concentration and provoked the inhibition of 1-oleoyl-2-acetyl-sn-glycerol (100 μM)-induced elevation of intracellular Ca²⁺ concentration in flexor digitorum brevis fibers isolated from Wild type, RYR1-pG2435R heterozygous, and RYR1-pG2435R homozygous mice. On the horizontal axis are indicated the experimental conditions used. For intracellular Ca²⁺ concentration measurements: n_mice= 3/experimental condition, n_cell= 10–14/genotype. Values are expressed as means ± S.D. for each condition. One-way ANOVA with Tukey’s post-test, *p≤0.05.

Blocking sarcolemmal calcium and sodium entry with gadolinium abolishes the increases in intracellular calcium and sodium concentrations elicited by 1-oleoyl-2-acetyl-sn-glycerol.

To gain further insight into molecular mechanisms for the increased sarcolemmal Ca²⁺ and Na⁺ entry responsible for the elevation in intracellular calcium and sodium concentrations after exposure to 1-oleoyl-2-acetyl-sn-glycerol, we measured intracellular calcium concentration and intracellular sodium concentration in isolated single wildtype and RYR1-p.G2435R muscle fibers before and after incubation in gadolinium and then in the presence of gadolinium, after exposure to 1-oleoyl-2-acetyl-sn-glycerol (100μM). Pretreatment with 25 μM gadolinium significantly lowered intracellular calcium concentration and intracellular sodium concentration by 15% and by 8% respectively in wildtype fibers; by 20% by 15% respectively in RYR1-p.G2435R heterozygous fibers; and by 27% and 29% respectively in RYR1-p.G2435R homozygous fibers (Figure 4A and B). Furthermore, gadolinium pretreatment prevented any significant increase in intracellular calcium and sodium concentrations during exposure to 1-oleoyl-2-acetyl-sn-glycerol in all genotypes (Figure 4A and B).

Figure 4. Gadolinium abolishes the 1-oleoyl-2-acetyl-sn-glycerol effects on intracellular Ca²⁺ and Na⁺ concentrations.

Preincubation of Wild type, RYR1-pG2435R heterozygous, and RYR1-pG2435R homozygous flexor digitorum brevis fibers in gadolinium (25 μM) reduced the intracellular Ca²⁺ and Na⁺ concentrations and eliminated the increase in intracellular Ca²⁺ (Fig. 4A) and Na⁺ concentrations (Fig. 4B) induced by 1-oleoyl-2-acetyl-sn-glycerol. Over the horizontal axis are indicated the experimental. For intracellular Ca²⁺ determinations: n_mice= 4/experimental condition, n_cell= 17–19/genotype. For intracellular Na⁺ determinations: n_mice= 4/experimental condition, n_cell= 13–15/genotype. Values are expressed as means ± S.D. for each condition. One-way ANOVA with Tukey’s post-test, *p≤0.05.

SAR7334 blocks the effects of 1-oleoyl-2-acetyl-sn-glycerol.

Because gadolinium is a non-specific sarcolemmal cation blocker, SAR7334 a newer more specific blocker of TRPC6 and TRPC3 channels²⁵ was used to further characterize the mechanism for the 1-oleoyl-2-acetyl-sn-glycerol mediated increase in intracellular calcium concentration. Single isolated wildtype and RYR1-p.G2435R muscle fibers were incubated in 1μM SAR7334 and then exposed to 1-oleoyl-2-acetyl-sn-glycerol (100μM). Pretreatment with SAR7334 significantly reduced the resting intracellular calcium concentration by 14% in wildtype fibers, by 19% in RYR1-p.G2435R heterozygous fibers and 34% in RYR1-p.G2435R homozygous fibers (Figure 5A). Furthermore, SAR7334 also prevented the 1-oleoyl-2-acetyl-sn-glycerol mediated elevation of intracellular calcium concentration in all three genotypes (Figure 5A).

Figure 5. SAR7334 blocked the elevation of intracellular Ca²⁺ concentration induced by 1-oleoyl-2-acetyl-sn-glycerol and partially inhibited the increase associated with a malignant hyperthermia episode.

Intracellular Ca²⁺ concentration was measured in Wild type, RYR1-pG2435R heterozygous, and RYR1-pG2435R homozygous flexor digitorum brevis fibers before and after incubation in SAR7374 (1μM), as well as after the incubation in SAR7374 when exposed to 1-oleoyl-2-acetyl-sn-glycerol (100 μM). Fig. 5A shows that preincubation in SAR7374 reduced. Intracellular Ca²⁺ concentration in all genotypes and prevent the increase in intracellular calcium concentration induced by 1-oleoyl-2-acetyl-sn-glycerol. Over the horizontal axis are indicated the experimental conditions. Fig. 5B shows a typical experiment carried out in vivo in RYR1-pG2435R homozygous mice anesthetized with ketamine100mg/kg/xylazine 50mg/kg. Intracellular calcium concentration was measured simultaneously in the superficial fibers of the vastus lateralis muscle in the right and left hind limb. Superficial muscle fibers were superfused with Ringer solution alone (left leg, control, red line) or SAR7374 (1μM) for 5 min (right leg, black line), and the mouse was then exposed to 2% halothane by inhalation (black arrow). The insert on the left upper corner shows the experimental arrangement: 1 and 2 Ca²⁺ microelectrodes, 3 and 4 grounds, 5 rectal temperature probe. For intracellular Ca²⁺ concentration measurements in Fig.5A: n_mice= 4/genotype, n_cell= 18–13 experimental condition. Values are expressed as means ± S.D. for each condition. One-way ANOVA with Tukey’s post-test, *p≤0.05.

SAR7334 partially blocked the elevation of intracellular calcium concentration associated with malignant hyperthermia episode

Having observed inhibition of the 1-oleoyl-2-acetyl-sn-glycerol induced increase in intracellular calcium concentration by SAR7334 in wildtype and RYR1-p.G2435R single muscle fibers, we then tested the activity of this TRPC3/6 channel blocker during a malignant hyperthermia episode. We, therefore, investigated the effect of SAR7334 on intracellular calcium concentration in malignant hyperthermia muscle in vivo during exposure to 2% halothane. intracellular calcium concentration was measured simultaneously in the right and left vastus lateralis muscles in wildtype and homozygous RYR1-p.G2435R mice before and after the addition of 2% halothane to their inspired gas (Figure 5B). The right vastus lateralis muscle was pretreated locally with 1 μM SAR7334 and the left muscle served as the control. Figure 5B shows a typical in vivo experiment carried out in a homozygous RYR1-p.G2435R mouse, data is reported as nanomolar (nM). Prior to drug treatment intracellular calcium concentration was very similar in muscles from both legs (265±9 nM left leg versus 275±9 nM right leg). 10 min after the application of 1μM SAR7334 on the exposed vastus lateralis fibers of the right leg reduced intracellular calcium concentration from 265±9 nM to 187±11 nM (Figure 5B). Inhalation of halothane elicited an elevation of intracellular calcium concentration in the untreated leg to 1,373±81 nM while in the SAR7334-treated leg the initial increase in intracellular calcium concentration was reduced by 62% (526±99 nM) after which intracellular calcium concentration declined to 453±32nM (Figure 5B). After 20 min of inhalation of halothane, the rectal temperature reached 42° C, and the mouse died.

Hyperforin induced elevation of intracellular calcium and sodium concentrations

Hyperforin is known to elevate the intracellular concentration of Ca²⁺ through the activation of di-acyl-glycerol sensitive TRPC6 channels without activating the other TRPC isoforms (TRPC1, TRPC3, TRPC4, TRPC5, and TRPC7)²⁶. Hyperforin (5 μM) caused a prominent sustained rise in intracellular calcium concentration, that was greater in RYR1-p.G2435R (homozygous>heterozygous) than wildtype muscle fibers (Figure 6A). In wildtype muscle fibers intracellular calcium concentration was enhanced from 124±3 nM (n=9) to 151±10 nM (n=10) upon incubation with Hyperforin while in RYR1-p.G2435R heterozygous fibers it increased from 164±22 nM (n=10) to 234±31 nM (n=10) and in RYR1-p.G2435R homozygous fibers it was elevated from 269±37 nM (n=10) to 441±41 nM (n=10) (Figure 6A). Removal of extracellular Ca²⁺ reduced pre exposure intracellular calcium concentration and blocked the hyperforin effect in all genotypes (Figure 6C). Similar to its effect on intracellular calcium concentration, hyperforin elevated intracellular sodium concentration in wildtype fibers from 8±0.1 mM (n=15) to 9.5±0.6 mM (n=10) (p<0.001), in RYR1-p.G2435R heterozygous fibers from 10±0.6 mM (n=15) to 13.5±1 (n=10) (p<0.001) and in RYR1-p.G2435R homozygous fibers from 14.5±1.2 mM (n=11) to 20.3±1.6 mM (n=13) (p<0.001) (Figure 6B).

Figure 6. Effect of hyperforin on intracellular Ca²⁺ and Na⁺ concentrations.

Hyperforin (5 μM) caused a sustained rise in intracellular Ca²⁺ and Na⁺ concentration in single flexor digitorum brevis muscle fibers that were greater in malignant hyperthermia (homozygous> heterozygous) than Wild type (Fig. 6A and 6B). Removal of extracellular Ca²⁺ inhibited the hyperforin effect on intracellular Ca²⁺ concentration (Fig. 6C). On the horizontal axis are indicated the experimental conditions. For intracellular Ca²⁺ concentration determinations in Fig 6A: n_mice= 3/experimental condition, n_cell= 9/10 genotype. For intracellular Na⁺ concentration determinations in Fig. 6B: n_mice= 3/experimental condition, n_cell= 15/10 genotype. For intracellular Ca²⁺ concentration determinations in Fig. 6C: n_mice= 3/experimental condition, n_cell= 12/10 genotype. Values are expressed as means ± S.D. for each condition. Paired and unpaired t-test and one-way ANOVA with Tukey’s post-test, *p≤0.05.

Effects of genotype and sarcolemmal cation entry agonists and antagonists on manganese quench of Fura2 in differentiated myotubes

When the buffer was changed from imaging buffer to manganese quench buffer baseline quench of Fura2 fluorescence was observed in all three genotypes (Figure 7A for representative traces reported as arbitrary fluorescence units) with the median quench rate being greater in malignant hyperthermia-homozygous (-0.23 arbitrary fluorescence units/s Interquartile range (IQR) -0.13 to −0.38 arbitrary fluorescence units/s) versus malignant hyperthermia-heterozygous (−0.18 arbitrary fluorescence units/s, IQR −0.11 to -0.32 arbitrary fluorescence units/s) and wildtype (-0.19 arbitrary fluorescence units/s, IQR −0.12 to −0.32 arbitrary fluorescence units/s) (Figure 7B). Qualitatively similar results were observed in isolated adult single muscle fibers (data not shown).

Figure 7: Resting sarcolemmal divalent cation permeability in myotubes.

(A) Representative traces indicating the change in the rate of the fura2 fluorescence that was quenched by Mn²⁺ in WT, heterozygous and homozygous myotubes from RYR1-pG2435R mice after changing from imaging buffer to manganese buffer. (B) Box-plots showing the quench rate (median and InterQuartileRange) in control, 25 μM gadolinium or 250 nM SAR7334 treated myotubes from each of the three genotypes. The whiskers represent the 10–90^th centile. A larger rate of quench is represented by a more negative number and this indicates a greater cationic entry through the sarcolemmal cation channels into the cell from the extracellular space which is an estimate of the sarcolemmal permeability to Ca²⁺. ^&p<0.05 in RYR1-pG2435R homozygous versus RYR1-pG2435R heterozygous and wild type. **p<0.005 and p<0.0001 in each genotype vs. their control conditions (Kruskall-Wallis test with Dunn’s multiple comparisons test, n≥35 myotubes for each condition).

We next determined the effect of sarcolemmal cation blockers 25 μM gadolinium (a nonspecific sarcolemmal cation entry blocker), and 250 nM SAR7334 (a TRPC specific blocker) on the rate of manganese quench of the fura2 fluorescence signal. Treatment with 25 μM gadolinium profoundly blocked manganese quench to near baseline levels in all three genotypes (p<0.0001, n=65 – 96, Figure 7B). The reduction in the median quench rate was 88% in wildtype myotubes (n=65), 100% in heterozygous RYR1-p.G2435R myotubes (n=72) and 95.6% in homozygous RYR1-p.G2435R myotubes (n= 96) (Figure 7B). After treatment with 250nM SAR7334 in wildtype myotubes the change in median quench rate was not significant (8%, p>0.999, n = 49), while in heterozygous RYR1-p.G2435R myotubes and in homozygous RYR1-p.G2435R myotubes there was a significant decrease (45%, p<0.005, n = 53 and 69%, p<0.001, n = 35 for heterozygous and homozygous RYR1-p.G2435R myotubes respectively) in the median manganese quench rate (Figure 7B).

Protein expression differences in wildtype and malignant hyperthermia muscle.

Western blot analysis showed that TRPC3 and TRPC6 were significantly increased in a gene dose dependent manner in malignant hyperthermia muscles (heterozygous (p<0.01) and homozygous (p<0.001)) compared to wildtype. Figure 8A shows representative Western blot analysis of the expression of TRPC3 and TRPC6 in wildtype, heterozygous RYR1-p.G2435R and homozygous RYR1-p.G2435R muscle; beta actin was used as a loading control. Densitometric analysis of 5 independent Western blots for each TRPC protein is shown in Figure 8B.

Figure 8.

Fig. 8A shows representative Western blots showing the expression of TRPC3, TRPC6, and ß-actin in Wild type, RYR1-pG2435R heterozygous, and RYR1-pG2435R homozygous gastrocnemius muscles. Densitometric analysis of 5 independent Western blots for each TRPC protein is shown in Fig. 8B. Data were normalized to ß-actin and are expressed as mean ± S.D. n_muscles=8. Student’s unpaired independent t-test, *p≤0.05

Discussion.-

The major findings of the present study are as follows.

1. We confirmed our previous finding⁹ that RYR1-pG2435R muscle cells have chronically elevated intracellular resting calcium and sodium concentrations (homozygous>heterozygous) and now show that this is associated with elevated manganese quench indicating increased sarcolemmal cation influx;

2. Compared to wildtype, incubation of RYR1-pG2435R muscle cells with 1-oleoyl-2-acetyl-sn-glycerol or the TRPC6 activator hyperforin significantly increased intracellular calcium and sodium concentrations (homozygous>heterozygous);

3. Increases in intracellular calcium and sodium concentrations induced by 1-oleoyl-2-acetyl-sn-glycerol are dependent on extracellular Ca²⁺ and are blocked by the nonspecific sarcolemmal cation channel blocker gadolinium; as well as the more specific TRPC3 and TRPC6 channel blocker SAR7334.

4. SAR7334 treatment also significantly reduced resting intracellular calcium and sodium concentrations, blocked manganese quench, and reduced the elevation of intracellular calcium in RYR1-p.G2435R fibers after exposure to halothane;

5. The observed changes in the above RYR1-pG2434R responses were accompanied by an increase in the protein expression of TRPC3, TRPC6 in RYR1-p.G2435R (homozygous>heterozygous) skeletal muscles compared to wildtype.

For some time it has been thought that the mechanism behind the increased resting intracellular calcium in malignant hyperthermia susceptible muscle is simply due to sarcoplasmic reticulum leak and that the massive increase in muscle intracellular calcium during an malignant hyperthermia episode comes from and is maintained by sarcoplasmic reticulum Ca²⁺ stores. Based on our studies in other animal models relevant to malignant hyperthermia we have previously suggested that sarcolemmal cation entry channels are at the very least, significant contributors to the chronically elevated intracellular calcium in quiescent malignant hyperthermia muscles and myotubes^21–22 and that they play a similarly significant role during a fulminant malignant hyperthermia episode²².

A primary finding was that in addition to increased resting intracellular calcium, that resting intracellular sodium is also elevated in RYR1pG2435R muscle cells (homozygous>heterozygous) compared to wildtype, both in vitro and in vivo^21–22. This suggests that at least one source of Ca²⁺ could be the extracellular pool and not the sarcoplasmic reticulum. In this study we confirmed this hypothesis showing that incubation of muscle with gadolinium, a potent non-specific inhibitor of sarcolemmal cation influx, significantly reduced the intracellular levels of both sodium and calcium and blocked manganese quench, a monitor of sarcolemmal cation entry.

Di-acyl-glycerol is a second messenger involved in key cellular processes in skeletal muscle and other excitable cells²⁷. In addition to its role as a lipid second messenger to recruit protein kinases C, it also activates the di-acyl-glycerol sensitive TRPC3/6 subgroup of TRPC channels in the sarcolemma^24,28. Here we utilized the membrane-permeable di-acyl-glycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol to determine if the pathway for sarcolemmal sodium and calcium influx was through TRPC3 and TRPC6²⁹. 1-oleoyl-2-acetyl-sn-glycerol induced an elevation in the resting intracellular calcium and sodium in all three genotypes, and this was greater in RyR1-p.G2435R (homozygous>heterozygous) muscle than wildtype (Figures 2A and 2B). The elevation of intracellular calcium, and the increase rate of manganese quench elicited by 1-oleoyl-2-acetyl-sn-glycerol was blocked either by removal of extracellular Ca²⁺ or by blocking sarcolemmal Ca²⁺ and Na⁺ entry with gadolinium or SAR7334 (Figures 3, 4 and 6). These data rule out any possibility that 1-oleoyl-2-acetyl-sn-glycerol increased intracellular calcium by releasing Ca²⁺ from intracellular stores, and confirmed that the source of the increase was through TRPC3/6 whose expression we showed was upregulated in RYR1-p.G2435R compared to wildtype muscles.

Incubation of wildtype and RYR1-p.G2435R muscle fibers with SAR7334, a much more specific blocker of TRPC6 and TRPC3 channels than gadolinium²⁵, reduced resting calcium in RYR1-p.G2435R cells (homozygous<heterozygous<wildtype) and blocked the effects of 1-oleoyl-2-acetyl-sn-glycerol. Local application of SAR7334 directly to the superficial muscle fibers in vivo reduced resting intracellular calcium in homozygous RYR1-p.G2435R fibers and partially inhibited the halothane-induced increase in intracellular calcium in RyR1-p.G2435R muscle cells. This finding in particular demonstrates that during the malignant hyperthermia episode there is an initial sarcoplasmic reticulum supported increase in intracellular calcium which soon appears to be quickly partially depleted, as demonstrated by the rapid fall in intracellular calcium from ~750nM to ~400nM in the SAR7334 treated leg during the same time intracellular calcium rose to 1,373±81 nM and then plateaus in the non-treated leg. All of these data together support the hypothesis that sarcolemmal Ca²⁺ entry plays a critical role in maintaining the new intracellular calcium steady-state that has been observed during an malignant hyperthermia crisis. Furthermore the observed immediate elevation of intracellular calcium in the SAR7334 treated leg, is most probably mediated by Ca²⁺ release from the sarcoplasmic reticulum and the decline in the intracellular calcium in this leg shows that that this level cannot be maintained by sarcoplasmic reticulum Ca²⁺ cycling due to Ca²⁺ extrusion and blockade of sarcolemmal Ca²⁺ entry.

Consistent with the 1-oleoyl-2-acetyl-sn-glycerol-mediated elevation of intracellular calcium and sodium in muscle cells plus the ability of the TRPC specific blocker SAR7334 to block both the pharmacologic and halothane induced increase in Ca²⁺, we also demonstrated that hyperforin, an activator of TRPC6 mediated inward cationic current^26,31,32, mimicked the action of 1-oleoyl-2-acetyl-sn-glycerol and its effect was greater in RYR1-p.G2435R (homozygous>heterozygous) than wildtype muscle cells. As observed with 1-oleoyl-2-acetyl-sn-glycerol removal of extracellular Ca²⁺ also eliminated the hyperforin-elicited increase in resting intracellular calcium³¹. The enhanced cation flux observed across the sarcolemma in RYR1-p.G2435R cells is consistent with the cell boundary theorem which states that the long term steady state of the free intracellular calcium concentration inside cells must be driven by changes with the outside through fluxes across the plasmalemma^22,33.

In summary, these results demonstrated that skeletal muscle of mice expressing the RYR1-p.G2435R have an elevated resting intracellular calcium and sodium and a significantly enhanced, extracellular Ca²⁺ dependent response to 1-oleoyl-2-acetyl-sn-glycerol and hyperforin. We further showed that the 1-oleoyl-2-acetyl-sn-glycerol response could be prevented by TRPC3 and TRPC6 blockers. These results provide further support to our working hypothesis that in response to partial depletion of sarcoplasmic reticulum calcium stores due to malignant hyperthermia causing mutations in RYR1, TRPC3 and TRPC6 channels play a critical role in producing the intracellular calcium and sodium overload both at rest and during the malignant hyperthermia crisis.