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Heat- and anesthesia-induced malignant hyperthermia in an RyR1 knock-in mouse

Mihail G. Chelu^*,1, Sanjeewa A. Goonasekera^,1, William J. Durham^*,1, Wei Tang^*, John D. Lueck, Joyce Riehl, Isaac N. Pessah, Pumin Zhang^*, Meenakshi B. Bhattacharjee, Robert T. Dirksen and Susan L. Hamilton^*,2

^* Department of Molecular Physiology and Biophysics and
Department of Pathology Baylor College of Medicine, Houston, Texas, USA;
Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York, USA; and
Department of VM: Molecular Biosciences and Center for Children’s Environmental Health and Disease Prevention, University of California, Davis, California, USA

² Correspondence: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. E-mail: susanh{at}bcm.tmc.edu

SPECIFIC AIMS

Mutation of tyrosine 522 of RyR1 (the predominant skeletal muscle calcium release channel) to serine has been associated with human malignant hyperthermia (MH), a life-threatening disorder characterized by skeletal muscle rigidity and elevated body temperature in response to halogenated anesthetics such as isoflurane or halothane. In the present study, we have created mice harboring this mutation to determine its effects on calcium homeostasis, skeletal muscle function, and in vivo sensitivity to anesthesia and heat.

PRINCIPAL FINDINGS

1. Heterozygous Y522S mice are a model for human MH
Heterozygous Y522S mutation of RYR1 in mice produces the signature characteristics of MH: increased sensitivity to caffeine-induced contractures in vitro (Fig. 1 A) and in vivo contractures and hyperthermia [mutant rectal temperature 39.5 ± 1.1°C (n=4) compared with 35.8 ± 0.5°C (n=4) in the wild-type animals] in response to halogenated anesthetics (e.g., isoflurane).

View larger version (39K): [in this window] [in a new window]   Figure 1. Caffeine and heat contractile sensitivity of skeletal muscle obtained from adult wild-type (Wt) and heterozygous (Ht) male mice. A) Caffeine sensitivity of soleus muscle from male adult mice at 25°C. Soleus muscles of male Ht mice (EC50=5±1 mM, n=5) are more sensitive to caffeine than soleus muscles of male Wt mice (EC50=1±1 mM, n=5). B) Heat challenge at 41°C in adult Ht and Wt mice. The Ht mice responded with fully body contractions as reflected in arching of their backs and extension of their legs. Identical heat challenge is well-tolerated in Wt mice. C) Stress-frequency relationship at 37°C in soleus muscles of adult male mice. Heterozygous solei exhibit a 35% reduction in maximal tetanic stress at 37°C. D) Stress-frequency relationship at 25°C in soleus muscles of adult male mice. No statistically significant difference in the stress-frequency relationship is observed in Ht and Wt mice. E) Temperature dependence of basal stress in Wt and Ht solei. Increases of bath temperature over 35°C induces sustained basal contraction in Ht solei while temperatures 41°C are required to induce basal contractions in Wt solei. F) Temperature dependence of basal stress in Wt and Ht diaphragms. The temperature window for enhanced basal stress occurs at lower temperatures in diaphragms of Ht mice. For all panels: *P < 0.05; **P < 0.01; filled circles, Ht; open circles, Wt.

2. Heterozygous Y522S mice are susceptible to heat-induced whole body contractures in vivo and heat-induced contractures in vitro
Heterozygous mice experience whole body contractures in response to heating (Fig. 1B ) and skeletal muscles from these mice generate less force (Fig. 1C ) and exhibit increased sensitivity to contractures (Fig. 1E-F ) in response to heating in vitro. At room temperature, force generation in mutant and wild-type muscle is not different (Fig. 1D ).

3. The heterozygous mutation enhances RyR1 sensitivity to activation by temperature, caffeine, and voltage but has no effect on calcium stores
In myotubes, the heterozygous mutation enhances sensitivity of calcium release in response to caffeine and voltage. Calcium store depletion and basal calcium leak are not detected (Fig. 2 ).

View larger version (28K): [in this window] [in a new window]   Figure 2. Maximal SR Ca2+ release in response to electrical stimulation, caffeine, and 4-cmc in intact wild-type, heterozygous, and homozygous (Hm) Y522S myotubes. A) Representative traces of sequential activation of Ca2+ release by electrical stimulation and 60 s exposures/washes to maximal activating concentrations of caffeine (30 mM) and 4-cmc (500 µM) in intact Wt (left), Ht (middle), and Hm (right) myotubes. Average (±SEM) values for resting indo-1 ratio (F405/F485) (B), the magnitude of Ca2+ release induced by electrical stimulation (C), caffeine (D), and 4-cmc (E) in Wt (n=5), Ht (n=7), and Hm (n=8) myotubes (*P <0.01).

CONCLUSIONS AND SIGNIFICANCE

In the present study, we investigated the effects of the Y522S mutation of RYR1, which has been linked to human MH. Our data indicate that heterozygous expression of the Y522S mutation enhances RyR1 sensitivity to activation by temperature, caffeine, and voltage and these effects lead to MHS, rhabdomyolysis, and temperature- and anesthetic-induced sudden death. In contrast to previous results in other cell systems, this mutation did not affect calcium stores or resting cytosolic Ca²⁺ levels. The Y522S mutation has also been associated with the occurrence of central cores (areas of myofibrillar disorganization lacking mitochondria). Central cores, however, are not detected in Het mice, suggesting that other factors or alterations (e.g., elevation in resting Ca²⁺) are required for core formation.

A novel finding in the present study was a slightly greater sensitivity to heat-induced contractures in the soleus, a slow-type muscle, relative to the diaphragm, which in the mouse is a predominantly fast-type muscle,a difference that was greatly exaggerated in the mutant mice. Increased sensitivity of the slow, and to a lesser extent, fast, muscles may be a significant contributor to the development of a hyperthermic crisis by positively reinforcing anesthesia-induced skeletal muscle contractures. The possible importance of temperature per se is underscored by the finding that isoflurane exposure did not trigger contractures when the body temperature of the mutant mice was lowered.

In summary, mice heterozygous for the Y522S mutation of RYR1, corresponding to the human occurrence of this mutation, are a model for human MH, exhibiting in vivo sensitivity to halogenated anesthetics and positive in vitro contracture responses to caffeine, both signature characteristics of the MH phenotype. These mice experience whole-body contractures in response to heating and their skeletal muscles are sensitive to temperature-induced contractures. Calcium measurements in myotubes from these mice support the conclusion that these responses are due to increased RYR1 sensitivity to stimuli that promote calcium release.

View larger version (31K): [in this window] [in a new window]   Figure 3. Summary of effects of Y522S mutation of RYR1. Speculated effects are indicated by "?."

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4497fje;

¹ These authors contributed equally to this work.

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