Pharmacologic and functional characterization of malignant hyperthermia in the R163C RyR1 knock-in mouse.

Department of Anesthesia, Perioperative and Pain Medicine, Brigham & Women's Hospital, Boston, Massachusetts 02115, USA.
Anesthesiology (Impact Factor: 5.88). 01/2007; 105(6):1164-75.
Source: PubMed


Malignant hyperthermia is a pharmacogenetic disorder affecting humans, dogs, pigs, and horses. In the majority of human cases and all cases in animals, malignant hyperthermia has been associated with missense mutations in the skeletal ryanodine receptor (RyR1).
The authors used a "knock-in" targeting vector to create mice carrying the RyR1 R163C malignant hyperthermia mutation.
Validation of this new mouse model of human malignant hyperthermia susceptibility includes (1) proof of transcription of the R163C allele and expression of ryanodine receptor protein in R163C heterozygous and R163C homozygous animals; (2) fulminant malignant hyperthermia episodes in R163C heterozygous mice after exposure to 1.25-1.75% halothane or an ambient temperature of 42 degrees C characterized by increased rectal temperature, respiratory rate, and inspiratory effort, with significant blood biochemical changes indicating metabolic acidosis, ending in death and hyperacute rigor mortis; (3) intraperitoneal pretreatment with dantrolene provided 100% protection from the halothane-triggered fulminant malignant hyperthermia episode; (4) significantly increased sensitivity (decreased effective concentration causing 50% of the maximal response) of R163C heterozygous and homozygous myotubes to caffeine, 4-chloro-m-cresol, and K-induced depolarization; (5) R163C heterozygous and homozygous myotubes have a significantly increased resting intracellular Ca concentration compared with wild type; (6) R163C heterozygous sarcoplasmic reticulum membranes have a twofold higher affinity (Kd = 35.4 nm) for [H]ryanodine binding compared with wild type (Kd = 80.1 nm) and a diminished inhibitory regulation by Mg.
Heterozygous R163C mice represent a valid model for studying the mechanisms that cause the human malignant hyperthermia syndrome.

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    • "MH in humans usually results from missense mutations in the type I ryanodine receptor (RyR1), which functions as the Ca2+ release channel in the sarcoplasmic reticulum (SR) of skeletal muscle [1]. Recently, multiple mouse models for heat- and halothane-induced sudden death were developed including knock-in of mutations in RyR1 linked to MH in humans [10,11] and knockout of calsequestrin1 (Casq1) [12,13], the primary SR Ca2+ binding protein in skeletal muscle. The Y524S mutation increases RyR1 Ca2+ leak and susceptibility to activation. "
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    ABSTRACT: Store-operated calcium entry (SOCE) channels play an important role in Ca(2+) signaling. Recently, excessive SOCE was proposed to play a central role in the pathogenesis of malignant hyperthermia (MH), a pharmacogenic disorder of skeletal muscle. We tested this hypothesis by characterizing SOCE current (ISkCRAC) magnitude, voltage dependence, and rate of activation in myotubes derived from two mouse models of anesthetic- and heat-induced sudden death: 1) type 1 ryanodine receptor (RyR1) knock-in mice (Y524S/+) and 2) calsequestrin 1 and 2 double knock-out (dCasq-null) mice. ISkCRAC voltage dependence and magnitude at -80 mV were not significantly different in myotubes derived from wild type (WT), Y524S/+ and dCasq-null mice. However, the rate of ISkCRAC activation upon repetitive depolarization was significantly faster at room temperature in myotubes from Y524S/+ and dCasq-null mice. In addition, the maximum rate of ISkCRAC activation in dCasq-null myotubes was also faster than WT at more physiological temperatures (35-37°C). Azumolene (50 µM), a more water-soluble analog of dantrolene that is used to reverse MH crises, failed to alter ISkCRAC density or rate of activation. Together, these results indicate that while an increased rate of ISkCRAC activation is a common characteristic of myotubes derived from Y524S/+ and dCasq-null mice and that the protective effects of azumolene are not due to a direct inhibition of SOCE channels.
    PLoS ONE 10/2013; 8(10):e77633. DOI:10.1371/journal.pone.0077633 · 3.23 Impact Factor
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    • "While the identification and further study of these mutants is primarily driven by the need to understand and prevent or alleviate the associated diseases, the study of the well-defined functional defects associated with each mutation is becoming a useful approach to unravel structure–function relationships in these large and complex channels. Several mice have been created with RyR1 mutations homologous to those associated with MH in humans (Yang et al. 2006; Chelu et al. 2006; Yuen et al. 2012). All show a MH-like hypermetabolic response to volatile anaesthetics and temperature. "
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    ABSTRACT: Malignant hyperthermia (MH) is linked to mutations in the type 1 ryanodine receptor, RyR1, the Ca(2+) channel of the sarcoplasmic reticulum (SR) of skeletal muscle. The Y522S MH mutation was studied for its complex presentation, which includes structurally and functionally altered cell "cores". Imaging cytosolic and intra-SR [Ca(2+)] in muscle cells of heterozygous YS mice we determined Ca(2+) release flux activated by clamp depolarization, permeability of the SR membrane- P, ratio of flux and [Ca(2+)] gradient- and SR Ca(2+) buffering power-B. In YS cells resting [Ca(2+)]SR was 45% of the value in normal littermates (WT). P was more than doubled, so that initial flux was normal. Measuring [Ca(2+)]SR(t) revealed dynamic changes in B(t). The alterations were similar to those caused by cytosolic BAPTA, which promotes release by hampering Ca(2+)-dependent inactivation (CDI). The [Ca(2+)] transients showed abnormal "breaks", decaying phases after an initial rise, traced to a collapse in flux and P. Similar breaks occurred in WT myofibers with calsequestrin reduced by siRNA; calsequestrin content, however, was normal in YS muscle. Thus, the Y522S mutation causes greater openness of the RyR1, lowers resting [Ca(2+)]SR and alters SR Ca(2+) buffering in a way that copies the functional instability observed upon reduction of calsequestrin content. The similarities with the effects of BAPTA suggest that the mutation, occurring near the cytosolic vestibule of the channel, reduces CDI as one of its primary effects. The unstable SR buffering, mimicked by silencing of calsequestrin, may help precipitate the loss of Ca(2+) control that defines a fulminant MH event.
    The Journal of Physiology 06/2013; 591(18). DOI:10.1113/jphysiol.2013.259572 · 5.04 Impact Factor
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    • "In comparison with cultured myotubes, knock-in animal models offer the advantage of assessing the consequences of the mutations in fully differentiated adult muscle fibres. Mouse models are currently available for three mutant forms of RyR1: Y522S (Chelu et al. 2006), R163C (Yang et al. 2006) and I4897T (Zvaritch et al. 2007) and intracellular Ca 2+ transients under voltage-clamp conditions have so far been investigated in adult muscle fibres isolated from the heterozygous Y522S and I4897T models (Andronache et al. 2009; Loy et al. 2011). Some of the results from these studies confirmed previous data from expression in myotubes, but also highlighted features specific to adult muscle fibres. "
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    ABSTRACT: Non-Technical Summary Calcium ions flowing through the type 1 ryanodine receptor (RyR1) calcium channel trigger contraction of skeletal muscle cells. Close to 300 mutations of the gene encoding RyR1 are responsible for several muscular diseases in human. Properties of pathological mutant RyR1s have so far been essentially assessed from studies in cultured cells and in differentiated native muscle fibres from a few available transgenic mouse models. We show that functional properties of mutant RyR1s can be reliably assessed following in vivo expression in adult mouse muscles. The Y523S, R615C and R2163H RyR1 mutants produce a similar over-sensitive activation of the calcium flux whereas I4897T RyR1 mutants are responsible for a depressed Ca2+ flux. The alterations appear to result from inherent modifications of RyR1 channel function and not from indirect changes in the muscle fibre homeostasis. The present strategy will help understand the physio-pathological defects underlying alterations of muscle function in affected patients. Abstract Mutations of the gene encoding the type 1 ryanodine receptor (RyR1) are associated with skeletal muscle disorders including malignant hyperthermia susceptibility (MHS) and central core disease (CCD). We used in vivo expression of EGFP-RyR1 constructs in fully differentiated mouse muscle fibres to characterize the function of several RyR1 mutants. Wild-type and Y523S, R615C, R2163H and I4897T mutants of RyR1 were separately expressed and found to be present within restricted regions of fibres with a pattern consistent with triadic localization. Confocal measurements of voltage-clamp-activated myoplasmic Ca2+ transients demonstrated alterations of sarcoplasmic reticulum (SR) Ca2+ release spatially correlated with the presence of exogenous RyR1s. The Y523S, R615C and R2163H RyR1 MHS-related mutants were associated with enhanced peak Ca2+ release for low and moderate levels of depolarization, whereas the I4897T CCD mutant produced a chronic reduction of peak SR Ca2+ release. For example, peak Ca2+ release in response to a depolarization to –20 mV in regions of fibres expressing Y523S and I4897T was 2.0 ± 0.3 (n= 9) and 0.46 ± 0.1 (n= 5) times the corresponding value in adjacent, non-expressing regions of the same fibre, respectively. Interestingly no significant change in the estimated total amount of Ca2+ released at the end of large depolarizing pulses was observed for any of the mutant RyR1 channels. Overall, results are consistent with an ‘inherent’ increase in RyR1 sensitivity to activation by the voltage sensor for the MHS-related RyR1 mutants and a partial failure of voltage-gated release for the CCD-related I4897T mutant, that occur with no sign of change in SR Ca2+ content. Furthermore, the results indicate that RyR1 channel density is tightly regulated even under the present conditions of forced exogenous expression.
    The Journal of Physiology 11/2011; 589(Pt 22):5361-82. DOI:10.1113/jphysiol.2011.216408 · 5.04 Impact Factor
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