Pharmacologic and functional characterization of malignant hyperthermia in the R163C RyR1 knock-in mouse.
ABSTRACT 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.
SourceAvailable from: Katharine J Goodall[Show abstract] [Hide abstract]
ABSTRACT: Heparanase is a β-d-endoglucuronidase that cleaves heparan sulphate, a key component of the ECM and basement membrane. The remodelling of the ECM by heparanase has been proposed to regulate both normal physiological and pathological processes, including wound healing, inflammation, tumour angiogenesis and cell migration. Heparanase is also known to exhibit non-enzymatic functions by regulating cell adhesion, cell signalling and differentiation. In this study, constitutive heparanase-deficient (Hpse(-/-) ) mice were generated on a C57BL/6 background using the Cre/loxP recombination system, with a complete lack of heparanase mRNA, protein and activity. Although heparanase has been implicated in embryogenesis and development, Hpse(-/-) mice are anatomically normal and fertile. Interestingly, consistent with the suggested function of heparanase in cell migration, the trafficking of dendritic cells from the skin to the draining lymph nodes was markedly reduced in Hpse(-/-) mice. Furthermore, the ability of Hpse(-/-) mice to generate an allergic inflammatory response in the airways, a process that requires dendritic cell migration, was also impaired. These findings establish an important role for heparanase in immunity and identify the enzyme as a potential target for regulation of an immune response.European Journal of Immunology 04/2014; 44(4). DOI:10.1002/eji.201343645 · 4.52 Impact Factor
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ABSTRACT: The advent of the polymerase chain reaction and the availability of data from various global human genome projects should make it possible, using a DNA sample isolated from white blood cells, to diagnose rapidly and accurately almost any monogenic condition resulting from single nucleotide changes. DNA-based diagnosis for malignant hyperthermia (MH) is an attractive proposition, because it could replace the invasive and morbid caffeine-halothane/in vitro contracture tests of skeletal muscle biopsy tissue. Moreover, MH is preventable if an accurate diagnosis of susceptibility can be made before general anesthesia, the most common trigger of an MH episode. Diagnosis of MH using DNA was suggested as early as 1990 when the skeletal muscle ryanodine receptor gene (RYR1), and a single point mutation therein, was linked to MH susceptibility. In 1994, a single point mutation in the α 1 subunit of the dihydropyridine receptor gene (CACNA1S) was identified and also subsequently shown to be causative of MH. In the succeeding years, the number of identified mutations in RYR1 has grown, as has the number of potential susceptibility loci, although no other gene has yet been definitively associated with MH. In addition, it has become clear that MH is associated with either of these 2 genes (RYR1 and CACNA1S) in only 50% to 70% of affected families. While DNA testing for MH susceptibility has now become widespread, it still does not replace the in vitro contracture tests. Whole exome sequence analysis makes it potentially possible to identify all variants within human coding regions, but the complexity of the genome, the heterogeneity of MH, the limitations of bioinformatic tools, and the lack of precise genotype/phenotype correlations are all confounding factors. In addition, the requirement for demonstration of causality, by in vitro functional analysis, of any familial mutation currently precludes DNA-based diagnosis as the sole test for MH susceptibility. Nevertheless, familial DNA testing for MH susceptibility is now widespread although limited to a positive diagnosis and to those few mutations that have been functionally characterized. Identification of new susceptibility genes remains elusive. When new genes are identified, it will be the role of the biochemists, physiologists, and biophysicists to devise functional assays in appropriate systems. This will remain the bottleneck unless high throughput platforms can be designed for functional work. Analysis of entire genomes from several individuals simultaneously is a reality. DNA testing for MH, based on current criteria, remains the dream.Anesthesia and analgesia 02/2014; 118(2):397-406. DOI:10.1213/ANE.0000000000000063 · 3.42 Impact Factor
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ABSTRACT: Passive SR Ca2+-leak through the ryanodine receptor (RyR) plays a critical role in the mechanisms that regulate intracellular resting Ca2+ concentration ([Ca2+]rest) in muscle. This process appears to be isoform specific as expression of either RyR1 or RyR3 confers myotubes with different [Ca2+]rest. Using chimeric RyR3/RyR1 receptors expressed in dyspedic myotubes here we show that isoform dependent regulation of [Ca2+]rest is primarily defined by a small region of the receptor encompassing amino acid 3770-4007 of RyR1 (3620-3859 of RyR3) named as the Ca2+ Leak Regulatory (CLR) region.[Ca2+]rest regulation by the CLR region was associated with alteration of RyRs Ca2+-activation profile and changes in SR Ca2+-leak rates. Biochemical analysis using Tb3+ binding assays and intrinsic tryptophan fluorescence spectroscopy of purified CLR domains revealed that this determinant of RyRs holds a novel Ca2+-binding domain with conformational properties that are distinctive of each isoform. Our data suggest that the CLR region provide channels with unique functional properties that modulate the rate of passive SR Ca2+-leak and confer RyR1 and RyR3 with distinctive [Ca2+]restregulatory properties. The identification of a new Ca2+-binding domain of RyRs with a key modulatory role in [Ca2+]restregulation provides new insights into Ca2+-mediated regulation of RyRs.Biochemical Journal 03/2014; DOI:10.1042/BJ20131553 · 4.78 Impact Factor