Gene dose influences cellular and calcium channel dysregulation in heterozygous and homozygous T4826I-RYR1 malignant hyperthermia-susceptible muscle

Malignant hyperthermia susceptibility (MHS) is primarily conferred by mutations within ryanodine receptor type 1 (RYR1). Here we address how the MHS mutation T4826I within the S4-S5 linker influences excitation-contraction coupling and resting myoplasmi

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Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Pharmacologic and Functional Characterization of Malignant Hyperthermia in the R163C RyR1 Knock-in Mouse

BACKGROUND: 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). METHODS: The authors used a "knock-in" targeting vector to create mice carrying the RyR1 R163C malignant hyperthermia mutation. RESULTS: 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°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. CONCLUSIONS: Heterozygous R163C mice represent a valid model for studying the mechanisms that cause the human malignant hyperthermia syndrome

Dynamic regulation of ryanodine receptor type 1 (RyR1) channel activity by Homer 1

Homer, a family of scaffolding proteins originally identified in neurons, is also expressed in skeletal muscle. Previous studies showed that splice variants of Homer 1 (H1) amplify the gain of the ryanodine receptor type 1 (RyR1) channel complex. Using [3H]ryanodine ([3H]Ry) to probe the conformational state of RyR1, the actions of long- and short-forms of H1 are examined singly and in combination. At ≤200 nM, H1 long-forms (H1b or H1c possessing coiled-coil (CC) domains) and short-forms (H1a or H1EVH1 lacking CC domains) enhance specific [3H]Ry binding to RyR1. However, at a concentration >200 nM, either H1 form completely inhibited [3H]Ry binding. Importantly, the combinations of H1c + H1EVH1, or H1b + H1a acted in an additive manner to enhance or inhibit [3H]Ry-binding activity. H1a and H1c individually or in combination produced the same dynamic pattern in regulating purified RyR1 channels reconstituted in planar lipid bilayers. In combination, their net action on RyR1 channels depends on total concentrations of H1. These data provide a mechanism by which constitutively and transiently expressed H1 forms can tightly regulate RyR1 channel activity in response to changing levels of expression and degradation of H1 proteins

Mice expressing T4826I-RYR1 are viable but exhibit sex- and genotype-dependent susceptibility to malignant hyperthermia and muscle damage

Mutation T4825I in the type 1 ryanodine receptor (RYR1T4825I/+) confers human malignant hyperthermia susceptibility (MHS). We report a knock-in mouse line that expresses the isogenetic mutation T4826I. Heterozygous RYR1T4826I/+ (Het) or homozygous RYR1T4826I/T4826I (Hom) mice are fully viable under typical rearing conditions but exhibit genotypeand sex-dependent susceptibility to environmental conditions that trigger MH. Hom mice maintain higher core temperatures than WT in the home cage, have chronically elevated myoplasmic[Ca2+]rest, and present muscle damage in soleus with a strong sex bias. Mice subjected to heat stress in an enclosed 37°C chamber fail to trigger MH regardless of genotype, whereas heat stress at 41°C invariably triggers fulminant MH in Hom, but not Het, mice within 20 min. WT and Het female mice fail to maintain euthermic body temperature when placed atop a bed whose surface is 37°C during halothane anesthesia (1.75%) and have no hyperthermic response, whereas 100% Hom mice of either sex and 17% of the Het males develop fulminant MH. WT mice placed on a 41°C bed maintain body temperature while being administered halothane, and 40% of the Het females and 100% of the Het males develop fulminant MH within 40 min. Myopathic alterations in soleus were apparent by 12 mo, including abnormally distributed and enlarged mitochondria, deeply infolded sarcolemma, and frequent Z-line streaming regions, which were more severe in males. These data demonstrate that an MHS mutation within the S4-S5 cytoplasmic linker of RYR1 confers genotype- and sex-dependent susceptibility to pharmacological and environmental stressors that trigger fulminant MH and promote myopathy