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Central core disease, one of the most common congenital myopathies in humans, has been linked to mutations in the RYR1 gene encoding the Ca(2+) release channel of the sarcoplasmic reticulum (RyR1). Functional analyses showed that disease-associated RYR1 mutations led to impairment of skeletal muscle Ca(2+) homeostasis; however, thorough understanding of the molecular mechanisms underlying central core disease and other RyR1-related conditions is still lacking. We screened by sequencing the complete RYR1 transcripts in ten unrelated patients with central core disease and identified five novel, p.M4640R, p.L4647P, p.F4808L, p.D4918N and p.F4941C, and four recurrent mutations. Four of the novel mutations involved amino acid residues that were positioned within putative transmembrane segments of the RyR1. The pathogenic character of the identified mutations was demonstrated by bioinformatic analyses and by the in vitro functional studies in HEK293 cells and RYR1-null (dyspedic) myotubes. Characterization of Ca(2+) channel properties of RyR1s carrying one recurrent and two novel mutations upholds the view that diminished intracellular Ca(2+) release caused by impaired Ca(2+) channel gating and/or Ca(2+) permeability is an important component of central core disease etiology. This study expands the list of functionally characterized disease-associated RyR1 mutations, increasing the value of genetic diagnosis for RyR1-related disorders., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Mice with a malignant hyperthermia mutation (Y522S) in the ryanodine receptor (RyR1) display muscle contractures, rhabdomyolysis, and death in response to elevated environmental temperatures. We demonstrate that this mutation in RyR1 causes Ca(2+) leak, which drives increased generation of reactive nitrogen species (RNS). Subsequent S-nitrosylation of the mutant RyR1 increases its temperature sensitivity for activation, producing muscle contractures upon exposure to elevated temperatures. The Y522S mutation in humans is associated with central core disease. Many mitochondria in the muscle of heterozygous Y522S mice are swollen and misshapen. The mutant muscle displays decreased force production and increased mitochondrial lipid peroxidation with aging. Chronic treatment with N-acetylcysteine protects against mitochondrial oxidative damage and the decline in force generation. We propose a feed-forward cyclic mechanism that increases the temperature sensitivity of RyR1 activation and underlies heat stroke and sudden death. The cycle eventually produces a myopathy with damaged mitochondria.

A heterozygous Ile4898 to Thr (I4898T) mutation in the human type 1 ryanodine receptor/Ca(2+) release channel (RyR1) leads to a severe form of central core disease. We created a mouse line in which the corresponding Ryr1(I4895T) mutation was introduced by using a "knockin" protocol. The heterozygote does not exhibit an overt disease phenotype, but homozygous (IT/IT) mice are paralyzed and die perinatally, apparently because of asphyxia. Histological analysis shows that IT/IT mice have greatly reduced and amorphous skeletal muscle. Myotubes are small, nuclei remain central, myofibrils are disarranged, and no cross striation is obvious. Many areas indicate probable degeneration, with shortened myotubes containing central stacks of pyknotic nuclei. Other manifestations of a delay in completion of late stages of embryogenesis include growth retardation and marked delay in ossification, dermatogenesis, and cardiovascular development. Electron microscopy of IT/IT muscle demonstrates appropriate targeting and positioning of RyR1 at triad junctions and a normal organization of dihydropyridine receptor (DHPR) complexes into RyR1-associated tetrads. Functional studies carried out in cultured IT/IT myotubes show that ligand-induced and DHPR-activated RyR1 Ca(2+) release is absent, although retrograde enhancement of DHPR Ca(2+) conductance is retained. IT/IT mice, in which RyR1-mediated Ca(2+) release is abolished without altering the formation of the junctional DHPR-RyR1 macromolecular complex, provide a valuable model for elucidation of the role of RyR1-mediated Ca(2+) signaling in mammalian embryogenesis.

Ca(2+) release from intracellular stores is controlled by complex interactions between multiple proteins. Triadin is a transmembrane glycoprotein of the junctional sarcoplasmic reticulum of striated muscle that interacts with both calsequestrin and the type 1 ryanodine receptor (RyR1) to communicate changes in luminal Ca(2+) to the release machinery. However, the potential impact of the triadin association with RyR1 in skeletal muscle excitation-contraction coupling remains elusive. Here we show that triadin binding to RyR1 is critically important for rapid Ca(2+) release during excitation-contraction coupling. To assess the functional impact of the triadin-RyR1 interaction, we expressed RyR1 mutants in which one or more of three negatively charged residues (D4878, D4907, and E4908) in the terminal RyR1 intraluminal loop were mutated to alanines in RyR1-null (dyspedic) myotubes. Coimmunoprecipitation revealed that triadin, but not junctin, binding to RyR1 was abolished in the triple (D4878A/D4907A/E4908A) mutant and one of the double (D4907A/E4908A) mutants, partially reduced in the D4878A/D4907A double mutant, but not affected by either individual (D4878A, D4907A, E4908A) mutations or the D4878A/E4908A double mutation. Functional studies revealed that the rate of voltage- and ligand-gated SR Ca(2+) release were reduced in proportion to the degree of interruption in triadin binding. Ryanodine binding, single channel recording, and calcium release experiments conducted on WT and triple mutant channels in the absence of triadin demonstrated that the luminal loop mutations do not directly alter RyR1 function. These findings demonstrate that junctin and triadin bind to different sites on RyR1 and that triadin plays an important role in ensuring rapid Ca(2+) release during excitation-contraction coupling in skeletal muscle.

The skeletal muscle Ca(2+)-release channel (ryanodine receptor type 1 (RyR1)) is a redox sensor, susceptible to reversible S-nitrosylation, S-glutathionylation, and disulfide oxidation. So far, Cys-3635 remains the only cysteine residue identified as functionally relevant to the redox sensing properties of the channel. We demonstrate that expression of the C3635A-RyR1 mutant in RyR1-null myotubes alters the sensitivity of the ryanodine receptor to activation by voltage, indicating that Cys-3635 is involved in voltage-gated excitation-contraction coupling. However, H(2)O(2) treatment of C3635A-RyR1 channels or wild-type RyR1, following their expression in human embryonic kidney cells, enhances [(3)H]ryanodine binding to the same extent, suggesting that cysteines other than Cys-3635 are responsible for the oxidative enhancement of channel activity. Using a combination of Western blotting and sulfhydryl-directed fluorescent labeling, we found that two large regions of RyR1 (amino acids 1-2401 and 3120-4475), previously shown to be involved in disulfide bond formation, are also major sites of both S-nitrosylation and S-glutathionylation. Using selective isotopecoded affinity tag labeling of RyR1 and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, we identified, out of the 100 cysteines in each RyR1 subunit, 9 that are endogenously modified (Cys-36, Cys-315, Cys-811, Cys-906, Cys-1591, Cys-2326, Cys-2363, Cys-3193, and Cys-3635) and another 3 residues that were only modified with exogenous redox agents (Cys-253, Cys-1040, and Cys-1303). We also identified the types of redox modification each of these cysteines can undergo. In summary, we have identified a discrete subset of cysteines that are likely to be involved in the functional response of RyR1 to different redox modifications (S-nitrosylation, S-glutathionylation, and oxidation to disulfides).

Mutations in the cardiac ryanodine receptor 2 (RyR2) have been associated with catecholaminergic polymorphic ventricular tachycardia and a form of arrhythmogenic right ventricular dysplasia. To study the relationship between RyR2 function and these phenotypes, we developed knockin mice with the human disease-associated RyR2 mutation R176Q. Histologic analysis of hearts from RyR2(R176Q/+) mice revealed no evidence of fibrofatty infiltration or structural abnormalities characteristic of arrhythmogenic right ventricular dysplasia, but right ventricular end-diastolic volume was decreased in RyR2(R176Q/+) mice compared with controls, indicating subtle functional impairment due to the presence of a single mutant allele. Ventricular tachycardia (VT) was observed after caffeine and epinephrine injection in RyR2(R176Q/+), but not in WT, mice. Intracardiac electrophysiology studies with programmed stimulation also elicited VT in RyR2(R176Q/+) mice. Isoproterenol administration during programmed stimulation increased both the number and duration of VT episodes in RyR2(R176Q/+) mice, but not in controls. Isolated cardiomyocytes from RyR2(R176Q/+) mice exhibited a higher incidence of spontaneous Ca(2+) oscillations in the absence and presence of isoproterenol compared with controls. Our results suggest that the R176Q mutation in RyR2 predisposes the heart to catecholamine-induced oscillatory calcium-release events that trigger a calcium-dependent ventricular arrhythmia.

Malignant hyperthermia (MH) is a life-threatening disorder characterized by skeletal muscle rigidity and elevated body temperature in response to halogenated anesthetics such as isoflurane or halothane. Mutation of tyrosine 522 of RyR1 (the predominant skeletal muscle calcium release channel) to serine has been associated with human malignant hyperthermia. In the present study, mice created harboring this mutation were found to represent the first murine model of human malignant hyperthermia. Mice homozygous for the Y522S mutation exhibit skeletal defects and die during embryonic development or soon after birth. Heterozygous mice, which correspond to the human occurrence of this mutation, are MH susceptible, experiencing whole body contractions and elevated core temperatures in response to isoflurane exposure or heat stress. Skeletal muscles from heterozygous mice exhibit increased susceptibility to caffeine- and heat-induced contractures in vitro. In addition, the heterozygous expression of the mutation results in enhanced RyR1 sensitivity to activation by temperature, caffeine, and voltage but not uncompensated sarcoplasmic reticulum calcium leak or store depletion. We conclude that the heterozygous expression of the Y522S mutation confers susceptibility to both heat- and anesthetic-induced MH responses.

Ca+-induced Ca2+ release (CICR) in the heart involves local Ca2+ signaling between sarcolemmal L-type Ca2+ channels (dihydropyridine receptors, DHPRs) and type 2 ryanodine receptors (RyR2s) in the sarcoplasmic reticulum (SR). We reconstituted cardiac-like CICR by expressing a cardiac dihydropyridine-insensitive (T1066Y/Q1070M) alpha1-subunit (alpha1CYM) and RyR2 in myotubes derived from RyR1-knockout (dyspedic) mice. Myotubes expressing alpha1CYM and RyR2 were vesiculated and exhibited spontaneous Ca2+ oscillations that resulted in chaotic and uncontrolled contractions. Coexpression of FKBP12.6 (but not FKBP12.0) with alpha1CYM and RyR2 eliminated vesiculations and reduced the percentage of myotubes exhibiting uncontrolled global Ca2+ oscillations (63% and 13% of cells exhibited oscillations in the absence and presence of FKBP12.6, respectively). alpha1CYM/RyR2/FKBP12.6-expressing myotubes exhibited robust and rapid electrically evoked Ca2+ transients that required extracellular Ca2+. Depolarization-induced Ca2+ release in alpha1CYM/RyR2/FKBP12.6-expressing myotubes exhibited a bell-shaped voltage dependence that was fourfold larger than that of myotubes expressing alpha1CYM alone (maximal fluorescence change was 2.10 +/- 0.39 and 0.54 +/- 0.07, respectively), despite similar Ca2+ current densities. In addition, the gain of CICR in alpha1CYM/RyR2/FKBP12.6-expressing myotubes exhibited a nonlinear voltage dependence, being considerably larger at threshold potentials. We used this molecular model of local alpha1C-RyR2 signaling to assess the ability of FKBP12.6 to inhibit spontaneous Ca2+ release via a phosphomimetic mutation in RyR2 (S2808D). Electrically evoked Ca2+ release and the incidence of spontaneous Ca2+ oscillations did not differ in wild-type RyR2- and S2808D-expressing myotubes over a wide range of FKBP12.6 expression. Thus a negative charge at S2808 does not alter in situ regulation of RyR2 by FKBP12.6.

Ca2+ ions play a pivotal role in a wide array of cellular processes ranging from fertilization to cell death. In skeletal muscle, a mechanical interaction between plasma membrane dihydropyridine receptors (DHPRs, L-type Ca2+ channels) and Ca2+ release channels (ryanodine receptors, RyR1s) of the sarcoplasmic reticulum orchestrates a complex, bi-directional Ca2+ signaling process that converts electrical impulses in the sarcolemma into myoplasmic Ca2+ transients during excitation-contraction coupling. Mutations in the genes that encode the two proteins that coordinate this electrochemical conversion process (the DHPR and RyR1) result in a variety of skeletal muscle disorders including malignant hyperthermia (MH), central core disease (CCD), multiminicore disease, nemaline rod myopathy, and hypokalemic periodic paralysis. Although RyR1 and DHPR disease mutations are thought to alter excitability and Ca2+ homeostasis in skeletal muscle, only recently has research begun to probe the molecular mechanisms by which these genetic defects lead to distinct clinical and histopathological manifestations. This review focuses on recent advances in determining the impact of MH and CCD mutations in RyR1 on muscle Ca2+ signaling and how these effects contribute to disease-specific aspects of these disorders., (Copyright 2004 Elsevier Inc.)