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1.
BMJ ; 383: 2738, 2023 11 27.
Article in English | MEDLINE | ID: mdl-38011917
2.
Biochem J ; 474(16): 2749-2761, 2017 08 07.
Article in English | MEDLINE | ID: mdl-28687594

ABSTRACT

Mutations in the skeletal muscle ryanodine receptor (RyR1) cause malignant hyperthermia (MH) and central core disease (CCD), whereas mutations in the cardiac ryanodine receptor (RyR2) lead to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most disease-associated RyR1 and RyR2 mutations are located in the N-terminal, central, and C-terminal regions of the corresponding ryanodine receptor (RyR) isoform. An increasing body of evidence demonstrates that CPVT-associated RyR2 mutations enhance the propensity for spontaneous Ca2+ release during store Ca2+ overload, a process known as store overload-induced Ca2+ release (SOICR). Considering the similar locations of disease-associated RyR1 and RyR2 mutations in the RyR structure, we hypothesize that like CPVT-associated RyR2 mutations, MH/CCD-associated RyR1 mutations also enhance SOICR. To test this hypothesis, we determined the impact on SOICR of 12 MH/CCD-associated RyR1 mutations E2347-del, R2163H, G2434R, R2435L, R2435H, and R2454H located in the central region, and Y4796C, T4826I, L4838V, A4940T, G4943V, and P4973L located in the C-terminal region of the channel. We found that all these RyR1 mutations reduced the threshold for SOICR. Dantrolene, an acute treatment for MH, suppressed SOICR in HEK293 cells expressing the RyR1 mutants R164C, Y523S, R2136H, R2435H, and Y4796C. Interestingly, carvedilol, a commonly used ß-blocker that suppresses RyR2-mediated SOICR, also inhibits SOICR in these RyR1 mutant HEK293 cells. Therefore, these results indicate that a reduced SOICR threshold is a common defect of MH/CCD-associated RyR1 mutations, and that carvedilol, like dantrolene, can suppress RyR1-mediated SOICR. Clinical studies of the effectiveness of carvedilol as a long-term treatment for MH/CCD or other RyR1-associated disorders may be warranted.


Subject(s)
Calcium Signaling , Malignant Hyperthermia/genetics , Models, Molecular , Myopathy, Central Core/genetics , Point Mutation , Ryanodine Receptor Calcium Release Channel/genetics , Adrenergic beta-Antagonists/pharmacology , Amino Acid Substitution , Animals , Calcium Signaling/drug effects , Carbazoles/pharmacology , Carvedilol , Dantrolene/pharmacology , Fluorescence Resonance Energy Transfer , Genetic Predisposition to Disease , HEK293 Cells , Humans , Malignant Hyperthermia/drug therapy , Malignant Hyperthermia/metabolism , Microscopy, Fluorescence , Muscle Relaxants, Central/pharmacology , Mutagenesis, Site-Directed , Myopathy, Central Core/metabolism , Propanolamines/pharmacology , Protein Conformation , Rabbits , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ryanodine Receptor Calcium Release Channel/chemistry , Ryanodine Receptor Calcium Release Channel/metabolism , Single-Cell Analysis
3.
Proc Natl Acad Sci U S A ; 112(23): 7165-70, 2015 Jun 09.
Article in English | MEDLINE | ID: mdl-26040000

ABSTRACT

Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA). Here, we examined PLN stability and degradation in primary cultured mouse neonatal cardiomyocytes (CMNCs) and mouse hearts using immunoblotting, molecular imaging, and [(35)S]methionine pulse-chase experiments, together with lysosome (chloroquine and bafilomycin A1) and autophagic (3-methyladenine and Atg5 siRNA) antagonists. Inhibiting lysosomal and autophagic activities promoted endogenous PLN accumulation, whereas accelerating autophagy with metformin enhanced PLN degradation in CMNCs. This reduction in PLN levels was functionally correlated with an increased rate of SERCA2a activity, accounting for an inotropic effect of metformin. Metabolic labeling reaffirmed that metformin promoted wild-type and R9C PLN degradation. Immunofluorescence showed that PLN and the autophagy marker, microtubule light chain 3, became increasingly colocalized in response to chloroquine and bafilomycin treatments. Mechanistically, pentameric PLN was polyubiquitinylated at the K3 residue and this modification was required for p62-mediated selective autophagy trafficking. Consistently, attenuated autophagic flux in HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1-null mouse hearts was associated with increased PLN levels determined by immunoblots and immunofluorescence. Our study identifies a biological mechanism that traffics PLN to the lysosomes for degradation in mouse hearts.


Subject(s)
Autophagy , Calcium-Binding Proteins/metabolism , Metformin/pharmacology , Myocytes, Cardiac/drug effects , Animals , HEK293 Cells , Humans , Lysosomes/metabolism , Mice , Mice, Knockout , Myocytes, Cardiac/metabolism , Proteolysis , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/physiology , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/physiology , Ubiquitination
4.
Biochem Biophys Res Commun ; 460(1): 34-9, 2015 Apr 24.
Article in English | MEDLINE | ID: mdl-25619131

ABSTRACT

Muscle spindles from the hind limb muscles of adult Ryr1(I4895T/wt) (IT/+) mice exhibit severe structural abnormalities. Up to 85% of the spindles are separated from skeletal muscle fascicles by a thick layer of connective tissue. Many intrafusal fibers exhibit degeneration, with Z-line streaming, compaction and collapse of myofibrillar bundles, mitochondrial clumping, nuclear shrinkage and pyknosis. The lesions resemble cores observed in the extrafusal myofibers of this animal model and of core myopathy patients. Spindle abnormalities precede those in extrafusal fibers, indicating that they are a primary pathological feature in this murine Ryr1-related core myopathy. Muscle spindle involvement, if confirmed for human core myopathy patients, would provide an explanation for an array of devastating clinical features characteristic of these diseases and provide novel insights into the pathology of RYR1-related myopathies.


Subject(s)
Muscle Fibers, Skeletal/pathology , Muscle Spindles/metabolism , Muscle Spindles/pathology , Muscular Diseases/genetics , Muscular Diseases/pathology , Ryanodine Receptor Calcium Release Channel/genetics , Animals , Disease Models, Animal , Humans , Mice , Muscle Fibers, Skeletal/metabolism
5.
Emerg Infect Dis ; 20(12): 1969-79, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25418327

ABSTRACT

Variably protease-sensitive prionopathy (VPSPr) can occur in persons of all codon 129 genotypes in the human prion protein gene (PRNP) and is characterized by a unique biochemical profile when compared with other human prion diseases. We investigated transmission properties of VPSPr by inoculating transgenic mice expressing human PRNP with brain tissue from 2 persons with the valine-homozygous (VV) and 1 with the heterozygous methionine/valine codon 129 genotype. No clinical signs or vacuolar pathology were observed in any inoculated mice. Small deposits of prion protein accumulated in the brains of inoculated mice after challenge with brain material from VV VPSPr patients. Some of these deposits resembled microplaques that occur in the brains of VPSPr patients. Comparison of these transmission properties with those of sporadic Creutzfeldt-Jakob disease in the same lines of mice indicated that VPSPr has distinct biological properties. Moreover, we established that VPSPr has limited potential for human-to-human transmission.


Subject(s)
Genetic Variation , Prion Diseases/genetics , Prion Diseases/transmission , Prions/genetics , Animals , Astrocytes/metabolism , Astrocytes/pathology , Brain/metabolism , Brain/pathology , Disease Models, Animal , Genotype , Gliosis/genetics , Gliosis/metabolism , Gliosis/pathology , Humans , Mice , Mice, Transgenic , Prion Diseases/metabolism , Prion Diseases/pathology , Prions/metabolism
6.
Mol Genet Genomic Med ; 2(6): 472-83, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25614869

ABSTRACT

Whole exome sequencing (WES) was used to determine the primary cause of muscle disorder in a family diagnosed with a mild, undetermined myopathy and malignant hyperthermia (MH) susceptibility (MHS). WES revealed the compound heterozygous mutations, p.Ile235Asn and p.Glu982Lys, in ATP2A1, encoding the sarco(endo)plasmic reticulum Ca(2+) ATPase type 1 (SERCA1), a calcium pump, expressed in fast-twitch muscles. Recessive mutations in ATP2A1 are known to cause Brody myopathy, a rare muscle disorder characterized by exercise-induced impairment of muscle relaxation and stiffness. Analyses of affected muscles showed the absence of SERCA1, but SERCA2 upregulation in slow and fast myofibers, suggesting a compensatory mechanism that partially restores the diminished Ca(2+) transport in Brody myopathy. This compensatory adaptation to the lack of SERCA1 Ca(2+) pumping activity within the muscle explains, in part, the mild course of disease in our patient. Diagnosis of MHS in this family was secondary to a loss of SERCA1 due to disease-associated mutations. Although there are obvious differences in clinical expression and molecular mechanisms between MH and Brody myopathy, a feature common to both conditions is elevated myoplasmic Ca(2+) content. Prolonged intracellular Ca(2+) elevation is likely to have led to MHS diagnosis in vitro and postoperative MH-like symptoms in Brody patient.

7.
J Mol Biol ; 425(21): 4034-46, 2013 Nov 01.
Article in English | MEDLINE | ID: mdl-23978697

ABSTRACT

Ryanodine receptors (RyRs) are large tetrameric calcium (Ca(2+)) release channels found on the sarcoplasmic reticulum that respond to dihydropyridine receptor activity through a direct conformational interaction and/or indirect Ca(2+) sensitivity, propagating sarcoplasmic reticulum luminal Ca(2+) release in the process of excitation-contraction coupling. There are three human RyR subtypes, and several debilitating diseases are linked to heritable mutations in RyR1 and RyR2 including malignant hypothermia, central core disease, catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Despite the recent appreciation that many disease-associated mutations within the N-terminal RyRABC domains (i.e., residues 1-559) are located in the putative interfaces mediating tetrameric channel assembly, the precise structural and dynamical consequences of the mutations are not well understood. We used solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography to examine the effect of ARVD2-associated (i.e., R176Q) and CPVT-associated [i.e., P164S, R169Q and delta exon 3 (Δ3)] mutations on the structure and dynamics of RyR2A. Our solution NMR data exposed a mobile α-helix, unique to type 2; further, this α2 helix rescues the ß-strand lost in RyR2A Δ3 but remains dynamic in the hot-spot loop (HS-loop) P164S, R169Q and R176Q mutant proteins. Docking of our X-ray crystal/NMR hybrid structure into the RyR1 cryo-electron microscopy map revealed that this RyR2A α2 helix is in close proximity to dense "columns" projecting toward the channel pore. This is in contrast to the HS-loop mutations that cause structural changes largely localized to the intersubunit interface between adjacent ABC domains. Taken together, our data suggest that ARVD2 and CPVT mutations have at least two distinct structural consequences linked to channel dysfunction: perturbation of the HS-loop (i.e., domain A):domain B intersubunit interface and disruption of the communication between the N-terminal region and the channel domain.


Subject(s)
Cardiomyopathies/genetics , Exons , Mutation , Ryanodine Receptor Calcium Release Channel/chemistry , Ryanodine Receptor Calcium Release Channel/genetics , Amino Acid Sequence , Animals , Mice , Molecular Sequence Data , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Alignment/methods
8.
Anesthesiology ; 118(2): 344-9, 2013 02.
Article in English | MEDLINE | ID: mdl-23460944

ABSTRACT

BACKGROUND: Malignant hyperthermia (MH, MIM# 145600) is a complex pharmacogenetic disorder that is manifested in predisposed individuals as a potentially lethal reaction to volatile anesthetics and depolarizing muscle relaxants. Studies of CASQ1-null mice have shown that CASQ1, encoding calsequestrin 1, the major Ca2+ binding protein in the lumen of the sarcoplasmic reticulum, is a candidate gene for MH in mice. The aim of this study was to establish whether the CASQ1 gene is associated with MH in the North American population. METHODS: The entire coding region of CASQ1 in 75 unrelated patients diagnosed by caffeine-halothane contracture test as MH susceptible (MHS) was analyzed by DNA sequencing. Subsequently, three groups of unrelated individuals (130 MHS, 100 MH negative, and 192 normal controls) were genotyped for a variant that was identified by sequencing. Levels of CASQ1 expression in the muscle from unrelated MHS and MH negative individuals were estimated by Western blotting. RESULTS: Screening of the entire coding sequence of the CASQ1 gene in 75 MHS patients revealed a single variant c.260T > C (p.Met87Thr) in exon 1. This variant is unlikely to be pathogenic, because its allele frequency in the MHS group was not significantly different from that of controls. There was also no difference in calsequestrin 1 protein levels between muscle samples from MHS and controls, including those carrying the p.Met87Thr variant. CONCLUSIONS: This study revealed a low level of protein coding sequence variability within the human CASQ1 gene, indicating that CASQ1 is not a major MHS locus in the North American population.


Subject(s)
Calcium-Binding Proteins/genetics , Malignant Hyperthermia/epidemiology , Malignant Hyperthermia/genetics , Mitochondrial Proteins/genetics , Amino Acid Sequence , Blotting, Western , Calcium Channels/genetics , Calcium Channels, L-Type , Calsequestrin , Computational Biology , DNA/genetics , Exons/genetics , Humans , Introns/genetics , Molecular Sequence Data , Muscle, Skeletal/metabolism , North America/epidemiology , RNA/genetics , Retrospective Studies , Ryanodine Receptor Calcium Release Channel/genetics , White People
9.
Neuromuscul Disord ; 23(2): 120-32, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23183335

ABSTRACT

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.


Subject(s)
Muscle Contraction/physiology , Mutation/genetics , Myopathy, Central Core/genetics , Myopathy, Central Core/physiopathology , Ryanodine Receptor Calcium Release Channel/genetics , Adolescent , Adult , Calcium/metabolism , Cells, Cultured , Child , Child, Preschool , Female , Genetic Testing , HEK293 Cells , Homeostasis/physiology , Humans , Infant , Infant, Newborn , Male , Muscle Fibers, Skeletal/metabolism , Myopathy, Central Core/diagnosis , Pedigree , Polymorphism, Genetic/genetics , Retrospective Studies , Ryanodine Receptor Calcium Release Channel/metabolism
10.
FEBS J ; 279(20): 3952-64, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22913516

ABSTRACT

The ryanodine receptor (RyR) is a large, homotetrameric sarcoplasmic reticulum membrane protein that is essential for Ca(2+) cycling in both skeletal and cardiac muscle. Genetic mutations in RyR1 are associated with severe conditions including malignant hyperthermia (MH) and central core disease. One phosphorylation site (Ser 2843) has been identified in a segment of RyR1 flanked by two RyR motifs, which are found exclusively in all RyR isoforms as closely associated tandem (or paired) motifs, and are named after the protein itself. These motifs also contain six known MH mutations. In this study, we designed, expressed and purified the tandem RyR motifs, and show that this domain contains a putative binding site for the Ca(2+)/calmodulin-dependent protein kinase ß isoform. We present a 2.2 Å resolution crystal structure of the RyR domain revealing a two-fold, symmetric, extended four-helix bundle stabilized by a ß sheet. Using mathematical modelling, we fit our crystal structure within a tetrameric electron microscopy (EM) structure of native RyR1, and propose that this domain is localized in the RyR clamp region, which is absent in its cousin protein inositol 1,4,5-trisphosphate receptor.


Subject(s)
Protein Structure, Tertiary , Ryanodine Receptor Calcium Release Channel/chemistry , Ryanodine Receptor Calcium Release Channel/metabolism , Amino Acid Motifs/genetics , Amino Acid Sequence , Animals , Binding Sites , Blotting, Western , Calcium-Calmodulin-Dependent Protein Kinase Type 2/chemistry , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cryoelectron Microscopy , Crystallography, X-Ray , HEK293 Cells , Humans , Models, Molecular , Molecular Sequence Data , Mutation , Phosphorylation , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Secondary , Rabbits , Ryanodine Receptor Calcium Release Channel/genetics , Sequence Homology, Amino Acid
11.
Proc Natl Acad Sci U S A ; 109(2): 610-5, 2012 Jan 10.
Article in English | MEDLINE | ID: mdl-22203976

ABSTRACT

The type 1 ryanodine receptor (RyR1) is expressed widely in the brain, with high levels in the cerebellum, hippocampus, and hypothalamus. We have shown that L-type Ca(2+) channels in terminals of hypothalamic magnocellular neurons are coupled to RyRs, as they are in skeletal muscle, allowing voltage-induced Ca(2+) release (VICaR) from internal Ca(2+) stores without Ca(2+) influx. Here we demonstrate that RyR1 plays a role in VICaR in nerve terminals. Furthermore, in heterozygotes from the Ryr1(I4895T/WT) (IT/+) mouse line, carrying a knock-in mutation corresponding to one that causes a severe form of human central core disease, VICaR is absent, demonstrating that type 1 RyR mediates VICaR and that these mice have a neuronal phenotype. The absence of VICaR was shown in two ways: first, depolarization in the absence of Ca(2+) influx elicited Ca(2+)syntillas (scintilla, spark, in a nerve terminal, a SYNaptic structure) in WT, but not in mutant terminals; second, in the presence of extracellular Ca(2+), IT/+ terminals showed a twofold decrease in global Ca(2+) transients, with no change in plasmalemmal Ca(2+) current. From these studies we draw two conclusions: (i) RyR1 plays a role in VICaR in hypothalamic nerve terminals; and (ii) a neuronal alteration accompanies the myopathy in IT/+ mice, and, possibly in humans carrying the corresponding RyR1 mutation.


Subject(s)
Calcium Channels, L-Type/metabolism , Calcium/metabolism , Hypothalamus/cytology , Myopathy, Central Core/genetics , Neurons/metabolism , Presynaptic Terminals/metabolism , Ryanodine Receptor Calcium Release Channel/genetics , Animals , Fluorescence , Gene Knock-In Techniques , Hypothalamus/metabolism , Mice , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Ryanodine Receptor Calcium Release Channel/metabolism
12.
Soc Sci Med ; 73(7): 1080-7, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21835523

ABSTRACT

Many studies suggest that quality childcare can positively influence children's outcomes in a wide range of domains, including mental health. While an extensive literature on the effects of childcare on individual children exists, how quality childcare programs contribute to trends at the population-level is yet to be established. In this study, we examine community differences in the quality of childcare and the mental health of children attending childcare centres in three communities in British Columbia, Canada. Previous research on Kindergarten children conducted in these communities indicated that two exhibited expected outcomes (based on socioeconomic criteria, these communities were classified as "better off" and "worse off"), and one exhibited better than expected outcomes and was therefore labeled "resilient." We hypothesized that the better than expected child outcomes in the resilient community were due to better quality childcare in this community. To test this hypothesis, we assessed 621 children and their 24 respective childcare centres, and conducted extensive observations of the three study communities. As expected, teachers (but not parents) from the resilient community reported fewer children's mental health problems and childcare quality was found to be higher in the resilient community than in the comparison communities. However, city differences were lost in the hierarchical linear regressions suggesting that the community effects were mediated through childcare quality. To interpret these findings we turned to our observations that indicated that the resilient community was markedly different from the other two in terms of the social capital and developmental assets that it possessed.


Subject(s)
Child Care/standards , Mental Health , Residence Characteristics , Resilience, Psychological , British Columbia , Child Day Care Centers , Child, Preschool , Female , Humans , Interviews as Topic , Male , Social Support , Surveys and Questionnaires
13.
Am J Physiol Cell Physiol ; 301(4): C841-9, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21697544

ABSTRACT

Sarcolipin (SLN) inhibits sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pumps. To evaluate the physiological significance of SLN in skeletal muscle, we compared muscle contractility and SERCA activity between Sln-null and wild-type mice. SLN protein expression in wild-type mice was abundant in soleus and red gastrocnemius (RG), low in extensor digitorum longus (EDL), and absent from white gastrocnemius (WG). SERCA activity rates were increased in soleus and RG, but not in EDL or WG, from Sln-null muscles, compared with wild type. No differences were seen between wild-type and Sln-null EDL muscles in force-frequency curves or maximum rates of force development (+dF/dt). Maximum relaxation rates (-dF/dt) of EDL were higher in Sln-null than wild type across a range of submaximal stimulation frequencies, but not during a twitch or peak tetanic contraction. For soleus, no differences were seen between wild type and Sln-null in peak tetanic force or +dF/dt; however, force-frequency curves showed that peak force during a twitch and 10-Hz contraction was lower in Sln-null. Changes in the soleus force-frequency curve corresponded with faster rates of force relaxation at nearly all stimulation frequencies in Sln-null compared with wild type. Repeated tetanic stimulation of soleus caused increased (-dF/dt) in wild type, but not in Sln-null. No compensatory responses were detected in analysis of other Ca(2+) regulatory proteins using Western blotting and immunohistochemistry or myosin heavy chain expression using immunofluorescence. These results show that 1) SLN regulates Ca(2+)-ATPase activity thereby regulating contractile kinetics in at least some skeletal muscles, 2) the functional significance of SLN is graded to the endogenous SLN expression level, and 3) SLN inhibitory effects on SERCA function are relieved in response to repeated contractions thus enhancing relaxation rates.


Subject(s)
Biological Transport/physiology , Calcium/metabolism , Muscle Proteins/genetics , Muscle Proteins/metabolism , Muscle Relaxation/physiology , Proteolipids/genetics , Proteolipids/metabolism , Animals , Gene Expression Regulation/physiology , Mice , Mice, Knockout , Muscle Contraction/physiology , Mutation , Myocardium/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism
14.
Can J Anaesth ; 58(6): 504-13, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21455645

ABSTRACT

PURPOSE: Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder that is manifested on exposure of susceptible individuals to halogenated anesthetics or succinylcholine. Since MH is associated primarily with mutations in the ryanodine receptor type 1 (RYR1) gene, the purpose of this study was to determine the distribution and frequency of MH causative RyR1 mutations in the Canadian MH susceptible (MHS) population. METHODS: In this study, we screened a representative cohort of 36 unrelated Canadian MHS individuals for RYR1 mutations by sequencing complete RYR1 transcripts and selected regions of CACNA1S transcripts. We then analyzed the correlation between caffeine-halothane contracture test (CHCT) results and RYR1 genotypes within MH families. RESULTS: Eighty-six percent of patients had at least one RyR1 mutation (31 out of 36), five of which were unrelated individuals who were double-variant carriers. Fifteen of the 27 mutations identified in RYR1 were novel. Eight novel mutations, involving highly conserved amino acid residues, were predicted to be causal. Two of the mutations co-segregated with the MHS phenotype within two large independent families (a total of 79 individuals). Fourteen percent of MHS individuals (five out of 36) carried neither RYR1 nor known CACNA1S mutations. CONCLUSIONS: The distribution and frequency of MH causative RyR1 mutations in the Canadian MHS population are close to those of European MHS populations. Novel mutations described in this study will contribute to the worldwide pool of MH-associated mutations in the RYR1 gene, ultimately increasing the value of MH genetic diagnostic testing.


Subject(s)
Malignant Hyperthermia/genetics , Mutation , Ryanodine Receptor Calcium Release Channel/genetics , Amino Acid Sequence , Genetic Association Studies , Humans , Molecular Sequence Data , Polymorphism, Genetic
15.
J Biol Chem ; 286(19): 17060-8, 2011 May 13.
Article in English | MEDLINE | ID: mdl-21454501

ABSTRACT

The ryanodine receptor type 1 (RyR1) is a homotetrameric Ca(2+) release channel located in the sarcoplasmic reticulum of skeletal muscle where it plays a role in the initiation of skeletal muscle contraction. A soluble, 6×-histidine affinity-tagged cytosolic fragment of RyR1 (amino acids 1-4243) was expressed in HEK-293 cells, and metal affinity chromatography under native conditions was used to purify the peptide together with interacting proteins. When analyzed by gel-free liquid chromatography mass spectrometry (LC-MS), 703 proteins were identified under all conditions. This group of proteins was filtered to identify putative RyR interacting proteins by removing those proteins found in only 1 RyR purification and proteins for which average spectral counts were enriched by less than 4-fold over control values. This resulted in 49 potential RyR1 interacting proteins, and 4 were selected for additional interaction studies: calcium homeostasis endoplasmic reticulum protein (CHERP), endoplasmic reticulum-Golgi intermediate compartment 53-kDa protein (LMAN1), T-complex protein, and phosphorylase kinase. Western blotting showed that only CHERP co-purified with affinity-tagged RyR1 and was eluted with imidazole. Immunofluorescence showed that endogenous CHERP co-localizes with endogenous RyR1 in the sarcoplasmic reticulum of rat soleus muscle. A combination of overexpression of RyR1 in HEK-293 cells with siRNA-mediated suppression of CHERP showed that CHERP affects Ca(2+) release from the ER via RyR1. Thus, we propose that CHERP is an RyR1 interacting protein that may be involved in the regulation of excitation-contraction coupling.


Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/physiology , Endoplasmic Reticulum/metabolism , Membrane Proteins/chemistry , Membrane Proteins/physiology , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/physiology , Ryanodine Receptor Calcium Release Channel/chemistry , Animals , Calcium Channels/chemistry , Female , Humans , Imidazoles/chemistry , Mannose-Binding Lectins/chemistry , Microscopy, Fluorescence/methods , Muscle, Skeletal/metabolism , Phosphorylase Kinase/metabolism , Protein Binding , Protein Interaction Mapping , Rabbits , Rats , Rats, Sprague-Dawley , Sarcoplasmic Reticulum/metabolism
16.
Biochim Biophys Acta ; 1813(5): 948-64, 2011 May.
Article in English | MEDLINE | ID: mdl-21118704

ABSTRACT

This review focuses on muscle disorders and diseases caused by defects in the Ca(2+) release channels of the sarcoplasmic reticulum, the ryanodine receptors, and in the luminal, low affinity, high capacity Ca(2+)-binding proteins, calsequestrins. It provides a time line over the past half century of the highlights of research on malignant hyperthermia (MH), central core disease (CCD) and catecholaminergic polymorphic ventricular tachycardia (CPVT), that resulted in the identification of the ryanodine receptor (RYR), calsequestrin (CASQ) and dihydropyridine receptor (CACNA1S) genes as sites of disease-causing mutations. This is followed by a description of approaches to functional analysis of the effects of disease-causing mutations on protein function, focusing on studies of how mutations affect spontaneous (store overload-induced) Ca(2+)-release from the sarcoplasmic reticulum, the underlying cause of MH and CPVT. Subsequent sections describe results obtained by analysis of knockin mouse lines carrying MH- and CCD-causing mutations, including a Casq1 knockout. The review concludes with the presentation of two mechanistic models. The first shows how dysregulation of Ca(2+) homeostasis can lead to muscle diseases involving both RyR and Casq proteins. The second describes a theory of central core formation wherein non-uniformity of Ca(2+) release, resulting in non-uniformity of muscle contraction, is presented as an intrinsic property of the specific tertiary structure of mutant heterotetrameric ryanodine receptors and as the underlying cause of core formation in skeletal muscle. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.


Subject(s)
Models, Biological , Muscular Diseases/pathology , Sarcoplasmic Reticulum/pathology , Animals , Calcium Signaling/genetics , Humans , Muscular Diseases/genetics , Mutation/genetics , Penetrance , Sarcoplasmic Reticulum/genetics
17.
J Gen Physiol ; 137(1): 43-57, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21149547

ABSTRACT

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca(2+) release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation-contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1(I4898T) mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca(2+) content, and RYR1 Ca(2+) release channel function using adult heterozygous Ryr1(I4895T/+) knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca(2+) content, both electrically evoked and 4-chloro-m-cresol-induced Ca(2+) release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4-6-mo-old IT/+ mice. The sensitivity of the SR Ca(2+) release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca(2+) permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca(2+) release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca(2+) ion permeation.


Subject(s)
Calcium/metabolism , Muscle Weakness/genetics , Muscle Weakness/metabolism , Ryanodine Receptor Calcium Release Channel/genetics , Sarcoplasmic Reticulum/metabolism , Animals , Calcium Channels/metabolism , Calcium Signaling/genetics , Cresols/metabolism , Gene Knock-In Techniques , HEK293 Cells , Humans , Male , Mice , Mice, Inbred Strains , Muscle Contraction/genetics , Muscle Contraction/physiology , Muscle Strength/genetics , Muscle Strength/physiology , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiopathology , Mutation , Ryanodine Receptor Calcium Release Channel/deficiency , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum/genetics
18.
Proc Natl Acad Sci U S A ; 107(43): 18481-6, 2010 Oct 26.
Article in English | MEDLINE | ID: mdl-20937869

ABSTRACT

Cardiac-specific overexpression of a constitutively active form of calcineurin A (CNA) leads directly to cardiac hypertrophy in the CNA mouse model. Because cardiac hypertrophy is a prominent characteristic of many cardiomyopathies, we deduced that delineating the proteomic profile of ventricular tissue from this model might identify novel, widely applicable therapeutic targets. Proteomic analysis was carried out by subjecting fractionated cardiac samples from CNA mice and their WT littermates to gel-free liquid chromatography linked to shotgun tandem mass spectrometry. We identified 1,918 proteins with high confidence, of which 290 were differentially expressed. Microarray analysis of the same tissue provided us with alterations in the ventricular transcriptome. Because bioinformatic analyses of both the proteome and transcriptome demonstrated the up-regulation of endoplasmic reticulum stress, we validated its occurrence in adult CNA hearts through a series of immunoblots and RT-PCR analyses. Endoplasmic reticulum stress often leads to increased apoptosis, but apoptosis was minimal in CNA hearts, suggesting that activated calcineurin might protect against apoptosis. Indeed, the viability of cultured neonatal mouse cardiomyocytes (NCMs) from CNA mice was higher than WT after serum starvation, an apoptotic trigger. Proteomic data identified α-crystallin B (Cryab) as a potential mediator of this protective effect and we showed that silencing of Cryab via lentivector-mediated transduction of shRNAs in NCMs led to a significant reduction in NCM viability and loss of protection against apoptosis. The identification of Cryab as a downstream effector of calcineurin-induced protection against apoptosis will permit elucidation of its role in cardiac apoptosis and its potential as a therapeutic target.


Subject(s)
Calcineurin/metabolism , Endoplasmic Reticulum/metabolism , Myocardium/metabolism , alpha-Crystallin B Chain/metabolism , Animals , Apoptosis/physiology , Calcineurin/genetics , Cardiomegaly/genetics , Cardiomegaly/metabolism , Cardiomegaly/pathology , Gene Expression , Gene Expression Profiling , Gene Knockdown Techniques , Mice , Mice, Transgenic , Myocardium/cytology , Protein Array Analysis , Proteomics , RNA, Small Interfering/genetics , Stress, Physiological , alpha-Crystallin B Chain/antagonists & inhibitors , alpha-Crystallin B Chain/genetics
19.
Proc Natl Acad Sci U S A ; 106(51): 21813-8, 2009 Dec 22.
Article in English | MEDLINE | ID: mdl-19959667

ABSTRACT

Ryr1(I4895T/wt) (IT/+) mice express a knockin mutation corresponding to the human I4898T EC-uncoupling mutation in the type 1 ryanodine receptor/Ca(2+) release channel (RyR1), which causes a severe form of central core disease (CCD). IT/+ mice exhibit a slowly progressive congenital myopathy, with neonatal respiratory stress, skeletal muscle weakness, impaired mobility, dorsal kyphosis, and hind limb paralysis. Lesions observed in myofibers from diseased mice undergo age-dependent transformation from minicores to cores and nemaline rods. Early ultrastructural abnormalities include sarcomeric misalignment, Z-line streaming, focal loss of cross-striations, and myofibrillar splitting and intermingling that may arise from defective myofibrillogenesis. However, manifestation of the disease phenotype is highly variable on a Sv129 genomic background. Quantitative RT-PCR shows an equimolar ratio of WT and mutant Ryr1 transcripts within IT/+ myofibers and total RyR1 protein expression levels are normal. We propose a unifying theory in which the cause of core formation lies in functional heterogeneity among RyR1 tetramers. Random combinations of normal and either leaky or EC-uncoupled RyR subunits would lead to spatial differences in Ca(2+) transients; the resulting heterogeneity of contraction among myofibrils would lead to focal, irreversible tearing and shearing, which would, over time, enlarge to form minicores, cores, and nemaline rods. The IT/+ mouse line is proposed to be a valid model of RyR1-related congenital myopathy, offering high potential for elucidation of the pathogenesis of skeletal muscle disorders arising from impaired EC coupling.


Subject(s)
Calcium/physiology , Myopathies, Nemaline/physiopathology , Ryanodine Receptor Calcium Release Channel/physiology , Animals , Mice , Microscopy, Electron, Transmission , Muscle, Skeletal/pathology , Muscle, Skeletal/ultrastructure , Phenotype , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Ryanodine Receptor Calcium Release Channel/genetics
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