Elena Zvaritch

Mechanistic models for muscle diseases and disorders originating in the sarcoplasmic reticulum.

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.

An Ryr1I4895T mutation abolishes Ca2+ release channel function and delays development in homozygous offspring of a mutant mouse line

A heterozygous Ile4898 to Thr (I4898T) mutation in the human type 1 ryanodine receptor/Ca2+ release channel (RyR1) leads to a severe form of central core disease. We created a mouse line in which the corresponding Ryr1I4895T 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 Ca2+ release is absent, although retrograde enhancement of DHPR Ca2+ conductance is retained. IT/IT mice, in which RyR1-mediated Ca2+ 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 Ca2+ signaling in mammalian embryogenesis.

Ca2+ dysregulation in Ryr1I4895T/wt mice causes congenital myopathy with progressive formation of minicores, cores, and nemaline rods

Ryr1I4895T/wt (IT/+) mice express a knockin mutation corresponding to the human I4898T EC-uncoupling mutation in the type 1 ryanodine receptor/Ca2+ 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 Ca2+ 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.

Muscle weakness in Ryr1I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca2+ ion permeation and release from the sarcoplasmic reticulum

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca2+ 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 RYR1I4898T 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 Ca2+ content, and RYR1 Ca2+ release channel function using adult heterozygous Ryr1I4895T/+ 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 Ca2+ content, both electrically evoked and 4-chloro-m-cresol–induced Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca2+ ion permeation.

Type 1 ryanodine receptor knock-in mutation causing central core disease of skeletal muscle also displays a neuronal phenotype

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 Ca2+ channels in terminals of hypothalamic magnocellular neurons are coupled to RyRs, as they are in skeletal muscle, allowing voltage-induced Ca2+ release (VICaR) from internal Ca2+ stores without Ca2+ influx. Here we demonstrate that RyR1 plays a role in VICaR in nerve terminals. Furthermore, in heterozygotes from the Ryr1I4895T/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 Ca2+ influx elicited Ca2+syntillas (scintilla, spark, in a nerve terminal, a SYNaptic structure) in WT, but not in mutant terminals; second, in the presence of extracellular Ca2+, IT/+ terminals showed a twofold decrease in global Ca2+ transients, with no change in plasmalemmal Ca2+ 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.

CASQ1 gene is an unlikely candidate for malignant hyperthermia susceptibility in the North American population.

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.

Novel excitation-contraction uncoupled RYR1 mutations in patients with central core disease.

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.

Exome analysis identifies Brody myopathy in a family diagnosed with malignant hyperthermia susceptibility

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 Ca2+ 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 Ca2+ transport in Brody myopathy. This compensatory adaptation to the lack of SERCA1 Ca2+ 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 Ca2+ content. Prolonged intracellular Ca2+ elevation is likely to have led to MHS diagnosis in vitro and postoperative MH‐like symptoms in Brody patient.

Muscle spindles exhibit core lesions and extensive degeneration of intrafusal fibers in the Ryr1 I4895T/wt mouse model of core myopathy *

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.

Response to “Malignant Hyperthermia — human stress triggering” in reference to original article “Mechanistic models for muscle diseases and disorders originating in the sarcoplasmic reticulum” http://dx.doi.org/10.1016/j.bbamcr.2010.11.009

We are grateful to Drs. Gronert, Tobin and Muldoon for pointing out the fact that MH-type reactions have been induced in humans by stress.Wewere aware of thework cited, but, in an effort to stress the point that there is duality to the cause of MH reactions (a point which is fully developed in other parts of the review), we stated “... triggering of an MH reaction, ... requires the presence of both a causal mutation and either a triggering anesthetic, or, in the case of pigs, stressful conditions.” Nevertheless, their commentary illuminates an area to which we had not given appropriate attention or clarity. In our further comments, wewill attempt to clarify the issue raised. Multiple ryanodine receptor isoforms are encoded by multiple genes, RYR1, RYR2 and RYR3; and this is also the case for RyR1-interrelated and -interacting muscle Ca regulatory proteins, including, dihydropyridine receptors, calsequestrin and Ca pumps and exchangers. Structure/function differences among RyR1, RyR2 and RyR3 proteins within any species aremuch greater than the differences amongRyR1s expressed in different species, such as humans and pigs, and this is also the case for the interacting Ca regulatory proteins. The potential for expression of different mixes of a whole variety of Ca regulatory proteins (Michael Berridge [1] has aptly described them as a “Ca signaling toolkit”) is what gives each muscle its special properties. Over time, evolution, separating humans and pigs as unique species, has altered the muscle milieu in which Ca regulatory proteins function, inevitably altering the precise manner in which their signaling functions are regulated. Accordingly, it is always a great relief to scientists tofind that knockout or knockinmouse lines, or lines over-expressing transgenes, display the characteristics of the human disease, since this is often not the case. In comparing RyR1-related diseases in pigs and humans, there are apparent differences. For example the halothane challenge test failed to identify heterozygous MH pigs, making it impossible to eliminate the gene from breeding stock until the precise mutation was identified [2]. By contrast, thevastmajority of heterozygousMHhumansare identified