Preclinical model systems of ryanodine receptor 1-related myopathies and malignant hyperthermia: a comprehensive scoping review of works published 1990–2019

Background Pathogenic variations in the gene encoding the skeletal muscle ryanodine receptor (RyR1) are associated with malignant hyperthermia (MH) susceptibility, a life-threatening hypermetabolic condition and RYR1-related myopathies (RYR1-RM), a spectrum of rare neuromuscular disorders. In RYR1-RM, intracellular calcium dysregulation, post-translational modifications, and decreased protein expression lead to a heterogenous clinical presentation including proximal muscle weakness, contractures, scoliosis, respiratory insufficiency, and ophthalmoplegia. Preclinical model systems of RYR1-RM and MH have been developed to better understand underlying pathomechanisms and test potential therapeutics. Methods We conducted a comprehensive scoping review of scientific literature pertaining to RYR1-RM and MH preclinical model systems in accordance with the PRISMA Scoping Reviews Checklist and the framework proposed by Arksey and O’Malley. Two major electronic databases (PubMed and EMBASE) were searched without language restriction for articles and abstracts published between January 1, 1990 and July 3, 2019. Results Our search yielded 5049 publications from which 262 were included in this review. A majority of variants tested in RYR1 preclinical models were localized to established MH/central core disease (MH/CCD) hot spots. A total of 250 unique RYR1 variations were reported in human/rodent/porcine models with 95% being missense substitutions. The most frequently reported RYR1 variant was R614C/R615C (human/porcine total n = 39), followed by Y523S/Y524S (rabbit/mouse total n = 30), I4898T/I4897T/I4895T (human/rabbit/mouse total n = 20), and R163C/R165C (human/mouse total n = 18). The dyspedic mouse was utilized by 47% of publications in the rodent category and its RyR1-null (1B5) myotubes were transfected in 23% of publications in the cellular model category. In studies of transfected HEK-293 cells, 57% of RYR1 variations affected the RyR1 channel and activation core domain. A total of 15 RYR1 mutant mouse strains were identified of which ten were heterozygous, three were compound heterozygous, and a further two were knockout. Porcine, avian, zebrafish, C. elegans, canine, equine, and drosophila model systems were also reported. Conclusions Over the past 30 years, there were 262 publications on MH and RYR1-RM preclinical model systems featuring more than 200 unique RYR1 variations tested in a broad range of species. Findings from these studies have set the foundation for therapeutic development for MH and RYR1-RM.


Introduction
Ryanodine receptor 1-related myopathies (RYR1-RM) are a diverse spectrum of rare monogenic neuromuscular disorders that manifest from variations in the RYR1 gene [1,2]. In total, > 700 RYR1 variations have been identified; many of which are private to an individual case or family [3]. RYR1 exhibits little functional variation (per a recently developed bioinformatic residual variance intolerance [RVIS] scoring system: − 8.29 [0.01%]) [4] and encodes a 2.2 megadalton homotetrameric calcium ion channel (RyR1) that is localized to the sarcoplasmic reticulum (SR) membrane in skeletal muscle [5]. The physical connection between the RyR1 cytosolic shell and dihydropyridine receptor (DHPR) enables a coordinated release of SR calcium to the muscle cell cytosol, a process that facilitates excitation-contraction coupling in response to depolarization of the transverse-tubule membrane [6,7]. ER/SR calcium concentration is an estimated 1000-10,000 times greater than cytosolic calcium concentration, and maintenance of this steep gradient is imperative to the health of the cell [8,9]. Preclinical studies have identified intracellular calcium dysregulation as the central pathomechanism resulting from RYR1 variations characterized by SR calcium leak or excitation-contraction uncoupling [10]. In addition, the presence of truncation variations often reported in compound heterozygous cases can lead to decreased RyR1 expression [11,12]. Owing to > 100 cysteine residues per subunit, RyR1 are susceptible to post-translational modifications, which in the case of mutant channels, further exacerbate intracellular calcium dysregulation though a previously reported feed-forward mechanism [13,14]. For example, an elevated level of S-nitrosylated cysteines greatly increases channel activity, thus perpetuating calcium release. RYR1-RM pathomechanisms have been reviewed in detail elsewhere [10].
RYR1-RM can be inherited in a dominant or recessive manner and are slowly progressive with clinical manifestations including proximal muscle and facial weakness, joint contractures, scoliosis, ophthalmoplegia, and respiratory muscle weakness [15]. Although presentation often occurs at birth or in early childhood, adult-onset cases have also been reported [16,17]. Affected individuals are considered at risk of malignant hyperthermia (MH) susceptibility. Genetic predisposition to MH can result in a potentially fatal hypermetabolic response and skeletal muscle rigidity upon exposure to triggers such as volatile anesthetics, exercise in the heat, and influenza [18,19]. In addition to myopathy, other clinical phenotypes attributed to RYR1 variations include rhabdomyolysis-myalgia syndrome and intermittent periodic paralysis [20,21]. Historically, RYR1-RM were sub-categorized based on skeletal muscle histopathology. This yielded subtypes such as central core disease, multiminicore disease, centronuclear myopathy, and congenital fiber-type disproportion [22]. Despite being the most frequently reported non-dystrophic neuromuscular disorder [23], there is currently no approved treatment for RYR1-RM.
A decade after the first report of central core disease in humans [24], Hall and colleagues observed a fatal hypermetabolic response to suxamethonium in pigs [25]. This was the first MH animal model system whose phenotype, also referred to as porcine stress syndrome, was later attributed to the R615C variation in RYR1 [26,27]. Since this landmark discovery, technological and scientific advances have led to the development of preclinical model systems that can be grouped into cell culture and animal categories, each with their own advantages and limitations [28][29][30][31][32].

Objective
The objective of this scoping review was to comprehensively review the scientific literature for MH and RYR1-RM preclinical model systems, thus generating a resource to guide future research.

Methods
The PRISMA extension for Scoping Reviews (PRISMA-ScR) Checklist and the framework proposed by Arksey and O'Malley [33] were used to guide this scoping review. The overarching research question was: what preclinical model systems have been reported for MH and RYR1-RM?

Identifying relevant studies
Two major electronic databases (PubMed and EMBASE) were searched without language restriction for articles and abstracts published between January 1, 1990 and July 3, 2019. The search strategy comprised the following a priori search terms present in the title or abstract

Study selection
Following removal of duplicates, titles and abstracts of all publications were reviewed independently by two of the authors and marked for inclusion if they discussed a MH or RYR1-RM preclinical model system. Publications were marked for exclusion if they were (1) not gene or isoform of interest (e.g. CACNA1S-related MH), (2) clinical report, (3) structural biology, (4) wild-type models and methods, (5) cardiac or smooth muscle, (6) review articles, or (7) categorized as miscellaneous. All publications were discussed with a third author who adjudicated when there was discordance between the first two authors over whether publication should be included or excluded.

Charting data and reporting the results
The following data were extracted from full text publications selected for inclusion in the review: first author, year of publication, title of the publication, variation(s) of the preclinical model system(s), and conclusions of the publication on the disease model system(s). Data were tabulated according to type of preclinical model system. Categories included transfected human embryonic kidney cell (HEK)-293 cells, transfected RYR1-null (dyspedic) myotubes, immortalized B-lymphocytes, primary cell culture, porcine model systems, and rodent model systems. Data on all other preclinical model systems, including zebrafish, avian, C. elegans, and drosophila, were combined and tabulated separately. Two authors reviewed data extracted for each article. To

Study characteristics
The search strategy utilized in this study yielded 5049 research publications between January 1, 1990 and July 3, 2019. Nine additional publications were retrieved through other information sources. Following removal of 2814 duplicates, 2284 abstracts were screened for inclusion. A total of 1956 publications were excluded at this point, leaving 328 for full text review. During full text review, 66 additional publications were excluded leaving 262 publications for inclusion in this review. An overview of this process is provided in Fig. 1.
The majority of publications that met inclusion criteria for this review focused on RYR1 cellular and rodent model systems (43 and 39%, and respectively), Fig. 2a. Wild-type/methods publications formed the largest group of those excluded (24%), followed by those focused on cardiac/smooth muscle (19%), not isoform/ gene of interest (16%), and clinical reports (13%), Fig. 2b.
The highest frequencies of variations reported in RYR1 preclinical model systems were localized to established MH/central core disease (MH/CCD) hot spots 1, 2, and 3 located between exons 1-17, 39-46, and 90-103, respectively, Fig. 3. At least one RYR1 preclinical model system was reported for every RyR1 structural region.
A total of 250 unique RYR1 variations were reported in human/mouse/porcine model systems with 95% being missense substitutions. The most frequently reported RYR1 variations reported across species were R614C/R615C (human/porcine total n = 39), Y523S/Y524S (rabbit/mouse total n = 30), I4898T/I4897T/I4895T (human/rabbit/mouse total n = 20), and R163C/R165C (human/mouse total n = 18). The dyspedic mouse was the most frequently reported mouse model system comprising 47% of publications in this category. The predominant type of RYR1 preclinical model system used has varied over time. From 1990 to 1994, the R615C porcine model system was most frequently reported. Cellular model systems were then most frequently reported until 2010, after which this transitioned to rodent model systems including RyR1-null (dyspedic) and Y524S, R163C, and I4895T mutant mice, Fig. 4.

Expression of recombinant RYR1 in dyspedic myotubes
Transfection of dyspedic myotubes with mutant RYR1 cDNA was reported in 25 publications,

Expression of endogenous mutant RYR1
Additionally, 16 publications reported immortalization of patient primary B-lymphocytes for downstream functional characterization, Table 3. These 16 publications included 32 unique missense substitutions, one deletion, and two deletion-insertions. A total of 50 unique RYR1 variants, all missense substitutions, were tested in 19 publications utilizing primary cell culture model systems, Table 4.

Animal model systems Mice
A total of 15 RYR1 rodent model systems were identified of which ten were heterozygous, three were compound heterozygous, and a further two were knockout, Table 5.
Variations discussed in this section are numbered according to the mouse sequence. Core formation was reported in three of the rodent model systems, excluding knockout (Y524S [158], Q1970fsX16 + A4329D [179],  Results demonstrate selective effects of dantrolene on skeletal muscle ryanodine receptors that are consistent with the actions of dantrolene in vivo and suggest a mechanism of action in which dantrolene may act directly at the skeletal muscle ryanodine receptor complex to limit its activation by calmodulin and C 2+ I4895T [168]). Overall, six of the ten heterozygous rodent model systems had missense substitutions affecting the RyR1 cytosolic shell domain. Two compound heterozygous model systems had a single missense substitution engineered into one allele with a frameshift leading to a deletion or truncation on the opposite allele [178,179]. In these model systems, one variation affected the RyR1 cytosolic shell and the other affected the RyR1 channel and activation core. An additional compound heterozygous model system had a single missense substitution affecting the RyR1 channel and activation core with a second missense substitution and deletion, on the opposite allele, affecting the RyR1 cytosolic shell [181]. Various forms of aberrant intracellular calcium dynamics were reported in all rodent systems (except knockout). This included evidence of increased resting cytosolic calcium and RyR1-open probability under resting conditions (SR calcium leak) [173] as well as decreased calcium permeation (excitation-contraction uncoupling) [30]. The two most frequently reported RYR1 rodent model systems were the dyspedic mouse, accounting for 47% of rodent publications [54,109,, and the Y524S knock-in mouse, which accounted for 22% of rodent publications, Table 5. Studies utilizing dyspedic mice/1B5 myotubes not transfected with mutant RYR1 cDNA, were primarily focused on elucidating the following: (a) relative importance and functional role of wildtype RyR isoforms [213,227,234], (b) fundamental   An abnormal ketamine response in mutants defective in the ryanodine receptor gene ryr-1 (unc-68) of Caenorhabditis elegans S1444N substitution is at a putative protein kinase C phosphorylation site in ryr-1 unc-68(e540) contains a splice acceptor mutation that creates a premature stop codon in the ryr-1 gene physiology of excitation-contraction coupling components [205,225,232], (c) roles of specific RyR1 structural regions on channel function [216,222,235]. The Y524S knock-in mouse has been utilized extensively to investigate the mechanisms behind several phenotypes on the RYR1-RM disease spectrum including MH susceptibility [162], statin-induced myopathy [152], and central core disease [158]. Y524S mice have also been used to test potential therapeutics for RYR1-RM including the antioxidant N-acetylcysteine [145,160] and the activator of the AMP-activated protein kinase 5aminoimidazole-4-carboxamide ribonucleoside (AICAR) [153].

Other animal model systems
The pathomechanism, diagnosis, and acute treatment of malignant hyperthermia was investigated in 24 publications that used the R615C porcine model system [27,, Table 6. A number of other preclinical model systems have been described including avian, zebrafish, C. elegans, canine, equine, and drosophila, Table 7. Six publications reported on a single recessive zebrafish model system of RYR1-RM termed the relatively relaxed (ryr mi340 ) mutant [28,[263][264][265][266][267] which was utilized for high-throughput drug screening [263] and testing of Nacetylcysteine as a potential therapeutic to address elevated oxidative stress [264]. A further six publications reported using Caenorhabditis elegans (C. elegans) with variants in unc68, the RYR1 ortholog [32,[280][281][282][283][284]. With 40% sequence homology to humans, C. elegans have been used to investigate RyR1 functional sites [281] and test the potential impact of RYR1 mutations on central nervous system function [32]. A single heterozygous canine model system of malignant hyperthermia was reported. The canine model system carried a single missense substitution, V547A, affecting the RyR1 cytosolic shell domain and was characterized by responsiveness to an in vivo halothane-succinylcholine challenge and having a positive in vitro contracture test [278]. Four publications described equine model systems of malignant hyperthermia and exertional rhabdomyolysis that carried variations in the RYR1 gene [274][275][276][277]. Two RYR1 variants were reported: (a) R2454G associated with fulfilment malignant hyperthermia and a high affinity for ryanodine binding [277] and (b) C7360G associated with both anesthetic-induced malignant hyperthermia and exertional/non-exertional rhabdomyolysis [276]. Three publications reported on drosophila with variations in the equivalent RYR1 gene (dRyr) [271][272][273]. A total of nine RYR1 variations were presented comprising eight missense substitutions and one insertion, Table 7. Missense substitutions in drosophila dRyr conferred halothane sensitivity [272], and drosophila with CRISPR/ Cas9 gene-edited dRyr have been used to investigate insecticide resistance [271]. Three publications utilized transfected wild-type rodent cells to generate RYR1

Discussion
This comprehensive scoping review of MH and RYR1-RM preclinical model systems identified 262 relevant published records and serves as a compendium to guide future research. During the period spanning January 1, 1990 to July 3, 2019 a diverse range of preclinical model systems were utilized to investigate the etiology, pathomechanisms, and potential treatments for MH and RYR1-RM. There has been sustained research output since 2010 with the predominant model system used varying over time between porcine, cellular, and rodent. A single missense substitution, R615C, was the sole porcine variant reported. As the first RYR1 preclinical model system, studies of R615C pigs led to fundamental discoveries including identification of 4-CmC as a potent RyR1 agonist and identification of RYR1 as a genetic locus for malignant hyperthermia [27,255]. The R615C porcine model system was also utilized to better understand the mechanism of dantrolene which remains the only approved treatment for MH crises [252].
The number of RYR1 variations reported in the literature (> 700) has been prohibitive in terms of developing in vivo model systems reflecting each variant. This review has outlined the extent to which cellular model systems, in particular transfected HEK-293 cells and dyspedic myotubes, have been versatile systems through which to investigate the pathogenicity of RYR1 variations and their impact on intracellular calcium homeostasis. However activity of the RyR1 protein complex is tightly regulated by coupling to the dihydropyridine receptor and by modulators of channel function such as 12-kDa FK506-binding protein (FKBP12) and calmodulin [10]. Absence of these components in the HEK-293 system may therefore affect the reliability of functional data for clinical translation. Epstein-Barr virus-driven immortalization of patient-derived lymphoblasts has also proven a valuable non-recombinant methodology when clinical biospecimens are available, although they also do not contain all elements of the skeletal muscle triad. Both HEK-293 cells and dyspedic myotubes have a standardized and well-characterized background and are therefore less likely, than immortalized patient cells, to be influenced by variations in other genes that may impact RyR1 function. Although patient tissue is not always readily available, it is important to recognize that functional studies of patient-derived primary myotubes can provide valuable supporting evidence of RYR1 variant pathogenicity. Indeed, such studies have been incorporated within the MH variant scoring matrix developed by the European Malignant Hyperthermia Group (EMHG) [289]. Despite the above limitations, recent advances in the engineering of skeletal muscle threedimensional systems using patient-derived induced pluripotent stem cells holds the prospect of providing a physiologically relevant cellular system through which to evaluate and screen potential treatments for skeletal muscle disorders, including RYR1-RM [290][291][292]. Our observation that the most frequently reported variants were localized to MH/CCD hotspot regions is consistent with the initial clinical focus to identify patients with variants in these distinct regions and perform functional characterization [293]. Common functional analyses identified in this review include RyR1 agonist sensitivity (caffeine, 4-CmC), 3[H]-ryanodine binding, halothane and/or isoflurane sensitivity, and intracellular calcium measurements via calcium-sensitive fluorescent dyes such as fluo-4.
The dyspedic mouse was utilized by 47% of publications in the rodent category and its RYR1-null myotubes were transfected in 23% of publications in the cellular model category, a testament to importance of the dyspedic mouse for both understanding the fundamental physiology of the ryanodine receptor and as a stable model system to characterize mutant RyR1 channels. Heterozygous knock-in rodent models have formed the basis of in vivo RYR1-RM preclinical testing. Y524S, I4895T, and R163C were the most extensively studied knock-in rodent models over the last 30 years. These mice have provided valuable insights into the effects of single missense substitutions on RyR1 dysfunction including channel leak and excitation-contraction uncoupling. Furthermore, these mice have enabled the identification downstream pathologic sequalae in vivo such as elevated oxidative/nitrosative/ER stress and an unfolded protein response. However the abovementioned knock-in mice do not necessarily mirror the phenotypes observed in autosomal dominant patients with equivalent RYR1 variants (reviewed in detail elsewhere [292]). Two recently published compound heterozygous RYR1-RM rodent models recapitulate clinical manifestations observed in recessive RYR1-RM patients, including decreased RyR1 protein expression, reduced muscle mass, and progressive muscle weakness [178,179]. An additional compound heterozygous mouse (T4706M/ S1669C + L1716del) included in this review is currently undergoing full characterization [181].
In contrast to rodent model systems, zebrafish are more cost-efficient, have transparent embryos that facilitate visualization of dynamic events, have a shorter lifecycle, have larger hatch sizes, and are easier to maintain [294,295]. Zebrafish are readily manipulated by chemical approaches because embryos can readily absorb compounds that they are exposed to in solution, therefore allowing for high-throughput chemical screening [296,297]. A recessive zebrafish model system of RYR1-RM, termed the relatively relaxed (ryr mi340 ) mutant, exhibits weak muscle contractions resulting in slow swimming, dramatically decreased Ca 2+ transients at the t-tubules of fast muscles due to defective E-C coupling, and small amorphous cores detectable by electron microscopy. Despite the abovementioned advantages over rodent model systems, a consideration is that relatively relaxed (ryr1b) zebrafish are homozygous with a truncated RyR1 channel (residual expression = 1-10% of normal RyR1). As such, its genetic defect and pathomechanism do not align with a majority of RYR1-RM clinical cases.
Consistent with the findings of this review, over 90% of animals used in research are mice or rats [298]. However, other animal model systems have also been developed and used to study the skeletal muscle ryanodine receptor and the consequences of genetic variations. C. elegans and drosophila have been primarily used for genetic and developmental biology studies, whereas the porcine, equine, and canine model systems have focused on understanding and characterizing the etiology of MH in these species. As an alternative to higher order RYR1-RM animal model systems, the use of simpler organisms (C. elegans, yeast, drosophila) and vertebrates (in particular zebrafish) with sufficient genome sequence homology to humans could be revitalized using more precise genome editing techniques such as prime editing [299]. However, due to evolutionary distance between DNA sequences, results from non-mammalian model systems should undergo further careful validation in mammals such as mice and pigs prior to translation to clinical studies. It is possible that records published in supplementary material may not have been captured by the search strategy used for this review and may be considered a limitation.
Advances in functional genomics coupled with the increase in demand for mice as a primary experimental system are expected to continue driving the need for additional transgenic, gene-edited and combinatorial breeding of different RYR1-RM model systems in the near future. Development of conditional targeted animal models (cre/lox, tet, and other similar approaches) can reduce generation and retention of extraneous animals and also allow for the conduct of developmental studies in late-onset myopathy subtypes in this heterogeneous group of disorders. Determining which murine model most closely represents a majority of either dominant or recessive cases of RYR1-RM remains an open question. Funding of research utilizing recently developed model systems is essential to translating these promising advances into clinical trials and treatment discoveries.

Conclusion
Over the past 30 years, there were 262 publications on MH and RYR1-RM preclinical model systems featuring more than 200 unique RYR1 variations tested in a broad range of species. Findings from these studies have set the foundation for therapeutic development for MH and RYR1-RM.