A unique cause of blepharoptosis associated with RYR1 mutation

111th ENMC International Workshop on Multi-minicore Disease. 2nd International MmD Workshop, 9–11 November 2002, Naarden, The Netherlands

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 β-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.

To report the clinical, pathological and genetic findings in a group of patients with a previously not described phenotype of congenital myopathy due to recessive mutations in the gene encoding the type 1 muscle ryanodine receptor channel (RYR1). Seven unrelated patients shared a predominant axial and proximal weakness of varying severity, with onset during the neonatal period, associated with bilateral ptosis and ophthalmoparesis, and unusual muscle biopsy features at light and electron microscopic levels. Muscle biopsy histochemistry revealed a peculiar morphological pattern characterized by numerous internalized myonuclei in up to 51% of fibres and large areas of myofibrillar disorganization with undefined borders. Ultrastructurally, such areas frequently occupied the whole myofibre cross section and extended to a moderate number of sarcomeres in length. Molecular genetic investigations identified recessive mutations in the ryanodine receptor (RYR1) gene in six compound heterozygous patients and one homozygous patient. Nine mutations are novel and four have already been reported either as pathogenic recessive mutations or as changes affecting a residue associated with dominant malignant hyperthermia susceptibility. Only two mutations were located in the C-terminal transmembrane domain whereas the others were distributed throughout the cytoplasmic region of RyR1. Our data enlarge the spectrum of RYR1 mutations and highlight their clinical and morphological heterogeneity. A congenital myopathy featuring ptosis and external ophthalmoplegia, concomitant with the novel histopathological phenotype showing fibres with large, poorly delimited areas of myofibrillar disorganization and internal nuclei, is highly suggestive of an RYR1-related congenital myopathy.

Malignant hyperthermia (MH) and central core disease (CCD) mutations were introduced into full-length rabbit Ca2+ release channel (RYR1) cDNA, which was then expressed transiently in HEK-293 cells. Resting Ca2+ concentrations were higher in HEK-293 cells expressing homotetrameric CCD mutant RyR1 than in cells expressing homotetrameric MH mutant RyR1. Cells expressing homotetrameric CCD or MH mutant RyR1 exhibited lower maximal peak amplitudes of caffeine-induced Ca2+ release than cells expressing wild type RyR1, suggesting that MH and CCD mutants might be "leaky." In cells expressing homotetrameric wild type or mutant RyR1, the amplitude of 10 mM caffeine-induced Ca2+ release was correlated significantly with the amplitude of carbachol- or thapsigargin-induced Ca2+ release, indicating that maximal drug-induced Ca2+ release depends on the size of the endoplasmic reticulum Ca2+ store. The content of endogenous sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b), measured by enzyme-linked immunosorbent assay, 45Ca2+ uptake, and confocal microscopy, was increased in HEK-293 cells expressing wild type or mutant RyR1, supporting the view that endoplasmic reticulum Ca2+ storage capacity is increased as a compensatory response to an enhanced Ca2+ leak. When heterotetrameric (1:1) combinations of MH/CCD mutant and wild type RyR1 were expressed together with SERCA1 to enhance Ca2+ reuptake, the amplitude of Ca2+ release in response to low concentrations of caffeine and halothane was higher than that observed in cells expressing wild type RyR1 and SERCA1. In Ca2+-free medium, MH/CCD mutants were more sensitive to caffeine than wild type RyR1, indicating that caffeine hypersensitivity observed with a variety of MH/CCD mutant RyR1 proteins is not dependent on extracellular Ca2+ concentration.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Skeletal muscles are a highly structured tissue responsible for movement and metabolic regulation, which can be broadly subdivided into fast and slow twitch muscles with each type expressing common as well as specific sets of proteins. Congenital myopathies are a group of muscle diseases leading to a weak muscle phenotype caused by mutations in a number of genes including RYR1. Patients carrying recessive RYR1 mutations usually present from birth and are generally more severely affected, showing preferential involvement of fast twitch muscles as well as extraocular and facial muscles. In order to gain more insight into the pathophysiology of recessive RYR1-congential myopathies, we performed relative and absolute quantitative proteomic analysis of skeletal muscles from wild-type and transgenic mice carrying p.Q1970fsX16 and p.A4329D RyR1 mutations which were identified in a child with a severe congenital myopathy. Our in-depth proteomic analysis shows that recessive RYR1 mutations not only decrease the content of RyR1 protein in muscle, but change the expression of 1130, 753, and 967 proteins EDL, soleus and extraocular muscles, respectively. Specifically, recessive RYR1 mutations affect the expression level of proteins involved in calcium signaling, extracellular matrix, metabolism and ER protein quality control. This study also reveals the stoichiometry of major proteins involved in excitation contraction coupling and identifies novel potential pharmacological targets to treat RyR1-related congenital myopathies. Editor's evaluation This is a fundamental study reporting a comprehensive proteomic analysis in three skeletal muscle types from wild-type and RYR1-related myopathy mice. It adds quantitative stoichiometry of several excitation-contraction coupling-related proteins. This valuable work compares the disease-related proteomes of the different skeletal muscle groups. https://doi.org/10.7554/eLife.83618.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Skeletal muscles constitute the largest organ, accounting for approximately 60% of the total body mass; they are responsible for movement and posture and additionally, play a fundamental role in regulating metabolism. Furthermore, skeletal muscles are plastic and can respond to physiological stimuli such as increased workload and exercise by undergoing hypertrophy. Broadly speaking muscles can be subdivided into different types depending on their speed of contraction, namely slow twitch muscles are characterized by level of oxidative activity, while fast twitch muscles show high content of enzymes involved in glycolytic activity. Fast- and slow-twitch muscle can be also identified based on the expression of specific myosin heavy chain (MyHC) isoforms (Lieber, 2010; Schiaffino and Reggiani, 2011). Fast twitch muscles, also known as type II fibers, are specialized for rapid movements, are mainly glycolytic contain large glycogen stores and few mitochondria, fatigue rapidly and characteristically express the MyHC isoforms 2 X, 2B, and 2 A. They are also the first muscles to appear during development and are more severely impacted in patients with congenital myopathies; they also undergo more prominent age-related atrophy or sarcopenia (Lieber, 2010; Schiaffino and Reggiani, 2011; Buckingham et al., 2003; Jungbluth et al., 2005; Lawal et al., 2018; Nilwik et al., 2013). Slow twitch muscles (type 1 fibers) are mainly oxidative, contain many mitochondria and are fatigue resistant. Slow twitch muscle, such as soleus, contain muscle fibers expressing the MyHC 1 isoform in addition of muscle fibers expressing MyHC 2 A (Schiaffino and Reggiani, 2011). Type 1 fibers are generally less severely affected in patients with neuromuscular disorders such congenital myopathies. Although such a general classification based on MyHC isoform expression was used for many years by biochemists and physiologists, it has been recently improved thanks to the implementation of 'omic' approaches which have helped refine the phenotypic signature at the single fiber level. A great deal of data has shown that type 2 A fast fibers display a protein profile similar to type I fibers, namely a remarkable level of enzymes involved in oxidative metabolism. Interestingly, type 2 X fibers apparently encode proteins annotated to both oxidative and glycolytic pathways (Eggers et al., 2021; Murgia et al., 2021). There are also a number of functionally specialized muscles including extraocular muscles (EOM), jaw muscles and inner ear muscles that have a different embryonic origin and are made up of atypical fiber types (Schiaffino and Reggiani, 2011). For example, EOMs are the fastest contracting muscles yet they are fatigue resistant, contain many mitochondria and express most MyHC isoforms including type 1, embryonic and neonatal MyHC as well as EO-MyHC (Porter et al., 1995). EOMs are also specifically spared in patients with Duchenne Muscular Dystrophy yet they are affected in patients with some congenital myopathies, including patients with recessive RYR1 myopathies carrying a hypomorphic or null allele (Porter et al., 1995; Fischer et al., 2002; Porter et al., 2003; Amburgey et al., 2013). Congenital Myopathies (CM) are a genetically heterogeneous group of early onset, non-dystrophic diseases preferentially affecting proximal and axial muscles. More than 20 genes have been implicated in CM, the most commonly affected being those encoding proteins involved in calcium homeostasis and excitation contraction coupling (ECC) and thin-thick filaments (Jungbluth et al., 2018). Mutations in RYR1, the gene encoding the ryanodine receptor 1 (RyR1) calcium channel of the sarcoplasmic reticulum, are found in approximately 30% of all CM patients, making it the most commonly mutated gene in human CM (Amburgey et al., 2013; Jungbluth et al., 2018). Within the group of patients carrying RYR1 mutations, those with the recessive form of the disease are more severely affected, present from birth, have axial and proximal muscle weakness as well as involvement of facial and EOM (Lawal et al., 2018; Amburgey et al., 2013; Jungbluth et al., 2018). A common finding is also the reduced content of RyR1 protein in muscle biopsies (Zhou et al., 2007; Monnier et al., 2008) which could be one of the causes leading to the weak muscle phenotype. To date, the pathomechanism of disease of recessive RYR1 mutations is not completely understood and for this reason we created a mouse model knocked in for compound heterozygous mutations identified in a severely affected child with RYR1-related congenital myopathy. The double knock in mouse, henceforth referred to as double heterozygous or dHT mouse, carries the RyR1 p.Q1970fsX16 mutation in one allele leading to the absence of a transcript due to nonsense-mediated decay of the allele carrying the frameshift mutation, and the mis-sense RyR1 p.A4329D mutation in the other allele (Elbaz et al., 2019). The muscle phenotype of the dHT mouse model closely resembles that of human patients carrying a hypomorphic allele plus a mis-sense RYR1 mutation, including reduced RyR1 protein content in skeletal muscles, the presence of cores and myofibrillar dis-array, mis-alignment of RyR1 and the dihydropyridine receptor and impaired EOM function (Elbaz et al., 2019; Eckhardt et al., 2020). Interestingly, beside a reduction in RyR1, the latter muscles also exhibited a significant decrease in mitochondrial number as well as changes in the expression and content of other proteins, including the almost complete absence of the EOM-specific MyHC isoform (Eckhardt et al., 2020). Such results imply that broad changes in protein expression caused by the mutation and/or reduced content of RyR1 channels, impact other signaling pathways, leading to altered muscle function. A corollary to this is that since not all muscles are equally affected (for example fast twitch muscles and EOMs are more affected than slow twitch muscles) there may be differences in how the RYR1 mutations affect the different muscle types. In order to establish how and if Ryr1 mutations differentially impinge on the expression and function of proteins specific for different muscle types, we performed qualitative and quantitative proteomic analysis of EDL, soleus and EOMs from wild-type and dHT mice. Results Figure 1 shows a diagram of our experimental workflow: three muscle types were isolated from 12 weeks old wild-type (WT)(n=5) and dHT (n=5) mice, samples were processed for Mass Spectrometry and the results obtained were analyzed against a protein database containing sequences of the predicted SwissProt entries of Mus musculus (https://www.ebi.ac.uk/, release date 2019/03/27), Myh2 and Myh13 from Trembl, the six calibration mix proteins (Ahrné et al., 2016) and commonly observed contaminants (in total 17,414 sequences) using the SpectroMine software. Results obtained from five muscles per group were averaged, filtered so that only changes in protein content ≥0.20 fold and showing a significance of q<0.05 or greater, were considered. In addition, proteins yielding only 1 peptide were not used for analysis and were filtered out. Figure 1 Download asset Open asset Schematic overview of the workflow. (A) Skeletal muscles from 12 weeks old WT (5 mice) and dHT littermates (5 mice) were isolated and flash frozen. Three different types of muscles were isolated per mouse, namely EDL, soleus and EOMs. On the day of the experiment, muscles were solubilized and processed for LC-MS. (B) For absolute protein quantification, synthetic peptides of RyR1, Cav1.1, Stim1 and Orai1 were used. (C) Protein content in different muscle types and in the different mouse genotypes were analyzed and compared. Comparison of the proteome of EDL, soleus and EOM muscles from WT mice In order to perform their specific physiological functions, different muscle types express different protein isoforms or different amounts of specific proteins. For example, slow twitch muscles contain large amounts of the oxygen binding protein myoglobin and of carbonic anhydrase III the enzyme catalyzing the conversion of CO2 to H2CO3 and HCO3- (Garry et al., 1996; Dowling et al., 2021), while fast twitch muscles express large amounts of the calcium buffer protein parvalbumin (Celio and Heizmann, 1982) additionally, each muscle type contains specific isoforms of contractile and sarcomeric proteins (Schiaffino and Reggiani, 2011). Our first aim was to analyze the proteomes of WT mouse EDL, soleus and EOM muscles to establish their most important qualitative differences (Figure 2). Figure 2 Download asset Open asset Proteomic analysis of EDL, soleus and EOM muscles from WT mice confirms the significant difference in content if proteins involved in the TCA cycle and electron transport chain, fatty acid metabolism and muscle contraction. (A) Hierarchically clustered heatmaps of the relative abundance of proteins in EDL (columns 1–5) and soleus muscles (columns 6–10) from five mice. Blue blocks represent proteins which are increased in content, yellow blocks proteins which are decreased in content in EDL versus soleus muscles. Right pie chart shows overall number of increased (blue) and decreased (yellow) proteins. Areas are relative to their numbers. (B) Volcano plot of a total of 1866 quantified proteins which showed significant increased (blue) and decreased (yellow) content. The horizontal coordinate is the difference multiple (logarithmic transformation at the base of 2), and the vertical coordinate is the significant difference p value (logarithmic transformation at the base of 10). The proteins showing major change in content are abbreviated. Soleus: condition 2; EDL: condition 1(C) Reactome pathway analysis showing major pathways which differ between EDL and soleus muscles. (D) Hierarchically clustered heatmaps of the relative abundance of proteins in EDL (columns 1–5) and EOM muscles (columns 6–10) from five mice. Blue blocks represent proteins which are increased in content, yellow blocks proteins which are decreased in content in EDL versus EOM muscles. Right pie chart shows overall number of increased (blue) and decreased (yellow) proteins. Areas are relative to their numbers. (E) Volcano plot of a total of 1866 quantified proteins which showed significant increased (blue) and decreased (yellow) content. The horizontal coordinate is the difference multiple (logarithmic transformation at the base of 2), and the vertical coordinate is the significant difference p value (logarithmic transformation at the base of 10). The proteins showing major change in content are abbreviated. EOM: condition 2; EDL: condition 1 (F) Reactome pathway analysis showing major pathways which differ between EDL and EOM muscles. (G) Hierarchically clustered heatmaps of the relative abundance of proteins in soleus muscles (columns 1–5) and EOM (columns 6–10) from five mice. Blue blocks represent proteins which are increased in content, yellow blocks proteins which are decreased in content in soleus muscles versus EOM. Right pie chart shows overall number of increased (blue) and decreased (yellow) proteins. Areas are relative to their numbers. (H) Volcano plot of a total of 1866 quantified proteins which showed significant increased (blue) and decreased (yellow) content. The horizontal coordinate is the difference multiple (logarithmic transformation at the base of 2), and the vertical coordinate is the significant difference p value (logarithmic transformation at the base of 10). The proteins showing major change in content are abbreviated. EOM: condition 2; soleus: condition 1 (I) Reactome pathway analysis showing major pathways which differ between soleus and EOM muscles. A q-value of equal or less than 0.05 was used to filter significant changes prior to the pathway analyses. An additional filter was applied to the Heatmaps and Piecharts and only proteins showing a significant change ≥0.2 fold are included. Figure 2A shows that the content of more than 1800 proteins are differentially expressed (q<0.05) in soleus compared to EDL muscles from WT mice, of these 547 are present in lower amounts and 1319 are present in higher amounts in soleus compared to EDL muscles; Figure 2B shows a volcano plot of the log2 fold change of proteins in slow (condition 2) versus fast (condition 1) muscles. Reactome pathway analysis (Figure 2C) revealed that the pathways showing the greatest number of changes in annotated genes are those encoding proteins associated with mitochondrial function (fatty acid metabolism, TCA cycle, electron transport chain, complex 1 biogenesis, and ß-oxidation) which are significantly reduced in EDL muscles compared to soleus muscles. This is not unexpected considering that slow twitch muscles are made up type I and type IIa/IIx fibers which contain more mitochondria and oxidative enzymes than fast twitch type IIb fibers of fast twitch muscles. On the other hand, EDL muscles are significantly enriched in proteins annotated to muscle contraction, carbohydrate metabolism and glycolysis as well as collagen, integrins and extracellular matrix proteins compared to soleus muscles. Figure 2D shows that the content of more than 2500 proteins are differentially expressed (q<0.05) in EOM compared to EDL from WT mice, of these 508 are present in lower amounts and 2074 are present in higher amounts in EOM compared to EDL muscles. The volcano plot in Figure 2E shows the log2 fold change of proteins in EOM (condition 2) versus fast EDL (condition 1) muscles. Interestingly, Reactome pathway analysis (Figure 2F) revealed that EDL muscles contain a larger number of proteins annotated to adaptive immunity and MHC class I antigen presentation compared to EOMs, while the classes of proteins annotated to the citric acid cycle, electron transport chain and fatty acid ß-oxidation are significantly lower in EDL compared to EOMs. This result is in line with the fact that like soleus muscles, or cardiac muscles, EOMs are enriched in mitochondria (Porter et al., 1995; Fischer et al., 2002) to support continuous movements of the eyes. Figure 2H shows that the content of more than 2000 proteins are differentially expressed (q<0.05) in EOM compared to soleus from WT mice, of these 521 are present in lower amounts and 1663 are present in higher amounts in EOM compared to soleus muscles. The volcano plot in Figure 2H shows the log2 fold change of proteins in EOM (condition 2) versus slow soleus (condition 1) muscles. Reactome pathway analysis (Figure 2I) revealed that the most affected category is that containing genes annotated to muscle contraction (that were both up- and downregulated) followed by genes involved in MHC class I antigen presentation, translation and ubiquitin/proteasome degradation that are upregulated in soleus muscles compared to EOM muscles. Reactome pathway analysis as well as Genome Ontology pathway analysis are not sufficiently informative and probably miss important groups of proteins specific to skeletal muscle function; this observation prompted us to select specific proteins whose expression level is known to be different between fast, slow and EOM muscles. Focusing on the relative change in protein content between EDL and soleus muscles of contractile and sarcomeric proteins, our results confirm that the slow muscle Troponin I and C1 isoforms as well as the slow-MyHC 1 (encoded by Myh7) are enriched between 32 and 197-fold in soleus muscles, whereas α-actinin 3 and 4 and myomesin 1 are more abundant in EDL muscles and desmin is enriched in soleus muscles (Supplementary file 1a). Analysis of sarcoplasmic reticulum proteins involved in ECC show that the content of calsequestrin 2 and SERCA2 is 11- and 22-fold higher in soleus muscles, whereas the relative content of proteins of the junctional face membrane of the sarcoplasmic reticulum involved in ECC (Treves et al., 2009) including RyR1, the dihydropyridine (DHPR) complex (including the α1, β1, and α2δ subunits), Stac3, junctophilin-1 and triadin is more than 50% higher in EDL muscles compared to soleus, as is FKBP12 which binds to and stabilizes the RyR1 complex (Brillantes et al., 1994). Fast twitch muscles are also enriched in SERCA1, calsequestrin 1 and junctophilin 2. The abundance of protein annotated to calcium signaling and sarcoplasmic reticulum in EDL is consistent with the larger membrane volume of sarcotubular membrane in fast-twitch muscles compared to slow twitch muscles (Luff and Atwood, 1971). A similar approach was used to compare the relative content of specific proteins changing between EDL and EOMs and soleus and EOMs. Importantly, the results of the mass spectroscopy approach reported here validate a great deal of experimental observations including the fact that EOMs express high levels of Myhc13, a specific extra-ocular muscle MyHC isoform (MyHC-EO), as well as more cardiac muscle specific protein isoforms. For example, within the contractile and sarcomeric protein category, compared to EDL muscles, EOMs are particularly enriched in MyHC-slow (24-fold), MyHC-EO (29-fold) and Troponin C1 (slow and cardiac muscle isoform, 31-fold), whereas they contain very low amounts of α-actinin 3 (0.02-fold), MyHC 2b (0.07-fold) and MyHC 2 X (0.61-fold). Within the ECC coupling category, EOMs are enriched in calsequestrin 2 (21-fold), SERCA2 (3.6-fold) and junctin/junctate/aspartyl-ß-hydroxylase (3.5-fold) whereas their content of RyR1, the α-1 subunit of the dihydropyridine receptor (DHPRα1s), calsequestrin 1, Stac3, junctophilin-1 and triadin is significantly reduced by more than 50% compared to EDL muscles (Supplementary file 1b). Similarly, soleus muscles and EOMs vary in their content of a large number of proteins. Within the contractile and sarcomeric protein category, EOMs are enriched in embryonic MyHC (Myhc3, 49-fold), MyHC-EO (20-fold) and cardiac troponin T (3.10-fold), whereas compared to soleus muscles they contain very low amounts of slow- MyHC (0.0096-fold), myosin light chain 2 (0.01-fold), myozenin-2 (0.017-fold) and α-actinin 2 (0.017-fold). In the ECC category, EOMs are enriched in a number of proteins including SERCA1 (eightfold) and SERCA3 (sevenfold), Stim1 (fourfold), Junctin/junctate/Aspartyl-ß-hydroxylase (threefold), DHPRα1s (1.4-fold) and junctophilin-1 (1.4-fold), whereas they contain very low amounts of SERCA2 and >50% lower amounts of Mitsugumin 53 (Supplementary file 1c). Interestingly compared to EDL and soleus muscles, EOMs are enriched more than twofold in Stim1, junctin/junctate/aspartylß-hydroxlase. Furthermore, compared to soleus muscles and EOMs, EDLs are enriched in parvalbumin and in proteins annotated to calcium-dependent signaling' via the calcium /calmodulin dependent protein kinase IIα and IIγ, whereas soleus and EOM muscles are enriched in S100A1. Altogether, the results of the mass spectrometry analysis not only confirm known differences between muscle types (Schiaffino and Reggiani, 2011; Porter et al., 1995; Fischer et al., 2002; Celio and Heizmann, 1982; Luff and Atwood, 1971) but also reveal new molecular signatures of EDL, soleus and EOMs. In this context, it is worth mentioning that more than 10 heat shock proteins are more abundant in soleus muscles and EOMs compared to EDL muscles, including Hspb6 (16-fold higher in soleus compared to EDL) and Hspa12a (7-fold higher in EOM vs soleus). Hspb6 has been implicated in protection against atrophy, ischemia, hypertensive stress, and metabolic dysfunction (Dreiza et al., 2010). Importantly, a great deal of data has shown that muscles from patients with several neuromuscular disorders including those caused by RYR1 mutations show fiber type 1 predominance (Jungbluth et al., 2005; Lawal et al., 2018) and heat shock proteins have been suggested to have a against muscle caused by calcium and of mitochondrial chain et al., as well as against in et al., Interestingly, the content of Mitsugumin 53 (encoded by a protein involved in muscle membrane et al., 2009) is higher in slow twitch muscles compared to fast twitch muscles. on the these observations we the that increased expression of Mitsugumin with a of heat shock proteins (Dreiza et al., 2010; et al., 2003; et al., et al., be in muscle fiber type 1 associated with the presence of recessive RYR1 mutations or with other type of To this we the proteome of fast and slow twitch muscles in a mouse model of congenital muscle disorders carrying the p.Q1970fsX16 mutation in one allele and the mis-sense p.A4329D mutation in the other allele (Elbaz et al., 2019). Comparison of muscles isolated from WT and RyR1 dHT mice In the the proteome of three different muscles from dHT mice vs those of WT mice were compared. Figure and shows that in EDL muscles a total of proteins are significantly (q<0.05) in dHT in and proteins are up- or only in the EDLs of dHT mice compared to WT mice, respectively. Reactome pathway (Figure analysis revealed that proteins involved in homeostasis of the extracellular matrix, including and chain and are in EDLs from WT compared to dHT mice. also compared the proteome of soleus muscles from WT and dHT mice. Figure and show that the overall number of proteins showing significant changes in their relative content between dHT and WT mice, is than that observed in EDL muscles. In we found that and proteins are up- or only in the soleus muscles of dHT mice compared to those from WT mice, respectively. to EDL muscles, Reactome pathway analysis to a preferentially affected Figure 3 with 1 all Download asset Open asset Proteomic analysis of muscles from dHT and WT mice. and Hierarchically clustered heatmaps of the relative abundance of proteins in EDL soleus muscles (C) and EOMs (E) from three to five mice. Blue blocks represent proteins which are increased in content, yellow blocks proteins which are decreased in content in WT (columns in A and in versus dHT in A and in Right pie chart shows overall number of increased and decreased (yellow) proteins. Areas are relative to their numbers. and Volcano of total quantified proteins showing significant increased (blue) and decreased (yellow) content in dHT (condition 2) versus WT (condition 1) EDL soleus (D) and EOMs The horizontal coordinate is the difference multiple (logarithmic transformation at the base of 2), and the vertical coordinate is the significant difference p value (logarithmic transformation at the base of 10). The proteins showing major change in content are abbreviated. A q-value of equal or less than 0.05 was used to filter significant changes prior to the pathway analyses. An additional filter was applied to the Heatmaps and Piecharts and only proteins showing a significant change are included. is a common observed in patients affect by congenital myopathies to recessive RYR1 mutations (Lawal et al., 2018; Amburgey et al., 2013; Jungbluth et al., we also the proteome of EOMs from dHT and WT mice. Figure and shows that and proteins are up- or only in the EOM of dHT mice compared to WT mice, respectively. Interestingly, Reactome pathway analysis that genes encoding proteins involved in the citric acid cycle and electron transport chain, and protein to heat are upregulated in dHT vs WT EOMs (Figure The diagram (Figure shows that the three muscle types from the dHT mice a number of proteins whose content or It also shows that there are a number of proteins whose content or in a specific muscle type namely proteins in EDL, proteins in soleus and proteins in EOMs. analyzed these proteins to they were annotated to specific pathways but the results were not sufficiently informative as as skeletal muscle ECC and calcium homeostasis are In analysis showed that the genes encoding the proteins that were or upregulated specifically in dHT EDL, soleus and EOM were annotated to the category, response to and process and metabolic response to and of process in EDL muscles from dHT mice (Figure and and metabolic process and metabolic process and metabolic process in soleus muscles from dHT mice (Figure and and metabolic process and process and metabolic process and of quality in EOM muscles from dHT mice (Figure and Figure 4 with 1 all Download asset Open asset in protein content in EDL, soleus and EOM between dHT vs WT mice. (A) diagram showing significantly decreased proteins and increased proteins in the three muscle types. (B) process analysis of common proteins that are and (C) upregulated in muscle from dHT mice. common proteins showing significant changes in content in both EDL and soleus muscles. common proteins showing significant changes in content in EDL and common proteins showing significant changes in content in EOM and soleus muscles. (D) of the proteins whose content is increased in EDL, soleus and EOMs in dHT mice. (E) analysis annotated to of the proteins that are increased in muscles from dHT mice. we and analyzed protein a role in skeletal muscle muscle contraction, and heat shock protein and calcium-dependent that a significantly different (q<0.05) content between the mouse 1 shows that several proteins involved in skeletal muscle ECC are in EDLs from dHT mice, including the RyR1 as well as binding protein and 1 whose relative content by more than and respectively. The content of which different proteins including and (Treves et al., almost whereas in EDLs from dHT mice. the expression of type 2 fibers is impacted since MyHC 2 X and 2B as well 3

Decision letter: Quantitative proteomic analysis of skeletal muscles from wild-type and transgenic mice carrying recessive Ryr1 mutations linked to congenital myopathies

Dominant mutations in the skeletal muscle ryanodine receptor (RYR1) gene are well-recognized causes of both malignant hyperthermia susceptibility (MHS) and central core disease (CCD). More recently, recessive RYR1 mutations have been described in few congenital myopathy patients with variable pathology, including multi-minicores. Although a clinical overlap between patients with dominant and recessive RYR1 mutations exists, in most cases with recessive mutations the pattern of muscle weakness is remarkably different from that observed in dominant CCD. In order to characterize the spectrum of congenital myopathies associated with RYR1 mutations, we have investigated a cohort of 44 patients from 28 families with clinical and/or histopathological features suggestive of RYR1 involvement. We have identified 25 RYR1 mutations, 9 of them novel, including 12 dominant and 13 recessive mutations. With only one exception, dominant mutations were associated with a CCD phenotype, prominent cores and predominantly occurred in the RYR1 C-terminal exons 101 and 102. In contrast, the 13 recessive RYR1 mutations were distributed evenly along the entire RYR1 gene and were associated with a wide range of clinico-pathological phenotypes. Protein expression studies in nine cases suggested a correlation between specific mutations, RyR1 protein levels and resulting phenotype: in particular, whilst patients with dominant or recessive mutations associated with typical CCD phenotypes appeared to have normal RyR1 expression, individuals with more generalized weakness, multi-minicores and external ophthalmoplegia had a pronounced depletion of the RyR1 protein. The phenomenon of protein depletion was observed in some patients compound heterozygous for recessive mutations at the genomic level and silenced another allele in skeletal muscle, providing additional information on the mechanism of disease in these patients. Our data represent the most extensive study of RYR1-related myopathies and indicate complex genotype-phenotype correlations associated with mutations differentially affecting assembly and function of the RyR1 calcium release channel.

Multiminicore disease is a recessive congenital myopathy characterized by the presence of small cores or areas lacking oxidative enzymes, in skeletal muscle fibres. From a clinical point of view, the condition is widely heterogeneous and at least four phenotypes have been identified; genetic analysis has revealed that most patients with the classical form of multiminicore characterized by rigidity of the spine, early onset and respiratory impairment harbour recessive mutations in the SEPN1 gene, whereas the majority of patients belonging to the other categories, including patients with ophthalmoplegia or patients with a phenotype similar to central core disease, carry recessive mutations in the RYR1. In the present review we discuss the most recent findings on the functional effect of mutations in SEPN1 and RYR1 and discuss how they may adversely affect muscle function and lead to the clinical phenotype.

The main histological abnormality in congenital fiber type disproportion (CFTD) is hypotrophy of type 1 (slow twitch) fibers compared to type 2 (fast twitch) fibers. To investigate whether mutations in RYR1 are a cause of CFTD we sequenced RYR1 in seven CFTD families in whom the other known causes of CFTD had been excluded. We identified compound heterozygous changes in the RYR1 gene in four families (five patients), consistent with autosomal recessive inheritance. Three out of five patients had ophthalmoplegia, which may be the most specific clinical indication of mutations in RYR1. Type 1 fibers were at least 50% smaller, on average, than type 2 fibers in all biopsies. Recessive mutations in RYR1 are a relatively common cause of CFTD and can be associated with extreme fiber size disproportion.

Estimation of the Allele Frequency of Type 1 Polysaccharide Storage Myopathy and Malignant Hyperthermia in Quarter Horses in Brazil

C.P.2 In silico analysis of recessive RYR1 mutations identifies novel potential disease mechanisms

The molecular basis of a number of inherited diseases that affect the eyelids has been elucidated over the last two decades. Due to the vast number of these diseases, a clinician may become overwhelmed by the volume of data, making it difficult to incorporate newer information into his or her clinical practice. This article intends to review the recent developments of inherited diseases that affect the eyelids that a typical oculoplastic surgeon will encounter. This review proposes categorizing genetic diseases affecting the eyelids on rarity and whether the disease manifests itself at birth or later in life. Based on this classification system the following 10 diseases (the first five manifesting at birth, the last five later in life) are considered more likely to be encountered by the typical oculoplastic surgeon and reviewed in detail: blepharophimosis-ptosis-epicanthus inversus syndrome, congenital fibrosis of the extraocular muscles, lymphedema-distichiasis syndrome, neurofibromatosis type 1, congenital myasthenic syndrome, oculopharyngeal muscular dystrophy, chronic progressive external ophthalmoplegia, myotonic dystrophy, neurofibromatosis type 2, and basal cell nevus syndrome. The remaining known genetic disorders that affect the eyelids are considered less likely to be encountered by the typical oculoplastic surgeon and are listed in tabular form. It is prudent for the oculoplastic surgeon to be knowledgeable of inherited disorders that affect the eyelids to aid in accurate diagnosis, counseling, and treatment. The development of future therapies may at some point make treatment of these diseases no longer surgical. http://links.lww.com/COOP/A4.

Bidirectional communication between the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor (RYR1) in the sarcoplasmic reticulum (SR) is responsible for both skeletal-type excitation–contraction coupling (voltage-gated Ca2+ release from the SR) and increased amplitude of L-type Ca2+ current via the DHPR. Because the DHPR and RYR1 are functionally coupled, mutations in RYR1 that are linked to malignant hyperthermia (MH) may affect DHPR activity. For this reason, we investigated whether cultured myotubes originating from mice carrying an MH-linked mutation in RYR1 (R163C) had altered voltage-gated Ca2+ release from the SR, membrane-bound charge movement, and/or L-type Ca2+ current. In myotubes homozygous (Hom) for the R163C mutation, voltage-gated Ca2+ release from the SR was substantially reduced and shifted (∼10 mV) to more hyperpolarizing potentials compared with wild-type (WT) myotubes. Intramembrane charge movements of both Hom and heterozygous (Het) myotubes displayed hyperpolarizing shifts similar to that observed in voltage-gated SR Ca2+ release. The current–voltage relationships for L-type currents in both Hom and Het myotubes were also shifted to more hyperpolarizing potentials (∼7 and 5 mV, respectively). Compared with WT myotubes, Het and Hom myotubes both displayed a greater sensitivity to the L-type channel agonist ±Bay K 8644 (10 µM). In general, L-type currents in WT, Het, and Hom myotubes inactivated modestly after 30-s prepulses to −50, −10, 0, 10, 20, and 30 mV. However, L-type currents in Hom myotubes displayed a hyperpolarizing shift in inactivation relative to L-type currents in either WT or Het myotubes. Our present results indicate that mutations in RYR1 can alter DHPR activity and raise the possibility that this altered DHPR function may contribute to MH episodes.

Human malignant hyperthermia (MH) is a clinical syndrome, not a single disease, triggered by exposure to volatile anesthetics and depolarizing skeletal muscle relaxants that is often, but not always, inherited as an autosomal dominant trait with incomplete penetrance and variable expressivity. MH arises from mutations that exhibit both locus heterogeneity (i.e., MH from different genes) and allelic heterogeneity (i.e., MH from different mutations in a single gene). In the present issue of the journal, Vukcevic et al.1 report testing for MH susceptibility using Epstein-Barr virus (EBV)-transformed lymphoblasts cultured from lymphocytes of humans known to be predisposed to MH by prior clinical event and skeletal muscle contracture test. The authors demonstrate greater cytoplasmic calcium (Ca2+) responses to 4-chlorocresol (4-cmc), a skeletal muscle Ca2+ release channel/ryanodine receptor (RYR1) agonist, in lymphoblasts from 5 MH patients with different mutations when compared with normal controls. The report is commendable in a number of respects including provision of detailed clinical summaries and in vitro contracture test (IVCT) data, the addition of corresponding genotypic information, the comparison of Ca2+ transients over a range of 4-cmc concentrations, and determination of EC50 values. In particular, the method described is noteworthy for its potential to address some of the challenges confronting MH diagnostic assays based on conventional contracture testing and genotyping. Independent of a clinical event, the most widely accepted method for ascertaining MH susceptibility is the ex vivo muscle biopsy configured for physiological testing of changes in contractility in the presence of volatile anesthetics (i.e., halothane) and the RYR1 agonist, caffeine. The tests, developed with different protocols in North America and Europe, are known as the caffeine halothane contracture test (CHCT) and the IVCT, respectively.2,3 Contracture strength thresholds that segregate normal, susceptible, and equivocal (in Europe) responses have been arrived at by consensus, based on data from patients with clinically documented MH in the absence of coincident myopathy (which may itself introduce bias4), and compared with the responses of normal individuals with an intentional bias favoring false-positive designations to not miss potentially susceptible individuals if a higher cutoff is chosen. The utility of the contracture test varies with its intended application, i.e., for differential diagnosis of a confusing event in the operating room, for MH research, and as a guide to drug selection in clinical care and genetic counseling. In the first case, if positive results are found, thereby indicating that an untoward perioperative event was most probably MH, the contracture test holds substantial value and may be lifesaving to a patient and family members. Negative results on a test performed for differential diagnosis are a greater challenge to interpretation, but may shift the focus away from a diagnosis of MH. For MH research, the value of the IVCT is unquestioned. Present-day knowledge of the molecular pathogenesis of human MH would be inconceivable without contracture test data to correlate with candidate genes and mutations in patients and pedigrees. However, as an instrument for genetic counseling, and as an aid to the caregiver tasked with selecting a clinical anesthetic regimen in a relative of an MH patient, contracture testing has well-recognized shortcomings. Contracture testing is invasive, scarring, expensive (approximately $5000), and is now performed at only 5 centers in North America (www.mhaus.org). There is not, and cannot be, experimental validation of contracture test results on children younger than 10 years of age, a population at higher risk for MH than adults. The interlaboratory replicability of the European IVCT (i.e., samples from the same patient tested in different laboratories using the same protocol) is not high (i.e., 56% between laboratories in the only published study5). Comparable results using the North American CHCT protocol and thresholds have yet to appear. Nor has contracture test reproducibility (i.e., samples from the same patient tested in the same laboratory on different days) been reported for either the European or North American test. Whereas a positive test result (and most probably an equivocal result), using whatever thresholds have been selected, provides evidence against the further use of trigger agents in a given patient or family member, the meaning of negative contracture tests in patients with enough indication to have the test in the first instance (e.g., the individual has a family member with MH) is not known. To measure the true incidence of false-negative contracture test results requires sufficient numbers of IVCT- and CHCT-negative patients to undergo subsequent anesthetics with trigger agents. Because an MH-susceptible individual may have as many as 30 uneventful anesthetics before experiencing a trigger,6 the number of anesthetics needed on contracture test–negative individuals to determine the false-negative incidence with precision is unsettled. Moreover, the incidence of false-negative (and false-equivocal) results is likely to vary between distinct MH-predisposing mutations in distinct genetic backgrounds. As the present study confirms, different RYR1 mutations confer varying degrees of cellular dysfunction, as measured by agonist-induced Ca2+ release, in samples from patients with distinct genetic backgrounds, thereby undermining confidence that 1 or 2 threshold contracture test values are sufficient to segregate all susceptibility to the MH syndrome into 2 or 3 categories (i.e., negative, positive, and/or equivocal) for the purposes of genetic counseling. Accordingly, no such investigation of the incidence of false-negative contracture test responses has been, or is likely to be conducted, and no peer-reviewed scientific data are available to the concerned patient, family member, or caregiver regarding the safety of administering drugs that trigger MH to patients who are contracture test negative (or equivocal). Although genetic testing for MH differential diagnosis and counseling is minimally invasive, requiring only a few drops of blood or a cheek swab for DNA isolation, is easily replicated and reproduced, and is less costly than contracture testing depending on the number of mutations sought, inherent deficiencies also constrain widespread MH genotyping. Only about 60% of human MH susceptibility may be correlated with DNA sequence variations in the RYR1 gene.7 Although >200 amino acid substitutions in RYR1 have been identified, only 26 have been experimentally classified as causal mutations (see list maintained at www.emhg.org). Even with full-length RYR1 sequencing, a number of mechanisms for MH genetic pathology may be missed, for example, copy number variations, epigenomic modifications, and polymorphisms and alternate splice sites in unsequenced introns and promoter regions. Most importantly, a full-length genomic characterization of RYR1 leaves the contribution of other known and suspected loci unscreened. At least 4 additional genetic loci have been associated with MH predisposition. With the exception of the skeletal muscle isoform of the α subunit of the voltage-dependent calcium channel (CACNA1S),8 also known as the dihydropyridine receptor (DHPR), other MH genes remain unknown. Polymorphisms at these loci may themselves act as Mendelian determinants of MH susceptibility, or they may serve in concert with known loci to amplify MH susceptibility. Thus, in the setting of both locus and allelic genetic heterogeneity, interpretation of MH genetic test data is not straightforward. Whereas the presence of a shared MH mutation in family members of a proband who has had a clear-cut clinical MH trigger permits identification of these individuals as MH susceptible, the absence of 1 or more genotypes in question from a partial panel (i.e., a negative genetic test result) is not interpretable. In most, if not all cases, a patient will not have been harmed by the genetic testing, and his or her a priori risk of MH will be somewhat less than that in the untested population. Indeed, it has been estimated that up to 1 of 2000 patients carry alleles that may predispose to MH upon exposure to trigger agents.9 Unfortunately, in view of this figure, genotyping for MH susceptibility will remain incomplete for the foreseeable future. Relying on earlier work,10 Vukcevic et al. reasoned that white cells from patients with previously uncharacterized RYR1 mutations and a history of MH susceptibility by clinical event and IVCT abnormalities would also respond to lower concentrations of 4-cmc than cells from normal individuals. The authors asked study participants' centers to have anticoagulated whole blood samples drawn by their primary care physicians and forwarded by mail to a testing site in Würzberg, Germany for white blood cell isolation. There, B-cell lymphocytes were purified, cultured, and transformed into patient-specific, lymphoblastoid (i.e., dedifferentiated) cell lines via EBV infection.11 The EBV-transformed lymphoblasts, which express the skeletal muscle RYR1 isoform, were then tested for intracellular Ca2+ release from endoplasmic reticulum stores using changes in Ca2+-sensitive fluorescent dye intensity in response to increasing concentrations of 4-cmc, and the responses were compared with those in cells from control participants. The authors report that cell lines from each MH-susceptible individual with a distinct mutation responded to 4-cmc with statistically significant lower EC50 values than cells from normal individuals, and that the magnitude of the responses differed between individuals with different mutations. If confirmed, it is possible to envision a test devised for both research and clinical applications based on the present data that may complement contracture testing and genotypic data for the diagnosis of MH susceptibility and counseling. Such a test holds promise to be noninvasive, inexpensive, patient- and family-specific, and amenable to a formal characterization for analytical and clinical validity including measures of both replicability and reproducibility. For this to occur, however, a number of questions must first be answered. The size of the present study is small, with just 3 of 5 MH patients lacking a coexisting myopathy, and the remaining 2 with central core disease and an undiagnosed myopathy, respectively. Will similar results be observed when more patients, some with the same mutation from the same family and some with the same mutation from different families, are tested? Additionally, the magnitudes of response differences between patients and normal controls are small in the reported Ca2+ release assays, but the variances are relatively large, particularly when the standard errors of the mean are corrected to standard deviations. Will statistical significance and clinical significance be maintained when variances between different patients with the same mutation are tested against normal controls, rather than variances between different samples of cells cultured from the same patient? Although EBV-transformed cells have many attractive properties as test vehicles, it is not known whether the RYR1 channel expressed in lymphoblastoid cells is identically regulated in comparison to that of skeletal muscle. Is the response of RYR1 in circulating white cells, therefore, a true surrogate for the MH responsiveness of skeletal myocytes? Of note, the authors have not provided positive control data taken, for example, from patients expressing mutations with clear-cut MH causality, nor have they provided the reader with information regarding selection of the normal control participants. How many individuals comprised the normal control group? Were the normal participants previously anesthetized without event? How often? Did they have contracture tests? What were the results? Did they have a relative with MH? Were they genotyped? If they were genotyped, how extensive was this evaluation? Nor do the authors justify selection of 4-cmc. Were other agents tried (e.g., halothane or caffeine) and failed? Was effort given to testing agents in combination to magnify differences between patient and normal responses, and to control the magnitude of variances? Answers to these and other methodological questions are critical to more widespread development of the present and similar assays that aim to fill in gaps in contracture testing and genotyping outlined above. The present investigation is not the first to correlate an MH genotype with an altered physiological phenotype in lymphoblastoid cells exposed to 4-cmc. Zullo et al.12 recently immortalized lymphoblastoid cell lines from normal and MH-susceptible patients with diverse RYR1 mutations, and reported enhanced 4-cmc–induced acidification of the cell medium in samples from patients with a history of MH when compared with normal controls. Ca2+ fluxes were not directly measured. Whereas these authors demonstrated that a subset of cell lines with specific mutations exhibit increments in extracellular acidification, other cell lines from MH-susceptible individuals, for example, those expressing the Cys4664Arg mutation, did not acidify the medium even to the same degree as observed in control cells, thereby suggesting that MH may not be the consequence of a single shared pathway and that not all steps in MH pathogenesis are modeled by tissues other than muscle. Although Ca2+ fluxes may be an appropriate index for MH susceptibility in patients with RYR1 mutations, it is not yet known whether Ca2+ fluxes play a role in MH in individuals who have mutations at other known or as yet unidentified genetic loci. Thus, the general applicability of the methods proposed by Zullo et al. and those of the present article awaits acquisition of further data. The future of phenotypic testing for uncommon genetic disorders, and in particular pharmacogenetic disorders such as MH, however, seems brighter to us than might be apparent from the above discussion. Notably, stem cell research and somatic nuclear transfer offer possibilities not dreamed of when contracture testing for MH was initially developed. It is now possible to isolate patient-specific nuclei carrying mutations of interest from such sources as white blood cells or skin fibroblasts, and transfer them into pluripotential cells. In turn, the modified cells may then be induced to form muscle cells upon which exposure to trigger agents and physiological testing (for example, by measuring Ca2+ transients and acidification in response to various MH triggers) can proceed.13,14 In addition, the ability to generate patient-specific induced pluripotent stem cells from readily available tissues and to transform them into differentiated cell lines for further investigation has recently been described.15–17 Thus, the panoply of human genetic heterogeneity is now amenable to quantification with rigorous control of the polymorphism of interest, the genetic background of the sample, and the cellular apparatus of the relevant differentiated cell. Correlation of data from these methods with detailed clinical information, genotyping, and with quantitative rather than categorical data derived from contracture testing and the cellular assays reported herein will be critical to their validation and refinement. Because the mutation itself need not be known to discriminate modified cells from MH and non-MH sources, phenotypes resolved in this fashion may also be fitting substrates for pedigree and genome-wide association studies aimed at discerning novel genetic loci capable of imparting MH susceptibility. Accordingly, we believe that comprehensive and well-validated preoperative testing for MH susceptibility is not an insurmountable aim, but rather one that will require the acquisition of an expanded and integrated test repertoire. AUTHOR CONTRIBUTIONS Both authors helped write the manuscript, and approved the final manuscript.

Distinct Effects on Ca 2+ Handling Caused by Malignant Hyperthermia and Central Core Disease Mutations in RyR1