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

Abstract

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 protective effect against muscle damage caused by calcium dysregulation and uncoupling of mitochondrial respiratory chain (Maglara et al., 2003) as well as protective effects against ischemic injury in cardiomyocytes (Martin et al., 1997). Interestingly, the content of Mitsugumin 53 (encoded by Trim72), a protein involved in muscle membrane repair (Cai et al., 2009) is 2.8-fold higher in slow twitch muscles compared to fast twitch muscles. Thus, on the basis these observations we cannot exclude the possibility that increased expression of Mitsugumin 53, along with a set of heat shock proteins (Dreiza et al., 2010; Maglara et al., 2003; Martin et al., 1997; Larkins et al., 2012), might be relevant in preventing muscle fiber type 1 damage associated with the presence of recessive RYR1 mutations or with other type of stressing events. To directly verify this hypothesis, we examined the proteome of fast and slow twitch muscles in a mouse model (RyR1 dHT) 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 next experiments, the proteome of three different muscles from dHT mice vs those of WT mice were compared. Figure 3A and B shows that in EDL muscles a total of 848 proteins are significantly (q<0.05) mis-regulated in dHT mice; in particular, 529 and 319 proteins are up- or downregulated only in the EDLs of dHT mice compared to WT mice, respectively. Reactome pathway (Figure 3—figure supplement 1A) analysis revealed that proteins involved in homeostasis of the extracellular matrix, including collagen assembly and chain formation, collagen degradation, ECM organization and integrin interaction, are up-regulated in EDLs from WT compared to dHT mice. We also compared the proteome of soleus muscles from WT and dHT mice. Figure 3C and D show that the overall number of proteins showing significant changes in their relative content between dHT and WT mice, is smaller than that observed in EDL muscles. In particular, we found that 339 and 170 proteins are up- or downregulated only in the soleus muscles of dHT mice compared to those from WT mice, respectively. Contrary to EDL muscles, Reactome pathway analysis failed to identify a preferentially affected cellular pathway. Figure 3 with 1 supplement see all Download asset Open asset Proteomic analysis comparison of muscles from dHT and WT mice. (A, C and E) Hierarchically clustered heatmaps of the relative abundance of proteins in EDL (A), 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 1–5 in A and E; 1–3 in C) versus dHT (5–10 in A and E; 4–8 in C). Right pie chart shows overall number of increased (purple) and decreased (yellow) proteins. Areas are relative to their numbers. (B, D and F) Volcano plots of total quantified proteins showing significant increased (blue) and decreased (yellow) content in dHT (condition 2) versus WT (condition 1) EDL (B), soleus (D) and EOMs (F). 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 ≥0.2-fold are included. Since ophthalmoplegia is a common clinical sign observed in patients affect by congenital myopathies linked to recessive RYR1 mutations (Lawal et al., 2018; Amburgey et al., 2013; Jungbluth et al., 2018), we also investigated the proteome of EOMs from dHT and WT mice. Figure 3E and F shows that 560 and 117 proteins are up- or downregulated only in the EOM of dHT mice compared to WT mice, respectively. Interestingly, Reactome pathway analysis indicated that genes encoding proteins involved in the citric acid cycle and electron transport chain, ATP synthesis and uncoupling protein complexes linked to heat formation are upregulated in dHT vs WT EOMs (Figure 3—figure supplement 1B). The Venn diagram (Figure 4) shows that the three muscle types from the dHT mice share a number of proteins whose content increases or decreases. It also shows that there are a number of proteins whose content increases or decreases in a specific muscle type only, namely 848 proteins in EDL, 677 proteins in soleus and 509 proteins in EOMs. We analyzed these proteins to verify whether they were annotated to specific cellular pathways but the results were not sufficiently informative as far as skeletal muscle function, ECC and calcium homeostasis are concerned. In fact, GO analysis showed that the genes encoding the proteins that were downregulated or upregulated specifically in dHT EDL, soleus and EOM were annotated to the Biological processes category, specifically: biological cellular processes, response to stimulus and multicellular organismal process (downregulated) and metabolic process, response to stimulus and positive regulation of biological process (upregulated) in EDL muscles from dHT mice (Figure 4—figure supplement 1A and D), cellular process, biological regulation and metabolic process (downregulated) and cellular process, metabolic process and organic substance metabolic process (upregulated) in soleus muscles from dHT mice (Figure 4—figure supplement 1B and E) and cellular process, cellular metabolic process and oxidation reaction process (downregulated) and cellular process, primary metabolic process and regulation of biological quality (upregulated) in EOM muscles from dHT mice (Figure 4—figure supplement 1C and F). Figure 4 with 1 supplement see all Download asset Open asset Changes in protein content in EDL, soleus and EOM between dHT vs WT mice. (A) Venn diagram showing significantly decreased proteins (left) and increased proteins (right) in the three muscle types. (B) GO biological process analysis of common proteins that are downregulated and (C) upregulated in muscle from dHT mice. Left panels, common proteins showing significant changes in content in both EDL and soleus muscles. Central panels, common proteins showing significant changes in content in EDL and EOMs; right panels, common proteins showing significant changes in content in EOM and soleus muscles. (D) List of the 39 proteins whose content is increased in EDL, soleus and EOMs in dHT mice. (E) GO analysis annotated to Biological processes of the 39 proteins that are increased in muscles from dHT mice. Thus, we selected and analyzed protein families playing a role in skeletal muscle ECC, muscle contraction, collagen and ECM, heat shock response/chaperones, protein synthesis and calcium-dependent regulatory functions that exhibit a significantly different (q<0.05) content between the two mouse genotypes. Table 1 shows that several proteins involved in skeletal muscle ECC are down-regulated in EDLs from dHT mice, including the RyR1 (60% decrease) as well as its stabilizing binding protein FKBP12, DHPRα1 and junctophillin 1 whose relative content decreases by more than 30%, 23%, and 40%, respectively. The content of Asph which encodes different proteins including junctin, junctate, humbug and aspartyl-ß-hydroxylase (Treves et al., 2000) increases almost twofold, whereas SRP-35 (Dhrs7c) increases 1.34-fold in EDLs from dHT mice. Additionally, the expression of type 2 fibers is impacted since MyHC 2 X and 2B as well asα-actinin 3 (wh