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.

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

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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

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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.

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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).

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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 (which is preferentially expressed in type 2 fibers) (Schiaffino and Reggiani, 2011) are decreased in the EDLs of dHT mice. The decrease of the fast isoforms of MyHC in dHT EDL muscles is accompanied by a decrease of many collagen isoforms. On the other hand, the content of several heat shock proteins as well as the content of 60 S and 40 S ribosomal proteins is increased in fast twitch fibers from the dHT. In addition, we found that the calcium/CaM-dependent protein kinases 1, 2α 2β and 2δ are increased in EDL from dHT mice.

Table 1

	Gene name	Protein*	Relative content	q value
ECC	Ryr1	Ryanodine receptor 1 (RyR1)	0.40	3.97x10–5
	Jph1	Junctophillin-1	0.64	0.025
	Cacna1s	Voltage dependent L type calcium channel subunit a1s (DHPR α1s)	0.73	0.018
	Dhrs7c	Dehydrogenase/reductase SDR family member 7 C (SRP-35)	1.34	0.0045
	Asph	Aspartyl/asparaginyl ß-hydroxylase (junctin/junctate/asp-ß-hydroxylase)	1.84	0.00095
Contractile proteins	Myh13	MyHC-EO	0.35	0.0063
	Myh1	Myosin-1 (MyHC-2x)	0.61	0.043
	Myh4	Myosin-4 (MyHC 2b)	0.71	0.018
	Actn3	α-actinin 3	0.74	0.012
Collagen and ECM proteins	Col2a1	Collagen (II)α–1 chain	0.18	0.0043
	Col1a2	Collagen (I) α –2 chain	0.25	0.027
	Col11a1	Collagen (XI) α –1 chain	0.35	0.0047
	Col5a2	Collagen (V)α –2 chain	0.37	0.00059
	Col5a1	Collagen (V) aα –1 chain	0.50	0.00156
	Col16a1	Collagen (XVI) α –1 chain	0.53	0.00154
	Col4a2	Collagen (IV) α –2 chain	0.7	0.040
	Itgav	Integrin α -V	0.77	0.044
	Itgb1bp2	Integrin ß–1- binding protein 2	1.3	0.045
Heat shock proteins	Hspb3	Hsp ß–3	0.73	0.00376
	Hspb8	Hsp ß–8 (a-crystallin C chain)	0.75	0.0160
	Hspa2	Heat shock related 70 kDa protein (Hsp70-2)	0.77	0.026
	Hspd1	60 kDa Hsp, mitochondrial (Chaperonin 60)	1.30	0.011
	Hspa5	Heat Shock Protein Family A (Hsp70) Member 5 (BiP)	1.41	0.00928
	Hsph1	Hsp 105 kDa (Hsp105, Hsp110)	1.47	0.0155
	Hspb6	Hsp ß–6 (HspB6)	1.5	0.0259
	Hspbp1	Hsp 70-binding protein	1.8	0.022
Ribosomal proteins	Rpl23	60 S Ribosomal protein L23	0.433	0.023
	Mrpl1	39 S ribosomal protein L1, mitochondrial	0.526	0.004
	Mrpl46	39 S ribosomal protein L46, mitochondrial	0.592	0.011
	Rpl34	60 S ribosomal protein L34	0.659	0.0042
	Rps15a	40 S ribosomal protein S15a	0.659	0.0056
	Mrpl43	39 S ribosomal protein L43, mitochondrial	0.684	0.029
	Mrps5	28 S ribosomal protein S5, mitochondrial	0.74	0.0021
	Rpl11	60 S ribosomal protein L11	1.265	0.013
	Rpl6	60 S ribosomal protein L6	1.273	0.012
	Rpl35	60 S ribosomal protein L35	1.290	0.034
	Mrpl19	39 S ribosomal protein L19, mitochondrial	1.346	0.028
	Rps25	40 S ribosomal protein S25	1.35	0.0076
	Rpl27a	60 S ribosomal protein L27a	1.365	0.02
	Rpl27	60 S ribosomal protein L27	1.374	0.016
	Rpl9	60 S ribosomal protein L9	1.374	0.015
	Rps2	40 S ribosomal protein S2	1.39	0.0065
	Rps8	40 S ribosomal protein S8	1.39	0.013
	Rplp2	60 S acidic ribosomal protein P2	1.403	0.0087
	Rps10	40 S ribosomal protein S10	1.41	0.033
	Rpl38	60 S ribosomal protein L38	1.431	0.025
	Rpl23a	60 S ribosomal protein L23a	1.459	0.005
	Rps12	40 S ribosomal protein S12	1.473	0.0128
	Rps9	40 S ribosomal protein S9	1.50	0.0278
	Rpl18	60 S ribosomal protein L18	1.491	0.017
	Mrps7	28 S ribosomal protein S7, mitochondrial	1.567	0.010
	Rpl10a	60 S ribosomal protein L10a	1.591	0.017
	Rpl22	60 S ribosomal protein L22	1.651	0.017
	Rps17	40 S ribosomal protein S17	1.661	0.0016
	Rps16	40 S ribosomal protein S16	1.82	0.0020
FK506 binding proteins	Fkbp1a	Peptidyl-prolyl cis-trans isomerase FKBP1A (FKBP12; calstabin-1)	0.64	0.0025
	Fkbp8	Peptidyl-prolyl cis-trans isomerase FKBP8 (38 kDa FKBP)	1.30	0.024
	Fkbp9	Peptidyl-prolyl cis-trans isomerase FKBP9 (63 kDa FK506-binding protein)	1.60	0.0057
Calcium-dependent protein kinases	Camk1	Calcium/Calmodulin-dependent protein kinase type 1 (CaM kinase I)	1.32	0.022
	Camk2a	Calcium/calmodulin-dependent protein kinase type II subunit α	1.40	0.0189
	Camk2b	Calcium/calmodulin-dependent protein kinase type II subunit ß	1.46	0.010
	Camk2d	Calcium/calmodulin-dependent protein kinase type II subunit δ	2.24	0.00025
Varia	Psmd7	26 S proteasome non-ATPase regulatory subunit 7	0.58	0.0016
	Psmg2	Proteasome assembly chaperone 2	1.66	0.038
	Fth1	Ferritin	1.69	0.0033

1. *

   The nomenclature of Proteins is based on that of the UniProtKB database.

We applied a similar approach as described above (i.e. proteins showing significantly different (q<0.05)≥0.2-fold change in content between the two mouse genotypes) to identify important components differing between dHT and WT soleus muscles (Table 2). In the ECC protein category, RyR1, DHPRα1s, and Junctophillin 1 are significantly decreased, as is triadin, whereas junctin/junctate/ß-hydroxylase and SERCA2 are increased in muscles from dHT mice. In the contractile protein group, significant changes are only observed for Troponin 3 whose content decreases by about 30%. Similar to what was observed in EDL muscles, we found that the content of calcium/calmodulin-dependent protein kinases IIδ and γ is increased. In addition, S100A1, a calcium binding protein which binds to and regulates RyR1 activity (Treves et al., 1997; Prosser et al., 2011), is significantly increased in soleus muscles from dHT mice. Finally, proteins constituting the 60 S and 40 S ribosomal subunits are increased in soleus muscles from dHT mice compared to WT.

Table 2

	Gene name	Protein*	Relative content	q value
ECC	Ryr1	Ryanodine receptor 1 (RyR1)	0.66	0.0080
	Jph1	Junctophillin 1	0.73	0.026
	Cacna1s	Voltage-dependent L type calcium channel subunit α1s (DHPR α1s)	0.67	0.017
	Trdn	Triadin	0.69	0.0352
	Asph	Aspartyl/asparaginyl ß-hydroxylase (junctin/junctate/aspß-hydroxylase)	1.31	0.045
	ATP2a2	Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2)	1.65	0.0256
Contractile proteins	Tnnt3	Troponin 3 (fast skeletal muscle type)	0.69	0.017
Calcium binding proteins	S100a1	Protein S100 A1	1.69	0.033
	Camk2d	Calcium/calmodulin-dependent protein kinase type II subunit δ	1.23	0.033
	Camk2g	Calcium/calmodulin-dependent protein kinase type II subunitγ	1.43	0.039
Ion Pumps	Atp1b1	Na+/K+ATPase ß1	0.77	0.047
	Atp1a1	Na+/K+ATPase α 1	1.41	0.017
Collagen and ECM proteins	Col11a1	Collagen (XI) α –1 chain	1.63	0.031
	Itgb5	Integrin a V/ß–5	11.95	0.044
Ribosomal proteins	Rpl36a	60 S ribosomal protein L36a	0.263	0.0021
	Mrpl10	39 S ribosomal protein L10, mitochondrial	0.577	0.040
	Rpl8	60 S ribosomal protein L8	0.661	0.022
	Rpl26	60 S ribosomal protein L26	0.695	0.022
	Mrpl42	39 S ribosomal protein L42, mitochondrial	0.788	0.026
	Rpl30	60 S ribosomal protein L30	1.259	0.020
	Rpl19	60 S ribosomal protein L19	1.260	0.050
	Mrpl41	39 S ribosomal protein L41, mitochondrial	1.289	0.033
	Rpl11	60 S ribosomal protein L11	1.325	0.026
	Rpl10	60 S ribosomal protein L10	1.432	0.013
	Rpl22	60 S ribosomal protein L22	1.436	0.017
	Rplp2	60 S acidic ribosomal protein P2	1.473	0.022
	Rpl35	60 S ribosomal protein L35	1.533	0.040
	Rpl23a	60 S ribosomal protein L23a	1.612	0.014
	Rpl23	60 S ribosomal protein L23	1.638	0.022
Varia	Psmg1	Proteasome Assembly Chaperone 1	0.488	0.024
	Dnajb6	DnaJ homolog subfamily B member 6 (Hsp J-2)	1.45	0.033
	Psma2	Proteasome 20 S Subunit α 2	1.51	0.011

1. *

   The nomenclature of Proteins is based on that of the UniProtKB database.

In EOMs, the proteins showing the greatest fold change (aside those involved in ECC and muscle contraction), are heat shock proteins, ribosomal proteins and proteins of the ECM, a variety of heat shock proteins and calcium/calmodulin-dependent protein kinases IIβ and IIδ and S100 family proteins (Table 3).

Table 3

	Gene name	Protein*	Relative content	q value
ECC	Ryr1	Ryanodine receptor 1 (RyR1)	0.42	1.73x10–6
	Asph	Aspartyl/asparaginyl ß-hydroxylase (junctin/junctate/aspß-hydroxylase)	1.35	0.00028
	Casq2	Calsequestrin-2	1.45	0.00031
	Casq1	Calsequestrin-1	1.55	0.0063
	ATP2a2	Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2)	1.55	0.00052
Contractile proteins	Myh13	MyHC-EO	0.27	1.01x10–5
	Actn2	α -actinin 2	1.36	0.0047
	Myh7b	Myosin-7B (MyH7B, cardiac musle ß isoform, MyHC14)	1.44	0.0038
	Actn1	α -actinin 1	1.45	0.015
	Tnnt2	Troponin T, cardiac isoform	1.45	0.0037
	Myot	Myotilin	1.61	0.0018
	Tnnt1	Troponin T slow, skeletal muscle (TnTs)	1.85	0.022
	Myoz3	Myozenin 3	2.03	1.29x10–5
	Myh6	Myosin 6 (MyHC cardiac muscle α-isoform)	2.22	1.01x10–5
	Tnnc1	Troponin C, slow skeletal and cardiac (TN-C)	2.61	8.1x10–5
Collagen and ECM proteins	Itga7	Integrin α 7	1.29	0.000191
	Col6a5	Collagen (VI) α –5 chain	1.32	0.0033
	Col6a6	Collagen (VI) α –6 chain	1.32	0.0087
	Col12a1	Collagen (XII) α –1 chain	1.34	0.012
	Col14a1	Collagen (XIV) α –1 chain	1.61	0.00072
	Col11a2	Collagen (XI) α –2 chain	4.46	1.27x10–5
Heat shock proteins	Hspa9	Mitochondrial, stress-70 protein	0.78	0.013
	Hsp90b1	Hsp 90b1 (GRP-94; 90 kDa glucose regulated protein)	1.26	0.0052
	Hspb3	Hsp ß- 3	1.27	0.0056
	Dnaja1	Dnaj homolog subfamily A member 1 (Hsp 40 kDa protein 4)	1.28	0.0067
	Hspb1	Hsp ß–1 (Hsp25)	1.29	0.00029
	Hspa5	Heat Shock Protein Family A (Hsp70) Member 5 (BiP)	1.32	0.00033
	Hspa1a	Heat shock 70 kDa protein 1 A	1.33	0.0069
	Hsp90aa1	Hsp 90 a	1.40	0.00094
	Dnajb1	Dnaj homolog subfamily B member 1 (Hsp40)	1.46	0.00021
	Dnajb4	DnaJ homolog subfamily B member 4 (Hsp40)	1.54	0.011
	Hspb6	Hsp ß- 6	1.61	4.05x10–5
Ribosomal Proteins	Rps2	40 S ribosomal protein S2	1.260	0.004
	Rpsa	40 S ribosomal protein SA	1.276	0.0035
	Rps11	40 S ribosomal protein S11	1.321	0.0016
	Rps20	40 S ribosomal protein S20	1.324	0.0007
	Rpl10	60 S ribosomal protein L10	1.332	0.0035
	Rplp2	60 S acidic ribosomal protein P2	1.332	0.00048
	Rpl11	60 S ribosomal protein L11	1.335	0.0077
	Rps28	40 S ribosomal protein S28	1.346	0.0041
	Rpl3	60 S ribosomal protein L13	1.385	1.42x10–5
	Rps7	40 S ribosomal protein S7	1.396	0.0125
	Rpl27a	60 S ribosomal protein L27a	1.461	0.00034
	Rps27a	40 S ribosomal protein S27a	1.570	0.038
FK506 binding proteins	Fkbp1a	Peptidyl-prolyl cis-trans isomerase FKBP1A (FKBP12; calstabin-1)	0.79	0.015
Calcium-dependent protein kinases	Camk2b	Calcium/Calmodulin-Dependent Protein Kinase IIß	1.30	0.0018
	Camk2d	Calcium/Calmodulin-Dependent Protein Kinase IIδ	2.32	8.35x10–6
Calcium binding proteins	S100a16	S100 A16	1.29	0.014
	S100a1	S100 A1	1.30	0.029

1. *

   The nomenclature of Proteins is based on that of the UniProtKB database.

Of note, the overall changes caused by Ryr1 mutations on the protein composition of muscles is more prominent in fast twitch muscles such as EOMs and EDLs compared to the slow twitch soleus muscle and the proteomic approach revealed that the content of many proteins differs between dHT and WT EDL, soleus and EOM muscles. We next refined our analysis and searched for protein whose content variation is most strongly associated with the dHT genotype. In particular, we searched for proteins which show significant changes in content in all three muscle types, namely EDL, Sol, and EOM. 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. The downregulation of RyR1 appears to be a unique a signature of the dHT phenotype, since its decrease is the only change shared between all muscle types investigated (Figure 4A). Biological process GO analysis revealed that the content of genes annotated to intracellular organelles, cytoplasm and ER (Figure 4B left panel, downregulated) and cellular process, metabolic process and organic substance metabolic process (Figure 4C left panel, upregulated) amongst others is changed in both EDL and soleus muscles from dHT mice. On the other hand, the content of genes annotated to supramolecular fiber, extrinsic component of membrane and myofibril (Figure 4B middle panel, downregulated), biological regulation, regulation of cellular process and organic substance metabolic process (Figure 4C middle panel, upregulated) amongst others is changed in both EDL and EOMs from dHT mice. While the content of genes annotated to cytoplasm, organelle membrane and mitochondrial membrane (Figure 4B right panel, downregulated) and cellular process, metabolic process and organonitrogen compound metabolic process (Figure 4C right panel, upregulated) amongst others is changed in both soleus and EOMs from dHT mice. Two heat shock proteins Hsp70 (BiP) and Hsp family B small member 6 (Hspb6) are increased only in EDL and EOMs. Interestingly, the content of 39 proteins including Kelch-like protein 41, two annotated to calcium signaling such as calmodulin kinase 2δ and aspartyl-ß -hydroxylase are increased in all three muscle types from dHT mice (Figure 4A and D), as are several proteins associated with the 40 S and 60 S ribosomal subunits. GO analysis of biological process revealed that the content of genes annotated to cellular process, cellular component organization or biogenesis and cellular component organization amongst others are upregulated in the three muscle types in dHT mice (Figure 4E).

Quantification and stoichiometry of ECC proteins in WT and dHT muscles

Skeletal muscle ECC relies on the highly ordered architecture of two intracellular membrane compartments, namely the transverse tubules which are invaginations of the plasma membrane containing the DHPR macromolecular complex and the sarcoplasmic reticulum containing the RyR1 macromolecular complex, as well as other proteins involved in calcium homeostasis and accessory structural proteins (Treves et al., 2009; Franzini-Armstrong and Jorgensen, 1994). The relative content of many of these proteins has been determined, nevertheless few studies have established their stoichiometry in relation to particular muscle types (Franzini-Armstrong and Jorgensen, 1994; Smith et al., 2013; Leberer and Pette, 1986). Within a total muscle homogenate, sarcoplasmic reticulum membrane proteins are of low abundance, thus, to quantify these proteins we performed high-resolution TMT mass spectrometry by using spiked-in labeled peptides from major protein involved in key steps of ECC calcium signaling to build a standard calibration curve. In particular, we used peptides from RyR1 and DHPRα1s, and Stim1 and Orai1, proteins which are involved in calcium release from the SR and in calcium entry across sarcolemma, respectively. The obtained protein concentrations showed a high correlation (R2=0.96; Figure 5) with the MS abundance estimates determined from the global proteomics analysis. Therefore, we used this curve to extrapolate the absolute amounts and stoichiometry of proteins whose values fall within the linear domain of the curve, namely, JP-45, triadin, junctophilin 1, Stac3 in addition to RyR1, DHPRα1s, Stim1 and Orai1. The content of the RyR1 protomer in WT fast twitch EDL muscles is 1.29±0.07 μmol/kg wet weight, whereby the calculated RyR1 tetrameric complex is 0.32 μmol/kg wet weight (Table 4) a value which is threefold lower compared to that determined in total muscle homogenates by[³H]-ryanodine equilibrium binding by Bers et al. (Bers and Stiffel, 1993). On the other hand, our RyR1 quantification results in mouse total muscle homogenates obtained by TMT mass spectrometry using labeled peptides is approximately fivefold higher compared to those obtained in rabbit and frog whole skeletal muscle homogenate preparations by Anderson et al., 1994 and by Margreth et al., 1993. Our results also show that the RyR1 concentration (in μmol/Kg) in soleus and EOM muscles from WT mice is approximately 38% and 46% of that found in EDL muscles of WT mice, respectively (Table 4). We found that the content of DHPRα1s in EDL muscles is 0.56±0.03 mol/kg wet weight, a value approx. 2.5-fold higher compared to that of soleus muscles (0.18±0.01 μmol/kg wet weight) and of EOMs (0.21±0.01 mol/kg wet weight). Thus, the calculated RyR1 tetramer to DHPRα1s ratio in EDL muscles from WT and dHT mice is 0.571 and 0.429, respectively (Table 5). Such a value appears to be slightly higher both in soleus and EOM muscles (0.667 and 0.625 in WT and dHT soleus muscles and 0.714 and 0.474 in WT and dHT EOMs, respectively, Table 5). The Stac3 content correlates with that of DHPRα1s, namely EDL is the muscle which is most enriched in Stac3 (0.62±0.07 μmol/kg wet weight); soleus and EOMs contain approximately one-third of the Stac 3 present in the EDL, namely 0.22±0.02 mol/kg wet weight and 0.17±0.01 mol/kg wet weight in soleus and EOMs, respectively. Stac3 content in muscles from dHT is similar to that of WT littermates. Interestingly, the content of Stim1 depends on the muscles type. Mass spectrometry quantification revealed that EOMs contain the highest amounts of Stim1 (1.35±0.03 μmol/kg wet weight) compared to soleus (0.55±0.03 μmol/kg wet weight) and EDL (0.46±0.02 μmol/kg wet weight). Western blot analysis of total muscle homogenates from WT mice confirmed that EOMs contain four times more Stim1 than EDL muscles Figure 6 and Supplementary file 1b and that equal proportions of Stim1 and Stim1L are present in the three muscle groups, with no preferential expression of the long isoform in any of the muscles investigated. As to WT EDL and soleus, we found no major differences in Stim1 expression, confirming previous data by Cully et al., 2016. The expression of Stim1 is accompanied by the expression of Orai1 in EDL and EOMs but not in soleus muscles. Indeed, mouse EOMs contain the highest amount of Orai1 monomer (0.16±0.03 μmol/kg wet weight) and EDLs contained approximately 68% of that (0.11±0.01 μmol/kg wet weight). To our surprise in soleus muscles, the content of Orai1 is below the detection level of mass spectrometry measurement, indicating that slow twitch (soleus) muscles express very little, if any, Orai1 compared to fast twitch EDL and EOMs.

Figure 5

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Table 4

Gene name	EDL		soleus		EOM
	WT	dHT	WT	dHT	WT	dHT
Ryr1 monomers (terameric channel)	1.29±0.07 (0.32)	0.86±0.01 (0.21)	0.49±0.02 (0.12)	0.40±0.002 (0.10)	0.59±0.02 (0.15)	0.35±0.01 (0.09)
Cacna1s	0.56±0.03	0.49±0.01	0.18±0.01	0.16±0.002	0.21±0.01	0.19±0.004
Stac3	0.62±0.07	0.53±0.06	0.22±0.02	0.20±0.01	0.17±0.01	0.15±0.01
Jsrp1	0.42±0.03	0.40±0.01	0.32±0.01	0.29±0.03	0.35±0.01	0.35±0.02
Asph	0.21±0.01	0.26±0.01	0.30±0.02	0.35±0.03	0.82±0.03	1.00±0.03
Trdn	0.96±0.18	0.79±0.06	0.16±0.03	0.13±0.01	0.23±0.01	0.22±0.01
Jph1	0.71±0.09	0.58±0.04	0.29±0.02	0.25±0.01	0.24±0.01	0.23±0.01
Stim1	0.46±0.02	0.48±0.03	0.55±0.03	0.56±0.03	1.35±0.03	1.42±0.09
Orai1 monomers (6-subunt complex)	0.11±0.01 (0.02)	0.13±0.02 (0.02)	Not detected	Not detected	0.16±0.03 (0.03)	0.17±0.01 (0.03)

Table 5

Gene name	EDL		soleus		EOM
	WT	dHT	WT	dHT	WT	dHT
Ryr1 complex/Cacna1s	0.571	0.429	0.667	0.625	0.714	0.474
Stac3/Cacna1s	1.11	1.08	1.22	1.25	1.67	1.84
Jsrp1/Cacna1s	0.75	0.82	1.78	1.81	0.95	1.00
Stim1/Orai1 complex	23.0	24.0	-	-	45.0	47.3

Figure 6

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Figure 6—source data 1

Bots refer to data shown in Figure 6A.

TOP: Original uncropped western blots showing immunoreactivity of STIM1L and STIM1 in EDL, Soleus and EOM muscles from WT mouse N° 5. BOTTOM: same blot re-probed with anti-MyHC recognizing all isoforms (loading normalization).

https://cdn.elifesciences.org/articles/83618/elife-83618-fig6-data1-v2.zip

Download elife-83618-fig6-data1-v2.zip

Figure 6—source data 2

Bots refer to data used for statistical analysis depicted in Figure 6B.

Original uncropped western blots showing immunoreactivity of STIM1L, STIM1 and MyHC in EDL, Soleus and EOM muscles from WT mouse N° 1, 2, 3 (TOP) and WT mouse N° 4 (BOTTOM).

https://cdn.elifesciences.org/articles/83618/elife-83618-fig6-data2-v2.zip

Download elife-83618-fig6-data2-v2.zip

To understand in greater detail the changes in skeletal muscle function in congenital myopathies caused by recessive RYR1 mutations, we performed an in-depth qualitative and quantitative analysis of protein content and abundance in EDL, soleus and EOMs from WT and dHT mice. The results of the proteomic analysis reveal that, asides the drastic reduction in RyR1 content, profound changes occur in the content of many proteins particularly in fast-, slow-twitch and EOM muscles. Namely, we found that recessive Ryr1 mutations lead to an increase content aspartyl-ß-hydroxylase (Asph), some ribosomal proteins and calmodulin kinase 2 delta. EDL and EOMs that are more severely affected, also share changes in the content of other proteins, including collagens, heat shock proteins, and CamK2b as well as additional ribosomal proteins. We believe that the reduced RyR1 calcium channel content has a domino effect leading to changes in content of many proteins, particularly in EDL and EOMs.

All data, code, and materials used in the analysis are available in some form to any researcher for purposes of reproducing or extending the analysis. Mass spectrometry data has been deposited to the ProteomeXchange Consortium via the Pride partner repository (http://ebi.ac.uk/pride) with the following access number PXD036789 (http://www.ebi.ac.uk/pride/archive/projects/PXD036789). Original western blot figures have been uploaded as a zipped file as source data 1 and 2.

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Author details

1. Jan Eckhardt

   Departments of Biomedicine and Neurology, Basel University Hospital, Basel, Switzerland

   Present address

   Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Writing – review and editing

   Contributed equally with

   Alexis Ruiz

   Competing interests

   No competing interests declared

2. Alexis Ruiz

   Departments of Biomedicine and Neurology, Basel University Hospital, Basel, Switzerland

   Contribution

   Data curation, Investigation, Methodology, Writing – review and editing

   Contributed equally with

   Jan Eckhardt

   Competing interests

   No competing interests declared

3. Stéphane Koenig

   Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland

   Contribution

   Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

4. Maud Frieden

   Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7135-0874

5. Hervé Meier

   Departments of Biomedicine and Neurology, Basel University Hospital, Basel, Switzerland

   Contribution

   Data curation, Software, Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

6. Alexander Schmidt

   Proteomics Core Facility, Biozentrum, Basel University, Basel, Switzerland

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Writing – original draft, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

7. Susan Treves

   1. Departments of Biomedicine and Neurology, Basel University Hospital, Basel, Switzerland
   2. Department of Life Science and Biotechnology, University of Ferrara, Ferrara, Italy

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   susan.treves@unibas.ch

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0007-9631

8. Francesco Zorzato

   1. Departments of Biomedicine and Neurology, Basel University Hospital, Basel, Switzerland
   2. Department of Life Science and Biotechnology, University of Ferrara, Ferrara, Italy

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   fzorzato@usb.ch

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8469-7065

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