Tubastatin A attenuates impaired autophagic degradation and promotes myogenic program in skeletal muscle following downhill running.

This study was designed to probe the effect of downhill running on microtubule acetylation and autophagic flux in rat skeletal muscle. Sprague-Dawley rats were subjected to an exercise protocol of a 90-min downhill run with a slope of -16° and a speed of 16 m·min-1, and then the soleus was sampled at 0, 12, 24, 48, and 72 h after exercise. Protein expression levels of microtubule-associated protein 1 light chain 3 (LC3), p62/sequestosome 1 (p62), α-tubulin, and acetylated α-tubulin (AcK40 α-tubulin) were detected by Western blotting. Alpha-tubulin was costained with AcK40 α-tubulin or cytoplasmic dynein intermediate chain in a single muscle fiber, and LC3 was costained with lysosomal-associated membrane protein 1 in cryosections. To assess autophagic flux in vivo, colchicine or vehicle was injected intraperitoneally 3 d before the exercise experiment, and the protein levels of LC3 and p62 were measured by Western blotting. Downhill running induced a significant increase in the protein levels of LC3-II and p62, whereas the level and proportion of AcK40 α-tubulin were markedly decreased. Furthermore, the amount of dynein on α-tubulin was decreased after downhill running, and autophagosomes accumulated in the middle of myofibrils. Importantly, LC3-II flux was decreased after downhill running compared with that in the control group. A bout of downhill running decreases microtubule acetylation, which may impair dynein recruitment and autophagosome transportation, causing blocked autophagic flux.

The purpose of this study was to determine if changes in oxidative stress biomarkers in blood and skeletal muscles are similar in normal and antioxidant supplemented rats after a downhill run. Sixty-six male Sprague-Dawley rats were pretreated with a normal rat diet or diet + antioxidants (2,000 mg vitamin C + 1,000 IU vitamin E/kg diet) for 2 weeks. Exercised rats ran 90 min on a rodent treadmill at a speed of 16 m/min at -16 degrees grade. Rats were sacrificed either at rest, immediately, 2 hrs, or 48 hrs postexercise. Malondialdehyde (MDA) and protein carbonyl (PC) concentrations and glutathione status in blood, vastus lateralis (white fast-twitch), vastus intermedius (red fast-twitch), and soleus (slow-twitch) muscles were determined. A significant increase from rest in PC occurred in plasma, vastus intermedius and soleus muscle 2 hrs after the downhill run (p < 0.05), with no changes observed at any other times postexercise. Antioxidant supplementation significantly decreased PC concentrations in both vastus intermedius and soleus muscles at all times combined (p < 0.05). MDA and glutathione status in blood and muscles were unaffected by either the downhill run or antioxidant treatment. For PC and MDA, the concentrations were lower in blood as compared to skeletal muscle, with the opposite finding for oxidized glutathione; however, the pattern of response postexercise was similar. These data indicate that (a) PC, but not MDA or oxidized glutathione, is elevated transiently following downhill running in male rats; (b) the elevation in PC postexercise occurs in plasma, vastus intermedius, and soleus muscles; (c) antioxidant therapy can attenuate PC in vastus intermedius, and soleus muscles; and (d) while the concentrations of oxidative stress biomarkers differ between blood and the various skeletal muscles, the pattern of response postexercise is similar.

Phytoecdysteroids are natural plant steroids synthesized by a variety of hardy plants. Phytoecdysteroids, such as 20‐hydroxyecdysone (20E), possess anabolic properties and previous research indicates that 20E stimulates protein synthesis in cultured muscle cells and increases grip strength in young rats after 28 days of supplementation. The purpose of this study was to investigate the extent to which 20E stimulates protein synthesis and Akt/mTOR signaling in skeletal muscle after an acute bout of downhill running (DHR). Male C57BL6 mice (3–6 mo old) were assigned to four groups. Mice in the DHR groups performed an acute bout of DHR to exhaustion on a rodent treadmill at 17 m•min−1 and −20° decline. After completion of the DHR bout and recovery, mice received an oral gavage treatment of either 50 mg•kg−1 body mass (BM) of 20E (DHR + 20E; n=5) or vehicle (DHR + vehicle; n=6), and then treated daily for the next four consecutive days. Two groups that did not perform DHR, but were treated for five consecutive days with either 50 mg•kg−1 BM of 20E (No DHR + 20E; n=8) or vehicle (No DHR + vehicle; n=6), were used as controls. On the fifth day post‐DHR, mice were not treated with 20E or vehicle; however, an IP injection of puromycin (0.040 μmol•g−1 BM) was administered 30 min prior to sacrifice to assess protein synthesis. Skeletal muscles were harvested and activation of protein synthesis was assessed using the SUnSET method and Western blot and Akt/mTOR signaling activation was measured via Western blot. Mice in the DHR + vehicle group ran for 58.5 min, while mice in the DHR + 20E group ran for 57.0 min. Compared to No DHR + vehicle, DHR + vehicle increased skeletal muscle puromycin incorporation 14%, but DHR + 20E increased puromycin incorporation 32%, five days post‐DHR. Puromycin incorporation was not different between No DHR + vehicle and No DHR + 20E groups. DHR resulted in ~25% increase in phosphorylation of both Akt and p70S6K, compared to No DHR + vehicle; but there was no difference in Akt or p70S6K phosphorylation between DHR + 20E and DHR + vehicle groups, five days post‐DHR. Compared to No DHR + vehicle, DHR + vehicle increased 4EBP1 phosphorylation 35%, but DHR + 20E increased 4EBP1 phosphorylation 70%, and DHR + vehicle increased ribosomal protein S6 (rpS6) phosphorylation 40%, but DHR + 20E increased rpS6 phosphorylation 119%, five days post‐DHR. In conclusion, these findings suggest that DHR stimulates protein synthesis and activates Akt/mTOR signaling five days after DHR, but 20E treatment enhances the pattern of response to an acute bout of DHR in skeletal muscle of young mice.

Expressions and effects of autophagy-related genes in bleomycin-induced skin fibrosis of mice

To study the effect of downhill running on glycogen metabolism, 94 rats were exercised by running for 3 h on the level or down an 18 degrees incline. Muscle and liver glycogen concentrations were measured before exercise and 0, 48 and 52 h postexercise. Rats were not fed during the first 48 h of recovery but ingested a glucose solution 48 h postexercise. Downhill running depleted glycogen in the soleus muscle and liver significantly more than level running (P less than 0.01). The amount of glycogen resynthesized in the soleus muscle and liver in fasting or nonfasting rats was not altered significantly by downhill running (P greater than 0.05). On every day of recovery the rats were injected with dexamethasone, which induced similar increases in glycogen concentration in the soleus muscle and liver after the 52nd h of the postexercise period in the case of downhill and level running. The glycogen depletion and repletion results indicated that, under our experimental conditions, downhill running in the rat, a known model of eccentric exercise, affected muscle glycogen metabolism differently from eccentric cycling in humans.

To identify factors that influence post-exercise muscle glycogen repletion, we compared the glycogen recovery after level running with downhill running, an experimental model of impaired post-exercise glycogen recovery. Male Institute of Cancer Research (ICR) mice performed endurance level running (no inclination) or downhill running (-5° inclination) on a treadmill. In Experiment 1, to determine whether these two types of exercise resulted in different post-exercise glycogen repletion patterns, tissues were harvested immediately post-exercise or 2 days post-exercise. Compared to the control (sedentary) group, level running induced significant glycogen supercompensation in the soleus muscle at 2 days post-exercise (p = 0.002). Downhill running did not induce glycogen supercompensation. In Experiment 2, mice were orally administered glucose 1 day post-exercise; this induced glycogen supercompensation in soleus and plantaris muscle only in the level running group (soleus: p = 0.005, plantaris: p = 0.003). There were significant positive main effects of level running compared to downhill running on the plasma insulin (p = 0.017) and C-peptide concentration (p = 0.011). There was no difference in the glucose transporter 4 level or the phosphorylated states of proteins related to insulin signaling and metabolism in skeletal muscle. The level running group showed significantly higher hexokinase 2 (HK2) protein content in both soleus (p = 0.046) and plantaris muscles (p =0.044) at 1 day after exercise compared to the downhill running group. Our findings suggest that post-exercise skeletal muscle glycogen repletion might be partly influenced by plasma insulin and skeletal muscle HK2 protein levels.

Histochemical, biochemical, and contractile properties of red, white, and intermediate fibers.

Objective Purpose：Downhill running can causes muscle damage, called delayed muscle damage and induced oxidative stress and inflammatory reaction, causing abnormity of skeletal muscle morphology, changing in blood biochemical indexes, and decreasing in function of skeletal muscle systolic. Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, is degraded by dimethylarginine dimethylaminohydrolase 1 (DDAH1). There were new evidences demonstrated that DDAH1 is an important regulator of cell redox state and apoptosis. In summary, the study shown that DDAH1 is an important regulator of cell redox state and apoptosis. Emerging evidences suggests that DDAH1 controls cellular oxidative stress and apoptosis via a miR-21-dependent pathway. However, the effect and mechanism of DDAH1 on damage of skeletal muscle caused by downhill running is not clear enough. Thus，the purpose of this experiment was to determine the effect and mechanism of DDAH1 in downhill running.&#x0D; Keys: downhill running; delayed onset muscle soreness(DOMS); eccentric exercise; skeletal muscle.&#x0D; Methods Method: The experimental mice were 24 female C57 mice of 10 weeks old and 24 female DDAH1 hybrid knockout mice of 10 weeks old. DDAH1 KO mice used for this study was knockout of dimethylarginine dimethylaminohydrolase 1 compared with WT mice. Animals were fed standard laboratory chow and had access to water ad libitum. C57 mice were divided into 3 groups: C57 control, C57 48H, C57 120H; DDAH1 KO mice were divided into 3 groups: DDAH1 control, DDAH1 48H, DDAH1 120H. C57 and DDAH1 KO mice used for this study completed a single bout of downhill running exercise (20°, 17 m/min, 60 min), and gastrocnemius muscle, soleus muscle and quadriceps femoris muscle were collected 48 and 120 hours (H) postexercise (PE). C57control group and DDAH1 KO control group dose not exercise. Speed on the treadmill was gradually increased from 10 to 17m/min during a 7-min warm-up period (increased of 1m/min every minute). All experiments were conducted at approximately the same time of day. Maximal grip strength was measured ifor each groups. Grip strength testing was completed to detect post-eccentric exercise injury in C57 and DDAH1 KO mice. All results were analyzed by means of methods of histological and molecular biological.&#x0D; Results Method: The experimental mice were 24 female C57 mice of 10 weeks old and 24 female DDAH1 hybrid knockout mice of 10 weeks old. DDAH1 KO mice used for this study was knockout of dimethylarginine dimethylaminohydrolase 1 compared with WT mice. Animals were fed standard laboratory chow and had access to water ad libitum. C57 mice were divided into 3 groups: C57 control, C57 48H, C57 120H; DDAH1 KO mice were divided into 3 groups: DDAH1 control, DDAH1 48H, DDAH1 120H. C57 and DDAH1 KO mice used for this study completed a single bout of downhill running exercise (20°, 17 m/min, 60 min), and gastrocnemius muscle, soleus muscle and quadriceps femoris muscle were collected 48 and 120 hours (H) postexercise (PE). C57control group and DDAH1 KO control group dose not exercise. Speed on the treadmill was gradually increased from 10 to 17m/min during a 7-min warm-up period (increased of 1m/min every minute). All experiments were conducted at approximately the same time of day. Maximal grip strength was measured ifor each groups. Grip strength testing was completed to detect post-eccentric exercise injury in C57 and DDAH1 KO mice. All results were analyzed by means of methods of histological and molecular biological.&#x0D; Conclusions Conclusion: The DDAH1 knockout has a protective effect on delayed onset muscle soreness(DOMS) caused by downhill running, and accelerate the injury recovery. &#x0D;

Experiments were conducted to test the hypothesis that injury to skeletal muscle in rats resulting from prolonged downhill running is prevented to a greater extent by prior downhill training than by either uphill or level training. Changes in plasma creatine phosphokinase (CPK) activity and glucose-6-phosphate dehydrogenase (G-6-PDase) activity in the soleus (S), vastus intermedius (VI), and medial head of triceps brachii (TM) muscles were evaluated as markers of muscle injury 48 h after 90 min of intermittent downhill running (16 m . min -1). Prior to this acute downhill run, groups of rats were trained by either downhill (-16 degrees), level (0 degrees), or uphill (+16 degrees) running (16 m . min -1) for 30 min/day. Training duration was either 5 days or 1 day. A training effect (i.e., reduced muscle injury) was indicated if muscle G-6-PDase or plasma CPK activity in a trained group following the 90-min downhill run was not different from that of nonexercised control animals and/or if it was lower than that of nontrained runners. A significant training effect was achieved in all three muscles with 5 days of either downhill or level training, but only in S after 5 days of uphill training. Elevation of plasma CPK activity was prevented by 5 days of training on all three inclines.(ABSTRACT TRUNCATED AT 250 WORDS)

What is the central question of this study? Exercise training by running has an effect on age-related muscle and bone wasting that improves physical activity and quality of life in the elderly. However, the effect of downhill running on age-related muscle and bone wasting, and its mechanisms, are unclear. What is the main finding and its importance? Gradual downhill running can improve skeletal muscle growth and bone formation by enhancing autophagy and bone morphogenetic protein signalling in aged rats. Therefore, downhill running exercise might be a practical intervention to improve skeletal muscle and bone protection in the elderly. Recent evidence suggests that autophagy and the bone morphogenetic protein (BMP) signalling pathway regulate skeletal muscle growth and bone formation in aged rats. However, the effect of downhill running on muscle growth and bone formation is not well understood. Thus, we investigated the effect of downhill and uphill running on age-related muscle and bone weakness. Young and late middle-aged rats were randomly assigned to control groups (young, YC; and late middle-aged, LMC) and two types of running training groups (late middle-aged downhill, LMD; and late middle-aged uphill, LMU). Training was progressively carried out on a treadmill at a speed of 21mmin-1 with a slope of +10deg for uphill training versus 16mmin-1 with a slope of -16deg for downhill training, both for 60minday-1 , 5 daysweek-1 for 8weeks. Downhill and uphill training increased autophagy-related protein 5, microtubule-associated protein light chain, Beclin-1 and p62 proteins in aged rats. In addition, superoxide dismutase, haem oxygenase-1 and the BMP signalling pathway were elevated. Phosphorylation of mammalian target of rapamycin and myogenic differentiation were increased significantly in the LMD and LMU groups. Consequently, in the femur, BMP-2, BMP-7 and autophagy molecules were highly expressed in the LMD and LMU groups. These results suggest that both downhill and uphill training appear to have a positive effect on expression of autophagy molecules and BMPs. In particular, these physiological adaptations from gradual downhill exercise have an effect on bone morphological changes and muscle quality similar to gradual uphill training interventions in ageing.

To observe the effects of electroacupuncture (EA) on the autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency (POI), and explore the mechanism of EA in improving POI. Thirty-two female SD rats were randomly divided into a blank group (n=8) and a model making group (n=24). The rats in the model making group were injected intraperitoneally with cyclophosphamide for 15 days to establish the POI model (the dosage on the 1st day was 50 mg/kg, and 8 mg/kg from the 2nd day to 15th day). The successfully modeled rats were then randomly divided into a model group, an EA group, and an estradiol (E2) group, with 8 rats in each group. Rats in the EA group received EA at bilateral "Gongsun" (SP 4) with continuous wave, frequency of 2 Hz, and current intensity of 0.1 to 1 mA, 20 min per treatment, once daily for 14 days. Rats in the E2 group were administered with E2 (0.01 mg/mL) by gavage (10 mL/kg), once daily for 14 days. The changes in estrous cycle were observed by rapid Giemsa staining before and after modeling. After intervention, ovarian tissue morphology was observed by HE staining; serum levels of follicle-stimulating hormone (FSH), E2, anti-Mullerian hormone (AMH), and inhibin B (INHB) were detected by ELISA; immunofluorescence staining was used to observe the expression of p62 in ovarian granulosa cells; the ultrastructure of ovarian granulosa cells was observed by transmission electron microscopy, and the number of autophagosomes and autolysosomes was compared; Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of p62, Beclin-1, and microtubule-associated protein 1A/1B-light chain 3 (LC3) in ovarian tissue. The results of vaginal smears in the blank group showed regular cyclical changes; the rats in the model group showed prolonged estrous cycle or cycle arrest, mostly in proestrus or metestrus, with overall ovarian atrophy, disordered structure, and decreased granulosa cells. Compared with the blank group, rats in the model group showed increased serum FSH level (P<0.01), decreased serum levels of E2, AMH, and INHB (P<0.01), decreased positive expression of p62 in ovarian granulosa cells (P<0.01), with obvious swelling of ovarian granulosa cells, mild to moderate swelling of mitochondria, slight expansion of rough endoplasmic reticulum, and hypertrophy of Golgi apparatus; the number of autophagosomes and autolysosomes in the ovaries was increased (P<0.01), the expression of p62 protein and mRNA was decreased (P<0.01), and the expression of Beclin-1 and LC3 protein and mRNA in ovarian tissue was increased (P<0.01). Compared with the model group, rats in the EA group and the E2 group showed decreased serum FSH levels (P<0.01), increased levels of E2, AMH, and INHB (P<0.01), increased positive expression of p62 in ovarian granulosa cells (P<0.01), alleviated degree of ovarian granulosa cell damage, with relatively intact organelle morphology, and decreased number of autophagosomes and autolysosomes in the ovaries (P<0.01); the rats also showed increased expression of p62 protein and mRNA (P<0.01), and decreased expression of Beclin-1 and LC3 protein and mRNA (P<0.01) in ovarian tissue. EA at "Gongsun" (SP 4) could improve ovarian reserve function in POI rats by reducing the number of autophagosomes and autolysosomes, up-regulating p62 expression, and down-regulating Beclin-1 and LC3 expression, thus inhibiting autophagy of ovarian granulosa cells, and regulating the serum levels of FSH, E2, AMH, and INHB.

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

Exercise.

Quantitative measurement of resting skeletal muscle [Ca 2+] i following acute and long-term downhill running exercise in mice