Predominant synthesis of giant myofibrillar proteins in striated muscles of the long-tailed ground squirrel Urocitellus undulatus during interbout arousal

Molecular mechanisms underlying muscle-mass retention during hibernation have been extensively discussed in recent years. This work tested the assumption that protein synthesis hyperactivation during interbout arousal of the long-tailed ground squirrel Urocitellus undulatus should be accompanied by increased calpain-1 activity in striated muscles. Calpain-1 is known to be autolysed and activated in parallel. Western blotting detected increased amounts of autolysed calpain-1 fragments in the heart (1.54-fold, p < 0.05) and m. longissimus dorsi (1.8-fold, p < 0.01) of ground squirrels during interbout arousal. The total protein synthesis rate determined by SUnSET declined 3.67-fold in the heart (p < 0.01) and 2.96-fold in m. longissimus dorsi (p < 0.01) during interbout arousal. The synthesis rates of calpain-1 substrates nebulin and titin in muscles did not differ during interbout arousal from those in active summer animals. A recovery of the volume of m. longissimus dorsi muscle fibres, a trend towards a heart-muscle mass increase and a restoration of the normal titin content (reduced in the muscles during hibernation) were observed. The results indicate that hyperactivation of calpain-1 in striated muscles of long-tailed ground squirrels during interbout arousal is accompanied by predominant synthesis of giant sarcomeric cytoskeleton proteins. These changes may contribute to muscle mass retention during hibernation.

. Animal body weight, heart weight and heart weight to animal body weight ratio. Values are means ± SD. SA summer activity, HIB hibernation, IBA interbout arousal. **Significant difference vs SA, p < 0.01.

Groups
Animal body weight, g Heart weight, g Heart weight/animal body weight, mg/g   Tables S8, S9). Image alignment was made using the IGL Align sEM Align program. The 3D images of muscle fibres were formed using IGL Trace software. Contours of muscle fibres were manually retouched in each image (Supplementary Figs. S1, S2). Quantitative parameters were calculated using 3D View 3.5 software. The statistical analysis of the results was carried out with SigmaPlot 11.0, from Systat Software, Inc., San Jose California USA, https ://www.systa tsoft ware.com. The data were analysed using nonparametric single-factor dispersion analysis for repeated measurements (Kruskal-Wallis One Way Analysis of Variance on Ranks) followed with the pairwise comparison by the Tukey's test. Values are means ± SEM (SD = 129.3 for SA, 119.9 for HIB and 113.3 for IBA). **p < 0.01 (vs SA), ## p < 0.01 (IBA vs HIB). Supplementary Tables S11, S13). A decrease in the total calpain-1 content was detected in the heart during torpor (by 24.7%, p < 0.01) (Fig. 2, Supplementary Table S10). Only a tendency to a decrease in the calpain-1 content (by 18.4%) was observed in the hearts of IBA ground squirrels (Fig. 2). The total calpain-1 contents in m. longissimus dorsi of ground squirrels from the three experimental groups were equal (Fig. 2, Supplementary Table S12). A 19.1% (p < 0.05) decrease in calpastatin content was found in the heart during torpor (Fig. 2, Supplementary Table S14). No statistically significant differences were revealed in calpastatin content in the heart of ground squirrels from the IBA and SA groups (Fig. 2). No seasonal differences were observed in the calpastatin contents in m. longissimus dorsi or in the Hsp 90α/ß content in the heart and m. longissimus dorsi of ground squirrels from the three experimental groups (Fig. 2, Supplementary Tables S15-S17).

SDS-PAGE analysis of titin and nebulin contents.
The content of intact titin-1 (T1) decreased in the heart (by 16%, p < 0.05) and m. longissimus dorsi (by 14.4%, p < 0.01) during torpor ( Fig. 3; Supplementary Tables S18, S20). The content of proteolytic fragments of titin (T2) also decreased in the muscles of HIB animals (by 28.8% and by 2.1 times, respectively, p < 0.01) ( Fig. 3; Supplementary Tables S19, S21). No reliable differences were found between T1 and T2 contents in striated muscles of ground squirrels from the SA and IBA groups (Fig. 3). The nebulin content was unchanged in m. longissimus dorsi of ground squirrels from the three experimental groups (Fig. 3, Supplementary Table S22).

Determination of titin phosphorylation level.
We observed a decreased T1 phosphorylation level (by 23%, p < 0.05) in the heart during hibernation (Fig. 4, Supplementary Table S23). An increased T1 phosphorylation level (by 31.6%, p < 0.05) was observed in m. longissimus dorsi during torpor (Fig. 4, Supplementary  Table S24). No reliable differences were found between T1 phosphorylation levels in striated muscles of summer active animals and ground squirrels during interbout arousal (Fig. 4). Since the T2 content was comparatively low during hibernation and its phosphorylation in striated muscles of ground squirrels was insignificant, seasonal changes in the phosphorylation level of T2 were not compared.
Electron microscopy of sarcomeric structure. We observed no changes in the sarcomeric structure of the heart from the three experimental groups (Fig. 5). Disorders of the sarcomeric structure were found in m. longissimus dorsi during hibernation ( Fig. 6C-F). About one third of m. longissimus dorsi muscle fibres during torpor were represented by straight ordered sarcomeres. Two thirds of the muscle fibres exhibited disordered sarcomeric structures with dramatic changes in sarcomeric length (cf. length S in Fig. 6A,C), disordering of myofilament directions (arrowheads in Fig. 6E), disappearance of I-, A-, H-zones and M-lines (see Fig. 6C,E,F), dissociation of Z-disk structure (Z in Fig. 6E,F), and detachment of myofilaments from Z-disks (asterisks in Fig. 6D,F) and from one another (arrowheads in Fig. 6D,F). No sarcomeric structure disorders in m. longissimus dorsi of IBA ground squirrels were found. protein synthesis rate. SUnSET measurements revealed a significant decrease in the rate of total protein synthesis in the heart (by 3.67 ± 0.8, p < 0.01) and m. longissimus dorsi (by 2.96, p < 0.01) during interbout arousal as compared to that in summer active animals (Fig. 7A,C; Supplementary Tables S25, S26). No reliable differences were found in the synthesis rates of titin and nebulin in ground squirrel muscles from the SA and IBA groups (Fig. 7B,D; Supplementary Tables S27-S29).

Discussion
Seasonal changes in the total content of calpain proteases (calpain-1, calpain-2) and their inhibitor calpastatin in striated muscles of such hibernators as the Daurian ground squirrel 7,22,36 and thirteen-lined ground squirrel 30,31 have been investigated earlier. Various changes have been found in the content of these proteins in different Figure 2. Changes in the contents of calpain-1, calpastatin and Hsp 90 in the hearts and m. longissimus dorsi of ground squirrels. SA summer activity, HIB hibernation, IBA interbout arousal. (A) Representative immunoblots of the proteins (contrast of all images, + 100; brightness, from -18 to -105). Full-length blot for cardiac calpain-1 is given in Supplementary Figs. S3, S4. The first (SA), third (HIB) and fourth (IBA) tracks from the full-length blot ( Supplementary Fig. S3) are shown in (A). Full-length blots for other proteins are in Supplementary Figs. S5, S6, S8-S13. The original images are at: https ://drive .googl e.com/open?id=1Xljy CN3sf Wktju HRPAC ab3eQ fOc6i 2AC. (B) Content of autolysed fragments of calpain-1 (as percentage of the total content of calpain-1). Increased content of autolysed calpain-1 fragments was found both in the heart (1.32-and 1.54-fold, p < 0.05) and m. longissimus dorsi (LD; 1.69-fold, p < 0.05 and 1.8-fold, p < 0.01) during hibernation and interbout arousal, respectively (Supplementary Tables S11, S13). (C) Bar graphs of the total contents of calpain-1, calpastatin and Hsp90α/ß (Supplementary Tables S10, S12, S14-S17). The statistical analysis of the results was carried out with SigmaPlot 11.0, from Systat Software, Inc., San Jose California USA, https :// www.systa tsoft ware.com. The data were analysed using nonparametric single-factor dispersion analysis for repeated measurements (Kruskal-Wallis One Way Analysis of Variance on Ranks) followed with the pairwise comparison by the Tukey's test. Values are means ± SD. *p < 0.05, **p < 0.01 (vs SA). n = 7/group. www.nature.com/scientificreports/ torpor-activity cycles in striated muscles of these hibernators. This research is the first to report on seasonal changes in the total content of calpain-1 and calpastatin in striated muscles of the long-tailed ground squirrel Urocitellus undulatus. No seasonal differences were observed in calpain-1 and calpastatin contents in m. longissimus dorsi of animals from the three experimental groups (Fig. 2). A comparatively minor but statistically significant decrease in the content of these proteins was found in the heart of HIB ground squirrels (Fig. 2). This article is also the first to communicate an increased autolysis of calpain-1 in striated muscles of a hibernating animal during interbout arousal (Fig. 2). These data are indicative of a hyperactivation of calpain-1 in the heart and m. longissimus dorsi of the long-tailed ground squirrel during this period. These changes were accompanied by the recovery of T1 content, which was reduced in striated muscles during hibernation (Fig. 3). Simultaneously, the content of titin T2 fragments significantly increased in striated muscles in the IBA group as  Tables S18-S22). The statistical analysis of the results was carried out with SigmaPlot 11.0, from Systat Software, Inc., San Jose California USA, https ://www.systa tsoft ware.com. The data were analysed using nonparametric single-factor dispersion analysis for repeated measurements (Kruskal-Wallis One Way Analysis of Variance on Ranks) followed with the pairwise comparison by the Tukey's test. Values are means ± SD. n = 7/ group. *p < 0.05, **p < 0.01 (vs SA). # p < 0.05 (IBA vs HIB).

Scientific RepoRtS
| (2020) 10:15185 | https://doi.org/10.1038/s41598-020-72127-y www.nature.com/scientificreports/ compared to the HIB group (Fig. 3). These results testify to an increased titin turnover in striated muscles during interbout arousal. The increase can be explained by the hyperactivation of calpain-1 because titin is substrate for this protease. Our SUnSET data are consistent with these results. A significant decrease was found in the rate of total protein synthesis (MW range, 10-200 kDa) in the heart and m. longissimus dorsi during interbout arousal, whereas no reliable differences were observed between the synthesis rates of titin and nebulin in the muscles of animals from the IBA and SA groups (Fig. 7). These changes were accompanied by the restoration of the highly ordered sarcomeric structure of m. longissimus dorsi (Fig. 6B), by a slight increase in the volume of muscle fibres of m. longissimus dorsi ( Fig. 1) and by a trend towards an increase in the mass of the heart muscle in the IBA group (Table 1). Titin and nebulin are giant proteins of thick and thin filaments and play the role of a sarcomeric cytoskeleton. The renewal of these proteins and the recovery of their content during interbout arousal shall, undoubtedly, contribute to an increase in muscle mass. Thus, our results show that the hyperactivation of calcium-activated protease calpain-1 in striated muscles of long-tailed ground squirrels during interbout arousals is accompanied by a recovery/renewal of the titin and nebulin contents and an increase in muscle mass. Data on Ca 2+ overload during interbout arousals in three skeletal muscles of Daurian ground squirrels 35 are consistent with our findings. An increased activity of calpain-1 may contribute to an activation of the NFAT-calcineurin pathway 30,31 , as well as to an increase in myofibrillar protein turnover, which is initiated by calpain-dependent proteolysis of titin and nebulin. These changes, in turn, may contribute to muscle mass retention during hibernation. The results obtained do not contradict the conclusion made by the authors of Ref. 33 that the myofibrillar remodelling is most likely responsible for preventing skeletal muscle atrophy during a prolonged disuse in hibernation.  Tables S23, S24). The statistical analysis of the results was carried out with SigmaPlot 11.0, from Systat Software, Inc., San Jose California USA, https ://www.systa tsoft ware.com. The data were analysed using nonparametric single-factor dispersion analysis for repeated measurements (Kruskal-Wallis One Way Analysis of Variance on Ranks) followed with the pairwise comparison using the Tukey's test. Values are means ± SD. n = 5/group. *p < 0.05 (vs SA).

Scientific RepoRtS
| (2020) 10:15185 | https://doi.org/10.1038/s41598-020-72127-y www.nature.com/scientificreports/ An increased content of autolysed fragments of calpain-1 was also observed in the heart and longissimus dorsi muscle of long-tailed ground squirrels during torpor (Fig. 2). There is no doubt that in hypothermia, when the muscle temperature is 1.5-2.5 °C, the activity of calpain-1 is considerably but, apparently, not completely, inhibited. The level of titin, especially its T2 fragments, reduced in muscles of long-tailed ground squirrels during torpor (Fig. 3). Similar changes were found in striated muscles of the brown bear (Ursus arctos) during winter sleep 52 . These results show that the proteolytic activity of calpains in striated muscles of hibernating animals is maintained during hibernation. These data are consistent with the results of our earlier research using casein zymography 53 . The ability of calpain-1 (μ-calpain) and calpain-2 (m-calpain) to proteolyse casein in gel was significantly higher in extracts prepared from striated muscles of long-tailed ground squirrels from the HIB group as compared to the SA group 53 . Incomplete inhibition of calpain proteolytic activity may be due to Ca 2+ overload observed, for instance, in the gastrocnemius muscle of Daurian ground squirrels during hibernation 35 . The authors of Ref. 35 have hypothesised that Ca 2+ overload may activate calpains and promote protein degradation during hibernation. In this context, it is necessary to discuss our data related to changes in the titin phosphorylation level in the heart and longissimus dorsi muscle of the long-tailed ground squirrel.
It is known that phosphorylation modifies the sensitivity of proteins to degradation by calpain-1 54 . We found no direct experimental evidence to confirm a change in the sensitivity of titin to proteolysis mediated by a change in its phosphorylation level. However, there are indirect data testifying that hyperphosphorylation of skeletal muscle titin enhances its sensitivity to cleavage by calpains (for references, see Ref. 55 ). Our data obtained in this work do not contradict this assumption. A decreased titin content was observed in longissimus dorsi muscle during hibernation against the background of the increased phosphorylation level of T1 (Figs. 3, 4). The hyperphosphorylation of skeletal muscle titin can be mediated by calcium-dependent protein kinases such as PKC, the activity of which should increase under Ca 2+ overload identified in striated muscles of Daurian ground squirrels during torpor 35 . A decrease in the T1 content in the skeletal muscle of long-tailed ground squirrels from the HIB group probably contributed to sarcomeric structure disorders (Fig. 6), because it is known that giant elastic protein titin (also known as connectin) plays an important role in maintaining an ordered sarcomeric structure 56,57 . An increase in the number of PO 3 groups in titin could also contribute to that of interfilament spacing within this muscle (Fig. 6D-F). These changes may increase the access of proteases to the most vulnerable parts of the titin molecule.
A decreased phosphorylation of titin was detected in the cardiac muscle of long-tailed ground squirrels during torpor (Fig. 4). The molecular mechanisms of these changes are not clear, but hypophosphorylation of titin www.nature.com/scientificreports/ might probably be responsible for the decrease in its sensitivity to proteolysis in cardiac muscle, the contractile activity of which is not completely suppressed during hibernation. This hypothesis needs to be proven. When discussing the results, the point to note is that there were no appreciable seasonal changes in calpastatin and Hsp 90 levels (Fig. 2). It was shown that Hsp 90 interacted with calpain-1, not with calpain-2, to form a discrete complex where the protease maintains its catalytic activity, though with a lower affinity for Ca 2+ ions 51 . Keeping in mind that the level of Hsp 70 was 1.7 times higher during hibernation and the early phase of arousal in skeletal muscles of the hibernating bat Murina leucogaster 58 , we expected to observe similar changes for Hsp 90, which in turn would have led to an increased activity of calpain-1. This assumption was not confirmed. No significant seasonal differences were found in the level of Hsp 90 in investigated striated muscles of long-tailed ground squirrels (Fig. 2). Our results provide evidence that calpain-1 is the most essential of the investigated proteins for turnover and de novo recovery of giant proteins of thick and thin filaments during interbout arousals.
In conclusion, the results of our study indicate that increased autolysis of calpain-1 was accompanied by predominant synthesis of giant myofibrillar proteins titin and nebulin with significant inhibition of synthesis of other proteins in striated muscles of long-tailed ground squirrels during interbout arousals. These changes were accompanied by a slight increase in the volume of muscle fibres of m. longissimus dorsi, by a trend towards an increase in heart muscle mass and recovery of a highly ordered sarcomeric structure of m. longissimus dorsi in the IBA group. Myofibrillar remodelling most likely plays an important role in long-tailed ground squirrels both in preventing skeletal muscle atrophy and in cardiac muscle mass retention during hibernation. However, it is to be verified if such changes would be observed in other skeletal muscles, e.g., hind-limb muscles, in this ground squirrel species and in other hibernators. It may be assumed that both atrophic changes and those of various molecular parameters can differ significantly depending on the type of muscles containing-to a greater or lesser extent-slow-or fast-twitch fibres. In this regard, it looks promising to compare two traditionally investigated skeletal muscles-slow-twitch m. soleus and fast-twitch m. gastrocnemius.

Materials and methods
Experimental animals. Long-tailed ground squirrels Urocitellus undulatus of both sexes (body mass, 450-750 g; estimated age, between 1 and 2 years old) were captured in early August 2017 and early August 2018 in Yakutiya (Siberia), transported by air to Pushchino (Moscow Region) and housed in individual cages (74 × 57 × 55 cm) in a specially equipped vivarium under natural photoperiodicity. Food was supplemented with sunflower seeds and carrots, and nesting material was provided ad libitum. Early in November, the animals were weighed, then the cages with the animals were transferred to a darkroom with a temperature of 1-3 °C for the onset of the hibernation season. The weights of the animals were 535-845 g (Supplementary Table S5).  Tables S25, S26). There was a significant decrease in the rate of total protein synthesis in the heart (by 3.67, p < 0.01) and m. longissimus dorsi (by 2.96, p < 0.01) during interbout arousal in comparison with those in summer active animals (n = 5/group). (D) Bar graphs of titin and nebulin syntheses in striated muscles (Supplementary Tables S27-S29). No significant differences were found between the synthesis rates of titin and nebulin in muscles of ground squirrels from the two groups (n = 3/group). The data were analysed using the nonparametric Mann-Whitney U criterion. Values are means ± SD. **p < 0.01. In December, hibernation bouts were 7-8 days; in January-February, 10-14 days. In March, hibernation bouts lasted for 8-10 days. Food was not provided during hibernation. "Deep torpor" samples were taken from animals sacrificed in the predicted mid-bout. Spontaneously aroused animals were placed into individual cages, which contained nesting material and ~ 100 g of cabbage as a source of moisture. After several hours these animals were sacrificed for taking samples. As wooden hibernation boxes became freed, they got occupied by other ground squirrels, which were preliminarily weighed.

Scientific RepoRtS
Experiments were carried out with three groups of animals taken at different phases of their annual cycle: (1) hibernation (HIB; hypothermia, myocardium temperature, 2.3 °C ± 0.1 °C; rectal temperature, 1.

SDS-PAGE analysis and Western blotting.
Extraction of calpain-1 from muscle tissues was based on a previously described method 59 . Muscle tissues were homogenised in lysis buffer (Tris buffer) containing 0.4 M Tris HCl, pH 6.8, and 25 mM EGTA. Following homogenization, 4% SDS was added to the buffer. The homogenates were then incubated at 4 °C for 20-40 min and centrifuged at 3,000×g for 5 min. Subsequently, the supernatant was collected and mixed (2:1 v/v) with SDS loading buffer (0.125 M Tris HCl, 10% glycerol, 4% SDS, 4 M urea, 10% ß-mercaptoethanol and 0.001% bromophenol blue, pH 6.8). The samples were heated at 95 °C for 4 min. Calpastatin, Hsp 90 and GAPDH (reference protein, Table 2) were extracted from muscles using lysis buffer (12 mM Tris HCl, 1.2% SDS, 5 mM EGTA, 10% glycerol, 2% ß-mercaptoethanol, 5 µg/ml leupeptin and E64, pH 6.8-7.0). Total protein concentrations in the samples were measured by the Bradford method according to the manufacturer's recommended protocol (Sileks, Russia). Bovine serum albumin was used as a standard. The protein samples were electrophoresed in 6.5% (for calpain-1) and 9.5% (for the other above-mentioned proteins) polyacrylamide slab gels 60 . Equal protein amounts were added to the gels (muscle samples of all experimental groups were run on the same gel).
Protein transfer to PVDF or nitrocellulose membranes was run for 2 h at 100 mA according to a previously described method 61 Fig. S24, pp. 50-51). Secondary antibodies conjugated to alkaline phosphatase (goat anti-rabbit Ig, 1:3,000; Abcam, ab6722 and goat anti-mouse Ig, 1:3,000; Abcam, ab6790) were used. Incubation with secondary antibodies proceeded at 21-23 °C for 1 h. Then the membranes were washed in buffer (PBS, pH 7.4; and 0.05% Tween 20) 5 times for 5 min. An NBT/BCIP substrate solution (Roche, Basel, Switzerland) was used to visualise the antibody-protein complexes. Incubation with this substrate was carried out at room temperature for 5-30 min. The GAPDH protein contents were used as loading controls. All stages of incubation of PVDF or nitrocellulose membranes were carried out on an MR-1 Mini-Rocker Shaker (Biosan, Latvia).

Determination of titin phosphorylation level.
The level of titin phosphorylation was determined using a previously described method 65 with minor modifications. The native level of protein phosphorylation was estimated in the gels using a Pro-Q Diamond fluorescent dye (ThermoFisher Scientific). The gels were incubated in an aqueous solution of 50% EtOH and 10% acetic acid for 12-18 h, washed with distilled water for 30 min and stained for 1.5 h. The gels were then washed with Pro-Q Diamond phosphoprotein gel destaining solution (ThermoFisher Scientific), and protein bands containing phosphate groups were visualised using a Bio- Table 2. GAPDH levels in the heart and m. longissimus dorsi of ground squirrels (Supplementary Tables S6,  S7). Values are means ± SD. SA summer activity, HIB hibernation, IBA interbout arousal.  Supplementary Fig. S1). At least 8-20 consecutive shots of one slice were taken by the checkerboard method. Then a panoramic image of each section was produced using PTGui 9.1.8 Pro; the contrast and brightness of the sections were changed using Adobe Photoshop. The scale of the object micrometer at the same magnification was photographed. The panoramic images of the serial sections were aligned relative to one another in IGL Align sEM Align. Each point marked on the reference image was indicated by a corresponding point on an image to be aligned 67 . In addition, the contours of external perimysium served as reference points. The outlines for each muscle fibre were constructed by manual segmentation in IGL Trace (version 1.20b) 67 . For calibration, the shrinkage coefficient of muscle tissue (5%) induced by postfixation procedures was taken into account (see Supplementary Fig. S2). The 3D images of muscle fibres were generated in the wrl/vrml format followed by their transformation into the raw format. The 3D images of muscle fibres were formed using IGL Trace software (see Supplementary Fig. S2). The quantitative parameters were calculated using the commercial program Actify's 3D View. The volumes of 300-375 muscle fibres (the number of fibres for each muscle sample was 75) were calculated for each animal group (see Supplementary Tables S8, S9). Since the distribution of the volume data was not normal (Shapiro-Wilk test), we estimated the significance of differences using nonparametric single-factor dispersion analysis for repeated measurements (Kruskal-Wallis One Way Analysis of Variance on Ranks) which was followed with the pairwise comparison by the Tukey's test. The values are given as M ± SEM or M ± SD, where M is the mean value, SEM is the standard error of the mean and SD is the standard deviation. Statistical significance was set at p ≤ 0.05. The dynamics of atrophic change development in the investigated skeletal muscle from December to March was not studied.
SUnSET technique for measuring the protein synthesis rate. SUnSET (surface sensing of translation) is a nonradioactive technique for in vivo measurement of protein synthesis in striated muscles 68 . The technique involves the use of the antibiotic puromycin (a structural analogue of tyrosyl-tRNA) and anti-puromycin antibodies to detect the amount of puromycin incorporation into nascent peptide chains. The SUnSET technique uses standard Western blotting and immunohistochemical technologies to visualise and quantify in vivo rates of protein synthesis [68][69][70] . In our experiments for in vivo measurements of protein synthesis, summer active (n = 5) and winter active (n = 5) ground squirrels were injected intraperitoneally with 40 nmol/g puromycin hydrochloride (Enzo Life Sciences, USA) diluted in normal saline (Solopharm, Russia). Exactly 20 min after the injection, the animals were anaesthetised with Zoletil. Exactly 25 min after the puromycin injection, the anaesthetised ground squirrels were euthanised by decapitation, striated muscle tissue was collected and frozen immediately in liquid nitrogen for Western blotting analysis.
Densitometry and statistical analysis. Gels and membranes were digitised, and the data were processed using Total Lab v1.11 software (Newcastle Upon Tyne, England). The levels of calpain-1, calpastatin and Hsp 90 were analysed relative to GAPDH (reference protein). Applying the SUnSET assay, the levels of proteins synthesised in vivo were identified relative to the total protein (measured by the Bradford method or stained with Ponceau S). To determine the titin and nebulin contents relative to the MyHC content, the total optical density (OD) of the MyHC peak, as well as the total OD of the nebulin and titin peaks (T1 isoforms (T1) and T2 fragments (T2)), were determined. There is evidence that the titin/myosin ratio within the A-band titin in Scientific RepoRtS | (2020) 10:15185 | https://doi.org/10.1038/s41598-020-72127-y www.nature.com/scientificreports/ the sarcomere is 6 titin molecules per half myosin filament 71 . A previously described method for estimating the titin and nebulin contents relative to the MyHC content is widely used 72 . This approach is more precise than that for measuring the titin content relative to the total protein content in a sample. The statistical analysis of the results was carried out with SigmaPlot 11.0, from Systat Software, Inc., San Jose California USA, www.systa tsoft ware.com. Since the distribution of some data samples was not normal (Shapiro-Wilk test), we estimated the significance of differences using nonparametric single-factor dispersion analysis for repeated measurements (Kruskal-Wallis One Way Analysis of Variance on Ranks) which was followed with the pairwise comparison by the Tukey's test. Data obtained while measuring the protein synthesis rates in striated muscles of ground squirrels from the SA and IBA groups were analysed using the nonparametric Mann-Whitney U criterion. The values are presented as M ± SD, where M is the mean value and SD is the standard deviation. The differences were considered to be statistically significant at p < 0.05.
Equipment and settings. Gels and blots were digitised at 1,200 dpi on an Epson Perfection 3,200 PHOTO scanner with Epson scan 3.0 software. Photoshop software was used to enhance the brightness/contrast levels across the entire field of each image. Minor manipulations were performed to remove contamination in the figures of the article. Densitometry was performed using Total Lab v1.11 software (Newcastle Upon Tyne, England). The measurement tracks were created manually. Background subtraction was done by the rubber-band tool. Protein bands containing phosphate groups were visualised using a Bio-Rad ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Gels were placed on the Chemi/UV/Stain-Free Tray, and measurements were carried out according to the following parameters: Application Category-Protein Gel-SYPRO Ruby. Exposure time was 10-15 s. For 3D reconstruction, exposure times during imaging varied from 1/160 to 1/640 s. The size of the images was 2464 × 1632 pixels (8 bits/pixel). The panoramic image of each section was produced using PTGui 9.1.8 Pro at 300 × 300 resolution. Alignment of panoramic image series was carried out using IGL Align sEM Align (version 1.20b). Muscle fibre 3D reconstructions were created using the IGL Trace software (version 1.20b), courtesy of Dr John Fiala (Boston University, USA, http//www.synap ses.bu.edu/). Muscle fibre volumes were measured using Actify's 3D View according to the following parameters: Model-Mass properties-Volume (render mode, smooth shading).