We investigated the potential mechanism of the SR in maintenance of calcium (Ca²⁺) homeostasis of slow-twitch muscle (soleus, SOL), fast-twitch muscle (extensor digitorum longus, EDL) and mixed muscle (gastrocnemius, GAS) in hibernating ground squirrels (Spermophilus dauricus). Results showed that cytosolic and SR Ca²⁺ concentrations in distinct skeletal muscle fibers increased and decreased during late torpor, respectively, but both returned to summer-active levels during early torpor. Ryanodine receptor1 (RyR1) and sarco/endoplasmic reticulum Ca²⁺ ATPase isoform 1 (SERCA1) protein expression increased during hibernation. Up-regulation factors of SERCA activity: Phospholamban phosphorylation increased in the SOL and GAS, β-adrenergic receptor-2 protein expression increased in the GAS, and calmodulin kinase-2 phosphorylation increased in the SOL during hibernation. Down-regulation factors of SERCA activity:  Sarcolipin and SERCA1 co-localization decreased in the EDL and GAS. These data suggest that SERCA activity in skeletal muscle fibers increases likely during hibernation. FKBP12/calsequestrin1 (negative regulatory factors of RyR1) and RyR1 co-localization decreased in the GAS, indicating that the RyR1 channel opening probability increased during hibernation. Dihydropyridine receptors protein expression and its co-localization with RYR1 decreased during hibernation prompts that the contractility of skeletal muscle was weakened. Protein expression of Ca²⁺-binding proteins calsequestrin1 and calmodulin increased indicating that the ability of intracellular free calcium binding increased during whole hibernation period. These findings confirm that the release, uptake, and binding of free Ca²⁺ in the SR were enhanced in different skeletal muscles during hibernation. Up-regulation of muscular sarcoplasmic reticulum function protects skeletal muscle fibers against cytoplasmic calcium overload during hibernation in ground squirrels.We investigated the potential mechanism of the SR in maintenance of calcium (Ca²⁺) homeostasis of slow-twitch muscle (soleus, SOL), fast-twitch muscle (extensor digitorum longus, EDL) and mixed muscle (gastrocnemius, GAS) in hibernating ground squirrels (Spermophilus dauricus). Results showed that cytosolic and SR Ca²⁺ concentrations in distinct skeletal muscle fibers increased and decreased during late torpor, respectively, but both returned to summer-active levels during early torpor. Ryanodine receptor1 (RyR1) and sarco/endoplasmic reticulum Ca²⁺ ATPase isoform 1 (SERCA1) protein expression increased during hibernation. Up-regulation factors of SERCA activity: Phospholamban phosphorylation increased in the SOL and GAS, β-adrenergic receptor-2 protein expression increased in the GAS, and calmodulin kinase-2 phosphorylation increased in the SOL during hibernation. Down-regulation factors of SERCA activity:  Sarcolipin and SERCA1 co-localization decreased in the EDL and GAS. These data suggest that SERCA activity in skeletal muscle fibers increases likely during hibernation. FKBP12/calsequestrin1 (negative regulatory factors of RyR1) and RyR1 co-localization decreased in the GAS, indicating that the RyR1 channel opening probability increased during hibernation. Dihydropyridine receptors protein expression and its co-localization with RYR1 decreased during hibernation prompts that the contractility of skeletal muscle was weakened. Protein expression of Ca²⁺-binding proteins calsequestrin1 and calmodulin increased indicating that the ability of intracellular free calcium binding increased during whole hibernation period. These findings confirm that the release, uptake, and binding of free Ca²⁺ in the SR were enhanced in different skeletal muscles during hibernation. Up-regulation of muscular sarcoplasmic reticulum function protects skeletal muscle fibers against cytoplasmic calcium overload during hibernation in ground squirrels.

Isolation of single muscle fibers. Animals were deeply anaesthetized with sodium pentobarbital (90 mg/kg). Muscle samples with tendons were dissected carefully from surrounding tissues and sarcolemma, ensuring intact nerves and blood supply. The muscles were separated into two complete strips along the longitudinal axis using tweezers, then rinsed with 20 mL of phosphate-buffered saline (PBS, 137 mM sodium chloride, 4.3 mM disodium chloride, 2.7 mM potassium chloride, 1.4 mM monopotassium phosphate, pH 7.4), acutely dissociated with 3 mL of enzymatic digestion solution consisting of 0.35% collagenase I and 0.17% neutral protease (Sigma-Aldrich, Saint Quentin Fallavier, France), and finally incubated at 33 °C on an orbital shaker for 2 h. The enzymatic digestion solution was saturated with 95% O₂ and 5% CO₂ gas mixture to ensure the muscle fibers were completely digested, after which the solution was removed with PBS and the muscles were agitated gently and repeatedly with pipettes [62]. The dissociated single muscle fibers were set onto culture chamber slides and finally observed under an inverted microscope (Olympus, IX2-ILL100, Japan).

Muscle samples for other experiments were subsequently stored in liquid nitrogen until further processing. At the end of surgical intervention, the animals were sacrificed by an overdose injection of sodium pentobarbital. The Northwest University Ethics Committee reviewed and approved all animal study procedures. All procedures were carried out in accordance with approved guidelines.

Measurement of cytoplasm Ca²⁺. Fluo-3-acetoxymethylester (Fluo-3/AM) (Invitrogen, Carlsbad, USA), which exhibits an increase in fluorescence upon Ca²⁺ binding, was used to measure cytosolic free Ca²⁺, as described previously [63]. In brief, the above isolated muscle fibers were incubated in glass petri dishes with Fluo-3/AM at a concentration of 5 mM for 30 min at 37 °C, after which the Fluo-3/AM-loaded muscle fibers were washed with fresh PBS and then scanned under a laser confocal microscope equipped with the Olympus FV10-ASW system (krypton/argon laser illumination at 488 nm and capture at 526 nm). A single muscle fiber with intact morphology and smooth cytomembrane was found at low magnification (100´), with continuous photographs taken of the middle two-thirds segment of the selected muscle fiber at high magnification (400´). Six different areas were randomly selected for fluorescence intensity measurements in each image. Total fluorescence intensity / total area of the selected region was used as the average fluorescence intensity of the muscle fiber, which represented the concentration of Ca²⁺ labeled. The average value of the measured result was taken as the fluorescence intensity of the muscle fiber cytosolic Ca²⁺ concentration. The average value of 10 muscle fibers was taken as the fluorescence intensity of the muscle fiber cytosolic Ca²⁺ concentration. Quantification analysis of the fluorescence intensity was performed with NIH Image J software (Image-ProPlus 6.0).

Measurement of SR Ca²⁺. Magnesium-Fluo-4-acetoxymethylester (mag-Fluo-4/AM) (#M14206, Thermo Fisher Scientific, Rockford, IL, USA), which exhibits an increase in fluorescence upon binding to Ca²⁺, was used to indicate SR free Ca²⁺, as described previously [64]. Briefly, single muscle fibers were incubated with mag-Fluo-4/AM (5 mM) and ER-Tracker Red dye (#E34250, Thermo Fisher Scientific) for 30 min at 37 °C. After incubation on glass petri dishes, the mag-Fluo-4/AM-loaded muscle fibers were washed with fresh PBS and then scanned under a laser confocal microscope equipped with the Olympus FV10-ASW system (Olympus, FV10-MCPSU, Japan) with krypton/argon laser illumination at 488 nm and capture at 526 nm. Average fluorescence intensity was used to indicate changes in SR Ca²⁺ in muscle fibers, with the specific method similar to measurement of cytoplasm Ca²⁺. Quantification analysis of fluorescence intensity was performed with NIH Image J software.

Co-localization analysis of immunohistochemistry. We cut 10-μm thick frozen muscle cross-sections from the mid-belly of each muscle at −20 °C with a cryostat (Leica, Wetzlar, CM1850, Germany), which were then stored at −80 °C for further staining. Immunohistochemistry was used to determine co-localization with DHPR/RyR1, CSQ1/RyR1, FKBP12/RyR1, SLN/SERCA1, and SLN/SERCA2. After air drying for 2 h, the sections were incubated in a blocking solution (5% BSA) (Boster, Wuhan, China) for 10 min at room temperature and, in turn, incubated in a primary antibody (Table 1) solution at 4 °C overnight. On the following day, the sections were incubated with secondary antibody at 37 °C for 2 h. After this, the sections were incubated with another primary antibody and secondary antibody under the same conditions. The details of primary and secondary antibodies are listed in Table 2. Finally, the glass slides were placed in 4’-6’-diamidino-2-phenylindole (DAPI)(1:100, # D9542, Sigma-Aldrich) at 37 °C for 30 min. Images were visualized using a confocal laser scanning microscope by krypton/argon laser illumination at 350 nm, 488 nm, and 647 nm emitted light, and capture at 461 nm, 526 nm, and 665 nm. Six figures were analyzed in each sample and eight samples were analyzed in each group. Pearson coefficient was used to measure the overlap level of two proteins [65], NIH Image software (Image-Proplus 6.0) was used to quantify the co-localization coefficient.

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Quantitative real-time PCR. Total RNA was routinely extracted from muscles using an RNAiso Plus kit (TaKaRa, Dalian, China) according to the manufacturer’s protocols. We determined RNA quality via the OD260/OD280 ratio; only samples with a ratio > 1.8 were reverse transcribed into cDNA using a TAKARA reagent (TaKaRa), then stored at − 20 °C for subsequent reactions. Quantitative real-time PCR (RT-PCR) was performed using a SYBR Premix Ex Taq II kit (TaKaRa). Amplification and dissolution curves were first observed, with the right curve then chosen. Here, α-tubulin (reference gene) and 2^−△△ct were used to analyze the relative concentrations of serca1, serca2, sln, plb, csq1, cam, fkbp12, and ryr1 mRNA.