10.5061/DRYAD.SBCC2FR2Q
Wang, Zhe
0000-0002-4950-9954
Northwest University
Up-regulation of sarcoplasmic reticulum function protects skeletal muscle
against cytoplasmic calcium overload during hibernation in ground
squirrels
Dryad
dataset
2019
National Natural Science Foundation of China
https://ror.org/01h0zpd94
31772459
2019-12-11T00:00:00Z
2019-12-11T00:00:00Z
en
77705223 bytes
1
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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. INSERT TABLE 1 HERE 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.