Materials
N-benzyl-p-toluene sulfonamide (BTS) was purchased from Tocris Biosciences (Bristol/UK, England), latrunculin A from AdipoGen (San Diego/California, United States), and 4-chloro-m-cresol (4CmC) from Pfaltz & Bauer (West Chester/Pennsylvania, United States). The calcium dyes fura-2 AM and mag-fluo-4 AM were purchased from Invitrogen (Grand Island/New York, United States). Molecular weight marker for western blotting was purchased from GenDEPOT (Barker/TX, United States). Every other reagent including Insulin, KN-92, KN-93, and the myristoylated autocamtide 2-related inhibitory peptide (AIP) were purchased from Sigma-Aldrich (St. Louis, United States).
Animals
In this study, male mice between six and 12 weeks old were used (The mice were generated by the Hamilton lab and backcrossed with the C57BL/6 J mice obtained from Jackson’s Laboratory (Bar Harbor/Maine, United States)), unless otherwise indicated. All mice were housed at room temperature with a 12:12 hour light-dark cycle and provided with food and water ad libitum. All procedures were approved by the Animal Care Committees at Baylor College of Medicine (Texas, United States) and the University of Rochester (New York, United States).
Creation of E1014K mice
The EK mutation was engineered on a genomic fragment of about 500 bp containing the exon-encoding residue E1014. A tetracycline resistance gene cassette (tet, for bacteria selection) and a neomycin cassette (neo, for ES cell selection) were inserted into the middle of the engineered fragment, flanked by about 250 bp homologies to CaV1.1. This selection marker-containing fragment was used to isolate a larger CaV1.1 genomic clone from a mouse 129 phage library via homologous recombination in Escherichia coli [20]. Several clones were isolated and we chose one with an appropriate length of homologies on each side of the two cassettes for electroporation into AB2.2 ES from the 129SvEv cells. Recombinant ES clones were identified using southern blot analysis, and one of the clones was injected into blastocysts derived from C57BL/6 J mice to produce chimeras. The targeted allele was germ-line transmitted and the two selection cassettes were removed through crosses with Meox2-Cre mice [21].
To expedite the transfer of the E1014K mutation from the 129SvEv mouse sub-strain background to a congenic C57BL/6 J background, speed congenics were used, in addition to conventional backcrossing. We identified 92 microsatellites of maximal base-pair length disparity between the 129SvEv and C57BL/6 J strains. These microsatellites were chosen to representatively span the entire genome and show distinct electrophoretic separation. Primer pairs were selected to PCR amplify the chosen microsatellites to be resolved by electrophoresis in Spreadex EL300 Wide Mini Gels (Elchrom Scientific, Cham, Switzerland). Finally, we compared 129SvEv and C57BL/6 J DNA standards to DNA from our backcrossed mice, and selected mice with the most sequence homology to C57BL/6 J DNA to be used in the next backcross. This method both ensured congenicity between the wild-type (WT) and EK mutant mice and provided a means to more quickly begin experimentation.
Isolation of flexor digitorum brevis muscle fibers
Skeletal muscle fibers were isolated from the flexor digitorum brevis (FDB) muscle obtained from WT and EK mice as described [22].
Whole-cell patch clamp recordings of CaV1.1 currents
The whole-cell patch clamp technique was used to assess CaV1.1 currents (ICa) in FDB fibers isolated from WT and EK mice. FDB fibers were bathed in an external recording solution containing (in mM): 157 TEA-methanesulfonate, 2 CaCl2, 10 HEPES, 0.5 anthracene-9-carboxylic acid (9-AC), and 0.1 BTS, at pH 7.4, adjusted with TEA-OH. The patch pipette internal solution contained (in mM): 140 Cs-methanesulfonate, 10 HEPES, 20 Na-EGTA, and 4 MgCl2, at pH 7.4, adjusted with CsOH. All reagents here were purchased from Sigma Aldrich (St. Louis/Missouri, United States). The patch pipette resistance when placed in the external solution was between 0.6 and 1.0 Mohm. Fibers were voltage-clamped at a holding potential of −80 mV. Series resistance was compensated up to 80%. Data were sampled every 120 μs and filtered using a low pass Bessel filter (Axon Instrument, Jakarta/Selatan, Indonesia) with a 2 kHz cut-off frequency. ICa was activated by 200 ms depolarizing pulses ranging from −40 mV to +60 mV in 10 mV increments delivered every 10 seconds. CaV1.1 current-voltage relationships (ICa-V) were obtained from peak currents measured during each depolarization normalized to cell capacitance and plotted against the corresponding test potential. ICa-V data were then fitted by the following modified Boltzman equation:
$$ \left(\mathrm{V}\right) = {\mathrm{G}}_{\max }*\left({\mathrm{V}\hbox{-} \mathrm{V}}_{\mathrm{rev}}\right)/\left(1 + \exp \left[\left({\mathrm{V}}_{0.5}\hbox{--}\ \mathrm{V}\right)/{\mathrm{k}}_{\mathrm{g}}\right]\right) $$
(1)
where G
max
is the maximal L-channel conductance, V is test potential, V
rev
is the L-channel reversal potential, V
0.5
is the potential for half-maximal activation of G
max
, and k
g
is a slope factor.
CaV1.1 currents were analyzed using Igor Pro 6 (Lake Oswego, Oregon, United States) and Clampfit 9 (Sunnyvale, California, United States) software.
Measurements of electrically-evoked Ca2+ release in flexor digitorum brevis muscle fibers stimulated during a single twitch
Acutely isolated FDB fibers were loaded for 20 minutes at room temperature with 4 μM mag-fluo-4 AM in a Kreb’s Ringer solution containing (in mM): 146 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES, at pH 7.4. Fibers were then washed and incubated for 20 minutes in dye-free Ringer’s solution supplemented with 20 μM BTS, a skeletal muscle myosin inhibitor, to block contraction. Mag-fluo-4 AM-loaded FDB fibers were excited at 480 ± 15 nm and fluorescence emission detected at 535 ± 20 nm was collected at 10 kHz using a photomultiplier system. Electrical field stimulation (8 V, 1 ms, and 10 stimuli at 1 Hz) was elicited using a glass electrode placed adjacent to the cell of interest. Peak changes in mag-fluo-4 fluorescence for all 10 stimuli were measured as (Fmax-F0)/F0 and then averaged to generate a single peak value for each fiber. The rate of mag-fluo-4 fluorescence decay for the second, third, and fourth twitches for each fiber was fitted to a first order exponential function and the resulting amplitude and tau values were averaged.
Mn2+ quench measurements
Mn2+ quench of fura-2 emission was measured in myotubes loaded with 5 μM fura-2 AM for 1 h at 37°C in Kreb’s Ringer solution. Briefly, prior to Mn2+ quench measurements, myotubes (primary cultured cells from muscle of mice) were treated with 100 μM ryanodine to block RyR1-mediated Ca2+ release during subsequent KCl application. Fura-2-loaded myotubes were excited at the experimentally determined fura-2 isosbestic point (362 nm) and emission monitored at 510 nm during perfusion of 50 mM KCl in the presence of 0.5 mM Mn2+. Maximum rates of fura-2 quench during KCl application were determined and evaluated for statistical significance.
Calcium imaging in confocal line scan mode in flexor digitorum brevis muscle fibers stimulated with a single 50 Hz train
To monitor Ca2+ release during electrical stimulation, FDBs fiber were loaded with 5 μM of mag-fluo-4 AM for 30 minutes at room temperature in the presence of 20 μM of BTS. Loaded FDBs were placed on the stage of a confocal microscope with an adapted perfusion system (tyrode with 20 μM BTS at 0.5 mL/min) and imaged in line scan mode using the 20x objective (EC Plan-Neofluar) mounted in the confocal microscope (Zeiss LSM 510 meta, California, United States), one line was acquired every 1.15 milliseconds (3.66 μsec/pixel time) using the 488 nm excitation laser and the LP 505 emission filter (Zeiss, California, United States). FDBs were stimulated with 50 square electrical pulses (200 μsec duration) at 50 Hz and the produced florescence transients was normalized (F/F0) and plotted.
Measurement of Ca2+ transients during repetitive stimulation with 100 Hz trains
Isolated FDB fibers were loaded with 5 μM of mag-fluo-4-AM for 30 minutes at room temperature, followed by washout with fresh DMEM (Life technologies, NY, United States). Electrical stimulation was performed using two platinum wires placed at each side of the fiber oriented longitudinally and fatigue was induced with uninterrupted application of electrical trains (100 Hz, 250 ms, every 1.5 seconds; 0.17 duty cycle) for 300 seconds. For evaluation of RyR1-releasable SR Ca2+ store content, 1 mM of 4CmC was perfused at 3.25 ml/min, applied after 60 trains of electrical stimulation. Mag-fluo-4 fluorescence was collected at 20 Hz. Data were collected and analyzed using Metafluor version 6.2 software (Molecular Devices, California, United States).
Western blotting
Muscles were homogenized and lysed in ice-cold RIPA buffer consisting of (mM): 25 Tris pH 7.6, 150 NaCl, 1 Na3VO4, 10 NaPyroPO4, 10 β-glycerophosphate, 10 NaF, PMSF, protease inhibitor cocktail (Santa Cruz), 1% NP40, 1% sodium deoxycholate, and 0.1% SDS (Every reagents came from Sigma Aldrich, St. Louis, United States). Equal amounts of total protein from whole muscle lysates were resolved by electrophoresis, transferred to PVDF (Millipore, Billercia, United States) membrane and western blot analyses were performed using antibodies shown in Additional file 1: Table S1. LI-COR IRDye™ infrared dyes were used as secondary antibodies and immunoreactive bands were visualized using the Odyssey Infrared Imaging System (LI-COR) (LI-COR Inc, Lincoln, United States). To allow the use of data from multiple western blots, the fluorescent band intensity of each band within a single western was first normalized to GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) as a loading control and then calculated as %WT average from that specific western blot. Data were then pooled to give %WT ± SEM.
Co-localization studies and single fiber immunocytochemistry
Single FDB fibers plated on glass slides were fixed with 2% paraformaldehyde in 0.1 M phosphate buffer (PB) (21.6 mM Na2HPO4 and 81.4 mM NaH2PO4, pH7.2) for one hour at room temperature, washed twice with phosphate buffered saline (PBS) (3.8 mM NaH2PO4, 16.2 mM Na2HPO4, 150 mM NaCl, pH 7.4), permeabilized, and blocked in PBS containing 0.5% Triton X-100 and 5% BSA overnight at 4°C (All reagents come from Sigma Aldrich as described in Materials). Primary antibodies diluted in PBST (PBS containing 0.5% TX-100) were added to slides and incubated overnight at 4°C. After washing twice with PBS, Alexa-fluor conjugated antibodies were added. Fibers were washed three times with PBS for 10 minutes each and mounted in Fluoromount-G (SouthernBiotech, Birmingham, United States). Fibers were imaged using a Zeiss LSM 510 META confocal microscope with a 100x/1.30NA oil lens, HeNe 543 nm laser, and Argon 458,477,488,514 laser (Zeiss, California, United States).
Proximity ligation assay
We used proximity ligation assays (PLAs) to identify proteins that are within 40 Å of each other. PLA was performed on single FDB fibers plated on glass disks. Fibers were kept at 37°C in a 95% O2-5% CO2 incubator in DMEM solution supplemented with 10% FBS. Fibers were then fixed with 2% paraformaldehyde in 0.1 M PB and incubated with the primary antibodies. The PLA was performed with the Duolink kit (Olink Biosciences, Uppsala, Sweden) according to the instruction of the user manual using an anti-goat MINUS PLA and anti-rabbit PLUS PLA probes and the orange detection reagent (Cy3) (Olink Biosciences, Uppsala, Sweden). Fibers were imaged with confocal microscopy (Zeiss LSM 510 META, with a 100x/1.30NA oil lens and HeNe 543 nm laser). For analysis, Z stacks were projected and saved as a single image. The positive spots were counted with Image J (8-bit images filtered with a Gaussian Blur filter (Rasband, W.S Image J, U.S. National Institutes of Health, Bethesda, Maryland, United States), σ = 1, and same threshold per set adjusted at 15-30). The number of spots counted was normalized to the area of the fiber estimated from the width and length of the fiber in the image. For each set of experiments, the average counts of WT fibers (control) were set to 100% and the number of spots in each experimental condition was calculated as percentage change.
Insulin treatment
Eight-week-old WT and EK mice were fasted for 12 hours and then given an intraperitoneal injection of insulin (1 U/kg) diluted in saline. Control mice were injected with saline. After 7.5 minutes mice were sacrificed and muscles (soleus and EDL (Extensor Digitorum Longus)) were isolated, frozen in liquid nitrogen, and stored at −80°C until use. Muscle levels of pAkt1/2 and pGSK3β in the presence and absence of insulin were measured as described in Butler et al. [23].
Detection of puromycin-labelled proteins
For measurement of protein synthesis we used an in vivo SUnSET technique [24,25]. Briefly, mice (13 weeks of age) were food deprived for eight hours. Propofol (18 μl/g) (Abbott Laboratories, North Chicago, United States) was administered via an intraperitoneal injection 15 minutes before the puromycin injection. The mice were then given an intraperitoneal injection of puromycin (0.04 μmol/g BW) and sacrificed 35 minutes later. At 10 minutes before sacrificing, insulin or saline (control) was administered via intraperitoneal injection. Muscles were isolated, homogenized, and prepared for western blotting with anti-puromycin antibody. For normalization to total protein, the same western blots were stained with Swift Membrane Stain™ kit (G-Biosciences, St. Louis, United States).
Ras activity
Ras activity was measured using a Ras activity assay kit (Cytoskeleton, Denver, United States). Briefly, muscle was lysed in buffer and protein concentration was measured. Raf-Ras binding domain (RBD) beads (50 μl) were added to the muscle lysates (total 500 μl of 2 mg/ml lysates) and the mixture was incubated at 4°C on a rotator for one hour. After incubation, Raf-RBD beads were pelleted by centrifugation at 5,000 × g at 4°C for one minute and washed with wash buffer. The bound active Ras was eluted in the two × sample buffer by boiling for three minutes. Eluted protein was run on 12% gel, transferred to PVDF membrane, and immunoblotted with Ras-specific antibody. The westerns were normalized to the amount of GAPDH in the muscle lysates (60 μg).
Sarco/endoplasmic reticulum Ca2+-ATPase activity
Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity in tissue homogenates was performed as described [26,27].
Electrical stimulation of isolated muscle for signaling changes
To assess stimulation-induced changes in signaling pathways we used the method of Sakamoto et al. [28]. Intact soleus and EDL muscles were removed and suspended between a force transducer and stationary anchor within a test chamber filled with warmed (35°C) Kreb’s Ringer solution (KRS) oxygenated with a 95/5% mixture of O2/CO2, as above. After a 30 minute resting equilibration period, muscles to be stimulated underwent a fatigue protocol (100 Hz, 200 ms train duration, one second intervals) for five minutes per muscle. At the completion of the stimulation protocol, rested and stimulated muscles were immediately frozen in liquid N2 and stored at −80°C. For muscles treated with KN-93, the drug was added to the chamber at the start of the 30 minutes equilibration period, at a final concentration of 5 μM.
Muscle force frequency and fatigue
Intact soleus and EDL muscles were removed and immediately immersed in incubation medium comprised of KRS (oxygenated with a 95/5% mixture of O2/CO2. Muscles were tied with sutures at the musculotendinous junction and suspended between a force transducer and stationary anchor within a test chamber filled with warmed (35°C), oxygenated incubation medium. After a 20 minutes rest to allow mounted muscles to equilibrate, muscle optimal length (l
o
) was determined via single-twitch force generation measurements. Next, force frequency measurements were obtained at l
o
using frequencies from 15 to 300 Hz at 200 ms/train followed by a fatigue protocol performed over five minutes per muscle. The specific fatigue protocol for each muscle used was for the soleus: 15 Hz, 200 ms duration, one second intervals) and for EDL: 60 Hz, 200 ms duration, one second intervals. Muscle stimulation occurred within the test chamber using platinum electrodes attached to a Grass S48 stimulator and recorded within Chart5 (version 5.2) software (eDAQ Inc, Colorado Springs, United States).
Fibertyping with cryosections and immunostaining
Skeletal muscles (soleus and EDL) were dissected, embedded in OCT compound (Tissue-Tek, Torrance, United States), and frozen in 2-methylbutane (Sigma Aldrich, St. Louis, United States) precooled in liquid nitrogen. The frozen muscles were sectioned with 10-μm thickness using a SHANDON cryostat microtome (Thermo Electron Corporation, Madison, United States). Immunofluorescent staining was performed using specific antibodies against myosin heavy chain I (MHCI), IIa (MHCIIa), and IIb (MHCIIb) (DSHB, Iowa City, USA). Briefly, sections were rehydrated with PBS for 10 minutes, followed by incubation at 4°C overnight with MHCI (BA-F8, IgG2b), MHCIIa (SC-71, IgG1), and/or MHCIIb (BF-F3, IgGM) antibodies diluted 1:50 in PBS. After washing with PBS, muscle sections were incubated at room temperature for 90 minutes with isotype-specific AlexaFluor-594-conjugated goat anti-mouse IgG2b, AlexaFluor-488-conjugated goat anti-mouse IgG1, and AlexaFluor-594-conjugated goat anti-mouse IgGM secondary antibodies diluted 1:200. After three consecutive washes with PBS, muscle slides were mounted with VECTASHIELD mounting media (Vector Laboratories, Burlingame, United States). Images were taken under a fluorescence microscope (Olympus America, Center Valley, United States). The relative numbers of the different fiber types were quantified and normalized by the total number of muscle fibers per field.
Fiber cross-sectional area
For cross-sectional area (CSA) calculations, 10-μm-thick frozen sections were obtained from the mid-belly area of the soleus and EDL muscles. Sections were immunostained for fiber type as described in the previous section, imaged at 10x magnification through an Olympus DP70 camera (Olympus America, Center Valley, Pennsylvania, United States), and saved in .tif format. Saved images were then imported into Photoshop CSE version 10.0 (Adobe Systems, San Jose, California, United States) for analysis. First, measurement scale was established by tracing a within-image scale bar (μm). Next, myofiber CSA was measured by tracing the external border of individual myofibers using the Magnetic Lasso tool. Myofibers exhibiting evidence of tears or processing artifacts were excluded from the analysis. Recorded measurements were then exported into Excel for analysis, with resulting CSA values reported in μm2.
Statistical analyses
We performed a statistical analyses of two groups using the Student’s t-test. P <0.05 was considered to be statistically significant. *P <0.05, **P <0.01, and ***P <0.001 were used to indicate statistical significance.