Animals, muscle tissues and bundle preparations
Fast- and slow- twitch skeletal muscles were collected from the hindlimbs of young Sprague Dawley rats, i.e., the fast-twitch extensor digitorum longus (EDL) muscle and the slow-twitch soleus (SOL) muscle. The animal experiments were carried out according to the guidelines of the Swedish Board of Agriculture and approved by the ethical committees at Karolinska Institutet. The EDL and SOL muscle tissues were dissected into bundles of approximately 50 fibers (-5 mm long) in relaxing solution containing 50% (vol/vol) glycerol at 4 °C and tied to glass capillaries, stretched to about 110% of their resting slack length. Afterwards the bundles were chemically skinned by treatment for 24 hours at 4 °C in a relaxing solution, and then stored at -20 °C. Within one week after above skinning treatment, the bundles were cryo-protected by transferring them to relaxing solutions containing increasing concentrations of sucrose (0, 0.5, 1.0, 1.5, and 2.0 M) at 30-minute intervals, and then frozen in liquid propane chilled by liquid nitrogen. The frozen bundles were stored at -140 °C. Before the experiment, the bundle was incubated in sucrose solutions with decreasing concentrations (2.0, 1.5, 1.0 and 0.5M) sequentially at 30-minute intervals and then kept in the skinning solution at -20 °C for 2 weeks or shorter prior to usage. [9, 18] The methods are presented in a schematic diagram (Figure 1)
The extraction of myosin and the spectrophotometric quantitation
A muscle mini bundle consisting of 10-20 fibers (that may be adjusted according to the measured concentration of extracted protein) was separated gently and incubated in the microtube with 20 μl a high‐salt buffer (0.5 m KCI, 25 mm Hepes, 4 mm MgCl2, 4 mm EGTA, pH adjusted to 7.6 before adding 2 mm ATP and 1% β‐mercaptoethanol) at 4 °C for 30 minutes. The solution containing extracted myosin and other minor proteins was kept on ice for the application to the different sections of the experiment, i.e., the spectrophotometric quantification of the concentration, QD-mediated thermometry and 12% SDS-PAGE to determine the relative content of myosin in the extracted proteins. The concentration of extracted protein was quantified by conventional spectrophotometry using absorbance at 280 nm (Nanodrop, Thermo Scientific), which does not require any standard curve, but the blank control (contains high-salt buffer only) and internal control (different concentration of BSA, such as 1, 2, 3 and 4 mg/ml) were applied for quality control. Every sample and control for A280 measurement have been briefly and sufficiently vortexed and the quantification was repeated at least 3 times for consistency. The extracted myosin was proportional to the extracted total protein (see the section of results). The A280 value of the extracted total protein was therefore used to represent the concentration of the extracted myosin in most situations unless specified otherwise.
The optimized QD-mediated thermometry of myosin ATPase enzymatic reaction
The assay for determining the saturated ATP concentration was performed firstly. Commercial control myosin protein (1 mg, Cytoskeleton, Inc., Denver, CO) was dissolved in 100 μL of myosin resuspension buffer (15 mM Tris HCl of pH 7.5, 0.2 M KCl and 1 mM MgCl2) then diluted to the concentration of 0.2 μM. For each measurement, 1 µL of the control myosin was pipetted to a well containing 30 µL low‐salt buffers (25mM KCI, 25 mm Hepes, 4 mm MgCl2, 1 mm EGTA, pH adjusted to 7.6 before adding 1% β‐mercaptoethanol) on a black 384‐well microtiter plate, followed by the addition of 1 µL Cadmium telluride core‐type QD (1 mg/mL) (Sigma‐Aldrich) and 30 µL of the low‐salt buffers containing different ATP concentrations (0, 3.5, 4.5, 5, and 6 mM ATP in low‐salt buffers) respectively. After the quantitation, the extracted myosin was optimally diluted for actual measurement, then 1 µL of extracted myosin was pipetted to a well containing 30 µL low‐salt buffer, followed by the addition of 1 µL QD (1 mg/mL) and 30 µL of the blank control (0mM ATP) or ATP solutions (5mM ATP), respectively. The addition of blank control/ATP solutions and the detection of fluorescence signal were performed at the same time to avoid any inconsistency with time course due to the instantaneous enzymatic reaction. The QD fluorescence signal was recorded every 15 seconds for 5 minutes by a fluorescence spectrophotometer (TECAN, Infinite M200, Switzerland), while the excitation and emission wavelengths were fixed at 310 and 530 nm, respectively. The measurements for each preparation were performed in sextuplicate with both the blank control and ATP solution at 25°C. The low salt buffer and QD were kept at room temperature (22°C) and the remaining solutions were kept on ice.
Myosin isoform expression and relative quantitation
After myosin extraction, the mini bundle was placed in SDS sample buffer in a microfuge tube and stored at −80 °C. The composition of MyHC isoforms was determined by 6% SDS-PAGE. The acrylamide concentration was 4% (wt/vol) in the stacking gel and 6% in the running gel, and the gel matrix included 30% glycerol. Sample loads were kept small to improve the resolution of the MyHC bands (type I, IIa, IIx and IIb). Electrophoresis was performed at 120 V for 22 h with a Tris-glycine electrode buffer (pH 8.3) at 10 °C (SE 600 vertical slab gel unit; Hoefer Scientific Instruments, Holliston, MA, USA). The gels were silver-stained and subsequently scanned in a GS-900 Calibrated Densitometer (Bio-Rad). The volume integration function (Image Lab software 6.0, Bio-Rad) was used to quantify the relative amount of each MyHC isoform when more than one isoform was expressed. After the QD-mediated thermometry assay, the remaining myosin preparations were kept in urea buffer in a microfuge tube and stored at −80 °C. The relative quantitation of MyHC contents in total extracted protein was determined by 12% SDS-PAGE. After centrifugation and heating (90°C for 2 minutes) a volume of 4 µl was loaded on 12% SDS-PAGE. The total acrylamide and Bis concentrations were 4% (wt/vol) in the stacking gel and 12% in the running gel. The gel matrix included 10% glycerol. Electrophoresis was performed for 5 h with a Tris–glycine electrode buffer (pH 8.3) at 15°C (SE 600 vertical slab gel unit, Hoefer Scientific Instruments). The gels were stained with Coomassie blue (SimplyBlue SafeStain, Invitrogen), as this staining shows high reproducibility and the ability to penetrate the gel and stain all proteins present, i.e., allowing accurate quantitative protein analyses. The gels were subsequently scanned to determine the relative contents of myosin heavy chain in total extracted protein [9, 19, 20].
Data analysis and statistic
QD fluorescence signals were detected and normalized to the starting fluorescent value and the corresponding relative fluorescence intensity formed a negative hyperbolic regression plotted over time. The linear part of the curve corresponding to the initial 60 seconds, was used to calculate the slope of the relative fluorescence intensity over time. The slope of the blank control subtracted from the slope of the “real” reaction then normalized to the concentration of the extracted myosin, indicating myosin efficiency. For the sextuplicate measurement of each preparation, the subtracted and normalized slope values were evaluated individually according to the criterion, i.e., calculated slopes which fell outside one standard deviation were excluded and the remaining qualified slopes were included, and the slopes in negative values (indicating null reactions) were excluded (Figure 2). Statistical analyses were performed by SigmaPlot software version14. The data were presented as mean ± standard deviation and analyzed by the Student's unpaired t test.