Bellinger et al. 10.1073/pnas.0711074105.
Fig. 5. Persistence of remodeling and functional impairment of the RyR1 channel after exercise. (A) RyR1 macromolecular complex constituents: RyR1 (total and RyR1-pS2844), bound PDE4D3, PKA, PP1, and calstabin1 after immunoprecipitation of RyR1 from mouse vastus muscle after the indicated exercise protocol [S = sedentary, L = low intensity (15 min twice daily) and H = high intensity (90 min twice daily)] and after the indicated number of days of rest. Developed with ECL. (B) Densitometric quantification of PKA and PP1 bound to RyR1 in A, normalized to the total amount of RyR1 immunoprecipitated. (C) Force-frequency curves of isolated intact EDL muscles from WT sedentary mice and mice after 28 days of swimming exercise and 0, 1, 3, or 7 days of subsequent rest (indicated as 28 Ex, 0 R, etc., n = 4 muscles for each condition, data presented as mean ±SEM). (D) H&E-stained, ´400 light microscopy of EDL muscle from sedentary mice and mice after a 28 day swimming exercise protocol. (E) Western blot for total calstabin1 in EDL muscle lysate from mice after various durations of exercise. (F) Western blot for eNOS (red) in EDL muscle lysate from either sedentary mice or mice after 21 days of fatiguing exercise. RyR1 (green) serves as a loading control.
Fig. 6. Lack of correlation between body weight and exercise capacity in WT and cal1-/- mice. (A) Body weights of cal1-/- mice were slightly smaller than those of WT littermates. (B) Treadmill running time plotted for each individual mouse against body weight on the day before the exercise protocol. WT (blue) and cal1-/- (green) both showed no significant correlation between body weight and treadmill running times. Least-squares linear regression lines are drawn in the corresponding color. Data are presented as mean ±SEM.
Fig. 7. S107 treatment does not effect fiber type during exercise. (A) Histogram of the relative frequencies of calcium transient decay rate t during 1-Hz single-twitch field stimulation of dissociated FDB single muscle fibers from mice after 21 days of exercise and treatment with vehicle (n = 41). (B) Similar histogram from mice after 21 days of exercise and treatment with S107 (n = 54).
Scheme 1. Chemical structure of S107.
Table 1
SI Methods
Synthesis of S107. S26 (180 mg, 0.92 mmol) in MeOH (20 ml) was mixed with a 30% CH2O solution (1.5 ml, excess) and sodium cyanoborohydride (NaBCNH3) (0.4 g, excess). The reaction mixture was stirred at room temperature, and the pH of the solution was maintained between 4 and 5 by addition of a few drop of 1 M HCl. After 3 h, the solvents were removed under reduced pressure. The residue was dissolved in 20 ml of ethyl acetate and washed with H2O and saturated NaHCO3 (2 ´ 10 ml). After removal of the solvents, the product S107 was purified by SiO2 column chromatography (yield, 170 mg, 93%). The structure of S107 was confirmed by NMR, MS, and Elemental Analysis (see below). The structure of S107 is shown in SI Fig. 7.
Elemental Analysis of S107-HCl salt. m.p.: 222°C decomposed; 1H-NMR (300 MHz, CD3OD): d 7.55 p.p.m. (d, 1H), 7.25 (s, 1H), 6.98 (d, 1H), 4.70 (s, broad, 2H), 3.85 (s, 3H), 3.70 (s, broad, 2H), 3.10 (s, broad 2H), 2.80 (s, 3H); 13C-NMR (300 MHz, CD3OD): d 161.4, 136.0, 134.0, 128.5, 119.5, 115.6, 61.2, 60.0, 59.7, 37.5, 31.5; MS (m/z): [M + 1]+ calculated for C11H16NOS, 210.09; found, 210.20; analysis (% calculated, % found for C11H16ClNOS): C (53.76, 53.42), H (6.56, 6.61), N (5.70, 5.67), S (13.05, 13.49).
Drug Delivery and Exercise Models. On day -3 of each trial, osmotic pumps (Alzet Model 2004, 200 ml total volume, 0.25 ml/hr delivery; Durect) filled with either 200 ml of PBS or 200 ml of S107 (10 mg/ul diluted in H2O) were implanted s.c. on the dorsal surface of each mouse by a horizontal incision at the neck. Mice were allowed to recover for 3 days before the initiation of exercise. Standard food and water were provided ad libitum through the experiment.
The mouse swimming protocol consisted of twice-daily swimming sessions of increasing duration separated by a 1-hour rest period. Exercise capacity was assessed by weekly treadmill run to exhaustion (Model: Exer-6M Treadmill with Treadmill Shock Detection Unit; Columbus Instruments). After an initial conditioning regimen lasting 5 days, during which the swimming sessions were increased in 10-min increments from 40 min each to 80 min each, the swimming sessions thereafter lasted 90 min each. A 30-cm-wide by 30-cm-long opaque acrylic tank was filled with tap water to a depth of at least 20 cm. Water was circulated and warmed to 32-34°C by using a separate reservoir with heating element, thermostat, and pump. Eight mice, balanced pairwise with respect to genotype and/or treatment group, swam at any one time in the tank. Littermates who did not exercise were reserved as sedentary controls. To confirm that uniform exercise conditions were achieved, pilot experiments were performed in which the motion of each individually identified mouse was tracked with a video tracking system (San Diego Instruments). Individual recorded tracks over the full 90 min of each swim were analyzed for distance swum, mean velocities over time, etc., by using the SMART 2.0 software with Social Behavior package (San Diego Instruments).
Treadmill run to exhaustion was performed on days 1, 7, 14, and 21 of the exercise protocol (model Exer-6M Treadmill with Treadmill Shock Detection Unit; Columbus Instruments). Mice were placed in their respective lanes with the shocking apparatus turned off and allowed to adjust to the surroundings for 10 min. The forward half of the treadmill was covered with aluminum foil to block out light, and a desk lamp illuminated the shocking area at the rear of the treadmill. After the adjustment period, the treadmill was set to 10 m/min, and the mice were trained to run with gentle prodding for 6 min. The electric current was then turned on, and the number of shocks delivered during the next two 3-minute intervals (training period) were recorded. The shock counter was then reset, and visits to the shocking area and shocks delivered to each mouse were recorded at 3-min intervals until the end of the experiment. At regular intervals, the speed of the treadmill was ramped up from the initial 10 m/min to 24 m/min. The speed was increased no more than 2 m/min every 6 min. Task failure was defined as when a mouse could not continue running despite gentle prodding.
Human Exercise Protocol. Three weeks before test sessions, subjects reported to the Human Performance Laboratory at Appalachian State University for baseline measurements of cardiorespiratory fitness and body composition. On three consecutive test session days, subjects ate a standardized breakfast (7-8:00 a.m.) and lunch (completed by 12:30 pm) and then reported to the ASU Human Performance Laboratory at 2:00 pm. Subjects exercised on exercise bikes at 70% VO2max from 3:00-6:00 pm. Test session days were Monday, Tuesday, and Wednesday. Oxygen consumption and other metabolic parameters were measured by using a metabolic cart (with a mouthpiece and nose clip) every 30 min, and blood lactate and glucose (via finger stick) every 60 min to verify that subjects were adhering to the prescribed exercise workloads. Subjects ingested 0.5-1.0 liters water every hour of exercise while avoiding all forms of ingested energy (e.g., bars, drinks). Resting control subjects sat in the laboratory during the exercise test sessions. Blood, urine, and saliva samples were collected 15-30 min before exercise/sitting, and then within 5-10 min after exercise on each of the three test sessions. Muscle biopsy samples were obtained 15-30 min before exercise/sitting and then within 5-10 min after exercise by using a needle biopsy procedure on days 1 and 3. Four samples were taken (two from each thigh), ≈2 inches apart. Biopsies were snap-frozen in liquid nitrogen and stored at -80°C (1).
Force Measurement. Intact EDL muscles were dissected free for isometric force measurements in a perfused tissue bath, FDB muscle fibers were enzymatically dissociated by standard methods (35) for confocal imaging of calcium by using the calcium indicator fluo4-AM, and muscle tissue and plasma were frozen in liquid nitrogen. 4-0 silk sutures were tied to the proximal and distal tendons of intact EDL muscles, and the muscles were dissected free and placed in a modified Ringer's solution (140 mM NaCl, 5 mM KCl, 2.0 mM CaCl2, 2 mM MgCl2, 10 mM Hepes, and10 mM glucose, pH 7.4) bubbled with 100% O2. Muscles were hung vertically in 50-ml Radnoti jacketed glass chambers with one tendon attached with 4-0 silk suture to an isometric force transducer (F30; Harvard Apparatus) and the other tendon attached by suture to a stationary arm with built-in platinum stimulating plate electrodes. The muscle was perfused with 35°C Ringer's solution bubbled with 100% O2 and equilibrated for 10 min at a resting tension of 1 cN, which was found to result in an optimal resting length for EDL from all groups examined. A brief potentiation protocol was performed, and force-frequency relationships were measured with 800-ms stimulations at 40-150 Hz, with a 60-second delay between tetani. DMCv4.1.6 (Aurora Scientific) was used to stimulate and record muscle responses, and DMAv3.2 (Aurora Scientific) was used to identify the peak force reached during each tetani. After stimulation, muscle length was determined at resting tension, and muscle weight was recorded after blotting the muscle dry. Muscle cross-sectional area was calculated with the formula, CSA (mm) = muscle weight (mg)/(0.75 ´ muscle length (mm) ´ 1.04).
Confocal Microscopy. Single flexor digitorum brevis (FDB) fibers were enzymatically dissociated by standard methods (2). Briefly, the muscle was dissected from the paw, placed in a modified Ringer's solution, and stripped of all fascia. Type 1 collagenase (2 mg/ml, Sigma) in Ringer's solution was prepared fresh, and the muscle was digested for 2 h at 37°C in an incubator shaking at 125 rpm. The muscle was placed in fresh Ringer's and gently triturated. Single fibers were collected and allowed to attach to glass coverslips coated in laminin (L-2020; Sigma). The cells were loaded with 2 mM fluo4-AM ester (Invitrogen) for 20 min, placed on the Zeiss LSM 5 LIVE microscope stage and superfused for 15 min with Ringer's. The fibers were paced at 1 Hz for 10 min before imaging baseline fiber properties. Line scan images were continuously acquired at 1-ms scan rate during a tetanic sequence consisting of 300-ms stimulation at 100 Hz with a 0.5-Hz train rate, for a total of 200 s. Linescan images were analyzed in ImageJ. A baseline F0 fluorescent signal immediately before stimulation was determined for each fiber, and an F/F0 ratio linescan image was calculated for each fiber.
Single Channel Recordings. RyR1 channels were reconstituted by spontaneous fusion of microsomes into the planar lipid bilayer (a mixture of phosphatidylethanolamine and phosphatidylserine in a 3:1 ratio; Avanti Polar Lipids). Planar lipid bilayers were formed across a 200-mm aperture in a polysulfonate cup (Warner Instruments), which separated two bathing solutions (1 mM EGTA, 250/125 mM Hepes/Tris, 50 mM KCl, 0.5 mM CaCl2, pH 7.35 as cis solution and 53 mM Ba(OH)2, 50 mM KCl, 250 mM Hepes, pH 7.35 as trans solution). After incorporation, RyR1 channel activity was recorded continuously for at least 10 min. The concentration of free Ca2+ in the cis chamber was calculated with WinMaxC program (version 2.50; www.stanford.edu/~cpatton/maxc.html). Single-channel currents were recorded at 0 mV by using the Axopatch 200A patch-clamp amplifier (Axon Instruments) in gap-free mode, filtered at 1 kHz, and digitized at 10 kHz. Data acquisition was performed by using Digidata 1322A and Axoscope 9 software (Axon Instruments). The recordings were analyzed by using Clampfit 10.1 (Molecular Devices) and Origin software (ver. 6.0, Microcal Software).
Analysis of Ryanodine Receptor Complex. Ten-milligram muscle samples were isotonically lysed. RyR1 was immunoprecipitated by incubating 250 mg of homogenate with anti-RyR antibody (2 ml 5029 Ab) in 0.5 ml of a modified RIPA buffer (50 mM Tris·HCl, pH 7.4, 0.9% NaCl, 5.0 mM NaF, 1.0 mM Na3VO4, 0.5% Triton X-100, and protease inhibitors) for 1 h at 4°C. The samples were incubated with protein A Sepharose beads (Amersham Pharmacia) at 4°C for 1 h, after which the beads were washed three times with buffer. Proteins were separated on SDS/PAGE gels (4-20% gradient) and transferred onto nitrocellulose membranes for 2 h at 200 mA (SemiDry transfer blot; Bio-Rad). After incubation with blocking solution (Li-Cor Biosciences) to prevent nonspecific antibody binding and a wash in Tris-buffered saline with 0.1% Tween-20, membranes were incubated for 1-2 h at room temperature with primary antibodies anti-calstabin (1:2,500 in blocking buffer), anti-RyR (5029; 1:5,000), or anti-phospho-RyR2-pSer2809 (1:5000), which detects PKA-phosphorylated mouse RyR1-pSer2844 and RyR2-pSer2808, anti-PDE4D3 (1:1,000), and anti-Cys-NO (1:1,000; Sigma). After three washes, membranes were incubated with infrared-labeled secondary antibodies (1:10,000 dilution, Li-Cor Biosystems). Band intensities were quantified by using the Odyssey Infrared Imaging System (Li-Cor Biosciences). Levels of RyR1 PKA phosphorylation, RyR1 S-nitrosylation, and bound PDE4D3 and calstabin1 are normalized to the total RyR1 immunoprecipitated (arbitrary units). Control samples were run on each gel to aid normalization. Total RyR1 levels did not significantly change under any exercise condition. SI Fig. 5A was developed by using a horseradish peroxidase-conjugated secondary antibody and ECL.
Calpain and Creatine PhosphoKinase Assays. Muscle homogenates were diluted to a final concentration of 600 mg/ml, and the calpain activity in the homogenate was measured as per kit manufacturer's instructions (Calbiochem) (based on the degradation of the fluorescent peptide substrate Suc-LLVY-AMC). Blood was removed by intracardiac aspiration and spun down, and plasma was eluted and frozen in liquid nitrogen. Plasma creatine phosphokinase (CPK) activity was assayed by using the reagent kit from Pointe Scientific. Plasma samples (duplicates, 5 ml each) were added to 200 ml of CPK reagent, and the change in absorbance at 340 nM was recorded over 4 min by using a plate reader. The average absorbance change per minute was used to determine the CPK levels as per manufacturer's instructions.
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