In situ detection and measurement of intracellular reactive oxygen species in single isolated mature skeletal muscle fibers by real time fluorescence microscopy.

Isolation of single skeletal muscle fibers

Experiments were performed in accordance with UK Home Office guidelines under the UK Animals (Scientific Procedures) Act 1986. Female C57BL/6 mice, 2–4 months old, were humanely killed and the flexor digitorum brevis muscles were removed and placed into 0.4% type H collagenase (EC 3.4.24.3) (Sigma–Aldrich Co. St. Louis, MO) solution in culture medium. This was composed of minimum essential medium Eagle (MEM) (Sigma–Aldrich Co.) supplemented with 10 % fetal bovine serum (FBS) (Invitrogen Ltd, Paisley, UK) containing 2 m M glutamine, 50 i.u. penicillin, and 50 μ g ml −1 streptomycin. Both FDB muscles from each mouse were incubated in collagenase solution at 37°C for 2 h, the mixture was manually shaken every 30 min to improve digestion of the connective tissue. Fiber bundles that had not been separated during the incubation were gently triturated by a wide-bore plastic pipette to separate the fibers. Free single muscle fibers were separated from damaged fibers and contaminating cells by centrifugation at low speed (600 g for 30 s). The fibers were washed four times in fresh culture medium. Cleaned fibers were plated onto 35-mm dishes pre-coated with 120 μ l of a 1:6 mixture of Vitrogen collagen (Cohesion Technologies Inc., Palo Alto, CA) in 7 × Dulbecco's modified Eagle's medium (D-MEM) (Invitrogen Ltd.). Fibers were allowed to attach for 30 min at 37°C, 1 ml of fresh culture medium was added to each plate, and fibers were cultured for 18 h at 37°C in a 5% CO 2 atmosphere ( 2 , 41 ). Viability of single skeletal muscle fibers The viability of the isolated muscle fibers in culture was determined by examination of their morphology by bright-field microscopy and by their ability to exclude the vital stain, trypan blue. Fibers were incubated in a 0.04 mg/ml solution of trypan blue (Sigma-Aldrich Co.) in culture medium and examined under bright-field microscopy. Loading of single skeletal muscle fibers with CM-DCFH DA A solution of 5- (and 6-) chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-DCFH DA) (Molecular Probes,™ Invitrogen, Eugene, OR) was prepared daily in absolute ethanol and kept at 4°C. Culture plates containing isolated fibers were washed with Dulbecco's Phosphate Buffered Saline (D-PBS) (Sigma-Aldrich Co.), and fibers were pre-incubated in D-PBS at 37°C for 30 min. The medium was then replaced by D-PBS containing CM-DCFH DA (17.5 μM ) and incubated for 30 min at 37°C. The fibers were then washed with D-PBS and covered with MEM without Phenol Red (Sigma-Aldrich Co.) for fluorescence microscopy. 5- (and 6-) chloromethyl-2′,7′-dichlorodihydrofluorescein (CM-DCFH) is a derivative of DCFH modified to improve retention by cells. The diacetate form (CM-DCFH DA) is a non-polar molecule that diffuses into the fibers, and within the cytosol the acetate group is cleaved by cytosolic esterases to yield CM-DCFH, a hydrophilic nonfluorescent molecule that is retained by the cell. Intracellular ROS, particularly hydrogen peroxide, convert CM-DCFH to its fluorescent derivate, CM-DCF ( 49 ).

Fluorescence microscopy and image analysis

The imaging system consisted of a Zeiss Axiovert 200M epifluorescence microscope equipped with X10 and X20 objectives and filter sets: a 450–490 nm excitation/515–565 nm emission filter set that was used for CM-DCF fluorescence, and a 365 nm excitation/420 nm emission filter set that was used for 4′,6-diamidino-2-phenylindole (DAPI) fluorescence (Carl Zeiss Microimaging GmbH, Jena, Germany). Images were acquired and analyzed using a computer-controlled Zeiss HRc charged-coupled device (CCD) camera (Carl Zeiss Microimaging GmbH) and by AxioVision 4.4 image capture and analysis software (Carl Zeiss Microimaging GmbH) for quantification of changes in emission fluorescence. This software allows measurements to be made from user-defined areas of the microscope field; in this case, the fluorescence measurements were localized to selected areas of the muscle fibers. CM-DCF fluorescence from fibers was recorded at 15 min intervals over 45 min at 25°C. Exposure of fibers to ultraviolet (UV) light was minimized by use of a 500 ms exposure time. Immediately after the measurement at 45 min, all remaining CM-DCFH in the fibers was photo-oxidized by continuous illumination with UV light for 15 min. During this time, images were recorded every 20 s. Overexposure of fibers to UV light caused oxidation of all remaining CM-DCFH and produced a maximum CM-DCF fluorescence emission that provides a measure of the total amount of available CM-DCFH in the portion of each fiber under study ( 31 ). The effect of temperature on endogenous CM-DCFH oxidation was examined by increasing the temperature of the cell culture to 37°C throughout the measurement. In some studies, the CM-DCF fluorescence in fibers was examined by confocal microscopy in comparison with the fluorescence of mitotracker red (MitoTracker® Red CMXRos, (Molecular Probes™, Invitrogen). Fibers loaded with CM-DCFH (17.5 μM ) and mitotracker red (20 n M ) were examined using a Zeiss LSM 510 confocal microscope with a 63X/1.4 oil DIC objective and an argon ion laser. Excitation of CM-DCF was at 488 nm and the emission was monitored through a 505–530 nm filter reflected from a 545 nm dichroic mirror. Excitation of mitotracker red was achieved using a 543 nm helium neon laser and the emission was collected through a 545 nm dichroic mirror and a 560 nm long pass filter. Image data acquisition and analysis were carried out with Carl Zeiss Laser Scanning Systems LSM 510 software, version 3.2 (Carl Zeiss Microimaging GmbH).

Experimental protocol Exposure of fibers to exogenous oxidant

The sensitivity of the experimental system to small changes in ROS was evaluated by addition of hydrogen peroxide to the culture wells at 15 min after the beginning of the experiment. Following preliminary titration experiments to determine the appropriate concentration of hydrogen peroxide for use (not shown in detail), a small volume of a concentrated solution of hydrogen peroxide was added to the 2 ml culture medium to produce a final concentration of 1 μM and the time-course and change in CM-DCF fluorescence monitored. Electrical stimulation of contractions by fibers Isolated fibers were stimulated to contract, as previously described for myotube cultures ( 31 , 37 ). In brief, platinum electrodes were placed into the well and provided trains of biphasic square wave pulses of 2 ms in duration for 0.5 s repeated every 5 s at 50 Hz and 30 V/well. The total stimulation time was 15 min and this commenced at 15 min following the beginning of the experiment. Fibers contracted throughout the 15 min period (see video in supplementary material ; for supplementary video , see www.liebertonline.com/ars ) * . Enhancement of intracellular glutathione content Isolated fibers were incubated with 5 m M glutathione ethyl ester (GSHEE) (Sigma-Aldrich Co.) in the culture medium for 2 h prior to loading with CM-DCFH DA.

Statistical analyses

Data are presented as mean ± S.E.M. For multiple comparisons at any time point, analysis was by one-way ANOVA followed by the post hoc LSD test. Single comparisons between two experimental conditions at a time point were undertaken using the unpaired Student's t test. Comparisons between data from individual fibers at different time points were undertaken using Student's paired t test. Statistical significance was set at the α level of 0.05 for all of the tests.

Experimental protocol Exposure of fibers to exogenous oxidant

The sensitivity of the experimental system to small changes in ROS was evaluated by addition of hydrogen peroxide to the culture wells at 15 min after the beginning of the experiment. Following preliminary titration experiments to determine the appropriate concentration of hydrogen peroxide for use (not shown in detail), a small volume of a concentrated solution of hydrogen peroxide was added to the 2 ml culture medium to produce a final concentration of 1 μM and the time-course and change in CM-DCF fluorescence monitored. Electrical stimulation of contractions by fibers Isolated fibers were stimulated to contract, as previously described for myotube cultures ( 31 , 37 ). In brief, platinum electrodes were placed into the well and provided trains of biphasic square wave pulses of 2 ms in duration for 0.5 s repeated every 5 s at 50 Hz and 30 V/well. The total stimulation time was 15 min and this commenced at 15 min following the beginning of the experiment. Fibers contracted throughout the 15 min period (see video in supplementary material ; for supplementary video , see www.liebertonline.com/ars ) * . Enhancement of intracellular glutathione content Isolated fibers were incubated with 5 m M glutathione ethyl ester (GSHEE) (Sigma-Aldrich Co.) in the culture medium for 2 h prior to loading with CM-DCFH DA.