Muscle isometric force measurement

In situ gastrocnemius isometric force measurement was followed previous study [25]. Isometric contractile properties of the gastrocnemius muscles were measured on days 3 and 7 after burn (ASI Dynamic Muscle Control v5.300, Aurora Scientific, Canada). Under general anesthesia, the mouse was laid in a prone position on the warm platform with 37°C water circulated. The gastrocnemius muscle was gently dissected free of surrounding musculature, skin, and fascia to maintain the neurovascular pedicle as well as the proximal and distal attachments. The Achilles tendon was sutured and attached to the lever arm of a dual-mode servo muscle lever system (mod 305c, Aurora Scientific, Canada). The femur was secured to the platform. Electrodes were implanted into the distal end of the severed sciatic nerve. A single 200 Hz twitch was stimulated with an impulse duration of 0.2 ms at 10 mA. The muscle was then stretched 0.2 mm and stimulated again with a 25-second break between stimulations. This pattern was continued until less than a 2% change between twitches was detected, indicating the optimal length (Lo) of the muscle. The maximum twitch (Pt), force frequency, tetanic (Po), and fatigue parameters were then consequently measured at Lo. Isometric tetanic function was stimulated at 20, 40, 80, 100, 150, 180, and 200 Hz with impulse duration of 0.2 ms, 75 pulses per train, at 10mA, for a total of 1 second. Po was measured three times with an off-tension recovery period of 2 minutes between stimulations. Relaxed muscle fiber at the baseline and applied mineral oil during the relaxation time.

Skeletal muscle mitochondrial respiratory capacity was measured via high-resolution respirometry as previously described [26]. In brief, about 20mg of fresh gastrocnemius tissue was immersed in preservation buffer on ice before being transferred into the O2K chamber with the polygraphic oxygen sensors controlled by DatLab software (Oroboros Instruments, Innsbruck, Austria) and containing 2 mL respiration buffer. Preservation buffer includes 2.77 mM CaK2EGTA, 7.23 mM K2EGTA, 50mM MES hydrate, 20mM imidazole, 20mM taurine, 15mM Na2Phosphocreatine, 6.56 mM MgCl2·6H20, 5.77 mM Na2ATP, and 0.5 mM dithiothreitol; pH 7.1. Respiration buffer contains 0.5mM EGTA, 60mM lactobionic acid, 3 mM MgCl2·6H20, 20 mM taurine, 10mM KH2PO4, 20 mM HEPES, 110 mM sucrose, and 1g/L bovine serum albumin. All experiments were performed within a range of 200–450μM of oxygen concentration to ensure that oxygen consumption and diffusion would not be limited to respiration. Mitochondrial respiratory capacity was determined by the sequential addition of substrates and uncouplers. State 2 (leak) respiration supported primarily by electron flow through complex I of the respiratory chain (LI) was achieved by the titration of 1.5mM octanoyl-l-carnitine, 5mM pyruvate, 2mM malate, and 10mM glutamate into the O2K chamber. Electron transfer was then coupled to phosphorylation by the addition of 5mM ADP, inducing coupled state 3i respiration with electron transfer supported by Complex I (PI). 10mM succinate was added to the O2K chamber to induce maximal state 3i+ii respiration with parallel electron input from Complex I and II (PI+II). 10μM cytochrome C was added to assess the competence of the outer mitochondrial membrane. The absence of a significant increase in respiratory flux following the addition of cytochrome C indicates that the outer mitochondrial membranes are intact. Finally, oxidative phosphorylation was uncoupled by the titration of carbonyl cyanide m-chlorophenylhydrazone (CCCP) to a final concentration of 5μM to assess maximal electron transfer capacity (E). All chemicals were purchased from Sigma-Adrich (St. Louis, MO) unless specified.

Western blot assay was followed by the previous publication [27]. Briefly, 20 mg of frozen gastrocnemius muscle tissue was homogenized with 0.1mm Zirconium Homogenizer Beads (CPI international) for protein extraction. Tissue lysates were prepared using T-PER tissue protein extraction reagent (Thermal Scientific, Rockford, IL). Protein concentrations were examined by the protein assay kit (BioRad, Hercules, CA) based on the Bradford dye-binding method. Using the Bio-Rad Mini-Protean system (Bio-Rad, Hercules, CA), approximate 20μg of protein samples were applied on Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 4–20%) and the separated protein components were transferred to a Polyvinylidene difluoride (PVDF) membrane. The membranes were then blocked by the nonspecific binding background with 5% bovine serum albumin (BSA) at room temperature for 1 hour and subsequently probed with primary antibodies at 4°C overnight. The membranes were then incubated with horse-radish peroxidase (HRP)-conjugated secondary antibodies for 1 hour. After rinsing off the secondary antibodies, the blots were applied for protein signal detection using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, Rockford, IL). The stained blots were examined using the ChemiDoc™ Touch Imaging System (BioRad, Hercules, CA). All of antibodies were from Cell Signaling Technology (CS#) (Danvers, MA) or Abcam Biotechnology (#ab)(Cambridge, United Kingdom), including Mruf1/2/3 (#ab172479), Fbx32 (#ab74023), caspase 3 (CS#9662), PCNA (CS#13110), MBP (#ab40390), and S100B (#ab52642). To analyze the blots, BioRad Image lab software was used to quantify band intensity and calculate the ratio of the target protein compared to the loading control GAPDH (CS#5174).