Age-related changes of skeletal muscle metabolic response to contraction are also sex-dependent-Journal of Physiology 2023

Published in the Journal of Physiology, 2023, PMID: 37742081Ethical ApprovalAll experiments in this study were reviewed and approved by the University of Washington Institutional Animal Care and Use Committee (IACUC) on protocol 4130-01. All experiments were designed and performed to eliminate unneeded pain and/or suffering, including following the principles of replacement, reduction, and refinement wherever possible to reduce total animal needs and usage.AnimalsFemale and male C57BL/6 mice were procured from the National Institute on Aging aged rodent colony. Young animals were between 5-7 months and aged animals were between 27-29 months old at the times of sacrifice. All animals were maintained on a 14/10 light/dark cycle at 21°C and given access to food and water ad libitum with no changes prior to experimentation.In vivo muscle contraction and mechanicsAnimals were given O2 at 1 L/min and induced for anesthesia using 4% isoflurane. Once surgical plane of anesthesia was reached animals were moved to a water heated circulating platform maintained at 37°C, the right hindlimb was fixed in place at the knee and the foot was secured to a servomotor (Aurora Scientific, Aurora, ON, CA). During all procedures animals were maintained between 2-2.5% isoflurane. The gastrocnemius was stimulated via the tibial nerve using a high-power, bi-phase stimulator (Aurora Scientific) between 3-5 volts optimized for maximum force generation. Animals were stimulated with either HII (150 Hertz (Hz) every 3 seconds for six stimuli, followed by 10 seconds of rest, repeated for 10 bouts) or LISS (30 Hz every 10 seconds for 20 minutes). All data was analyzed using Dynamic Muscle Analysis Software (v 5.300 Aurora Scientific) and Prism 9.51. Maximum force comparisons were made using one-way ANOVA and a Tukey’s multiple comparisons test. Fatigue curves were compared using two-way repeated measures ANOVA with Šídák’s multiple comparisons test, n=5-9 mice.Tissue dissection and partitioningImmediately following in vivo muscle stimulation animals were euthanized using cervical dislocation. The stimulated and non-stimulated gastrocnemius were dissected and placed on ice. Each gastrocnemius was split into three parts. An approximate 3-6 mg portion of the red gastrocnemius was taken for mitochondrial respiration and the remaining muscle was uniformly split in two and snap frozen in liquid N2 for metabolomics or biochemical follow up assays.Mitochondrial respirationFollowing dissection 3-6 mg of red gastrocnemius was separated into two fiber bundles and manually teased apart on ice in BIOPS (10 mM Ca-EGTA buffer, 0.1 µM free calcium,20 mM imidazole, 20 mM taurine, 50 mM K-MES, 6.56 mM MgCl2, 5.77 mM ATP, 15 mM phosphocreatine, pH 7.1) for 5 minutes or until visible fibers were loosely separated from adjacent fibers. Following manual teasing fiber bundles were permeabilized on ice in BIOPS with saponin (50 ug/ml) for 40 minutes with gentle rocking. Following permeabilization fiber bundles were washed for 5 minutes in BIOPS, followed by 5 minutes and 15 minutes in respiration buffer (RB, 0.5 mM EGTA, 20 mM taurine, 3 mM MgCl2, 110 mM Sucrose, 60 mM K-MES, 20 mM Hepes, 10 mM KH2PO4, 1mg/ml BSA, pH 7.1) on ice with gentle rocking. Following wash steps, fiber bundles were placed in RB in an Oxygraph 2-K dual respirometer/fluorometer (Oroboros Instruments, Innsbruck, AT) at 37°C, with 750 rpm stirring. Oxygen concentration was maintained between 250-450 uM. Respiration was stimulated with titrations to final concentration of 0.1 mM malate, 50uM ADP, 2.5 mM ADP, and 1 mM steps up to 10 mM glutamate; or with titrations to final concentration of 0.1 mM malate, 50 uM ADP, 2.5 mM ADP, and 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, and 70 uM palmitoyl carnitine. All data was analyzed using Datlab 7.4.0.4 (Oroboros Instruments) and Graphpad Prism 9.51. Respirometry values were compared using repeated measures one-way ANOVA with a Tukey’s multiple comparisons test n=5-8 mice.MetabolomicsAqueous metabolites for targeted LC-MS profiling of 70 skeletal muscle samples were extracted using a protein precipitation method as previously described (Meador et al., 2020; Kurup et al., 2021; Mhatre et al., 2023). Samples were first homogenized in 200 µL purified deionized water at 4 ˚C, and then 800 µL of cold methanol containing 124 µM 6C13-glucose and 25.9 µM 2C13-glutamate was added (reference internal standards were added to the samples in order to monitor sample prep). Afterwards samples were vortexed, stored for 30 minutes at -20 ˚C, sonicated in an ice bath for 10 minutes, centrifuged for 15 min at 14,000 rpm and 4 ˚C, and then 600 µL of supernatant was collected from each sample (precipitated protein pallets were saved for BCA assay). Lastly, recovered supernatants were dried on a SpeedVac and reconstituted in 0.5 mL of LC-matching solvent containing 17.8 µM 2C13-tyrosine and 39.2 3C13-lactate (reference internal standards were added to the reconstituting solvent in order to monitor LC-MS performance). Samples were transferred into LC vials and placed into a temperature controlled autosampler for LC-MS analysis.Targeted LC-MS metabolite analysis was performed on a duplex-LC-MS system composed of two Shimadzu UPLC pumps, CTC Analytics PAL HTC-xt temperature-controlled auto-sampler and AB Sciex 6500+ Triple Quadrupole MS equipped with ESI ionization source. UPLC pumps were connected to the auto-sampler in parallel and were able to perform two chromatography separations independently from each other. Each sample was injected twice on two identical analytical columns (Waters Xbridge BEH Amide XP) performing separations in hydrophilic interaction liquid chromatography (HILIC) mode. While one column was performing separation and MS data acquisition in ESI+ ionization mode, the other column was getting equilibrated for sample injection, chromatography separation and MS data acquisition in ESI- mode. Each chromatography separation was 18 minutes (total analysis time per sample was 36 minutes). MS data acquisition was performed in multiple-reaction-monitoring (MRM) mode. LC-MS system was controlled using AB Sciex Analyst 1.6.3 software. Measured MS peaks were integrated using AB Sciex MultiQuant 3.0.3 software. The LC-MS assay was targeting 361 metabolites (plus four spiked reference internal standards). Up to 182 metabolites (plus four spiked standards) were measured across the study set, and over 95% of measured metabolites were measured across all the samples. In addition to the study samples, two sets of quality control (QC) samples were used to monitor the assay performance as well as data reproducibility. One QC [QC(I)] was a pooled human serum sample used to monitor system performance and the other QC [QC(S)] was pooled study samples and this QC was used to monitor data reproducibility. Each QC sample was injected per every 10 study samples. The data were well reproducible with a median CV of 5.4 % over 2.5 days of non-stop data acquisition. Targeted metabolomics was examined using MetaboAnalyst 5.0 One Factor Statistical and Pathway Analysis. Features with >50% missing values were removed, and remaining missing values were excluded. Data was normalized using sample protein concentrations, mean-centered, and autoscaled. Metabolite changes were analyzed using paired students t-test between the contracted and non-stimulated muscle, and pathway changes were analyzed using enrichment analysis. All metabolite comparisons include a Holm-Bonferroni correction for multiple testing, n=5-8 mice.Surgery and elamipretide treatmentAnimals were induced for surgery using 4% isoflurane in 1 L/min O2. Once surgical plane of anesthesia was reached as determined by absence of a toe pinch reflex, isoflurane was reduced to 2-2.5% and maintained at this level throughout surgical intervention. Animals were given a subcutaneous dose of 5 mg/kg meloxicam to reduce post-operative pain and both eyes were fully covered using artificial tears ocular lubricant. For surgical implantation, the midback was depilated using Nair and fully cleaned using gauze and alcohol wipes. To sterilize the incision site, alternating application of alcohol followed by povidone/iodine was applied 3 times. An approximate 1 cm incision was made along the midback, and a subcutaneous pocket was created using blunt ended scissors. Osmotic pumps (Alzet 1004) were pre-loaded to deliver ELAM for 4 weeks at 3 mg/kg/day and inserted into the subcutaneous pocket. Following pump implantation, the incision was closed and secured using 2-3 5mm wound clips and a drop of vet bond surgical glue. The entire surgery from anesthesia to wound closure was performed in approximately 10 minutes. Following recovery from surgery animals were monitored for 5 hours and then every day for 5 days observing for signs of pain or distress and given supplemental meloxicam (5 mg/kg) as needed. Following 4 weeks of ELAM delivery, animals were anesthetized and prepared for surgery as described above, and the original osmotic pump was surgically removed and replaced with a freshly loaded and primed pump to deliver ELAM for an additional 4 weeks.