Greater Oxidative Capacity in Primary Myotubes from Endurance-trained Women

Abstract

Purpose Exercise training promotes skeletal muscle mitochondrial biogenesis and an increase in maximal oxygen consumption. Primary myotubes retain some metabolic properties observed in vivo but it is unknown whether this includes exercise-induced mitochondrial adaptations. The goal of this study was to test if primary myotubes from exercise-trained women have higher mitochondrial content and maximal oxygen consumption compared to untrained women. Methods Six trained and nine untrained Caucasian women participated in this study. Muscle biopsies from the vastus lateralis muscle of the right leg were obtained and primary muscle cells were isolated. Maximal respiration rates, mitochondrial mRNA and protein content, and succinate dehydrogenase activity were measured in skeletal muscle and primary myotubes from trained and untrained women. Results Trained women, compared to untrained women, had higher maximal whole-body oxygen consumption (+18%, P = 0.03), in vivo maximal skeletal muscle oxidative capacity measured with near infrared spectroscopy (+48%, P < 0.01), and maximal oxygen consumption in permeabilized muscle fibers (+38%, P = 0.02), which coincided with higher protein levels of muscle mitochondrial enzymes. Primary myotubes from trained women had higher maximal oxygen consumption (+38%, P = 0.03) suggesting that some elements of exercise-induced metabolic programming persists ex vivo. Consistent with this idea, myotubes from trained women had higher mRNA levels of transcriptional regulators of mitochondrial biogenesis in addition to higher protein levels of mitochondrial enzymes. Conclusion These data suggest the existence of an “exercise metabolic program”, where primary myotubes isolated from exercise-trained individuals exhibit greater mitochondrial content and oxidative capacity compared to untrained individuals. These myotubes may be a useful model to study molecular mechanisms relevant to exercise adaptations in human skeletal muscle.

Extensive skeletal muscle cell mitochondriopathy distinguishes critical limb ischemia patients from claudicants

“…Isolating these cells from PAD patients provided us an opportunity to examine oxidative metabolism under tightly controlled normoxic and nutrient-rich conditions, effectively minimizing the influence of differing local environments created by PAD clinical manifestations. MPCs appear to be uniquely prone to mirroring the local metabolic environment from which they were isolated (51,52). CLI patient MPCs retained the following characteristics: stable myotube mitochondrial content, attenuated oxygen consumption, and a suppressed bioenergetic mRNA profile.…”

Mitochondrial PE potentiates respiratory enzymes to amplify skeletal muscle aerobic capacity

“…Respiration in permeabilized muscle fiber bundles and isolated mitochondria was performed as previously described. 46,47 Briefly, a small portion of freshly dissected red gastrocnemius muscle tissue was placed in Buffer X (7.23 mM K 2 EGTA, 2.77 mM Ca K 2 EGTA, 20 mM imidazole, 20 mM taurine, 5.7 mM ATP, 14.3 mM phosphocreatine, 6.56 mM MgCl 2 .6H 2 O, and 50 mM K-MES, pH = 7.1), Fiber bundles were separated and permeabilized for 30 min at 4°C with saponin (30 µg/ml) and immediately washed in Buffer Z (105 mM K-MES, 30 mM KCl, 10 mM K 2 HPO 4 , 5 mM MgCl 2 .6H 2 O, 0.5 mg/ml BSA, and 1 mM EGTA, pH = 7.4) for 15 min. After washing, high-resolution respiration rates were measured using an OROBOROS Oxygraph-2k.…”

“…On the day of the experiment, medium was switched to XF Assay Medium Modified DMEM (pH = 7.4) containing added glucose (10 mM), pyruvate (200 mM), and glutamine (200 mM) for 1 h. Subsequently, basal and maximal respiration rates were measured as previously described. 46…”

The Lands cycle modulates plasma membrane lipid organization and insulin sensitivity in skeletal muscle

“…Primary human skeletal muscle cells (HSkMC) were isolated from fresh muscle biopsies as previously described (15, 56). HSkMC were cultured in growth media containing low glucose DMEM, 10x FBS, 0.5 mg/mL BSA, 0.5 mg/mL fetuin, 10 ng/mL human EGF, 1 µM dexamethasone, and 0.1% penicillin-streptomycin.…”

“…Whole muscle or cells were homogenized and Western blots were performed as previously described (56). Protein homogenates were analyzed for abundance of phosphorylated(Tyr972)-insulin receptor (Invitrogen: 44-800G), insulin receptor-β (Cell Signaling: 3020S), phosphorylated(Thr308)-Akt (Cell Signaling: 9275S), phosphorylated(Ser472)-Akt (Cell Signaling: 9271L), Akt (Cell Signaling: 9272S), phosphorylated(Thr642)-AS160 (Cell Signaling: 8881), AS160 (Millipore Sigma: 07-741), MyoD (DSHB: D7F2), mitochondrial complexes I-V (Abcam: ab110413), MHC type I (DSHB: A4.840), MHC type IIa (DSHB: SC-71), MHC type IIx (DSHB: 6H1), MHC type IIb (DSHB: BF-F3), MHC neo (DSHB: N1.551), MHC emb (DSHB: BF-G6), Caveolin-3 (BD Biosciences: 610-420), Na/K ATPase (Cell Signaling: 3010S), Flotillin-1 (Cell Signaling: 3253), and actin (Millipore Sigma: A2066).…”