Project description:The annotation of the Affymetrix HTA 2.0 array was updated to optimise the detection of RNA in human skeletal muscle samples by removing invalid and low signal-high-variance probes Human primary skeletal muscle cells were isolated from muscle biopsies from healthy adults as previously described (Crossland et al. 2016; Crossland et al. 2017). Myogenic cell enrichment used magnetic-activated cell sorting (MACS) and anti-CD56 microbeads (130-050-401; Miltenyi Biotec). Myogenic purity was assessed through measurement of fractional desmin positivity (Crossland et al. 2016; Crossland et al. 2017). Cells were washed in PBS and fixed in ice-cold 1:1 acetone/methanol, before being blocked in 5% (v/v) goat serum for 30 min at room temperature. Cells were subsequently incubated with rabbit anti-desmin monoclonal antibody (ab32362; Abcam) for 1h at room temperature, washed, and incubated with anti-rabbit TRITC-conjugated secondary antibody as previously detailed (Crossland et al. 2016; Crossland et al. 2017). Finally, cells were washed in PBS and mounted/DAPI-stained using FluoroshieldTM mounting medium with DAPI. Myoblasts at passage 5-6 were cultured on Collagen Type I-coated 6-well dishes in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; Life Technologies) containing 20% (v/v) fetal bovine serum (FBS, Sigma-Aldrich), 1% (v/v) antibiotic-antimycotic (AbAm) solution and 4mM L-glutamine (Life Technologies). Cells were maintained at 37oC with 5% CO2. Once cells reached ~95% confluency, differentiation was induced by switching the medium to DMEM/F-12 containing 2% (v/v) horse serum (Sigma-Aldrich), 4mM L-glutamine and 1% (v/v) AbAm solution (Life Technologies). Six days following the initiation of differentiation, a media change was carried out, using differentiation media supplemented with long R3 IGF-1 (10 ng/ml; Sigma-Aldrich), with or without 100 nM rapamycin (Sigma-Aldrich). DMSO (final concentration 0.01% (v/v)) was used as a vehicle control for rapamycin. Samples were collected at 0h baseline and following 4h and 24h treatment.
Project description:Phosphorylation of skeletal muscle proteins mediates cellular signaling and adaptive responses to exercise. Bioinformatic and machine learning approaches identified preclinical models that recapitulate human exercise responses. Feature selection showed that muscles from treadmill running mice and maximum intensity contractions shared the most differentially phosphorylated phosphosites (DPPS) with human exercise. Benefits of exercise in chronic diseases may be reduced by hyperammonemia, a consistent perturbation in chronic diseases and a muscle cytotoxin generated during contractile activity. Comparative analysis of experimentally validated molecules identified 63 DPPS on 265 differentially expressed phosphoproteins (DEpP) shared between hyperammonemia in myotubes and skeletal muscle from exercise models. Functional enrichment analyses revealed distinct temporal patterns of enrichment shared between hyperammonemia and exercise models including protein kinase A(PKA), calcium signaling, mitogen activated protein kinase(MAPK) signaling, and protein homeostasis. Our approach of feature extraction of comparative unbiased data allows for model selection and target identification to optimize responses to interventions.
Project description:Activation of the sympathetic nervous system causes pronounced metabolic changes that are mediated by multiple adrenergic receptor subtypes. Systemic treatment with β<sub>2-</sub>adrenergic receptor agonists results in multiple beneficial metabolic effects, including improved glucose homeostasis. To elucidate the underlying cellular and molecular mechanisms, we chronically treated wild-type mice and several newly developed mutant mouse strains with clenbuterol, a selective β<sub>2</sub>-adrenergic receptor agonist. Clenbuterol administration caused pronounced improvements in glucose homeostasis and prevented the metabolic deficits in mouse models of β-cell dysfunction and insulin resistance. Studies with skeletal muscle-specific mutant mice demonstrated that these metabolic improvements required activation of skeletal muscle β<sub>2</sub>-adrenergic receptors and the stimulatory G protein, G<sub>s</sub>. Unbiased transcriptomic and metabolomic analyses showed that chronic β<sub>2</sub>-adrenergic receptor stimulation caused metabolic reprogramming of skeletal muscle characterized by enhanced glucose utilization. These findings strongly suggest that agents targeting skeletal muscle metabolism by modulating β<sub>2</sub>-adrenergic receptor-dependent signaling pathways may prove beneficial as antidiabetic drugs.
Project description:Cytochrome c (Cytc) is essential for mitochondrial respiration and apoptosis. Tissue specific post-translational modifications of Cytc play an important regulatory role in these processes. Here, we describe a new acetylation site, lysine 39, which was mapped in ischemic porcine skeletal muscle. Recombinant acetylmimetic K39Q protein demonstrated increased cytochrome c oxidase (COX) activity and decreased caspase-3 and cardiolipin peroxidase activities. Cytc double knockout cells expressing acetylmimetic K39Q Cytc showed an increase in mitochondrial respiration, mitochondrial membrane potential, and mitochondrial ROS production and a decrease in cell death. These results are discussed in the context of X-ray crystallography structures of K39 acetylated (1.50 Å) and acetylmimetic K39Q Cytc (1.36 Å) and NMR dynamics. We propose that lysine 39 acetylation is an adaptive response allowing skeletal muscle to meet heightened energy demand while simultaneously providing the tissue with robust resilience to ischemia-reperfusion injury.