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Hyperammonemia-dependent and independent skeletal muscle phosphoproteomic responses during exercise

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Myoblast

SUBMITTER: Ling Li

LAB HEAD: Srinivasan Dasarathy

PROVIDER: PXD031372 | Pride | 2022-10-25

REPOSITORIES: Pride

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Project description:Plastics are persistent synthetic polymers that accumulate in the marine environment as waste. Microplastic (MP) particles are derived from the breakdown of larger debris or can enter the environment as microscopic fragments. Filter-feeder organisms ingest MP while feeding and are likely to be impacted by MP pollution. To assess the impact of polystyrene microspheres (PS) on the physiology of the Pacific oyster, adult oysters were experimentally exposed to virgin micro-PS (2 and 6 Âµm in diameter; 32 Âµg L-1) for two months during a reproductive cycle. Effects were investigated on transcriptomic responses, in digestive gland gonads and oocytes of exposed oysters. Transcriptomic profiles in the tissues of the exposed oyster showed endocrine disrupting signals, notably highlighting alteration in glucocorticoid response, insulin pathway and fatty-acid metabolism in response to micro-PS exposition. In oocytes from exposed females, several transcripts coding for proteins involved in Ca2+ binding were differentially expressed suggesting a disruption of the Ca2+ signaling pathway with crucial consequences on oocyte maturation. To assess the impact of polystyrene microspheres (PS) on the physiology of the Pacific oyster, adult oysters were experimentally exposed to virgin micro-PS (2 and 6 Âµm in diameter; 32 Âµg L-1) for two months during a reproductive cycle and compared to control oysters. Adults were sampled 2 and 8 weeks after the beginning of exposure (corresponding to T1 and T3, respectively, 8-9 replicates per time of sampling and condition for a total of 56). Tissues were immediately dissected from each oyster, frozen in liquid nitrogen, then crushed to a fine powder at -196Â°C with an oscillating mill mixer and stored in liquid nitrogen until RNA extraction. Oocytes were collected from 5 females per condition, filtered in a 40 Âµm sieve, counted and transferred into 1.5 mL of Extract-all reagent (Eurobio, Courtaboeuf, France) (20,000 oocytes). Total RNA was isolated using 1.5 mL of Extract-all Reagent per 50 mg of gonad powder. For microarray hybridizations, 200 ng of total RNA were indirectly labeled with Cy3 using the Low Input Quick Amp Labeling kit One-Color. Hybridization was performed using the Agilent Gene expression hybridization kit (5188-5242), with 1.65 Î¼g of labeled RNA, for 16 h at 65 Â°C. The employed arrays were Agilent 60-mer 4x44K custom microarrays, containing 31,918 C. gigas ESTs, designed by Dheilly et al. (2011). Slides were scanned on an Agilent Technologies G2565AA Microarray Scanner system at 5 Î¼m resolution, using default parameters. Features were extracted using the Agilent Feature Extraction software 6.1.

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Hyperammonemia-dependent and independent skeletal muscle phosphoproteomic responses during exercise

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