Muscle Proteome Research Articles

Remodeling of the Human Skeletal Muscle Proteome Found After Long-Term Endurance Training but Not After Strength Training

Regular exercise training has tremendous systemic tissue-specific health benefits, but the molecular adaptations to long-term exercise training are not completely understood. The aim of the current study was to investigate the skeletal muscle proteome of highly endurance-trained, strength-trained and untrained individuals and performed exercise- and sex-specific analyses. As a previous investigation from our lab showed limited baseline differences at the gene expression level between highly strength-trained males and untrained males, we hypothesized that the that long-term resistance training results in baseline differences in protein abundances. Additionally, we hypothesized that endurance-trained individuals would have a greater abundance of mitochondrial proteins compared with the untrained and the strength-trained individuals. Liquid-chromatography followed by mass-spectrometry (LC-MS/MS) was performed on skeletal muscle tissue from all individuals. A total of 6713 proteins were detected, with 4269 proteins being expressed in at least 50% of the samples. Over 650 proteins were differentially expressed in the skeletal muscle of endurance-trained individuals compared with controls. Strikingly, 92% of the shared proteins with higher expression in both the male and female endurance groups were known mitochondrial proteins. In contrast to the findings in endurance trained individuals, minimal differences were found in strength-trained individuals and between females and males. Lastly, to determine the preventive-medicine capabilities of long-term training, a co-expression network and comparative literature analysis revealed key proteins and pathways related to the health benefits of exercise, which were primarily related to differences in mitochondrial protein abundance. This network is available as an interactive database resource ( https://inetmodels.com ) where investigators can correlate clinical data with global gene and protein expression data for hypothesis generation. In conclusion, we suggest that long-term endurance training but not strength training results in a r emodeling of the skeletal muscle proteome in humans. This work was financially supported by grants from the Swedish Research Council (2018-02932), the Swedish Center for Sports Research (2020-D0007, P2022-0082), the National Science Foundation (Award Number: 1951792), the Whitaker International Program and the Karolinska Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A novel deep proteomic approach in human skeletal muscle unveils distinct molecular signatures affected by aging and resistance training.

The skeletal muscle proteome alterations to aging and resistance training have been reported in prior studies. However, conventional proteomics in skeletal muscle typically yields wide protein abundance ranges that mask the detection of lowly expressed proteins. Thus, we adopted a novel deep proteomics approach whereby myofibril (MyoF) and non-MyoF fractions were separately subjected to protein corona nanoparticle complex formation prior to digestion and Liquid Chromatography Mass Spectrometry (LC-MS). Specifically, we investigated MyoF and non-MyoF proteomic profiles of the vastus lateralis muscle of younger (Y, 22±2 years old; n=5) and middle-aged participants (MA, 56±8 years old; n=6). Additionally, MA muscle was analyzed following eight weeks of resistance training (RT, 2d/week). Across all participants, the number of non-MyoF proteins detected averaged to be 5,645±266 (range: 4,888-5,987) and the number of MyoF proteins detected averaged to be 2,611±326 (range: 1,944-3,101). Differences in the non-MyoF (8.4%) and MyoF (2.5%) proteomes were evident between age cohorts, and most differentially expressed non-MyoF proteins (447/543) were more enriched in MA versus Y. Biological processes in the non-MyoF fraction were predicted to be operative in MA versus Y including increased cellular stress, mRNA splicing, translation elongation, and ubiquitin-mediated proteolysis. RT in MA participants only altered ~0.3% of MyoF and ~1.0% of non-MyoF proteomes. In summary, aging and RT predominantly affect non-contractile proteins in skeletal muscle. Additionally, marginal proteome adaptations with RT suggest more rigorous training may stimulate more robust effects or that RT, regardless of age, subtly alters basal state skeletal muscle protein abundances.

Identification of gravity-responsive serum proteins in spaceflight mice using a quantitative proteomic approach with data-independent acquisition mass spectrometry.

Physical inactivity associated with gravity unloading, such as microgravity during spaceflight and hindlimb unloading (HU), can cause various physiological changes. In this study, we attempted to identify serum proteins whose levels fluctuated in response to gravity unloading. First, we quantitatively assessed changes in the serum proteome profiles of spaceflight mice using mass spectrometry with data-independent acquisition. The serum levels of several proteins involved in the responses to estrogen and glucocorticoid, blood vessel maturation, osteoblast differentiation, and ossification were changed by microgravity exposure. Furthermore, a collective evaluation of serum proteomic data from spaceflight and HU mice identified 30 serum proteins, including Mmp2, Igfbp2, Tnc, Cdh5, and Pmel, whose levels varied to a similar extent in both gravity unloading models. These changes in serum levels could be involved in the physiological changes induced by gravity unloading. A collective evaluation of serum, femur, and soleus muscle proteome data of spaceflight mice also showed 24 serum proteins, including Igfbp5, Igfbp3, and Postn, whose levels could be associated with biological changes induced by microgravity. This study examined serum proteome profiles in response to gravity unloading, and may help deepen our understanding of microgravity adaptation mechanisms during prolonged spaceflight missions.

Towards a comprehensive understanding of the muscle proteome in Schizothorax labiatus: Insights from seasonal variations, metabolic responses, and reproductive signatures in the River Jhelum

Proteomics is a very advanced technique used for defining correlations, compositions and activities of hundreds of proteins from organisms as well as effectively used in identifying particular proteins with varying peptide lengths and amino acid counts. In the present study, an endeavour has been put forth to create muscle proteome expression of snow trout, Schizothorax labiatus. Liquid chromatography-mass spectrometry (LC-MS) using label free quantification (LFQ) technique has extensively been carried out to explore changes in protein metabolism and its composition to discriminate across species, clarify functions and pinpoint protein biomarkers from organisms. In LFQ technique, the abundances of proteins are determined based on the signal intensities of their corresponding peptides in mass spectrometry. The main benefit of using this method is that it doesn't require pre-labelling proteins with isotopic tags, which streamlines the experimental procedure and gets rid of any bias that might have been caused by the labelling process. LFQ techniques frequently offer a wider dynamic range, making it possible to detect and quantify proteins over a broad range of abundances obtained from the complex biological materials including fish muscle. The results of proteomic analysis could provide an insight in understanding about how various proteins are expressed in response to environmental challenges. For proteomic study, two different weight groups of S. labiatus were taken from River Jhelum based on biological, physiological and logistical factors. These groups corresponded to different life stages, such as younger size and adults/brooders in order to capture potential variations in the muscle proteome related to growth and development. The proteomic analysis of S. labiatus depicted that an overall of 220 proteins in male and 228 in female fish of group 1 were noted. However, when male and female S. labiatus were examined based on spectral count and peptide abundance using ProteinLynx Global Software, a total of 10 downregulated and 32 upregulated proteins were found. In group 2 of S. labiatus, a total of 249 proteins in male and 301 in female fish were documented. When the two genders of S. labiatus were likened to one another by LFQ technique, a total of 41 downregulated and 06 upregulated proteins were identified. The variability in the protein numbers between two fish weight groups reflected biological differences, influenced by factors such as age, developmental stages, physiological condition and reproductive activities. During the study, it was observed that S. labiatus exhibited downregulated levels of proteins that were involved in feeding and growth. The contributing factors to this manifestation could be explained by lower feeding and metabolic activity of fish and decreased food availability during winter in River Jhelum. Contrarily, the fish immune response proteins were found to be significantly over-expressed in S. labiatus, indicating that the environment was more likely to undergo increased microbial infection, pollution load and anthropogenic activities. In addition, it was also discovered that there was an upregulated expression of the reproductive proteins in S. labiatus, which could be linked to the fish's pre-spawning time as the fish used in this study was collected in the winter season which is the pre-spawning period of the fish. Therefore, the present study would be useful in obtaining new insights regarding the molecular makeup of species, methods of adaptation and reactions to environmental stresses. This information contributes to our understanding of basic science and may have applications in environmental monitoring, conservation and preservation of fish species.

Robust, Precise, and Deep Proteome Profiling Using a Small Mass Range and Narrow Window Data-Independent-Acquisition Scheme.

In recent years, a plethora of different data-independent acquisition methods have been developed for proteomics to cover a wide range of requirements. Current deep proteome profiling methods rely on fractionations, elaborate chromatography, and mass spectrometry setups or display suboptimal quantitative precision. We set out to develop an easy-to-use one shot DIA method that achieves high quantitative precision and high proteome coverage. We achieve this by focusing on a small mass range of 430-670 m/z using small isolation windows without overlap. With this new method, we were able to quantify >9200 protein groups in HEK lysates with an average coefficient of variance of 3.2%. To demonstrate the power of our newly developed narrow mass range method, we applied it to investigate the effect of PGC-1α knockout on the skeletal muscle proteome in mice. Compared to a standard data-dependent acquisition method, we could double proteome coverage and, most importantly, achieve a significantly higher quantitative precision, as compared to a previously proposed DIA method. We believe that our method will be especially helpful in quantifying low abundant proteins in samples with a high dynamic range. All raw and result files are available at massive.ucsd.edu (MSV000092186).

Proteomics and bioinformatics analyses based on two-dimensional electrophoresis and LC-MS/MS for the primary characterization of protein changes in chicken breast meat from divergent farming systems: Organic versus antibiotic-free

Proteomics is a key analytical method in meat research thanks to its potential in investigating the proteins at interplay in post-mortem muscles. This study aimed to characterize for the first time the differences in early post-mortem muscle proteomes of chickens raised under two farming systems: organic versus antibiotic-free. Forty post-mortem Pectoralis major muscle samples from two chicken strains (Ross 308 versus Ranger Classic) reared under organic versus antibiotic-free farming systems were characterized and compared using two-dimensional electrophoresis and LC-MS/MS mass spectrometry. Within antibiotic-free and organic farming systems, 14 and 16 proteins were differentially abundant between Ross 308 and Ranger Classic, respectively. Within Ross 308 and Ranger Classic chicken strains, 12 and 18 proteins were differentially abundant between organic and antibiotic-free, respectively. Bioinformatics was applied to investigate the molecular pathways at interplay, which highlighted the key role of muscle structure and energy metabolism. Antibiotic-free and organic farming systems were found to significantly impact the muscle proteome of chicken breast meat. This paper further proposes a primary list of putative protein biomarkers that can be used for chicken meat or farming system authenticity.

Shatavari supplementation in postmenopausal women alters the skeletal muscle proteome and pathways involved in training adaptation

PurposeShatavari is an understudied, widely available herbal supplement. It contains steroidal saponins and phytoestrogens. We previously showed that six weeks of shatavari supplementation improved handgrip strength and increased markers of myosin contractile function. Mechanistic insights into shatavari’s actions are limited. Therefore, we performed proteomics on vastus lateralis (VL) samples that remained from our original study.MethodsIn a randomised double-blind trial, women (68.5 ± 6 years) ingested either placebo or shatavari (equivalent to 26,500 mg/d fresh weight) for six weeks. Tandem mass tag global proteomic analysis of VL samples was conducted (N = 7 shatavari, N = 5 placebo). Data were normalized to total peptides and scaled using a reference sample. Data were filtered using a 5% FDR. For each protein, the pre to post supplementation difference was expressed as log2 fold change. Welch’s t tests with Benjamini–Hochberg corrections were performed for each protein. Pathway enrichment (PADOG, CAMERA) was interrogated in Reactome (v85).ResultsNo individual protein was significantly different between supplementation conditions. Both PADOG and CAMERA indicated that pathways related to (1) Integrin/MAPK signalling, (2) metabolism/insulin secretion; (3) cell proliferation/senescence/DNA repair/cell death; (4) haemostasis/platelets/fibrin; (5) signal transduction; (6) neutrophil degranulation and (7) chemical synapse function were significantly upregulated. CAMERA indicated pathways related to translation/amino acid metabolism, viral infection, and muscle contraction were downregulated.ConclusionOur analyses indicate that shatavari may support muscle adaptation responses to exercise. These data provide useful signposts for future investigation of shatavari’s utility in conserving and enhancing musculoskeletal function in older age. Trial registrationNCT05025917 30/08/21, retrospectively registered.

Early postmortem beef muscle proteome and metabolome variations due to supranutritional zinc and ractopamine hydrochloride supplementation.

It was hypothesized that the longissimus thoracis (LT) muscle proteome, phosphoproteome, and metabolome could explain postmortem metabolism and tenderness differences in muscle from cattle supplemented zinc (Zn) and/or ractopamine hydrochloride (RH). High percentage Angus steers (N = 20) were fed in a 2 × 2 factorial assigned to Zn and RH treatments: control (CON; n = 10; analyzed 36mg Zn/kg dry matter [DM]) or supranutritional Zn supplementation (SUPZN; n = 10; control diet + 60mg Zn/kg DM [from ZnSO4] + 60mg Zn/kg DM [from Zn-amino acid complex]) for the entire 89-d trial. During the 28 d before harvest, steers were blocked by body weight within Zn treatments to RH treatments of 0 (NO; n = 10) or 300mg (RAC; n = 10) per steer per day. Steers were harvested at the Iowa State Meat Laboratory, where pH decline (1, 3, 6, and 24h postmortem) was measured. At 24h postmortem, LT muscle sections were removed from carcasses, and steaks were analyzed for Warner-Bratzler shear force (WBSF) values at 1, 3, 7, and 14 d postmortem. Muscle samples were taken at 1h, 1, 3, 7, and 14 d postmortem for the following analysis: troponin-T degradation (1, 3, 7, and 14 d postmortem), myosin heavy chain analysis (1h postmortem), sarcoplasmic proteome analysis through tandem mass tagging analysis (1h and 1 d postmortem), metabolome analysis (1h and 1 d postmortem), and phosphoproteome analysis (1h postmortem). SUPZN-NO tended to have a lower (P = 0.06) pH at 6h postmortem and a lower WBSF value (P = 0.06) at 1 d postmortem. CON-RAC had a higher (P = 0.04) pH at 6h postmortem and WBSF value (P < 0.01) at 1 d postmortem. A lower pH at 6h postmortem and lower WBSF value at 1 d postmortem in the SUPZN-NO treatment was accompanied by more sorbitol and fructose at 1 d postmortem, and less myosin regulatory light chain 2 at 1h postmortem, and less adenosine monophosphate deaminase 1 (AMPD1) at 1 d postmortem than all other treatments. A higher pH at 6h postmortem and higher WBSF value at 1 d postmortem in CON-RAC and SUPZN-RAC was accompanied by more soluble structural proteins (troponin-T and myosin-7) at 1h postmortem than CON-NO. At 1h postmortem, CON-RAC had more glyceraldehyde-3-phosphate dehydrogenase than CON-NO or SUPZN-RAC. Differences in energy metabolism enzymes, metabolites, and structural proteins may affect ATP production, rigor development, and lactate buildup which may explain the differences in postmortem metabolism and tenderness development at 1 d postmortem.

Multi-omics reveals changed energy metabolism of liver and muscle by caffeine after mice swimming.

In recent years, numerous studies have investigated the effects of caffeine on exercise, and provide convincing evidence for its ergogenic effects on exercise performance. However, the precise mechanisms underlying these ergogenic effects remain unclear. In this study, an exercise swimming model was conducted to investigate the effects of orally administered with caffeine before swimming on the alterations of proteome and energy metabolome of liver and muscle after swimming. We found proteins in liver, such as S100a8, S100a9, Gabpa, Igfbp1 and Sdc4, were significantly up-regulated, while Rbp4 and Tf decreased after swimming were further down-regulated in caffeine group. The glycolysis and pentose phosphate pathways in liver and muscle were both significantly down-regulated in caffeine group. The pyruvate carboxylase and amino acid levels in liver, including cysteine, serine and tyrosine, were markedly up-regulated in caffeine group, exhibiting a strong correlation with the increased pyruvic acid and oxaloacetate levels in muscle. Moreover, caffeine significantly decreased the lactate levels in both liver and muscle after swimming, potentially benefiting exercise performance.

Data-independent acquisition-based SWATH-MS proteomics profiling to decipher the impact of farming system and chicken strain and discovery of biomarkers of authenticity in organic versus antibiotic-free chicken meat

In the literature, there is a paucity of methods and tools that allow the identification of biomarkers of authenticity to discriminate organic and non-organic chicken meat products. Shotgun proteomics is a powerful tool that allows the investigation of the entire proteome of a muscle and/or meat sample. In this study, a shotgun proteomics approach using Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) has been applied for the first time to characterize and identify candidate protein biomarkers of authenticity in post-mortem chicken Pectoralis major muscles produced under organic and non-organic farming systems (antibiotic-free). The proteomics characterization was further performed within two chicken strains, these being Ross 308 and Ranger Classic, which differ in their growth rate. From the candidate protein biomarkers, the bioinformatics enrichment analyses revealed significant differences in the muscle proteome between the two chicken strains, which may be related to their genetic background and rearing conditions. The results further provided novel insights on the potential interconnected pathways at interplay that are associated with the differences as a consequence of farming system of chicken strain, such as muscle contraction and energy metabolism. This study could pave the way to more in-depth investigations in proteomics applications to assess chicken meat authenticity and better understand the impact of farming systems on the chicken muscle and meat quality.

Remodeling of the human skeletal muscle proteome found after long-term endurance training but not after strength training

Exercise training has tremendous systemic tissue-specific health benefits, but the molecular adaptations to long-term exercise training are not completely understood. We investigated the skeletal muscle proteome of highly endurance-trained, strength-trained, and untrained individuals and performed exercise- and sex-specific analyses. Of the 6,000+ proteins identified, >650 were differentially expressed in endurance-trained individuals compared with controls. Strikingly, 92% of the shared proteins with higher expression in both the male and female endurance groups were known mitochondrial. In contrast to the findings in endurance-trained individuals, minimal differences were found in strength-trained individuals and between females and males. Lastly, a co-expression network and comparative literature analysis revealed key proteins and pathways related to the health benefits of exercise, which were primarily related to differences in mitochondrial proteins. This network is available as an interactive database resource where investigators can correlate clinical data with global gene and protein expression data for hypothesis generation.

Relative quantification of progressive changes in healthy and dysferlin-deficient mouse skeletal muscle proteomes.

Individuals with dysferlinopathies, a group of genetic muscle diseases, experience delay in the onset of muscle weakness. The cause of this delay and subsequent muscle wasting are unknown, and there are currently no clinical interventions to limit or prevent muscle weakness. To better understand molecular drivers of dysferlinopathies, age-dependent changes in the proteomic profile of skeletal muscle (SM) in wild-type (WT) and dysferlin-deficient mice were identified. Quadriceps were isolated from 6-, 18-, 42-, and 77-wk-old C57BL/6 (WT, Dysf+/+ ) and BLAJ (Dysf-/- ) mice (n = 3, 2 male/1 female or 1 male/2 female, 24 total). Whole-muscle proteomes were characterized using liquid chromatography-mass spectrometry with relative quantification using TMT10plex isobaric labeling. Principle component analysis was utilized to detect age-dependent proteomic differences over the lifespan of, and between, WT and dysferlin-deficient SM. The biological relevance of proteins with significant variation was established using Ingenuity Pathway Analysis. Over 3200 proteins were identified between 6-, 18-, 42-, and 77-wk-old mice. In total, 46 proteins varied in aging WT SM (p < .01), while 365 varied in dysferlin-deficient SM. However, 569 proteins varied between aged-matched WT and dysferlin-deficient SM. Proteins with significant variation in expression across all comparisons followed distinct temporal trends. Proteins involved in sarcolemma repair and regeneration underwent significant changes in SM over the lifespan of WT mice, while those associated with immune infiltration and inflammation were overly represented over the lifespan of dysferlin-deficient mice. The proteins identified herein are likely to contribute to our overall understanding of SM aging and dysferlinopathy disease progression.

Mass Spectrometry-Based Proteomic Technology and Its Application to Study Skeletal Muscle Cell Biology.

Voluntary striated muscles are characterized by a highly complex and dynamic proteome that efficiently adapts to changed physiological demands or alters considerably during pathophysiological dysfunction. The skeletal muscle proteome has been extensively studied in relation to myogenesis, fiber type specification, muscle transitions, the effects of physical exercise, disuse atrophy, neuromuscular disorders, muscle co-morbidities and sarcopenia of old age. Since muscle tissue accounts for approximately 40% of body mass in humans, alterations in the skeletal muscle proteome have considerable influence on whole-body physiology. This review outlines the main bioanalytical avenues taken in the proteomic characterization of skeletal muscle tissues, including top-down proteomics focusing on the characterization of intact proteoforms and their post-translational modifications, bottom-up proteomics, which is a peptide-centric method concerned with the large-scale detection of proteins in complex mixtures, and subproteomics that examines the protein composition of distinct subcellular fractions. Mass spectrometric studies over the last two decades have decisively improved our general cell biological understanding of protein diversity and the heterogeneous composition of individual myofibers in skeletal muscles. This detailed proteomic knowledge can now be integrated with findings from other omics-type methodologies to establish a systems biological view of skeletal muscle function.

Skeletal muscle proteome analysis underpins multifaceted mitochondrial dysfunction in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is a severe multisystemic disorder caused by a deficiency of the mitochondrial protein frataxin. While some aspects of FRDA pathology are developmental, the causes underlying the steady progression are unclear. The inaccessibility of key affected tissues to sampling is a main hurdle. Skeletal muscle displays a disease phenotype and may be sampled in vivo to address open questions on FRDA pathophysiology. Thus, we performed a quantitative mass spectrometry-based proteomics analysis in gastrocnemius skeletal muscle biopsies from genetically confirmed FRDA patients (n = 5) and controls. Obtained data files were processed using Proteome Discoverer and searched by Sequest HT engine against a UniProt human reference proteome database. Comparing skeletal muscle proteomics profiles between FRDA and controls, we identified 228 significant differentially expressed (DE) proteins, of which 227 were downregulated in FRDA. Principal component analysis showed a clear separation between FRDA and control samples. Interactome analysis revealed clustering of DE proteins in oxidative phosphorylation, ribosomal elements, mitochondrial architecture control, and fission/fusion pathways. DE findings in the muscle-specific proteomics suggested a shift toward fast-twitching glycolytic fibers. Notably, most DE proteins (169/228, 74%) are target of the transcription factor nuclear factor-erythroid 2. Our data corroborate a mitochondrial biosignature of FRDA, which extends beyond a mere oxidative phosphorylation failure. Skeletal muscle proteomics highlighted a derangement of mitochondrial architecture and maintenance pathways and a likely adaptive metabolic shift of contractile proteins. The present findings are relevant for the design of future therapeutic strategies and highlight the value of skeletal muscle-omics as disease state readout in FRDA.

Lentiviral gene therapy with IGF2-tagged GAA normalizes the skeletal muscle proteome in murine Pompe disease

Pompe disease is a lysosomal storage disorder caused by deficiency of acid alpha-glucosidase (GAA), resulting in glycogen accumulation with profound pathology in skeletal muscle. We recently developed an optimized form of lentiviral gene therapy for Pompe disease in which a codon-optimized version of the GAA transgene (LV-GAAco) was fused to an insulin-like growth factor 2 (IGF2) peptide (LV-IGF2.GAAco), to promote cellular uptake via the cation-independent mannose-6-phosphate/IGF2 receptor. Lentiviral gene therapy with LV-IGF2.GAAco showed superior efficacy in heart, skeletal muscle, and brain of Gaa−/− mice compared to gene therapy with untagged LV-GAAco. Here, we used quantitative mass spectrometry using TMT labeling to analyze the muscle proteome and the response to gene therapy in Gaa−/− mice. We found that muscle of Gaa−/− mice displayed altered levels of proteins including those with functions in the CLEAR signaling pathway, autophagy, cytoplasmic glycogen metabolism, calcium homeostasis, redox signaling, mitochondrial function, fatty acid transport, muscle contraction, cytoskeletal organization, phagosome maturation, and inflammation. Gene therapy with LV-GAAco resulted in partial correction of the muscle proteome, while gene therapy with LV-IGF2.GAAco resulted in a near-complete restoration to wild type levels without inducing extra proteomic changes, supporting clinical development of lentiviral gene therapy for Pompe disease. SignificanceLysosomal glycogen accumulation is the primary cause of Pompe disease, and leads to a cascade of pathological events in cardiac and skeletal muscle and in the central nervous system. In this study, we identified the proteomic changes that are caused by Pompe disease in skeletal muscle of a mouse model. We showed that lentiviral gene therapy with LV-IGF2.GAAco nearly completely corrects disease-associated proteomic changes. This study supports the future clinical development of lentiviral gene therapy with LV-IGF2.GAAco as a new treatment option for Pompe disease.

Molecular fingerprint of gilthead seabream physiology in response to pollutant mixtures in the wild

The increase in trace element concentrations in the aquatic environment due to anthropogenic activities, urges the need for their monitoring and potential toxicity, persistence, bioaccumulation, and biomagnification at different trophic levels. Gilthead seabream is a species of commercial importance in the Mediterranean Sea, both for the aquaculture and fisheries sectors, however very little is known about their trace element contamination accumulation and the resulting effect on their health status. In the present study, 135 juveniles were collected from seven coastal lagoons known to be essential nursery areas for this species. We measured seventeen different inorganic contaminants at the individual level in fish muscle (namely Al, As, Be, Bi, Cd, Cr, Cu, Hg, Li, Ni, Pb, Rb, Sb, Sr, Ti, Tl and Zn). Our results revealed the accumulation of multiple trace elements in individuals and distinct contamination signatures between lagoons which might lead to contrasted quality as nurseries for juveniles of numerous ecologically and economically relevant fish species in addition to seabreams. We further evaluated the potential adverse effect of these complex contamination mixtures on the liver (the main organ implicated in the metabolism of xenobiotics) and red muscle (a highly metabolic organ) using a proteomic approach. Alterations in cellular organization pathways and protein transport were detected in both tissues (albeit they were not similarly regulated). Chromosome organization and telomere maintenance in the liver appeared to be affected by contaminant mixture which could increase mortality, age-related disease risk and shorter lifetime expectancy for these juveniles. Red muscle proteome also demonstrated an upregulation of pathways involved in metabolism in response to contamination which raises the issue of potential energy allocation trade-offs between the organisms’ main functions such as reproduction and growth. This study provides new insights into the cellular and molecular responses of seabreams to environmental pollution and proposed biomarkers of health effects of trace elements that could serve as a starting point for larger-scale biomonitoring programs.

First comparison of early post-mortem proteomes in two goat muscle types: M. Longissimus thoracis and M. semitendinosus

This study aimed to characterize and compare the muscle proteomes of goat Longissimus thoracis (LT) and Semitendinosus (ST) muscles taken early post-mortem from young crossbred males. Thanks to a quantitative proteomics approach, 129 differentially expressed proteins (DEPs) were identified. The proteins belong to five interconnected biological pathways: muscle contraction and structure (n = 47), catalytic, metabolism & ATP processes (n = 33), binding and transport (n = 23), oxidative stress and cell redox homeostasis (n = 10), response to stress and protein folding (n = 6). An additional group of miscellaneous proteins (n = 10) grouped proteins playing other functions. The chemometrics analyses allowed clear separations of the muscles and evidenced 14 common proteins (ACTB, ACTN3, TMOD4, TPM3, TNNT1, AK1, AKR1B1, ACAA2, GPI, AGL, DNPEP, RTN4, ATP2A1, and CAVIN4) that could be considered as potential biomarkers. Overall, the study provided the first insights on the differences between goat LT and ST muscles and the basis related to the regulation of the skeletal muscle. Moreover, it resulted that a better understanding of the muscle characteristics would help in developing practices/processes to monitor meat quality and reduce the variations in fresh quality of meat, aged meat, and/or processed goat meat products.

SAT012 In Vivo Mapping Of Subcellular Proteomes In Mice

Abstract Disclosure: K. Kim: None. I. Park: None. J. Kim: None. J. Kim: None. H. Rhee: None. J. Suh: None. To facilitate the understanding of metabolic changes associated with aging and disease processes we have developed new in vivo tools that enable tissue-specific profiling of subcellular proteomes. First we describe a method to profile in vivo mitochondrial proteomes utilizing transgenic mice expressing mitoAPEX, a peroxidase-based proximity labeling enzyme containing a mitochondrial matrix targeting sequence. Upon label activating conditions, mitoAPEX rapidly (&amp;lt;1 min) catalyzes production of biotin radicals which biotinylate proteins within a 20 nm radius. Mass analysis of biotinylated proteomes from proximity labeled mitoAPEX mouse tissues confirmed specific and efficient labeling of the mitochondrial proteome and revealed tissue-specific patterns of the matrix proteome. The labeled muscle proteomes from young and old mitoAPEX mice revealed significant changes in the quantity and composition of protein species. Of these, RTN4IP1, was shown to be downregulated in muscle tissue of old mice. However, in contrast to previous reports, our analysis of RTN4IP1 shows RTN4IP1 is localized to the mitochondrial matrix and not the outer membrane. In addition to the mitoAPEX mice, we generated another in vivo proximity labeling tool iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which labels secretory pathway proteins, via proximity labeling activity by ER lumen targeted TurboID, an engineered biotin ligase. We expressed iSLET in the mouse liver and demonstrate efficient labeling of the liver secreted proteome which could be tracked and identified within circulating blood plasma. We expect mitoAPEX and iSLET mice will facilitate our understanding of mitochondrial function and interorgan communication networks in aging and associated disease processes. Presentation: Saturday, June 17, 2023

Impact of Bmal1 Rescue and Time-Restricted Feeding on Liver and Muscle Proteomes During the Active Phase in Mice

Molecular clocks and daily feeding cycles support metabolism in peripheral tissues. Although the roles of local clocks and feeding are well defined at the transcriptional level, their impact on governing protein abundance in peripheral tissues is unclear. Here, we determine the relative contributions of local molecular clocks and daily feeding cycles on liver and muscle proteomes during the active phase in mice. LC-MS/MS was performed on liver and gastrocnemius muscle harvested 4 h into the dark phase from WT, Bmal1 KO, and dual liver- and muscle-Bmal1-rescued mice under either ad libitum feeding or time-restricted feeding during the dark phase. Feeding-fasting cycles had only minimal effects on levels of liver proteins and few, if any, on the muscle proteome. In contrast, Bmal1 KO altered the abundance of 674 proteins in liver and 80 proteins in muscle. Local rescue of liver and muscle Bmal1 restored ∼50% of proteins in liver and ∼25% in muscle. These included proteins involved in fatty acid oxidation in liver and carbohydrate metabolism in muscle. For liver, proteins involved in de novo lipogenesis were largely dependent on Bmal1 function in other tissues (i.e., the wider clock system). Proteins regulated by BMAL1 in liver and muscle were enriched for secreted proteins. We found that the abundance of fibroblast growth factor 1, a liver secreted protein, requires BMAL1 and that autocrine fibroblast growth factor 1 signaling modulates mitochondrial respiration in hepatocytes. In liver and muscle, BMAL1 is a more potent regulator of dark phase proteomes than daily feeding cycles, highlighting the need to assess protein levels in addition to mRNA when investigating clock mechanisms. The proteome is more extensively regulated by BMAL1 in liver than in muscle, and many metabolic pathways in peripheral tissues are reliant on the function of the clock system as a whole.

Sex differences in muscle protein expression and DNA methylation in response to exercise training

BackgroundExercise training elicits changes in muscle physiology, epigenomics, transcriptomics, and proteomics, with males and females exhibiting differing physiological responses to exercise training. However, the molecular mechanisms contributing to the differing adaptations between the sexes are poorly understood.MethodsWe performed a meta-analysis for sex differences in skeletal muscle DNA methylation following an endurance training intervention (Gene SMART cohort and E-MTAB-11282 cohort). We investigated for sex differences in the skeletal muscle proteome following an endurance training intervention (Gene SMART cohort). Lastly, we investigated whether the methylome and proteome are associated with baseline cardiorespiratory fitness (maximal oxygen consumption; VO2max) in a sex-specific manner.ResultsHere, we investigated for the first time, DNA methylome and proteome sex differences in response to exercise training in human skeletal muscle (n = 78; 50 males, 28 females). We identified 92 DNA methylation sites (CpGs) associated with exercise training; however, no CpGs changed in a sex-dependent manner. In contrast, we identified 189 proteins that are differentially expressed between the sexes following training, with 82 proteins differentially expressed between the sexes at baseline. Proteins showing the most robust sex-specific response to exercise include SIRT3, MRPL41, and MBP. Irrespective of sex, cardiorespiratory fitness was associated with robust methylome changes (19,257 CpGs) and no proteomic changes. We did not observe sex differences in the association between cardiorespiratory fitness and the DNA methylome. Integrative multi-omic analysis identified sex-specific mitochondrial metabolism pathways associated with exercise responses. Lastly, exercise training and cardiorespiratory fitness shifted the DNA methylomes to be more similar between the sexes.ConclusionsWe identified sex differences in protein expression changes, but not DNA methylation changes, following an endurance exercise training intervention; whereas we identified no sex differences in the DNA methylome or proteome response to lifelong training. Given the delicate interaction between sex and training as well as the limitations of the current study, more studies are required to elucidate whether there is a sex-specific training effect on the DNA methylome. We found that genes involved in mitochondrial metabolism pathways are differentially modulated between the sexes following endurance exercise training. These results shed light on sex differences in molecular adaptations to exercise training in skeletal muscle.