Protein Degradation In Skeletal Muscle Research Articles

352 Supplementing bovine satellite cells with anabolic hormones and/or zinc at differentiation impacts abundance of mRNA involved in growth, mineral transport, and oxidative stress

Abstract Zinc (Zn) is the most utilized metal in biological processes and supports growth through critical roles in protein turnover. The Zn requirements of skeletal muscle may be augmented when growth promoting technologies, such as anabolic hormones are being utilized. Both anabolic hormones and Zn affect protein synthesis and degradation in skeletal muscle. However, the molecular mechanisms through which Zn may interact with anabolic hormones to support skeletal muscle growth remains unknown. The objective of this study was to determine whether trenbolone acetate (TBA), estradiol (E2) and Zn interact to promote differentiation of bovine satellite cell (BSC) cultures by assessing abundance of mRNA involved in growth, mineral transport, differentiation, protein turnover and oxidative stress. To do this, primary BSC cultures were isolated from three different steers and grown to 80% confluency and induced to differentiate in 3% horse serum, at which time a factorial design was employed to assess the impact of Zn (0, 10, 20 or 40 μM) as ZnCl2 and/or the impact of hormone (10 nM TBA,10 nM E2, 10 nM TBA and 10 nM E2). Zinc concentrations represent physiologically relevant plasma Zn concentrations. Total mRNA was isolated from the treated cultures at 4 h, 12 h and 24 h post-treatment. Assays utilizing cultures from each steer were run in duplicate (n = 6). The mRNA abundance of 45 genes were examined and analyzed using the PROC MIXED procedure of SAS with time as a repeated measure to evaluate the fixed effects of hormone, mineral, time, and all interactions. No effects (P ≥ 0.19) of hormone x mineral x time, hormone x time, hormone x mineral or hormone were noted. A mineral x time effect (P ≤ 0.04) was noted for the igf1r, slc39a7, mapk1, mmp2, and G6pd. A mineral effect was found for hormone receptors (ir, ar, esr1, esr2, and gper; P ≤ 0.03), mRNA involved in mineral transport (slc30a10, slc30a7, sod1, sod2, slc2a4, and slc39a8; P ≤ 0.009), and genes related to skeletal muscle differentiation (pax7, myf5, myf6, spry1, myog, mef2a, and myod; P ≤ 0.02) and protein turnover (akt1, fox01, fox04, mstn,fbox32, fox03, mtor, trim63, and CKM; P ≤ 0.03). These differences are generally driven by abundance being increased (P ≤ 0.05) in cultures treated with 40 or 20 μM Zn compared with cultures receiving 0 or 10 μM Zn. These results indicate that while Zn and hormone do not appear to interact to influence gene expression of differentiating primary BSC cultures at the time points measured, increased concentrations of Zn appear to increase mRNA abundance of hormone receptors, mineral transporters, and genes involved in skeletal muscle differentiation and protein turnover.

Correction to “α‐Ketoglutarate prevents skeletal muscle protein degradation and muscle atrophy through PHD3/ADRB2 pathway”

Correction to “α‐Ketoglutarate prevents skeletal muscle protein degradation and muscle atrophy through <scp>PHD3</scp>/<scp>ADRB2</scp> pathway”

Myogenic microRNAs as Therapeutic Targets for Skeletal Muscle Mass Wasting in Breast Cancer Models.

Breast cancer is the type of cancer with the highest prevalence in women worldwide. Skeletal muscle atrophy is an important prognostic factor in women diagnosed with breast cancer. This atrophy stems from disrupted skeletal muscle homeostasis, triggered by diminished anabolic signalling and heightened inflammatory conditions, culminating in an upregulation of skeletal muscle proteolysis gene expression. The importance of delving into research on modulators of skeletal muscle atrophy, such as microRNAs (miRNAs), which play a crucial role in regulating cellular signalling pathways involved in skeletal muscle protein synthesis and degradation, has been recognised. This holds true for conditions of homeostasis as well as pathologies like cancer. However, the determination of specific miRNAs that modulate skeletal muscle atrophy in breast cancer conditions has not yet been explored. In this narrative review, we aim to identify miRNAs that could directly or indirectly influence skeletal muscle atrophy in breast cancer models to gain an updated perspective on potential therapeutic targets that could be modulated through resistance exercise training, aiming to mitigate the loss of skeletal muscle mass in breast cancer patients.

Correction to “α‐Ketoglutarate prevents skeletal muscle protein degradation and muscle atrophy through PHD3/ADRB2 pathway”

Correction to “α‐Ketoglutarate prevents skeletal muscle protein degradation and muscle atrophy through <scp>PHD3</scp>/<scp>ADRB2</scp> pathway”

The Influence of Stress and Binge-Patterned Alcohol Drinking on Mouse Skeletal Muscle Protein Synthesis and Degradation Pathways.

Adverse experiences (e.g., acute stress) and alcohol misuse can both impair skeletal muscle homeostasis, resulting in reduced protein synthesis and greater protein breakdown. Exposure to acute stress is a significant risk factor for engaging in alcohol misuse. However, little is known about how these factors together might further affect skeletal muscle health. To that end, this study investigated the effects of acute stress exposure followed by a period of binge-patterned alcohol drinking on signaling factors along mouse skeletal muscle protein synthesis (MPS) and degradation (MPD) pathways. Young adult male C57BL/6J mice participated in the Drinking in the Dark paradigm, where they received 2-4 h of access to 20% ethanol (alcohol group) or water (control group) for four days to establish baseline drinking levels. Three days later, half of the mice in each group were either exposed to a single episode of uncontrollable tail shocks (acute stress) or remained undisturbed in their home cages (no stress). Three days after stress exposure, mice received 4 h of access to 20% ethanol (alcohol) to model binge-patterned alcohol drinking or water for ten consecutive days. Immediately following the final episode of alcohol access, mouse gastrocnemius muscle was extracted to measure changes in relative protein levels along the Akt-mTOR MPS, as well as the ubiquitin-proteasome pathway (UPP) and autophagy MPD pathways via Western blotting. A single exposure to acute stress impaired Akt singling and reduced rates of MPS, independent of alcohol access. This observation was concurrent with a potent increase in heat shock protein seventy expression in the muscle of stressed mice. Alcohol drinking did not exacerbate stress-induced alterations in the MPS and MPD signaling pathways. Instead, changes in the MPS and MPD signaling factors due to alcohol access were primarily observed in non-stressed mice. Taken together, these data suggest that exposure to a stressor of sufficient intensity may cause prolonged disruptions to signaling factors that impact skeletal muscle health and function beyond what could be further induced by periods of alcohol misuse.

Differential analysis of ubiquitin-proteomics in skeletal muscle of Duroc pigs and Tibetan fragrant pigs.

Understanding the differences in ubiquitination-modified proteins between Duroc pigs and Tibetan fragrant pigs is crucial for comprehending the growth and development of their skeletal muscles. In this study, skeletal muscle samples from 30-day-old Duroc pigs and Tibetan fragrant pigs were collected. Using ubiquitination 4D-Label free quantitative proteomics, we analyzed and identified ubiquitination-modified peptides, screening out 109 differentially expressed ubiquitination-modified peptides. Further enrichment analysis was conducted on the proteins associated with these differential peptides. GO analysis results indicated that the differential genes were primarily enriched in processes such as regulation of protein transport, motor activity, myosin complex, and actin cytoskeleton. KEGG pathway analysis revealed significant enrichment in pathways such as Glycolysis/Gluconeogenesis and Hippo signaling pathway. The differentially expressed key ubiquitinated proteins, including MYL1, MYH3, TNNC2, TNNI1, MYLPF, MYH1, MYH7, TNNT2, TTN, and TNNC1, were further identified. Our analysis demonstrates that these genes play significant roles in skeletal muscle protein synthesis and degradation, providing new insights into the molecular mechanisms of muscle development in Duroc pigs and Tibetan fragrant pigs, and offering theoretical support for breeding improvements in the swine industry.

Bckdk-Mediated Branch Chain Amino Acid Metabolism Reprogramming Contributes to Muscle Atrophy during Cancer Cachexia.

Branched chain amino acids (BCAAs) are essential amino acids and important nutrient signals for energy and protein supplementation. The study uses muscle-specific branched-chain α-keto acid dehydrogenase kinase (Bckdk) conditional knockout (cKO) mice to reveal the contribution of BCAA metabolic dysfunction to muscle wasting. Muscle-specific Bckdk-cKO mice are generated through crossbreeding of Bckdkf/f mice with Myf5Cre mice. Lewis lung cancer (LLC) tumor transplantation is used to establish the cancer cachexia model. The occurrence of cancer cachexia is accelerated in the muscle-specific Bckdk-cKO mice after bearing LLC tumor. Wasting skeletal muscle is characterized by increased protein ubiquitination degradation and impaired protein synthesis. The wasting muscle gastrocnemius is mechanized as a distinct BCAA metabolic dysfunction. Based on the atrophy phenotype resulting from BCAA metabolism dysfunction, the optimized BCAA supplementation improves the survival of cancer cachexia in muscle-specific Bckdk-cKO mice bearing LLC tumors, and improves the occurrence of cancer cachexia. The mechanism of BCAA supplementation on muscle mass preservation is based on the promotion of protein synthesis and the inhibition of protein ubiquitination degradation. Dysfunctional BCAA metabolism contributes to the inhibition of protein synthesis and increases protein degradation in the cancer cachexia model of muscle-specific Bckdk-cKO mice bearing LLC tumors. The reprogramming of BCAA catabolism exerts therapeutic effects by stimulating protein synthesis and inhibiting protein degradation in skeletal muscle.

Effects of pre-slaughter fasting on antemortem skeletal muscle protein degradation levels and postmortem muscle free amino acid concentrations in broiler chickens

This study investigated the effects of pre-slaughter fasting time on the relationship between skeletal muscle protein degradation levels at slaughter and chicken meat quality after 48 h of postmortem aging. Twenty-four broiler chicks at 0 d of age were used in this study until 28 d of age. At 27 d of age, the chickens were assigned to 4 treatment groups: 0 h of fasting (0H), 8 h of fasting (8H), 16 h of fasting (16H), or 24 h of fasting (24H). They were slaughtered at 28 d of age. Blood samples were collected before fasting and immediately before slaughter. Plasma Nτ-methylhistidine concentration, an index of skeletal muscle protein degradation level, and muscle free amino acid concentration were analyzed. Antemortem changes in individual plasma Nτ-methylhistidine concentrations were significantly increased in 8H, 16H, and 24H compared to that in 0H (P < 0.05). After 48 h of postmortem storage, the glutamic acid content in the pectoralis major muscles increased with fasting time (P < 0.05), and the umami taste of chicken soup in the fasting groups (8H, 16H, 24H) was higher than that in the 0H group (P < 0.05). The antemortem changes in plasma Nτ-methylhistidine concentrations were correlated with glutamic acid content in the pectoralis major muscles (r = 0.57, P < 0.05) and umami taste (r = 0.66, P < 0.05). These results suggest that skeletal muscle protein degradation levels at slaughter are related to postmortem chicken meat quality, especially glutamic acid content and umami taste.

Protective effect of Luffa cylindrica Roemer against dexamethasone-induced muscle atrophy in primary rat skeletal muscle cells

Glucocorticoids (GCs) are commonly used in the treatment of chronic inflammatory conditions. However, the administration of high doses and long-term use of GCs can induce muscle atrophy (MA) in patients, leading to a decline in quality of life and increased mortality. MA leads to protein degradation in skeletal muscle, resulting in a reduction of muscle mass. This process is triggered by GCs like dexamethasone (DEX), which induce the expression of E3 ubiquitin ligases, namely Atrogin-1 and muscle RING-finger protein-1 (MuRF1). In this study, we examined the anti-MA potential of Luffa cylindrica Roemer (LCR) on DEX-treated primary skeletal myotubes. Primary skeletal myotubes stimulated with LCR alone resulted in a significant upregulation of myotube development, characterized by an increase in both the number and diameter of myotubes. Contrastingly, combined treatment with LCR and DEX reduced the expression of Atrogin-1, while treatment with DEX alone induced the expression of MuRF1. Furthermore, LCR treatment successfully restored the number and diameter of myotubes that had been diminished by DEX treatment. These findings suggest that LCR holds potential for treating MA, as an accelerating effect on muscle development and anti-MA effects on primary skeletal muscle cells were observed.

Vitamin D Supplementation Influences Ultramarathon-Induced Changes in Serum Amino Acid Levels, Tryptophan/Branched-Chain Amino Acid Ratio, and Arginine/Asymmetric Dimethylarginine Ratio.

Exercise affects serum levels of amino acids and their metabolites, with important metabolic consequences. Since vitamin D impacts skeletal muscle protein degradation, we hypothesised that it would also impact exercise-induced changes in serum amino acid levels and the serum levels of arginine metabolites, influencing the body's ability to synthesise NO. Accordingly, we analysed the effect of a single high-dose vitamin D supplementation on the serum levels of various amino acids in ultramarathon runners. Thirty-five male amateur runners were assigned to the supplemented group, administered 150,000 IU vitamin D in vegetable oil 24 h before the run (n = 16), or the control (placebo) group (n = 19). Blood was sampled 24 h before, immediately after, and 24 h after the run. Changes in the serum levels of some amino acids were distinct in the two groups. The asymmetric dimethyl arginine levels were significantly decreased immediately after the run and increased 24 h later and were not affected by the supplementation. The symmetric dimethyl arginine levels were increased after the run in both groups but were lower in the supplemented group than in the placebo group 24 h after the run. The dimethylamine levels increased significantly in the supplemented group as compared to the placebo group. In conclusion, vitamin D impacts exercise-induced changes in serum amino acids and methylated arginine metabolites.

Effect of temporary freezing on postmortem protein degradation patterns

BackgroundA precise determination of time since death plays a major role in forensic routine. Currently available techniques for estimating the postmortem interval (PMI) are restricted to specific time periods or cannot be applied for individual case-specific reasons. During recent years, it has been repeatedly demonstrated that Western blot analysis of postmortem muscle protein degradation can substantially contribute to overcome these limitations in cases with different background. Enabling to delimit time points at which certain marker proteins undergo distinct degradation events, the method has become a reasonable new tool for PMI delimitation under various forensic scenarios. However, additional research is yet required to improve our understanding of protein decomposition and how it is affected by intrinsic and extrinsic factors.Since there are temperature limits for proteolysis, and investigators are confronted with frozen corpses, investigation of the effects of freezing and thawing on postmortem protein decomposition in the muscle tissue is an important objective to firmly establish the new method. It is also important because freezing is often the only practical means to intermittently preserve tissue samples from both true cases and animal model research.MethodsSets of dismembered pig hind limbs, either freshly detached non-frozen, or thawed after 4 months of freeze-storage (n = 6 each), were left to decompose under controlled conditions at 30 °C for 7 days and 10 days, respectively. Samples of the M. biceps femoris were regularly collected at predefined time points. All samples were processed via SDS-PAGE and Western blotting to identify the degradation patterns of previously characterized muscle proteins.ResultsWestern blots show that the proteins degrade predictably over time in precise patterns that are largely unaffected by the freeze-and-thaw process. Investigated proteins showed complete degradation of the native protein band, partly giving rise to degradation products present in distinct time phases of the decomposition process.ConclusionThis study provides substantial new information from a porcine model to assess the degree of bias that freezing and thawing induces on postmortem degradation of skeletal muscle proteins. Results support that a freeze–thaw cycle with prolonged storage in frozen state has no significant impact on the decomposition behavior. This will help to equip the protein degradation–based method for PMI determination with a robust applicability in the normal forensic setting.

Morphological changes and protein degradation during the decomposition process of pig cadavers placed outdoors or in tents—a pilot study

The delimitation of the postmortem interval (PMI) is of utmost importance in forensic science. It is especially difficult to determine the PMI in advanced decomposition stages and/or when dead bodies are found under uncommon circumstances, such as tents, or other (semi-) enclosed environments. In such cases, especially when insect access is restricted, morphological assessment of body decomposition is one of the remaining approaches for delimitation of the PMI. However, as this method allows only vague statements/indications about the PMI, it is required to develop new and more reliable methods. One of the most important candidates is the biochemical analysis of protein degradation. In this regard, it has been demonstrated that specific skeletal muscle protein degradation patterns characterize certain time points postmortem and thus can be used as markers for PMI estimation. In order to test this method in different micro-environments, a pilot study using ten pig carcasses was conducted in summer in Northern Germany. The cadavers were openly placed outside (freely accessible for insects), as well as enclosed in tents nearby, and left to decompose to investigate decomposition processes over a time course of 10 days. Muscle samples of the M. biceps femoris were collected on a regular basis and processed via SDS-PAGE and degradation patterns of selected proteins identified by Western blotting. In addition, morphological changes of the cadavers during decomposition were assessed using the total body score (TBS). Results showed that postmortem protein degradation patterns are largely consistent between treatment groups (open field versus tents) despite major morphological differences in the decomposition rate. This field study provides evidence that muscle protein degradation is mostly unaffected by different levels of exposure, making it a sufficient candidate for PMI delimitation under various circumstances.

Assessing Gluten-Free Soy Bread Quality and Amino Acid Content.

The nutritional and palatability relevance of bread prepared with soy flour was examined. There are a few effective nutritional measures that combine palatability, convenience, and functionality in the suppression of muscle loss (contributing to the improvement and prevention of sarcopenia). Therefore, in the present study, we attempted to produce bread using soybeans, which are rich in amino acids involved in the synthesis and degradation of skeletal muscle proteins. Rice flour was also used to avoid gluten intolerance. The bread was baked in an automatic bread maker, and the rheological properties of its breadcrumbs were determined using a creep meter. We found that a 70 g slice of soy bread satisfied approximately one-fifth of the daily nutritional requirement for leucine. Although soy decreased the specific volume of bread by preventing starch construction, the use of preprocessed rice flour recovered the volume, and corn starch improved the taste. We propose that the addition of soy bread to the daily diet may be an effective protein source.

Mechanism of skeletal muscle atrophy after spinal cord injury: A narrative review.

Spinal cord injury leads to loss of innervation of skeletal muscle, decreased motor function, and significantly reduced load on skeletal muscle, resulting in atrophy. Factors such as braking, hormone level fluctuation, inflammation, and oxidative stress damage accelerate skeletal muscle atrophy. The atrophy process can result in skeletal muscle cell apoptosis, protein degradation, fat deposition, and other pathophysiological changes. Skeletal muscle atrophy not only hinders the recovery of motor function but is also closely related to many systemic dysfunctions, affecting the prognosis of patients with spinal cord injury. Extensive research on the mechanism of skeletal muscle atrophy and intervention at the molecular level has shown that inflammation and oxidative stress injury are the main mechanisms of skeletal muscle atrophy after spinal cord injury and that multiple pathways are involved. These may become targets of future clinical intervention. However, most of the experimental studies are still at the basic research stage and still have some limitations in clinical application, and most of the clinical treatments are focused on rehabilitation training, so how to develop more efficient interventions in clinical treatment still needs to be further explored. Therefore, this review focuses mainly on the mechanisms of skeletal muscle atrophy after spinal cord injury and summarizes the cytokines and signaling pathways associated with skeletal muscle atrophy in recent studies, hoping to provide new therapeutic ideas for future clinical work.

Hyperglycaemia Attenuated C2C12 Myoblast Proliferation and Induced Skeletal Muscle Atrophy via Modulating Myogenic Regulatory Factors Genes Expression in Diabetic Rats

Diabetes mellitus is characterised by high blood glucose level termed hyperglycaemia (HG). It has been reported to affect skeletal muscle by inducing skeletal muscle atrophy and skeletal muscle protein degradation leading to impairment of muscle function. This study aimed to investigate the effects of HG on the expression of myogenic regulatory factor genes in muscle progenitor cells and in skeletal muscle. The number of C2C12 myoblasts cultured in HG condition was significantly decreased compared to control in dose and time dependent manner. In addition, the number of Ki-67 positive nuclei was significantly decreased after treatment under HG condition. Real time PCR showed significant suppression of MyoD and myogenin, while Myf5 gene expression was significantly enhanced, compared to control. Furthermore, histological examinations of muscle fibres showed atrophy of tibialis anterior (TA) muscle from diabetic rats. The frequency of distribution of muscle fibre cross-sectional area (MCA) in diabetic rats was shifted leftward from that of normal control rats. In contrast, the MyoD and myogenin expression in TA muscle of diabetic rats were significantly increased compared to normal control rats. This study provides novel knowledge on the changing myogenic regulatory factor gene expression in hyperglycaemic condition, both in vitro and in vivo, leading to skeletal muscle atrophy.

Mealworm Ethanol Extract Enhances Myogenic Differentiation and Alleviates Dexamethasone-Induced Muscle Atrophy in C2C12 Cells.

Aging, and other disease-related muscle disorders are serious health problems. Dexamethasone (DEX), a synthetic glucocorticoid, can trigger skeletal muscle atrophy. This study examined the effects of mealworm (Tenebrio molitor larva) ethanol extract (TME) on C2C12 myoblast differentiation and DEX-induced myotube atrophy. TME induced myotube formation compared to the differentiation medium (DM) group. TME also significantly increased the mRNA expression of muscle creatine kinase (CKm) and myogenic regulatory factors (MRFs), such as myogenin (MyoG), myogenic factor (Myf)5, and MRF4 (Myf6). TME dramatically increased the muscle-specific protein, MyoG, compared to the control, whereas the expression of myogenic differentiation 1 (MyoD) remained unchanged. It also activated the mammalian target of rapamycin (mTOR) signaling pathway. In the DEX-induced muscle atrophy C2C12 model, TME reduced the gene expression of atrogin-1, muscle RING finger protein-1 (MuRF-1), and myostatin, which are involved in protein degradation in skeletal muscles. Furthermore, TME elevated the phosphorylation of forkhead box O3 (FoxO3α) and protein kinase B (Akt). These findings suggest that TME can enhance myotube hypertrophy by regulating the mTOR signaling pathway, and can rescue DEX-induced muscle atrophy by alleviating atrophic muscle markers mediated by Akt activation. Thus, TME can be a potential therapeutic agent for treating muscle weakness and atrophy.

The Role of Skeletal Muscle Mitochondria in Colorectal Cancer Related Cachexia: Friends or Foes?

Up to 60% of colorectal cancer (CRC) patients develop cachexia. The presence of CRC related cachexia is associated with more adverse events during systemic therapy, leading to a high mortality rate. The main manifestation in CRC related cachexia is the loss of skeletal muscle mass, resulting from an imbalance between skeletal muscle protein synthesis and protein degradation. In CRC related cachexia, systemic inflammation, oxidative stress, and proteolytic systems lead to mitochondrial dysfunction, resulting in an imbalanced skeletal muscle metabolism. Mitochondria fulfill an important function in muscle maintenance. Thus, preservation of the skeletal muscle mitochondrial homeostasis may contribute to prevent the loss of muscle mass. However, it remains elusive whether mitochondria play a benign or malignant role in the development of cancer cachexia. This review summarizes current (mostly preclinical) evidence about the role of skeletal muscle mitochondria in the development of CRC related cachexia. Future human research is necessary to determine the physiological role of skeletal muscle mitochondria in the development of human CRC related cachexia.

The accumulation of muscle RING finger-1 in regenerating myofibers: Implications for muscle repair in immune-mediated necrotizing myopathy.

Muscle RING finger-1 (MuRF-1) plays a key role in the degradation of skeletal muscle proteins. We hypothesize the involvement of MuRF-1 in immune-mediated necrotizing myopathy (IMNM). Muscle biopsies from patients with IMNM (n = 37) were analyzed and compared to biopsies from patients with dermatomyositis (DM, n = 13), dysferlinopathy (n = 9) and controls (n = 7) using immunostaining. MuRF-1 staining could be observed in IMNM, DM and dysferlinopathy biopsies, whereas the percentage of MuRF-1 positive myofibers was significantly higher in IMNM than in dysferlinopathy (p = 0.0448), and positively correlated with muscle weakness and disease activity in IMNM and DM. Surprisingly, MuRF-1 staining predominantly presented in regenerating fibers but not in atrophic fibers. Moreover, MuRF-1-positive fibers tended to be distributed around necrotic myofibers and myofibers with sarcolemma membrane attack complex deposition. Abundant MuRF-1 expression in IMNM and DM was associated with rapid activation of myogenesis after muscle injury, whereas relatively low expression of MuRF-1 in dysferlinopathy may be attributed to damaged muscle regeneration. MuRF-1 accumulated in regenerating myofibers, which may contribute to muscle injury repair in IMNM and DM. MuRF-1 staining may help clinicians differentiate IMNM and dysferlinopathy.

Abstract A062: Skeletal Muscle Selective Autophagy Receptors are induced PDAC Cachexia

Abstract Cancer-induced cachexia is a hypermetabolic condition characterized by the unintentional wasting of muscle and adipose tissue, affecting over 80% of patients with pancreatic ductal adenocarcinoma (PDAC). Muscle wasting during cachexia is due to increased skeletal muscle protein degradation via ubiquitin-proteasome and autophagy-lysosome pathways. Autophagy-lysosome degradation requires delivery of cargo to the lysosome for destruction and recycling. Macroautophagy is the most prevalent component of autophagy, encompassing bulk and selective autophagy, and it requires the de novo synthesis of an autophagosome. Bulk autophagy randomly engulfs portions of the cytoplasm. Selective autophagy is mediated through selective autophagy receptors (SAR), which bind and couple cargo to the autophagosome via the general autophagy ligands LC3B and GABARAP. While an increase in the general autophagy machinery is well described in cachectic muscle, much less is known about how complexes and organelles are selectively targeted for degradation. Methods: Here, 12-week-old male C57BL/6J mice were orthotopically implanted with 1x105 KPC cells; controls underwent sham surgery. Half of the tumor-bearing mice were treated with 120 mg/kg gemcitabine and 10 mg/kg nab-paclitaxel (GemNP) at 4 and 10 days. For endpoint analysis, mice were euthanized at 14 days when KPC mice had significant body weight, muscle mass and muscle protein loss compared to SHAM controls. In our in vitro model of PDAC cachexia, KPC-conditioned media (CM) induces C2C12 myotube wasting; thus, myotubes were treated with 50% KPC-CM or control for 48hrs. Results: GemNP reduced end tumor mass by nearly 25% and prevented body weight and muscle loss. mRNAseq of gastrocnemius muscle demonstrated induction of ribosomal component gene expression, while deep proteomics revealed reduction of 30 ribosomal component proteins in KPC mice, consistent with ongoing destruction of ribosomes. Gene expression of general autophagy ligands, LC3B (2.9-fold) and GABARAP (1.6-fold), were increased in KPC mice. Gene expression for SARs associated with ribophagy (NUFIP1, 2.6-fold) and reticulophagy (Fam134b, 11.3-fold) were increased in KPC. FAM134b protein (1.8-fold) was also increased in KPC mice. SARs for lipophagy (PNPLA2, 3.2- and PNPLA8, 1.8-fold) and mitophagy (BNIP3, 4.5-fold) were induced in KPC versus SHAM, while SARs for glycophagy and ferritinophagy were similar. SQSTM1 (3.9-fold) and NBR1 (1.9-fold), general SARs for multiple organelles including aggrephagy, lysophagy, proteaphagy and pexophagy, were increased in KPC. This activation of SARs was due to tumor-induced wasting and not chemotherapy as SAR gene expression in KPC-GemNP mice was similar to SHAM controls. Finally, we also observe markers of SAR-mediated autophagy in our in vitro model of PDAC cachexia. Conclusion: These data indicate that muscle wasting in PDAC cachexia is through activation of selective autophagy of ribosomes, mitochondria, lipid droplets, endoplasmic reticulum, protein aggregates, lysosomes, peroxisomes, and proteasomes. Citation Format: Brittany R. Counts, Ashok Narasimhan, Tara S. Umberger, Emma H. Doud, Amber L. Mosley, Teresa A. Zimmers. Skeletal Muscle Selective Autophagy Receptors are induced PDAC Cachexia [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A062.

4-Oxo-2-nonenal (4-ONE)-Induced Degradation of Bovine Skeletal Muscle Proteins.

Lipids are an important component of meat, as they provide desirable sensory characteristics and nutritional benefits. However, lipids are susceptible to degradation through oxidation and produce toxic oxidative byproducts. 4-Oxo-2-nonenal (4-ONE) is an oxidative byproduct that is highly reactive and cytotoxic. In this study, we investigated the influence of 4-ONE-induced protein degradation on fresh and gastric digested bovine skeletal muscle proteins. The results indicated that 4-ONE naturally forms in fresh muscle proteins. We report here for the first time that 4-ONE causes severe degradation of bovine skeletal muscle proteins. An SDS-PAGE gel analysis showed evidence that the skeletal muscle proteins attenuated over the incubation time, as the density of the protein bands faded significantly after 120 h. Additionally, protein and band density analyses showed a significant decrease in protein abundance and band densities throughout the incubation time. This study revealed that the lipid oxidation byproduct, 4-oxo-2-nonenal (4-ONE) is responsible for causing skeletal muscle protein degradation. Future studies should assess the bioprotective role of antioxidants and other food ingredients for their potential to prevent the formation and/or detoxification of 4-ONE in meat.