Effet de la protéine de morue sur la régénération musculaire consécutive à une blessure chez le rat

Cette thèse visait à déterminer les effets de la protéine de morue sur la régénération post-blessure du muscle squelettique chez le rat. Nous avons observé que la croissance et la récupération de la masse musculaire étaient meilleures lorsque les animaux consommaient de la protéine de morue, conduisant à une augmentation de l’aire des fibres musculaires comparée à la caséine. Ces effets bénéfiques de la protéine de morue étaient accompagnés d’un niveau plus élevé de myogénine et d’une réduction de noyaux centraux et de l’espace interstitiel. Plus spécifiquement, cette thèse consistait à déterminer des mécanismes et à identifier un groupe d’acides aminés présent dans la protéine de morue pouvant expliquer ses effets bénéfiques. Nos résultats ont démontré que la protéine de morue, grâce à ses niveaux élevés d’arginine, de glycine, de taurine et de lysine, a réduit la densité des macrophages pro-inflammatoire (ED1+) et le niveau de COX-2 tandis qu’elle a augmenté la densité des macrophages anti-inflammatoires (ED2+) comparée à la caséine. Cependant, cette réponse anti-inflammatoire ne pouvait expliquer que partiellement l’effet positif de la protéine de morue. En effet, l’ajout d’arginine, de glycine, de taurine et de lysine à la caséine, bien que simulant adéquatement l’effet anti-inflammatoire, reproduisait en partie seulement la croissance et la régénération musculaire accrues observées avec la protéine de morue. De plus, nous avons observé que la protéine de morue diminuait le niveau de MuRF1 à la phase inflammatoire, indiquant une réduction de dégradation des protéines musculaires comparée à la caséine. La protéine de morue pourrait avoir aussi favorisé la protéosynthèse musculaire lors de la phase avancée du processus puisque les niveaux de phospho-Akt-Ser473 étaient augmentés. Par ailleurs, les effets hypertrophiques et anti-cataboliques de la protéine de morue n’étaient que partiellement expliqués par ses niveaux élevés d’arginine, de glycine, de taurine et de lysine. En conclusion, les effets anti-inflammatoires de la protéine de morue sont attribués à ses niveaux élevés d’arginine, de glycine, de taurine et de lysine alors que ces acides aminés contribuent partiellement aux effets bénéfiques observés avec la protéine de morue sur la récupération de la masse musculaire. Ces données pourront servir de tremplin vers des traitements nutritionnels efficaces pour optimiser la régénération musculaire post-blessure.The overall aim of this thesis was to study the effects of cod protein on regeneration of skeletal muscle following injury in rats. We observed that recovery of muscle mass and/or growth were higher in animals consuming the cod protein regimen, leading to larger fiber size compared with those consuming the casein diet. The beneficial effects of cod protein on muscle regeneration were also shown by higher level of myogenin, lower number of centrally-nucleated fibers and reduced interstitial space. Specifically, the current thesis was designed to identify which specific amino acids in cod protein could underly its impact on muscle repair and to investigate the pathways supporting these effects. Our results showed that cod protein reduced the density of pro-inflammatory macrophages (ED1+) and the level of COX-2 while increasing the density of anti-inflammatory macrophages (ED2+) compared to casein, due to its high levels of arginine, glycine, taurine and lysine. However, this anti-inflammatory action could only partially explain the positive effect seen with cod protein on muscle recovery because the addition of arginine, glycine, taurine and lysine to casein, although it closely mimicked the anti-inflammatory effect of cod protein, did not support muscle growth and regeneration as did cod protein. When examining the IGF1-Akt/PKB signaling during the recovery period, we observed that cod protein decreased the level of MuRF1 early after the injury, indicating a reduced muscle protein degradation compared to casein. Data also suggest that cod protein might have increased muscle protein synthesis during the later phase of the recovery process based on an increased phospho-Akt-Ser473. Hypertrophic and anti-catabolic effects exerted by cod protein were only partially driven by its high levels of arginine, glycine, taurine and lysine. Through this work in rats, we have demonstrated that while the beneficial effects of consuming cod protein on inflammation are driven by its high levels of arginine, glycine, taurine and lysine, these amino acids only partly contribute to the effect seen with cod protein on muscle mass recovery following injury. These data could help elaborate more efficient nutritional strategies in order to optimize muscle recovery after injury

Gpr18 agonist dampens inflammation, enhances myogenesis, and restores muscle function in models of Duchenne muscular dystrophy

Introduction: Muscle wasting in Duchenne Muscular Dystrophy is caused by myofiber fragility and poor regeneration that lead to chronic inflammation and muscle replacement by fibrofatty tissue. Our recent findings demonstrated that Resolvin-D2, a bioactive lipid derived from omega-3 fatty acids, has the capacity to dampen inflammation and stimulate muscle regeneration to alleviate disease progression. This therapeutic avenue has many advantages compared to glucocorticoids, the current gold-standard treatment for Duchenne Muscular Dystrophy. However, the use of bioactive lipids as therapeutic drugs also faces many technical challenges such as their instability and poor oral bioavailability.Methods: Here, we explored the potential of PSB-KD107, a synthetic agonist of the resolvin-D2 receptor Gpr18, as a therapeutic alternative for Duchenne Muscular Dystrophy.Results and discussion: We showed that PSB-KD107 can stimulate the myogenic capacity of patient iPSC-derived myoblasts in vitro. RNAseq analysis revealed an enrichment in biological processes related to fatty acid metabolism, lipid biosynthesis, small molecule biosynthesis, and steroid-related processes in PSB-KD107-treated mdx myoblasts, as well as signaling pathways such as Peroxisome proliferator-activated receptors, AMP-activated protein kinase, mammalian target of rapamycin, and sphingolipid signaling pathways. In vivo, the treatment of dystrophic mdx mice with PSB-KD107 resulted in reduced inflammation, enhanced myogenesis, and improved muscle function. The positive impact of PSB-KD107 on muscle function is similar to the one of Resolvin-D2. Overall, our findings provide a proof-of concept that synthetic analogs of bioactive lipid receptors hold therapeutic potential for the treatment of Duchenne Muscular Dystrophy

Shrimp Protein Hydrolysate Modulates the Timing of Proinflammatory Macrophages in Bupivacaine-Injured Skeletal Muscles in Rats

This study was designed to determine whether marine-derived proteins other than cod could have beneficial effects on inflammation following muscle injury. Macrophage and neutrophil densities were measured from bupivacaine-injured tibialis anterior muscle of rats fed isoenergetic diets containing either shrimp hydrolysate (Shr), casein hydrolysate (CaH), or whole casein (Ca). In this study, Shr reduced ED1+-macrophages at day 2 (p=0.013), day 5 (p=0.006), and day 14 after injury (p=0.038) compared with Ca, indicating faster resolution of inflammation in Shr. Except for day 2 after injury where Shr led to lower ED1+-macrophages compared with CaH (p=0.006), both Shr and CaH responded similarly at days 5, 14, and 28 after injury. This findings suggest that beneficial effects of Shr on ED1+-cells might be related to generation of anti-inflammatory peptides through the hydrolysis process, in addition to its high content of anti-inflammatory amino acids. However, while increasing myofiber cross-sectional area in noninjured muscles compared with both Ca and CaH, Shr failed to have a positive effect in corresponding injured muscles. These data indicate that shrimp hydrolysate can facilitate resolution of inflammation after muscle injury mainly through modulating proinflammatory macrophage accumulation but have less effect on optimal recovery in terms of muscle mass and fiber size

A n-3 PUFA depletion applied to rainbow trout fry (Oncorhynchus mykiss) does not modulate its subsequent lipid bioconversion capacity

Nutritional strategies are currently developed to produce farmed fish rich in n-3 long-chain PUFA (LC-PUFA) whilst replacing fish oil by plantderived oils in aquafeeds. The optimisation of such strategies requires a thorough understanding of fish lipid metabolism and its nutritional modulation. The present study evaluated the fatty acid bioconversion capacity of rainbow trout (Oncorhynchus mykiss) fry previously depleted in n-3 PUFA through a 60-d pre-experimental feeding period with a sunflower oil-based diet (SO) followed by a 36-d experimental period during which fish were fed either a linseed oil-based diet (LO) (this treatment being called SO/LO) or a fish oil-based diet (FO) (this treatment being called SO/FO). These treatments were compared with fish continuously fed on SO, LO or FO for 96 d. At the end of the 36-d experimental period, SO/LO and SO/FO fish recovered >80% of the n-3 LC-PUFA reported for LO and FO fish, respectively. Fish fed on LO showed high apparent in vivo elongation and desaturation activities along the n-3 biosynthesis pathway. However, at the end of the experimental period, no impact of the fish n-3 PUFA depletion was observed on apparent in vivo elongation and desaturation activities of SO/LO fish as compared with LO fish. In contrast, the fish n-3 PUFA depletion negatively modulated the n-6 PUFA bioconversion capacity of fish in terms of reduced apparent in vivo elongation and desaturation activities. The effects were similar after 10 or 36 d of the experimental period, indicating the absence of short-term effects

Inhibition of type I PRMTs reforms muscle stem cell identity enhancing their therapeutic capacity

In skeletal muscle, muscle stem cells (MuSC) are the main cells responsible for regeneration upon injury. In diseased skeletal muscle, it would be therapeutically advantageous to replace defective MuSCs, or rejuvenate them with drugs to enhance their self-renewal and ensure long-term regenerative potential. One limitation of the replacement approach has been the inability to efficiently expand MuSCs ex vivo, while maintaining their stemness and engraftment abilities. Herein, we show that inhibition of type I protein arginine methyltransferases (PRMTs) with MS023 increases the proliferative capacity of ex vivo cultured MuSCs. Single cell RNA sequencing (scRNAseq) of ex vivo cultured MuSCs revealed the emergence of subpopulations in MS023-treated cells which are defined by elevated Pax7 expression and markers of MuSC quiescence, both features of enhanced self-renewal. Furthermore, the scRNAseq identified MS023-specific subpopulations to be metabolically altered with upregulated glycolysis and oxidative phosphorylation (OxPhos). Transplantation of MuSCs treated with MS023 had a better ability to repopulate the MuSC niche and contributed efficiently to muscle regeneration following injury. Interestingly, the preclinical mouse model of Duchenne muscular dystrophy had increased grip strength with MS023 treatment. Our findings show that inhibition of type I PRMTs increased the proliferation capabilities of MuSCs with altered cellular metabolism, while maintaining their stem-like properties such as self-renewal and engraftment potential

Muscle mass recovery of regenerating tibialis anterior muscle in rats.

<p>Both <i>sham</i> and bupivacaine-injured muscles were collected at days 0, 2, 5, 14, and 28 post-injury, and weighed. Muscle weights are presented for injured (A) and <i>sham</i> (B) muscles. A recovery index, as the percentage of injured relative to <i>sham</i> values, was calculated and presented for muscle mass (C). Values are mean ± SEM (n = 7-8 rats per dietary group/time point). Groups bearing different letters for a given time point are significantly different (p≤0.05). C, casein; CP, cod protein; C⁺, casein supplemented with L-arginine, glycine, L-taurine and L-lysine.</p

Expression level of MyoD and myogenin, performed by immunoblotting.

<p>The expression of MyoD (A) and myogenin (B), both are usually up-regulated during muscle regeneration, was measured in the injured muscles at day 2 and day 5, respectively. All values were corrected for GAPDH as a protein loading control. Results are expressed as a percentage of values obtained at day 0 (mean ± SEM, n = 3/group). Groups bearing different letters for a given time point are significantly different (p≤0.05). C, casein; CP, cod protein; C⁺, casein supplemented with L-arginine, glycine, L-taurine and L-lysine.</p

Time course of inflammatory cell accumulation in rat tibialis anterior muscle following bupivacaine injection.

<p>Transverse sections (10 µm) were immunoassayed with specific antibodies against neutrophils (W3/13) (A), ED1⁺-macrophages (B), or ED2⁺-macrophages (C). Labeled cells were counted at 400X magnification, and expressed as a number of cells/mm³. Values are mean ± SEM (n = 7-8 rats per dietary group/time point). As a control, values for neutrophils and macrophages at T0 are indicated by the dotted line. Groups bearing different letters for a given time point are significantly different (p≤0.05). C, casein; CP, cod protein; C⁺, casein supplemented with L-arginine, glycine, L-taurine and L-lysine.</p

Representative H/E stained cross-sections and percentage of centrally nucleated myofibers.

<p>The number of centrally nucleated myofibers was counted in two non crossing H/E-stained sections of injured muscles collected at days 5, 14, and 28 post-injury. Representative H/E stained cross-sections and the percentages of centrally nucleated myofibers are shown in panels A and B, respectively. Data from panel B are expressed as a percentage of the total myofibers in the cross-sections. Values are mean ± SEM (n = 7-8 rats per dietary group/time point). Groups bearing different letters for a given time point are significantly different (p≤0.05). C, casein; CP, cod protein; C⁺, casein supplemented with L-arginine, glycine, L-taurine and L-lysine.</p