The aim of this study was to evaluate the effects of a Gallium Arsenide (GaAs) laser, using a high final energy of 4.8 J, during muscle regeneration after cryoinjury. Thirty Wistar rats were divided into three groups: Control (C, n = 10); Injured (I, n = 10) and Injured and laser treated (Injured/LLLT, n = 10). The cryoinjury was induced in the central region of the tibialis anterior muscle (TA). The applications of the laser (904 nm, 50 mW average power) were initiated 24 h after injury, at energy density of 69 J cm(-1) for 48 s, for 5 days, to two points of the lesion. Twenty-four hours after the final application, the TA muscle was removed and frozen in liquid nitrogen to assess the general muscle morphology and the gene expression of TNF-α, TGF-β, MyoD, and Myogenin. The Injured/LLLT group presented a higher number of regenerating fibers and fewer degenerating fibers (P < 0.05) without changes in the collagen remodeling. In addition, the Injured/LLLT group presented a significant decrease in the expression of TNF-α and myogenin compared to the injured group (P < 0.05). The results suggest that the GaAs laser, using a high final energy after cryoinjury, promotes muscle recovery without changing the collagen remodeling in the muscle extracellular matrix.

Skeletal muscle is capable of phenotypic adaptation to environmental factors, such as nutrient availability, by altering the balance between muscle catabolism and anabolism that in turn coordinates muscle growth. Small noncoding RNAs, known as microRNAs (miRNAs), repress the expression of target mRNAs, and many studies have demonstrated that miRNAs regulate the mRNAs of catabolic and anabolic genes. We evaluated muscle morphology, gene expression of components involved in catabolism, anabolism and energetic metabolism and miRNAs expression in both the fast and slow muscle of juvenile pacu (Piaractus mesopotamicus) during food restriction and refeeding. Our analysis revealed that short periods of food restriction followed by refeeding predominantly affected fast muscle, with changes in muscle fiber diameter and miRNAs expression. There was an increase in the mRNA levels of catabolic pathways components (FBXO25, ATG12, BCL2) and energetic metabolism-related genes (PGC1α and SDHA), together with a decrease in PPARβ/δ mRNA levels. Interestingly, an increase in mRNA levels of anabolic genes (PI3K and mTORC1 complex: mTOR, mLST8 and RAPTOR) was also observed during food restriction. After refeeding, muscle morphology showed similar patterns of the control group; the majority of genes were slightly up- or down-regulated in fast and slow muscle, respectively; the levels of all miRNAs increased in fast muscle and some of them decreased in slow muscle. Our findings demonstrated that a short period of food restriction in juvenile pacu had a considerable impact on fast muscle, increasing the expression of anabolic (PI3K and mTORC1 complex: mTOR, mLST8 and RAPTOR) and energetic metabolism genes. The miRNAs (miR-1, miR-206, miR-199 and miR-23a) were more expressed during refeeding and while their target genes (IGF-1, mTOR, PGC1α and MAFbx), presented a decreased expression. The alterations in mTORC1 complex observed during fasting may have influenced the rates of protein synthesis by using amino acids from protein degradation as an alternative mechanism to preserve muscle phenotype and metabolic demand maintenance.

Pacu (Piaractus mesopotamicus) is a Brazilian fish with a high economic value in pisciculture due to its rusticity and fast growth. Postnatal growth of skeletal muscle in fish occurs by hyperplasia and/or hypertrophy, processes that are dependent on the proliferation and differentiation of myoblasts. A class of small noncoding RNAs, known as microRNAs (miRNAs), represses the expression of target mRNAs, and many studies have demonstrated that miR-1, miR-133, miR-206 and miR-499 regulate different processes in skeletal muscle through the mRNA silencing of hdac4 (histone deacetylase 4), srf (serum response factor), pax7 (paired box 7) and sox6 ((sex determining region Y)-box 6), respectively. The aim of our work was to evaluate the expression of these miRNAs and their putative target mRNAs in fast- and slow-twitch skeletal muscle of pacu during growth. We used pacus in three different development stages: larval (aged 30 days), juvenile (aged 90 days and 150 days) and adult (aged 2 years). To complement our study, we also performed a pacu myoblast cell culture, which allowed us to investigate miRNA expression in the progression from myoblast proliferation to differentiation. Our results revealed an inverse correlation between the expression of the miRNAs and their target mRNAs, and there was evidence that miR-1 and miR-206 may regulate the differentiation of myoblasts, whereas miR-133 may regulate the proliferation of these cells. miR-499 was highly expressed in slow-twitch muscle, which suggests its involvement in the specification of the slow phenotype in muscle fibers. The expression of these miRNAs exhibited variations between different development stages and between distinct muscle twitch phenotypes. This work provides the first identification of miRNA expression profiles in pacu skeletal muscle and suggests an important role of these molecules in muscle growth and in the maintenance of the muscle phenotype.

The postembryonic growth of skeletal muscle in teleost fish involves myoblast proliferation, migration and differentiation, encompassing the main events of embryonic myogenesis. Ascorbic acid plays important cellular and biochemical roles as an antioxidant and contributes to the proper collagen biosynthesis necessary for the structure of connective and bone tissues. However, whether ascorbic acid can directly influence the mechanisms of fish myogenesis and skeletal muscle growth remains unclear. The aim of our work was to evaluate the effects of ascorbic acid supplementation on the in vitro myoblast proliferation and migration of pacu (Piaractus mesopotamicus). To provide insight into the potential antioxidant role of ascorbic acid, we also treated myoblasts in vitro with menadione, which is a powerful oxidant. Our results show that ascorbic acid-supplemented myoblasts exhibit increased proliferation and migration and are protected against the oxidative stress caused by menadione. In addition, ascorbic acid increased the activity of the antioxidant enzyme superoxide dismutase and the expression of myog and mtor, which are molecular markers related to skeletal muscle myogenesis and protein synthesis, respectively. This work reveals a direct influence of ascorbic acid on the mechanisms of pacu myogenesis and highlights the potential use of ascorbic acid for stimulating fish skeletal muscle growth.