RESUMEN

Muscle fibres are classified as fast, intermediate and slow. In vitro myoblast cell culture model from fast muscle is a very useful tool to study muscle growth and development; however, similar models for slow muscle do not exist. Owing to the compartmentalization of fish muscle fibres, we have developed a slow myoblast cell culture for rainbow trout (Oncorhynchus mykiss). Slow and fast muscle-derived myoblasts have similar morphology, but with differential expression of slow muscle markers such as slow myhc, sox6 and pgc-1α We also characterized the mir-133 and mir-499 microRNA families in trout slow and fast myoblasts as a case study during myogenesis and in response to electrostimulation. Three mir-133 (a-1a, a-1b and a-2) and four mir-499 (aa, ab, ba and bb) paralogues were identified for rainbow trout and named base on their phylogenetic relationship to zebrafish and Atlantic salmon orthologues. Omy-mir-499ab and omy-mir-499bb had 0.6 and 0.5-fold higher expression in slow myoblasts compared with fast myoblasts, whereas mir-133 duplicates had similar levels in both phenotypes and little variation during development. Slow myoblasts also showed increased expression for omy-mir-499b paralogues in response to chronic electrostimulation (7-fold increase for omy-mir-499ba and 2.5-fold increase for omy-mir-499bb). The higher expression of mir-499 paralogues in slow myoblasts suggests a role in phenotype determination, while the lack of significant differences of mir-133 copies during culture development might indicate a different role in fish compared with mammals. We have also found signs of sub-functionalization of mir-499 paralogues after electrostimulation, with omy-mir-499b copies more responsive to electrical signals.

Asunto(s)

RESUMEN

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 PPARß/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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Skeletal muscle growth in the pirarucu (Arapaima gigas) is highly interesting to fish farmers because it provides information about how the mechanism in muscle mass increase, characteristic of the pecies, is regulated. Pirarucu has specific muscle growth that highlights the species’s significance and commercial value. Current research evaluates the morphology and the growth-related gene expression in the red and white skeletal muscles of the pirarucu. Muscle samples were collected from the lateral anterior region and frozen in liquid nitrogen. Histological sections were performed and stained by HE for morphological analysis. Red and white muscle samples were used to determine MyoD, myogenin, and myostatin genes expression by Real-time Polymerase Chain Reaction. Although MyoD and myogenin were not statistically different in the two types of muscles, myostatin was significantly higher in the white rather than in the red muscle. Results show the muscle growth characteristics of the species and may be helpful for improving aquaculture management programs.

A caracterização do crescimento muscular no pirarucu (Arapaima gigas) é de elevado interesse para os piscicultores, pois fornece informações de extrema importância sobre como esse mecanismo é regulado e permite o rápido aumento na massa muscular característico da espécie. O pirarucu possui um crescimento muscular típico, o que destaca sua importância e seu valor comercial. O objetivo do presente trabalho foi avaliar a morfologia e a expressão de genes relacionados ao crescimento da musculatura esquelética vermelha e branca do pirarucu. As amostras de músculo vermelho e branco foram obtidas da região lateral anterior superficial e profunda, respectivamente, e estas foram congeladas em nitrogênio líquido. Para análise morfológica, cortes histológicos obtidos em criostato foram submetidos à coloração com HE. A expressão dos genes MyoD, miogenina e miostatina foi feita por PCR em tempo real após transcrição reversa (RT-qPCR). Em relação à expressão de MyoD e miogenina, não houve diferença estatística na comparação entre os músculos; por outro lado, a expressão da miostatina foi significativamente maior no músculo branco, em comparação com o músculo vermelho. Estes resultados refletem as características de crescimento muscular do pirarucu e podem ser úteis na tentativa de melhorar as condições de criação e a sobrevivência da espécie.

Asunto(s)

RESUMEN

Skeletal muscle growth in the pirarucu (Arapaima gigas) is highly interesting to fish farmers because it provides information about how the mechanism in muscle mass increase, characteristic of the pecies, is regulated. Pirarucu has specific muscle growth that highlights the speciess significance and commercial value. Current research evaluates the morphology and the growth-related gene expression in the red and white skeletal muscles of the pirarucu. Muscle samples were collected from the lateral anterior region and frozen in liquid nitrogen. Histological sections were performed and stained by HE for morphological analysis. Red and white muscle samples were used to determine MyoD, myogenin, and myostatin genes expression by Real-time Polymerase Chain Reaction. Although MyoD and myogenin were not statistically different in the two types of muscles, myostatin was significantly higher in the white rather than in the red muscle. Results show the muscle growth characteristics of the species and may be helpful for improving aquaculture management programs.(AU)

A caracterização do crescimento muscular no pirarucu (Arapaima gigas) é de elevado interesse para os piscicultores, pois fornece informações de extrema importância sobre como esse mecanismo é regulado e permite o rápido aumento na massa muscular característico da espécie. O pirarucu possui um crescimento muscular típico, o que destaca sua importância e seu valor comercial. O objetivo do presente trabalho foi avaliar a morfologia e a expressão de genes relacionados ao crescimento da musculatura esquelética vermelha e branca do pirarucu. As amostras de músculo vermelho e branco foram obtidas da região lateral anterior superficial e profunda, respectivamente, e estas foram congeladas em nitrogênio líquido. Para análise morfológica, cortes histológicos obtidos em criostato foram submetidos à coloração com HE. A expressão dos genes MyoD, miogenina e miostatina foi feita por PCR em tempo real após transcrição reversa (RT-qPCR). Em relação à expressão de MyoD e miogenina, não houve diferença estatística na comparação entre os músculos; por outro lado, a expressão da miostatina foi significativamente maior no músculo branco, em comparação com o músculo vermelho. Estes resultados refletem as características de crescimento muscular do pirarucu e podem ser úteis na tentativa de melhorar as condições de criação e a sobrevivência da espécie.(AU)

Asunto(s)