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Prolonged fasting and refeeding modifies the proteome of slow-twitch skeletal muscle fibres in Piaractus mesopotamicus

ABSTRACT: In fish, the slow-twitch skeletal muscle fibres have an oxidative metabolism, present peripherical location in the red muscle, and are used for sustained movements. They may respond to alterations in food availability differently than the fast-twitch muscle. The aim of this manuscript was to study the slow-twitch muscle fibres of Piaractus Mesopotamicus, a neotropical fish, submitted to 30 days of fasting (D30) followed by one day (D31) or 30 days of refeeding (D60). The treated animals were compared with regularly fed fish. To verify the presence of atrophy and hypertrophy, we performed histological analysis of muscle fibre diameter in D30 and D60, and RT-qPCR gene expression analysis of catabolic (murfa, murfb, mafbx) and anabolic genes (igf-1, mTOR) in D30, D31 and D60. The gene expression of the allergen and Ca2+-carrier parvalbumin (pvalb) was also measured in D30, D31 and D60. The proteome of slow-twitch fibres at D30 and D60 was obtained by shotgun proteomics (digestion of proteins with trypsin followed by LC-MS/MS identification), and the proteins differentially expressed were used to construct protein interaction networks. The histological analysis showed no difference between treated fish in relation to the control. The expression of catabolic and anabolic genes was not changed, except for the negative regulation of igf-1 in D30 and of mtor in D31. The expression of pvalb was not changed in D30 and D60, but it was decreased in D31. The proteomic analysis identified 169 proteins in D30 (24 upregulated and 18 downregulated) and 170 proteins in D60 (17 upregulated and 21 downregulated). Many of them were related to energetic metabolism, including lipid metabolism, as shown by the protein network analysis. These results enlarge our understanding of the acclimation of slow-twitch fibres to changes in nutrient availability and set a path for future research.

INSTRUMENT(S): Q-Tof Premier

ORGANISM(S): Piaractus Mesopotamicus

TISSUE(S): Slow Muscle

SUBMITTER: Maria Laura Gabriel Kuniyoshi

LAB HEAD: Maeli Dal-Pai-Silva

PROVIDER: PXD014320 | Pride | 2019-11-28

REPOSITORIES: Pride

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			Action	DRS
	05junho_Rafaela_1_1.zip	Other
	05junho_Rafaela_1_2.zip	Other
	05junho_Rafaela_1_3.zip	Other
	05junho_Rafaela_2_1.zip	Other
	05junho_Rafaela_2_2.zip	Other

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Project description:The zebrafish u-boot (ubo) gene encodes the transcription factor Prdm1, which is essential for the specification of the primary slow twitch muscle fibres that derive from adaxial cells. Here we show that Prdm1 executes this function by acting as a transcriptional repressor. Expression of the slow twitch isoform of the myosin heavy chain in adaxial cells is achieved by Prdm1 mediated repression of the transcriptional repressor Sox6. Using Chromatin immuno precipitation (ChIP) we found that genes encoding fast specific isoforms of sarcomeric proteins are direct targets of Prdm1. These genes are ectopically expressed in the adaxial cells of ubotp39 mutant embryos, suggesting that Prdm1 promotes slow twitch fibre differentiation by acting both as a global repressor of fast fibre specific genes as well as of a repressor of slow specific genes. Keywords: ChIP-chip Genomic array design. Microarrays were designed as described below and manufactured by Agilent Technologies (www.agilent.com). We have previously described the ChIP-chip technique in zebrafish and the design of promoter microarrays which cover a 2kb region around the transcription start sites (TSSs) of 11,512 zebrafish genes based on Zv4 (Wardle et al, 2006. Genome Biology 7:R71). In order to capture additional regulatory information further from the basal promoter region we also designed an expanded set of 60mer probes that cover 9kb upstream and 3kb downstream of the TSS of these genes using the same parameters as for the 2kb microarrays. These probe sequences were re-mapped to Zv6 over the course of the project and the coordinates given are for Zv6. We also incorporated several sets of control probes, both positive and negative. On each array there are 769 negative probes designed against zebrafish gene desert regions and Arabidopsis thaliana genes. In addition we included duplicates of promoter probes for several genes that we anticipated may be bound by factors of interest for this and other studies (tnnt3a, mylz2, myhz2, wnt11, flh, vent, msgn1, myod, fgf8, pcdh8) and these probes are arrayed on both slides slide. Finally on each array there are 1804 controls added by Agilent. The final design contained 352,490 probes divided between two microarray slides. Further information on design and manufacture of the microarrays can be found at the Agilent Technologies website.

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Prolonged fasting and refeeding modifies the proteome of slow-twitch skeletal muscle fibres in Piaractus mesopotamicus

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