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In vitro Protein Expression Profile of Cultivated Muscle Cells from Labeo rohita

ABSTRACT: A quantitative proteomics approach was used to investigate in Labeo rohita cell line across three passages P1, P15 and P25. A new cell line developed from the muscle tissue of L. rohita (LR) was utilized to understand the proteome in-depth. Comparative proteome analysis was performed for LR muscle cells at different passages (1, 15 and 25) using label free quantitative proteomics. High-resolution mass spectrometry was used for data using label-free quantification. Data were further analysed using a statistical tool across passages of rohu muscle cell line. Functional analysis of differentially expressed proteins was marked as glycolytic (eno1 and eno3), metabolic (pgm, smyd1, ak1, aldob and bpgm), cytoplasmic ribosomal (rpl27, rpl3 and eifa5) and carbon metabolic (pdh) pathways across the three passages. The proteins observed across the pathways were also correlated with the differentiation of LR muscle cells during myogenesis. The current study provides insight to determine the different groups of proteins that are expressed during the development of myotubes.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Labeo Rohita (indian Major Carp)

TISSUE(S): Cell Culture, Skeletal Muscle Satellite Cell

SUBMITTER: Nevil Pinto

LAB HEAD: Nevil Pinto

PROVIDER: PXD059501 | Pride | 2025-02-19

REPOSITORIES: Pride

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Project description:Because of its high plasticity and rapid growth rate, turkey skeletal muscle is an important model for studying mechanisms responsible for vertebrate skeletal muscle development and function. Skeletal muscle is composed of metabolically heterogeneous myofibers that exhibit high plasticity at both the morphological and transcriptional levels. The objective of this study was to employ microarray analysis to elucidate the differential gene expression between the tonic- “red” Anterior latissimus dorsi (ALD) muscle, the phasic- “white” Posterior latissimus dorsi (PLD), and “mixed”-phenotype Biceps femoris (BF) in 1-week and 19-week old male turkeys. A total of 170 differentially expressed genes were identified in the analyzed muscle samples (P<0.05). Gene Go analysis software was utilized to identify top gene networks and metabolic pathways involving differentially expressed genes. The largest differences were observed between ALD and PLD muscles, where 32 genes were over-expressed and 82 genes were under-expressed in ALD1-PLD1 comparison; and 70 genes were over-expressed and 70 under-expressed in ALD19-PLD19 comparison. The largest number of genes over-expressed in ALD muscles as compared to other muscles, code for extracellular matrix proteins such as dystroglycan and collagen. Furthermore, a number of genes involved in glycolytic metabolism were under-expressed in ALD muscles as compared to BF and PLD muscles. The gene analysis revealed that phenotypically “red” BF muscle has high expression of glycolytic genes usually associated with “white” muscle phenotype. Muscle-specific differences were observed in expression levels of genes coding for proteins involved in mRNA processing and translation regulation, proteosomal degradation, apoptosis, and insulin resistance. The study utilized Nicholas turkey males at two different stages of growth (1 week old: high satellite cell mitotic activity; 19 weeks old: low satellite cell mitotic activity, hypertrophy, market age). Six 1-week-old and six 19-week-old Nicholas turkey males (Meleagris gallopavo) were randomly selected from a single flock. The experiments were designed to compare genes characterizing muscle fiber phenotype at each time point, and to elucidate age-related differences in gene expression levels. Each analyzed cDNA sample was derived from six pooled RNA samples, resulting with six cDNA samples per muscle (one cDNA per bird). To minimize dye bias, the cDNA samples were labeled either with Cy3 or Cy5. A total of 17 Turkey Skeletal Muscle Long Oligo (TKSMLO) microarrays were utilized. Birds were randomly assigned to each array and the cDNA hybridizations we performed at random. The array data was Log2 transformed and then normalized by using locally weighed regression and smoothing within each array and across all arrays. Mixed model ANOVA with Bonferroni correction of P=0.05 for multiple testing was performed to increase the sensitivity of detected differences in gene expression levels.

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In vitro Protein Expression Profile of Cultivated Muscle Cells from Labeo rohita

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