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Multi-omics Insights into Postnatal Skeletal Muscle Development in Duroc Pigs

ABSTRACT:

Skeletal muscles, accounting for 40% of mammalian body mass, exhibit pronounced heterogeneity due to distinct anatomical locations. Animal husbandry excessively focuses on longissimus dorsi (LDM) development, while neglecting other muscles. In this study, we integrated Bulk RNA Sequencing (bulk RNA-seq) and Liquid Chromatography-Mass Spectrometery (LC-MS) analyses of Soleus (SOL), Gastrocnemius (GAS), and Psoas major (PMM) across three key stages in Duroc pigs. As a result, we identified 9 critical genes (S100A1, MBOAT2, CA3, GYG2, ACTN3, ENO3, SLC3A2, SLC16A10, and GAPDH) and 8 metabolites potentially involved in regulating both skeletal muscle development and fiber-type transformation. The heterogeneity between SOL and GAS was low at birth but increased gradually during development. In contrast, PMM exhibited high heterogeneity compared to SOL and GAS from birth. Notably, expression levels of MYH7, MYH1, and MYH4 displayed stage-specific and muscle type-dependent variations. Moreover, we observed a developmental shift from the MAPK signaling pathway (1-21 d) to the regulation of actin cytoskeleton (21-120 d). Pairwise comparisons between SOL, GAS, and PMM revealed signaling pathways enriched in muscle fiber-type switching. Collectively, through the integration of bulk RNA-seq and LC-MS data, this study provides novel molecular breeding strategies for genetic improvement in meat-producing animals.

INSTRUMENT(S): Liquid Chromatography MS - positive - HILIC, Liquid Chromatography MS - negative - HILIC

PROVIDER: MTBLS12963 | MetaboLights | 2025-09-09

REPOSITORIES: MetaboLights

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			Action	DRS
	a_MTBLS12963_LC-MS_negative_hilic_metabolite_profiling.txt	Txt
	a_MTBLS12963_LC-MS_positive_hilic_metabolite_profiling.txt	Txt
	i_Investigation.txt	Txt
	m_MTBLS12963_LC-MS_negative_hilic_metabolite_profiling_v2_maf.tsv	Tabular
	m_MTBLS12963_LC-MS_positive_hilic_metabolite_profiling_v2_maf.tsv	Tabular

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Project description:Background: skeletal muscle is a complex, versatile tissue composed of a variety of functionally diverse fiber types. Although the biochemical, structural and functional properties of myofibers have been the subject of intense investigation for the last decades, understanding molecular processes regulating fiber type diversity is still complicated by the heterogeneity of cell types present in the whole muscle organ. Methodology/Principal Findings: we have produced a first catalogue of genes expressed in mouse slow-oxidative (type 1) and fast-glycolytic (type 2B) fibers through transcriptome analysis at the single fiber level (microgenomics). Individual fibers were obtained from murine soleus and EDL muscles and initially classified by myosin heavy chain isoform content. Gene expression profiling on high density DNA oligonucleotide microarrays showed that both qualitative and quantitative improvements were achieved, compared to results with standard muscle homogenate. First, myofiber profiles were virtually free from non-muscle transcriptional activity. Second, thousands of muscle-specific genes were identified, leading to a better definition of gene signatures in the two fiber types as well as the detection of metabolic and signaling pathways that are differentially activated in specific fiber types. Several regulatory proteins showed preferential expression in slow myofibers. Discriminant analysis revealed novel genes that could be useful for fiber type functional classification. Conclusions/Significance: as gene expression analyses at the single fiber level significantly increased the resolution power, this innovative approach would allow a better understanding of the adaptive transcriptomic transitions occurring in myofibers under physiological and pathological conditions. EDL and soleus muscles were incubated with type I collagenase to dissociate intact myofibres that were separated under stereo microscope from the bulk of hyper contracted fibres. Isolated myofibres were divided in two parts: one was immersed in Laemmli buffer for fibre typing; the other was placed in RNA extraction buffer for RNA amplification. We analyzed the transcription profiles of 10 biological replicas of type 2B single muscle fibres from EDL and 10 biological replicas of type 1 single muscle fibres from soleus. Microarray competitive hybridizations were carried out against an artificial control with a balanced composition of type 1 and type 2B fibres (20 hybridizations). Each oligonucleotide is spotted in two replicates on the glass slide, so for every data two intra-slide replicas are present. The control was created as follows: three couples of soleus and EDL muscles were removed from 3 different mice and treated with collagenase. Total RNA was extracted separately from EDL and soleus muscles. By mixing about 1/3 RNA from EDL and 2/3 RNA from soleus muscles the control had approximately the same contributions of type 1 and type 2B fibres. Purified RNA samples from single fibres and from control were amplified twice using the Amino Allyl MessageAmpM-bM-^DM-" II aRNA Amplification Kit (Ambion).

Project description:Purpose: Despite demonstrating that the overall effect of long-term rapamycin-treatment is overwhelmingly positive in aging skeletal muscle, we observed muscle-specificity in the responsiveness to rapamycin, leading us to hypothesize that the primary drivers of age-related muscle loss and therefore effective intervention strategies may differ between muscles. To address this question and dissect the key signaling nodes associated with mTORC1-driven muscle aging, we created a comprehensive multi-muscle gene expression atlas in adult, sarcopenic and rapamycin-treated mice using RNA-seq. Methods: To examine the impact of long-term rapamycin treatment, male C57BL/6 mice were fed encapsulated rapamycin incorporated into a standardized AIN93M diet at 42 ppm (i.e. mg per kg of food), corresponding to a dose of ~4 mg·kg-1·day-1, from 15- to 30-months of age. This dose of rapamycin has been shown to extend lifespan maximally in male C57BL/6 mice. We performed RNA-seq on gastrocnemius (GAS), tibialis anterior (TA), triceps brachii (TRI) and soleus (SOL) muscles from each of six mice for 10m, 30m and 30mRM groups. The four muscle types were chosen to encompass fore- and hindlimb locations (e.g. TRI and GAS); slow and fast contraction properties (e.g. SOL and GAS); anterior and posterior positioning (e.g. TA and GAS) and the extent of protection by rapamycin (TA and TRI: protected; SOL: partiallly protected; GAS: not protected). Results: Age-related gene expression changes were remarkably consistent across the four different muscles, varying only in magnitude. Despite having the smallest age-related muscle loss of the four muscles, TA had the strongest age-related gene expression response which was associated with an increased reinnervation response. Despite the strong between-muscle commonality of age-related changes, gene expression responses to prolonged rapamycin treatment differed substantially between muscles. Rapamycin partially reversed many age-related changes in mRNA expression in the TA and TRI, but not in SOL or GAS muscle. Principle component analysis (PCA) showed that rapamycin had common 'anti-aging' effects on all muscles, while also exerting muscle-specific pro-aging effects on muscles not protected by rapamycin. Conclusions: Sustained, muscle fiber-specific mTORC1 activity drives sarcopenia-like gene expression programs, and the hyperactive mTORC1 seen in sarcopenic muscle may therefore contribute to sarcopenia.

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Multi-omics Insights into Postnatal Skeletal Muscle Development in Duroc Pigs

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