Effects of intrauterine growth restriction (IUGR) on porcine muscle development
Item statusRestricted Access
Embargo end date08/12/2022
Cortés Araya, Yennifer
Intrauterine growth restriction (IUGR) is a leading cause of neonatal morbidity and mortality in humans. Preferential allocation of available resources to vital tissues in the IUGR foetus occurs at the expense of skeletal muscle, resulting in reduced tissue growth and metabolic adaptations that increase the risk of premature muscle loss and metabolic disease later in life. Several animal models have been used to study the effects of IUGR on skeletal muscle. However, the developmental mechanisms driving impaired myogenesis in IUGR have only been partly elucidated. IUGR occurs spontaneously in the pig, generating severe consequences on muscle development, providing a convenient, physiologically-relevant large animal model for studying developmental aspects of IUGR. The studies in this thesis aimed to identify genome-wide transcriptional signatures associated with the adaptive response of foetal skeletal muscle to IUGR in the pig. Another aim was to establish a suitable in vitro system and to use this to functionally target identified candidate genes to elucidate their involvement in the IUGR muscle phenotype. Using RNA-sequencing of muscle from IUGR and normal-weight (NW) pig littermates at a late foetal stage (Day 90 of gestation), a total of 1031 genes were identified to be differentially expressed (DE). Those DE transcripts mapped to multiple biological pathways involved in Development, Tissue injury (Inflammation, Coagulation and Anti-oxidation/Detoxification) and Metabolism (Glucose metabolism, Lipid biosynthesis/transport, and Amino acid degradation). To establish an in vitro system for investigating the involvement of some of the consequences identified in IUGR, progenitor muscle cells (PMCs) from IUGR and normal weight (NW) littermates were obtained and were differentiated under established myogenic or adipogenic conditions, followed by analyses of lineage markers by immunochemistry and RT-qPCR. IUGR PMCs displayed reduced myogenic capacity and an increased ability to undergo adipogenic differentiation compared to PMCs from NW littermates. These results demonstrated that PMCs are developmentally programmed in utero by IUGR. At the same time, they provided a powerful tool that was later used to elucidate some of the mechanisms involved in the IUGR phenotype. Among the up-regulated transcripts identified in IUGR muscle was the global metabolic regulator, KLB, a co-receptor for FGF21. In addition, FGF21 levels in plasma were increased in IUGR relative to NW littermates. Functional targeting of KLB was performed by determining myogenesis and mTOR signalling responses to transfection with KLB siRNA and treatment with the KLB ligand, FGF21 (0-100 ng/ml), using porcine PMCs as well as PMCs isolated from human foetuses. Transfection of PMCs with KLB siRNA promoted myogenesis and mTOR activation, whereas treatment with FGF21 had opposite and dose-dependent effects in porcine and human PMCs. This showed that the effects of KLB on muscle growth occur through inhibition of mTOR signalling and may involve both direct and indirect mechanisms. Importantly, these results indicate that the effects of KLB on muscle cells are conserved in pigs and humans. Finally, some of the identified DE transcripts were analysed on foetal muscle samples from IUGR and NW littermates at gestational days (GD) 45, 60 and 90. RT-qPCR analysis showed that, in general, altered expression of developmental transcripts in IUGR foetal muscle started on GD45, whereas for tissue injury and metabolic transcripts, changes were observed starting on GD60 and GD90, respectively. These results indicate that different functional gene categories are sequentially affected in muscle by IUGR during foetal development and that interventions at the sow level aimed at preventing or ameliorating the IUGR phenotype in offspring should be implemented early during gestation. Altogether, this thesis provides new insight into the molecular mechanisms underlying the effects of IUGR on muscle development. The results may aid the development of novel strategies to ameliorate the effects of IUGR in the pig industry.