Reduced TGFβ Responsiveness in Skeletal Muscle Satellite Cells From Lambs With Fetal Growth Restriction

Abstract

Placental insufficiency (PI) restricts the nutrient and oxygen transfer to the fetus and causes fetal growth restriction (FGR) and compromises skeletal muscle growth. Consequently, infants with FGR have reduced skeletal muscle mass throughout life, increasing their risk for developing metabolic diseases. Fetal adaptation to PI-FGR disrupt postnatal skeletal muscle metabolism and growth. Postnatal muscle growth is regulated by growth factors such as transforming growth factor beta (TGFβ) and insulin-like growth factor 1 (IGF-1). TGFβ functions in skeletal muscle cell differentiation and fusion of myofibers by inhibiting the expression of multiple muscle-specific proteins like MyoG. We show precocious fusion rates in satellite cells stimulated with TGFβ, suggesting impaired TGFβ activity. IGF-1 regulates both anabolic and catabolic pathways in skeletal muscle. IGF-1 increases skeletal muscle protein synthesis via PI3K/AKT/mTOR pathways. Also, PI3K/AKT can inhibit FoxOs and suppress transcription of E3 ubiquitin ligases that regulate ubiquitin proteasome system mediated protein degradation. Together, TGFβ regulate cellular differentiation whereas IGF-1 regulate both protein synthesis and degradation pathways and gene expression changes in signaling can greatly affect skeletal muscle myofiber size and function. Another key regulator of gene expression is DNA methylation. Methylation of cytosines change DNAs hydrophobic property and inhibit interactions with transcriptional activators and repressors. We hypothesize that reduced TGFβ responsiveness in satellite cells from lambs with FGR is due to promoter DNA methylation inducing higher expression of genes in PI3K/AKT signaling pathways leading to impaired satellite cell differentiation. Lambs with PI-induced FGR were generated by exposing pregnant ewes to environmental hyperthermia during mid-gestation. At birth, FGR (3.2± 0.3 kg) lambs were lighter (P<0.05) than control (CON; 4.5 ± 0.2 kg) lambs. At 30 days old FGR (10±1 kg) lambs weighed less than CON (12.8±0.4 kg) lambs and no changes in semitendinosus muscle weight relative to body weight was found. Satellite cells were isolated from 30-day old lambs. To test their responsiveness to TGFβ, we measured cell fusion rates on control and FGR satellite cells treated with either TGFβ inhibitor (A8301) or untreated during differentiation. Additionally, dose-response curves were performed on control and FGR satellite cells with increasing concentrations of TGFβ (0-10ng/ml). To test TGFβ-induced transcriptional activity, we performed RNA seq analysis to satellite cells treated with either TGFβ (3ng) or no TGFβ (0ng) and RNA was collected. Then, to capture epigenetic changes, we collected genomic DNA from CON and FGR satellite cells to perform DNA methylation analysis on differentially promoter regions. Lastly, we performed ChIP-qPCR against RNA polymerase II to validate gene transcription. The percent of cellular fusion was higher (P<0.01) in satellite cells treated with a TGFβ inhibitor (A 83-01; 64.2±3.8%) compared to untreated cells (42.3±4.4%) from control lambs. A greater percent of cell fusion was found in cells from FGR lambs (55.4±1.7%), which was not affected with the TGFβ inhibitor. Dose-response curves for TGFβ showed that satellite cells from GGR lambs were less responsive to TGFβ. RNAseq identified 1,033 DE genes in CON-0ng vs CON-3ng and 1163 DE genes in FGR-0ng vs FGR-3ng. DNA methylation analysis identified 2,372 differentially methylated promoter regions. Transcriptomic and DNA methylation data identified similar pathways such as TGFβ, PI3K/AKT, WNT, RAS signaling and cell cycle and apoptosis. Dose-response curves revealed that TGFβ responsiveness was blunted in satellite cells from FGR lambs leading to increased fusion rates by impaired TGFβ activity. Transcriptome data from satellite cells stimulated with TGFβ revealed increased gene expression of PI3K/AKT pathways suggesting it as a main regulator of muscle differentiation after TGFβ stimulation. The TGFβ-SMAD4 signaling in FGR satellite cells was not different from controls leading to the assumption that stimulation with TGFβ increased PI3K/AKT gene expression. Therefore, we evaluated epigenetic changes in CON and FGR satellite cells which resulted in hypomethylated promoters of genes in PI3K/AKT signaling. These data suggest that reduced TGFβ responsiveness in FGR satellite cells is due to enhancement of PI3K/AKT signaling pathway. TGFβ regulates fundamental processes in lowering skeletal muscle mass during FGR by increasing differentiation rates and lowering satellite cell pools. This represents a critical mechanism that is responsible for mediating programming events in satellite cells from FGR lambs.