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Maternal inflammation at mid-gestation impairs subsequent fetal myoblast function and skeletal muscle growth in rats, resulting in intrauterine growth restriction at term

Maternal inflammation at mid-gestation impairs subsequent fetal myoblast function and skeletal... Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Maternal inflammation at mid-gestation impairs subsequent fetal myoblast function and skeletal muscle growth in rats, resulting in intrauterine growth restriction at term. C.N. Cadaret*, R.J. Posont*, K.A. Beede*, H.E. Riley*, J.D. Loy†, and D.T. Yates* *Department of Animal Science, University of Nebraska-Lincoln, Lincoln, NE 68583 †School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 Corresponding Author: Dustin Yates PO Box 830908 Lincoln, NE 68583 dustin.yates@unl.edu This manuscript is based on research that was partially supported by the National Institute of General Medical Sciences Grant 1P20GM104320 (J. Zempleni, Director), the Nebraska Agricultural Experiment Station with funding from the Hatch Act (NEB-26-224) and Hatch Multistate Research capacity funding program (NEB-26-226, NEB-26-225) through the USDA National Institute of Food and Agriculture. The Biomedical and Obesity Research Core (BORC) in the Nebraska Center for Prevention of Obesity Diseases (NPOD) receives partial support from NIH (NIGMS) COBRE IDeA award NIH 1P20GM104320. The contents of this publication are © The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non- Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 the sole responsibility of the authors and do not necessarily represent the official views of the NIH or NIGMS. The authors have no conflicts of interest to declare. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 ABSTRACT: Maternal inflammation induces intrauterine growth restriction (MI-IUGR) of the fetus, which compromises metabolic health in human offspring and reduces value in livestock. The objective of this study was to determine the effect of maternal inflammation at mid-gestation on fetal skeletal muscle growth and myoblast profiles at term. Pregnant Sprague-Dawley rats th were injected daily with bacterial endotoxin (MI-IUGR) or saline (controls) from the 9 to the th 11 day of gestational age (dGA; term = 21 dGA). At necropsy on dGA 20, average fetal mass and upper hindlimb cross-sectional areas were reduced (P < 0.05) in MI-IUGR fetuses compared + + + to controls. MyoD and myf5 myoblasts were less abundant (P < 0.05) and myogenin myoblasts were more abundant (P < 0.05) in MI-IUGR hindlimb skeletal muscle compared to controls, indicating precocious myoblast differentiation. Type I and Type II hindlimb muscle fibers were smaller (P < 0.05) in MI-IUGR fetuses than in controls, but fiber type proportions did not differ between experimental groups. Fetal blood plasma TNFα concentrations were below detectable amounts in both experimental groups, but skeletal muscle gene expression for the cytokine receptors TNFR1, IL6R, and FN14 was greater (P < 0.05) in MI-IUGR fetuses than controls, perhaps indicating enhanced sensitivity to these cytokines. Maternal blood glucose concentrations at term did not differ between experimental groups, but MI-IUGR fetal blood contained less (P < 0.05) glucose, cholesterol, and triglycerides. Fetal-to-maternal blood glucose ratios were also reduced (P < 0.05), which is indicative of placental insufficiency. Indicators of protein catabolism, including blood plasma urea nitrogen and creatinine kinase were greater (P < 0.05) in MI-IUGR fetuses than in controls. From these findings, we conclude that maternal inflammation at mid-gestation causes muscle-centric fetal programming that impairs myoblast function, increases protein catabolism, and reduces skeletal muscle growth near term. Fetal muscle sensitivity to inflammatory cytokines appeared to be enhanced after maternal Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 inflammation, which may represent a mechanistic target for improving these outcomes in MI- IUGR fetuses. Key words: Adaptive fetal programming, developmental origins, inflammatory regulation, maternofetal stress, thrifty phenotype Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 INTRODUCTION Maternal stress during gestation induces intrauterine growth restriction (IUGR), which is linked to lifelong deficits in skeletal muscle growth and metabolic dysfunction in humans and livestock (Yates et al., 2016; Yates et al., 2018). Late prenatal and postnatal muscle growth requires proliferation, differentiation, and fusion of myoblasts with existing muscle fibers (Zhu et al., 2004), and this rate-limiting process can be altered by inflammation (Langen et al., 2001; Langen et al., 2004; Posont et al., 2018). Inflammatory changes within the skeletal muscle niche can arise from systemic inflammation and from resident macrophages within the tissue itself (Kharraz et al., 2013). Acute inflammation impairs adult myoblast function in vitro (Frost et al., 1997; Langen et al., 2001), and we hypothesized that maternal inflammation may likewise disrupt fetal myoblast function and muscle growth in utero. The programming effects of fetal stress on muscle development and growth have implications for long-term metabolic health in IUGR-born humans and growth performance in livestock (Zhu et al., 2004; Yates et al., 2011; Yates et al., 2012), but the underlying mechanisms are not well understood. Our previous studies in placental insufficiency-induced IUGR fetal sheep have identified intrinsically impaired myoblast function and restricted muscle growth as outcomes of unidentified molecular adaptations (Yates et al., 2012; Yates et al., 2014; Yates et al., 2016). Therefore, the objective of this study was to determine the effects of sustained maternal inflammation at mid-gestation on myoblast profiles, muscle growth, and metabolic indicators at term. MATERIALS AND METHODS Animals and experimental design Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Nebraska-Lincoln. Animal studies were performed at the University of Nebraska-Lincoln Animal Science Complex, which is accredited by AAALAC International. Timed-pregnant Sprague-Dawley rats (Envigo, Indianapolis, IN) were purchased and delivered th on the 6 day of gestational age (dGA). Beginning at dGA 8, rats were individually housed, fed th th commercial rat chow ad libitum, and weighed daily. From the 9 to the 11 dGA, rats were randomly assigned to be injected (i.p.) daily with 250 μl physiological saline (control, n = 9) or 0.1 μg/kg BW of lipopolysaccharide (LPS) endotoxin from E. coli O55:B5 (Sigma-Aldrich, St. Louis, MO) in 250 μl physiological saline to create maternal inflammation-induced IUGR (MI- IUGR, n = 10). Maternal blood samples were collected via saphenous vein puncture and rectal temperatures were recorded at 3, 6, and 24 hours after each of the daily injections. On dGA 20 (term = 21 dGA), rats were euthanized by decapitation under heavy isoflurane anesthesia and maternal blood samples were collected. Litter size and individual fetal weights were recorded, and skinned hindlimbs were collected from the three fetuses in each litter located closest to the uterine bifurcation. The right hindlimb from each fetus was snap frozen and the left was fixed in 4% PFA. Pooled blood samples were collected from the remaining fetuses in each litter via exsanguination. Blood analysis Glucose concentrations were determined from maternal and fetal whole blood in duplicate with a Contour next EZ glucose meter (Bayer Corp., Whippany, NJ). Blood plasma was then isolated by centrifugation (14,000 x g, 2 minutes, 4°C) and TNFα concentrations were determined in duplicate by Rat TNFα Quantikine ELISA kit (R&D Systems, Minneapolis, MN) Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 as previously described (hui Seo et al., 2017). Inter-assay and intra-assay CV was less than 10%, and the detection limit was 5 pg/ml. Fetal blood plasma concentrations of urea nitrogen (BUN), high-density lipoprotein cholesterol (HDLC), total cholesterol, triglycerides, and total protein were determined via colorimetric assays with a Vitros-250 Chemistry Analyzer (Ortho Clinical Diagnostics, Linden, NJ) according to manufacturer recommendations. Concentrations of γ- glutamyltransferase (GGT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were determined via multiple-point rate assays with the Vitros-250 Chemistry Analyzer. Gene expression RNA isolation. RNA was extracted from frozen fetal hindlimb muscle (~30 mg) via RNeasy Fibrous Tissue Mini Kit (Qiagen, Germantown, MD). For all samples, isolated RNA was quantified on a NanoDrop microvolume spectrophotometer (Thermo Fisher; Waltham, MA), and reverse transcribed into cDNA via QuantiTect Reverse Transcription Kit (Qiagen). Droplet digital PCR. Primer pairs for PCR were designed for the genes of interest listed in Table 1. Droplet digital PCR (ddPCR) was performed in duplicate with the QX200 ddPCR System (BioRad, Hercules, CA). Each reaction contained Evagreen Supermix, 10 μM of each primer, and equivalent amounts of cDNA template. Droplets were generated in a QX200 Droplet Generator with Droplet Generator Oil, transferred to a PCR plate, sealed, and placed in a C1000 Touch Thermal Cycler. Samples were activated (95°C for 5 minutes), denatured for 40 cycles (95°C for 30 seconds), annealed and extended for 40 cycles (60°C for 1 minute), and stabilized (4°C for 5 minutes and 90°C for 5 minutes). Finally, droplets were read on the QX200 Droplet Reader and results were analyzed with QuantaSoft Software (BioRad) to obtain transcripts/μl for Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 genes of interest. Results were normalized to transcripts/µl of YWHAZ, which was stable across treatment groups. Immunohistochemistry Myoblast profiles. Fixed fetal hindlimbs were embedded in OCT Compound (Thermo Fisher). Tissue cross sections cut at a thickness of 8 µm were taken at the mid-point of the femur, prepared with a CryoStar NX50 (Thermo Fisher), and mounted on glass microscope slides as previously described (Yates et al., 2014). Slides were dried at 37°C for 30 minutes and then washed three times in PBS + 0.5% Triton-X-100. Antigen retrieval was performed by boiling slides in 10 mM citric acid for 20 minutes and allowing them to cool slowly to room temperature. Non-specific binding was blocked by incubating slides with 0.5% NEN blocking buffer (Perkin-Elmer, Waltham, MA) in PBS at room temperature for 1 hour. Slides were then incubated overnight at 4°C with primary antibodies diluted in PBS + 1% bovine serum albumin (Sigma-Aldrich). Negative controls were incubated in PBS + 1% bovine serum albumin without primary antibody. Sections were stained with antibodies raised in the rabbit against myf5 (1:100; Santa Cruz, Dallas, TX), in the mouse against myoD (1:200; Dako, Santa Clara, CA), and in the mouse against myogenin (1:250, Abcam, Cambridge, MA) to identify nuclei expressing these myogenic factors. Immunocomplexes were detected with affinity-purified immunoglobulin antiserum conjugated to Alexa Fluor 594 (1:2000; Cell Signaling, Danvers, MA) or Alexa Fluor 488 (1:1000; Cell Signaling). Myoblast profiles were assessed in the medial and lateral muscle groups, which included the adductor and gracilis muscles and the semitendinosus, semimembranosus, and biceps femoris, respectively. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Muscle fiber profiles. A second set of hindlimb cross sections were stained to identify fiber type ratios and fiber size as previously described (Yates et al., 2016). Briefly, fibers in medial and lateral muscle groups expressing myosin heavy chain (MyHC) isoforms specific for Type I and Type II fibers were identified with antibodies raised in the mouse against MyHC-I (BA-D5, 1:20; Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Iowa City, IA) and MyHC-II (F18, 1:25; DSHB), respectively. Muscle sections were counterstained with antibodies raised in the rabbit against desmin (1:1000, Sigma-Aldrich) to identify all fibers. Immunocomplexes were detected with affinity-purified immunoglobulin antiserum conjugated to Alexa Fluor 594 (1:2000) or Alexa Fluor 488 (1:1000). All fluorescent images were visualized on an Olympus IX73 and digital micrographs were captured with a DP80 microscope camera (Olympus, Center Valley, PA). Images were analyzed with Olympus cellsSens Dimension software to determine proportions of positive nuclei or fibers within fetal skeletal muscle sections. Animal identifications and treatments were de-identified prior to analyses. Statistical analysis All data were analyzed by ANOVA using the mixed procedure of SAS 9.4 (SAS Institute, Cary, NC) to determine the effect of experimental group. Repeated measures were used for serial maternal measurements. Skeletal muscle gene expression data are expressed as transcript copies per µl, normalized to copies per µl of YWHAZ. Proportions of nuclei staining positive for myogenic regulatory factors were determined from a minimum of 250 positive nuclei, counted across 6 non-overlapping fields of view per fetus. Skeletal muscle fiber type proportions were determined from a minimum of 2,500 total fibers counted across 3 fields of view per fetus. Average fiber size was determined from a minimum of 250 fibers across 3 fields Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 of view per fetus. Rat (dam) was considered the experimental unit for all variables, and values were averaged across the fetuses analyzed in each dam’s litter. All data are expressed as group mean ± standard error. RESULTS Maternal responses to lipopolysaccharide administration. Experimental group x time point interactions were observed (P < 0.05) for maternal rectal temperatures and plasma TNFα concentrations, but not for maternal plasma glucose concentrations. Maternal rectal temperatures in MI-IUGR dams were greater (P < 0.05) than in control dams at 6, 30, 54, and 72 hours after beginning the daily injections on dGA 9 (Figure 1A), but were not different from control temperatures at the other measured time points. Maternal blood glucose did not differ between experimental groups (Figure 1B). Maternal plasma TNFα concentrations in control dams were below the detectable limit (5 pg/ml) at all time points except for 24 hours, when 5 of the 9 controls exhibited plasma TNFα concentrations between 5 and 16.5 pg/ml. Plasma TNFα concentrations were increased (P < 0.05) in MI-IUGR dams at 3, 6, 24, 27, and 51 hours after beginning daily injections (Figure 1C). Maternal weights and daily feed intake did not differ between the two experimental groups on any day. Fetal morphometrics and blood chemistry. At necropsy, the number of fetuses per litter did not differ between controls and MI-IUGR (14.2 ± 0.6 and 13.1 ± 0.6, respectively), but average fetal mass and upper hindlimb area were reduced (P < 0.05) in MI-IUGR fetuses compared to controls (Figure 2). Plasma TNFα concentrations at necropsy were below detectable limits (5 pg/ml) in all fetuses and in all dams with the exception of two MI-IUGR dams that exhibited 8 and 24 pg/ml, respectively. Maternal blood glucose concentrations did not differ between experimental groups at necropsy, but fetal blood glucose was decreased (P < Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 0.05) in MI-IUGR fetuses compared to controls (Table 2). Fetal BUN concentrations were increased (P < 0.05) in MI-IUGR fetuses compared to controls. Plasma HDLC concentrations did not differ between control and MI-IUGR fetuses. Plasma cholesterol and triglyceride concentrations were reduced (P < 0.05) in MI-IUGR fetuses compared to controls. Plasma total protein content did not differ between control and MI-IUGR fetuses. Plasma AST, ALT, and creatine kinase were greater (P < 0.05) in MI-IUGR fetuses compared to controls. Fetal plasma GGT did not differ between experimental groups. Skeletal muscle gene expression. Skeletal muscle TNFR1 (TNFα receptor 1), FN14 (TWEAK receptor), and IL6R (IL-6 receptor) expression was increased (P < 0.05) in MI-IUGR fetuses compared to controls (Table 3). Skeletal muscle ADRB1 (β1 adrenoceptor) mRNA was lower (P < 0.05) in MI-IUGR fetuses compared to controls, but skeletal muscle ADRB2 (β2 adrenoceptor) and ADRB3 (β3 adrenoceptor) mRNA content did not differ between experimental groups. Skeletal muscle mRNA expression for the pan macrophage marker CD68 (cluster of differentiation 68) was decreased (P < 0.05) in MI-IUGR fetuses compared to controls. Fetal skeletal muscle mRNA expression for the M2-macrophage marker CD163 (cluster of differentiation 163) did not differ between experimental groups. Skeletal muscle myoblast profiles. Representative micrographic images are shown for myoblast populations staining positive for myf5 (Figure 3A), myoD (Figure 3B), and myogenin (Figure 3C) in fetal hindlimb skeletal muscle. At necropsy, MI-IUGR decreased (P < 0.05) the + + number of myf5 and myoD nuclei (Figure 3D and Figure 3E, respectively) and increased (P < 0.05) the number of myogenin nuclei (Figure 3F) in fetal hindlimb muscle compared to controls. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Skeletal muscle fiber type proportions and sizes. Representative micrographic images are shown for muscle fiber populations staining positive for MyHC-I (Type I; Figure 4A) and MyHC-II (Type II; Figure 4B) in fetal hindlimb skeletal muscle. At necropsy, the average cross sectional area of Type I and Type II fibers in hindlimb skeletal muscle were smaller (P < 0.05) in MI-IUGR fetuses compared to controls (Figure 4C and Figure 4D, respectively). The proportions of Type I and Type II fibers in fetal hindlimb skeletal muscle did not differ between experimental groups. DISCUSSION In this study, we found that maternal inflammation at mid-gestation impaired subsequent fetal myoblast function, reduced fetal muscle growth, and led to an IUGR phenotype at term. Systemic inflammation was not apparent at term in these dams or their fetuses. However, the sensitivity of fetal skeletal muscle to inflammatory cytokines appeared to be enhanced due to greater gene expression for cytokine receptors. This coincided with indicators of reduced myoblast proliferation and precocious differentiation, diminished muscle fiber hypertrophy, and smaller hindlimb areas. Fetal programming by maternal inflammation also increased indicators of protein catabolism in fetal blood plasma at term. Fetal hypoglycemia and reduced fetal-to- maternal glycemic ratios were indicative of placental insufficiency, which would help to explain how maternal inflammation at mid-gestation resulted in the fetal changes observed near term. The evidence for enhanced cytokine sensitivity, impaired myoblast function, and increased protein catabolism in this study provide insight into a potential programming mechanism for the predisposition of IUGR-born offspring to poor muscle growth and metabolism. The first of the three daily injection of LPS caused a temporal increase in maternal circulating TNFα and body temperatures that peaked at 3 to 6 hours and was gone by 24 hours. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Interestingly, the maternal response to the subsequent injections was less profound, perhaps due to a reduced sensitivity of the immune system to the endotoxin. Furthermore, the impact of the endotoxin on maternal wellbeing beyond the period of administration appeared to be minimal, as daily intake, average daily gain, glycemic levels, and circulating TNFα concentrations subsequent to the final injection were comparable to controls. Conversely, maternal inflammation at mid-gestation caused changes in fetal skeletal muscle that remained present at term, well after the inflammation had subsided. Greater gene expression for cytokine receptors in MI-IUGR fetal muscle are evidence of enhanced sensitivity to inflammatory cytokines despite low circulating TNFα that suggests an absence of systemic fetal inflammation near term. Skeletal muscle is highly responsive to inflammatory cytokines (Frost et al., 1997; Cadaret et al., 2017), and increased inflammatory activity can disrupt muscle growth. Thus, it is possible that greater inflammatory tone caused by increased cytokine sensitivity contributed to reduced fetal muscle growth in this study, although a more in-depth evaluation of this potential mechanism is warranted. Smaller fetal myofiber size following maternal inflammation was coincident with impaired fetal myoblast function, which is consistent with our previous findings in placental insufficiency-induced IUGR fetal sheep (Yates et al., 2014; Posont et al., 2018). At term, medial and lateral hindlimb muscles of MI-IUGR fetal rats had fewer nuclei expressing myoD and myf5, which are indicative of myoblasts in the proliferative stage (Kitzmann et al., 1998) prior to differentiating (Rudnicki et al., 1993). Additionally, more nuclei stained positive for myogenin, which is expressed by myoblasts that have exited the cell cycle and begun differentiating (Rawls et al., 1995). These transcription factor profiles indicate precocious entry of fetal myoblasts into terminal differentiation at the expense of proliferation, which would conform with greater Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 sensitivity to inflammatory cytokines. The effect of cytokines on myoblast function is complex, but increased activity of IL-6/IL6R pathways has been shown to increase myoblast differentiation and correlates with greater myogenin expression (Hoene et al., 2013). Although TNFα/TNFR1 and TWEAK/Fn14 pathways antagonize myoblast differentiation in many cases, their increased activity has also been shown to diminish myoD in myoblasts (Langen et al., 2004; Dogra et al., 2006), as observed in the present study. Paradoxical reductions in gene expression for macrophage indicators in this study may be indicative of a compensatory reduction in resident macrophages, particularly M1 macrophages, which are important contributors to the proliferation-inducing myoblast niche (Du et al., 2017). The lack of changes in β2 and β3 adrenoceptor gene expression following maternal inflammation was somewhat surprising, as both have anti-inflammatory properties (Lirussi et al., 2008; Kolmus et al., 2014). Previous studies have shown that less β1 adrenergic activity (indicated by reduced receptor gene expression is our study) could be associated with greater inflammation (Ardestani et al., 2017), but it would be unlikely that less β1 activity would affect myoblast function directly (Church et al., 2014). Greater BUN concentrations indicate that protein catabolism was increased near term in MI-IUGR fetuses (Depner, 2001), perhaps to provide an energy substrate to compensate for hypoglycemia and reduced circulating triglycerides (Lemons and Schreiner, 1983). Indeed, a previous study in IUGR fetal sheep, which were hypoglycemic by ~50%, found that fetal urea production was ~2.5-fold greater than in controls (Jensen et al., 1999). In IUGR-born rats, BUN continued to be greater at 3 months of age (He et al., 2015), indicating that it is a programmed change. Higher circulating creatine kinase, AST, and ALT concentrations in our MI-IUGR fetuses are further evidence of increased muscle protein catabolism (Depner, 2001; Nie et al., Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 2011; Bodie et al., 2016). Greater protein catabolic rates together with reductions in protein accretion observed in IUGR fetal sheep (Rozance et al., 2018) would presumably contribute to the reduction in hindlimb muscle fiber size in our MI-IUGR fetuses. Mechanisms linking maternal inflammation and muscle-centric fetal programming were not directly evaluated in this study, but fetal hypoglycemia in the presence of maternal euglycemia would indicate that placental insufficiency was present. A previous study in rats rd showed that daily maternal LPS injections in the early 3 trimester reduced trophoblast invasion and spiral artery remodeling in the placenta (Cotechini et al., 2014b). These changes were accompanied by reductions in placental weight, area, and thickness (Cotechini et al., 2014a). Enhanced gene expression for cytokine receptors observed in our MI-IUGR fetuses could have also been the result of direct fetal inflammation, as chronic inflammatory events have been shown to increase TNFR1 and IL6R in other tissues (Lam et al., 2008; Liu et al., 2016). Placental permeability to maternal inflammatory cytokines is low (Aaltonen et al., 2005), but the rodent placenta is at least somewhat permeable to LPS (Kohmura et al., 2000). Thus, it is possible that the fetus produced its own inflammatory response to LPS transferred from maternal to fetal circulation. However, other studies indicate that the placenta itself is a more likely source for fetal inflammation, as maternal LPS administration has been shown to increase placental cytokine production substantially (Bloise et al., 2013). From these findings, we can conclude that muscle-centric fetal programming by maternal inflammation at mid-gestation impairs fetal myoblast function and increases fetal protein catabolism. These changes were associated with impaired skeletal muscle growth, which contributed to IUGR at term. The role of these changes in postnatal muscle mass deficits of IUGR-born offspring was not clear from the present study but may be a reasonable aim for future Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 studies with this model. Likewise, it was unclear if the muscle-centric fetal programming events observed in this study were survival adaptations by the fetus, but reduced skeletal muscle would allow re-appropriation of limited fetal nutrients to neural and cardiac tissues. Thus, it is important for future studies seeking to moderate inflammation-induced fetal programming during maternal stress conditions to consider the impact on fetal survival mechanisms in addition to potential benefits for postnatal health and growth performance. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 LITERATURE CITED Aaltonen, R., T. Heikkinen, K. Hakala, K. Laine, and A. Alanen. 2005. Transfer of proinflammatory cytokines across term placenta. Obstet Gynecol 106: 802-7. 10.1097/01.AOG.0000178750.84837.ed. Ardestani, P. M., A. K. Evans, B. Yi, T. Nguyen, L. Coutellier, and M. Shamloo. 2017. 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The muscle regulatory factors MyoD and myf-5 undergo distinct cell cycle-specific expression in muscle cells. J Cell Biol 142: 1447-59. Kohmura, Y., T. Kirikae, F. Kirikae, M. Nakano, and I. Sato. 2000. Lipopolysaccharide (LPS)- induced intra-uterine fetal death (IUFD) in mice is principally due to maternal cause but not fetal sensitivity to LPS. Microbiol Immunol 44: 897-904. Kolmus, K., M. Van Troys, K. Van Wesemael, C. Ampe, G. Haegeman, J. Tavernier, and S. Gerlo. 2014. beta-agonists selectively modulate proinflammatory gene expression in skeletal muscle cells via non-canonical nuclear crosstalk mechanisms. PLoS One 9: e90649. 10.1371/journal.pone.0090649. Lam, S. Y., G. L. Tipoe, E. C. Liong, and M. L. Fung. 2008. Chronic hypoxia upregulates the expression and function of proinflammatory cytokines in the rat carotid body. Histochem Cell Biol 130: 549-59. 10.1007/s00418-008-0437-4. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Langen, R., A. Schols, M. Kelders, E. Wouters, and Y. Janssen-Heininger. 2001. Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor-κB. The FASEB Journal 15: 1169-80. Langen, R. C., J. L. Van Der Velden, A. M. Schols, M. C. Kelders, E. F. Wouters, and Y. M. Janssen-Heininger. 2004. Tumor necrosis factor-alpha inhibits myogenic differentiation through MyoD protein destabilization. FASEB J 18: 227-37. 10.1096/fj.03-0251com. Lemons, J. A., and R. L. Schreiner. 1983. Amino acid metabolism in the ovine fetus. Am J Physiol 244: E459-66. 10.1152/ajpendo.1983.244.5.E459. Lirussi, F., Z. Rakotoniaina, S. Madani, F. Goirand, M. Breuiller-Fouche, M. J. Leroy, P. Sagot, J. J. Morrison, M. Dumas, and M. Bardou. 2008. ADRB3 adrenergic receptor is a key regulator of human myometrial apoptosis and inflammation during chorioamnionitis. Biol Reprod 78: 497-505. 10.1095/biolreprod.107.064444. Liu, L., J. A. Fissel, A. Tasnim, J. Borzan, A. Gocke, P. A. Calabresi, and M. H. Farah. 2016. Increased TNFR1 expression and signaling in injured peripheral nerves of mice with reduced BACE1 activity. Neurobiol Dis 93: 21-7. 10.1016/j.nbd.2016.04.002. Nie, J., T. K. Tong, K. George, F. H. Fu, H. Lin, and Q. Shi. 2011. Resting and post-exercise serum biomarkers of cardiac and skeletal muscle damage in adolescent runners. Scand J Med Sci Sports 21: 625-9. 10.1111/j.1600-0838.2010.01096.x. Posont, R., K. Beede, S. Limesand, and D. Yates. 2018. Changes in myoblast responsiveness to TNFa and IL-6 contribute to decreased skeletal muscle mass in intrauterine growth restricted fetal sheep. . Translational Animal Science 2: S44-S7. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Rawls, A., J. H. Morris, M. Rudnicki, T. Braun, H. H. Arnold, W. H. Klein, and E. N. Olson. 1995. Myogenin's functions do not overlap with those of MyoD or Myf-5 during mouse embryogenesis. Dev Biol 172: 37-50. 10.1006/dbio.1995.0004. Rozance, P. J., L. Zastoupil, S. R. Wesolowski, D. A. Goldstrohm, B. Strahan, M. Cree-Green, M. Sheffield-Moore, G. Meschia, W. W. Hay, Jr., R. B. Wilkening, and L. D. Brown. 2018. Skeletal muscle protein accretion rates and hindlimb growth are reduced in late gestation intrauterine growth-restricted fetal sheep. J Physiol 596: 67-82. 10.1113/JP275230. Rudnicki, M. A., P. N. J. Schnegelsberg, R. H. Stead, T. Braun, H. H. Arnold, and R. Jaenisch. 1993. Myod or Myf-5 Is Required for the Formation of Skeletal-Muscle. Cell 75: 1351-9. Doi 10.1016/0092-8674(93)90621-V. Yates, D. T., C. N. Cadaret, K. A. Beede, H. E. Riley, A. R. Macko, M. J. Anderson, L. E. Camacho, and S. W. Limesand. 2016. Intrauterine growth-restricted sheep fetuses exhibit smaller hindlimb muscle fibers and lower proportions of insulin-sensitive Type I fibers near term. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 310: R1020-R9. Yates, D. T., D. S. Clarke, A. R. Macko, M. J. Anderson, L. A. Shelton, M. Nearing, R. E. Allen, R. P. Rhoads, and S. W. Limesand. 2014. Myoblasts from intrauterine growth-restricted sheep fetuses exhibit intrinsic deficiencies in proliferation that contribute to smaller semitendinosus myofibres. J Physiol 592: 3113-25. 10.1113/jphysiol.2014.272591. Yates, D. T., A. S. Green, and S. W. Limesand. 2011. Catecholamines mediate multiple fetal adaptations during placental insufficiency that contribute to intrauterine growth restriction: lessons from hyperthermic sheep. Journal of pregnancy 2011. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Yates, D. T., A. R. Macko, M. Nearing, X. Chen, R. P. Rhoads, and S. W. Limesand. 2012. Developmental programming in response to intrauterine growth restriction impairs myoblast function and skeletal muscle metabolism. J Pregnancy 2012: 631038. 10.1155/2012/631038. Yates, D. T., J. L. Petersen, T. B. Schmidt, C. N. Cadaret, T. L. Barnes, R. J. Posont, and K. A. Beede. 2018. ASAS-SSR Triennnial Reproduction Symposium: Looking Back and Moving Forward-How Reproductive Physiology has Evolved: Fetal origins of impaired muscle growth and metabolic dysfunction: Lessons from the heat-stressed pregnant ewe. J Anim Sci 96: 2987- 3002. 10.1093/jas/sky164. Zhu, M. J., S. P. Ford, P. W. Nathanielsz, and M. Du. 2004. Effect of maternal nutrient restriction in sheep on the development of fetal skeletal muscle. Biol Reprod 71: 1968-73. 10.1095/biolreprod.104.034561. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Tables Table 1. Primers for ddPCR. Product Accession Gene Protein Primer Sequence Size Number GGGAGTACGGCTCCTTCTTC ADRB1 β1 Adrenoceptor 45 NM_012701 CGTCTACCGAAGTCCAGAGC AGCCACCTACGGTCTCTGAA ADRB2 β2 Adrenoceptor 208 NM_012492.2 GTCCCGTTCCTGAGTGATGT TTGCCTCCAATATGCCCTAC ADRB3 β3 Adrenoceptor 46 NM_013108 AAGGAGACGGAGGAGGAGAG CACTGATCCAGTGAGGAGCA FN14 TWEAK Receptor 88 NM_181086 GGCAATTAGACACCCTGGAA CACGAGCCATCATGAAGAGA IL6R IL-6 Receptor 96 NM_017020 GCCAAGGTGCTTGGATTTTA TTGTAGGATTCAGCTCCTGTC TNFR1 TNFα Receptor 1 109 NM_013091 CTCTTACAGGTGGCACGAAGTT CCGAGCTGTCTAACGAGGAG YWHAZ 14-3-3 protein ζ 88 NM_013011 GAGACGACCCTCCAAGATGA Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Table 2. Blood components in MI-IUGR fetal rats. Parameter Control MI-IUGR P-value Blood Glucose, mM Maternal 6.7 ± 0.3 7.2 ± 0.3 NS Fetal 2.5 ± 0.2 2.0 ± 0.2 0.01 Maternal-Fetal Ratio 2.8 ± 0.3 3.6 ± 0.4 0.01 Fetal Blood Plasma BUN, mg/dl 20.0 ± 0.2 23.9 ± 0.8 <0.01 Cholesterol, mg/dl 51.0 ± 0.3 45.0 ± 0.2 <0.01 HDLC, mg/dl 20.1 ± 1.4 18. ± 1.1 NS Triglycerides, mg/dl 71.3 ± 1.9 61.5 ± 1.5 <0.01 Total Protein, g/dl 1.77 ± 0.09 1.71 ± 0.08 NS AST, IU/l 124.8 ± 8.9 159.8 ± 13.5 0.02 ALT, IU/l 56.9 ± 1.4 66.2 ± 5.2 <0.01 Creatine Kinase, IU/l 5355 ± 393 9045 ± 1001 <0.01 GGT, IU/l 15.9 ± 0.3 16.4 ± 1.0 NS NS, not significant; BUN, blood plasma urea nitrogen; HDLC, high density lipoprotein cholesterol; AST, aspartate transaminase; ALT, alanine transaminase; GGT, gamma- glutamyltransferase. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Table 3. Gene expression in skeletal muscle from MI-IUGR fetal rats. Gene Control MI-IUGR P-value TNFR1 0.50 ± 0.05 0.86 ± 0.22 0.01 IL6R 0.42 ± 0.06 0.62 ± 0.09 <0.01 FN14 0.13 ± 0.01 0.21 ± 0.03 0.04 ADRB1 0.46 ± 0.09 0.30 ± 0.05 0.09 ADRB2 0.21 ± 0.02 0.21 ± 0.01 NS ADRB3 0.18 ± 0.04 0.24 ± 0.07 NS CD68 0.23 ± 0.04 0.13 ± 0.02 0.03 CD163 0.16 ± 0.03 0.15 ± 0.04 NS transcripts / YWHAZ transcripts. NS, not significant; TNFR1, TNFα receptor 1; IL6R, IL-6 receptor; FN14, TWEAK receptor; ADRB1, β1 adrenoceptor; ADRB2, β2 adrenoceptor; ADRB3, β3 adrenoceptor; CD68, cluster of differentiation 68; CD163, cluster of differentiation 163. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 List of figure captions Figure 1. Maternal responses to daily LPS endotoxin injection (0.1 µg/kg BW; i.p.). Pregnant th th rats were injected with LPS (MI-IUGR; n = 10) or saline (control; n = 9) from the 9 to the 11 day of gestation (shown by arrows). Rectal temperatures (A) were measured by digital thermometer. Glucose concentrations (B) were measured in whole blood via glucose meter and TNFα concentrations (C) were measured in blood plasma via ELISA. *means differed (P < 0.05) between experimental groups for the time period. Figure 2. Average fetal mass (A) and upper hindlimb area (B) for MI-IUGR (n = 10) and control th (n = 9) fetal rats on the 20 day of gestation. Average fetal mass was determined by weighing all fetuses in each litter. Upper hindlimb cross-sectional area at the mid-point of the femur was averaged for the 3 fetuses in each litter located closest to the uterine bifurcation. *means differed (P < 0.05) between experimental groups. th Figure 3. Myoblast profiles in MI-IUGR (n = 10) and control (n = 9) fetal rats on the 20 day of gestation. Representative micrographs are shown for fetal myoblasts staining positive for myf5 (A; red), myoD (B; red), and myogenin (C; red), and counterstained with DAPI (blue). The number of nuclei staining positive for myf5 (D), myoD (E), and myogenin (F) per µm were determined from fixed cross sections of the upper hindlimb of the 3 fetuses in each litter located closest to the uterine bifurcation. *means differed (P < 0.05) between experimental groups. Figure 4. Skeletal muscle fiber sizes in MI-IUGR (n = 10) and control (n = 9) fetal rats on the th 20 day of gestation. Representative micrographs are shown for fetal hindlimb muscle fibers Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 staining positive for MyHC-1 (A; Type I fibers; red) and MyHC-2 (B; Type II fibers; red), and counterstained for desmin (green). The average cross-sectional areas of Type I (C) and Type II (E) muscle fibers were determined from fixed cross sections of the upper hindlimb of the 3 fetuses in each litter located closest to the uterine bifurcation. *means differed (P < 0.05) between experimental groups. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Figure 1 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Figure 2 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Figure 3 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Figure 4 Accepted Manuscript http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Translational Animal Science Oxford University Press

Maternal inflammation at mid-gestation impairs subsequent fetal myoblast function and skeletal muscle growth in rats, resulting in intrauterine growth restriction at term

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Abstract

Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Maternal inflammation at mid-gestation impairs subsequent fetal myoblast function and skeletal muscle growth in rats, resulting in intrauterine growth restriction at term. C.N. Cadaret*, R.J. Posont*, K.A. Beede*, H.E. Riley*, J.D. Loy†, and D.T. Yates* *Department of Animal Science, University of Nebraska-Lincoln, Lincoln, NE 68583 †School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583 Corresponding Author: Dustin Yates PO Box 830908 Lincoln, NE 68583 dustin.yates@unl.edu This manuscript is based on research that was partially supported by the National Institute of General Medical Sciences Grant 1P20GM104320 (J. Zempleni, Director), the Nebraska Agricultural Experiment Station with funding from the Hatch Act (NEB-26-224) and Hatch Multistate Research capacity funding program (NEB-26-226, NEB-26-225) through the USDA National Institute of Food and Agriculture. The Biomedical and Obesity Research Core (BORC) in the Nebraska Center for Prevention of Obesity Diseases (NPOD) receives partial support from NIH (NIGMS) COBRE IDeA award NIH 1P20GM104320. The contents of this publication are © The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non- Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 the sole responsibility of the authors and do not necessarily represent the official views of the NIH or NIGMS. The authors have no conflicts of interest to declare. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 ABSTRACT: Maternal inflammation induces intrauterine growth restriction (MI-IUGR) of the fetus, which compromises metabolic health in human offspring and reduces value in livestock. The objective of this study was to determine the effect of maternal inflammation at mid-gestation on fetal skeletal muscle growth and myoblast profiles at term. Pregnant Sprague-Dawley rats th were injected daily with bacterial endotoxin (MI-IUGR) or saline (controls) from the 9 to the th 11 day of gestational age (dGA; term = 21 dGA). At necropsy on dGA 20, average fetal mass and upper hindlimb cross-sectional areas were reduced (P < 0.05) in MI-IUGR fetuses compared + + + to controls. MyoD and myf5 myoblasts were less abundant (P < 0.05) and myogenin myoblasts were more abundant (P < 0.05) in MI-IUGR hindlimb skeletal muscle compared to controls, indicating precocious myoblast differentiation. Type I and Type II hindlimb muscle fibers were smaller (P < 0.05) in MI-IUGR fetuses than in controls, but fiber type proportions did not differ between experimental groups. Fetal blood plasma TNFα concentrations were below detectable amounts in both experimental groups, but skeletal muscle gene expression for the cytokine receptors TNFR1, IL6R, and FN14 was greater (P < 0.05) in MI-IUGR fetuses than controls, perhaps indicating enhanced sensitivity to these cytokines. Maternal blood glucose concentrations at term did not differ between experimental groups, but MI-IUGR fetal blood contained less (P < 0.05) glucose, cholesterol, and triglycerides. Fetal-to-maternal blood glucose ratios were also reduced (P < 0.05), which is indicative of placental insufficiency. Indicators of protein catabolism, including blood plasma urea nitrogen and creatinine kinase were greater (P < 0.05) in MI-IUGR fetuses than in controls. From these findings, we conclude that maternal inflammation at mid-gestation causes muscle-centric fetal programming that impairs myoblast function, increases protein catabolism, and reduces skeletal muscle growth near term. Fetal muscle sensitivity to inflammatory cytokines appeared to be enhanced after maternal Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 inflammation, which may represent a mechanistic target for improving these outcomes in MI- IUGR fetuses. Key words: Adaptive fetal programming, developmental origins, inflammatory regulation, maternofetal stress, thrifty phenotype Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 INTRODUCTION Maternal stress during gestation induces intrauterine growth restriction (IUGR), which is linked to lifelong deficits in skeletal muscle growth and metabolic dysfunction in humans and livestock (Yates et al., 2016; Yates et al., 2018). Late prenatal and postnatal muscle growth requires proliferation, differentiation, and fusion of myoblasts with existing muscle fibers (Zhu et al., 2004), and this rate-limiting process can be altered by inflammation (Langen et al., 2001; Langen et al., 2004; Posont et al., 2018). Inflammatory changes within the skeletal muscle niche can arise from systemic inflammation and from resident macrophages within the tissue itself (Kharraz et al., 2013). Acute inflammation impairs adult myoblast function in vitro (Frost et al., 1997; Langen et al., 2001), and we hypothesized that maternal inflammation may likewise disrupt fetal myoblast function and muscle growth in utero. The programming effects of fetal stress on muscle development and growth have implications for long-term metabolic health in IUGR-born humans and growth performance in livestock (Zhu et al., 2004; Yates et al., 2011; Yates et al., 2012), but the underlying mechanisms are not well understood. Our previous studies in placental insufficiency-induced IUGR fetal sheep have identified intrinsically impaired myoblast function and restricted muscle growth as outcomes of unidentified molecular adaptations (Yates et al., 2012; Yates et al., 2014; Yates et al., 2016). Therefore, the objective of this study was to determine the effects of sustained maternal inflammation at mid-gestation on myoblast profiles, muscle growth, and metabolic indicators at term. MATERIALS AND METHODS Animals and experimental design Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Nebraska-Lincoln. Animal studies were performed at the University of Nebraska-Lincoln Animal Science Complex, which is accredited by AAALAC International. Timed-pregnant Sprague-Dawley rats (Envigo, Indianapolis, IN) were purchased and delivered th on the 6 day of gestational age (dGA). Beginning at dGA 8, rats were individually housed, fed th th commercial rat chow ad libitum, and weighed daily. From the 9 to the 11 dGA, rats were randomly assigned to be injected (i.p.) daily with 250 μl physiological saline (control, n = 9) or 0.1 μg/kg BW of lipopolysaccharide (LPS) endotoxin from E. coli O55:B5 (Sigma-Aldrich, St. Louis, MO) in 250 μl physiological saline to create maternal inflammation-induced IUGR (MI- IUGR, n = 10). Maternal blood samples were collected via saphenous vein puncture and rectal temperatures were recorded at 3, 6, and 24 hours after each of the daily injections. On dGA 20 (term = 21 dGA), rats were euthanized by decapitation under heavy isoflurane anesthesia and maternal blood samples were collected. Litter size and individual fetal weights were recorded, and skinned hindlimbs were collected from the three fetuses in each litter located closest to the uterine bifurcation. The right hindlimb from each fetus was snap frozen and the left was fixed in 4% PFA. Pooled blood samples were collected from the remaining fetuses in each litter via exsanguination. Blood analysis Glucose concentrations were determined from maternal and fetal whole blood in duplicate with a Contour next EZ glucose meter (Bayer Corp., Whippany, NJ). Blood plasma was then isolated by centrifugation (14,000 x g, 2 minutes, 4°C) and TNFα concentrations were determined in duplicate by Rat TNFα Quantikine ELISA kit (R&D Systems, Minneapolis, MN) Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 as previously described (hui Seo et al., 2017). Inter-assay and intra-assay CV was less than 10%, and the detection limit was 5 pg/ml. Fetal blood plasma concentrations of urea nitrogen (BUN), high-density lipoprotein cholesterol (HDLC), total cholesterol, triglycerides, and total protein were determined via colorimetric assays with a Vitros-250 Chemistry Analyzer (Ortho Clinical Diagnostics, Linden, NJ) according to manufacturer recommendations. Concentrations of γ- glutamyltransferase (GGT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were determined via multiple-point rate assays with the Vitros-250 Chemistry Analyzer. Gene expression RNA isolation. RNA was extracted from frozen fetal hindlimb muscle (~30 mg) via RNeasy Fibrous Tissue Mini Kit (Qiagen, Germantown, MD). For all samples, isolated RNA was quantified on a NanoDrop microvolume spectrophotometer (Thermo Fisher; Waltham, MA), and reverse transcribed into cDNA via QuantiTect Reverse Transcription Kit (Qiagen). Droplet digital PCR. Primer pairs for PCR were designed for the genes of interest listed in Table 1. Droplet digital PCR (ddPCR) was performed in duplicate with the QX200 ddPCR System (BioRad, Hercules, CA). Each reaction contained Evagreen Supermix, 10 μM of each primer, and equivalent amounts of cDNA template. Droplets were generated in a QX200 Droplet Generator with Droplet Generator Oil, transferred to a PCR plate, sealed, and placed in a C1000 Touch Thermal Cycler. Samples were activated (95°C for 5 minutes), denatured for 40 cycles (95°C for 30 seconds), annealed and extended for 40 cycles (60°C for 1 minute), and stabilized (4°C for 5 minutes and 90°C for 5 minutes). Finally, droplets were read on the QX200 Droplet Reader and results were analyzed with QuantaSoft Software (BioRad) to obtain transcripts/μl for Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 genes of interest. Results were normalized to transcripts/µl of YWHAZ, which was stable across treatment groups. Immunohistochemistry Myoblast profiles. Fixed fetal hindlimbs were embedded in OCT Compound (Thermo Fisher). Tissue cross sections cut at a thickness of 8 µm were taken at the mid-point of the femur, prepared with a CryoStar NX50 (Thermo Fisher), and mounted on glass microscope slides as previously described (Yates et al., 2014). Slides were dried at 37°C for 30 minutes and then washed three times in PBS + 0.5% Triton-X-100. Antigen retrieval was performed by boiling slides in 10 mM citric acid for 20 minutes and allowing them to cool slowly to room temperature. Non-specific binding was blocked by incubating slides with 0.5% NEN blocking buffer (Perkin-Elmer, Waltham, MA) in PBS at room temperature for 1 hour. Slides were then incubated overnight at 4°C with primary antibodies diluted in PBS + 1% bovine serum albumin (Sigma-Aldrich). Negative controls were incubated in PBS + 1% bovine serum albumin without primary antibody. Sections were stained with antibodies raised in the rabbit against myf5 (1:100; Santa Cruz, Dallas, TX), in the mouse against myoD (1:200; Dako, Santa Clara, CA), and in the mouse against myogenin (1:250, Abcam, Cambridge, MA) to identify nuclei expressing these myogenic factors. Immunocomplexes were detected with affinity-purified immunoglobulin antiserum conjugated to Alexa Fluor 594 (1:2000; Cell Signaling, Danvers, MA) or Alexa Fluor 488 (1:1000; Cell Signaling). Myoblast profiles were assessed in the medial and lateral muscle groups, which included the adductor and gracilis muscles and the semitendinosus, semimembranosus, and biceps femoris, respectively. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Muscle fiber profiles. A second set of hindlimb cross sections were stained to identify fiber type ratios and fiber size as previously described (Yates et al., 2016). Briefly, fibers in medial and lateral muscle groups expressing myosin heavy chain (MyHC) isoforms specific for Type I and Type II fibers were identified with antibodies raised in the mouse against MyHC-I (BA-D5, 1:20; Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Iowa City, IA) and MyHC-II (F18, 1:25; DSHB), respectively. Muscle sections were counterstained with antibodies raised in the rabbit against desmin (1:1000, Sigma-Aldrich) to identify all fibers. Immunocomplexes were detected with affinity-purified immunoglobulin antiserum conjugated to Alexa Fluor 594 (1:2000) or Alexa Fluor 488 (1:1000). All fluorescent images were visualized on an Olympus IX73 and digital micrographs were captured with a DP80 microscope camera (Olympus, Center Valley, PA). Images were analyzed with Olympus cellsSens Dimension software to determine proportions of positive nuclei or fibers within fetal skeletal muscle sections. Animal identifications and treatments were de-identified prior to analyses. Statistical analysis All data were analyzed by ANOVA using the mixed procedure of SAS 9.4 (SAS Institute, Cary, NC) to determine the effect of experimental group. Repeated measures were used for serial maternal measurements. Skeletal muscle gene expression data are expressed as transcript copies per µl, normalized to copies per µl of YWHAZ. Proportions of nuclei staining positive for myogenic regulatory factors were determined from a minimum of 250 positive nuclei, counted across 6 non-overlapping fields of view per fetus. Skeletal muscle fiber type proportions were determined from a minimum of 2,500 total fibers counted across 3 fields of view per fetus. Average fiber size was determined from a minimum of 250 fibers across 3 fields Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 of view per fetus. Rat (dam) was considered the experimental unit for all variables, and values were averaged across the fetuses analyzed in each dam’s litter. All data are expressed as group mean ± standard error. RESULTS Maternal responses to lipopolysaccharide administration. Experimental group x time point interactions were observed (P < 0.05) for maternal rectal temperatures and plasma TNFα concentrations, but not for maternal plasma glucose concentrations. Maternal rectal temperatures in MI-IUGR dams were greater (P < 0.05) than in control dams at 6, 30, 54, and 72 hours after beginning the daily injections on dGA 9 (Figure 1A), but were not different from control temperatures at the other measured time points. Maternal blood glucose did not differ between experimental groups (Figure 1B). Maternal plasma TNFα concentrations in control dams were below the detectable limit (5 pg/ml) at all time points except for 24 hours, when 5 of the 9 controls exhibited plasma TNFα concentrations between 5 and 16.5 pg/ml. Plasma TNFα concentrations were increased (P < 0.05) in MI-IUGR dams at 3, 6, 24, 27, and 51 hours after beginning daily injections (Figure 1C). Maternal weights and daily feed intake did not differ between the two experimental groups on any day. Fetal morphometrics and blood chemistry. At necropsy, the number of fetuses per litter did not differ between controls and MI-IUGR (14.2 ± 0.6 and 13.1 ± 0.6, respectively), but average fetal mass and upper hindlimb area were reduced (P < 0.05) in MI-IUGR fetuses compared to controls (Figure 2). Plasma TNFα concentrations at necropsy were below detectable limits (5 pg/ml) in all fetuses and in all dams with the exception of two MI-IUGR dams that exhibited 8 and 24 pg/ml, respectively. Maternal blood glucose concentrations did not differ between experimental groups at necropsy, but fetal blood glucose was decreased (P < Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 0.05) in MI-IUGR fetuses compared to controls (Table 2). Fetal BUN concentrations were increased (P < 0.05) in MI-IUGR fetuses compared to controls. Plasma HDLC concentrations did not differ between control and MI-IUGR fetuses. Plasma cholesterol and triglyceride concentrations were reduced (P < 0.05) in MI-IUGR fetuses compared to controls. Plasma total protein content did not differ between control and MI-IUGR fetuses. Plasma AST, ALT, and creatine kinase were greater (P < 0.05) in MI-IUGR fetuses compared to controls. Fetal plasma GGT did not differ between experimental groups. Skeletal muscle gene expression. Skeletal muscle TNFR1 (TNFα receptor 1), FN14 (TWEAK receptor), and IL6R (IL-6 receptor) expression was increased (P < 0.05) in MI-IUGR fetuses compared to controls (Table 3). Skeletal muscle ADRB1 (β1 adrenoceptor) mRNA was lower (P < 0.05) in MI-IUGR fetuses compared to controls, but skeletal muscle ADRB2 (β2 adrenoceptor) and ADRB3 (β3 adrenoceptor) mRNA content did not differ between experimental groups. Skeletal muscle mRNA expression for the pan macrophage marker CD68 (cluster of differentiation 68) was decreased (P < 0.05) in MI-IUGR fetuses compared to controls. Fetal skeletal muscle mRNA expression for the M2-macrophage marker CD163 (cluster of differentiation 163) did not differ between experimental groups. Skeletal muscle myoblast profiles. Representative micrographic images are shown for myoblast populations staining positive for myf5 (Figure 3A), myoD (Figure 3B), and myogenin (Figure 3C) in fetal hindlimb skeletal muscle. At necropsy, MI-IUGR decreased (P < 0.05) the + + number of myf5 and myoD nuclei (Figure 3D and Figure 3E, respectively) and increased (P < 0.05) the number of myogenin nuclei (Figure 3F) in fetal hindlimb muscle compared to controls. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Skeletal muscle fiber type proportions and sizes. Representative micrographic images are shown for muscle fiber populations staining positive for MyHC-I (Type I; Figure 4A) and MyHC-II (Type II; Figure 4B) in fetal hindlimb skeletal muscle. At necropsy, the average cross sectional area of Type I and Type II fibers in hindlimb skeletal muscle were smaller (P < 0.05) in MI-IUGR fetuses compared to controls (Figure 4C and Figure 4D, respectively). The proportions of Type I and Type II fibers in fetal hindlimb skeletal muscle did not differ between experimental groups. DISCUSSION In this study, we found that maternal inflammation at mid-gestation impaired subsequent fetal myoblast function, reduced fetal muscle growth, and led to an IUGR phenotype at term. Systemic inflammation was not apparent at term in these dams or their fetuses. However, the sensitivity of fetal skeletal muscle to inflammatory cytokines appeared to be enhanced due to greater gene expression for cytokine receptors. This coincided with indicators of reduced myoblast proliferation and precocious differentiation, diminished muscle fiber hypertrophy, and smaller hindlimb areas. Fetal programming by maternal inflammation also increased indicators of protein catabolism in fetal blood plasma at term. Fetal hypoglycemia and reduced fetal-to- maternal glycemic ratios were indicative of placental insufficiency, which would help to explain how maternal inflammation at mid-gestation resulted in the fetal changes observed near term. The evidence for enhanced cytokine sensitivity, impaired myoblast function, and increased protein catabolism in this study provide insight into a potential programming mechanism for the predisposition of IUGR-born offspring to poor muscle growth and metabolism. The first of the three daily injection of LPS caused a temporal increase in maternal circulating TNFα and body temperatures that peaked at 3 to 6 hours and was gone by 24 hours. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Interestingly, the maternal response to the subsequent injections was less profound, perhaps due to a reduced sensitivity of the immune system to the endotoxin. Furthermore, the impact of the endotoxin on maternal wellbeing beyond the period of administration appeared to be minimal, as daily intake, average daily gain, glycemic levels, and circulating TNFα concentrations subsequent to the final injection were comparable to controls. Conversely, maternal inflammation at mid-gestation caused changes in fetal skeletal muscle that remained present at term, well after the inflammation had subsided. Greater gene expression for cytokine receptors in MI-IUGR fetal muscle are evidence of enhanced sensitivity to inflammatory cytokines despite low circulating TNFα that suggests an absence of systemic fetal inflammation near term. Skeletal muscle is highly responsive to inflammatory cytokines (Frost et al., 1997; Cadaret et al., 2017), and increased inflammatory activity can disrupt muscle growth. Thus, it is possible that greater inflammatory tone caused by increased cytokine sensitivity contributed to reduced fetal muscle growth in this study, although a more in-depth evaluation of this potential mechanism is warranted. Smaller fetal myofiber size following maternal inflammation was coincident with impaired fetal myoblast function, which is consistent with our previous findings in placental insufficiency-induced IUGR fetal sheep (Yates et al., 2014; Posont et al., 2018). At term, medial and lateral hindlimb muscles of MI-IUGR fetal rats had fewer nuclei expressing myoD and myf5, which are indicative of myoblasts in the proliferative stage (Kitzmann et al., 1998) prior to differentiating (Rudnicki et al., 1993). Additionally, more nuclei stained positive for myogenin, which is expressed by myoblasts that have exited the cell cycle and begun differentiating (Rawls et al., 1995). These transcription factor profiles indicate precocious entry of fetal myoblasts into terminal differentiation at the expense of proliferation, which would conform with greater Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 sensitivity to inflammatory cytokines. The effect of cytokines on myoblast function is complex, but increased activity of IL-6/IL6R pathways has been shown to increase myoblast differentiation and correlates with greater myogenin expression (Hoene et al., 2013). Although TNFα/TNFR1 and TWEAK/Fn14 pathways antagonize myoblast differentiation in many cases, their increased activity has also been shown to diminish myoD in myoblasts (Langen et al., 2004; Dogra et al., 2006), as observed in the present study. Paradoxical reductions in gene expression for macrophage indicators in this study may be indicative of a compensatory reduction in resident macrophages, particularly M1 macrophages, which are important contributors to the proliferation-inducing myoblast niche (Du et al., 2017). The lack of changes in β2 and β3 adrenoceptor gene expression following maternal inflammation was somewhat surprising, as both have anti-inflammatory properties (Lirussi et al., 2008; Kolmus et al., 2014). Previous studies have shown that less β1 adrenergic activity (indicated by reduced receptor gene expression is our study) could be associated with greater inflammation (Ardestani et al., 2017), but it would be unlikely that less β1 activity would affect myoblast function directly (Church et al., 2014). Greater BUN concentrations indicate that protein catabolism was increased near term in MI-IUGR fetuses (Depner, 2001), perhaps to provide an energy substrate to compensate for hypoglycemia and reduced circulating triglycerides (Lemons and Schreiner, 1983). Indeed, a previous study in IUGR fetal sheep, which were hypoglycemic by ~50%, found that fetal urea production was ~2.5-fold greater than in controls (Jensen et al., 1999). In IUGR-born rats, BUN continued to be greater at 3 months of age (He et al., 2015), indicating that it is a programmed change. Higher circulating creatine kinase, AST, and ALT concentrations in our MI-IUGR fetuses are further evidence of increased muscle protein catabolism (Depner, 2001; Nie et al., Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 2011; Bodie et al., 2016). Greater protein catabolic rates together with reductions in protein accretion observed in IUGR fetal sheep (Rozance et al., 2018) would presumably contribute to the reduction in hindlimb muscle fiber size in our MI-IUGR fetuses. Mechanisms linking maternal inflammation and muscle-centric fetal programming were not directly evaluated in this study, but fetal hypoglycemia in the presence of maternal euglycemia would indicate that placental insufficiency was present. A previous study in rats rd showed that daily maternal LPS injections in the early 3 trimester reduced trophoblast invasion and spiral artery remodeling in the placenta (Cotechini et al., 2014b). These changes were accompanied by reductions in placental weight, area, and thickness (Cotechini et al., 2014a). Enhanced gene expression for cytokine receptors observed in our MI-IUGR fetuses could have also been the result of direct fetal inflammation, as chronic inflammatory events have been shown to increase TNFR1 and IL6R in other tissues (Lam et al., 2008; Liu et al., 2016). Placental permeability to maternal inflammatory cytokines is low (Aaltonen et al., 2005), but the rodent placenta is at least somewhat permeable to LPS (Kohmura et al., 2000). Thus, it is possible that the fetus produced its own inflammatory response to LPS transferred from maternal to fetal circulation. However, other studies indicate that the placenta itself is a more likely source for fetal inflammation, as maternal LPS administration has been shown to increase placental cytokine production substantially (Bloise et al., 2013). From these findings, we can conclude that muscle-centric fetal programming by maternal inflammation at mid-gestation impairs fetal myoblast function and increases fetal protein catabolism. These changes were associated with impaired skeletal muscle growth, which contributed to IUGR at term. The role of these changes in postnatal muscle mass deficits of IUGR-born offspring was not clear from the present study but may be a reasonable aim for future Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 studies with this model. Likewise, it was unclear if the muscle-centric fetal programming events observed in this study were survival adaptations by the fetus, but reduced skeletal muscle would allow re-appropriation of limited fetal nutrients to neural and cardiac tissues. Thus, it is important for future studies seeking to moderate inflammation-induced fetal programming during maternal stress conditions to consider the impact on fetal survival mechanisms in addition to potential benefits for postnatal health and growth performance. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 LITERATURE CITED Aaltonen, R., T. Heikkinen, K. Hakala, K. Laine, and A. Alanen. 2005. Transfer of proinflammatory cytokines across term placenta. Obstet Gynecol 106: 802-7. 10.1097/01.AOG.0000178750.84837.ed. Ardestani, P. M., A. K. Evans, B. Yi, T. Nguyen, L. Coutellier, and M. Shamloo. 2017. 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Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Yates, D. T., A. R. Macko, M. Nearing, X. Chen, R. P. Rhoads, and S. W. Limesand. 2012. Developmental programming in response to intrauterine growth restriction impairs myoblast function and skeletal muscle metabolism. J Pregnancy 2012: 631038. 10.1155/2012/631038. Yates, D. T., J. L. Petersen, T. B. Schmidt, C. N. Cadaret, T. L. Barnes, R. J. Posont, and K. A. Beede. 2018. ASAS-SSR Triennnial Reproduction Symposium: Looking Back and Moving Forward-How Reproductive Physiology has Evolved: Fetal origins of impaired muscle growth and metabolic dysfunction: Lessons from the heat-stressed pregnant ewe. J Anim Sci 96: 2987- 3002. 10.1093/jas/sky164. Zhu, M. J., S. P. Ford, P. W. Nathanielsz, and M. Du. 2004. Effect of maternal nutrient restriction in sheep on the development of fetal skeletal muscle. Biol Reprod 71: 1968-73. 10.1095/biolreprod.104.034561. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Tables Table 1. Primers for ddPCR. Product Accession Gene Protein Primer Sequence Size Number GGGAGTACGGCTCCTTCTTC ADRB1 β1 Adrenoceptor 45 NM_012701 CGTCTACCGAAGTCCAGAGC AGCCACCTACGGTCTCTGAA ADRB2 β2 Adrenoceptor 208 NM_012492.2 GTCCCGTTCCTGAGTGATGT TTGCCTCCAATATGCCCTAC ADRB3 β3 Adrenoceptor 46 NM_013108 AAGGAGACGGAGGAGGAGAG CACTGATCCAGTGAGGAGCA FN14 TWEAK Receptor 88 NM_181086 GGCAATTAGACACCCTGGAA CACGAGCCATCATGAAGAGA IL6R IL-6 Receptor 96 NM_017020 GCCAAGGTGCTTGGATTTTA TTGTAGGATTCAGCTCCTGTC TNFR1 TNFα Receptor 1 109 NM_013091 CTCTTACAGGTGGCACGAAGTT CCGAGCTGTCTAACGAGGAG YWHAZ 14-3-3 protein ζ 88 NM_013011 GAGACGACCCTCCAAGATGA Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Table 2. Blood components in MI-IUGR fetal rats. Parameter Control MI-IUGR P-value Blood Glucose, mM Maternal 6.7 ± 0.3 7.2 ± 0.3 NS Fetal 2.5 ± 0.2 2.0 ± 0.2 0.01 Maternal-Fetal Ratio 2.8 ± 0.3 3.6 ± 0.4 0.01 Fetal Blood Plasma BUN, mg/dl 20.0 ± 0.2 23.9 ± 0.8 <0.01 Cholesterol, mg/dl 51.0 ± 0.3 45.0 ± 0.2 <0.01 HDLC, mg/dl 20.1 ± 1.4 18. ± 1.1 NS Triglycerides, mg/dl 71.3 ± 1.9 61.5 ± 1.5 <0.01 Total Protein, g/dl 1.77 ± 0.09 1.71 ± 0.08 NS AST, IU/l 124.8 ± 8.9 159.8 ± 13.5 0.02 ALT, IU/l 56.9 ± 1.4 66.2 ± 5.2 <0.01 Creatine Kinase, IU/l 5355 ± 393 9045 ± 1001 <0.01 GGT, IU/l 15.9 ± 0.3 16.4 ± 1.0 NS NS, not significant; BUN, blood plasma urea nitrogen; HDLC, high density lipoprotein cholesterol; AST, aspartate transaminase; ALT, alanine transaminase; GGT, gamma- glutamyltransferase. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Table 3. Gene expression in skeletal muscle from MI-IUGR fetal rats. Gene Control MI-IUGR P-value TNFR1 0.50 ± 0.05 0.86 ± 0.22 0.01 IL6R 0.42 ± 0.06 0.62 ± 0.09 <0.01 FN14 0.13 ± 0.01 0.21 ± 0.03 0.04 ADRB1 0.46 ± 0.09 0.30 ± 0.05 0.09 ADRB2 0.21 ± 0.02 0.21 ± 0.01 NS ADRB3 0.18 ± 0.04 0.24 ± 0.07 NS CD68 0.23 ± 0.04 0.13 ± 0.02 0.03 CD163 0.16 ± 0.03 0.15 ± 0.04 NS transcripts / YWHAZ transcripts. NS, not significant; TNFR1, TNFα receptor 1; IL6R, IL-6 receptor; FN14, TWEAK receptor; ADRB1, β1 adrenoceptor; ADRB2, β2 adrenoceptor; ADRB3, β3 adrenoceptor; CD68, cluster of differentiation 68; CD163, cluster of differentiation 163. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 List of figure captions Figure 1. Maternal responses to daily LPS endotoxin injection (0.1 µg/kg BW; i.p.). Pregnant th th rats were injected with LPS (MI-IUGR; n = 10) or saline (control; n = 9) from the 9 to the 11 day of gestation (shown by arrows). Rectal temperatures (A) were measured by digital thermometer. Glucose concentrations (B) were measured in whole blood via glucose meter and TNFα concentrations (C) were measured in blood plasma via ELISA. *means differed (P < 0.05) between experimental groups for the time period. Figure 2. Average fetal mass (A) and upper hindlimb area (B) for MI-IUGR (n = 10) and control th (n = 9) fetal rats on the 20 day of gestation. Average fetal mass was determined by weighing all fetuses in each litter. Upper hindlimb cross-sectional area at the mid-point of the femur was averaged for the 3 fetuses in each litter located closest to the uterine bifurcation. *means differed (P < 0.05) between experimental groups. th Figure 3. Myoblast profiles in MI-IUGR (n = 10) and control (n = 9) fetal rats on the 20 day of gestation. Representative micrographs are shown for fetal myoblasts staining positive for myf5 (A; red), myoD (B; red), and myogenin (C; red), and counterstained with DAPI (blue). The number of nuclei staining positive for myf5 (D), myoD (E), and myogenin (F) per µm were determined from fixed cross sections of the upper hindlimb of the 3 fetuses in each litter located closest to the uterine bifurcation. *means differed (P < 0.05) between experimental groups. Figure 4. Skeletal muscle fiber sizes in MI-IUGR (n = 10) and control (n = 9) fetal rats on the th 20 day of gestation. Representative micrographs are shown for fetal hindlimb muscle fibers Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 staining positive for MyHC-1 (A; Type I fibers; red) and MyHC-2 (B; Type II fibers; red), and counterstained for desmin (green). The average cross-sectional areas of Type I (C) and Type II (E) muscle fibers were determined from fixed cross sections of the upper hindlimb of the 3 fetuses in each litter located closest to the uterine bifurcation. *means differed (P < 0.05) between experimental groups. Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Figure 1 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Figure 2 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Figure 3 Accepted Manuscript Downloaded from https://academic.oup.com/tas/advance-article-abstract/doi/10.1093/tas/txz037/5426689 by Ed 'DeepDyve' Gillespie user on 09 April 2019 Figure 4 Accepted Manuscript

Journal

Translational Animal Science – Oxford University Press

Published: Apr 3, 2019

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