Recipient Organization
UNIVERSITY OF ARIZONA
888 N EUCLID AVE
TUCSON,AZ 85719-4824
Performing Department
(N/A)
Non Technical Summary
Fetal growth restriction (FGR) caused by placental insufficiency (PI) leads to poor skeletal muscle growth and performance. This study aims to understand the mechanisms that limit muscle growth in FGR and explore the potential for reversing FGR-related deficiencies through oxygen and glucose supplementation. In FGR fetal sheep created with maternal heat stress, we have identified mitochondrial defects that lower oxidative metabolism and muscle growth. We plan to test the hypothesis that correcting oxygen and glucose concentrations in FGR fetuses will promote protein accretion, restore mitochondrial activity, and stimulate muscle growth. The proposed objectives will test the combined effect of sustained oxygen and glucose correction on net fetal protein accretion rates, mitochondrial metabolism, and the plasticity of skeletal muscle growth. The findings will provide fundamental knowledge on the mechanisms that obstruct skeletal muscle metabolism and growth in FGR. Understanding how nutrient replacement approaches, combined with oxygen supplementation, can improve the health of FGR fetuses will inform future studies and help establish effective strategies for managing FGR and promoting health.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Placental insufficiency (PI) is a common cause of fetal growth restriction (FGR) that creates an adverse fetal environment resulting in poor skeletal muscle growth and performance. Skeletal muscle remains a central component in the maintenance of glucose homeostasis and, following FGR reductions, increases the risk for future metabolic diseases. Although no treatments are presently available for FGR, the plasticity of the perinatal period may provide opportunities to increase lean mass. However, fundamental knowledge on the mechanisms that limit muscle growth is required. PI lowers fetal oxygen and glucose concentrations, which act to lower anabolic hormones and raise catecholamines. Together, these adverse FGR conditions create major obstacles for the treatment of FGR because of their inhibitory actions on insulin secretion and oxidative metabolism.Our PI-FGR sheep model recapitulates all the reported conditions of FGR identified in mammalian fetuses, including late gestation reductions in skeletal muscle growth and amino acid utilization for protein accretion. We have identified mitochondrial defects that limit skeletal muscle oxidative metabolism and lower the energy production required for normal net protein accretion and growth. Of importance, quantitative proteomic studies on isolated mitochondria from FGR fetuses identified that the hypoxia-inducible protein NADH dehydrogenase (ubiquinone) 1α subcomplex 4-like 2 (NDUFA4L2) was upregulated. NDUFA4L2 lowers oxygen consumption rates by inhibiting mitochondrial complex 1, thus serving as a key factor to lower oxidative metabolism and slow growth. The capacity for reversing FGR remains undetermined, particularly after the fetus has adapted to nutrient deprivation, representing a clinically relevant scenario. We provide compelling evidence that shows normalizing oxygen and glucose concentrations in the FGR fetus for five days normalizes IGF-1 concentrations and glucose homeostasis. Additionally, NDUFA4L2 abundance in the skeletal muscle declined in the treated FGR fetuses. We hypothesize that the correction of oxygen and glucose concentrations in the FGR fetus to normal, control fetal values will promote anabolic and metabolic responses that increased fetal net protein accretion rates and skeletal muscle growth. Furthermore, oxygen will subvert inhibitory activities that reduce mitochondrial oxidative capacity, and glucose will serve as a major nutrient for oxidative metabolism to allow amino acids to be used for protein accretion in FGR fetuses. The following aims will test the central hypothesis:Objective 1: Test the combined effect of sustained oxygen and glucose correction on net protein accretion rates in FGR fetuses. We hypothesize that restoration of oxygen and glucose supply to FGR fetuses will alleviate nutrient and anabolic hormone insufficiency and promote net protein accretion. We will determine leucine uptake and metabolism in the FGR fetus and measure fetal skeletal muscle fractional protein synthetic rates with stable isotope tracers following a five-day correction of their blood oxygen and glucose concentrations during late gestation. Objective 2: Determine whether oxygen supplementation lowers NDUFA4L2 expression and increases mitochondrial complex 1 activity in PI-FGR skeletal muscle. We hypothesize that hypoxia-induced NDUFA4L2 attenuates mitochondrial Complex I activity in FGR skeletal muscle and that oxygen and glucose correction lowers NDUFA4L2 and restores Complex I activity. We will examine mitochondrial oxidative metabolism of skeletal muscle of normal, FGR, and FGR with oxygen and glucose supplemented fetuses.Objective 3. Evaluate the plasticity in skeletal muscle growth and cellular composition following oxygen and glucose correction of FGR fetuses. We hypothesize that oxygen and glucose correction in FGR fetuses will stimulate myoblast proliferation and increase muscle fiber size. We will measure myoblast proliferation and myofiber morphology histologically. Proliferation rates will be determined in isolated myoblasts.The proposed experiments will provide fundamental new knowledge defining mechanisms for how conditions causing FGR affect skeletal muscle metabolism and program deficiencies in mitochondrial metabolism. Additionally, this work will provide the first evidence for the mixed oxygen and glucose supplementation approach to alleviate fetal responses to PI that lower amino acid uptake and accretion, disrupt mitochondrial function, and slow myoblast proliferation. Uniquely, we will test how repletion of oxygen and glucose in FGR fetuses will normalize substrate utilization and increase lean mass. Uncertainty surrounds nutrient replacement strategies in the clinical management of FGR, as some evidence suggests that nutrient supplementation might even cause harm to the fetus, limiting feasibility of the application. However, the combined nutrient with oxygen replacement model in a clinically and agriculturally relevant species will provide key insight into the mechanistic regulators of oxidative metabolism and muscle development under adverse fetal conditions. These innovative experiments, as well as the expertise of the team assembled, will combine to determine the potential benefit for supplementing oxygen in nutrient replacement strategies for IUGR fetuses and limit negative side effects, thus establishing the knowledge required for further longitudinal studies that seek to safely promote perinatal health of FGR fetuses and potentially prevent development of their later life disorders.
Project Methods
Objective 1 Experimental MethodsAnimal Preparations: PI-FGR fetuses will be generated by exposing pregnant ewes to a warm environment from 40 to 90 dGA. PI-FGR fetuses will be compared to pair-fed, age-matched controls maintained at 20°C. At ~120 dGA, catheters will be surgically placed into the fetal femoral artery and vein, umbilical vein, and ewe's femoral artery and vein. The pregnant ewe will be fitted with a trachea catheter.Experimental Groups: We plan to study the combined and individual effects of O2 correction with glucose in PI-FGR sheep fetuses ~0.8 of gestation. At ~125 dGA, humidified O2 will be bled into the ewe's trachea and dextrose infused into either the maternal or fetal femoral vein. Flow rates for each infusion will be adjusted to increase FGR fetal arterial pO2 to >20 mmHg and plasma glucose to 1 mM. Fetal oxygenation and glycemia will be monitored 2-3 times per day and infusions adjusted to maintain target values. Experimental controls will include FGR-AS fetuses that receive humidified air and saline at equivalent rates to FGR-OG fetuses. We will also investigate O2 + saline (FGR-OS) to FGR fetuses to determine the physiological contribution of O2 alone. FGR groups will also be compared to control fetuses that receive air and saline (Control-AS). Fetal blood or plasma samples will be collected to measure blood gasses, glucose, lactate, amino acids, insulin, and IGF-1 concentrations.Fetal leucine kinetic rates: At ~132 dGA (≥5 days of treatment), net umbilical (fetal) uptakes for amino acids will be determined under basal conditions for the treatment group. During the in vivo study, 3H2O (0.15 µCi/min/kg) and [1-13C]-L-leucine (0.5 µmol/min/kg) will be intravenously infused into the fetus to obtain steady state fluxes for leucine disposal rates, oxidation rates, and fluxes into and out of fetal and placental tissues. Analytical methods are described in detail in. Additional measurements: We will determine: umbilical blood flow; fetal concentrations and uptake rates of O2, glucose, and amino acids; and plasma concentrations of insulin, IGF-1, and NE.Skeletal Muscle Protein Synthesis with Phenylalanine: Phenylalanine is not metabolized in muscle; thus, its only fates are incorporation into or release from protein, making it well suited to measure fractional protein synthetic rates in muscle. An arterial blood sample will be obtained at time zero for baseline measurement. Following the leucine tracer study, two isotopomers of phenylalanine, ring 2,3,3 2H8 (m+8) and ring 2H5 (m+5) will be started 6 hours apart and infused overnight at a rate of 0.3 μmol/min/kg based on estimated fetal weight. Muscle biopsies from the biceps femoris muscle will be collected at necropsy.Postmortem Measurements: The placenta and fetus will be weighed. Muscles (semitendinosus, biceps femoris, & soleus) will be collected for objective 2 & 3.Objective 2 Experimental MethodRespiration of Mitochondria: To examine complex 1 mediated OCR, isolated mitochondria from the skeletal muscle will be placed in an oxygen-sensing chamber (Instech Laboratories) that is specifically design for mitochondrial and cellular OCR measurements. Maximal complex 1 mediated OCR (i.e. NADH mediated, State 3) will be measured in 5 mM glutamate, 5 mM malate and 100 µM ADP and compared to State 4 (proton leak) with the subsequent addition of 5 µg/mL oligomycin A. Then maximal uncoupled OCR will be determined with 5 μM carbonyl cyanide m-chlorophenylhydrazone. OCR will be determined from delta pO2 and expressed as nM O2 consumed per minute per mg of protein (or COXIV). Respiratory control ratios will be calculated as the ratio of the OCR in State 3 to State 4. For every preparation, mitochondrial integrity will be assessed by the addition of 10 μM cytochrome c (see Authentication of Key Biological Resources). Alternatively, soleus and semitendinosus fibers will be isolated, permeabilized with saponin, and placed in oxygen-sensing chambers to measure OCR under identical conditions. Mitochondrial density for normalization will be determined with citrate synthase, COXIV, or mitochondrial-to-genomic gene (DNA) ratios.Mitochondrial Quantitative Proteomics: Quantitative proteomics on isolated mitochondrial protein lysates will be performed as described previously. Tandem mass spectrometry using a Thermo Scientific Fusion Lumos will be accomplished in the University of Arizona, College of Medicine Quantitative Proteomics Laboratory. Ion intensity-based label-free quantitative proteomics will be performed with Progenesis QI for Proteomics software (Nonlinear Dynamics Ltd.) in tandem with the database searching engine Mascot (Matrix Science) loaded with the Ovis aris UniProt database (27,372 entries). This procedure is robust and will enable us to detect differences in relative abundance of mitochondrial proteins that are involved in the TCA cycle and electron transport chain in the OG group.Immunoblots and Enzyme Activities: Immunoblots for OXPHOS Antibody Cocktail, NDUFA4L2, or other proteomic-defined enzymes will be performed. NADH Dehydrogenase activity (Complex 1) will be measured with standardized enzymatic assays (Sigma-Aldrich).Objective 3 Experimental Methods Histological Evaluation: Central, cross-sectional biopsies of the semitendinosus muscles collected in Aim 1 will be used for morphological evaluation because it is a representative hindlimb muscle with mixed fiber types. Myofiber diameter and fiber type will be measured as reported previously. We will determine the abundance of fetal myoblasts (Pax7+) and the proportion of myoblasts undergoing proliferation (Ki67+). Treatment identities will be masked to prevent evaluator bias.Myoblast Cultures: Myoblasts will be isolated from the biceps femoris muscle and frozen for in vitro analysis. Myoblast proliferation rates for all groups will be measured at day 3 from an initial plating density of 3000 cells/cm2 when complications (e.g. plating, overcrowding) are minimal (CyQUANT Proliferation Assay). Cells will be grown in medium containing 10% FBS. In addition to intrinsic proliferation rates, we will evaluate extrinsic (serum) factors with 10% FSS collected from all four groups and used in place of FBS to stimulate proliferation of myoblast from controls.Statistical Analysis: Power calculations for a simple group mean comparison were performed for all measures to ensure an adequate sample size. To observe a difference (P<0.05) for a 25% increase in leucine disposal rates or 34% increase in fractional protein synthetic rates, we will need 8 fetuses/group (power >0.8; 2-tailed t-test). Evidence shows sex differences as a relevant biological variable for growth parameters. We anticipate a male-to-female ratio of 50%, doubling the number of fetuses (n=16/group). We have included two additional fetuses per group (environmental and correction variables) to account for expected experimental or treatment losses. To ensure an unbiased approach, all animals will be randomly assigned to experimental groups and where possible researchers will be blinded from treatment conditions. Statistical analyses will be conducted on parametric or log-transformed data using mixed model ANOVA procedures in SAS Software. Significant effects (P<0.05) of group, sex, and their interaction will be determined by the post hoc Tukey's (HSD) test.