Abstract Although intravenous lipid emulsions are routinely administered to preterm infants, their specific effects on skeletal muscle development remain unclear. In this study, a soybean oil‐based lipid emulsion (Intralipid 20®) was administered via intravenous infusion to fetal sheep (gestational day 88–90) at a dose rising from 1 g/kg/day (day 0) to 3 g/kg/day (days 2–8). Intralipid infusion did not alter overall fetal body weight, tibialis anterior (TA) muscle mass or serum testosterone levels. Histological analyses revealed no significant differences in muscle fibre diameter or collagen content in TA muscles between groups. However, Intralipid significantly upregulated the expression of key myogenic regulatory genes, including Myog (myogenin) and Myod (myogenic differentiation 1), while downregulating the expression of several genes associated with fibrogenesis: Col1a1 (collagen type I α1 chain), Col3a1 (collagen type III α1 chain), Lh2b (lysyl hydroxylase 2b) and P4ha (prolyl 4‐hydroxylase α). In contrast, Intralipid had no significant effect on the expression of genes associated with intramuscular adipogenesis, including Pparg (peroxisome proliferator‐activated receptor γ), Pdgfra (platelet‐derived growth factor receptor α), Zfp423 (zinc finger protein 423), Slc27a1 (solute carrier family 27 member 1), C/ebpa (CCAAT/enhancer‐binding protein α) and Fasn (fatty acid synthase). Similarly, genes related to inflammation, such as Tnfa (tumour necrosis factor α), Il‐6 (interleukin 6), Tlr4 (Toll‐like receptor 4) and Tlr2 (Toll‐like receptor 2), were unaffected. In conclusion, these findings indicate that short‐term lipid exposure alters gene expression patterns without measurable structural changes, suggesting that transcriptional responses may precede overt morphological remodelling in fetal skeletal muscle.

Transition of the fetus to extrauterine life requires increased cardiac workload and skeletal muscle activity, yet little is known about microvasculature growth during the perinatal period. We collected hindlimb skeletal muscles and cardiac left (LV) and right ventricles (RV) from fetal (135 days of gestational age; 135D) and neonatal (postnatal days 1 and 5; PD1 and PD5) lambs to measure vascular structures by immunofluorescence and expression of angiogenesis regulators. Heart and skeletal muscle weights and myofibre cross-sectional areas were greater in neonatal compared to fetal lambs. The proportion of slow-twitch oxidative myofibres in tibialis anterior (TA) and flexor digitorum superficialis (FDS) was greater in neonatal compared to fetal lambs. Vascularity in TA was 34% lower on PD1 (P = 0.0005) and 26% lower on PD5 (P = 0.00522) compared to 135D, and capillary density was 36% lower on PD5 compared to 135D (P = 0.0007). Similarly, vascularity in FDS was 40% lower on PD1 (P = 0.0003) and 45% lower on PD5 (P = 0.0001) compared to 135D. In RV and LV, vascularity was similar among age groups, but vessel density was 29% lower in LV on PD1 (P = 0.0001) and 40% lower on PD5 (P < 0.0001) compared to 135D. Several genes involved in angiogenesis were downregulated in neonatal compared to fetal muscle and LV, though VEGFA and VEGFR1 protein expression was higher. Striated muscle growth across the perinatal period is equivalent or greater than its microvascular expansion. Postnatal VEGFA protein expression may herald an increase in angiogenesis known to occur beyond the first week of life to meet ongoing striated muscle demand. KEY POINTS: Physiological changes at birth support increased cardiac workload and skeletal muscle activity in the neonate. Previous work in vivo showed that striated muscle perfusion was reduced in neonatal lambs compared to late gestation fetuses in the context of a marked increase in the partial pressure of oxygen upon breathing. Despite an increase in striated muscle size and a greater proportion of slow-twitch oxidative myofibres across the perinatal period, vascularity and microvessel density were either unchanged or reduced in several skeletal muscles and left and right cardiac ventricles of neonatal compared to late gestation fetal lambs. Our results indicate that under normal physiological conditions, striated muscle growth across the perinatal period is equivalent or greater than its microvascular expansion. Future investigations are warranted to determine how an adverse intrauterine environment or an abnormal birth transition may impact skeletal and cardiac microvascular growth.

Fetal circulating lipids are low but rise precipitously following birth. It is unknown how prematurely elevated lipids affect the fetal heart, which primarily uses carbohydrates for energy. Fetal sheep were surgically instrumented and received Intralipid 20® or Lactated Ringer's Solution intravenously. After 8 days, myocardial biopsies were taken, and cardiomyocytes were dispersed. Lipid uptake was assessed by labeled saturated long‐chain fatty acids (LCFA) and very long‐chain fatty acids (VLCFA) incorporation. Maximal oxygen consumption rates (OCR) were measured. Gene and protein expression levels were measured by quantitative PCR and Western blotting. Intralipid treatment increased LCFA ( p < 0.001) and VLCFA ( p < 0.001) lipid droplet number, and LCFA (males p = 0.002) and VLCFA ( p = 0.018) droplet size. Fetal Intralipid treatment reduced maximal OCR in basal media ( p = 0.005). Palmitic acid decreased maximal OCR regardless of fetal treatment or length of in vitro exposure ( p = 0.006). Fetal Intralipid upregulated genes included CD36 ( p = 0.001), CPT1A ( p < 0.001), CPT1B ( p < 0.001), VLCAD ( p < 0.001), and PDK4 ( p < 0.001), with no differences in protein expression. There were no effects on ER stress, apoptosis, or autophagy markers. Extended elevated lipid levels in the fetus increased lipid uptake and may have shifted substrate preference towards lipids, but all lipid exposure depressed fetal cardiac metabolism. Prematurely elevated lipids mature but suppress oxidative metabolism.

Robust preclinical models of asymmetric ventricular loading in late gestation reflecting conditions such as hypoplastic left heart syndrome are lacking. We characterized the morphometry and microvascular function of the hypoplastic left ventricle (LV) and remaining right ventricle (RV) in a sham-controlled late gestation fetal lamb model of impaired left ventricular inflow (ILVI). Singleton fetuses were instrumented at similar to 120 days gestational age (dGA; term is similar to 147 days) with vascular catheters, an aortic flow probe and a deflated left atrial balloon. Balloons in ILVI fetuses were inflated over the 8 day study until aortic output was eliminated; Sham balloons remained deflated. At the study end-point (similar to 134 dGA), cardiac function was assessed by echocardiography, microvascular perfusion of each free wall was measured by myocardial contrast echocardiography (MCE) and terminal morphometric data were collected. During the chronic study, flow through the ascending aorta of ILVI fetuses fell from 389 to -48 mL min(-1) with minimal changes to other haemodynamics or blood chemistry. End-point echocardiography and morphometry similarly showed significant and meaningful reductions in ILVI LV chamber volume and wall mass without statistically significant changes in RV size relative to Shams. MCE revealed modestly increased LV perfusion and profoundly increased RV perfusion in ILVI fetuses. Our model displays effective LV hypoplasia with preserved overall fetal health, and our finding of increased RV myocardial perfusion may indicate active vascular remodelling in response to the experimental lesion.

Fetal circulating fatty acids are typically low, and the fetal heart primarily relies on carbohydrates to meet energy demands. Preterm infants sometimes require parenteral nutrition, including Intralipid 20®, a lipid emulsion used to support growth and prevent essential fatty acid deficiency. While a critical intervention, lipid infusion at an early post-conceptional age exposes the developing heart to unexpectedly high levels of circulating fatty acids. The objective of this study was to understand how high circulating lipid concentrations at mid-gestation, a comparable age to preterm infants needing parenteral nutrition, affects cardiomyocyte lipid handling. We hypothesized that chronic exposure to high circulating lipid levels would promote cardiomyocyte maturation and increase capacity to uptake and process fatty acids. Fetal sheep from twin pregnancies were randomized and infused with either Intralipid 20® or Lactated Ringer’s Solution (control) for 8 days starting at gestational day 89±1. Fetuses were euthanized on gestational day 97±1 (term 147 days). Control N=8 (2 female, 6 male), Intralipid 20® N=9 (6 female, 3 male). All animal studies were performed at Oregon Health & Science University and approved by the Institutional Animal Care and Use Committee (#IP0007). Lipid droplets were imaged in live, freshly isolated cardiomyocytes through the incorporation of exogenous fatty acids Bodipy C12 (LCFA, 18 carbon) and Bodipy C16 (VLCFA, 22 carbon) on the Zeiss LSM 880 with Airyscan microscope. Images were analyzed in ImageJ. In utero Intralipid 20® exposed cardiomyocytes had a more robust response to in vitro Bodipy-labeled lipids, culminating in significantly higher lipid droplet density of both LCFA (p=0.0282) and VLCFA (p=0.0054), and significantly larger VLCFA-containing lipid droplets (p=0.0305). Data were analyzed by 2-way ANOVA. Significant p-values reported are for treatment main effects; there was no main effect of sex. There was no interaction between sex and treatment. These changes indicate that lipid exposure can mature cardiomyocytes in mid-gestation. National Heart, Lung, and Blood Institute: R01HL146997 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

KNDy neurons, located in the hypothalamic arcuate nucleus, coexpress kisspeptin (Kiss), neurokinin B, and dynorphin and play a crucial role in regulating GnRH/LH secretion in midgestation sheep fetuses. We hypothesize that KNDy-GnRH signaling is established during midgestation, with negative feedback acting through KNDy neurons regulating testosterone levels needed for brain masculinization in male fetuses. We used immunofluorescence histochemistry to assess the effect of chemical castration with the GnRH antagonist degarelix on arcuate KNDy neurons in fetal sheep. Fluorescent in situ hybridization demonstrated the presence of steroid receptors in untreated midgestation fetal kisspeptin neurons. Additionally, unanesthetized cannulated midgestation fetal sheep were used to examine the effects of KNDy peptides on LH secretion and characterize receptor specificity. Treatment of male lamb fetuses with degarelix on day 62 of gestation resulted in significantly decreased plasma LH and testosterone concentrations (P < .05), accompanied by a significant increase in arcuate Kiss neurons (P < .05). In unanesthetized cannulated fetuses, bolus administration of KP-10 (a Kiss receptor agonist) and senktide (NK3 receptor agonist) elicited robust LH release within 15 minutes. Pretreatment with the NK3 receptor antagonist SB222200 blocked the LH response to senktide, whereas P271 (Kiss receptor antagonist) did not affect basal LH or block the LH response to KP-10. Blocking κ-opiate receptor with PF4455242 significantly increased LH release. These results support the hypothesis that KNDy neurons regulate GnRH and gonadotropin secretion in midgestation sheep fetuses, acting as targets for negative feedback to maintain a stable androgen environment crucial for brain masculinization.

Intralipid is a lipid emulsion used for preterm infants, but its biological effects on adipose development remain poorly examined. We investigated the effects of intravenous Intralipid on the adipose tissue development of midgestation fetal sheep. Intralipid20 infusion was started in fetuses at 88-90 days of gestation (dG) following clinical infant dosing guidance (0.5-1 g/kg/d estimated body weight, increasing daily by 0.5-1 g/kg/d to a maximum of 3 g/kg/d). Intralipid increased perirenal fat weight, adipocyte size, and lipid accumulation. Expression of key adipogenic genes was upregulated, including Pparg (Peroxisome Proliferator-Activated Receptor Gamma), Fasn (Fatty Acid Synthase), Fabp4 (Adipocyte Fatty Acid Binding Protein 4), Acca (Acetyl-CoA Carboxylase α), and Cebpa (CCAAT/Enhancer-Binding Protein Alpha), while downregulating Pdgfra (Platelet-Derived Growth Factor Receptor Alpha), Cidea (Cell death-inducing DNA fragmentation factor α-like effector A), Ppargc1a (Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-Alpha), and Ucp1 (Uncoupling Protein 1). Similar changes were confirmed in omental fat. Intralipid elicited mild inflammation in both perirenal and omental fat by increasing the expression of Tnfa (Tumor Necrosis Factor-Alpha), with upregulation of Tlr4 (Toll-Like Receptor-4), and Tlr2 (Toll-Like Receptor-2) specifically in perirenal fat. Notably, this inflammatory response occurred without elevation of Il-6 (Interleukin-6) expression or NF-κB activation. Moreover, Intralipid treatment suppressed the expression of fibrosis-related genes, including Lh2b (lysyl oxidase-like 2b) and P4ha (prolyl 4-hydroxylase). These findings suggest that Intralipid administration at a developmental stage prior to normal term enhances ovine adipogenic differentiation and lipid accumulation which might be protective to premature infants, but it might slightly induce inflammation in the adipose tissues.

Pregnancies affected with placental insufficiency and fetal growth restriction (FGR) are characterized by reduced umbilical blood flow, decreased nutrient and oxygen supply to the fetus, and impaired fetal skeletal muscle growth. Vascular development within FGR skeletal muscle has not been well described. We hypothesized that chronic placental insufficiency impairs microvascular architecture in FGR fetal skeletal muscle, resulting in decreased vascularity. We used a sheep model of placental insufficiency-induced FGR by exposing pregnant ewes to elevated temperature. Four muscles from the hindlimb were obtained in late gestation from FGR and control fetal sheep for histological and molecular analyses. The proportion of slow-twitch oxidative fibers was 22% lower in the tibialis anterior (TA) and 32% lower in the extensor digitorum longus (EDL) muscles in FGR fetuses compared with controls. Total cross-sectional area was 20%-45% lower in biceps femoris (BF), TA, EDL, and soleus (SOL) muscles. The capillary number per myofiber was 34% lower in BF, 51% lower in TA, and 21% lower in SOL FGR muscles compared with controls. Capillary area was also 44% lower in the FGR TA muscle. Taken together, late gestation fetuses with placental insufficiency-induced FGR had smaller myofibers, fewer slow-twitch oxidative myofibers, and defects in angiogenesis and capillary formation.NEW & NOTEWORTHY Pregnancies affected by placental insufficiency-induced FGR result in fewer slow-twitch oxidative myofibers and lower capillary number per myofiber in fetal skeletal muscle by late gestation.

Methodological status quo is often closely guarded in animal research because changes are seen as a threat to approaches that have proven successful. Current practices are often considered within the group as "best practice." Perioperative analgesia is an important consideration in humane animal research to prevent central sensitization and can contribute to the benefits of multimodal anesthesia, but many research groups do not provide preoperative analgesia to pregnant ewes. We conducted this study to challenge the belief that preoperative buprenorphine negatively impacts the recovery of the ewe and therefore fetal health. Pregnant ewes at approximately 85 days of gestation were divided into 2 groups (each n = 6) that all had the same hysterotomy and fetal catheterization surgery performed. The first group received buprenorphine (0.3 mg, SC) preoperatively, and the second group received the buprenorphine postoperatively. Isoflurane use, time to each step of the recovery process, intraoperative maternal plasma cortisol, and fetal arterial blood values after 4 days of recovery were compared between groups. Equivalence of outcomes between groups was assessed while controlling for potential confounding variables (maternal body weight and length of isoflurane) using 2 one-sided tests with regression adjustment. Average isoflurane concentration after induction, maternal cortisol levels, fetal blood pH, and fetal blood pO2 were equivalent between the groups. The time from cessation of isoflurane to the time of spontaneous breath or extubation and the time from extubation to time of eating or standing were all shorter in the preoperative buprenorphine group. Fetal hematocrit was also lower in the preoperative buprenorphine group. Our study not only refutes that preoperative buprenorphine causes prolonged recovery of the pregnant ewe and detrimental health effects to the fetus but also describes the benefits of preoperative buprenorphine.

Cardiomyocyte proliferative maturation and metabolic maturation occur in the same perinatal period. Intrauterine conditions influence developmental trajectories of both processes; however, their interconnectedness is unknown. Circulating fetal lipid levels are typically low, but the heart may be prematurely exposed to elevated lipids. We experimentally increased fetal lipid levels to determine the impact on cardiomyocyte proliferative maturation. Fetal sheep were surgically instrumented with catheters. After recovery, Intralipid 20 or Lactated Ringer's Solution were infused according to a clinical g/kg schedule for 8 days until 133 ± 1 days of gestation. A left ventricular biopsy was fixed, and remaining cardiomyocytes were enzymatically dissociated. Myocardial composition was measured from Masson's Trichrome-stained sections. Cardiomyocyte length, width, nucleation, and Ki-67+ were studied in dispersed cells. Myocardial composition, cardiomyocyte dimensions, and Ki-67+ were not found to be different between groups. Cardiomyocytes of the Intralipid-treated fetuses were 14% more terminally differentiated than controls (p = 0.025). Early developmental exposure to circulating lipid accelerates fetal cardiomyocyte terminal differentiation and may contribute to fewer cardiomyocytes for life.

Circulating lipid levels are typically low in fetuses, and exposure to high lipid levels at developmental stages prior to term birth is sometimes associated with pathology. Experimentally, near-term fetuses tolerate one week of high lipid concentrations; it is unknown whether this brief exposure to elevated circulating lipids is pathological at an earlier developmental age. We studied the physiological response to intravenous lipid emulsion during mid-gestation. Fetal sheep received intravenous Intralipid 20® (n = 9) or Lactated Ringer's Solution (n = 8) from 85.0 ± 0.7 to 97.0 ± 0.7 days of gestation (term = 147 days). Intralipid was administered according to manufacturer's recommendations, with an initial dose of 0.5-1 g/kg/d that increased daily to a maximum of 3 g/kg/d. Hemodynamic and arterial blood parameters were assessed throughout the study. Fetal growth, liver function, and lipid droplet accumulation were measured on the final day. Fetal hemodynamics and blood gases did not change as a result of the treatment. Compared with Controls, Intralipid fetuses had lower blood lactate concentrations (1.3 ± 0.2 vs. 1.0 ± 0.2 mmol/l, P=0.009) after eight days of treatment. Conjugated (0.4 ± 0.1 vs. 0.6±0.1 mg/dl, P<0.001) and unconjugated (0.3 ± 0.1 vs. 1.2 ± 0.5 mg/dl, P<0.001) bilirubin levels were higher in Intralipid-infused fetuses than in Controls. Fetal somatic growth was unchanged, but heart weight was lower in fetuses receiving Intralipid (6.9 ± 0.7 vs. 6.1±0.7 g, P=0.008). Compared with Controls, Oil Red O staining was elevated in the liver and heart of Intralipid-infused fetuses (liver score: 18.9 ± 17.2 vs. 371.7±44.2, P<0.0001; heart score: 1.8 ± 2.8 vs. 97.6 ± 60.1, P=0.0006). Our findings suggest that mid-gestation fetal sheep can tolerate intravenous lipid emulsion. Lipid accumulation in the liver and heart may precede pathologies associated with ectopic lipid storage, but further research is needed to understand the long-term consequences of Intralipid infusion at this developmental stage.

Although the unfolded protein response (UPR) contributes to survival by removing misfolded proteins, endoplasmic reticulum (ER) stress also activates proapoptotic pathways. Changed sensitivity to normal developmental stimuli may underlie observed cardiomyocyte apoptosis in the healthy perinatal heart. We determined in vitro sensitivity to thapsigargin in sheep cardiomyocytes from four perinatal ages. In utero cardiac activation of ER stress and apoptotic pathways was determined at these same ages. Thapsigargin-induced phosphorylation of eukaryotic initiation factor 2 (EIF2A) was decreased by 72% between 135 and 143 dGA (P = 0.0096) and remained low at 1 dPN (P = 0.0080). Conversely, thapsigargin-induced caspase cleavage was highest around the time of birth: cleaved caspase 3 was highest at 1 dPN (3.8-fold vs. 135 dGA, P = 0.0380; 7.8-fold vs. 5 dPN, P = 0.0118), cleaved caspase 7 and cleaved caspase 12 both increased between 135 and 143 dGA (25-fold and 6.9-fold respectively, both P < 0.0001) and remained elevated at 1 dPN. Induced apoptosis, measured by TdT-mediated dUTP nick-end labeling (TUNEL) assay, was highest around the time of birth (P < 0.0001). There were changes in myocardial ER stress pathway components in utero. Glucose (78 kDa)-regulated protein (GRP78) protein levels were high in the fetus and declined after birth (P < 0.0001). EIF2A phosphorylation was profoundly depressed at 1 dPN (vs. 143 dGA, P = 0.0113). In conclusion, there is dynamic regulation of ER proteostasis, ER stress, and apoptosis cascade in the perinatal heart. Apoptotic signaling is more readily activated in fetal cardiomyocytes near birth, leading to widespread caspase cleavage in the newborn heart. These pathways are important for the regulation of normal maturation in the healthy perinatal heart.

Elevated cardiac troponin I (cTnI), a myocardial damage biomarker, has been reported in cord blood of neonates delivered vaginally or by cesarean section. Although the neonatal peak likely reflects the physiological adjustment to extrauterine life, a better understanding of serial prepartum changes is required to determine physiological causes of fetal cTnI release. We longitudinally sampled eight healthy lambs (20 days before spontaneous birth to 5 days postnatal), and from three fetuses receiving intravenous IGF-1. Samples were collected into heparin, and the plasma was stored at -80 degrees C for later determination of high-sensitivity (hs) cTnI levels (BeckmanCoulter UniCel DxI Access IA; log transformed detection limit = 0.30, quantification limit = 0.78, 99th percentile = 1.78). Positive and negative control samples were drawn from an adult ewe during a terminal experiment (myocardial ischemia) and similarly assessed. hs-cTnI data were log transformed from ng/L. Log(hs-cTnI) was 1.47 +/- 0.30 (means +/- SD) at 20 days before birth and declined to 1.02 +/- 0.65 in fetuses 12 +/- 4 h before birth (P < 0.0001, R-2 = 0.7869). Birth stimulated a delayed, transient peak in hs-cTnI (P = 0.0058). Newborn (43 +/- 19 min postnatal) levels were 1.39 +/- 0.40 (P = 0.0650 vs. fetus on day of birth) and 2.14 +/- 0.63 the day after birth (P = 0.0331 vs. newborn). The second day after birth, levels declined to 1.65 +/- 0.48 (P = 0.0238 vs. day 1). IGF-1 infusion increased hs-cTnI levels 25-50% over baseline (P = 0.0252, R-2 = 0.9938). Baseline adult ewe log(hs-cTnI) was below the limit of detection; 3 h following coronary artery ligation, levels were 3.21. In conclusion, we newly report that fetal hs-cTnI levels decline concomitantly with reduced proliferation of cardiomyocytes toward term. NEW & NOTEWORTHY Serial blood samples were collected from catheterized, normally developing fetal and newborn lambs and high-sensitivity cardiac troponin I (hs-cTnI) levels were assessed, providing unprecedented insight into the physiological processes leading to high levels in the perinatal period. Moderately high levels of hs-cTnI found in the normally developing fetus declined toward term. An elevation to high levels peaked the day after birth, after which hs-cTnI declined again. Stimulation of fetal cardiomyocyte proliferation with IGF-1 also elevated hs-cTnI.

INTRODUCTION: Circulating biomarkers such as Troponin I (TnI) that are typically indicative of myocardial damage in adults have been reported to be detectable in cord blood of healthy infants delivered both vaginally and by cesarean section prior to labor. We performed serial sampling of healthy fetal sheep from ~20 days before spontaneous birth to 5 days postnatal in order to describe normal developmental values of circulating TnI. METHODS: Seven fetal sheep were surgically instrumented with indwelling catheters advanced into the ascending aorta and the superior vena cava. Following surgical recovery, daily blood samples were taken first thing in the morning into heparin, and the plasma was frozen for later determination of high-sensitivity (hs) TnI levels (BeckmanCoulter UniCel DxI Access IA; log transformed limit of detection =0.30, limit of quantification =0.78, and 99th%ile =1.78). Positive and negative control samples were drawn from an adult ewe during a terminal experimental ligation of the left anterior descending coronary artery and similarly assessed. hs-TnI data were log transformed from ng/L, visually assessed, and outliers removed by the ROUT method. RESULTS: Log(hs-TnI) was 1.47±0.30 at 20 days before birth and declined to 0.88±0.36 in fetuses 12±4 hour before birth (p<0.0001, R2=0.8444). Birth stimulated a delayed, transient peak in hs-TnI (P=0.0058). In the newborn, 43±19 min after birth, levels were 1.39±0.40 (P=0.0650 vs. fetus on day of birth). The day after birth, log(hs-TnI) increased to 2.14±0.63 (P=0.0331 vs. newborn). The second day after birth, levels declined to 1.65±0.48 (P=0.0238 vs. day 1). Adult ewe log(hs-TnI) was below the limit of detection; three hours following coronary artery ligation, levels were 3.21. CONCLUSIONS: This study confirms that hs-TnI levels are moderate to high in healthy sheep fetuses, and reports for the first time that they decline towards term. Why TnI is elevated in the healthy fetus is unknown. If cardiomyocyte TnI is leaked during cytokinesis, the decline towards birth may reflect the diminishing rate of proliferation in this same period. The postnatal hs-TnI peak, above the clinical threshold for evidence of myocardial ischemia in adult humans, may result from hemodynamic, oxidative, or metabolic stresses associated with the establishment of postnatal physiology. This work was funded by an award from the NHLBI (R01HL142483). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Many genes used as internal controls for mRNA expression studies are unstable (change) over development. This study determined an approach to validate reference genes for mRNA studies spanning the fetal period to adulthood in sheep hearts.•We determined the mRNA expression of 12 candidate reference genes (ACTB, GAPDH, H3-3A, HYAL2, PPIA, RNA18S1, RPL32, RPL37A, RPL41, RPLP0, RPS15, and YWHAZ) via RT-qPCR. Per RefFinder, which incorporates computational algorithms by BestKeeper, comparative delta Ct, GeNorm, and NormFinder, RPL32, RPL37A, HYAL2, ACTB and GAPDH were the most stable reference genes, although none were unchanged across all ages.•Systematical calculation of the geometric means of 3 reference genes revealed the combination of HYAL2, RPL32, and RPL37A was unchanged across the 5 fetal, neonatal, and adult ages.•We determined the most stable combination of reference genes for cardiac gene expression studies in sheep from fetus to newborn to adult; these steps are applicable to determine internal controls for mRNA studies in other organs, other species, and periods in which reference gene instability is high.

In preterm neonates unable to obtain sufficient oral nutrition, intravenous lipid emulsion is life-saving. The contribution of post-conceptional level of maturation to pathology that some neonates experience is difficult to untangle from the global pathophysiology of premature birth. In the present study, we determined fetal physiological responses to intravenous lipid emulsion. Fetal sheep were given intravenous Intralipid 20 (R) (n = 4 females, 7 males) or Lactated Ringer's Solution (n = 7 females, 4 males) between 125 +/- 1 and 133 +/- 1 d of gestation (term = 147 d). Manufacturer's recommendation for premature human infants was followed: 0.5-1 g/kg/d initial rate, increased by 0.5-1 to 3 g/kg/d. Hemodynamic parameters and arterial blood chemistry were measured, and organs were studied postmortem. Red blood cell lipidomics were analyzed by LC-MS. Intravenous Intralipid did not alter hemodynamic or most blood parameters. Compared with controls, Intralipid infusion increased final day plasma protein (P=0.004; 3.5 +/- 0.3 vs. 3.9 +/- 0.2 g/dL), albumin (P = 0.031; 2.2 +/- 0.1 vs. 2.4 +/- 0.2 g/dL), and bilirubin (P<0.001; conjugated: 0.2 +/- 0.1 vs. 0.6 +/- 0.2 mg/dL; unconjugated: 0.2 +/- 0.1 vs. 1.1 +/- 0.4 mg/dL). Circulating IGF-1 decreased following Intralipid infusion (P<0.001; 66 +/- 24 vs. 46 +/- 24 ng/mL). Compared with control Oil Red O liver stains (median score 0), Intralipid-infused fetuses scored 108 (P=0.0009). Lipidomic analysis revealed uptake and processing of infused lipids into red blood cells, increasing abundance of saturated fatty acids. The near-term fetal sheep tolerates intravenous lipid emulsion well, although lipid accumulates in the liver. Increased levels of unconjugated bilirubin may reflect increased red blood cell turnover or impaired placental clearance. Whether Intralipid is less well tolerated earlier in gestation remains to be determined.

INTRODUCTION: One-third of cardiomyocyte are lost just before birth in the normally developing heart. The underlying reasons for this cell loss are unknown. We studied the role of the endoplasmic reticulum (ER) stress response in regulation of cardiomyocyte apoptosis in perinatal sheep hearts. We hypothesized that the signaling balance from survival to apoptosis changes just before the cardiomyocyte numbers decline, and that the ER stress response contributes. METHODS: Snap-frozen left ventricular (LV) samples and cultured cardiomyocytes from normally-growing lambs at 135 d of gestational age (dGA; term=147 dGA), 143 dGA and 1 d postnatal age (dPN) were used for this study (n=6 each age).Gene and protein regulation was studied by RT-PCR and western blot analysis. Parameters were compared by age by 1-way ANOVA followed, if justified, by Šídák's multiple comparisons test. RESULTS: In the LV, the ER stress master regulatory protein GRP78 was similar within fetal ages, but declined 32% between 143 dGA and 1 dPN (P= 0.0005), indicating less capacity for responding to misfolded proteins. eIF2α, downstream of GRP78, is critical for deciding the survival/apoptosis outcome following ER stress. Phosphorylation of eIF2α doubled between 135 dGA and 143 dGA (P=0.0015), implying increased ER stress in this period, and declined again at 1 dPN (p<0.0001 vs. 143 dGA). Cleaved apoptosis effector caspase 3 protein peaked after birth; levels at 1 dPN were 1- to 2-fold higher than at other ages (p<0.0001 vs. 135 dGA, P=0.002 vs. 143 dGA). Together, these data suggest that survival signaling in the ER stress pathway may be blunted between 135 and 143 dGA, as evidenced by diminished GRP78 expression and elevated eIF2α phosphorylation, resulting in activation of the apoptosis pathway. To better understand the mechanisms regulating dynamic perinatal changes in the ER stress response pathway, we studied sensitivity to ER stress (thapsigargin: 5 μM, 12 h) in cultured LV cardiomyocytes. In response to thapsigargin, GRP78 protein levels rose ~3-fold higher in cells from 135 dGA compared to 143 dGA or 1 dPN hearts (p<0.0001), supporting diminishing survival signaling closer to the time of birth. Phosphorylation of eIF2α followed a similar pattern and was highest in 135 dGA cells (p<0.0001 vs. 143 dGA and 1 dPN). This contrasts to the greater levels of phospho-eIF2α at 143 dGA in the tissue analysis. Thapsigargin stimulated 2-fold more cleavage of caspase 3 at 1 dPN than 135 dGA (P=0.0243), a similar timing as found in LV tissue. CONCLUSION: Findings suggest that at two weeks before birth, heart and cardiomyocytes are resilient to ER stress and stimulation of apoptosis, whereas nearer the time of birth there is a blunted ER stress response and increased activation of apoptotic pathways. R01HL142483 (Jonker), Collins Foundation (Bose). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

AbstractMicrovascular perfusion of striated muscle is an important determinant of health throughout life. Birth is a transition with profound effects on the growth and function of striated muscle, but the regulation of microvascular perfusion around this transition is poorly understood. We used contrast‐enhanced ultrasound perfusion imaging (CEUS) to study the perfusion of left ventricular myocardium and hindlimb biceps femoris, which are populations of muscle with different degrees of change in pre‐ to postnatal workloads and different capacities for postnatal proliferative growth. We studied separate groups of lambs in late gestation (135 days’ gestational age; 92% of term) and shortly after birth (5 days’ postnatal age). We used CEUS to quantify baseline perfusion, perfusion during hyperaemia induced by adenosine infusion (myocardium) or electrically stimulated unloaded exercise (skeletal muscle), flow reserve and oxygen delivery. We found heart‐to‐body weight ratio was greater in neonates than fetuses. Microvascular volume and overall perfusion were lower in neonates than fetuses in both muscle groups at baseline and with hyperaemia. Flux rate differed with muscle group, with myocardial flux being faster in neonates than fetuses, but skeletal muscle flux being slower. Oxygen delivery to skeletal muscle at baseline was lower in neonates than fetuses, but was not significantly different in myocardium. Flow reserve was not different between ages. Given the significant somatic growth, and the transition from hyperplastic to hypertrophic myocyte growth occurring in the perinatal period, we postulate that the primary driver of lower neonatal striated muscle perfusion is faster growth of myofibres than their associated capillary networks.