Single Antenatal Exposure to Ciclesonide Reduces Long-Term Cardiac Structural and Functional Alterations Compared to Currently Approved Synthetic Corticosteroids

Single Antenatal Exposure to Ciclesonide Reduces Long-Term Cardiac Structural and Functional Alterations Compared to Currently Approved Synthetic Corticosteroids | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Single Antenatal Exposure to Ciclesonide Reduces Long-Term Cardiac Structural and Functional Alterations Compared to Currently Approved Synthetic Corticosteroids Shekhar Gugnani, Tooba Fida, Rachel Tao, Keya Panchal, Julian Vallejo, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9283462/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Synthetic corticosteroids (sCS), Dexamethasone (Dex) or Betamethasone (Beta) are administered to mothers at risk of preterm birth to promote fetal organ maturation and reduce neonatal morbidity. Repeated prenatal or postnatal sCS exposure is associated with long term negative cardiovascular and neurologic outcomes. We previously demonstrated that repeated postnatal exposure to Ciclesonide (Cic) promotes lung maturation and minimizes birthweight or white matter reductions observed with repeated Dex administration. The long-term cardiac effects of a single antenatal sCS exposure are poorly understood. This study demonstrates that a single prenatal exposure to sCS reduces birth weight in a graded manner, with Dex>Beta > Cic. In aged animals Dex exposure led to an increase in body weight, Beta showed a trend towards a decrease, while Cic was indistinguishable from controls. Structural, histological, and electrophysiological cardiac abnormalities consistent with bradycardia and QTc prolongation were exclusively observed in aged Dex-exposed animals. Adult cardiac ion channel expression was decreased with Dex>Beta > Cic, indicating that the antenatal environment plays a pivotal role in short-term and long-term cardiac reprogramming. A single antenatal exposure to Cic minimizes adverse cardiometabolic effects compared to Dex or Beta suggesting that Cic may be a safer alternative for preterm birth compared to the current sCS clinical regimen. Health sciences/Cardiology Health sciences/Medical research Biological sciences/Physiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Preterm birth and its associated postnatal complications are one of the leading causes of death in children under the age of five, affecting more than 1 in 8 babies in the United States and almost one million babies per year globally 1 – 3 . Preterm birth poses significant short-term and long-term risks to both maternal and neonatal well-being 4 – 6 . In 2019, the rate of preterm birth in the United States rose to 10.23%, marking it the fifth consecutive year with an annual increased rate and the highest level of preterm birth in more than a decade 7 – 9 . The current clinically approved regimen for mothers at risk of preterm birth in the United States is the administration of synthetic corticosteroids (sCS); among these, the most widely used are Dexamethasone (Dex) and Betamethasone (Beta) 10 – 12 . These drugs accelerate fetal lung maturation, reducing the risk of intraventricular hemorrhage; necrotizing enterocolitis; and bronchopulmonary dysplasia, and significantly reducing neonatal death and morbidity as well as the need for intensive postnatal care 13 – 16 . While the short-term benefits of antenatal sCS in improving neonatal outcomes are well-established, their usage has been associated with adverse long-term systemic effects. Antenatal corticosteroid exposure has been linked to intrauterine growth restriction (characterized by low birth weight and multi-organ system dysfunction), with negative effects often persisting beyond birth 17 , 18 . Considerable evidence indicates that repeated antenatal exposure to sCS can cause neurological abnormalities, including increased risk for cerebral palsy, abnormal neurologic examinations, higher Major Depression Inventory scores, and cortical white matter deficits 17 , 19 . One area of concern is the impact of corticosteroids on the cardiovascular system. Prolonged antenatal, perinatal and adult exposure to sCS is associated with the cardinal features of metabolic disease, such as insulin resistance, hypertension, dyslipidemia, and obesity; these factors increase the risk for cardiovascular disease and ultimately stroke, arrhythmia, or heart failure 20 – 22 . Evidence in human and animal models indicates that repeated antenatal exposure to sCS leads to abnormal myocardial function, hypertension, and lasting cardiac dysfunction that is present not only at the time of birth but also throughout life 17 , 23 – 26 . Adult pregnant rats repeatedly exposed with up to 0.5 mg/kg Dex antenatally exhibit myocardial cell hypertrophy and a significant decrease in ventricular weight 17 . Antenatal sCS-induced cardiovascular dysfunction is associated with the generation of oxidative stress at birth 26 . Corticosteroids are potent inducers of reactive oxygen species (ROS), which can trigger hypertension and endothelial dysfunction (effects that can be prevented by antioxidant treatment). Therefore, mechanisms mediating off-target adverse effects of sCS may relate to the induction of excess ROS production and associated intracellular signaling, including the activation of stress kinases, cell cycle changes, and the induction of cell senescence 21 , 26 . The American College of Obstetricians and Gynecologists recommends a single course of sCS for pregnant women between 23 and 36 gestation weeks who are at risk for preterm birth within 7 days 27 . Given the potential systemic and cardiac side effects of traditional sCS administration, there is an urgent need to identify the long term consequences of a single clinically relevant exposure to sCS and to identify alternative treatments that can provide similar developmental and pathophysiological benefits while minimizing adverse long-term systemic effects. Ciclesonide (Cic, ALVESCO) is a prodrug that is converted into the metabolically active form desisobutyryl-ciclesonide (Des) by endogenous carboxylesterase enzymes predominantly expressed in the liver, intestine, respiratory tract, and placenta 28 – 30 . Cic is currently clinically approved as an inhaled preparation for the treatment of asthma in children over the age of 12 and as a nasal spray for allergic rhinitis in children greater than 6 years of age and adults 30 , 31 . Cic may be a safer alternative to Dex or Beta due to its high plasma protein binding affinity (> 99%) and high pulmonary lipid affinity; the drug has rapid clearance of its active form, with limited systemic circulation of unbound fractions 32 – 34 . Previous studies demonstrate that Cic administration in post-natal pups activates pulmonary corticosteroid responses without leading to the adverse effects on body growth, brain weight, or white matter loss observed with Dex exposure 34 , 35 . This suggests that Cic could be a safer sCS therapy for prematurity, potentially limiting long-term cardiovascular effects 34 . Although extensive research has focused on the immediate benefits and risks of repeated antenatal sCS administration, few studies address the potential long-term effects of these drugs on offspring. Published long-term studies use multiple or high dose sCS administration. Additionally, there is limited research on the underlying mechanisms and long-term consequences of a single-dose antenatal clinically relevant sCS exposure. This study compared the molecular, pathophysiological and functional consequences of a single antenatal Dex, Beta or Cic exposure on both the immediate and long-term metabolic or cardiac function. Furthermore, this study seeks to evaluate whether Cic could serve as a safer alternative for the management of preterm birth limiting cardiovascular dysfunction. Materials & Methodology Animal Protocols All animal experiments were performed according to approved Institutional Animal Care and Use Committee protocols at the University of Missouri – Kansas City (UMKC), conforming to relevant federal guidelines. The UMKC animal facility is operated as a specific pathogen free, AAALAC accredited, PHS assured facility. Animal care and husbandry meets the requirements in the Guide for the Care and Use of Laboratory Animals (8th edition), National Research Council. Animals are group housed and maintained on a 12 hour light/dark cycle with ad libitum food and water at a constant temperature of 70-72 o F and humidity of 30–70%. Daily health check inspections are performed by qualified veterinary staff and/or animal care technicians. Timed pregnant mice were purchased from Charles River, arrived at embryonic day E12, and were housed for two days in the animal facility before exposure to drugs. Males were singly housed while females were housed in groups of four. The Institutional Animal Care and Use Committee (IACUC) at the University of Missouri-Kansas City (Protocol #45543) approved all experimental animal procedures which were performed in accordance with institutional, federal, and ARRIVE guidelines. Animals were euthanized according to the 2020 American Veterinary Medical Association guidelines for CO2 asphyxiation and cervical dislocation. Males and females were randomly assigned to each treatment or control group. Sample size was determined in previous studies using a power analysis indicating that a size of 4–7 animals per group would be sufficient to achieve the expected effect size at an α of 0.05 17,19,34 . Drug dose and tissue collection: Timed pregnant CD1 mice were antenatally injected with a single intraperitoneal administration of vehicle (Veh, 0.001% Ethanol in phosphate buffered saline pH 7.2 (PBS), Dex, Beta, or Cic (0.4mg/kg, Millipore Sigma) at Embryonic Day 14.5 (E14.5). This approximates the minimal dose used clinically in humans (0.35 mg/ kg) and as previously reported 19 , 36 . Body weight was monitored in the first week after birth (Postnatal Day 1–7, P1-P7) until 92 weeks of age. Weight curves were generated and statistically compared in GraphPad and curve intersection was calaculated using Desmos online graphing calculator. For metabolic analyses, serum glucose and cholesterol levels were measured at the end of life using a OneTouch AimStrip Tandem Lipid profile and Glucose Measuring System (Ermaine Laboratories). Histological analyses: Hearts were collected at birth or adulthood and weighed. Adult hearts were immersed in cardioplegic solution (20mM KCl) and manually pumped for 30 seconds and fixed in Carnoy’s fixative (60% Ethanol, 30% Chloroform, and 10% Glacial Acetic Acid). P1 hearts were immediately fixed and not placed into cardioplegic solution due to their minute size. Hearts were embedded in paraffin and sectioned at 15 micrometers. Every 4th section was stained with Hematoxylin & Eosin (H&E) for structural analysis (N = 4–6 per treatment) and every 5th section stained with Masson’s Trichrome (N = 4–7 per treatment) for quantitative collagen analysis. The results were examined histologically using both a light microscope and an Invitrogen EVOS™ FL Auto 2 Imaging System by an observer blind to the treatment. For H&E-stained sections, four high-resolution images per slide at 20x magnification were taken of ventricle muscle and stitched together via Auto 2 Imaging Software. For Trichrome-stained sections, three high-resolution transverse and sagittal images per slide were taken at 20x magnification of ventricle muscle with peripheral collagen. Coronal and horizontal cross-sections were imaged for P1 and adult hearts, respectively; left and right ventricular free wall diameter, left and right ventricular chamber size, and septal thickness were digitally measured using QuPath Open-Source Software 37 . Cardiac collagen was quantified using the National Institute of Health ImageJ Open-Source Software with the Color Deconvolution 2 Plugin 38 . Electrocardiogram Analyses: From 72–76 weeks of age, electrocardiogram (ECG) analysis was performed using ECGenie (MouseSpecifics) in conscious animals (N = 6–8 per treatment). Animals were placed on an elevated platform and allowed to acclimatize for 10 minutes before the collection of baseline data. Recordings were gathered over a 15-to-60-minute period until 12–15 distinct consecutive episodes of five or more identifiable QRS peaks of electrophysiological activity were recorded. Data was analyzed using e-MOUSE™ digital software. The cardiac heart rate (HR), RR, PR, QRS, ST, and corrected QT (QTc) intervals were recorded and averaged across all ECGs per mouse per treatment. Heart rate was measured in beats per millisecond (bpms). RNA Isolation and Quantitative Polymerase Chain Reaction: Following ECG analysis, adult hearts were excised, and horizontal sections isolated from the middle widest cross-sectional area of the heart and used for RNA isolation using Trizol Extraction Reagent per the manufacturer’s instructions (Invitrogen by Thermo Fisher Scientific). RNA was converted to cDNA using ThermoFisher high-capacity RNA-to-cDNA kits, and Quantitative Polymerase Chain Reaction (qPCR) was performed using SYBR™ Green Master Mix with primers for key cardiac potassium and calcium ion channel genes (N = 3–5 per treatment) (Supplementary Table S1 ). Statistical Analyses : GraphPad Prism 10 statistical software was used to calculate significance and generate graphs (GraphPad Software Inc., La Jolla, CA). Data are presented as mean ± standard error of the mean (SEM) where indicated. Differences between control and experimental groups (N = 4–12 per experimental group) were compared using the following tests: One-Way ANOVA per time period with Dunnett’s post-hoc tests (body weight, ECG analyses, and PCR analyses), Log-Rank Mantel-Cox test (survival), and unpaired T-Tests (heart dimensions and collagen quantification). Mean and standard error of the mean were calculated for body weight and collagen quantification. To identify outliers, a Grubbs Outlier Test with an α of 0.05 was used. Additionally, a Welch’s One-Way ANOVA was used when Bartlett’s test of homogeneity indicated unequal variances (heart rate in ECG analyses). Results A single antenatal exposure to Dex, Beta, or Cic leads to distinct birth weight and growth trajectory : It is well-established in both rodent and human studies that sCS administration for the management of prematurity results in a significant decrease in birth weight 19 , 39 – 42 . From birth to week one, in utero exposure to Dex, Beta, or Cic led to a graded reduction in birth weight compared to controls (Dex 34% decrease: p = 0.0003; Beta 31% decrease: p = 0.0008; Cic 26% decrease: p = 0.0116) (Supplemental Table S2). By postnatal day 7 (week 1), body weights remained significantly lower in Dex- (25%, p = 0.0008) and Beta-treated groups (19%, p = 0.0044), Cic-exposed animals were indistibguishable from controls (p = 0.1126). By week 5, weights were similar to controls in all treatment groups. Between weeks 10 and 17, significant elevation in body weights became apparent in Dex and Cic treatment groups relative to controls (Dex: p = 0.0189; Cic: p = 0.0332). At the end of life (92 weeks), Dex animals maintained significantly higher body weights compared to Veh (Dex 72g, Veh 54.57; 32% increase; p = 0.0023). Beta-treated animals showed a trend towards a decrease (44.2g; 19% decrease; p = 0.0509), while Cic-treated animals showed no significant difference (49.8g; p = 0.5291). The increase in weight in Dex exposed animals was due to an observed increase in abdominal fat deposition in Dex versus controls. These findings demonstrate a biphasic growth response: early postnatal growth suppression followed by progressive weight gain in adulthood, which is particularly pronounced in the Dex group (Fig. 1 ). Blood glucose and cholesterol levels are normal in experimental versus control groups: Previous studies in humans have shown that antenatal sCS administration is associated with an increased risk for metabolic disease in adulthood, characterized by obesity, insulin resistance, hypertension, and cardiovascular disease 18 , 41 , 43 – 45 . Considering the increased weight in aged Dex-treated animals, we measured blood glucose and cholesterol levels in adults. Blood glucose concentrations were not significantly different between experimental and control free-eating groups: Veh (9.6 mmol/L), Dex (9.0 mmol/L), Beta (9.0 mmol/L), and Cic (11.0 mmol/L) (N = 5–7 per treatment; Dex: p = 0.82; Beta p = 0.38, Cic p = 0.17). Additionally, cholesterol levels remained within the normal physiological range for all groups, indicating that single-dose antenatal sCS exposure did not result in notable metabolic disturbances under the conditions tested. Antenatal exposure to sCS does not alter long-term survival: To determine if antenatal exposure to sCS altered longevity, survival rates of the animals were monitored over a 92 week period. From birth to 92 weeks no significant difference in survival was observed (p = 0.64 for all groups). At birth, the number of animals in each group was: Veh (N = 16), Dex (N = 13), Beta (N = 13), and Cic (N = 12); by 92 weeks, the number of surviving animals in each group was: Veh (N = 7), Dex (N = 4), Beta (N = 5), and Cic (N = 5) (Fig. 2 ). When separated by sex, no significant differences in survival were observed between treatment groups. These findings suggest that antenatal exposure to sCS does not significantly impact long-term survival, despite the observed physiological and cardiac alterations described below. Antenatal Dex exposure leads to cardiac pathology from birth to adulthood: To evaluate potential short-term cardiac effects of antenatal sCS exposure, hearts were examined at postnatal day 1 (P1). In light of the observed significant differences in birth weight and growth trajectory in Dex-exposed compared to Cic-exposed, these studies focused on Dex versus Cic comparisons. Previous studies have indicated that while repeated antenatal sCS exposure stimulates cardiomyocyte proliferation and energy production, adverse side effects are also observed leading to cardiac alterations at birth 41 , 46 – 48 . Few studies have investigated the consequences of a single physiologically relevant sCS on the neonatal heart. To examine the effects of sCS exposure on the heart’s anatomical structure at P1, we measured wall thicknesses and ventricular diameters of both the left and right ventricles. At birth, the left ventricle chamber size in Dex-exposed animals was decreased by 64% (p = 0.042), while the septal thickness increased by 41% compared to Veh (N = 4; p = 0.031). These structural changes were not observed in Cic-exposed animals (N = 4; left ventricle chamber: p = 0.662; septum: p = 0.231, Fig. 3 ). Previous studies highlighted cardiac alterations after repeated antenatal exposure to CS’s 41,45 . To determine whether a single exposure to antenatal CS’s led to cardiac abnormalities in adults, hearts were examined at 92 weeks. Heart wall and chamber sizes were measured, and structural analysis revealed a significant difference in heart size and chamber dimension in Dex-exposed animals versus controls. In adult animals, a marked increase in the proportional ratio of the right ventricular chamber to the diameter of the heart in the Dex-exposed groups was observed. Specifically, Dex-treated animals exhibited a 1.5-fold proportional increase (50% enlargement) in right ventricular chamber compared to controls (N = 4, p = 0.043). Beta- or Cic-exposed did not show statistical differences in right ventricular chamber size (N = 6 per treatment, p = 0.543 and p = 0.733 respectively, Fig. 4 a-e) or any other measurement. These findings suggest that antenatal exposure to Dex, may lead to substantial alterations in right heart chamber size, reflecting the long-term effects of these drugs on cardiovascular development. Alteration in muscle or extracellular matrix content can lead to changes in ventricular size. Collagen is one of the major structural proteins in the heart and is required to maintain the structural integrity of tissue, with its abnormal distribution associated with the onset of heart disease 49 . Previous studies have shown that Dex administration decreases collagen type IV synthesis in lung in postnatal animals 50 . To determine whether additional structural differences were present in sCS-exposed hearts, we examined cardiac collagen content. Trichrome staining revealed a significant reduction in cardiac collagen content in Dex-exposed animals, with a 56% decrease in collagen deposition compared to Veh-exposed (N = 5, Signal intensity/unit area = Vehicle 3.46 ± 0.62; Dex, 1.53 ± 0.36; p = 0.034). Statistical differences in collagen content were not observed in Beta-exposed hearts (N = 6, Signal intensity/unit area = 2.47 ± 0.36) or Cic-exposed hearts (N = 7, Signal intensity/unit area = 2.67 ± 0.45) (p = 0.348 and p = 0.205, respectively, Fig. 5 ). This reduction suggests that a single antenatal exposure to Dex leads to long-term changes in cardiac extracellular matrix, potentially compromising structural integrity and contributing to long-term cardiovascular dysfunction. Antenatal exposure to sCSs leads to distinct electrophysiological alterations and cardiac channel gene alterations in adults : Considering the structural changes observed in the Dex hearts compared to Beta, Cic, or Veh, we assessed the functional impact of antenatal exposure to sCSs on heart function at 72–76 weeks of age. Electrocardiogram (ECG) recordings were conducted, and significant changes in electrophysiological parameters were observed (Fig. 6 ). Dex-exposed adult animals exhibited a decreased HR (Veh 754 bpms versus Dex 681 bpms, p = 0.006) with a prolonged RR (p = 0.0016), PR (P = 0.0273), QRS (p = 0.0001), ST (p = 0.002), and QTc (p = 0.0009) intervals compared to Veh, indicating altered cardiac conduction (N = 5–8 per treatment). Beta and Cic-exposed adults did not show any significant changes in electrophysiological parameters compared to Veh, suggesting minimal impact on conduction (Fig. 6 ). To gain an insight into potential molecular underpinnings of the electrophysical alterations induced by Dex, we examined the expression of cardiac ion channel genes that play central roles in regulating rhythmicity and contractility 51 , 52 . Cardiac depolarization/repolarizaiton and contraction/relaxation is regulated by conductance of sodium, potassium and calcium ion channels, we therefore performed quantitative polymerase chain reaction (qPCR) on tissue isolated from adult chamber walls to analyze the expression of select genes encoding cardiac ion channels. Dex-exposed animals exhibited a marked decrease in the expression of several channel genes examined. KCNN2 is a subtype of small-conductance calcium-activated potassium channels that plays a crucial role in the repolarization of cardiac cells. Its expression was reduced by approximately 99% with Dex, (N = 6, p < 0.0001), 58% with Beta (N = 3, p = 0.001), and 38% with Cic (N = 4, p = 0.016). KCNJ2, an inward rectifier potassium channel that helps to establish the resting membrane potential during repolarization, expression was reduced by 64% with Beta (N = 6, p = 0.001) and 39% with Cic (N = 5, p = 0.042); no significant difference with Dex (N = 4, p = 0.375) was observed. CACNB2, a subunit of L-type voltage-dependent calcium channels that plays a role in voltage sensitivity for activation and peak calcium entry for depolarization and contractility, decreased by 84% with Dex (N = 4, p < 0.0001) and by 37% with Beta (N = 3, p = 0.031); expression was unaltered with Cic (N = 3, p = 0.990). CACNA1H, a Cav3.2 T-type calcium channel found in pacemaker cells and contributes to pacemaker activity as well as ventricular cells and contributes to excitation contraction coupling, was reduced by 42% with Dex (N = 3, p = 0.0063), 71% with Beta (N = 4, p < 0.0001), and 50% with Cic (N = 3, p = 0.002) (Fig. 7 ). These results indicate that a single antenatal exposure to Dex, Beta or Cic leads to graded CS-specific alterations in the expression of genes implicated in regulating cardiac electrophysiological and functional parameters in adults. Discussion sCS are extensively used to prevent adverse pulmonary, gastrointestinal and neurovascular complications associated with prematurity, however several studies have identified negative secondary consequences, with the pace of these studies accelerating dramatically over the last decade. Our study contributes to this growing body of research by focusing on aspects that remain largely unexplored. While most prior studies have concentrated on the antenatal administration of high-dose or repeated dose sCS exposure, effects of lower clinically relevant single antenatal doses have not been thoroughly investigated. This gap in research is particularly important given that clinical and translational studies have largely focused on the immediate effects of these drugs. However, the long-term consequences, especially in terms of cardiovascular health, also remain underexplored. While short-term studies have provided valuable insights, there is a pressing need for further research to uncover the full spectrum of long-term consequences associated with antenatal sCS exposure, particularly in areas like heart development, heart function, and metabolic health. This study provides new insights into the long-term effects of antenatal sCS exposure on growth, cardiovascular health, and cardiac development in an animal model. Our findings align with and extend previous research, demonstrating that antenatal sCS exposure significantly impacts birth weight, growth trajectories, and cardiovascular function and electrophysiology. Notably, we observed both recovery and persistent alterations in various physiological parameters, which highlight the complexities of sCS exposure during pregnancy. It is well-established that repeated antenatal administration of sCS leads to a significant reduction in birth weight of the offspring in both humans and rodent models 19 , 40 – 42 . Our data similarly confirms that a single antenatal exposure to sCSs significantly reduces birth weight. Specifically, Dex, Beta, and Cic exhibited lower birth weights compared to the Veh group. Interestingly, while all sCS-exposed groups showed reduced birth weights, Cic exposure led to a less pronounced weight reduction compared to Dex and Beta exposed, suggesting that Cic may be less impactful on fetal growth. Importantly, we have previously shown that repeated Cic administration in postnatal pups does not lead to the growth reduction observed with Dex exposure 34 . This observation may reflect differences in the route of exposure, potency, or pharmacological actions of these drugs in utero ; this could have important implications for clinical treatment protocols, particularly in premature births. Though the birth weight differences were significant, we observed full recovery in growth by week 5 with no differences between groups. This phenomenon is supported by research based on the ACTORDS randomized trial, which found that despite the initial reduction in weights at the time of birth, babies exposed to repeated sCS showed a postnatal growth acceleration 3–5 weeks after birth 42 . However, as animals aged, sCS-exposed pups exhibited significant increases in weight compared to controls. By week 92, Dex-exposed animals were significantly heavier than controls that was largely due to an increase in observed abdominal adipose content, Cic eposed were indistinguishable from controls, while Beta-exposed subjects showed a trend toward lower weight. These findings are interesting because of the significant role that both endogenous and exogenous corticosteroids play in lipid metabolism, promoting both lipogenic and lipolytic activities depending on context 53 – 57 . Studies in humans and animal models have also shown that repeated exposure to sCS in adults leads to fat deposition 58 , and antenatal exposure to sCSs or to factors that elevate maternal cortisol levels (such as maternal stress) are associated with an increased risk for metabolic disease in offspring later in life 59 . Both prolonged endogenous cortisol exposure or sCS exposure has been shown to lead to both acute and long-term epigenetic changes, such as histone modification and DNA methylation that can persist through generations 59 – 61 . The early effects of sCS exposure on growth may be mediated by epigenetic changes in-utero in genes that regulate lipid metabolism and may contribute to long-term alterations in body composition 44 . The lack of significant weight differences in the Cic exposed adults relative to controls indicates that Cic may differentially alter lipid metabolic targets, leading to minimal impact on long-term growth compared to Dex and Beta. No differences in postprandial blood glucose, cholesterol levels, or survival rates were observed in any of the adult experimental groups compared to controls, despite the known associations between sCS exposure and metabolic disturbances like insulin resistance and hyperglycemia. This supports previous research, suggesting that lower dose sCS exposure has fewer metabolic effects than repeated or high-dose exposure 62 . However, our study's assessment of metabolic parameters was limited to one timepoint in adulthood, and additional studies are needed to detect more subtle metabolic changes. Corticosteroid signaling during fetal development is critical for structural and functional maturation of cardiomyocytes that depend on the timing and dose of administration 13 . While previous studies have shown that multiple and continuous antenatal doses of Dex in animals from E12.5 to E15.5 resulted in transient cardiac growth restriction 41 , our studies demonstrate that even a single antenatal dosage of Dex leads to alterations in cardiac morphology at birth. At P1, Dex-treated animals showed an increase in septal thickness associated with a decrease in left ventricle chamber diameter, indicative of hypertrophic cardiomyopathy with potential diastolic dysfunction at birth and in early periods of life. These findings are consistent with previous findings demonstrating that repeated antenatal Dex treatment transiently decreases the myocardial deceleration index, a marker of diastolic function at birth 63 . Interestingly, studies in both both animals and children exposed to sCS in utero exhibit transient hypertension at birth, possibly associated with the transient decreased ventricular chamber size observed in our studies in animals 64 – 71 . One notable limitation of this study is that echocardiograms were not performed in these mice, which could have provided an accurate assessment of ventricular function. By 92 weeks of age, marked differences in heart structure between sCS exposures was observed that were distinct from findings at birth. Dex-exposed animals exhibited a 50% increase in size of the right ventricular chamber (RVC) and a 54% decrease in extracellular matrix collagen content in cardiac muscle compared to control, Beta-, or Cic-exposed. The decrease in collagen was not associated with a significant decrease in the LVC size, which could be due to anatomical differences as the left ventricular wall is larger than the right in healthy mice and humans 72 , 73 . The increase in the RVC in Dex-exposed animals suggests long-term alterations in cardiac structure that may be indicative of susceptibility to dilated cardiomyopathy. We cannot rule out that changes in the RVC were not due to primary alterations in pulmonary vasculature, which were not examined in this study. The observed alterations are consistent with findings from studies that link antenatal stress and corticosteroid exposure to alterations in cardiovascular development 58 . These findings are supported in human studies, which demonstrate that exposure to corticosteroids is linked to decreased fibroblast activity and decreased tissue collagen deposition in skin 74 . The identification of several corticosteroid receptor binding sites in genes implicated in regulating collagen further support these hypotheses 75 – 77 . The alteration of the extracellular matrix may contribute to the observed right ventricle chamber structural changes which could have functional implications for the heart’s susceptibility to chamber dilation, cardiomyopathy, and heart failure in adulthood. The lack of pronounced changes in heart structure in the Beta and Cic groups indicates limited long-term reprogramming of cardiac pathology. Electrophysiological assessments revealed cardiac conduction deficits in Dex-exposed animals, including prolonged RR, QRS, ST, and QTc intervals, findings that are consistent with cardiac conductance changes. Specifically, prolonged RR interval indicates a lower resting heart rate as observed, possibly due to decreased excitability in the sinoatrial node pacemaker cells. Longer QRS complexes indicate prolonged atrial repolarization and ventricular depolarization, and prolonged corrected QT interval indicates longer time to repolarize the ventricles. Cic and Beta antenatal exposure did not lead to long-term differences in the ECG waveforms. Preliminary insight into the molecular underpinning of the ECG changes induced by Dex demonstrates changes in gene expression for specific cardiac ion channels. Dex, Beta, and Cic exposure led to decreases in expression of key potassium and calcium ion channel genes. These include KCNN2 that encodes a small-conductance calcium-activated potassium channel, essential for late-phase repolarization and hyperpolarization in cardiomyocytes and CACNA1H that encodes the α1H subunit of the T-type calcium channel required for pacemaker cell depolarization and ventricular physiology. KCNN2 is decreased in a graded manner for KCNN2 with Dex − 97% >Beta − 58%>Cic − 38%, and for CACNA1H with Dex − 42% >Beta − 71%>Cic − 50%, CACNB2’s expression is reduced by both Dex and Beta but not by Cic. The almost complete absence of KCNN2 (-96%) and CACNB2 (-84%) with the reduced CACNA1H (-42%) observed in adults exposed to Dex in utero compared to Beta and Cic may explain the dramatic changes in electrical conductance seen in the ECGs. Furthermore, these findings suggest that individuals exposed to Dex in utero may be at increased risk for arrhythmia’s, particularly bradyarrhythmia’s and Torsade’s de Pointes 78 – 82 . Beta and Cic uniquely decreased KCNJ1, a mitochondrial ATP-sensitive potassium channel, in a graded manner with Beta − 64% > Cic − 39%. While functional ECG alterations were not observed with Beta or Cic exposure, a more detailed analysis may reveal other susceptibilities, for example in cardiac injury or fluid homeostasis 83 . While these studies have focused on the primary cardiac effects of prenatal sCS, the systemic effects of these drugs have not been examined. An important target of sCSs is the kidney, alterations in cardiac conduction may be due to renal electrolyte dysfunction. Interestingly, KCNJ1 inhibitors are currently being examined as therapeutic targets in heart failure to manage fluid retention 82 . Concluding, our study provides insights into the immediate and long-term effects of a single clinically relevant antenatal sCS exposure. While we observed significant early developmental changes, including reduced birth weight and altered cardiac structure, these effects were not consistently linked to metabolic disturbances or survival outcomes in adulthood. Both Dex and Beta are used to reduce risks associated with perterm birth; of importance, they promote lung maturation and reduce the risk of respiripatory distress syndrome 12 . Cic has been shown to promte postnatal lung maturation in term born animals, both in an animal model of bronchopulmonary dysplasia and in an in-utero enterotoxin lung injury model 34 , 84 . This indicates that Cic comparatively activates similar pulmonary pathways. Despite the changes noted in cardiac gene expression with exposure to Cic, we noticed that these changes were much less profound compared to the electrophysiological gene profile implicated by Dex or Beta administration, suggesting that Cic may be a superior alternative for antenatal use in minimizing long-term health risks of premature children. While our studies focus on exposure to sCS in-utero, it is important to note that infants born preterm are also at significant risk for secondary pulmonary complications such as bronchopulmonary dysplasia. As noted in the DART trial, one treatment option for this pathology is the administration of tapering doses of Dex 85 , which has the potential to lead to additional cardiac pathological or conductance abnormalities. Interestingly, sinus bradycardia has been shown to be a common early side effect associated with prednisone treatment in children 52 . These findings highlight the importance of the antenatal environment in cardiovascular programming and suggest that individuals with known antenatal sCS exposure may benefit from long-term cardiac monitoring and risk stratification. The deficits observed can be consequence of a primary effect from sCS on distinct molecular targets, such as KCNN2 86 ; or, they could be a secondary consequence of epigenetic modification induced in the microenvironment 60 , 61 . The identified molecular targets (KCNN2, CACNB2, CACNA1H) may represent potential therapeutic pathways for intervention in affected individuals. Notably, recent studies have linked the impact of premature birth itself to long-term cardiovascular alterations, possibly due to structural limitations imposed at birth 87 . Inherently, preterm birth may cause changes to both cardiac and vasculature structure and function, and it has been shown to specifically increase the long-term risk of cardiovascular disease, cardiometabolic disease, diabetes, hypertension, atrial fibrillation, and heart failure 88 – 92 . Ultimately, these studies highlight the need for continued research into the long-term consequences of antenatal sCS exposure, particularly regarding cardiovascular and electrophysiological health. Overall the results support the proposal that Cic may be a safer option for future use in managing preterm births than the current clinical regimen of Dex or Beta administration. Declarations Funding sources: This work was funded by the National Institutes of Health grants R01 HD087288 to APMN and DBD, by UMKC’s start up funds to APMN, and by the Sarah Morrison Research funds and UMKC’s School of Medicine Student Research Fund to SG and RT. This project was supported by funds to in part funds to MJW, JAV and PMN by funds from the Health Resources and Services Administration of the U.S. Department of Health and Human Services Administration (HRSA) of the U.S. Department of Health and Human Services (HHS) grant T99HP52109 for $ 16,000,000 with 10% financed with non-governmental sources. The contents are those of the authors and do not necessarily represent the official views of, nor an endorsement, by HRSA, HHS, or the US Government. Author Contribution Shekhar Gugnani: Performed Collagen assessment, a subset of electrophysiology, quantitative gene expression studies, cardiac anatomy assessment, co-wrote the manuscript and figures. Tooba Fida: Performed quantitative gene expression studies, cardiac anatomy assessment, co-wrote the manuscript and figures. Rachel Tao: Performed cardiac anatomy assessment in P1 animals. Keya Panchal: Performed cardiac anatomy assessment in P1 animals, body weight, glucose and cholesterol measurements. Julian Vallejo: Trained authors on electrophysiology studies and analyses. Manuscript review and revision. Donald Defranco: Provided expert guidance on synthetic corticosteroid biological and physiological function. Manuscript review and revision Michael Wacker: Provided expert guidance on cardiac structural analyses and physiological function. Manuscript review and revision. Ann Paula Monagan-Nichols: Project conceptualization and oversight, directed studies and evaluated results, project principle investigator, provided funding, manuscript draft and final edit. Acknowledgement Tianhua Lei is thanked for technical assistance: Performed animal studies, drug administration and animal monitoring. Collected and processed tissue for analyses. Akshay Kannan assisted with Desmos online graphing calculator tool for growth curve treatment intersection. Data Availability The data collected during experimental work performed for the purposes of this study are available from the corresponding author upon request. The authors declare no competing interests. References Walani, S. R. Global burden of preterm birth. Int. J. Gynaecol. Obstet. 150 , 31–33 (2020). Frey, H. A. & Klebanoff, M. A. The epidemiology, etiology, and costs of preterm birth. Semin Fetal Neonatal Med. 21 , 68–73 (2016). Fee, E. L., Stock, S. J. & Kemp, M. W. Antenatal steroids: benefits, risks, and new insights. J. Endocrinol. 258 , e220306 (2023). Chen, X. et al. Iatrogenic vs. Spontaneous Preterm Birth: A Retrospective Study of Neonatal Outcome Among Very Preterm Infants. Front. Neurol. 12 , 649749 (2021). Crump, C. An overview of adult health outcomes after preterm birth. Early Hum. Dev. 150 , 105187 (2020). Martin, J. A., Hamilton, B. E., Osterman, M. J. K. & Driscoll, A. K. Births: Final Data for 2019. Natl. Vital Stat. Rep. 70 , 1–51 (2021). Nelson, D. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9283462","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":640296149,"identity":"fdd02113-05aa-4c38-8a65-d07db404d8f2","order_by":0,"name":"Shekhar Gugnani","email":"","orcid":"","institution":"University of Missouri–Kansas City","correspondingAuthor":false,"prefix":"","firstName":"Shekhar","middleName":"","lastName":"Gugnani","suffix":""},{"id":640296150,"identity":"8bc7af38-5217-46f5-bf68-9faba18c9836","order_by":1,"name":"Tooba Fida","email":"","orcid":"","institution":"University of Missouri–Kansas City","correspondingAuthor":false,"prefix":"","firstName":"Tooba","middleName":"","lastName":"Fida","suffix":""},{"id":640296151,"identity":"5ae54573-f903-4aed-b278-d25206fcc56b","order_by":2,"name":"Rachel Tao","email":"","orcid":"","institution":"University of Missouri–Kansas City","correspondingAuthor":false,"prefix":"","firstName":"Rachel","middleName":"","lastName":"Tao","suffix":""},{"id":640296152,"identity":"18987880-bbac-4458-b167-009c4b78c04d","order_by":3,"name":"Keya Panchal","email":"","orcid":"","institution":"University of Missouri–Kansas City","correspondingAuthor":false,"prefix":"","firstName":"Keya","middleName":"","lastName":"Panchal","suffix":""},{"id":640296153,"identity":"5a81edd6-20c7-4241-a2db-b10acc9a512e","order_by":4,"name":"Julian Vallejo","email":"","orcid":"","institution":"University of Missouri–Kansas City","correspondingAuthor":false,"prefix":"","firstName":"Julian","middleName":"","lastName":"Vallejo","suffix":""},{"id":640296154,"identity":"6256c3aa-d46f-424e-b21a-9dea013f9d11","order_by":5,"name":"Donald DeFranco","email":"","orcid":"","institution":"University of Pittsburgh School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Donald","middleName":"","lastName":"DeFranco","suffix":""},{"id":640296155,"identity":"6c419493-1bef-435a-875b-2d8149ff337f","order_by":6,"name":"Michael Wacker","email":"","orcid":"","institution":"University of Missouri–Kansas City","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Wacker","suffix":""},{"id":640296156,"identity":"c70e18f0-e848-42ea-b8ae-86792d40c59a","order_by":7,"name":"A.Paula Monaghan Nichols","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYDACZgY2BoYCIIO9gYGxASRygCgtBkAGzwFitTDAtEgkEKnFnJ352YMPBnXy5pJvTDfOqGGQ47uRgF+LZTObueEMg8OGO2fnmN3ccIzBWJKQFoPDPGzSPAYHGDfcBmp52MCQuIFILXX2G26eAWupJ1YLM9BwHrObGxsYEgwIa2EzkwT6JXlnT1rZzRnHJAxnnnlAQMv5w88kPlTU2W5nP7ztZk+NjTzfcQK2IPRCKAkilSNpGQWjYBSMglGACQC+TUYRP8OMjAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Missouri–Kansas City","correspondingAuthor":true,"prefix":"","firstName":"A.Paula","middleName":"Monaghan","lastName":"Nichols","suffix":""}],"badges":[],"createdAt":"2026-03-31 18:24:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9283462/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9283462/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109434886,"identity":"4c7bc099-6b5f-424e-8047-1b5fde70bf70","added_by":"auto","created_at":"2026-05-18 05:55:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3429441,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth Curves in Mice Prenatally Exposed to Distinct sCS.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGrowth Curves from birth to end of life (~ 92 weeks) of sCS-exposed CD1 mice. Prenatal exposure to a single dose of any sCS (Dex, Beta, or Cic) led to a significant decrease in birth weights compared to control (Dex: p = 0.0003; Beta: p = 0.0008; Cic: p = 0.0116), but by week 10 this difference was no longer statistically significant. After 35 weeks, Dex-, Beta-, and Cic-treated mice showed an increase in weight compared to controls. By week 92, Dex animals were significantly obese (72g) compared to controls (54.57g, p = 0.0023). Beta trended toward a decrease in body weight (44.2g, “~” p-value of 0.0509), while Cic demonstrated no significant difference (49.8g, p = 0.5291). Significance values: * = p \u0026lt; 0.05, ** = p \u0026lt; 0.01, *** = p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1GrowthCurves.png","url":"https://assets-eu.researchsquare.com/files/rs-9283462/v1/93ab1be148fa6acadfcb6cc1.png"},{"id":109434974,"identity":"a2f94f92-a92a-41df-8699-01f4c5f8a2e2","added_by":"auto","created_at":"2026-05-18 05:55:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":349250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival Curve Comparisons in Mice Prenatally Exposed to Distinct sCS.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSurvival Curves from birth to end of life (92 weeks) in sCS-exposed CD1 mice. Mantel-Cox Log-Rank test revealed no significant observed difference in survival between treated groups. At birth, N=Veh 16, Dex 13, Beta 13, Cic 12. At end of life, N=Veh 7, Dex 4, Beta 5, Cic 5.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9283462/v1/62c14bc3f39b70880f3d3e68.png"},{"id":109434937,"identity":"f0280706-f529-411e-805e-3113bc656ed8","added_by":"auto","created_at":"2026-05-18 05:55:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3202509,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological and Structural Cardiac Alterations of P1 Mice Prenatally Exposed to Distinct sCS.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea,b,c: Hematoxylin \u0026amp; Eosin-stained 4x coronal images of P1 hearts exposed to (a) Veh, (b), Dex, or (c) Cic at birth.\u003c/p\u003e\n\u003cp\u003ed: Violin plots of Right Ventricular Free Wall (RVFW), Right Ventricular Chamber (RVC), Septum, Left Ventricular Chamber (LVC), and Left Ventricular Free Wall (LVFW) measurement comparisons on postnatal day 1. Left ventricular chamber size decreased by 64% and septum size increased by 41% in Dex-exposed mice compared to controls at birth (N=4; p = 0.042, p = 0.031 respectively).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9283462/v1/c5b09506630d134e4582b075.png"},{"id":109434877,"identity":"01ede0c7-0e27-4e7b-9225-ade99bf53418","added_by":"auto","created_at":"2026-05-18 05:55:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3857801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological and Structural Cardiac Alterations of Adult Mice Prenatally Exposed to Distinct sCS.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViolin plots of Right Ventricular Free Wall (RVFW), Right Ventricular Chamber (RVC), Septum, Left Ventricular Chamber (LVC), and Left Ventricular Free Wall (LVFW) measurement comparisons in adult mice. Dex-treated mice had an approximate 50% increase in the size of the right ventricle compared to control (N=4; p = 0.043).\u003c/p\u003e","description":"","filename":"FIGURE4AD1.png","url":"https://assets-eu.researchsquare.com/files/rs-9283462/v1/c14ed7e6e90b23a2d72f0c1d.png"},{"id":109434892,"identity":"80b913b7-cd08-4248-be8a-75de1b55cfd7","added_by":"auto","created_at":"2026-05-18 05:55:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6686189,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCardiac Collagen Quantification and Analysis in Adult Mice Prenatally Exposed to Distinct sCS.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea-h: Longitudinal (a-d) and transverse (e-h) 20x images of hearts stained with Masson-Trichrome to detect collagen in adult mice prenatally exposed to Veh (a,e), Dex (b,f), Beta (c,g), and Cic (d,h). Blue-stained collagen fibers were less abundant in Dex-exposed mice. Muscle fibers were stained red.\u003c/p\u003e\n\u003cp\u003e5I: Quantification of collagen content in sectioned adult hearts. A 56% decrease in connective tissue surface area was observed in Dex-exposed mice compared to control. (N=5, p = 0.034). Scale Bar= 65 μM\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9283462/v1/37d85f8946522d294e2a7f55.png"},{"id":109434891,"identity":"07ed3321-aa7f-4775-a140-8496c912a15b","added_by":"auto","created_at":"2026-05-18 05:55:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2704748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectrocardiogram Analyses in Adult Mice Prenatally Exposed to Distinct sCS.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViolin plots comparing electrocardiogram (ECG) recordings of adult mice prenatally exposed to sCS compared to control. Dex-exposed adult animals exhibited prolonged heart rate (HR; p = 0.006), RR (p = 0.0016), PR (p = 0.0273), QRS (p = 0.0001), ST (p = 0.002), and QTc (p = 0.0009) intervals compared to Veh (N=5-8 per treatment; 681 bpms vs. 754 bpms respectively). Beta- and Cic-exposed mice did not demonstrate significant changes in ECG intervals compared to control.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-9283462/v1/a3ef1481b07286625d04a703.png"},{"id":109434879,"identity":"906826a1-cf2f-44fa-b66f-5fd40c420ea2","added_by":"auto","created_at":"2026-05-18 05:55:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2158573,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantitative Analysis of Selective Cardiac Ion Channel Gene Expression via qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative Polymerase Chain Reaction (qPCR) expression levels of several key genes encoding cardiac ion channels. Compared to control, Dex-exposed mice had significantly decreased expression of KCNN2 (N=6, p \u0026lt; 0.0001), CACNB2 (N=4, p \u0026lt; 0.0001), and CACNA1H (N=3, p \u0026lt; 0.0063). Beta-exposed mice had significantly decreased KCNN2 (N=3, p = 0.001), KCNJ2 (N=6, p = 0.001), CACNB2 (N=2, p = 0.031), and CACNA1H (N=4, p \u0026lt; 0.0001). Cic-exposed mice had significantly decreased KCNN2 (N=4, p = 0.016), KCNJ2 (N=5, p = 0.042), and CACNA1H (N=3, p = 0.002).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-9283462/v1/b2e67bed3dedda795f08960b.png"},{"id":109435118,"identity":"e64ff506-6899-495c-94c2-1bf7bdfcc57a","added_by":"auto","created_at":"2026-05-18 05:56:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":22093298,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9283462/v1/b587aab6-3791-4eaa-9557-d0a678efa71b.pdf"},{"id":109434867,"identity":"cdb4d52d-d4a4-4b40-8f21-338d19806e97","added_by":"auto","created_at":"2026-05-18 05:55:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18374,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables121.docx","url":"https://assets-eu.researchsquare.com/files/rs-9283462/v1/153de64c0dcb60a1c98a7574.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Single Antenatal Exposure to Ciclesonide Reduces Long-Term Cardiac Structural and Functional Alterations Compared to Currently Approved Synthetic Corticosteroids","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePreterm birth and its associated postnatal complications are one of the leading causes of death in children under the age of five, affecting more than 1 in 8 babies in the United States and almost one million babies per year globally\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Preterm birth poses significant short-term and long-term risks to both maternal and neonatal well-being\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In 2019, the rate of preterm birth in the United States rose to 10.23%, marking it the fifth consecutive year with an annual increased rate and the highest level of preterm birth in more than a decade\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The current clinically approved regimen for mothers at risk of preterm birth in the United States is the administration of synthetic corticosteroids (sCS); among these, the most widely used are Dexamethasone (Dex) and Betamethasone (Beta)\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These drugs accelerate fetal lung maturation, reducing the risk of intraventricular hemorrhage; necrotizing enterocolitis; and bronchopulmonary dysplasia, and significantly reducing neonatal death and morbidity as well as the need for intensive postnatal care\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. While the short-term benefits of antenatal sCS in improving neonatal outcomes are well-established, their usage has been associated with adverse long-term systemic effects.\u003c/p\u003e \u003cp\u003eAntenatal corticosteroid exposure has been linked to intrauterine growth restriction (characterized by low birth weight and multi-organ system dysfunction), with negative effects often persisting beyond birth\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Considerable evidence indicates that repeated antenatal exposure to sCS can cause neurological abnormalities, including increased risk for cerebral palsy, abnormal neurologic examinations, higher Major Depression Inventory scores, and cortical white matter deficits\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. One area of concern is the impact of corticosteroids on the cardiovascular system. Prolonged antenatal, perinatal and adult exposure to sCS is associated with the cardinal features of metabolic disease, such as insulin resistance, hypertension, dyslipidemia, and obesity; these factors increase the risk for cardiovascular disease and ultimately stroke, arrhythmia, or heart failure\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEvidence in human and animal models indicates that repeated antenatal exposure to sCS leads to abnormal myocardial function, hypertension, and lasting cardiac dysfunction that is present not only at the time of birth but also throughout life\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Adult pregnant rats repeatedly exposed with up to 0.5 mg/kg Dex antenatally exhibit myocardial cell hypertrophy and a significant decrease in ventricular weight\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Antenatal sCS-induced cardiovascular dysfunction is associated with the generation of oxidative stress at birth\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Corticosteroids are potent inducers of reactive oxygen species (ROS), which can trigger hypertension and endothelial dysfunction (effects that can be prevented by antioxidant treatment). Therefore, mechanisms mediating off-target adverse effects of sCS may relate to the induction of excess ROS production and associated intracellular signaling, including the activation of stress kinases, cell cycle changes, and the induction of cell senescence\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe American College of Obstetricians and Gynecologists recommends a single course of sCS for pregnant women between 23 and 36 gestation weeks who are at risk for preterm birth within 7 days\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Given the potential systemic and cardiac side effects of traditional sCS administration, there is an urgent need to identify the long term consequences of a single clinically relevant exposure to sCS and to identify alternative treatments that can provide similar developmental and pathophysiological benefits while minimizing adverse long-term systemic effects. Ciclesonide (Cic, ALVESCO) is a prodrug that is converted into the metabolically active form desisobutyryl-ciclesonide (Des) by endogenous carboxylesterase enzymes predominantly expressed in the liver, intestine, respiratory tract, and placenta\u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Cic is currently clinically approved as an inhaled preparation for the treatment of asthma in children over the age of 12 and as a nasal spray for allergic rhinitis in children greater than 6 years of age and adults\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Cic may be a safer alternative to Dex or Beta due to its high plasma protein binding affinity (\u0026gt;\u0026thinsp;99%) and high pulmonary lipid affinity; the drug has rapid clearance of its active form, with limited systemic circulation of unbound fractions\u003csup\u003e\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Previous studies demonstrate that Cic administration in post-natal pups activates pulmonary corticosteroid responses without leading to the adverse effects on body growth, brain weight, or white matter loss observed with Dex exposure\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This suggests that Cic could be a safer sCS therapy for prematurity, potentially limiting long-term cardiovascular effects\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough extensive research has focused on the immediate benefits and risks of repeated antenatal sCS administration, few studies address the potential long-term effects of these drugs on offspring. Published long-term studies use multiple or high dose sCS administration. Additionally, there is limited research on the underlying mechanisms and long-term consequences of a single-dose antenatal clinically relevant sCS exposure. This study compared the molecular, pathophysiological and functional consequences of a single antenatal Dex, Beta or Cic exposure on both the immediate and long-term metabolic or cardiac function. Furthermore, this study seeks to evaluate whether Cic could serve as a safer alternative for the management of preterm birth limiting cardiovascular dysfunction.\u003c/p\u003e"},{"header":"Materials \u0026 Methodology","content":"\u003cp\u003e \u003cstrong\u003eAnimal Protocols\u003c/strong\u003e \u003cp\u003e All animal experiments were performed according to approved Institutional Animal Care and Use Committee protocols at the University of Missouri \u0026ndash; Kansas City (UMKC), conforming to relevant federal guidelines. The UMKC animal facility is operated as a specific pathogen free, AAALAC accredited, PHS assured facility. Animal care and husbandry meets the requirements in the Guide for the Care and Use of Laboratory Animals (8th edition), National Research Council. Animals are group housed and maintained on a 12 hour light/dark cycle with ad libitum food and water at a constant temperature of 70-72\u003csup\u003eo\u003c/sup\u003e F and humidity of 30\u0026ndash;70%. Daily health check inspections are performed by qualified veterinary staff and/or animal care technicians. Timed pregnant mice were purchased from Charles River, arrived at embryonic day E12, and were housed for two days in the animal facility before exposure to drugs. Males were singly housed while females were housed in groups of four. The Institutional Animal Care and Use Committee (IACUC) at the University of Missouri-Kansas City (Protocol #45543) approved all experimental animal procedures which were performed in accordance with institutional, federal, and ARRIVE guidelines. Animals were euthanized according to the 2020 American Veterinary Medical Association guidelines for CO2 asphyxiation and cervical dislocation. Males and females were randomly assigned to each treatment or control group. Sample size was determined in previous studies using a power analysis indicating that a size of 4\u0026ndash;7 animals per group would be sufficient to achieve the expected effect size at an α of 0.05\u003csup\u003e17,19,34\u003c/sup\u003e.\u003c/p\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDrug dose and tissue collection:\u003c/h2\u003e \u003cp\u003eTimed pregnant CD1 mice were antenatally injected with a single intraperitoneal administration of vehicle (Veh, 0.001% Ethanol in phosphate buffered saline pH 7.2 (PBS), Dex, Beta, or Cic (0.4mg/kg, Millipore Sigma) at Embryonic Day 14.5 (E14.5). This approximates the minimal dose used clinically in humans (0.35 mg/ kg) and as previously reported\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Body weight was monitored in the first week after birth (Postnatal Day 1\u0026ndash;7, P1-P7) until 92 weeks of age. Weight curves were generated and statistically compared in GraphPad and curve intersection was calaculated using Desmos online graphing calculator. For metabolic analyses, serum glucose and cholesterol levels were measured at the end of life using a OneTouch AimStrip Tandem Lipid profile and Glucose Measuring System (Ermaine Laboratories).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistological analyses:\u003c/h3\u003e\n\u003cp\u003eHearts were collected at birth or adulthood and weighed. Adult hearts were immersed in cardioplegic solution (20mM KCl) and manually pumped for 30 seconds and fixed in Carnoy\u0026rsquo;s fixative (60% Ethanol, 30% Chloroform, and 10% Glacial Acetic Acid). P1 hearts were immediately fixed and not placed into cardioplegic solution due to their minute size. Hearts were embedded in paraffin and sectioned at 15 micrometers. Every 4th section was stained with Hematoxylin \u0026amp; Eosin (H\u0026amp;E) for structural analysis (N\u0026thinsp;=\u0026thinsp;4\u0026ndash;6 per treatment) and every 5th section stained with Masson\u0026rsquo;s Trichrome (N\u0026thinsp;=\u0026thinsp;4\u0026ndash;7 per treatment) for quantitative collagen analysis. The results were examined histologically using both a light microscope and an Invitrogen EVOS\u0026trade; FL Auto 2 Imaging System by an observer blind to the treatment. For H\u0026amp;E-stained sections, four high-resolution images per slide at 20x magnification were taken of ventricle muscle and stitched together via Auto 2 Imaging Software. For Trichrome-stained sections, three high-resolution transverse and sagittal images per slide were taken at 20x magnification of ventricle muscle with peripheral collagen. Coronal and horizontal cross-sections were imaged for P1 and adult hearts, respectively; left and right ventricular free wall diameter, left and right ventricular chamber size, and septal thickness were digitally measured using QuPath Open-Source Software\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Cardiac collagen was quantified using the National Institute of Health ImageJ Open-Source Software with the Color Deconvolution 2 Plugin\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eElectrocardiogram Analyses:\u003c/h3\u003e\n\u003cp\u003eFrom 72\u0026ndash;76 weeks of age, electrocardiogram (ECG) analysis was performed using ECGenie (MouseSpecifics) in conscious animals (N\u0026thinsp;=\u0026thinsp;6\u0026ndash;8 per treatment). Animals were placed on an elevated platform and allowed to acclimatize for 10 minutes before the collection of baseline data. Recordings were gathered over a 15-to-60-minute period until 12\u0026ndash;15 distinct consecutive episodes of five or more identifiable QRS peaks of electrophysiological activity were recorded. Data was analyzed using e-MOUSE\u0026trade; digital software. The cardiac heart rate (HR), RR, PR, QRS, ST, and corrected QT (QTc) intervals were recorded and averaged across all ECGs per mouse per treatment. Heart rate was measured in beats per millisecond (bpms).\u003c/p\u003e\n\u003ch3\u003eRNA Isolation and Quantitative Polymerase Chain Reaction:\u003c/h3\u003e\n\u003cp\u003eFollowing ECG analysis, adult hearts were excised, and horizontal sections isolated from the middle widest cross-sectional area of the heart and used for RNA isolation using Trizol Extraction Reagent per the manufacturer\u0026rsquo;s instructions (Invitrogen by Thermo Fisher Scientific). RNA was converted to cDNA using ThermoFisher high-capacity RNA-to-cDNA kits, and Quantitative Polymerase Chain Reaction (qPCR) was performed using SYBR\u0026trade; Green Master Mix with primers for key cardiac potassium and calcium ion channel genes (N\u0026thinsp;=\u0026thinsp;3\u0026ndash;5 per treatment) (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical Analyses\u003c/b\u003e: GraphPad Prism 10 statistical software was used to calculate significance and generate graphs (GraphPad Software Inc., La Jolla, CA). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) where indicated. Differences between control and experimental groups (N\u0026thinsp;=\u0026thinsp;4\u0026ndash;12 per experimental group) were compared using the following tests: One-Way ANOVA per time period with Dunnett\u0026rsquo;s post-hoc tests (body weight, ECG analyses, and PCR analyses), Log-Rank Mantel-Cox test (survival), and unpaired T-Tests (heart dimensions and collagen quantification). Mean and standard error of the mean were calculated for body weight and collagen quantification. To identify outliers, a Grubbs Outlier Test with an α of 0.05 was used. Additionally, a Welch\u0026rsquo;s One-Way ANOVA was used when Bartlett\u0026rsquo;s test of homogeneity indicated unequal variances (heart rate in ECG analyses).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eA single antenatal exposure to Dex, Beta, or Cic leads to distinct birth weight and growth trajectory\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eIt is well-established in both rodent and human studies that sCS administration for the management of prematurity results in a significant decrease in birth weight\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. From birth to week one, in utero exposure to Dex, Beta, or Cic led to a graded reduction in birth weight compared to controls (Dex 34% decrease: p\u0026thinsp;=\u0026thinsp;0.0003; Beta 31% decrease: p\u0026thinsp;=\u0026thinsp;0.0008; Cic 26% decrease: p\u0026thinsp;=\u0026thinsp;0.0116) (Supplemental Table S2). By postnatal day 7 (week 1), body weights remained significantly lower in Dex- (25%, p\u0026thinsp;=\u0026thinsp;0.0008) and Beta-treated groups (19%, p\u0026thinsp;=\u0026thinsp;0.0044), Cic-exposed animals were indistibguishable from controls (p\u0026thinsp;=\u0026thinsp;0.1126). By week 5, weights were similar to controls in all treatment groups. Between weeks 10 and 17, significant elevation in body weights became apparent in Dex and Cic treatment groups relative to controls (Dex: p\u0026thinsp;=\u0026thinsp;0.0189; Cic: p\u0026thinsp;=\u0026thinsp;0.0332). At the end of life (92 weeks), Dex animals maintained significantly higher body weights compared to Veh (Dex 72g, Veh 54.57; 32% increase; p\u0026thinsp;=\u0026thinsp;0.0023). Beta-treated animals showed a trend towards a decrease (44.2g; 19% decrease; p\u0026thinsp;=\u0026thinsp;0.0509), while Cic-treated animals showed no significant difference (49.8g; p\u0026thinsp;=\u0026thinsp;0.5291). The increase in weight in Dex exposed animals was due to an observed increase in abdominal fat deposition in Dex versus controls. These findings demonstrate a biphasic growth response: early postnatal growth suppression followed by progressive weight gain in adulthood, which is particularly pronounced in the Dex group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBlood glucose and cholesterol levels are normal in experimental versus control groups:\u003c/h2\u003e \u003cp\u003ePrevious studies in humans have shown that antenatal sCS administration is associated with an increased risk for metabolic disease in adulthood, characterized by obesity, insulin resistance, hypertension, and cardiovascular disease\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Considering the increased weight in aged Dex-treated animals, we measured blood glucose and cholesterol levels in adults. Blood glucose concentrations were not significantly different between experimental and control free-eating groups: Veh (9.6 mmol/L), Dex (9.0 mmol/L), Beta (9.0 mmol/L), and Cic (11.0 mmol/L) (N\u0026thinsp;=\u0026thinsp;5\u0026ndash;7 per treatment; Dex: p\u0026thinsp;=\u0026thinsp;0.82; Beta p\u0026thinsp;=\u0026thinsp;0.38, Cic p\u0026thinsp;=\u0026thinsp;0.17). Additionally, cholesterol levels remained within the normal physiological range for all groups, indicating that single-dose antenatal sCS exposure did not result in notable metabolic disturbances under the conditions tested.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntenatal exposure to sCS does not alter long-term survival:\u003c/h3\u003e\n\u003cp\u003eTo determine if antenatal exposure to sCS altered longevity, survival rates of the animals were monitored over a 92 week period. From birth to 92 weeks no significant difference in survival was observed (p\u0026thinsp;=\u0026thinsp;0.64 for all groups). At birth, the number of animals in each group was: Veh (N\u0026thinsp;=\u0026thinsp;16), Dex (N\u0026thinsp;=\u0026thinsp;13), Beta (N\u0026thinsp;=\u0026thinsp;13), and Cic (N\u0026thinsp;=\u0026thinsp;12); by 92 weeks, the number of surviving animals in each group was: Veh (N\u0026thinsp;=\u0026thinsp;7), Dex (N\u0026thinsp;=\u0026thinsp;4), Beta (N\u0026thinsp;=\u0026thinsp;5), and Cic (N\u0026thinsp;=\u0026thinsp;5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). When separated by sex, no significant differences in survival were observed between treatment groups. These findings suggest that antenatal exposure to sCS does not significantly impact long-term survival, despite the observed physiological and cardiac alterations described below.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAntenatal Dex exposure leads to cardiac pathology from birth to adulthood:\u003c/h3\u003e\n\u003cp\u003eTo evaluate potential short-term cardiac effects of antenatal sCS exposure, hearts were examined at postnatal day 1 (P1). In light of the observed significant differences in birth weight and growth trajectory in Dex-exposed compared to Cic-exposed, these studies focused on Dex versus Cic comparisons. Previous studies have indicated that while repeated antenatal sCS exposure stimulates cardiomyocyte proliferation and energy production, adverse side effects are also observed leading to cardiac alterations at birth\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Few studies have investigated the consequences of a single physiologically relevant sCS on the neonatal heart. To examine the effects of sCS exposure on the heart\u0026rsquo;s anatomical structure at P1, we measured wall thicknesses and ventricular diameters of both the left and right ventricles. At birth, the left ventricle chamber size in Dex-exposed animals was decreased by 64% (p\u0026thinsp;=\u0026thinsp;0.042), while the septal thickness increased by 41% compared to Veh (N\u0026thinsp;=\u0026thinsp;4; p\u0026thinsp;=\u0026thinsp;0.031). These structural changes were not observed in Cic-exposed animals (N\u0026thinsp;=\u0026thinsp;4; left ventricle chamber: p\u0026thinsp;=\u0026thinsp;0.662; septum: p\u0026thinsp;=\u0026thinsp;0.231, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious studies highlighted cardiac alterations after repeated antenatal exposure to CS\u0026rsquo;s\u003csup\u003e41,45\u003c/sup\u003e. To determine whether a single exposure to antenatal CS\u0026rsquo;s led to cardiac abnormalities in adults, hearts were examined at 92 weeks. Heart wall and chamber sizes were measured, and structural analysis revealed a significant difference in heart size and chamber dimension in Dex-exposed animals versus controls. In adult animals, a marked increase in the proportional ratio of the right ventricular chamber to the diameter of the heart in the Dex-exposed groups was observed. Specifically, Dex-treated animals exhibited a 1.5-fold proportional increase (50% enlargement) in right ventricular chamber compared to controls (N\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;=\u0026thinsp;0.043). Beta- or Cic-exposed did not show statistical differences in right ventricular chamber size (N\u0026thinsp;=\u0026thinsp;6 per treatment, p\u0026thinsp;=\u0026thinsp;0.543 and p\u0026thinsp;=\u0026thinsp;0.733 respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-e) or any other measurement. These findings suggest that antenatal exposure to Dex, may lead to substantial alterations in right heart chamber size, reflecting the long-term effects of these drugs on cardiovascular development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlteration in muscle or extracellular matrix content can lead to changes in ventricular size. Collagen is one of the major structural proteins in the heart and is required to maintain the structural integrity of tissue, with its abnormal distribution associated with the onset of heart disease\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that Dex administration decreases collagen type IV synthesis in lung in postnatal animals\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. To determine whether additional structural differences were present in sCS-exposed hearts, we examined cardiac collagen content. Trichrome staining revealed a significant reduction in cardiac collagen content in Dex-exposed animals, with a 56% decrease in collagen deposition compared to Veh-exposed (N\u0026thinsp;=\u0026thinsp;5, Signal intensity/unit area\u0026thinsp;=\u0026thinsp;Vehicle 3.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62; Dex, 1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36; p\u0026thinsp;=\u0026thinsp;0.034). Statistical differences in collagen content were not observed in Beta-exposed hearts (N\u0026thinsp;=\u0026thinsp;6, Signal intensity/unit area\u0026thinsp;=\u0026thinsp;2.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36) or Cic-exposed hearts (N\u0026thinsp;=\u0026thinsp;7, Signal intensity/unit area\u0026thinsp;=\u0026thinsp;2.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45) (p\u0026thinsp;=\u0026thinsp;0.348 and p\u0026thinsp;=\u0026thinsp;0.205, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This reduction suggests that a single antenatal exposure to Dex leads to long-term changes in cardiac extracellular matrix, potentially compromising structural integrity and contributing to long-term cardiovascular dysfunction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAntenatal exposure to sCSs leads to distinct electrophysiological alterations and cardiac channel gene alterations in adults\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eConsidering the structural changes observed in the Dex hearts compared to Beta, Cic, or Veh, we assessed the functional impact of antenatal exposure to sCSs on heart function at 72\u0026ndash;76 weeks of age. Electrocardiogram (ECG) recordings were conducted, and significant changes in electrophysiological parameters were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Dex-exposed adult animals exhibited a decreased HR (Veh 754 bpms versus Dex 681 bpms, p\u0026thinsp;=\u0026thinsp;0.006) with a prolonged RR (p\u0026thinsp;=\u0026thinsp;0.0016), PR (P\u0026thinsp;=\u0026thinsp;0.0273), QRS (p\u0026thinsp;=\u0026thinsp;0.0001), ST (p\u0026thinsp;=\u0026thinsp;0.002), and QTc (p\u0026thinsp;=\u0026thinsp;0.0009) intervals compared to Veh, indicating altered cardiac conduction (N\u0026thinsp;=\u0026thinsp;5\u0026ndash;8 per treatment). Beta and Cic-exposed adults did not show any significant changes in electrophysiological parameters compared to Veh, suggesting minimal impact on conduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo gain an insight into potential molecular underpinnings of the electrophysical alterations induced by Dex, we examined the expression of cardiac ion channel genes that play central roles in regulating rhythmicity and contractility\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Cardiac depolarization/repolarizaiton and contraction/relaxation is regulated by conductance of sodium, potassium and calcium ion channels, we therefore performed quantitative polymerase chain reaction (qPCR) on tissue isolated from adult chamber walls to analyze the expression of select genes encoding cardiac ion channels. Dex-exposed animals exhibited a marked decrease in the expression of several channel genes examined. KCNN2 is a subtype of small-conductance calcium-activated potassium channels that plays a crucial role in the repolarization of cardiac cells. Its expression was reduced by approximately 99% with Dex, (N\u0026thinsp;=\u0026thinsp;6, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), 58% with Beta (N\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.001), and 38% with Cic (N\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;=\u0026thinsp;0.016). KCNJ2, an inward rectifier potassium channel that helps to establish the resting membrane potential during repolarization, expression was reduced by 64% with Beta (N\u0026thinsp;=\u0026thinsp;6, p\u0026thinsp;=\u0026thinsp;0.001) and 39% with Cic (N\u0026thinsp;=\u0026thinsp;5, p\u0026thinsp;=\u0026thinsp;0.042); no significant difference with Dex (N\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;=\u0026thinsp;0.375) was observed. CACNB2, a subunit of L-type voltage-dependent calcium channels that plays a role in voltage sensitivity for activation and peak calcium entry for depolarization and contractility, decreased by 84% with Dex (N\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and by 37% with Beta (N\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.031); expression was unaltered with Cic (N\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.990). CACNA1H, a Cav3.2 T-type calcium channel found in pacemaker cells and contributes to pacemaker activity as well as ventricular cells and contributes to excitation contraction coupling, was reduced by 42% with Dex (N\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.0063), 71% with Beta (N\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and 50% with Cic (N\u0026thinsp;=\u0026thinsp;3, p\u0026thinsp;=\u0026thinsp;0.002) (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results indicate that a single antenatal exposure to Dex, Beta or Cic leads to graded CS-specific alterations in the expression of genes implicated in regulating cardiac electrophysiological and functional parameters in adults.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003esCS are extensively used to prevent adverse pulmonary, gastrointestinal and neurovascular complications associated with prematurity, however several studies have identified negative secondary consequences, with the pace of these studies accelerating dramatically over the last decade. Our study contributes to this growing body of research by focusing on aspects that remain largely unexplored. While most prior studies have concentrated on the antenatal administration of high-dose or repeated dose sCS exposure, effects of lower clinically relevant single antenatal doses have not been thoroughly investigated. This gap in research is particularly important given that clinical and translational studies have largely focused on the immediate effects of these drugs. However, the long-term consequences, especially in terms of cardiovascular health, also remain underexplored. While short-term studies have provided valuable insights, there is a pressing need for further research to uncover the full spectrum of long-term consequences associated with antenatal sCS exposure, particularly in areas like heart development, heart function, and metabolic health.\u003c/p\u003e \u003cp\u003eThis study provides new insights into the long-term effects of antenatal sCS exposure on growth, cardiovascular health, and cardiac development in an animal model. Our findings align with and extend previous research, demonstrating that antenatal sCS exposure significantly impacts birth weight, growth trajectories, and cardiovascular function and electrophysiology. Notably, we observed both recovery and persistent alterations in various physiological parameters, which highlight the complexities of sCS exposure during pregnancy.\u003c/p\u003e \u003cp\u003eIt is well-established that repeated antenatal administration of sCS leads to a significant reduction in birth weight of the offspring in both humans and rodent models\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Our data similarly confirms that a single antenatal exposure to sCSs significantly reduces birth weight. Specifically, Dex, Beta, and Cic exhibited lower birth weights compared to the Veh group. Interestingly, while all sCS-exposed groups showed reduced birth weights, Cic exposure led to a less pronounced weight reduction compared to Dex and Beta exposed, suggesting that Cic may be less impactful on fetal growth. Importantly, we have previously shown that repeated Cic administration in postnatal pups does not lead to the growth reduction observed with Dex exposure\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. This observation may reflect differences in the route of exposure, potency, or pharmacological actions of these drugs \u003cem\u003ein utero\u003c/em\u003e; this could have important implications for clinical treatment protocols, particularly in premature births.\u003c/p\u003e \u003cp\u003eThough the birth weight differences were significant, we observed full recovery in growth by week 5 with no differences between groups. This phenomenon is supported by research based on the ACTORDS randomized trial, which found that despite the initial reduction in weights at the time of birth, babies exposed to repeated sCS showed a postnatal growth acceleration 3\u0026ndash;5 weeks after birth\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. However, as animals aged, sCS-exposed pups exhibited significant increases in weight compared to controls. By week 92, Dex-exposed animals were significantly heavier than controls that was largely due to an increase in observed abdominal adipose content, Cic eposed were indistinguishable from controls, while Beta-exposed subjects showed a trend toward lower weight. These findings are interesting because of the significant role that both endogenous and exogenous corticosteroids play in lipid metabolism, promoting both lipogenic and lipolytic activities depending on context\u003csup\u003e\u003cspan additionalcitationids=\"CR54 CR55 CR56\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Studies in humans and animal models have also shown that repeated exposure to sCS in adults leads to fat deposition\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, and antenatal exposure to sCSs or to factors that elevate maternal cortisol levels (such as maternal stress) are associated with an increased risk for metabolic disease in offspring later in life \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Both prolonged endogenous cortisol exposure or sCS exposure has been shown to lead to both acute and long-term epigenetic changes, such as histone modification and DNA methylation that can persist through generations\u003csup\u003e\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. The early effects of sCS exposure on growth may be mediated by epigenetic changes in-utero in genes that regulate lipid metabolism and may contribute to long-term alterations in body composition\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The lack of significant weight differences in the Cic exposed adults relative to controls indicates that Cic may differentially alter lipid metabolic targets, leading to minimal impact on long-term growth compared to Dex and Beta.\u003c/p\u003e \u003cp\u003eNo differences in postprandial blood glucose, cholesterol levels, or survival rates were observed in any of the adult experimental groups compared to controls, despite the known associations between sCS exposure and metabolic disturbances like insulin resistance and hyperglycemia. This supports previous research, suggesting that lower dose sCS exposure has fewer metabolic effects than repeated or high-dose exposure\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. However, our study's assessment of metabolic parameters was limited to one timepoint in adulthood, and additional studies are needed to detect more subtle metabolic changes.\u003c/p\u003e \u003cp\u003eCorticosteroid signaling during fetal development is critical for structural and functional maturation of cardiomyocytes that depend on the timing and dose of administration\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. While previous studies have shown that multiple and continuous antenatal doses of Dex in animals from E12.5 to E15.5 resulted in transient cardiac growth restriction\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, our studies demonstrate that even a single antenatal dosage of Dex leads to alterations in cardiac morphology at birth. At P1, Dex-treated animals showed an increase in septal thickness associated with a decrease in left ventricle chamber diameter, indicative of hypertrophic cardiomyopathy with potential diastolic dysfunction at birth and in early periods of life. These findings are consistent with previous findings demonstrating that repeated antenatal Dex treatment transiently decreases the myocardial deceleration index, a marker of diastolic function at birth\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Interestingly, studies in both both animals and children exposed to sCS \u003cem\u003ein utero\u003c/em\u003e exhibit transient hypertension at birth, possibly associated with the transient decreased ventricular chamber size observed in our studies in animals\u003csup\u003e\u003cspan additionalcitationids=\"CR65 CR66 CR67 CR68 CR69 CR70\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. One notable limitation of this study is that echocardiograms were not performed in these mice, which could have provided an accurate assessment of ventricular function.\u003c/p\u003e \u003cp\u003eBy 92 weeks of age, marked differences in heart structure between sCS exposures was observed that were distinct from findings at birth. Dex-exposed animals exhibited a 50% increase in size of the right ventricular chamber (RVC) and a 54% decrease in extracellular matrix collagen content in cardiac muscle compared to control, Beta-, or Cic-exposed. The decrease in collagen was not associated with a significant decrease in the LVC size, which could be due to anatomical differences as the left ventricular wall is larger than the right in healthy mice and humans\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. The increase in the RVC in Dex-exposed animals suggests long-term alterations in cardiac structure that may be indicative of susceptibility to dilated cardiomyopathy. We cannot rule out that changes in the RVC were not due to primary alterations in pulmonary vasculature, which were not examined in this study. The observed alterations are consistent with findings from studies that link antenatal stress and corticosteroid exposure to alterations in cardiovascular development\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. These findings are supported in human studies, which demonstrate that exposure to corticosteroids is linked to decreased fibroblast activity and decreased tissue collagen deposition in skin\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. The identification of several corticosteroid receptor binding sites in genes implicated in regulating collagen further support these hypotheses\u003csup\u003e\u003cspan additionalcitationids=\"CR76\" citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. The alteration of the extracellular matrix may contribute to the observed right ventricle chamber structural changes which could have functional implications for the heart\u0026rsquo;s susceptibility to chamber dilation, cardiomyopathy, and heart failure in adulthood. The lack of pronounced changes in heart structure in the Beta and Cic groups indicates limited long-term reprogramming of cardiac pathology.\u003c/p\u003e \u003cp\u003eElectrophysiological assessments revealed cardiac conduction deficits in Dex-exposed animals, including prolonged RR, QRS, ST, and QTc intervals, findings that are consistent with cardiac conductance changes. Specifically, prolonged RR interval indicates a lower resting heart rate as observed, possibly due to decreased excitability in the sinoatrial node pacemaker cells. Longer QRS complexes indicate prolonged atrial repolarization and ventricular depolarization, and prolonged corrected QT interval indicates longer time to repolarize the ventricles. Cic and Beta antenatal exposure did not lead to long-term differences in the ECG waveforms. Preliminary insight into the molecular underpinning of the ECG changes induced by Dex demonstrates changes in gene expression for specific cardiac ion channels. Dex, Beta, and Cic exposure led to decreases in expression of key potassium and calcium ion channel genes. These include KCNN2 that encodes a small-conductance calcium-activated potassium channel, essential for late-phase repolarization and hyperpolarization in cardiomyocytes and CACNA1H that encodes the α1H subunit of the T-type calcium channel required for pacemaker cell depolarization and ventricular physiology. KCNN2 is decreased in a graded manner for KCNN2 with Dex\u0026thinsp;\u0026minus;\u0026thinsp;97% \u0026gt;Beta\u0026thinsp;\u0026minus;\u0026thinsp;58%\u0026gt;Cic\u0026thinsp;\u0026minus;\u0026thinsp;38%, and for CACNA1H with Dex\u0026thinsp;\u0026minus;\u0026thinsp;42% \u0026gt;Beta\u0026thinsp;\u0026minus;\u0026thinsp;71%\u0026gt;Cic\u0026thinsp;\u0026minus;\u0026thinsp;50%, CACNB2\u0026rsquo;s expression is reduced by both Dex and Beta but not by Cic. The almost complete absence of KCNN2 (-96%) and CACNB2 (-84%) with the reduced CACNA1H (-42%) observed in adults exposed to Dex \u003cem\u003ein utero\u003c/em\u003e compared to Beta and Cic may explain the dramatic changes in electrical conductance seen in the ECGs. Furthermore, these findings suggest that individuals exposed to Dex in utero may be at increased risk for arrhythmia\u0026rsquo;s, particularly bradyarrhythmia\u0026rsquo;s and Torsade\u0026rsquo;s de Pointes\u003csup\u003e\u003cspan additionalcitationids=\"CR79 CR80 CR81\" citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBeta and Cic uniquely decreased KCNJ1, a mitochondrial ATP-sensitive potassium channel, in a graded manner with Beta\u0026thinsp;\u0026minus;\u0026thinsp;64% \u0026gt; Cic\u0026thinsp;\u0026minus;\u0026thinsp;39%. While functional ECG alterations were not observed with Beta or Cic exposure, a more detailed analysis may reveal other susceptibilities, for example in cardiac injury or fluid homeostasis\u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. While these studies have focused on the primary cardiac effects of prenatal sCS, the systemic effects of these drugs have not been examined. An important target of sCSs is the kidney, alterations in cardiac conduction may be due to renal electrolyte dysfunction. Interestingly, KCNJ1 inhibitors are currently being examined as therapeutic targets in heart failure to manage fluid retention\u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConcluding, our study provides insights into the immediate and long-term effects of a single clinically relevant antenatal sCS exposure. While we observed significant early developmental changes, including reduced birth weight and altered cardiac structure, these effects were not consistently linked to metabolic disturbances or survival outcomes in adulthood. Both Dex and Beta are used to reduce risks associated with perterm birth; of importance, they promote lung maturation and reduce the risk of respiripatory distress syndrome\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Cic has been shown to promte postnatal lung maturation in term born animals, both in an animal model of bronchopulmonary dysplasia and in an in-utero enterotoxin lung injury model\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. This indicates that Cic comparatively activates similar pulmonary pathways. Despite the changes noted in cardiac gene expression with exposure to Cic, we noticed that these changes were much less profound compared to the electrophysiological gene profile implicated by Dex or Beta administration, suggesting that Cic may be a superior alternative for antenatal use in minimizing long-term health risks of premature children. While our studies focus on exposure to sCS in-utero, it is important to note that infants born preterm are also at significant risk for secondary pulmonary complications such as bronchopulmonary dysplasia. As noted in the DART trial, one treatment option for this pathology is the administration of tapering doses of Dex\u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e, which has the potential to lead to additional cardiac pathological or conductance abnormalities. Interestingly, sinus bradycardia has been shown to be a common early side effect associated with prednisone treatment in children\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. These findings highlight the importance of the antenatal environment in cardiovascular programming and suggest that individuals with known antenatal sCS exposure may benefit from long-term cardiac monitoring and risk stratification. The deficits observed can be consequence of a primary effect from sCS on distinct molecular targets, such as KCNN2\u003csup\u003e86\u003c/sup\u003e; or, they could be a secondary consequence of epigenetic modification induced in the microenvironment\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. The identified molecular targets (KCNN2, CACNB2, CACNA1H) may represent potential therapeutic pathways for intervention in affected individuals. Notably, recent studies have linked the impact of premature birth itself to long-term cardiovascular alterations, possibly due to structural limitations imposed at birth\u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e. Inherently, preterm birth may cause changes to both cardiac and vasculature structure and function, and it has been shown to specifically increase the long-term risk of cardiovascular disease, cardiometabolic disease, diabetes, hypertension, atrial fibrillation, and heart failure\u003csup\u003e\u003cspan additionalcitationids=\"CR89 CR90 CR91\" citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e. Ultimately, these studies highlight the need for continued research into the long-term consequences of antenatal sCS exposure, particularly regarding cardiovascular and electrophysiological health. Overall the results support the proposal that Cic may be a safer option for future use in managing preterm births than the current clinical regimen of Dex or Beta administration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding sources:\u003c/h2\u003e \u003cp\u003eThis work was funded by the National Institutes of Health grants \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eR01 HD087288\u003c/span\u003e to APMN and DBD, by UMKC\u0026rsquo;s start up funds to APMN, and by the Sarah Morrison Research funds and UMKC\u0026rsquo;s School of Medicine Student Research Fund to SG and RT. This project was supported by funds to in part funds to MJW, JAV and PMN by funds from the Health Resources and Services Administration of the U.S. Department of Health and Human Services Administration (HRSA) of the U.S. Department of Health and Human Services (HHS) grant T99HP52109 for \u003cspan\u003e$\u003c/span\u003e16,000,000 with 10% financed with non-governmental sources. The contents are those of the authors and do not necessarily represent the official views of, nor an endorsement, by HRSA, HHS, or the US Government.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eShekhar Gugnani: Performed Collagen assessment, a subset of electrophysiology, quantitative gene expression studies, cardiac anatomy assessment, co-wrote the manuscript and figures. Tooba Fida: Performed quantitative gene expression studies, cardiac anatomy assessment, co-wrote the manuscript and figures. Rachel Tao: Performed cardiac anatomy assessment in P1 animals. Keya Panchal: Performed cardiac anatomy assessment in P1 animals, body weight, glucose and cholesterol measurements. Julian Vallejo: Trained authors on electrophysiology studies and analyses. Manuscript review and revision. Donald Defranco: Provided expert guidance on synthetic corticosteroid biological and physiological function. Manuscript review and revision Michael Wacker: Provided expert guidance on cardiac structural analyses and physiological function. Manuscript review and revision. Ann Paula Monagan-Nichols: Project conceptualization and oversight, directed studies and evaluated results, project principle investigator, provided funding, manuscript draft and final edit.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eTianhua Lei is thanked for technical assistance: Performed animal studies, drug administration and animal monitoring. Collected and processed tissue for analyses. Akshay Kannan assisted with Desmos online graphing calculator tool for growth curve treatment intersection.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data collected during experimental work performed for the purposes of this study are available from the corresponding author upon request.\u003c/p\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWalani, S. R. Global burden of preterm birth. \u003cem\u003eInt. J. Gynaecol. Obstet.\u003c/em\u003e \u003cb\u003e150\u003c/b\u003e, 31\u0026ndash;33 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrey, H. A. \u0026amp; Klebanoff, M. A. The epidemiology, etiology, and costs of preterm birth. \u003cem\u003eSemin Fetal Neonatal Med.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, 68\u0026ndash;73 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFee, E. L., Stock, S. 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Adverse Pregnancy Outcomes and Long-Term Risk of Atrial Fibrillation. \u003cem\u003eJAMA Cardiol.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 1285 (2025).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9283462/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9283462/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSynthetic corticosteroids (sCS), Dexamethasone (Dex) or Betamethasone (Beta) are administered to mothers at risk of preterm birth to promote fetal organ maturation and reduce neonatal morbidity. Repeated prenatal or postnatal sCS exposure is associated with long term negative cardiovascular and neurologic outcomes. We previously demonstrated that repeated postnatal exposure to Ciclesonide (Cic) promotes lung maturation and minimizes birthweight or white matter reductions observed with repeated Dex administration. The long-term cardiac effects of a single antenatal sCS exposure are poorly understood. This study demonstrates that a single prenatal exposure to sCS reduces birth weight in a graded manner, with Dex\u0026gt;Beta\u0026thinsp;\u0026gt;\u0026thinsp;Cic. In aged animals Dex exposure led to an increase in body weight, Beta showed a trend towards a decrease, while Cic was indistinguishable from controls. Structural, histological, and electrophysiological cardiac abnormalities consistent with bradycardia and QTc prolongation were exclusively observed in aged Dex-exposed animals. Adult cardiac ion channel expression was decreased with Dex\u0026gt;Beta\u0026thinsp;\u0026gt;\u0026thinsp;Cic, indicating that the antenatal environment plays a pivotal role in short-term and long-term cardiac reprogramming. A single antenatal exposure to Cic minimizes adverse cardiometabolic effects compared to Dex or Beta suggesting that Cic may be a safer alternative for preterm birth compared to the current sCS clinical regimen.\u003c/p\u003e","manuscriptTitle":"Single Antenatal Exposure to Ciclesonide Reduces Long-Term Cardiac Structural and Functional Alterations Compared to Currently Approved Synthetic Corticosteroids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-18 05:54:17","doi":"10.21203/rs.3.rs-9283462/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"83532365990097713352286168574364969460","date":"2026-05-21T07:19:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"243456548685792586089753758844952660759","date":"2026-05-06T16:00:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-06T14:12:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-06T13:18:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-05-05T01:32:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-23T20:14:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-23T20:09:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e93251ca-1a12-4855-939d-7f72df283342","owner":[],"postedDate":"May 18th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"83532365990097713352286168574364969460","date":"2026-05-21T07:19:22+00:00","index":62,"fulltext":""},{"type":"reviewerAgreed","content":"243456548685792586089753758844952660759","date":"2026-05-06T16:00:31+00:00","index":37,"fulltext":""},{"type":"reviewersInvited","content":"23","date":"2026-05-06T14:12:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-06T13:18:06+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":68142449,"name":"Health sciences/Cardiology"},{"id":68142450,"name":"Health sciences/Medical research"},{"id":68142451,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-05-18T05:54:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-18 05:54:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9283462","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9283462","identity":"rs-9283462","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}