Obesity remains a critical global public health concern, with its prevalence continuing to rise at an alarming rate^[1-6]. In the United States, over 30% of population is classified as obese, with data from the National Health and Nutrition Examination Survey (NHANES) highlighting an alarming obesity rate of 35.5% among females over 20 years of age^[7]. This trend is mirrored globally, where the increasing incidence of obesity among women of reproductive age poses significant risks to both maternal and fetal health^[4,8,9]. Gestational overnutrition often precipitates severe complications, including gestational diabetes mellitus, preeclampsia, and fetal macrosomia^[8,10]. Beyond immediate perinatal outcomes, the “Developmental Origins of Health and Disease” (DOHaD) hypothesis posits that the intrauterine environment serves as a critical window for physiological programming. Consequently, offspring born to obese mothers face a 2-fold greater risk of developing insulin resistance, childhood obesity, and type 2 diabetes later in life^[11].

Obesity, particularly when driven by high-fat, high-energy dietary intake, is inextricably linked to chronic low-grade inflammation. This systemic state is characterized by the elevation of proinflammatory cytokines across metabolic hubs, including the liver, adipose tissue, and skeletal muscle^[1,4,12,13]. These inflammatory responses are pivotal in the pathogenesis of insulin resistance and cardiovascular disease in both mothers and their offspring^[8,14]. A key mediator of this process is the elevation of circulating free fatty acids (FFAs), which function as endogenous ligands for proinflammatory toll-like receptors (TLRs), specifically TLR4^[15]. Upon activation by saturated fatty acids, TLR4 recruits downstream adaptor proteins, such as MyD88, to trigger the nuclear factor κB (NFκB) signaling cascade. Activation of this pathway drives the transcription of diverse proinflammatory mediators that can traverse the placental barrier, potentially programming the fetal immune system toward a state of chronic inflammatory dysfunction^[16,17].

Emerging evidence suggests that maternal overnutrition significantly impairs fetal cardiovascular development^[18-22], though the precise molecular drivers remain poorly defined. A novel and potentially critical mechanism involves the induction of regulated cell death pathways, most notably ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation. Ferroptosis has been increasingly implicated in metabolic and cardiovascular pathologies and may act in concert with inflammatory signaling to exacerbate maladaptive fetal cardiac remodeling^[23,24]. In obese pregnancies, excessive lipid accumulation within fetal myocardial tissues not only amplifies inflammatory flux but also sensitizes cardiomyocytes to ferroptotic injury by overwhelming the glutathione-dependent antioxidant defense system^[25-27]. This dual burden of inflammation and ferroptosis likely drives myocardial remodeling and impairs cardiomyocyte contractility, establishing a pathological substrate for long-term cardiovascular risk.

To elucidate these mechanisms, we utilized a well-established gestational overfeeding model in multiparous ewes^[28,29]. Sheep represent an excellent translational model for studying developmental programming, as their gestational milestones and frequent singleton or twin pregnancies closely parallel to human physiology. This model enabled the precise characterization of inflammatory signaling and regulated cell death pathways in fetal left ventricular tissues during late gestation^[30]. In this study, we evaluated cardiac geometry, histomorphology, lipid deposition, and immune cell infiltration (neutrophils and monocytes), alongside proinflammatory cytokine profiles and cardiomyocyte mechanical properties in fetal hearts. Furthermore, to determine the specific role of ferroptotic stress in mediating these morphometric and functional alterations, we analyzed key markers for ferroptosis resistance, including glutathione peroxidase 4 (GPX4) and SLC7A11, in fetal cardiac tissues.

This study was designed to elucidate the mechanistic pathways underlying fetal cardiac alterations in the setting of maternal overnutrition, utilizing a well-established pregnant sheep model. Previous investigations have identified various adverse sequelae of maternal obesity affecting the fetal heart^[33], placenta^[34], skeletal muscle^[35,36], and gut^[37], as well as broader metabolic programming^[35,38,39]. Our findings extend these observations by offering novel insights into how maternal overnutrition specifically drives inflammatory signaling and ferroptotic stress within the fetal myocardium. These results indicate that maternal overnutrition, both prior to and during gestation, triggers systemic inflammation, immune cell infiltration, and ferroptosis, which collectively compromise fetal heart homeostasis. These data highlight a critical pathophysiological link between maternal obesity, inflammation, and ferroptosis during fetal cardiac development.

While our study did not include longitudinal maternal metabolic profiling, such as blood glucose or insulin levels, several key physiological indicators confirm the successful establishment of the maternal overnutrition model. Specifically, ewes receiving the 150% NRC diet exhibited a significant increase in maternal liver weight, a hallmark of metabolic redirection and hepatic fat accumulation in overfed ruminants. Correspondingly, fetuses from overnourished ewes displayed a significant increase in subcutaneous fat and evident myocardial lipid deposition. These systemic and tissue-specific changes, combined with the significant upregulation of fetal myocardial TLR4, a receptor known to be activated by saturated fatty acids during nutritional stress, provide compelling evidence that the overnutrition regimen induced a robust state of maternal and fetal metabolic excess, thereby establishing a pathological substrate for developmental programming.

Our findings of localized inflammation in the fetal heart are further corroborated by evidence of systemic fetal programming at the progenitor cell level. Indeed, transcriptomic analysis from a nonhuman primate model suggests that maternal dietary stress fundamentally reprograms fetal hematopoietic stem cells - the precursors to infiltrating immune cells - by suppressing essential pathways, including ribosomal biogenesis [Figure 1B and C]. This cross-species evidence suggests that the pro-inflammatory environment within myocardium may be amplified by a pre-existing, systemic priming of the fetal immune system, originating at the earliest stages of hematopoietic development.

Building on our previous report of impaired myocardial contractility in the late-gestation offspring of obese ewes^[29], our current study identifies a comprehensive profile of maladaptive remodeling. While biometric data revealed restricted fetal growth and cardiac atrophy (although with unchanged heart size), many organ weights and crown-rump lengths remained unchanged, reflecting complex compensatory transplacental adjustments to maternal metabolic stress. At the cellular level, we observed a “latent” structural remodeling phase, where histological and molecular damage precedes overt changes in total cardiac mass. Specifically, fetal hearts exhibited cardiomyocyte atrophy, interstitial fibrosis, myofibrillar disarray, and myocardial steatosis, alongside robust neutrophil and monocyte infiltration. These structural abnormalities were mechanistically linked to compromised contractile capacity, intracellular Ca²⁺ derangement, and the potent activation of the TLR4/NFκB inflammatory axis - evidenced by the significant upregulation of iNOS, IL1α (IL1A), IL1β (IL1B), and IL6. It is noteworthy that the fibrotic remodeling in obese offspring may represent a proportional substitution of collagen for atrophying contractile units, a compensatory mechanism to maintain structural integrity amidst intrauterine growth restriction (IUGR). This pathological adaptation, characterized by reduced fetal organ weights despite stable body length, underscores a critical window of subclinical remodeling that may dictate long-term cardiovascular vulnerability.

Crucially, the discrepancy between stable heart-to-body weight ratio and profound internal changes observed here suggests that morphological indices, such as cardiomyocyte cross-sectional area, are far more sensitive markers of programming-induced injury than gross biometry. This lag identifies a critical window of subclinical pathological transition driven by oxidative stress and impaired nutrient exchange - often rooted in diminished placental proliferation and angiogenic factor expression during mid-gestation^[40]. In this context, maternal overnutrition disrupts transplacental nutrient exchange by impairing both placental growth and vascular development, exerting a profound negative impact on fetal growth. Ultimately, the morphological integrity of the sheep myocardium is compromised far earlier than gross heart size suggests, highlighting the subtle yet profound impact of maternal nutritional status on fetal cardiac development.

Prolonged maternal overnutrition and metabolic excess induce inflammatory responses across multiple tissues, including adipose tissue, skeletal muscle, and the gastrointestinal tract, in both mothers and their offspring^[41,42]. Evidence from human and animal studies has revealed pronounced elevations in proinflammatory cytokines and mediators - such as C-reactive protein, TLR4, IL-6, CD14 and CD68 - across various organs including gut, liver, adipose tissues, skeletal muscles, and placenta^[15,35]. Notably, our present study revealed elevated mRNA and protein expression of CD14 and CD68 in overnourished fetuses, corresponding with our IHC results. TLR4, a key pattern-recognition receptor involved in initiation of innate immune responses, was also significantly upregulated. TLR4 is uniquely positioned at the intersection of metabolism and immunity, as it is activated not only by bacterial lipopolysaccharides but also by saturated fatty acids, which are likely elevated under conditions of nutritional stress^[43,44]. Upon ligand binding, TLR4, in conjunction with its co-receptor CD14 (cluster of differentiation 14), recruits the adaptor protein MyD88 to activate the NFκB signaling cascade^[16]. This cascade prompts the transcriptional activation of genes implicated in inflammation, immunity, cell proliferation, and regulated cell death. Proinflammatory cytokines, including IL1α, IL1β, IL6, and IL18, further amplify this response by acting as activators of the NFκB pathway, which is initiated by the phosphorylation of the NFκB inhibitory protein IκB. This mechanism is strongly supported by our experimental findings of increased NFκB activity (demonstrated by elevated IκB phosphorylation) despite downregulated pan NFκB protein level in overnourished fetus [Figure 7M-N].

A novel and significant contribution of our study is the identification of ferroptosis, a recently characterized regulated cell death modality, as a critical mediator of cardiac maladaptation following maternal overnutrition^[27]. While ferroptosis has not been classically considered in the framework of the DOHaD fetal programming hypothesis, emerging evidence has implicated this iron-dependent pathway in myocardial injury, particularly within metabolically stressed or lipid-rich environments^[24]. Excessive lipid accumulation within fetal cardiomyocytes, as observed in our overnourished group, creates a biochemical substrate highly susceptible to ferroptotic damage, particularly when antioxidant defenses (e.g., GPX4) are overwhelmed. Furthermore, the chronic inflammatory milieu and sustained TLR4 activation observed in these fetuses may enhance iron uptake and lipid peroxidation, further facilitating the ferroptotic cascade. These findings suggest that maternal obesity does not merely drive structural and inflammatory remodeling, but actively modulates programmed cell death pathways in the fetal heart. This molecular switch toward ferroptosis likely contributes to the impaired contractile function observed in late gestation and provides a novel therapeutic target for mitigating long-term myocardial dysfunction in offspring.

Several experimental limitations warrant consideration. First, while the ovine model offers significant developmental parallels to human pregnancy (particularly when compared to rodents), species differences in placental architecture and gestational timeline must be acknowledged when translating these findings into clinical contexts. Second, the integration of transcriptomic data from a nonhuman primate (rhesus macaque) model with the physiological findings from fetal sheep may introduce a layer of interspecies variability. We leveraged the macaque dataset (GSE219095) specifically to delineate how maternal dietary stress reprograms fetal hematopoietic stem and progenitor cells, precursors to the immune cells infiltrating sheep myocardium. This cross-species approach facilitated the identification of pathways such as "Ribosomal Biogenesis" by utilizing the superior gene annotation of the macaque genome. However, potential divergence in inflammatory responses and developmental kinetics between species may limit the generalizability of this integrated analysis. Third, this observational approach lacks direct animal rescue or cellular experiments to confirm specific causal mechanisms. While the correlation between maternal diet and fetal proinflammatory activation is robust, mechanistic causality among inflammation, ferroptosis, and long-term cardiac function in offspring remains to be established. Fourth, despite evidence of immune activation, we observed suppressed IL-18 expression during late gestation. Given IL-18’s role in macrophage recruitment and systemic inflammation, its reduction may reflect a stage-dependent shift in immune dynamics, exhaustion of compensatory signaling, or feedback suppression by ferroptosis-related oxidative stress^[45,46]. Regarding our electrophysiological methodology, cells were loaded with Fura-2 AM and excited at 360/380 nm. Although the 340/380 nm ratio is the conventional standard, 360 nm was utilized as it sits near the Fura-2 isosbestic point. In this configuration, the 360 nm signal remains relatively insensitive to [Ca²⁺] fluctuations, effectively normalizing for dye loading and cell thickness, while the 380 nm signal captures calcium binding. While this approach may provide a different dynamic range than the 340/380 nm ratio, results remained consistent across all experimental groups analyzed under identical optical conditions. Finally, we acknowledge that our cardiomyocyte functional analysis utilized a cell-based sample size derived from a smaller biological cohort of three fetuses per group. While this captures cellular heterogeneity and contractile distribution, treating individual cells as independent observations may introduce pseudoreplication. We recognize the fetus as the primary biological unit; therefore, statistical power may be overestimated in cell-based analysis compared to animal-level analyses. To mitigate this, results were cross-referenced with tissue-level molecular data. Future studies should employ nested or mixed-effect models to more rigorously account for animal-to-animal variability.

In conclusion, our findings demonstrate that maternal overnutrition imposes multifaceted stress on the developing fetal heart by triggering inflammatory signaling, promoting oxidative damage, and disrupting cardiac architecture. These alterations culminate in a heightened myocardial vulnerability to ferroptosis. These pathophysiological alterations likely carry enduring consequences for cardiovascular health from the postnatal period into adulthood. Given that maternal obesity remains a critical global health concern, our data suggest that excessive gestational nutrient intake amplifies redox-immune crosstalk within the fetal myocardium, potentially driving pathological remodeling and intrinsic contractile dysfunction. These insights underscore the necessity of further research into the molecular mechanisms of developmental programming and highlight ferroptosis-targeted interventions as a promising therapeutic strategy to mitigate the cardiovascular risks associated with maternal obesity. Ultimately, extending these observations into neonatal and adult offspring stages is essential to confirm the persistence of these defects and solidify the clinical relevance of these findings within the framework of the developmental origins of adult heart disease.