Characterizing Early Cardiac Metabolic Programming via 30% Maternal Nutrient Reduction during Fetal Development in a Non-Human Primate Model

Intra-uterine growth restriction (IUGR) is a common cause of fetal/neonatal morbidity and mortality and is associated with increased offspring predisposition for cardiovascular disease (CVD) development. Mitochondria are essential organelles in maintaining cardiac function, and thus, fetal cardiac mitochondria could be responsive to the IUGR environment. In this study, we investigated whether in utero fetal cardiac mitochondrial programming can be detectable in an early stage of IUGR pregnancy. Using a well-established nonhuman IUGR primate model, we induced IUGR by reducing by 30% the maternal diet (MNR), both in males (MNR-M) and in female (MNR-F) fetuses. Fetal cardiac left ventricle (LV) tissue and blood were collected at 90 days of gestation (0.5 gestation, 0.5 G). Blood biochemical parameters were determined and heart LV mitochondrial biology assessed. MNR fetus biochemical blood parameters confirm an early fetal response to MNR. In addition, we show that in utero cardiac mitochondrial MNR adaptations are already detectable at this early stage, in a sex-divergent way. MNR induced alterations in the cardiac gene expression of oxidative phosphorylation (OXPHOS) subunits (mostly for complex-I, III, and ATP synthase), along with increased protein content for complex-I, -III, and -IV subunits only for MNR-M in comparison with male controls, highlight the fetal cardiac sex-divergent response to MNR. At this fetal stage, no major alterations were detected in mitochondrial DNA copy number nor markers for oxidative stress. This study shows that in 90-day nonhuman primate fetuses, a 30% decrease in maternal nutrition generated early in utero adaptations in fetal blood biochemical parameters and sex-specific alterations in cardiac left ventricle gene and protein expression profiles, affecting predominantly OXPHOS subunits. Since the OXPHOS system is determinant for energy production in mitochondria, our findings suggest that these early IUGR-induced mitochondrial adaptations play a role in offspring’s mitochondrial dysfunction and can increase predisposition to CVD in a sex-specific way.


Introduction
Cardiovascular disease (CVD) incidence is increasing worldwide at an alarming rate, especially among the young adult population [1,2].In comparison with >50-year-old adults, the incidence of CVD for younger adults for the same period is either similar or has increased [1].CVD risk is sex-specific, since men present an increased risk to develop CVD at an earlier stage in life than women [3].
It is now well accepted that maternal nutrition influences the intrauterine environment and, consequently, the offspring's short-and long-term health [4,5].Maternal nutrient restriction is a common cause of intrauterine growth restriction (IUGR) [6], in which the fetus might not reach its full growth potential [7].Multiple studies have provided evidence that fetuses experiencing nutrient deprivation during development exhibit a phenotype characterized by being born small for gestational age (SGA) [8].These infants often display cardiovascular abnormalities in comparison to infants who have achieved appropriate size for gestational age.Additionally, these individuals commonly exhibit impaired heart systolic and diastolic functions [9].Animal studies support progeny's cardiac remodeling in IUGR conditions characterized by reduced cardiomyocyte maturation [10], and increased apoptotic rates [9].
Using a well-established nonhuman IUGR primate model of moderate maternal nutrient reduction (MNR), which consisted of a 30% nutrient reduction in comparison with control mothers, and that shares a high level of gene homology with humans (94%) [11], we previously showed that term IUGR offspring (165 days old-0.9gestation, 0.9 G) [12] exhibited disrupted cardiac mitochondrial fitness, with a two-fold increase in fetal cardiac left ventricle (LV) mitochondrial DNA (mtDNA), increased transcripts levels for several respiratory chain subunits and increased abundance for the mitochondrial proteins NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 8 (NDUFB8), ubiquinol-cytochrome c reductase core protein I (UQCRC1), and cytochrome c and adenosine triphosphate (ATP) synthase.However, IUGR fetal cardiac mitochondria displayed significantly decreased complex-I and -II/III activities, possibly contributing to the 73% decreased ATP content and increased lipid peroxidation.The impairment of oxidative phosphorylation (OXPHOS) due to mitochondrial oxidative damage can contribute to an exacerbation of reactive oxygen species (ROS) production and oxidative disturbances [13].This creates a detrimental feedback loop, where increased ROS production further damages mitochondria and perpetuates oxidative stress [14].MNR fetal left ventricle (LV) also showed mitochondrial dysmorphology with sparse and disarranged cristae.Furthermore, our findings demonstrated that MNR induced sex-specific adaptations in cardiac mitochondrial biology among term fetuses in this animal model [12].This suggests that an early-impaired cardiac mitochondrial function could predispose MNR offspring to an increased CVD risk later in life [12].Right before birth, term MNR fetuses presented cardiac structural and functional alterations (i.e., extracellular fibrosis, miRNA expression levels, lipid metabolism [12]), which could be indicative of MNR-induced early cardiac adverse adaptations in this animal model [12].After birth, MNR offspring presented 11% less weight than control offspring [15].In fact, in this non-human primate (NHP) model, 3.5-year-old MNR offspring fed a control diet postnatally presented cardiac abnormalities, such as myocardial remodeling, impaired physiological function in both ventricles and altered diastolic and systolic functions [12,16].During the young and adult phases of the offspring, mitochondrial dysfunction can become accentuated, leading to an increased occurrence of mitophagy (selective degradation of damaged mitochondria [17]) and mitochondrial membrane permeabilization [18].This process enables the release of mitochondrial DNA (mtDNA) into the cytosol [19], which, in turn, activates inflammatory [20] and apoptotic pathways [21].In utero programming of mitochondrial function likely contributes to the documented developmental programming of adult cardiac dysfunction, indicating a programmed mitochondrial inability to deliver sufficient energy to cardiac tissues as a chronic mechanism for later-life heart failure [22].However, little is known about the in utero fetal cardiac mitochondrial nutritional plasticity and how early these metabolic adaptations can be detected.This information is relevant to understanding the etiology of several mitochondrial-related human diseases.
Despite the glycolytic dependence of fetal cardiomyocytes [23] and that during early embryonic stages, oxygen availability is reduced in comparison with postnatal stages [24] (highly aggravated by IUGR, which has been suggested to induce a hypoxic environment) and mitochondrial OXPHOS system activity is decreased [25], mitochondria play a crucial role supporting early-stage fetal growth [26] and cardiac development [27].For instance, mitochondrial oxidative metabolism is essential for cardiac differentiation from embryonic stem cells (i.e., cardiac specification and contraction ability) [28,29], which starts in the first few weeks of pregnancy both in humans and in NHP [30,31].Currently, it still remains unknown at which fetal stage MNR-induced cardiac mitochondrial-related alterations first occur.Thus, this study aimed to determine whether a 30% global MNR induces detectable in utero adaptations on fetal baboon left cardiac ventricular mitochondrial transcripts and protein content at the mid-gestation period (0.5 gestation, 0.5 G) and whether these are sexspecific.We hypothesized that maternal malnutrition during pregnancy impairs in utero fetal cardiac mitochondrial regulation at an early stage during fetal development, imprinting cardiac adaptations during the prenatal period that may impact postnatal mitochondrial cardiac function, contributing to offspring's increased risk of CVD later in life.
The cluster heatmap analysis identified a reasonable separation between the experimental groups' amino acid blood levels, resulting in well-formed clusters as seen in the heatmap (Figure 1).In general, MNR amino acid blood levels were increased in comparison with the C group.The differences between both groups were more pronounced in alanine, glycine, and taurine, all of which were increased in MNR vs. C, in agreement with published data for this model [6].
pronounced in alanine, glycine, and taurine, all of which were increased in MNR v agreement with published data for this model [6].To evaluate MNR-induced effects on mitochondrial biology, mtDNA copy number was determined via qRT-PCR, which was calculated by the ratio between the absolute amount of mitochondrial gene ND1 versus the absolute amount of the B2M nuclear gene for each sample.No statistically significant differences were detected for LV cardiac mtDNA copy number at 0.5 G between C and MNR offspring (Figure S1).

No Alterations Were Detected in Cardiac Mitochondrial DNA Copy Number
To evaluate MNR-induced effects on mitochondrial biology, mtDNA copy number was determined via qRT-PCR, which was calculated by the ratio between the absolute amount of mitochondrial gene ND1 versus the absolute amount of the B2M nuclear gene for each sample.No statistically significant differences were detected for LV cardiac mtDNA copy number at 0.5 G between C and MNR offspring (Figure S1).

MNR-Induced Mitochondrial Transcriptional Alteration in the Fetal Cardiac Left Ventricle at 0.5 Gestation
Human Mitochondrial Energy Metabolism and the Human Mitochondria Pathway Arrays were used to evaluate MNR-induced in utero fetal cardiac RNA transcriptional changes at 0.5 G.The expression levels of all of the evaluated genes are summarized in the heatmap and the scatter plot in Figure 2A and Table 3.The clustering analysis shows a clear separation between MNR and C groups.The fold-regulation between MNR and Control gene expression levels (Figure 2A-C, and Table 3) showed that transcripts for HSPD1 (fold-regulation: −1.6229, p = 0.0097), BCS1L (fold-regulation: −1.3446, p = 0.0089, LHPP (fold-regulation: −1.4705, p = 0.0026, ATP4A (fold-regulation: −1.5006, p = 0.0199) were downregulated, whereas the genes NDUFB6 (fold-regulation: 1.2714, p = 0.0474), NDUFB3 (fold-regulation: 1.2952, p = 0.0125), UQCRC2 (fold-regulation: 1.3692, p = 0.0274), ATP5O (fold-regulation: 1.1510, p = 0.0373), and ATP5A1 (fold-regulation: 2.2; p = 0.0089) were upregulated.Moreover, two genes had a tendency to be downregulated (CDKN2A, fold-regulation: p = 0.0558; NDUFB7 (p = 0.0587)), and five genes had a tendency to be upregulated (COX6C (p = 0.0714), BAK1 (p = 0.0856), ATP5J (p = 0.0588), TIMM10 (p = 0.0762), ATP5F1 (p = 0.0595)).3, in blue-unaltered gene expression, in red-lower gene expression, in green-higher gene expression; (C) variation in fold-regulation of gene expression in fetal cardiac left ventricle tissue of fetuses in MNR conditions relative to the Control group.PCR arrays were used to evaluate mRNA abundance of mitochondrial transcripts.Values were normalized to endogenous controls (hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L13a (RPL13A), and Beta-actin (ACTB)) and are expressed relative to their normalized values.The mean of gene expression for each group is represented (n ≥ 3 per group).Orange software package (version 3.32.0)was used for the computational data analysis and visualization.Clustering (opt ordering) was applied to both columns and rows.Data were normalized for value ranges between −1 and 1.The clustering and heatmap analysis highlight the transcriptional changes.The fold-regulation was calculated between the normalized gene expression of MNR samples and the normalized gene expression of control samples.All of the shown transcripts have a p-value < 0.1 vs. the control group.Comparison between groups was evaluated using a non-parametric Mann-Whitney test.The mean of gene expression is represented (n ≥ 3 per group).A p-value ≤ 0.05 was considered statistically significant (*), and gene expression was considered altered (upregulated or downregulated when comparing C vs. MNR).

Discussion
During pregnancy, the mother's nutritional intake plays a vital role in supporting fetal growth and development [32][33][34].Inadequate maternal nutrition, such as limiting the availability of essential fetal building blocks, can have detrimental effects on fetal growth [35,36] leading to IUGR and the development of SGA.SGA refers to infants who have a birth weight below the 10th percentile for their gestational age [37,38].MNR is a recognized risk factor for IUGR and SGA [39,40].However, it is not the sole cause, as other factors, such as maternal health conditions (e.g., hypertension, diabetes), smoking, drug use, and genetic factors, can also contribute to these conditions [41,42].
IUGR is a major obstetric condition that prompts the fetus and, later on, the offspring, for increased CVD risk [43].We have previously reported that a 30% MNR has an adverse impact on cardiac left ventricle (LV) mitochondria in a sex-dependent way in term NHP fetuses (0.9 of gestation) [12].Given the crucial role of mitochondria for cardiac differentiation and contraction in the first trimester of gestation [44], the objective of this study was to investigate whether the in utero programming of fetal cardiac mitochondria could be discernible during the early stage of pregnancy in the NHP animal model (90 days of gestation, 0.5 G).We aimed to assess the effects of a 30% maternal nutrient restriction (MNR) on the mitochondrial biology of the fetal heart during mid-gestation, using the same NHP animal model.In this study, the analysis of the cardiac LV of fetuses at 0.5 G showed that MNR already induced cardiac mitochondrial biology alterations during fetal development detectable at this time point.Thus, we here provide significant clues about MNR-induced early modulation of cardiac LV mitochondrial biology, allowing a timedependent characterization and comprehension of cardiac metabolic adaptations in MNR fetuses that programs for increased CVD risk [16].
In humans, SGA fetuses are usually hypoglycemic [45].Our study corroborates these findings with 0.5 NHP MNR fetuses showing decreased levels of glucose in comparison with C fetuses.Hypoglycemia during fetal development adversely impacts cardiogene-sis [46,47], the process of cardiomyocyte differentiation and maturation, since it mainly relies on glycolysis and lactate production until the postnatal period [48].In the postnatal stage, fuel for cardiac metabolism shifts to primarily fatty-acid oxidation which is dependent on cardiac mitochondria [49].This metabolic shift is necessary to induce cardiomyocyte proliferation [50].Despite not being the primary source of energy in the cardiomyocytes during fetal development, mitochondria play an essential role in fetal cardiac development [51].Mitochondria are important organelles for ventricular morphogenesis [51], through the regulation of apoptotic mechanisms [52], as well as in the regulation of cardiac differentiation markers, through mitochondrial fusion [53], and by the production of ROS that can act as signaling molecules [51].Hence, impaired mitochondrial function during the early stages of development may lead to detrimental adaptations in the developing heart, giving rise to a variety of anomalies in cardiac growth and development.We here show, for the first time, that 30% MNR induces mitochondrial-related transcriptional and protein abundance levels alterations as early as the mid-gestation period (0.5 G) in NHP fetuses cardiac LV, which may become relevant to understanding the mechanisms by which MNR affects cardiac function postnatally.
Cardiac gene expression analysis of mitochondria-related genes revealed that MNRinduced gene downregulation involves genes that encode for a phosphatase enzyme (LHPP) and proteins involved in macromolecular assembly (HSPD1 and BCS1L).It is worth mentioning that BCS1L plays an important role in MRC-complex III assembly [54].Interestingly, the downregulation of BCS1L and OXPHOS impairment has been proposed as a mechanistic link in prostate cancer-related fatigue development; however, more studies are needed to fully understand if this link between BCS1L gene expression and complexes activities is verified for other conditions.Nevertheless, transcripts for UQCRC2, a gene encoding for complex-III subunit, was upregulated for MNR fetuses, along with other OXPHOS complex subunits: complex-I (NDUFB6, NDUFB3), -IV (COX6C), and ATP synthase (ATP5A1, ATP5F1, ATP5J, ATP5O).This represents more than half of the measured genes whose expression was altered by MNR in the cardiac LV.In addition, and highlighting MNRinduced mitochondrial alterations, the protein amount of citrate synthase, a mitochondrial matrix enzyme commonly used as a mitochondrial marker [55], was decreased for MNR mid-gestation fetuses, suggesting a decreased mitochondrial number.Most transcripts for OXPHOS subunit genes were upregulated, and the same was observed in our previous study for cardiac tissue from term fetuses (0.9 G) [12].In both mid-gestation and term fetuses, transcripts for complex I subunits NDUFB6, and ATP synthase ATP5A1 were upregulated.These transcript alterations did not result in many protein content alterations for the same subunits, in both fetal time points (with the exception of COX6C for MNR mid-gestation fetuses), possibly due to the dynamic processes between the genome and the protein levels, which include transcription, splicing, and translation [12].In spite of this, in term fetuses, MRC complex-I, -II/III, and -IV activities were altered [12].This was also observed in a diet-induced animal model of IUGR in adult mice offspring (14 weeks old).Protein content for OXPHOS subunits was unaltered by IUGR; however, mitochondrial respiration was decreased in IUGR-offspring cardiac LV muscle [56].Therefore, to comprehensively assess the true impact of MNR on OXPHOS and its subsequent effects on cardiac mitochondria, it is essential to measure either respiratory rates or the activity of OXPHOS.However, conducting such studies can be challenging and often impractical due to limited sample availability and the significant amount of biological material required.The impact of fetal sex on mitochondrial metabolism-related gene expression and protein content was evident.The fold-regulation of MNR-M vs. MNR-F reveals a sex-specific pattern of gene expression induced by MNR.For MNR-F, the majority of upregulated transcripts in comparison with the respective C include those encoding for OXPHOS subunits, while for MNR-M, this was more evident for transcripts for other mitochondrial proteins that are involved in ornithine transport (SLC25A2), mitochondrial ADP/ATP exchange (SLC25A31), uncoupling of mitochondrial oxidative phosphorylation (UCP2), molecular folding (HSP90AA1), removal of the mitochondrial targeting pre-sequence of nuclear-encoded proteins (IMMP1L), and protein translocation (TIMM22, TOMM70A).Interestingly, both complex III-core 2 subunit gene expression (UQCRC2) and protein content were increased for MNR-M vs. C-M.Moreover, complex-I (NDUFB8) and -IV (COXII) subunit protein contents were increased for MNR-M vs. C-M, whereas no differences were detected in MNR-F.Thus, MNR seems to impact more severely male fetuses' OXPHOS protein content, whereas for female offspring, these alterations may become more pronounced at a later developmental stage or even at a postnatal stage, since we did not detect differences in these subunits' protein content for term female fetuses in our previous study [12].These sex-specific differences may be explained by, e.g., epigenetic regulation, or by the action of sex-steroid hormones, given that estrogen plays an active role in protein modification, gene regulation, and cellular process modulation [57].Nevertheless, further studies are needed to understand how sex influences in utero cardiac programming and adult offspring disease risk.
Another implication of the in utero MNR-induced OXPHOS alterations may be the impact in the production of ROS [4].Our results showed that during mid-gestation, oxidative stress markers' protein content was not altered, whereas we detected MNR-induced increased levels of MDA in term-fetuses [12], indicating that oxidative damage may only become evident during the second half of pregnancy in this NHP MNR model.This can relate to the unaltered mtDNA copy number, suggesting that perhaps mitochondrial mass and biogenesis are still preserved since lack of significant oxidative damage occurring at this fetal stage.Indeed, in a rabbit animal model of IUGR, in the LVs of 30-day-old fetuses (corresponding to late gestation period), increased expression of genes that modulate OXPHOS, including cardiac mitochondrial respiratory chain complex I, NADH dehydrogenase activity was detected [58].These alterations were accompanied by decreased cardiac enzymatic activities of complex-II, -IV, and -II + III [59].Nevertheless, these mitochondrial respiratory chain adaptations did not produce alterations in cardiac cellular ATP levels, nor in the antioxidant enzyme SOD2, nor in mitochondrial copy number [58], in accordance with our findings.Guitart-Mampel et al. suggest that the preservation of ATP levels and SOD2 protein expression levels and activity can be attributable to increased levels of Sirtuin 3 [59], acting as a compensatory mechanism, due to its action on promoting mitochondrial energy production, on the inhibition of oxidative stress, and on autophagy regulation [60].We recognize the possibility of Sirtuin 3 acting as a compensatory mechanism in the hearts of our animal model as well, however, it must be taken into consideration that in Guitart et.al.'s study, IUGR was achieved surgically and the animal models are very distinct from each other (i.e., gestation period, diet, litter size).Highlighting this, in our animal model, term fetuses display signs of oxidative damage [12].To our knowledge, no other study has explored Sirtuin-3 levels in the context of IUGR fetal programming of CVD, and thus, no major conclusions can be drawn.
Cardiomyocyte proliferation in the neonatal period depends mainly on oxidative metabolism [51,61].Even though we show here that some MNR-induced alterations begin to be detectable at mid-gestation in cardiac mitochondria, these become more pronounced at late gestation, e.g., increased mtDNA content and oxidative stress in comparison with C [12].We must consider that at 0.5 G, mitochondria are smaller and less mature [50].Thus, it is possible that MNR-induced adaptations in fetal cardiac mitochondrial function become significant enough to impair cardiac function only at a stage where cardiomyocytes mainly rely on mitochondrial respiration as a fuel source.However, we cannot underestimate the impact of mitochondrial dysfunction and signaling dysregulation in driving proper cardiomyocyte maturation, cardiac differentiation, and heart organogenesis that may only be perceivable in advanced postnatal life stages [49,62,63].To our knowledge, our study is the only one reporting IUGR-induced mitochondrial-related genes' expression alterations in fetal hearts at this stage in development, and thus, we can only raise hypotheses.It has been suggested that maternal nutrient reduction leads to a hypoxic environment in fetal organs [64,65].The literature has suggested that hypoxia alters the expression of transcription factors involved in the replication of mtDNA-encoded genes [66], which include genes from the subunits of the mitochondrial respiratory chain complexes.On top of that, it is widely accepted that hypoxia activates an oxygen-sensing transcription factor, the hypoxia-inducible factor 1α (HIF-1α) [67], which is responsible for modulating the expression of microRNAs that regulate MRC complex subunits' assembly proteins' expression [67], highlighting the potential role of epigenetic remodeling.In addition, because oxygen is the final electron acceptor of the mitochondrial electron transport chain (ETC), a lack of oxygen availability affects ETC function, resulting in an imbalance between oxygen and electron flow, leading to an overproduction of ROS [67], which can affect the expression of genes that encode for ETC complex subunits [68].This is less likely to occur in an early fetal stage because, in this study, we did not find any significant alterations related to markers of oxidative stress, but this can occur in later stages since these alterations were verified in the hearts of 165-day-old fetuses [12].The initial changes that we here report, sustained throughout an individual's lifetime, can potentially lead to a progressive disruption of cardiac metabolic functions.In spite of that, the sex-specific response is already clear at this stage, and the different patterns of gene expression according to fetal sex are significant, highlighting the need to explore CVD programming according to fetal offspring sex, highlighting the need for effective sex-specific treatment in disease management and mitigation.

Animal Care and Maintenance
The Animal Care and Use Committees of the Texas Biomedical Research Institute and the University of Texas Health Science Center at San Antonio, TX (no.1134PC) approved all animal procedures, including pain relief.These were conducted in the Association for Assessment and Accreditation of Laboratory Animal Care-approved facilities and NIH Guide for the Care and Use of Laboratory Animals.
As previously described [69], maternal morphometrics were determined pre-pregnancy to guarantee weight consistency and general morphometrics in the animals used in the present study.Non-pregnant outbred female baboons (Papio spp.) of a similar morphometric phenotype were selected for the study.Animals were housed at the Southwest National Primate Research Center at the Texas Biomedical Research Institute (TBRI) in the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)approved facilities.Housing conditions and animal caging were performed as previously described [12].Each baboon's weight was obtained while crossing an electronic scale (GSE 665; GSE Scale Systems, Milwaukee, WI, USA), and as described elsewhere [12].

Experimental Design
Normally cycling female baboons from 8 to 15 years old were observed twice a day for well-being and three times a week for turgescence (genital organ's skin swelling) and signs of vaginal bleeding to assess their reproductive cycle and enable determining the timing of pregnancy [69].After a 30-day adaptation to the feeding system, a fertile male was introduced into each breeding cage.On day 30 of pregnancy, which was determined by following the changes in the swelling of the sex skin and by ultrasonography, twentyfour female baboons were randomly assigned to eat standard primate chow ad libitum (control diet) or to receive 70% of the average daily amount of food eaten by the female control baboons (MNR group) on a body weight-adjusted basis at same gestational age.Cesarean section was performed at 90 days gestation (0.5 G) (Figure 6).Each fetus from a singleton pregnant female baboon is considered an experimental unit; in some cases, the pregnant female baboon was also assumed as the experimental unit when maternal data are presented (12 baboons/dietary group; 6 male control fetuses-C-M; 6 female control fetuses-C-F; 6 MNR male fetuses-MNR-M; and 6 MNR female fetuses-MNR-F).Purina Monkey Diet 5038, standard biscuits were provided once a day.The biscuit is described as a "complete life-cycle diet for all Old-World Primates" and contains stabilized vitamin C as well as all other required vitamins.The basic composition includes crude protein (≥15%), crude fat (≥5%), crude fiber (≤6%), ash (≤5%), and added minerals (≤3%) [69].
After the confirmation of pregnancy, food intake was recorded in 8 female baboons fed ad libitum and was calculated as 50.61 ± 3.61 kcal/kg of body weight per day.Before the start of the controlled diet, baboons were fed the same diet without a biscuit limit.Water was continuously available in the feeding cages via individual waterers (Lixit, Napa, CA, USA) and at several locations in the group housing.Animal food consumption, weights, and health status were recorded daily.More details regarding housing and environmental enrichment have been previously published [69].

Cesarean Section, Fetal and Maternal Morphometry, and Blood Sampling
Mothers were fasted from their last feeding time the day before, until the cesarean section [69].A fully certified M.D. or D.V.M. performed surgical procedures, and postsurgical care was prescribed and monitored by an accredited veterinarian.Cesarean section and fetal necropsy were performed under isoflurane anesthesia (2%, 2 L/min oxygen, tracheal intubation), followed by tranquilization with ketamine hydrochloride (10 mg/kg intramuscularly injection) at 90 days of gestation (0.5 G) using standard sterile techniques as previously described [69].Following hysterotomy, fetal exsanguination was performed with maternal and fetal baboons under general anesthesia as approved by the American Veterinary Medical Association Panel on Euthanasia [69].Fetal hearts were collected.Cardiac samples were taken from the free wall of the left cardiac ventricle that was cut transversely.Some pieces were flash-frozen and stored at −80 °C until analysis.Postoperatively, mothers were placed in individual cages and observed until they were upright under their power and returned to their group cage.More details regarding maternal post-partum handling have been previously described [12].

Analysis of mtDNA Copy Number via Quantitative Real-Time PCR
DNA extraction was performed as previously described [12].RT-PCR was performed using the SsoFast Eva Green Supermix (Bio-Rad, Hercules, CA, USA), in a CFX96 realtime PCR system (Bio-Rad), with the primers for ND1 (accession code NC_001992.1;sense sequence CCTATGAATCCGAGCAGCGT; antisense sequence GCTGGA-GATTGCGATGGGTA) and for B2M (accession code NC_018158.1,sense sequence CAGGGCCCAGGACAGTTAAG; antisense sequence GGGATGGGACTCATTCAGGG) at 500 nM each.The amplification of 25 ng DNA was performed with an initial cycle of 2 min at 98 °C, followed by 40 cycles of 5 s at 98 °C plus 5 s at 60 °C.At the end of each cycle, Eva Green fluorescence was recorded to allow Ct determination.For quality control, the Purina Monkey Diet 5038, standard biscuits were provided once a day.The biscuit is described as a "complete life-cycle diet for all Old-World Primates" and contains stabilized vitamin C as well as all other required vitamins.The basic composition includes crude protein (≥15%), crude fat (≥5%), crude fiber (≤6%), ash (≤5%), and added minerals (≤3%) [69].
After the confirmation of pregnancy, food intake was recorded in 8 female baboons fed ad libitum and was calculated as 50.61 ± 3.61 kcal/kg of body weight per day.Before the start of the controlled diet, baboons were fed the same diet without a biscuit limit.Water was continuously available in the feeding cages via individual waterers (Lixit, Napa, CA, USA) and at several locations in the group housing.Animal food consumption, weights, and health status were recorded daily.More details regarding housing and environmental enrichment have been previously published [69].

Cesarean Section, Fetal and Maternal Morphometry, and Blood Sampling
Mothers were fasted from their last feeding time the day before, until the cesarean section [69].A fully certified M.D. or D.V.M. performed surgical procedures, and postsurgical care was prescribed and monitored by an accredited veterinarian.Cesarean section and fetal necropsy were performed under isoflurane anesthesia (2%, 2 L/min oxygen, tracheal intubation), followed by tranquilization with ketamine hydrochloride (10 mg/kg intramuscularly injection) at 90 days of gestation (0.5 G) using standard sterile techniques as previously described [69].Following hysterotomy, fetal exsanguination was performed with maternal and fetal baboons under general anesthesia as approved by the American Veterinary Medical Association Panel on Euthanasia [69].Fetal hearts were collected.Cardiac samples were taken from the free wall of the left cardiac ventricle that was cut transversely.Some pieces were flash-frozen and stored at −80 • C until analysis.Postoperatively, mothers were placed in individual cages and observed until they were upright under their power and returned to their group cage.More details regarding maternal post-partum handling have been previously described [12].

Analysis of mtDNA Copy Number via Quantitative Real-Time PCR
DNA extraction was performed as previously described [12].RT-PCR was performed using the SsoFast Eva Green Supermix (Bio-Rad, Hercules, CA, USA), in a CFX96 real-time PCR system (Bio-Rad), with the primers for ND1 (accession code NC_001992.1;sense sequence CCTATGAATCCGAGCAGCGT; antisense sequence GCTGGAGATTGCGATGGGTA) and for B2M (accession code NC_018158.1,sense sequence CAGGGCCCAGGACAGTTAAG; antisense sequence GGGATGGGACTCATTCAGGG) at 500 nM each.The amplification of 25 ng DNA was performed with an initial cycle of 2 min at 98 • C, followed by 40 cycles of 5 s at 98 • C plus 5 s at 60 • C. At the end of each cycle, Eva Green fluorescence was recorded to allow Ct determination.For quality control, the melting temperature of the PCR products was determined after amplification by performing melting curves, and no template controls were run.
For absolute quantification and amplification efficiency, standards at known copy numbers were produced by purifying PCR products.After optimizing the annealing temperature, products were amplified for each primer pair using the HotstarTaq Master Mix Kit (#203445 Qiagen, Hilden, Germany).Briefly, 1 µL of a DNA sample was added to a PCR tube containing the HotStar Taq Master Mix and the specific primers and placed in a CFX96 real-time PCR system.The amplification protocol started with an initial activation step of 15 min at 95 • C degrees, followed by 35 cycles of 1 min at 94 • C (denaturation) plus 1 min at 60 • C (annealing), plus 1 min at 72 • C (extension), and a final extension step of 10 min at 72 • C.After amplification, the products were purified using the MiniElute PCR purification kit (#280006 Qiagen) following the manufacturer's instructions.Eluted DNA was quantified in a Nanodrop 2000 device, the copy numbers were adjusted to 5 × 10 9 copies/µL, and tenfold serial dilutions were prepared.mtDNA copy number was determined by the ratio between the absolute amounts of mitochondrial gene ND1 versus the absolute amount of the B2M nuclear gene in each sample, using the CFX96 Manager software (v.3.0; Bio-Rad).

Gene Expression Analysis by PCR Array
RNA extraction was performed following the protocol previously described by Cox et al. [70].RNA was quantified spectrophotometrically using Thermo Scientific NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) and stored at −80 • C. The RNA purity and quality were checked by Ultraviolet spectrophotometry as described elsewhere [12].After RNA preparation, the samples were treated as previously described [12].The RT 2 Profiler polymerase chain reaction (PCR) Array System (SuperArray Bioscience (Frederick, MD, USA), SA Biosciences (Frederick, MD, USA), Qiagen (Hilden, Germany)), was used to evaluate the different cardiac mitochondrial transcripts between control and MNR fetuses as previously described [12].Each PCR array contained 84 transcripts of the corresponding signaling pathway, a set of five reference genes as internal controls, and additional controls for efficiency of reverse transcription, PCR, and the absence of contaminating genomic DNA.Data were normalized with three endogenous controls that did not differ between groups hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L13a (RPL13A), and Beta-actin (ACTB) and analyzed with the ∆∆Ct method (where Ct is threshold cycle) using the PCR Array Data Analysis Web Portal (SA Biosciences).The transcripts used in this study are listed in Tables 4 and 5.
Table 4. Panel of gene expression analyzed using the Human Mitochondrial Energy Metabolism RT 2 Profiler PCR Array.This array profiled the expression of 84 key genes involved in mitochondrial energy metabolism, including genes encoding components of the electron transport chain and oxidative phosphorylation complexes.Position indicates the location in the 96-well plate where the transcripts were assessed, Symbol denotes the gene identification, RefSeq denotes the Reference Sequence from the National Center for Biotechnology Information collection, and Description gives summary information about the gene identification and/or function.Protein analyses via Western blotting were performed following standard protocols [71].Sample preparation and protein extraction were performed according to previously described protocols [12].Extracted proteins were solubilized to achieve a concentration of 1 mg/mL or 2 mg/mL of protein with Laemmli buffer (62.5 mM Tris pH 6.8 (HCl), 50% glycerol, 2% SDS, 0.005% bromophenol blue, supplemented with 5% β-mercaptoethanol) and boiled for 5 min in a water bath and then centrifuged at 14,000× g for 5 min.Equivalent amounts of total protein (10 µg per lane) were loaded in a 10-20% gradient Tris-HCl polyacrylamide gel as well as two different standards for molecular weight estimation and for monitoring electrophoresis progress, the Precision Plus Protein Dual Color Standards (Bio-Rad) and the SeeBlue Plus2 Pre-Stained Standard (ThermoFisher Scientific (Waltham, MA, USA), Invitrogen (Waltham, MA, USA)).Electrophoresis was carried out at room temperature in a Criterion system (Bio-Rad) using 150 V until the sample buffer (blue) reaches the bottom of the gel (≈90 min).After separation by SDS-PAGE, proteins were electrophoretically transferred in a TransBlot Cell system (Bio-Rad) to a polyvinylidene difluoride (PVDF) membrane previously activated, a constant amperage (0.5 A) for 2 h at 4 • C using a CAPS transfer buffer (10 mM 3-(Cyclohexylamino)-1-propanesulfonic acid pH 11 (NaOH), 10% methanol).The quality of the electrophoretic transfer was evaluated by the complete transfer of pre-stained molecular weight markers below 100 kDa and via Ponceau staining.Ponceau results were also used to confirm an equal amount of protein loading and to normalize band density.After Ponceau removal, the membranes were blocked in 5% non-fat milk/PBS overnight at 4 • C with agitation.Before incubation with primary antibodies, the membrane was washed for 10 min in PBS 0.05% Tween-20 (PBS-T).Primary antibodies were prepared in 1% non-fat milk/PBS to a final volume of 5 mL and incubated overnight at 4 • C.After incubation with primary antibodies, membranes were washed with PBS-T solution three times, 5 min each, and incubated with the correspondent alkaline phosphatase-conjugated secondary antibodies for 2 h at room temperature with stirring.For immunodetection, membranes were washed three times for 5 min each with PBS-T, rinsed in PBS to remove any Tween-20, which can be inhibitory to the detection method, dried, and incubated with an enhanced chemifluorescence (ECF) system (#RPN5785, GE Healthcare, Little Chalfont, Buckinghamshire, UK) during a maximum of 5 min.Density analysis of bands was carried out with VisionWorks LS Image Acquisition and Analysis Software (UVP).The resulting images were analyzed and densities were normalized to Ponceau.The average value of the C-Males (M) group was assumed as one unit, and the values of each sample were determined proportionally.All of the primary antibodies used in this experiment were purchased from abcam: NDUFB8 (1:500, ab110242), UQCRC2 (1:500, ab14745), MT-CO2 (1:500, ab110258), COX6C (1:1000, ab150422), ATP5A (1:500, ab110273), TOMM20 (1:500, sc11415), TFAM (1:00, sc23588), and citrate synthase (1:1000, ab129088).All of the secondary antibodies were purchased from Santa Cruz and were used in a 1:5000 dilution: rabbit (sc-2007), mouse (sc-2008), goat (sc-2771).

Data Analysis and Statistics
The software GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA) was used for data analysis.Each pregnant baboon and the corresponding fetus were considered an experimental unit.Outbred pregnant female baboons were randomly assigned to control or MNR groups.Data are expressed as mean or as mean ± SD.Normality was assessed via the Kolmogorov-Smirnov or Shapiro-Wilk tests.To assess the effect of MNR on fetal cardiac mitochondrial parameters, the following comparisons were made: (1) only to evaluate diet-induced alterations, independently of fetal sex: between Control (C) vs. MNR; (2) to simultaneously assess the impact of diet and sex: between male control (C-M) vs. female control (C-F), male MNR (MNR-M) vs. C-M, female MNR (MNR-F) vs. C-F, and MNR-M vs. MNR-F.The orange software (version 3.32.0)was used for the computational data analysis and visualization.Clustering (opt ordering) was applied to both columns and rows.Data were normalized for value ranges between −1 and 1.

Conclusions
In this study, 30% MNR led to sex-specific alterations on the NHP fetal cardiac leftventricle mitochondrial-related gene expression at 90 days old (0.5 G), some of which persisted until near term (0.9 G).More than half of the MNR-induced gene expression alterations encode for OXPHOS complex subunits.Given that the OXPHOS system is primarily responsible for energy production in the mitochondria, early IUGR-induced mitochondrial adaptations could play a role in IUGR offspring's increased predisposition to CVD.The fact that we here showed that MNR can induce fetal cardiac mitochondrial function alterations as early as the mid-gestation period highlights the need to better understand the early origins of CVD, which can provide new targets for disease prevention and mitigation.

Figure 2 .
Figure 2. Cardiac left ventricle (LV) tissue gene expression of 90-day-old control (C) fetuses and fetuses born from maternal nutrient reduction (MNR) conditions.(A) Gene expression heatmap view of Control fetuses born from mothers fed a control diet and MNR fetuses born from mothers fed a 30% nutrient-reduced diet; (B) differences in gene expression of fetal cardiac left ventricle (LV) tissue of fetuses in MNR conditions relative to the Control group, dots represent the plot between log10(2 −Delta(Ct) ) for the control group and MNR for each gene represented in Table 3, in blue-unaltered gene expression, in red-lower gene expression, in green-higher gene expression; (C) variation in fold-regulation of gene expression in fetal cardiac left ventricle tissue of fetuses in MNR conditions relative to the Control group.PCR arrays were used to evaluate mRNA abundance of mitochondrial transcripts.Values were normalized to endogenous controls (hypoxanthine

Figure 2 .
Figure 2. Cardiac left ventricle (LV) tissue gene expression of 90-day-old control (C) fetuses and fetuses born from maternal nutrient reduction (MNR) conditions.(A) Gene expression heatmap view of Control fetuses born from mothers fed a control diet and MNR fetuses born from mothers fed a 30% nutrient-reduced diet; (B) differences in gene expression of fetal cardiac left ventricle (LV) tissue of fetuses in MNR conditions relative to the Control group, dots represent the plot between log 10 (2 −Delta(Ct) ) for the control group and MNR for each gene represented in Table3, in blue-unaltered gene expression, in red-lower gene expression, in green-higher gene expression; (C) variation in fold-regulation of gene expression in fetal cardiac left ventricle tissue of fetuses in MNR conditions relative to the Control group.PCR arrays were used to evaluate mRNA abundance

Figure 3 .
Figure 3. Fold-regulation of 90-day-old fetal cardiac left ventricle (LV) tissues in fetuses in maternal nutrient reduction (MNR) conditions relative to the Control (C) group of the respective sex.Control (C): fetuses born from mothers fed a control diet; MNR: fetuses born from mothers fed a 30% nutrient-restricted diet; C-M/C-F: male/female fetuses born from mothers fed a control diet; MNR-M/F: fetuses born from mothers fed a 30% nutrient-restricted diet.(A) Diagram representing the number of genes from LV of MNR fetuses that were upregulated and downregulated relative to the C group of the same sex; (B,C) Fold-regulation of gene expression analysis of fetal cardiac LV tissue from MNR conditions relative to the C group of the respective sex.PCR arrays were used to evaluate mRNA abundance of mitochondrial transcripts.Values were normalized to endogenous controls (hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L13a (RPL13A), and Betaactin (ACTB)) and are expressed relative to their normalized values.The fold-regulation was calculated between the normalized gene expression of MNR samples and the normalized gene expression of control samples, according to sex ((B) MNR-M vs. C-M; (C) MNR-F vs. C-F)).All of the present transcripts have a p-value < 0.1 vs. the control group.Comparison between groups was evaluated using a non-parametric Mann-Whitney test (n ≥ 3 per group)).The mean of gene expression is represented.A p-value ≤ 0.05 was considered statistically significant (*), and gene expression was considered altered (upregulated or downregulated).When comparing the fold-regulation of gene expression in MNR-M vs. MNR-F, Figure 4A and Table3(section MNR-M vs. MNR-F) show that the upregulated transcripts for MNR-M were ATP5A1, ATP5G3, HSPD90AA1, IMMP1L, SLC25A2, SLC25A31, TIMM22, TOMM70A, and UCP2, with ATP5C1, COX6A2, NDUFV1, CPT2, DNM1L, MFN1, OPA1, and TP53, being tendentially upregulated, and for MNR-F, the upregulated genes were COX4I2, NDUFV3, CYC1, MIPEP, TOMM40, ATP4A, UQCRC1, UQCRFS1, SDHB, SDHD, NDUFA11, NDUFC1, NDUFS2, NDUFS3, TOMM20, BCS1L, NDUFA1, NDUFA10,

Figure 3 .
Figure 3. Fold-regulation of 90-day-old fetal cardiac left ventricle (LV) tissues in fetuses in maternal nutrient reduction (MNR) conditions relative to the Control (C) group of the respective sex.Control (C): fetuses born from mothers fed a control diet; MNR: fetuses born from mothers fed a 30% nutrient-restricted diet; C-M/C-F: male/female fetuses born from mothers fed a control diet; MNR-M/F: fetuses born from mothers fed a 30% nutrient-restricted diet.(A) Diagram representing the number of genes from LV of MNR fetuses that were upregulated and downregulated relative to the C group of the same sex; (B,C) Fold-regulation of gene expression analysis of fetal cardiac LV tissue from MNR conditions relative to the C group of the respective sex.PCR arrays were used to evaluate mRNA abundance of mitochondrial transcripts.Values were normalized to endogenous controls (hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L13a (RPL13A), and Beta-actin (ACTB)) and are expressed relative to their normalized values.The fold-regulation was calculated between the normalized gene expression of MNR samples and the normalized gene expression of control samples, according to sex ((B) MNR-M vs. C-M; (C) MNR-F vs. C-F)).All of the present transcripts have a p-value < 0.1 vs. the control group.Comparison between groups was evaluated using a non-parametric Mann-Whitney test (n ≥ 3 per group)).The mean of gene expression is represented.A p-value ≤ 0.05 was considered statistically significant (*), and gene expression was considered altered (upregulated or downregulated).

Figure 4 .Figure 4 .
Figure 4. Sex-specific differences in gene expression fold-regulation of 90-day-old fetal cardiac left ventricle (LV) tissue.Control (C): fetuses born from mothers fed a control diet; MNR: fetuses born from mothers fed a 30% nutrient-restricted diet; C-M/C-F: male/female fetuses born from mothers fed a control diet; MNR-M/F: fetuses born from mothers fed a 30% nutrient-restricted diet.Foldregulation of gene expression analysis of fetal cardiac LV tissue from control and MNR groups, according to fetal sex.PCR arrays were used to evaluate mRNA abundance of mitochondrial transcripts.Values were normalized to endogenous controls (hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L13a (RPL13A), and Beta-actin (ACTB)) and are expressed relative to their normalized values.(A) The fold-regulation was calculated between the normalized gene expression of MNR-M samples and the normalized gene expression of MNR-F samples; (B) The fold-Figure 4. Sex-specific differences in gene expression fold-regulation of 90-day-old fetal cardiac left ventricle (LV) tissue.Control (C): fetuses born from mothers fed a control diet; MNR: fetuses born from mothers fed a 30% nutrient-restricted diet; C-M/C-F: male/female fetuses born from mothers fed a control diet; MNR-M/F: fetuses born from mothers fed a 30% nutrient-restricted diet.Fold-regulation of gene expression analysis of fetal cardiac LV tissue from control and MNR groups, according to fetal sex.PCR arrays were used to evaluate mRNA abundance of mitochondrial transcripts.Values were normalized to endogenous controls (hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L13a (RPL13A), and Beta-actin (ACTB)) and are expressed relative to their normalized values.(A) The fold-regulation was calculated between the normalized gene expression of MNR-M samples and the normalized gene expression of MNR-F samples; (B) The fold-regulation was calculated between the normalized gene expression of MNR-M samples and the normalized gene expression of C-M samples (represented in blue) or between the normalized gene expression of MNR-F samples and the normalized gene expression of C-F samples (represented in pink).Comparison between groups was evaluated using a non-parametric Mann-Whitney test.The mean of gene expression is represented (n ≥ 3 per group).A p-value ≤ 0.05 was considered statistically significant (*), and gene expression was considered altered (upregulated or downregulated).

Figure 5 .Figure 5 .
Figure 5. Cardiac relative band density of mitochondrial function-associated proteins in the left ventricles of 90-day-old fetuses.Control (C): fetuses born from mothers fed a control diet; MNR: fetuses born from mothers fed a 30% nutrient-restricted diet; C-M/C-F: male/female fetuses born from mothers fed a control diet (filled symbols); MNR-M/MNR-F: fetuses born from mothers fed a 30% nutrient-restricted diet (open symbols).(A) Complex I (NADH dehydrogenase) subunit NDUFB8; (B) complex III (cytochrome c reductase) subunit UQCRC2; (C) complex IV subunit COXII; (D) translocase of outer mitochondrial membrane 20 (TOMM20); (E) complex IV (cytochrome c oxidase) Figure 5. Cardiac relative band density of mitochondrial function-associated proteins in the left ventricles of 90-day-old fetuses.Control (C): fetuses born from mothers fed a control diet; MNR: fetuses born from mothers fed a 30% nutrient-restricted diet; C-M/C-F: male/female fetuses born from mothers

Figure 6 .
Figure 6.Timeline of maternal nutrition during fetal development.Control group-C; maternal nutrient reduction-MNR.

Figure 6 .
Figure 6.Timeline of maternal nutrition during fetal development.Control group-C; maternal nutrient reduction-MNR.

Table 1 .
Summary of gestational parameters.Maternal and fetal morphological parameters at 0.5 gestation in control pregnancies and in maternal nutrient reduction (MNR) conditions via a 30% reduction of the food eaten by control mothers on a weight-adjusted basis.

Table 2 .
Maternal and fetal blood biochemical parameters at 50% gestation in control pregnancies and in the presence of 30% maternal nutrient restriction (MNR) of the food eaten by control mothers on a weight-adjusted basis.
Gene Expression Fold Regulation and p-Value
Gene Expression Fold Regulation and p-Value

Table 5 .
Panel of gene expression analyzed using the Human Mitochondria RT 2 Profiler PCR Array.This array profiled the expression of 84 genes involved in diverse mitochondrial function.The transcripts monitored by this array encoded proteins which are regulators of mitochondrial biogenesis, regulators and mediators of mitochondrial molecular transport, and genes involved in apoptosis.Position indicates the location in the 96-well plate where the gene was assessed, Symbol denotes the gene identification, RefSeq denotes the Reference Sequence from the National Center for Biotechnology Information collection, and Description gives summary information about the gene identification and/or function.