Perinatal iron restriction is associated with changes in neonatal cardiac function and structure in a sex-dependent manner.

Iron deficiency (ID) is common during gestation and in early infancy and can alter developmental trajectories with lasting consequences on cardiovascular health. While the effects of ID and anemia on the mature heart are well documented, comparatively little is known about their effects and mechanisms on offspring cardiac development and function in the neonatal period. Female Sprague-Dawley rats were fed an iron-restricted or iron-replete diet before and during pregnancy. Cardiac function was assessed in a cohort of offspring on postnatal days (PD) 4, 14, and 28 by echocardiography; a separate cohort was euthanized for tissue collection and hearts underwent quantitative shotgun proteomic analysis. ID reduced body weight and increased relative heart weights at all time points assessed, despite recovering from anemia by PD28. Echocardiographic studies revealed unique functional impairments in ID male and female offspring, characterized by greater systolic dysfunction in the former and greater diastolic dysfunction in the latter. Proteomic analysis revealed down-regulation of structural components by ID, as well as enriched cellular responses to stress; in general, these effects were more pronounced in males. ID causes functional changes in the neonatal heart, which may reflect an inadequate or maladaptive compensation to anemia. This identifies systolic and diastolic dysfunction as comorbidities to perinatal ID anemia which may have important implications for both the short- and long-term cardiac health of newborn babies. Furthermore, therapies which improve cardiac output may mitigate the effects of ID on organ development.

Perinatal iron deficiency combined with a high salt diet in adulthood causes sex-dependent vascular dysfunction in rats.

KEY POINTS: Perinatal iron deficiency causes changes in offspring mesenteric artery function in adulthood, particularly in males, which can be exacerbated by chronic intake of a high salt diet. Perinatal iron deficient male offspring exhibit enhanced conversion of big endothelin-1 to active endothelin-1, coinciding with decreased nitric oxide levels. Perinatal iron deficient male offspring have reduced nitric oxide-mediated endothelial-dependent vasodilatation coincident with increased vascular superoxide levels following consumption of a high salt diet. Perinatal iron deficiency has no apparent effects on vascular function in female offspring, even when fed a high salt diet. These results help us better understand underlying vascular mechanisms contributing to increased cardiovascular risk from perinatal stressors such as iron deficiency. ABSTRACT: Pre- and immediate postnatal stressors, such as iron deficiency, can alter developmental trajectories and predispose offspring to long-term cardiovascular dysfunction. Here, we investigated the impact of perinatal iron deficiency on vascular function in the adult offspring, and whether these long-term effects were exacerbated by prolonged consumption of a high salt diet in adulthood. Female Sprague Dawley rats were fed either an iron-restricted or -replete diet prior to and throughout pregnancy. Six weeks prior to experimentation at 6 months of age, adult offspring were fed either a normal or high salt diet. Mesenteric artery responses to vasodilators and vasoconstrictors were assessed ex vivo by wire myography. Male perinatal iron deficient offspring exhibited decreased reliance on nitric oxide with methacholine-induced vasodilatation (interaction P = 0.03), coincident with increased superoxide levels when fed the high salt diet (P = 0.01). Male perinatal iron deficient offspring exhibit enhanced big endothelin-1 conversion to active endothelin-1 (P = 0.02) concomitant with decreased nitric oxide levels (P = 0.005). Female offspring vascular function was unaffected by perinatal iron deficiency, albeit the high salt diet was associated with impaired vasodilation and decreased nitric oxide production (P = 0.02), particularly in the perinatal iron deficient offspring. These findings implicate vascular dysfunction in the sex-specific programming of cardiovascular dysfunction in the offspring by perinatal iron deficiency.

Prenatal iron deficiency causes sex-dependent mitochondrial dysfunction and oxidative stress in fetal rat kidneys and liver.

Prenatal iron deficiency alters fetal developmental trajectories, which results in persistent changes in organ function. Here, we studied the effects of prenatal iron deficiency on fetal kidney and liver mitochondrial function. Pregnant Sprague-Dawley rats were fed partially or fully iron-restricted diets to induce a state of moderate or severe iron deficiency alongside iron-replete control rats. We assessed mitochondrial function via high-resolution respirometry and reactive oxygen species generation via fluorescence microscopy on gestational d 21. Hemoglobin levels were reduced in dams in the moderate (-31%) and severe groups (-54%) compared with controls, which was accompanied by 55% reductions in fetal hemoglobin levels in both moderate and severe groups versus controls. Male iron-deficient kidneys exhibited globally reduced mitochondrial content and respiration, as well as increased cytosolic superoxide and decreased NO. Female iron-deficient kidneys exhibited complex II down-regulation and increased mitochondrial oxidative stress. Male iron-deficient livers exhibited reduced complex IV respiration and increased cytosolic superoxide, whereas female liver tissues exhibited no alteration in oxidant levels or mitochondrial function. These findings indicate that prenatal iron deficiency causes changes in mitochondrial content and function as well as oxidant status in a sex- and organ-dependent manner, which may be an important mechanism that underlies the programming of cardiovascular disease.-Woodman, A. G., Mah, R., Keddie, D., Noble, R. M. N., Panahi, S., Gragasin, F. S., Lemieux, H., Bourque, S. L. Prenatal iron deficiency causes sex-dependent mitochondrial dysfunction and oxidative stress in fetal rat kidneys and liver.

The Dictyostelium discoideum FimA protein, unlike yeast and plant fimbrins, is regulated by calcium similar to mammalian plastins.

Plastins, also known as fimbrins, are highly conserved eukaryotic multidomain proteins that are involved in actin-bundling. They all contain four independently folded Calponin Homology-domains and an N-terminal headpiece that is comprised of two calcium-binding EF-hand motifs. Since calcium-binding has been shown to be integral to regulating the activity of the three mammalian plastin proteins, we decided to study the properties of the headpiece regions of fimbrins from the model plant Arabidopsis thaliana, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and the amoeba Dictyostelium discoideum. Of these protein domains only the FimA headpiece from the amoeba protein possesses calcium binding properties. Structural characterization of this protein domain by multidimensional NMR and site-directed mutagenesis studies indicates that this EF-hand region of FimA also contains a regulatory 'switch helix' that is essential to regulating the activity of the human L-plastin protein. Interestingly this regulatory helical region seems to be lacking in the plant and yeast proteins and in fimbrins from all other nonmotile systems. Typical calmodulin antagonists can displace the switch-helix from the FimA headpiece, suggesting that such drugs can deregulate the Ca2+-regulation of the actin-bunding in the amoeba, thereby making it a useful organism for drug screening against mammalian plastins.

Perinatal iron deficiency causes sex-dependent alterations in renal retinoic acid signaling and nephrogenesis.

Long-term alterations in kidney structure and function have been observed in offspring exposed to perinatal stressors such as iron deficiency (ID), albeit the mechanisms underlying these changes remain unclear. Here, we assessed how perinatal ID alters renal vitamin A metabolism, an important contributor to nephrogenesis, in the developing kidney. Pregnant Sprague Dawley rats were fed either an iron-restricted or -replete diet throughout gestation, and offspring were studied on postnatal day (PD)1 and 28. Maternal iron restriction results in reduced renal retinoid concentrations in male and female offspring on PD1 (P=.005). Nephron endowment was reduced by 21% in male perinatal ID offspring (P<.001), whereas it was unaffected in perinatal ID females. Perinatal ID resulted in sex-dependent changes in kidney retinoid synthesis and metabolism, whereby male offspring exhibited increased expression of Raldh2 and Rar/Rxr isoforms, while females exhibited unchanged or decreased expression (all interaction P<.05). Male perinatal ID offspring exhibit sex-specific enhancements of retinoic acid pathway signaling components on PD1, including Gdnf (P<.01) and Ctnnb1 (P<.01), albeit robust upregulation of RA transcriptional target Stra6 was observed in both sexes (P=.006). On PD28, perinatal ID resulted in elevated renal retinoid concentrations (P=.02) coinciding with enhanced expression of Raldh2 (P=.04), but not any Rar isoform or Rxr. Further, perinatal ID resulted in robust upregulation of Gdnf, Ret, Ctnnb1, associated with further increases in both Cxcr4 and Stra6 (all P<.01) at PD28. Together, these data suggest perinatal ID results in sustained sex-dependent perturbations in vitamin A metabolism, which likely underlie sex-specific reductions in nephron endowment.

Maternal Iron Deficiency Alters Trophoblast Differentiation and Placental Development in Rat Pregnancy.

Iron deficiency, which occurs when iron demands chronically exceed intake, is prevalent in pregnant women. Iron deficiency during pregnancy poses major risks for the baby, including fetal growth restriction and long-term health complications. The placenta serves as the interface between a pregnant mother and her baby, and it ensures adequate nutrient provisions for the fetus. Thus, maternal iron deficiency may impact fetal growth and development by altering placental function. We used a rat model of diet-induced iron deficiency to investigate changes in placental growth and development. Pregnant Sprague-Dawley rats were fed either a low-iron or iron-replete diet starting 2 weeks before mating. Compared with controls, both maternal and fetal hemoglobin were reduced in dams fed low-iron diets. Iron deficiency decreased fetal liver and body weight, but not brain, heart, or kidney weight. Placental weight was increased in iron deficiency, due primarily to expansion of the placental junctional zone. The stimulatory effect of iron deficiency on junctional zone development was recapitulated in vitro, as exposure of rat trophoblast stem cells to the iron chelator deferoxamine increased differentiation toward junctional zone trophoblast subtypes. Gene expression analysis revealed 464 transcripts changed at least 1.5-fold (P < 0.05) in placentas from iron-deficient dams, including altered expression of genes associated with oxygen transport and lipoprotein metabolism. Expression of genes associated with iron homeostasis was unchanged despite differences in levels of their encoded proteins. Our findings reveal robust changes in placentation during maternal iron deficiency, which could contribute to the increased risk of fetal distress in these pregnancies.

Maternal and Cord Blood Hemoglobin as Determinants of Placental Weight: A Cross-Sectional Study.

BACKGROUND: Both high and low placental weights are associated with adverse pregnancy outcomes. Maternal hemoglobin levels can influence placental weight, but the evidence is conflicting. Since maternal hemoglobin does not invariably correlate with fetal/neonatal blood hemoglobin levels, we sought to determine whether cord blood hemoglobin or maternal hemoglobin status more closely associates with placental weight in women undergoing elective cesarean section at term. METHODS: This was a cross-sectional study conducted at the Royal Alexandra Hospital, Edmonton, Canada, involving 202 women with term singleton pregnancies undergoing elective cesarean section. Maternal blood and mixed cord blood hemoglobin levels were analyzed using a HemoCue Hb201+ system. Birth weight, placental weight, one- and five-minute APGAR scores, American Society of Anesthesiologists physical state classification, maternal age, and maternal height were also recorded. Relationships between maternal and cord blood hemoglobin levels with placental weight, birth weight, and birth weight to placental weight ratio were the main outcome measures. RESULTS: A total of 182 subjects were included in the analysis. Regression analysis showed that cord blood hemoglobin, but not maternal hemoglobin, was inversely related with placental weight (ß = -2.4, p = 0.001) and positively related with the birth weight to placental weight ratio (ß = 0.015, p = 0.001 and p = 0.63, respectively). CONCLUSIONS: Our findings suggest that measuring cord blood hemoglobin levels, rather than maternal hemoglobin levels, may provide important diagnostic information about in utero fetal adaptation to suboptimal placental function and neonatal health.

A systematic review and meta-analysis of the correlation between maternal and neonatal iron status and haematologic indices.

BACKGROUND: Iron deficiency (ID) is the leading single-nutrient deficiency in the world. Anaemia is a common outcome of ID that affects half of pregnancies worldwide with serious consequences for child development. Whether haematologic indices and biomarkers of iron status in pregnant women correlate with those of their neonates is unclear. This systematic review evaluated studies comparing haematologic and iron status indices in pregnant women and their newborns/neonates. METHODS: We searched MEDLINE, EMBASE, CINAHL, and Web of Science from database inception until March 2020 for primary studies comparing haematologic and iron status indices between women and their newborns up to 48 h after birth. We summarized the results descriptively and calculated pooled correlation coefficients in mothers and newborns/neonates using the Schmidt-Hunter method. The protocol was registered at PROSPERO International Prospective Register of Systematic Reviews (Registration number: CRD42018093094). FINDINGS: Sixty-five studies were included. Pooled correlation coefficients for biomarkers of iron status in mothers and newborns/neonates were 0.13 (ferritin), 0.42 (hepcidin), 0.30 (serum/plasma iron), 0.09 (transferrin), 0.20 (transferrin saturation), and 0.16 (total iron binding capacity). Pooled correlation coefficients for haematological indices in mothers and newborns/neonates were 0.15 (haemoglobin), 0.15 (haematocrit), 0.25 (mean cell/corpuscular haemoglobin), 0.22 (mean cell/corpuscular volume). INTERPRETATION: Maternal biomarkers of iron and haematologic status correlate poorly with those in newborns/neonates. These results underscore a need for alternative approaches to estimate foetal/neonatal iron status and haematological indices. FUNDING: MBO and SLB hold Canada Research Chairs, and grants from the Women and Children's Health Research Institute and Canadian Institutes of Health Research.

Perinatal iron deficiency and a high salt diet cause long-term kidney mitochondrial dysfunction and oxidative stress.

AIMS: Perinatal iron deficiency (ID) alters developmental trajectories of offspring, predisposing them to cardiovascular dysfunction in later life. The mechanisms underlying this long-term programming of renal function have not been defined. We hypothesized perinatal ID causes hypertension and alters kidney metabolic function and morphology in a sex-dependent manner in adult offspring. Furthermore, we hypothesized these effects are exacerbated by chronic consumption of a high salt diet. METHODS AND RESULTS: Pregnant Sprague Dawley rats were fed either an iron-restricted or replete diet prior to and throughout pregnancy. Adult offspring were fed normal or high salt diets for 6 weeks prior to experimentation at 6 months of age. Blood pressure (BP) was assessed via indwelling catheters in anaesthetized offspring; kidney mitochondrial function was assessed via high-resolution respirometry; reactive oxygen species and nitric oxide were quantified via fluorescence microscopy. Adult males, but not females, exhibited increased systolic BP due to ID (P = 0.01) and high salt intake (P = 0.02). In males, but not in females, medullary mitochondrial content was increased by high salt (P = 0.003), while succinate-dependent respiration was reduced by ID (P < 0.05). The combination of perinatal ID and high salt reduced complex IV activity in the cortex of males (P = 0.01). Perinatal ID increased cytosolic superoxide generation (P < 0.001) concomitant with reduced nitric oxide bioavailability (P < 0.001) in male offspring, while high salt increased mitochondrial superoxide in the medulla (P = 0.04) and cytosolic superoxide within the cortex (P = 0.01). Male offspring exhibited glomerular basement membrane thickening (P < 0.05), increased collagen deposition (P < 0.05), and glomerular hypertrophy (interaction, P = 0.02) due to both perinatal ID and high salt. Female offspring exhibited no alterations in mitochondrial function or morphology due to either high salt or ID. CONCLUSION: Perinatal ID causes long-term sex-dependent alterations in renal metabolic function and morphology, potentially contributing to hypertension and increased cardiovascular disease risk.

Characterization of the EF-Hand Calcium-Binding Domains of Human Plastins.

The three human plastins (L-plastin, T-plastin, and I-plastin) are important regulatory Ca2+-binding proteins that belong to the family of actin-binding proteins. Plastins are involved in the regulation of the actin cytoskeleton as well as the cross-linking of actin filaments. In addition to four calponin-homology (CH) domains, all three plastins contain two N-terminal EF-hand Ca2+-binding motifs which together are homologous to a single lobe of the well-known calcium-regulatory protein calmodulin. This part of the protein allows for the regulation of the actin bundling activity in response to elevated calcium levels. In this protocol, we describe the purification of the EF-hand headpiece domains of all three plastins, as well as SPR studies, ITC studies, and NMR interaction studies with different peptides and calcium. In combination, these three experimental techniques provide detailed insights into a novel regulatory mechanism, involving the linker region between the EF-hand domain and the first CH domain of the plastins.

The Calcium-Dependent Switch Helix of L-Plastin Regulates Actin Bundling.

L-plastin is a calcium-regulated actin-bundling protein that is expressed in cells of hematopoietic origin and in most metastatic cancer cells. These cell types are mobile and require the constant remodeling of their actin cytoskeleton, where L-plastin bundles filamentous actin. The calcium-dependent regulation of the actin-bundling activity of L-plastin is not well understood. We have used NMR spectroscopy to determine the solution structure of the EF-hand calcium-sensor headpiece domain. Unexpectedly, this domain does not bind directly to the four CH-domains of L-plastin. A novel switch helix is present immediately after the calcium-binding region and it binds tightly to the EF-hand motifs in the presence of calcium. We demonstrate that this switch helix plays a major role during actin-bundling. Moreover a peptide that competitively inhibits the association between the EF-hand motifs and the switch helix was shown to deregulate the actin-bundling activity of L-plastin. Overall, these findings may help to develop new drugs that target the L-plastin headpiece and interfere in the metastatic activity of cancer cells.

Modest and Severe Maternal Iron Deficiency in Pregnancy are Associated with Fetal Anaemia and Organ-Specific Hypoxia in Rats.

Prenatal iron-deficiency (ID) is known to alter fetal developmental trajectories, which predisposes the offspring to chronic disease in later life, although the underlying mechanisms remain unclear. Here, we sought to determine whether varying degrees of maternal anaemia could induce organ-specific patterns of hypoxia in the fetuses. Pregnant female Sprague Dawley rats were fed iron-restricted or iron-replete diets to induce a state of moderate (M-ID) or severe ID (S-ID) alongside respective controls. Ultrasound biomicroscopy was performed on gestational day (GD)20 to assess uterine and umbilical artery blood flow patterns. On GD21, tissues were collected and assessed for hypoxia using pimonidazole staining. Compared to controls, maternal haemoglobin (Hb) in M- and S-ID were reduced 17% (P < 0.01) and 48% (P < 0.001), corresponding to 39% (P < 0.001) and 65% (P < 0.001) decreases in fetal Hb. Prenatal ID caused asymmetric fetal growth restriction, which was most pronounced in S-ID. In both severities of ID, umbilical artery resistive index was increased (P < 0.01), while pulsatility index only increased in S-ID (P < 0.05). In both M-and S-ID, fetal kidneys and livers showed evidence of hypoxia (P < 0.01 vs. controls), whereas fetal brains and placentae remained normoxic. These findings indicate prenatal ID causes organ-specific fetal hypoxia, even in the absence of severe maternal anaemia.