Near to One's Heart: The Intimate Relationship Between the Placenta and Fetal Heart

The development of the fetal heart is exquisitely controlled by a multitude of factors, ranging from humoral to mechanical forces. The gatekeeper regulating many of these factors is the placenta, an external fetal organ. As such, resistance within the placental vascular bed has a direct influence on the fetal circulation and therefore, the developing heart. In addition, the placenta serves as the interface between the mother and fetus, controlling substrate exchange and release of hormones into both circulations. The intricate relationship between the placenta and fetal heart is appreciated in instances of clinical placental pathology. Abnormal umbilical cord insertion is associated with congenital heart defects. Likewise, twin-to-twin transfusion syndrome, where monochorionic twins have unequal sharing of their placenta due to inter-twin vascular anastomoses, can result in cardiac remodeling and dysfunction in both fetuses. Moreover, epidemiological studies have suggested a link between placental phenotypic traits and increased risk of cardiovascular disease in adult life. To date, the mechanistic basis of the relationships between the placenta, fetal heart development and later risk of cardiac dysfunction have not been fully elucidated. However, studies using environmental exposures and gene manipulations in experimental animals are providing insights into the pathways involved. Likewise, surgical instrumentation of the maternal and fetal circulations in large animal species has enabled the manipulation of specific humoral and mechanical factors to investigate their roles in fetal cardiac development. This review will focus on such studies and what is known to date about the link between the placenta and heart development.


INTRODUCTION
Since David Barker first documented the relationship between infant birth weight and adult onset disease (Barker and Osmond, 1986;Barker et al., 1989), there has been a revolutionary shift in thinking about how the early environment can impact life-long health and susceptibility to disease. The contribution of the placenta to this association and as an independent risk factor for future cardiovascular risk has more recently been identified. One of the first studies to link the placenta to cardiovascular disease was by Martyn and colleagues who identified that both the highest and lowest quintiles for placental efficiency (placenta weight as a proportion of birth weight) were associated with a greater number of deaths due to coronary heart disease in men born in the UK (Martyn et al., 1996). More recent studies of men born in Helsinki identified that a combination of maternal height, body mass index (BMI) and the shape of the placenta predict coronary heart disease in men (Eriksson et al., 2011). Specifically, short women who had a greater difference between the length and breadth of their placental surface were more likely to have a son with an increased risk of coronary heart disease. Likewise, tall women that either had a greater BMI and a placenta with a small surface area, or a lower BMI and reduced placental efficiency, conferred an elevated risk of coronary heart disease to their sons (Eriksson et al., 2011). Furthermore, having a thin placenta, or a large placenta area relative to birth weight, is associated with a greater incidence of sudden cardiac death in men and women, respectively (Barker et al., 2012). Altered placenta size and shape may be a reflection of a poor maternal environment, but may also contribute to a poor fetal environment. The placenta is the main interface between the mother and fetus, and regulates intrauterine development by supplying oxygen and nutrients required for fetal growth. There is now clear evidence that the placenta can adapt morphologically and functionally to supply signals arising from the mother, and demand signals from the fetus (Sferruzzi-Perri and Camm, 2016). The intricate relationship between the placenta and fetal heart is appreciated in instances of clinical placental pathology. Abnormal umbilical cord insertion (when the umbilical cord inserts abnormally into the fetal membranes instead of the center of the placenta), is associated with congenital heart defects (Albalawi et al., 2017). Likewise, twin-to-twin transfusion syndrome, where monochorionic twins have unequal sharing of their placenta due to inter-twin vascular anastomoses, results in one twin being under-perfused with blood and the other being over-perfused, can result in cardiac remodeling and dysfunction in both fetuses (Delabaere et al., 2016;Albalawi et al., 2017). Normally the placental circulation is considered one of low vascular resistance (Trudinger et al., 1985;Thompson and Trudinger, 1990), but in instances of poor placental development associated with fetal growth restriction, deficient remodeling of uterine spiral arteries can lead to malperfusion of the placenta and an increase in placental vascular resistance, which impair the placenta's endocrine and nutrient transport functions (For review, Chaddha et al., 2004;Burton and Jauniaux, 2018). As ∼45% of the combined ventricular output from the fetal heart is directed toward the placenta, an increase in placental vascular resistance may also increase cardiac afterload, thus increasing the mechanical force that the heart beats against. To date, the mechanistic basis of the relationships between the placenta, fetal Abbreviations: BMI, body mass index; CaMKII, calcium/calmodulin-dependent protein kinase II; E, embryonic day; ERK, extracellular signal-regulated kinase; GLUT4, glucose transport protein 4; HIF, hypoxia-inducible factor; IGF-1, insulin-like growth factor-1; IGF-1R, type 1 IGF receptor; IGF-2R type 2 IGF receptor/mannose-6-phosphate receptor; IR, insulin receptor; IUGR, intrauterine growth restriction; Jz, junctional zone; LV, left ventricle; LVDP, left ventricular developed pressure; LVEDP, left ventricular end diastolic pressure; Lz, labyrinthine zone; MAP, mean arterial pressure; NO, nitric oxide; NOS, nitric oxide synthase; PI, pulsitility index; PI3K, phosphoinositol-3 kinase; RV, right ventricle; SUAL, single umbilical artery ligation; T3, thyroid hormone (triiodothyronine); T4, thyroid hormone (thyroxine); TGF-β, transforming growth factor-beta; UPE, umbilico-placental embolization; VEGF, vascular endothelial growth factor. heart development and later risk of cardiac dysfunction have not been fully elucidated. However, studies using environmental exposures and gene manipulations in experimental animals are providing insights into the pathways involved.

ANIMAL MODELS OF ALTERED PLACENTATION, HYPOXAEMIA AND NUTRIENT RESTRICTION
Animal studies allow for manipulation of the placenta and the maternal and fetal environments to understand the mechanisms that underlie the placenta's influence on heart health. Of the animal models that describe both placental and heart phenotypes, there are broadly three groups that best categorize them: 1. Those that reduce oxygen and nutrient delivery to the fetus (carunclectomy, umbilico-placental embolization (UPE), single umbilical artery ligation (SUAL), maternal hyperthermia in sheep, unilateral uterine artery ligation in guinea pigs and bilateral uterine artery and vein ligation in rats; 2. Those that reduce oxygen availability for transfer to the fetus (maternal inhalation hypoxia in sheep, guinea pigs and rats); and 3. Those that alter nutrient availability for the fetus (global calorie restriction, low protein diet and high-fat/high-sugar diet). However, maternal hypoxia may result in reduced maternal food intake or alterations in the placenta's ability to deliver nutrients to the fetus. Likewise, altering the maternal diet may affect placenta development and thus decrease oxygen transfer capacity. For simplicity sake, the animal models have been divided into those that report fetal hypoxaemia ( Table 1) and those that do not ( Table 2). A key point highlighted by both tables is that changes to placental weight do not predict cardiac outcome as reduced, increased and unaltered placenta weight may all be associated with an altered cardiac phenotype in the offspring. It is clear that work is still required to characterize the morphometry and resource supply capacity of the placenta in animal models with cardiac phenotypes, and likewise cardiac phenotype in models with altered placentation.

ANIMAL MODELS WITH REPORTED FETAL HYPOXAEMIA
The most common consequence of complicated pregnancy is fetal hypoxaemia, which has been reported in human intrauterine growth restriction (IUGR) (Economides et al., 1991;Mori et al., 1993;Baschat et al., 2000). One of the more comprehensive sets of paired placenta and cardiac data in a model of altered placentation comes from the carunclectomy model in sheep. The removal of the majority of endometrial caruncles from the nonpregnant uterus results in the formation of less placentomes, and reduced total placental weight and uterine blood flow in subsequent pregnancies (Robinson et al., 1979;Jones et al., 1988). This model is one of placental insufficiency from conception, and results in fetal hypoxaemia, hypoglycaemia, hypoinsulinaemia, hypercortisolaemia, hypothyroidism [reduced thyroid hormones triiodothyronine (T3) and thyroxine (T4)], elevated catecholamines (noradrenaline and adrenaline) and reduced plasma insulin-like growth factor-1 (IGF-1; for review see Morrison, 2008). T3, cortisol and IGF-1 are important modulators of cardiomyocyte growth and maturation, which are discussed below. In this model, each placentome is modified to increase its surface area for exchange between the maternal and fetal circulations, however, the fetus is still growth restricted in late gestation (Zhang et al., 2016). The fetus has reduced umbilical blood flow (although an equivalent umbilical blood flow per kg of fetus compared to controls), and is normotensive (Owens et al., 1989;Edwards et al., 1999). One of the most notable changes in fetal heart development in the carunclectomy model is a reduction in the number of cardiomyocytes in late gestation (Botting et al., 2014). Sheep, like humans, begin the transition from proliferative cardiomyocytes to terminally differentiated cardiomyocytes in late gestation (Kim et al., 1992;Burrell et al., 2003;Jonker et al., 2007b). Consequently, changes to the endowment, but also in the function of cardiomyocytes in late gestation, may have consequences throughout life. Interestingly, despite the fetus being hypoxaemic in late gestation, the fetal heart is not hypoxic, nor does it have a greater percentage of apoptotic cardiomyocyte or a diminished percentage of cardiomyocytes in the cell cycle (Botting et al., 2014). This may be due to the adaptive increase in capillary density in the fetal heart, which may increase local oxygen supply to compensate for the placental insufficiency (Botting et al., 2014). There may also be other alterations in response to placental insufficiency that decrease oxygen demand by the fetal heart, such as an increase in anaerobic metabolism, thereby protecting it from further damage. Despite there being fewer and smaller cardiomyocytes in late gestation, each cardiomyocyte is larger relative to heart weight (Morrison et al., 2007). This may suggest an alteration in the regulation of cardiomyocyte hypertrophy in late gestation due to carunclectomy. After birth, lambs born with a low birth weight develop left ventricular hypertrophy by 3 weeks of age, and have a greater signaling through the pathological hypertrophy pathway, specifically the type 2 IGF/mannose-6-phosphate receptor (IGF-2R)/Gαq/calcium calmodulin-dependent protein kinase II (CaMKII) (Wang et al., 2011(Wang et al., , 2015b. Furthermore, the number of cardiomyocytes in the adult heart is positively correlated to birth weight (Vranas et al., 2017). Similarly, female guinea pigs exposed to maternal hypoxia have reduced cardiomyocyte number at 4 months of age, which highlights the life-long impact a reduction in cardiomyocyte endowment in utero may have (Botting et al., 2018). The consequence of these placenta-mediated changes on the function of the postnatal heart is yet to be determined. Further work is required to identify the contribution of low oxygen and glucose, as well as alterations in humoral factors, on the development of heart pathology in the offspring. By comparing the carunclectomy model to UPE, which is typically induced in the last trimester in sheep, a greater understanding of the specific role of humoral and haemodynamic factors that influence the fetal heart can be determined. Infusion of insoluble microspheres into the fetal descending aorta between the renal artery and common umbilical artery results in the blockage of vessels in the fetal portion of placentomes (cotyledons), which subsequently increases placental vascular resistance and reduces gas and nutrient exchange between the fetal and maternal circulations (Trudinger et al., 1987). Similar to the carunclectomy model, UPE results in fetal hypoxaemia and elevated plasma noradrenaline concentrations, but does not lead to a persistent elevation in fetal cortisol concentrations (Louey et al., 2000;Thompson et al., 2011) or hypoglycaemia (Thompson et al., 2011). UPE results in decreased total placenta weight and IUGR. In some studies, UPE results in an increase (Murotsuki et al., 1997) or no change (Louey et al., 2000;Thompson et al., 2011Thompson et al., , 2013 in fetal mean arterial pressure (MAP), which may be due to the timing and severity of UPE, as well as, the degree to which placental vascular resistance is increased.
Interestingly, UPE studies that report elevated fetal MAP also show an increase in relative heart weight (Murotsuki et al., 1997). This is in contrast to UPE studies that report normotensive fetuses, whose heart weights may be reduced but proportional to the reduction in fetal mass (Duncan et al., 2000;Thompson et al., 2013). The difference in fetal MAP between these UPE studies highlights that elevations in fetal MAP can promote cardiac hypertrophy (discussed below). Of note, even in the absence of an elevation in fetal MAP, UPE results in an increase in fetal cardiac fibrosis and an upregulation of collagen synthesis, likely through the transforming growth factor (TGF)-β pathway . Similar to the carunclectomy model, UPE results in delayed cardiomyocyte maturation, indicated by a reduced percentage of binucleated cardiomyocytes (Bubb et al., 2007;Morrison et al., 2007). However, unlike the carunclectomy model, UPE in late gestation does not alter cardiomyocyte size but decreases cell cycle activity, indicating that UPE may instead impair the proliferation of cardiomyocytes in the fetus 20 days after embolization commences (Louey et al., 2007). The differences in the cardiac phenotype between the carunclectomy and UPE models suggest that the timing of the insult in relation to placental development and cardiomyocyte maturation may help to identify the pathogenesis of cardiac pathology. Currently, the fetal and postnatal consequences of UPE on cardiac function are unknown. However, chronic fetal hypoxaemia in late gestation in sheep due to either maternal hypoxia or SUAL results in systolic and diastolic dysfunction in the isolated fetal heart (Brain et al., 2015), and greater infarct size due to ischaemia/reperfusion in the isolated newborn heart (Tare et al., 2014), respectively. Furthermore, an increase in collagen synthesis and evidence of left ventricular (LV) hypertrophy is seen in juvenile guinea pigs exposed to unilateral umbilical artery ligation from mid-gestation (Briscoe et al., 2004). Interestingly, if the insult to the placenta and fetus is shorter, but more severe, as is the case in the rat model of bilateral uterine artery and vein ligation, the cardiac phenotype with regards to the emergence of postnatal cardiac hypertrophy is less severe (heart weight relative to body weight is equivalent to controls at 2 and 6 months of age; Wadley et al., 2016). However, caution must be taken when making direct comparisons between sheep, guinea pigs, and humans relative to rats, given rat cardiomyocytes do not mature to become binucleated until after birth (binucleation occurring from 4 to 12 days after birth; Li et al., 1996). As such, rat cardiomyocytes may have a greater capacity to repair damage caused by in utero insults. For example, additional evidence from the same rat model of bilateral uterine vessel ligation demonstrates that a deficit in cardiomyocyte number may be corrected if a newborn is cross fostered onto a normal (sham) mother (Black et al., 2012).
Of the various sheep models of fetal hypoxaemia, one of the most documented placental phenotypes is that resulting from maternal hyperthermia. Housing pregnant ewes at 35-40 • C from ∼80 to 120 days of gestation results in reduced placental weight, decreased angiogenic signaling within the fetal portion of the placenta, and increased placental apoptosis compared to controls (Regnault et al., 2003;Monson et al., 2017). The maternal hyperthermia-induced placental restriction is likely due to the reported decrease in maternal uterine blood flow compared to controls. Interestingly, uterine blood flow is similar to controls when expressed per 100 g of placental and fetal weight . Fetuses whose mothers were exposed to hyperthermia in pregnancy have reduced umbilical blood flow when expressed as absolute or relative to either placental or fetal weight and have increased umbilical artery pulsatility index (PI) and resistance , likely due to increased placental vascular resistance. This results in either no change (Barry et al., 2016) or an increase (Galan et al., 2005;Regnault et al., 2007) in fetal MAP. Consequently, fetuses whose mothers were exposed to hyperthermia in pregnancy are hypoxaemic, hypoglycaemic, hypoinsulinaemic, hypercortisolaemic [males], and have elevated plasma noradrenaline levels compared to controls (Walker et al., 1990;Regnault et al., 1999Regnault et al., , 2007; a humoral profile much like the carunclectomy fetus in late gestation. The hyperthermia model also induces IUGR. To date, not much is known about the impact of placental changes induced by maternal hyperthermia on the fetal heart. However, the LV of fetuses whose mothers were exposed to hyperthermia in pregnancy have a greater insulin stimulated blood flow per gram of LV tissue and insulin stimulated glucose delivery and uptake, which relates to an increase in insulin receptor (IR) and glucose transport protein 4 (GLUT4) abundance [right ventricle (RV) not reported] (Barry et al., 2016). These adaptations may increase the chance of survival in a hypoxaemic, hypoglycaemic, and hypoinsulinaemic environment, but the consequence to the postnatal heart is currently not known.
The majority of information known about the effect of hypoxia on postnatal cardiac structure and function has been obtained from rodent studies, for which information on placental phenotype is also available. Reducing the fraction of oxygen in maternal inspired air from 21% to 10.5% from days 6 and 20 of gestation in rats and guinea pigs, respectively, results in decreased trophoblast invasion and spiral artery remodeling, coupled with an increase in maternal MAP (Zhou et al., 2013;Thompson et al., 2016). However, there are beneficial changes in placental morphology that would be expected to optimize oxygen delivery to the fetus in hypoxic dams, such as an increase in vascular density and a reduction in the barrier to oxygen diffusion (Thompson et al., 2016). Despite these adaptations, the fetal heart appears to remain hypoxic as indicated by the upregulation of hypoxia-inducible factors (HIFs), markers of nitrosative damage, and reduced mitochondrial function (Thompson et al., 2009;Evans et al., 2012a;Al-Hasan et al., 2013). This fetal cardiac phenotype could also be related to impaired coronary artery function due to altered nitric oxide (NO) availability [disturbed expression of cardiac NO synthase (NOS)] (Thompson et al., 2004(Thompson et al., , 2009Thompson and Dong, 2005;Dong and Thompson, 2006). Maternal hypoxia (10.5%) in rats from 15 to 21 day gestation induces IUGR, decreases fetal heart weight and cardiomyocyte cell cycle activity, increases cardiomyocyte apoptosis, and prematurely promotes cardiomyocyte quiescence (binucleation) (Bae et al., 2003;Paradis et al., 2014Paradis et al., , 2015. Furthermore, offspring of mothers exposed to hypoxia in pregnancy are more susceptible to myocardial infarction and ischaemia/reperfusion injury Xu et al., 2006;Xue and Zhang, 2009;Rueda-Clausen et al., 2011;Shah et al., 2017).
Interestingly, the presence of IUGR is not required for maternal hypoxia in rats to programme altered cardiac phenotypes. Maternal hypoxia (13%) from 6 to 20 days of gestation does not alter fetal weight compared to normoxic controls, but results in differences in LV contractility and responsiveness to α 1 -adrenergic and muscarinic agonists at 4 months of age . Additionally, maternal hypoxia results in programmed vascular dysfunction in the offspring irrespective of birth weight (Morton et al., 2010;Giussani et al., 2012;Bourque et al., 2013;Brain et al., 2015).
From the aforementioned studies, oxygen clearly plays an important role in both placental and heart development, however, more information is needed to understand the direct effect oxygen plays in these associations. Moreover, the role of changes in both oxygen and nutrient availability on the placenta and heart may be more informative in the context of complicated human pregnancies, as both substrates are often altered.

ANIMAL MODELS WITH FETAL NUTRIENT ALTERATIONS, BUT NO REPORTED FETAL HYPOXAEMIA
An observation from the Dutch Hunger Winter Study has been the importance of timing in the programming of adult disease (Roseboom et al., 2006). Babies exposed to the famine during late gestation were born small and remained small throughout their lives, with lower rates of obesity as adults than those born before and after the famine. Conversely, babies exposed during early gestation experienced elevated rates of obesity and cardiovascular disease in later life. The Dutch Hunger Winter therefore provided valuable insight into how dietary manipulation during specific periods of development can influence subsequent health. This concept of "critical windows" during development has been tested in several different animal experimental models. Experimental studies that have manipulated maternal calorie intake or quality during pregnancy, and show alterations in both placenta and cardiovascular morphology and function, are outlined in Table 2. Overall, these studies show that the specific effects on the placenta or fetal heart depend on the type of challenge, as well as, the duration, severity and timing relative to the formation of these two organs.
Maternal calorie restriction (10-50%) and low-protein diets (6-9%) in mice, rats and guinea pigs, typically reduce placenta weight, as well as regional weights and/or volumes of the transport labyrinthine zone (Lz) and endocrine junctional zone (Jz) ( Table 2). These changes are related to reduced formation of maternal blood spaces and fetal vasculature in the exchange region (Roberts et al., 2001a,b;Rutland et al., 2007), potentially mediated through vascular endothelial growth factor (VEGF) signaling (Liu et al., 2014) and/or an increase in apoptosis in the Lz (Belkacemi et al., , 2011b. In addition to the effects on placental morphology, maternal undernutrition induces mitochondrial abnormalities in the placenta (Belkacemi et al., 2011b;Mayeur et al., 2013;Rebelato et al., 2013). Mitochondria are implicated in numerous critical functions for feto-placental development, including ATP production for placental growth, production of oxidative stress and hormones, and control of apoptosis (Myatt, 2006;Wakefield et al., 2011). Mitochondrial defects may modify placental activity, and could therefore contribute to the restriction of both fetal and placental growth following calorie restriction. The expression of nutrient transporters (Lesage et al., 2002;Belkacemi et al., 2011c;Reynolds et al., 2015), growth factors (Woodall et al., 1996a;Gao et al., 2012a), appetite-and metabolism-regulating peptides (Caminos et al., 2008;Mayeur et al., 2016), angiotensin-converting enzymes (Gao et al., 2012b) are also altered by calorie and protein restriction and may contribute to suboptimal fetal growth and the associated programming of adulthood hypertension in these models. The ability of the placenta to act as a barrier to circulating maternal hormones is also affected by the maternal environment. Both calorie and protein restriction in rodents alters the placental expression of 11β-hydroxysteroid dehydrogenases type 1 and 2 (Langley-Evans et al., 1996;Bertram et al., 2001;Lesage et al., 2001;Belkacemi et al., 2011c), which activate and inactivate circulating glucocorticoids, respectively. Glucocorticoids have direct effects on the heart and vasculature (Walker, 2007). Therefore, increased fetal glucocorticoid exposure due to loss of the placenta glucocorticoid barrier will adversely affect both fetal growth and cardiovascular development before birth. Maternal low protein diets or global calorie restriction, have been shown to increase systolic blood pressure or MAP in adult offspring ( Table 2). The degree to which blood pressure is elevated varies with the specific nutritional challenge and potentially the extent of remodeling of the aorta and extracellular matrix (Khorram et al., 2007a(Khorram et al., ,b,c, 2010, impairment in mitochondrial oxidative phosphorylation (Nascimento et al., 2014) and changes in the expression of genes and miRNAs involved in cardiac energy metabolism (Slater-Jefferies et al., 2011). Further, adult offspring who are hypertensive may also be more vulnerable to ischaemia/reperfusion injury, as seen in the 9% protein restriction model (Elmes et al., 2008). Alterations in the reactivity of resistance arteries to vasodilators or constrictors, may also contribute to elevated blood pressure in adult offspring Torrens et al., 2003Torrens et al., , 2006Torrens et al., , 2008Sathishkumar et al., 2009Sathishkumar et al., , 2015. A maternal low protein diet results in a reduction in heart weight and endowment of cardiomyocytes at birth (Corstius et al., 2005). However, if protein restriction continues throughout lactation, during the period of cardiomyocyte maturation in rats, cardiomyocyte endowment is similar to controls at weaning (Lim et al., 2010).
In addition to undernutrition, excess calories during pregnancy can also affect the placenta and offspring heart. Maternal high-fat or high-fat/high-sugar diets have been associated with both unchanged (Fernandez-Twinn et al., 2006Blackmore et al., 2014;Reynolds et al., 2015) and reduced fetal and placental weights (Reynolds et al., 2014, depending on the length of exposure to the obesogenic diet. A maternal high-fat/high-sugar diet increases placental lipid deposition (Fernandez-Twinn et al., 2017), expression of HIF1α (Fernandez-Twinn et al., 2017) and pro-inflammatory mediators (Reynolds et al., 2014) and alters nutrient transport in a sex-specific manner (Reynolds et al., 2014). While inflammatory processes are essential for pregnancy progression and maintenance, dysregulation of immune function is a major contributor to pregnancy-related disorders (Denison et al., 2010). However, feeding an obesogenic diet during pregnancy has been shown to result in a reduced placental fetal capillary volume (Sferruzzi-Perri et al., 2013), which would impair fetal oxygen delivery (Kulandavelu et al., 2013), thereby contributing to the hypoxia-mediated response to maternal obesity. The increase in the expression of glucose and fatty acid transporters in only male fetuses by Reynolds et al. (2014), suggests an attempt to compensate for the diet-induced placental insufficiency. A maternal high fat or high-fat/high-sugar diet is associated with increases in systolic and diastolic blood pressure, left ventricular end diastolic pressure (LVEDP), and a decrease in left ventricular developed pressure (LVDP), in young adolescent and adult offspring. A decreased LVDP and increased LVEDP, indicative of decreased ventricular compliance and impaired relaxation, respectively, is most likely related to cardiac hypertrophy (Fernandez-Twinn et al., 2012;Blackmore et al., 2014), which have been determined in the high-fat/high-sugar murine model using molecular and stereological techniques. Further work is required to characterize the fetal origins of the cardiac abnormalities observed in adult offspring of high-fat/high-sugar fed dams.

GENETIC MODELS
Studies performed in genetically-modified mice have started to provide novel insights into the regulation of, and relationship between, fetal heart development and placental formation (Table 3). Indeed, findings of mutant mice suggest that the formation of the fetal heart requires many of the same genes that regulate the development of the placenta (e.g., Hand1, Firulli et al., 1998;Riley et al., 1998). The Mouse Genome Informatics database identifies 329 genes with both placental morphology and cardiovascular defects (search identifies 754 mutants when using broader term, extraembryonic tissue morphology in conjunction with cardiovascular; conducted on 04 February, 2018). A selection of these genes are listed in Table 3 (e.g., Hey1/2, Mekk3, Gab1, Hai1, Flrt2, Phd2, Cited2, Ovol1, Vcam1, Mmp14/16). Malformations of the heart and placenta are the most commonly cited reasons for mid-gestational lethality. Heart defects also arise at around day 10 of pregnancy, when organogenesis becomes highly dependent on placental function. Previous work has largely focussed on assessing the impact of a genetic manipulation on either the formation of the placenta or the fetal heart, rather than considering an interaction between the two. In spite of several of the genes listed in Table 3 being expressed in both the fetal heart and the placental cell lineages, the temporal expression and order of developmental defects have not always been accurately determined. However, some findings in mice comparing the fetal heart and placental expression of genes with respect to the time scale of development of defects, as well as, selective gene targeting strategies, have highlighted that fetal heart defects may arise secondary to placental abnormalities and/or insufficiency.
Loss of the homeobox gene transcription factor, Hoxa13, results in defective vascularization and formation of the placental labyrinthine (exchange) zone (Shaut et al., 2008), lethality from days 11 of gestation (Shaut et al., 2008;Scotti and Kmita, 2012) and thinning of the fetal ventricle walls. Interestingly, Hoxa13 is expressed in cell lineages that will form the placenta, but is absent from the fetal heart (Shaut et al., 2008). A deficiency in the zinc finger transcription factor, Ovol2 also causes abnormalities in both placenta and fetal heart development (Unezaki et al., 2007). Although, the Ovol2 gene is primarily expressed by the chorion and placental trophoblast and only lowly expressed by the fetal heart when cardiac abnormalities arise (Unezaki et al., 2007). Collectively, these data suggest that malformations of the fetal heart may be a consequence of defects in placental development.
During the establishment of normal circulation, myocardial development and cardiac morphogenesis depend on the patterns of blood flow returning from the yolk sac and chorioallantoic placenta (Linask et al., 2014). Therefore, placental abnormalities may disrupt cardiac and vascular development by altering the haemodynamic forces of blood returning to the heart and result in fetal demise (Linask et al., 2014). In support of this, retaining expression of RNA binding gene, Ott1/Rbm15 or the transcriptional regulator Rb gene in the placenta is sufficient to rescue the lethality of null fetuses (Wu et al., 2003;Raffel et al., 2009). Furthermore, the loss of placental, but not fetal  Barak et al., 1999 (Continued) Frontiers in Physiology | www.frontiersin.org Frontiers in Physiology | www.frontiersin.org expression of the transcription factor genes, E2f7 and E2f8, leads to fetal vascular dilatation, multifocal hemorrhages and lethality (Ouseph et al., 2012). However, the placenta is also thought to be responsive to blood flow forces in the fetal circulation (Linask et al., 2014). Although, very little is known about the importance of the developing fetal heart for the formation of the placenta (e.g., the consequence of cardiac-specific deficiency for placentation).

HOW HAEMODYNAMIC CHANGES INFLUENCE THE HEART
Studies in fetal sheep have investigated the specific effects of altered load on the fetal heart in normoxic and euglycaemic fetuses. Specifically, increasing left ventricular afterload by partially obstructing the ascending aorta results in a thicker LV/RV wall and smaller LV chamber volume compared to control (Fishman et al., 1978). This phenomenon of left ventricular hypertrophy in response to increased afterload is seen in adults, and is a mechanism to normalize wall stress according to the law of LaPlace. In adults who have quiescent cardiomyocytes, this increase in cardiac mass is predominantly due to an increase in cardiomyocyte hypertrophy (for review, Samuel and Swynghedauw, 2008). Initially it was proposed that the increase in fetal heart mass in response to an increase in afterload was due to an increase the number of cardiomyocytes (hyperplasia) (Fishman et al., 1978). Further investigations by Jonker and colleagues determined that cardiac growth in response to increased fetal MAP and venous pressure is biphasic, initially due to cardiomyocyte hyperplasia and elongation and subsequently due to hyperplasia, premature binucleation and hypertrophy of binucleated cardiomyocytes (Jonker et al., 2007a). This phenomenon is not isolated to the LV, with an increase in pulmonary artery pressure resulting in an increase in RV weight (Segar et al., 1997). The converse is also true-obstructing blood flowing into the LV (decreasing preload) results in a smaller heart with a reduced LV/RV weight (Jonker et al., 2007a). By decreasing fetal systolic pressure with an angiotensin-converting enzyme inhibitor, O'Tierney and colleagues determined that the fetal heart is reduced in size due to a decrease in hyperplasia and not due to alteration in cardiomyocyte size (O'Tierney et al., 2010).

HOW HUMORAL FACTORS INFLUENCE CARDIOMYOCYTES EITHER IN VIVO OR IN VITRO
Treatment of fetuses in vivo or isolated fetal cardiomyocytes with growth factors and hormones, whose concentrations may be altered by the placenta, allows for greater understanding of how the placenta may influence the fetal heart.

IGF-1
IGF-1 is an important growth-promoting hormone that is produced by many tissues and functions throughout fetal and FIGURE 1 | Schematic summary showing how the placenta and fetal heart may influence each other and how the maternal environment may modulate the placenta-fetal heart axis, and the structure and function of both organs. Examples of genes that regulate the development of both the placenta and heart are listed.
postnatal development in an autocrine/paracrine fashion. IGF-1 primarily promotes growth through the type 1 IGF receptor (IGF-1R) and downstream signaling pathways, including extracellular signal-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K). The carunclectomy model in sheep (Jones et al., 1988) and undernutrition across gestation in rats (Woodall et al., 1996a), decreases fetal plasma IGF-1 concentration in late gestation. Varying results from in vivo experiments in fetal sheep suggest that IGF-1 can either promote cardiac growth by hypertrophy (Lumbers et al., 2009) or hyperplasia (Sundgren et al., 2003). Likewise, treating fetal sheep cardiomyocytes with a form of IGF-1 in vitro results in either greater (Wang et al., 2012) or equivalent (Sundgren et al., 2003) cardiomyocyte hypertrophy compared to serum-free controls. Treating neonatal rat cardiomyocytes with IGF-1 results in a similar variation of results with either cardiomyocyte hypertrophy (Bass et al., 2012) or hyperplasia (Kajstura et al., 1994) reported. Despite the inconsistency between results, IGF-1 has consistently been reported to promote fetal cardiac growth, therefore, reduced plasma concentration may in part contribute to the smaller hearts observed in fetuses from the carunclectomy and undernutrition animal models.

Cortisol
Cortisol is an important regulatory signal during fetal development, which amongst other important roles, acts to mature the cardiovascular system in preparation for birth (for review, Fowden and Forhead, 2015). Fetuses exposed to placental insufficiency due to carunclectomy  or maternal hyperthermia [males only] (Walker et al., 1990), have increased plasma cortisol concentrations compared to controls in late gestation. Cortisol infusion to fetal sheep in late gestation results in a greater heart weight accompanied by either an increase in cell cycle activity (Giraud et al., 2006;Feng et al., 2013), increased cardiomyocyte hypertrophy (Lumbers et al., 2005), or decreased DNA content in the left ventricle (Rudolph et al., 1999). Due to the inconsistency in results, it is currently unclear how an increase in cortisol may affect the fetal heart in models of placental insufficiency. However, research into the effect of other humoral factors that are regulated by cortisol, such as thyroid hormone, appear clearer.

Thyroid Hormone
Thyroid hormones, especially T3, promote the maturation of a range of organs (For review, Forhead and Fowden, 2014). T4 is produced by the fetal thyroid gland and is converted to the more active T3 in late gestation. The conversion of T4 to T3 is catalyzed by deiodinases, which are upregulated by cortisol. As such, the surge in plasma T3 concentration is concurrent with the prepartum surge in plasma cortisol concentrations. T3 infusion to fetal sheep prior to the prepartum surge in T3, results in increased cardiomyocyte binucleation [a sign of increased maturation] and decreased cardiomyocyte cell cycle activity compared to controls (Chattergoon et al., 2012a). Furthermore, surgical ablation of the fetal thyroid gland results in reduced fetal cardiomyocyte binucleation and cell cycle activity (Chattergoon et al., 2012a). In vitro, T3 inhibits the proliferation of cardiomyocytes isolated from hearts either before or during the prepartum surge in T3 concentration (Chattergoon et al., 2007(Chattergoon et al., , 2012b). The carunclectomy model in sheep results in reduced fetal T3 and T4 plasma concentrations in late gestation (Harding et al., 1985). These studies provide evidence that the decreased percentage of binucleated cardiomyocytes observed in the fetal heart from the carunclectomy (Morrison et al., 2007) and UPE (Bubb et al., 2007) models may be due to reduced plasma T3 concentrations.

CONCLUSION
Epidemiological and clinical studies suggest a link between placental morphology and increased risk of cardiovascular disease in adult life. The mechanistic basis of this relationship has not been fully elucidated. However, experimental animal models and studies in genetically-modified mice, have provided novel insights into the relationship between placental formation and fetal heart development and the role humoral and mechanical forces play in the development of both of these organs (Figure 1). Further work characterizing placental morphology (e.g., surface area, thickness) and function (e.g., umbilical blood flow, oxygen and nutrient delivery) during complicated pregnancy, alongside echocardiographic measures of fetal cardiac structure, and function, will provide valuable insights into the placenta-heart axis. Such research may aid in the early diagnosis and monitoring of complicated pregnancies thus enabling timely interventions to modify longterm cardiovascular risk.

AUTHOR CONTRIBUTIONS
EC, KB, and AS-P contributed equally to reviewing the literature and writing and editing the manuscript.

AS-P is supported by a Royal Society Dorothy Hodgkin Research
Fellowship.