maternal nutrient restriction alters gene expression in the ovine fetal heart

J Physiol 558.1 (2004) pp 111–121 111

Maternal nutrient restriction alters gene expressionin the ovine fetal heart

Hyung-Chul Han1, Kathleen J. Austin1, Peter W. Nathanielsz2, Stephen P. Ford1,Mark J. Nijland2 and Thomas R. Hansen1

1Center for the Study of Fetal Programming and Department of Animal Science, University of Wyoming, Laramie, WY 82071, USA2Department of Obstetrics and Gynecology, School of Medicine, New York University, New York, NY 10016, USA

Adequate maternal nutrient supply is critical for normal fetal organogenesis. We previouslydemonstrated that a global 50% nutrient restriction during the first half of gestation causescompensatory growth of both the left and right ventricles of the fetal heart by day 78 ofgestation.Thus,itwashypothesizedthatmaternalnutrientrestrictionsignificantlyalteredgeneexpression in the fetal cardiac left ventricle (LV). Pregnant ewes were randomly grouped intocontrol (100% national research council (NRC) requirements) or nutrient-restricted groups(50% NRC requirements) from day 28 to day 78 of gestation, at which time fetal LV werecollected. Fetal LV mRNA was used to construct a suppression subtraction cDNA library fromwhich 11 cDNA clones were found by differential dot blot hybridization and virtualNorthern analysis to be up-regulated by maternal nutrient restriction: caveolin, stathmin,G-1 cyclin, α-actin, titin, cardiac ankyrin repeat protein (CARP), cardiac-specificRNA-helicase activated by MEF2C (CHAMP), endothelial and smooth muscle derivedneuropilin (ESDN), prostatic binding protein, NADH dehydrogenase subunit 2, and anunknown protein. Six of these clones (cardiac α-actin, cyclin G1, stathmin, NADHdehydrogenase subunit 2, titin and prostatic binding protein) have been linked to cardiachypertrophy in other species including humans. Of the remaining clones, caveolin, CARP andCHAMP have been shown to inhibit remodelling of hypertrophic tissue. Compensatory growthof fetal LV in response to maternal undernutrition is concluded to be associated with increasedtranscription of genes related to cardiac hypertrophy, compensatory growth or remodelling.Counter-regulatory gene transcription may be increased, in part, as a response to moderatingthe degree of cardiac remodelling. The short- and long-term consequences of these changes infetal heart gene expression and induction of specific homeostatic mechanisms in response tomaternal undernutrition remain to be determined.

(Received 21 January 2004; accepted after revision 4 May 2004; first published online 7 May 2004)Corresponding author T. Hansen: Department of Animal Science, University of Wyoming, Laramie, WY 82071, USA.Email: [email protected]

Compelling epidemiological and animal studies havedemonstrated that nutrient supply during early gestationis critical for the development of fetal organogenesis(Reynolds & Redmer, 1995). Undernutrition duringthe first half of gestation impairs fetal and placentalgrowth and alters the trajectory of development inmammalian species (Barker, 1995; Godfrey & Robinson,1998; Godfrey & Barker, 2000). In humans (Barker, 1994;Godfrey & Barker, 2000) and rodents (Langley & Jackson,1994), offspring from undernourished mothers have apredisposition to obesity, diabetes and cardiovasculardisease in adult life. Low weight or thinness at birthin human neonates is associated with increased risk

of cardiovascular and metabolic disorders in later life(Barker et al. 1993; Martyn et al. 1996; Stein et al. 1996).The ‘fetal origins’ hypothesis proposes that suboptimalconditions experienced by the fetus (e.g. nutritionaldeprivation or excess hormonal exposure) result in analtered trajectory of development that can permanentlychange structure, physiology and metabolism, therebypredisposing individuals to cardiovascular, metabolic andendocrine disease in adult life (Barker, 1995). The processwhereby the fetus compensates for a maternal insult(undernutrition, stress, etc.) at a sensitive or critical periodof fetal development with consequential long-term effectshas been termed fetal programming (Lucas, 1991). In

C© The Physiological Society 2004 DOI: 10.1113/jphysiol.2004.061697

112 H.-C. Han and others J Physiol 558.1

evolutionary terms, this phenomenon is likely to reflectthe benefits of developmental flexibility by the fetus,allowing for short-term survival. Such adaptations that arebeneficial for short-term fetal survival may be detrimentalto health in later life (Stein et al. 1996; Barker, 1998). Theconcept of fetal programming arose from epidemiologicalstudies carried out by Barker and his coworkers whostudied birth records of babies born in the United Kingdombetween 1910 and 1930 and related their weight andphysical characteristics at birth to their subsequent healthstatus in later life (Barker et al. 1989, 1993). Early workfocused on the cardiovascular system, where they foundincreased risk of cardiovascular disease was associatedwith low birth weight. Experimental studies using animalshave documented the effects of restricting the placentalnutrient supply during gestation on fetal organ systemsand cardiovascular responsiveness (Thureen et al. 1992;Galan et al. 1998; Symonds et al. 2003; Vonnahme et al.2003). Studies of the cardiovascular system have tendedto focus on the peripheral vasculature (Ozaki et al. 2000;Nishina et al. 2003; Brawley et al. 2004) but there is verylittle information available on the effects of maternalundernutrition on the heart itself. In the present study weconducted dietary restriction to 50% NRC requirement(50% total digestible nutrient (TDN)) in pregnant ewesfrom early (day 28) to mid (day 78) gestation, the timeof organ system development, differentiation and rapidplacental growth. We have previously demonstrated thatthis level of maternal undernutrition caused compensatorygrowth of the left ventricular heart by day 78 of gestationwhen compared to controls (Vonnahme et al. 2003).To further understand the impact of maternal under-nutrition on the developing fetal heart, the presentstudy focuses on the results of a global screen of genetranscription in the ovine fetal cardiac left ventricle (LV)by utilizing subtractive cDNA library and differentialscreening approaches.

Methods

Animal care and tissue collection

All animal procedures were approved by the Universityof Wyoming Animal Care and Use Committee. Thirteenmultiparous ewes of mixed breeding were housed inindividual pens in a confinement building with controlledtemperature (13–16◦C) and light (12 h light/day). Eweswere synchronized for oestrus with two injections ofLutalyse (UpJohn, Kalamazoo, MI, USA) 10 days apartand bred at 12 h intervals from the beginning ofoestrus by one of two intact rams. On day 20 of

gestation, ewes were weighed so that individual dietscould be calculated based on metabolic body weight(weight0.75). The diet consisted of a pelleted beet pulp(79.7% TDN, 93.5% dry matter (DM) and 10.0% crudeprotein). Final diets were calculated on a DM basis forTDN required for maintenance for an early pregnantewe (metabolic weight × 3.07% NRC requirements). Amineral–vitamin mixture consisting of 51.43% sodiumtriphosphate, 47.62% potassium chloride, 0.39% zincoxide, 0.06% cobalt acetate and 0.50% ADE vitaminpre-mix (17 636 800 IU vitamin A, 1 763 680 IU vitaminD3 and 898 400 IU vitamin E per kg; amount ofvitamin pre-mix was formulated to meet the vitamin Arequirements) was included with the beet pulp pellets tomeet requirements. On day 21 of gestation, all ewes wereplaced in individual pens and fed maintenance diets. Onday 28, ewes were weighed and then randomly assignedto the control-fed (n = 7; fed 100% NRC requirementswhich included 100% mineral–vitamin pre-mix) and thenutrient-restricted (n = 6; fed 50% NRC requirementswhich included 50% mineral–vitamin pre-mix) groups.At 7-day intervals, ewes were weighed and rations wereadjusted for weight gain or loss. On day 45 of gestation,the numbers of fetuses carried by each ewe was determinedby ultrasonography (Ausonics Microimager 1000 sectorscanning instrument; Ausonics Pty Ltd, Sydney, Australia).On day 78 of gestation, gravid uterine tissue and fetuseswere removed. Data were collected for each fetus andincluded total weight, crown-rump length, abdominalcircumference, sex and weights of liver, pancreas, lung,kidney, adrenal, LV, right ventricle (RV) and septum ofthe heart. The LV heart sample was snap frozen in liquidnitrogen.

Control fed ewes produced four sets of twins and threesingles. One set of control fed twins was deleted fromthe analysis because fetal LV and RV weights were > 3.3standard deviations from the mean. Also, one fetus froma different set of control fed twins was included fordetermination of heart weights (Table 1), but was deletedfor RNA processing because of a contaminated LV sample.Thus, five twins and three singles provided LV RNA fromcontrol fed ewes for the subtractive cDNA library (n = 8).Two sets of twins and four singles provided fetal LV RNAfrom nutrient-restricted ewes for the subtractive cDNAlibrary (n = 8).

Total RNA extraction

Total RNA was extracted from 100 mg of LV homogenizedin 1 ml Tri Reagent (Molecular Research Inc., Cincinnati,OH, USA). Homogenates were incubated at 25◦C for

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J Physiol 558.1 Undernutrition alters fetal heart gene expression 113

Table 1. Fetal heart weights (g) from conceptuses on day 78 of gestation from control-fed andnutrient-restricted ewes (mean ± S.E.M.)

Fetal heart weight (g) Organ wt/fetus wt (%)

Control Restricted Control RestrictedItem n = 9 n = 8 P n = 9 n = 8 ∗P

Fetus 319.1 ± 10.78 221.2 ± 11.96 0.0002 — — —LV 0.94 ± 0.07 0.94 ± 0.07 NS 0.29 ± 0.02 0.42 ± 0.02 0.0041RV 0.66 ± 0.04 0.61 ± 0.05 NS 0.21 ± 0.02 0.27 ± 0.02 0.0245Septum 0.43 ± 0.04 0.39 ± 0.04 NS 0.14 ± 0.01 0.17 ± 0.01 0.0773

∗P-value control versus restricted. Data were adopted, in part, from Vonnahme et al. (2003).

10 min, mixed with 0.2 ml chloroform, and incubated at25◦C for an additional 15 min. The homogenate was thencentrifuged at 12 000 g , for 15 min, at 4◦C, to removethe cellular debris. The RNA in the supernatant was pre-cipitated by adding isopropanol and incubated for 10 minat 25◦C. RNA was pelleted by centrifugation at 12 000 g , for15 min, at 4◦C. Pellets were washed with cold 75% ethanol,dried and resuspended in sterile diethylpyrocarbonate-treated water.

Construction of a suppression subtractioncDNA library

Total LV RNA was pooled by treatment group. Pooledfetal LV in each treatment group included four fromtwins and four from singles. Pooled nutrient-restrictedRNA served as tester and control-fed RNA served asa driver for construction of the subtractive cDNAlibrary. First strand cDNA was prepared using theSMART PCR cDNA synthesis kit (Clontech, Palo Alto,CA, USA), including SMART II A Oligonucleotide(5′-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3′)and 3′-SMART CDS Primer II A(5′-AAGCAGTGGTATCAACGCAGAGTACT(30)N−1N-3′;N = A, C, G or T; N−1 = A, G or C). Reverse transcriptionwas done using PowerScript reverse transcriptase andtemperatures of 70◦C for 2 min followed by 42◦C for 1 h.PCR for cDNA amplification was performed using 95◦C15 s, 60◦C 30 s and 68◦C 6 min. The number of PCR cycles(18 cycles) was optimized to ensure that cDNA productwas in the exponential phase of amplification. OptimizedcDNA was collected for generation of the library following18 cycles because this represented one cycle prior to theplateau in amplification. PCR-amplified cDNAs werepurified using CHROMA SPIN-1000 columns (Clontech,Palo Alto, CA, USA) and were then digested with RsaIat 37◦C for 3 h. Restriction of cDNA was confirmedby electrophoretic resolution on 1.5% agarose gels.Blunt-end-digested cDNA was purified by extraction withphenol–chloroform–isoamyl alcohol followed by ethanol

precipitation. Tester and driver cDNAs were ligated withtwo adaptors in separate reactions. An excess of drivercDNA was added to each adaptor-ligated tester cDNA,heat denatured at 98◦C for 1.5 min and allowed to annealat 68◦C for 8 h. Enriched cDNAs were then subjectedto a second hybridization at 68◦C overnight with freshdenatured driver cDNA to further enrich for differentiallyexpressed genes. Subtracted genes were amplified by twosubsequent PCR reactions. Primary PCR was performedat 94◦C 30 s, 66◦C 30 s, and 72◦C 1.5 min. Secondary PCRwas performed with 10× diluted primary PCR productsat 94◦C 30 s, 68◦C 30 s and 72◦C 1.5 min

Screening of differentially expressed cDNA

Amplified cDNA products from the cDNA subtractionwere ligated into pCR II vector (Invitrogen, Carlsbad, CA,USA) at 14◦C for 16 h. One Shot competent cells weretransformed with the ligation mixture and incubatedat 37◦C for 24 h on LB agar plates containing X-galand isopropyl-1-thio-β-d-galactopyranoside (IPTG).Colonies were randomly picked, grown in 96-well platescontaining 100 µl of LB-amp medium for 2 h at 37◦C withshaking. Subtracted cDNA in each bacterial culture wasamplified by PCR with corresponding primers generatedfrom the adapters used in hybridization. Five microlitresof PCR product was denatured by adding 0.6 N NaOH.Then, 1 µl of denatured PCR product was transferred tofour Nytran membranes (Schleicher and Schuell, Keene,NH, USA). The blots were neutralized in 0.5 m Tris-HCl(pH 7.5) and washed in water. Blots were baked at 80◦Cfor 2 h. Blots were hybridized with control subtracted andnutrient-restricted subtracted 32P-labelled cDNA at 42◦Cfor 18 h, and after stringent washing were placed on film(Bioworld, Atlanta, GA, USA).

Virtual Northern blot analysis

To confirm the dot blot screening, cDNA was generated asmentioned above and 5 µg of cDNA was loaded onto 1.5%



agarose gels in Tris-acetate EDTA (TAE) buffer. The cDNAwas transferred to 0.2 µm Nytran membranes by capillarymethod in 10 × SSC at 25◦C for 24 h. Blots were baked at80◦C for 2 h, prehybridized for 3 h in 50% formamide, 5 ×SSC, 0.05 m sodium phosphate, 5 × Denhardt’s solution,0.1% SDS, 0.1 mg ml−1 salmon sperm DNA, and thenhybridized with radiolabelled cDNA probes at 42◦C for18 h. Membranes were washed 2 × for 15 min in 2 ×SSC–0.1% SDS, 2 × for 15 min in 1 × SSC–0.1% SDS at42◦C, and then placed on X-ray film.

DNA sequencing and analysis

Differentially expressed clones were confirmed by virtualNorthern blot, amplified and then sequenced using anautomatic sequencer (3100 Genetic Analyser, AppliedBiosystems Inc., Foster City, CA, USA).

Results

Fetal weight and ventricular weight

As previously reported (Vonnahme et al. 2003), at the timeof tissue collection the degree of nutrient restriction hadelicited a 7.4% decrease in maternal weight while controlfed ewes exhibited a 7.5% increase in body weight. In thesame report we have shown that, while absolute left andright ventricle weight at day 78 gestation were not affectedby undernutrition, relative to body weight, both left andright ventricular weight were greater in the nutrient-restricted fetuses (Table 1; P < 0.05; from Vonnahme et al.2003). Fetal LV in the current study were collected fromthese same animals.

Figure 1. Differential screening of fetal LV nutrient-restricted subtraction cDNA libraryThis nutrient-restricted (tester) cDNA library was generated by ‘subtracting’ common cDNAs found in fetal LV fromcontrol-fed ewes. The same volume of PCR-amplified cDNA library from fetal LV derived from nutrient-restrictedewes was dot blotted onto nylon membranes. 32P-labelled nutrient-restricted (tester) or control-fed (driver) cDNAswere then hybridized with the nutrient-restricted (tester) cDNA library. Differentially expressed cDNAs are identifiedwith the arrows.

Subtractive library and differential screening

Generation of a subtractive cDNA library enriched forfetal LV cDNA from nutrient-restricted ewes on day 78 ofgestation and differential hybridization using subtractivecDNA probes revealed 41 differentially expressed genes(Fig. 1). Virtual Northern blot analysis confirmed that 11of the 41 clones were differentially expressed in fetal LVfrom nutrient-restricted when compared to control-fedewes (Table 2).

Discussion

We have previously reported that a 50% global nutrientrestriction between days 28 and 78 of ovine gestation leadsto an increase in the organ to body weight ratio of day78 fetal hearts (Vonnahme et al. 2003). In the presentstudy we report possible molecular systems involvedin compensatory growth of the fetal heart (Fig. 2).There have been several attempts to discover the genesresponsible for cardiac hypertrophy in adults. Presentedhere is the first report of genes that are up-regulatedduring increased growth in fetal heart in response tomaternal undernutrition. A subtractive cDNA library inwhich LV cDNAs from nutrient-restricted fetuses wereenriched was constructed. Screening of this library usingsubtracted cDNA probes followed by virtual Northernanalysis identified 11 enriched clones: cardiac α-actin,cardiac ankyrin repeating protein (CARP), caveolin-1,cyclin G1, NADH dehydrogenase subunit 2, cardiac-specific helicase activated by MEF2 (CHAMP), stathmin,titin, prostatic binding protein, endothelial and smoothmuscle derived neuropilin (ESDN) and a clone (3F2) that



Table 2. Differentially expressed genes in ovine fetal heart from conceptuses on day 78 of gestation from control-fed and nutrient-restricted ewes

VirtualGenBank Northerna Fold

Name accession no. C NR change Identity (%)c

α-Actin, cardiac BC009978 2.2 435/466 (93%)

Cardiac ankyrin repeating protein HSRNACINP 2.7 156/166 (93%)

Caveolin-1 NM 001753 1.1b 202/238 (84%)

Cyclin G1 NM 004060 5.3 162/184 (88%)

NADH dehydrogenase subunit 2 AF493542 1.5 161/189 (85%)

RNA helicase AF015812 36.0b 293/372 (78%)

Stathmin HRSNSTTH 4.9 190/208 (91%)

Titin NM 133437 1.7 432/464 (93%)

Prostatic binding protein NM 002567 1.5 339/373 (90%)

Neuropilin-like molecule NM 080927 3.1b 94/104 (90%)

Unknown (3F2) AC103987 3.2b 117/136 (86%)

aC, Control-Fed; NR, nutrient restricted. bNote that this fold increase is based on signal over background. The mRNA could not bedetected in fetal LV from controls. cThe percentage identity was calculated against human except NADH dehydrogenase subunit 2.

has 86% identity with Homo sapiens chromosome 18, cloneRP11-268I9.

Nutrient restriction during the first half of gestationinduced differential transcription of genes in fetal LVthat are heavier per unit fetal body weight comparedto controls. Cardiac hypertrophy is known to be aresult of up-regulation of protein synthesis and over-transcription of fetal genes that serve as markers of cardiachypertrophy such as skeletal α-actin, β-myosin heavychain, and atrial natriuretic factor (Adachi et al. 1995). Ofthe 11 differentially expressed clones confirmed by virtualNorthern analysis in the present study, eight genes (cardiacα-actin, cyclin G1, stathmin, NADH dehydrogenasesubunit 2, titin, CARP, ESDN and prostatic bindingprotein) have previously been identified in hypertrophiedadult heart tissue (Baumeister et al. 1997; Adachi et al.1998; Satoh et al. 1999; Aihara et al. 2000; Nozato et al.2000; Taimor et al. 2001; Pelletier, 2002; Egnaczyk et al.2003). Notably, of the remaining clones, caveolin andcardiac-specific helicase activated by MEF2 (CHAMP)have been reported to inhibit hypertrophic growth inadults. Finally, one gene product identified in fetal LVhad 86% sequence identity to Homo sapiens chromosome18, clone RP11-268I9, and will be studied in futureexperiments designed to define the coding region, inferredamino acid sequence, and its function in the fetalheart.

Participation of cell cycle regulation molecules inthe development of cardiac hypertrophy has beenstudied extensively (Li et al. 1998; Tamamori et al. 1998;Nozato et al. 2000; Busk & Hinrichsen, 2003). In adultanimals, LV hypertrophy is a compensatory growth ofcardiomyocytes as a result of increased working pressureof the heart. During fetal development, cardiomyocytesgrow actively by cell division and by increases in cell size(hypertrophy). However, the growth of cardiomyocytesby cell division is lost in the peripartum period (Li et al.1998). Cyclin-dependent kinases (CDKs) are requiredwith cyclins for mitosis to occur. Because cardio-myocytes undergo irreversible termination of mitosisafter birth, increased levels of these cyclins may lead toincreased synthesis of RNA and protein that can causehypertrophy in the heart. Cyclin G1 expression is increasedin cardiac myocytes from neonatal rats exposed toangiotensin II (Ang II) (Nozato et al. 2000). Whenthese cardiac myocytes were treated with CDK inhibitors(CDKI), growth was effectively inhibited. It was suggestedfrom these results that certain cell cycle regulators areassociated with hypertrophic growth of the heart. Inaddition, the G1 cyclin/cyclin dependent kinase pathwayinduces phosphorylation of pRb. Nozato et al. (2000)have proposed that G1 cyclin/CDK and/or phosphor-pRb initiate protein synthesis to cause hypertrophy ratherthan DNA synthesis through entry into the cell cycle.



In addition, the ability of Ang II to induce cardiachypertrophy through tyrosine kinase-mediated signaltransduction pathways such as JAK/STAT (Kodamaet al. 1998), mitogen activated protein kinase (MAPK)(Takahashi et al. 1997), PI 3-K (Rabkin et al. 1997), andprotein kinase C (PKC) pathways is well documented.However, it is not clear whether cyclins are involvedin hypertrophic growth in LV during fetal developmentbecause the heart is still undergoing mitotic growth. Non-etheless, it is likely that maternal nutrient restrictionresulted in increased cyclin G1 transcription that, in turn,contributed to the increase in fetal LV weight observed.

Fetal LV stathmin, titin, CARP, α-actin and ESDNwere also up-regulated in response to maternalundernutrition. The up-regulation of stathmin may leadto cardiac remodelling through up-regulation of bothcardiac titin and α-actin (Fig. 2). Stathmin binds to theC-terminal domain of p27 and phosphorylates serine 10,which results in deactivation of this CDKI. Inhibition

Figure 2. A model describing up-regulation of fetal LV mRNAs in response to hypertrophy induced bymaternal undernutritionBinding of MT1-MMP activates proMMP-2, which is then cleaved to form activated MMP-2. Caveolin is also requiredfor the AT2-induced activation of AT1 to initiate signal transduction. Stathmin inhibits CDKI p27, which allowsthe cell cycle to proceed. These molecules are involved in inducing hypertrophy and mediate cell proliferation andcardiac remodelling. Cardiac α-actin and titin are up-regulated in response to these processes. Caveolin is alsoknown to block G1 cyclin and binds to eNOS to inhibit its activity. CARP is activated by SAPKs in response tostress, which prevents protein synthesis. RNA helicase, also known as CHAMP, inhibits the cell cycle by activationof CDKI p21. These molecules are up-regulated and have inhibitory effects during hypertrophy. It is proposed thathypertrophy of fetal LV in response to maternal undernutrition is a homeostatic response between stimulatoryand inhibitory signal transduction pathways. The abbreviations used are: MT1-MMP, membrane type 1 matrixmetalloproteinase; AT1, angiotensin II type 1 receptor; ESDN, endothelial and smooth muscle derived neuropilin;eNOS, endothelial NO synthase; SAPKs, stress activated protein kinases; CHAMP, cardiac-specific helicase activatedby MEF2. Continuous arrows denote activation and dotted arrows denote inhibition of signalling pathways.

of p27 leads to cell cycle progression (Boehm et al.2002). Endothelin-1-induced hypertrophic injury causesover-expression of proteins involved in cytoskeletalreorganization including stathmin (Egnaczyk et al. 2003).Up-regulation of stathmin in the present experiments islikely to play a role in cell remodelling by inactivation ofCDKI during the fetal hypertrophic response. Mutations inthe muscle protein titin have been linked to dilated cardio-myopathy, a condition in which the heart chambers areenlarged, in humans and in other animal models (Huqet al. 2002). This giant sarcomere protein functions asa molecular spring, the properties of which define thepassive mechanical properties of cardiomyocytes, as wellas providing essential support for other muscle proteins(Granzier et al. 2002). Interestingly, a recent report haslinked CARP and its broader family of muscle ankyrinrepeat proteins to titin-based stress/strain signals (Milleret al. 2003). For example, CARP is up-regulated by cardiacmuscle stretch and colocalizes with titin N2A epitopes in



adult rat heart muscle tissues. These investigators furthersuggested that the muscle ankyrin repeat protein familyprovides a link between muscle stretch signal pathwaysand muscle gene expression. It appears feasible thereforethat CARP expression would be increased in the presenceof up-regulated titin. There are 23 titin exons unique tofetal and neonatal human myocardium, most of whichare highly conserved across species and down-regulated inthe adult (Lahmers et al. 2004). Since the PEVK repeatswithin titin have been suggested to function as proteininteraction sites (Gutierrez-Cruz et al. 2001), the increasednumber of PEVK repeats in fetal titin may increase its rolein mediating protein interactions (Lahmers et al. 2004).Which fetal titin isoforms were detected in the presentstudy remains to be determined. It is likely, however, thatthe up-regulated transcription of titin and α-actin in theovine fetal LV following maternal undernutrition is, atthe very least, an indicator of increased protein synthesisand increased growth. The up-regulation of CARP mayindicate that some of the increased growth may involveincreased cardiac muscle stretch. The impact of thesechanges on fetal and postnatal myocardial stiffness andcontractile function would provide a direction for futureinvestigation.

In this experiment, fetal LV caveolin and CHAMP havebeen identified as up-regulated in response to maternalnutrient restriction. Caveolin functions as a general kinaseinhibitor (Li et al. 1995; Garcia-Cardena et al. 1997; Ghoshet al. 1998), arresting the cell cycle at G0/1 phase througha p53/p21-dependent mechanism, negatively regulatingthe cell cycle (Galbiati et al. 2001) and repressing cyclinD1 (Hulit et al. 2000; Peterson et al. 2003). Caveolin 3,and caveolin 1/3 knockout mice induced the activationof G-proteins or Ras signalling and induced dilated LVcardiomyopathy and RV hypertrophy (Park et al. 2002;Woodman et al. 2002). Furthermore, it has been suggestedthat higher production of NO because of the loss ofcaveolin can be a contributing factor for these cardio-pulmonary defects. Interestingly, Molnar and coworkers(Molnar et al. 2002; Molnar et al. 2003) and others(Ozaki et al. 2000; Brawley et al. 2004; Nishina et al. 2003)have shown alterations in vascular smooth muscle NOsignalling in fetal, neonatal and adult sheep exposed tosuboptimal pregnancy environments including maternalundernutrition. Increased NO-induced vasodilatorycapacity may also contribute to the observation that fetalblood pressure near term is lower in fetuses exposedto early pregnancy nutrient restriction followed byre-alimentation (Hawkins et al. 2000). The role thatcaveolin regulation plays in altered ovine fetal vascularsmooth muscle responsiveness is intriguing and warrants

further examination. Since ESDN is thought to play a rolein the regulation of vascular cell growth (Kobuke et al.2001), and CARP has also been implicated in arteriogenesis(Boengler et al. 2003), the up-regulation of caveolin,together with that of ESDN and CARP, indicates increasedcardiac vascular smooth muscle growth and/or the pre-sence of increased vascularity. We have confirmed thispossibility by noting an increase in mRNA expression ofvascular endothelial growth factor (VEGF) and its receptorFlk in the heart tissue from the undernourished group(authors’ unpublished observations).

Liu et al. (2001) first described a MEF2C-dependentcardiac specific protein that was expressed in the heartthroughout prenatal and postnatal development in themouse. This protein contained seven conserved motifscharacteristic of helicases involved in RNA processing,DNA replication, and transcription and was namedCHAMP. CHAMP was later found to inhibit hypertrophicgrowth and the induction of fetal genes in both prenataland adult cardiomyocytes in culture (Liu & Olson, 2002).The anti-hypertrophic activity of CHAMP was shown torequire a conserved ATPase motif that characterizes theRNA helicase family and the up-regulation of the cell cycleCDKI p21CIP1. CHAMP is probably up-regulated due toincreased cyclin G1 transcription, and may act to moderatethe impact of maternal undernutrition on myocyte growthand remodelling.

Many of the factors discussed above are influencedby Ang II. Kingdom et al. (1993) reported that in agroup of intrauterine growth restriction (IUGR) humanfetuses, Ang II concentrations in umbilical venous bloodwere elevated when compared to uncomplicated termpregnancies, suggesting that the fetal renin–angiotensinsystem is activated in IUGR fetuses. The role of Ang IIreceptors in hypertensive adult offspring of rats exposed toprenatal undernutrition is well documented (Sherman &Langley-Evans, 1998; Langley-Evans et al. 1999). There aretwo known receptors for Ang II in sheep, type 1 (AT1) andtype 2 (AT2) with AT1 being constituently expressed whileAT2 is transiently expressed primarily during development(Burrell et al. 2001). Global maternal undernutrition(50%) during the last 30 days of pregnancy increasedfetal arterial blood pressure in the sheep fetus duringlate gestation (Edwards & McMillen, 2001). The arterialblood pressure response to Ang II was also higher in theundernutrition group, implying increased AT1 receptorexpression, most likely in the feto-placental vascular circuit(Yoshimura et al. 1990). Elevated Ang II concentrationsinduce a selective increase in LV mass in fetal sheep(Segar et al. 2001). Ang II acts on cardiac remodellingthrough the AT1 receptor and activates growth pathways in



adult animals. AT1 regulates accumulation of extracellularmatrix that induces the development of cardiac hyper-trophy (Brilla et al. 1995; Weber et al. 1995), perhaps asa result of accumulation of cardiac fibroblasts causingthe build up of collagen (Brilla et al. 1995). As alreadymentioned, Ang II has been implicated in cardiac hyper-trophy via cyclin G1 activation in rats (Nozato et al. 2000).Ang II also promotes interaction of AT1 with caveolin(Ushio-Fukai et al. 2001). Ang II also increases caveolin-1mRNA in rat vascular smooth muscle cells (Ishizaka et al.1998). These studies were interpreted to mean that caveolinis required for the mechanism of action of Ang II andactivation of associated signal transduction pathways. Theimpact of maternal nutrient restriction on fetal Ang IIlevels and direct consequences on fetal LV have yet to beascertained in the present model.

To our knowledge, this is the first animal model thatreproduces observations in the human fetus of increasedleft and right ventricular hypertrophy under conditions ofmaternal stress (Samson et al. 2000). Other experimentsusing the fetal lamb have typically resulted in LV hyper-trophy without enlargement of the RV (Burrington,1978; Fishman et al. 1978). Thus, this model of earlyfetal nutrient deprivation in the sheep may also berelevant for studying the aetiology and consequencesof human fetal ventricular hypertrophy. We have foundthat genes associated with inducing and regulatingcellular growth and remodelling in the LV were up-regulated. Changes in expression of these genes can beaffected by several factors such as genotype, sex, bodysize (De Simone et al. 2001) and environmental factorssuch as nutritional status (Aguilera et al. 2002). The LVundergoes pathological structural changes that go beyondthe compensatory needs with increases in extracellularmatrix and fibroblast invasion as a consequence (Arnett,2000). However, limited information is available thatdescribes the genetic basis for LV hypertrophy in the fetusat this early stage of development. In this study, under-nutrition caused increased fetal cardiac growth at midgestation. It is not clear yet whether the increase in growthof heart is compensatory growth, hypertrophy or hyper-plasia. As noted above, at mid gestation the fetal heart isstill capable of significant increase in cell number.

In the current study, confirmation that clones wereup-regulated in nutrient-restricted fetal LV for individualanimals was not possible due to limited availability oftissue. Also some of the up-regulated genes in nutrient-restricted fetal LV (i.e. CARP, ESDN and Caveolin), whichseems to inhibit hypertrophic growth, suggest that down-regulated genes due to undernutrition also may providehelpful information. Identification of genes that are down-

regulated in fetal LV because of maternal undernutritionis the focus of future experiments. Whether the changesdiscussed above are a cardio-protective response in the faceof limited nutrient supply, a response to increased systemicvascular resistance and myocyte stretch, or a response to analtered endocrine milieu also remains the focus of futureinvestigation. More specifically, the encoded proteins needto be studied to determine if they function as a cause or aconsequence of altered LV growth.

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Acknowledgements

The research was supported, in part, by a grant to T.R.H.,S.P.F. and P.J.N. through the University of Wyoming NIH BRIN1P20RR16474-01, and by an NICHD grant HD21350 to P.J.N.The authors thank Dr K. A. Vonnahme for management ofexperimental animals and tissue collection and Dr D. J. Perry,Dr B. R. Francis and Dr T. E. Spencer for helpful discussions.


maternal nutrient restriction alters gene expression in the ovine fetal heart

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