Journal of Molecular and Cellular Cardiology 66 (2014) 1–11

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Journal of Molecular and Cellular Cardiology

j ourna l homepage: www.e lsev ie r .com/ locate /y jmcc

Original article

Impact of gestational chronodisruption on fetal cardiac genomics

Hugo A. Galdames 1, Claudia Torres-Farfan 1, Carlos Spichiger, Natalia Mendez,Lorena Abarzua-Catalan, Pamela Alonso-Vazquez, Hans G. Richter ⁎Laboratory of Developmental Chronobiology, Institute of Anatomy, Histology and Pathology, Faculty of Medicine, Universidad Austral de Chile, Valdivia, Chile

⁎ Corresponding author at: Institute of Anatomy, HistoMedicine, Universidad Austral de Chile, PO box 567, ZipTel.: +56 63 293022; fax: +56 63 221604.

E-mail addresses: hugogaldames168@gmail.com (H.Aclaudia.torres@uach.cl (C. Torres-Farfan), cspichiger@uachnatamendezc@gmail.com (N. Mendez), lorena.abarzua@gpamelalonso.1@gmail.com (P. Alonso-Vazquez), hrichter@

1 Both authors contributed equally.

0022-2828/$ – see front matter © 2013 Elsevier Ltd. All rihttp://dx.doi.org/10.1016/j.yjmcc.2013.10.020

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 June 2013Received in revised form 20 October 2013Accepted 27 October 2013Available online 4 November 2013

Keywords:Cardiac gene networksWhole transcriptomeGestational chronodisruptionFetal programming of adult diseaseLeft ventricle hypertrophy

We recently reported that gestational chronodisruption induces fetal growth restriction andmarked effects on fetaladrenal physiology. Here, whole-transcriptome profiling was used to test whether gestational chronodisruptionmodifies gene expression in the fetal heart, potentially altering cardiac development. At day 10 of gestation(E10), pregnant ratswere randomized in two groups: constant light (LL) and control 12 h light/12 h dark photope-riod (LD). RNA isolated fromE18heartwas subjected tomicroarray analysis (Affymetrix platform for 28,000 genes).Integrated transcriptional changes were assessed by gene ontology and pathway analysis. Significant differentialexpression was found for 383 transcripts in LL relative to LD fetal heart (280 up-regulated and 103 down-regulated); with 42 of them displaying a 1.5-fold or greater change in gene expression. Deregulated markers ofcardiovascular disease accounted for alteration of diverse gene networks in LL fetal heart, including local steroido-genesis and vascular calcification, as well as cardiac hypertrophy, stenosis and necrosis/cell death. DNA integritywas also overrepresented, including a 2.1-fold increase of Hmga1mRNA, which encodes for a profuse architecturaltranscription factor. microRNA analysis revealed up-regulation of miRNAs 218-1 and 501 and concurrent down-regulation of their validated target genes. In addition, persistent down-regulation of Kcnip2mRNAand hypertrophyof the left ventricle were found in the heart from 90 days-old offspring from LLmothers. The dysregulation of a rel-evant fraction of the fetal cardiac transcriptome, together with the diversity and complexity of the gene networksaltered by gestational chronodisruption, suggest enduring molecular changes which may shape the hypertrophyobserved in the left ventricle of adult LL offspring.

© 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Themain cause of death continues to be cardiovascular disease [1]. Itis well accepted that environmental risk factors, including smoking andobesity, interact with our genetic background to increase susceptibilityto cardiovascular dysfunction [2]. However, it is now clear that earlyinsults at critical stages of development may also lead to permanentchanges in cardiac structure and function. Thus, a number of studieshave established a relationship between antenatal deleterious environ-ments (for instance, fetal undernutrition and/or hypoxia) and the onsetof adult diseases including hypertension, coronary heart disease, strokeas well as metabolic and neurologic disorders [3–5]. Prenatal adverseconditions often translate not only into preterm delivery and low birthweight but also intrauterine growth retardation, one of the primarykillers in obstetric medicine, occurring in 7–10% of pregnancies [6,7].

logy and Pathology, Faculty ofCode 5090000, Valdivia, Chile.

. Galdames),.cl (C. Spichiger),mail.com (L. Abarzua-Catalan),uach.cl (H.G. Richter).

ghts reserved.

Shift work affects one fourth to fifth of the workforce; whichmeans that even a modest detrimental impact on health may have im-portant public health implications [8]. Shift work is interlocked withchronodisruption, which in turn may be defined as a significant distur-bance of the temporal organization of endocrinology, physiology, metab-olism and behavior [9]. Mounting epidemiological and experimentalevidence suggests that chronodisruption is indeed detrimental for adulthuman beings, increasing the risk of cardiovascular and several otherdiseases [8–13]. Regarding the effects of chronodisruption on pregnancyoutcome, besides increased risk of miscarriage, preterm delivery and lowbirth weight have been consistently reported in shift worker women[14–19]; both are strong predictors of chronic disease later in life [3–5].As per animal models, a recent report using mice subjected to repeatedshifting of the light/dark cycle showed both, increased nonproductivemating and decreased term pregnancies [20]. Likewise, we recentlyreported that exposure of pregnant rats to constant light induced intra-uterine growth retardation, as well as changes in gene expression andreduced content of corticosterone (which in turn did not have acircadian rhythm) in the fetal adrenal [21]. This study also indicatedan altered in vitro fetal adrenal response to ACTH of both, corticosteroneproduction and relative expression of clock genes and steroidogenicgenes. Meanwhile, the probable long-term consequences of gestationalchronodisruption are just beginning to be addressed. In a rat model ofchronic phase shift along pregnancy and early postnatal life, Varcoe

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and colleagues [22] assessed an array of metabolic parameters inthe adult offspring and found increased adiposity, hyperleptinaemia,hyperinsulinaemia and poor glucose tolerance. However, despite thevast number of shift worker pregnant women worldwide, the impactof developmental chronodisruption on fetal and postnatal physiologyremains largely under-studied.

Here we investigated the effects of gestational chronodisruptionon global gene expression in the fetal heart using a microarray-basedapproach. Considering the lack of previous evidence, the advantagesof microarray (likewise RNA sequencing) are evident: it is unbiased,transcriptome-wide and its emphasis is on discovery rather thanhypothesis testing; with the aim being to generate hypotheses worthyof further investigation. Hence, our aim was to begin defining fetal car-diac alterations imposed by gestational chronodisruption through iden-tification and validation of deregulated genes, together with mappingto integrated gene networks by means of functional genomics. Further-more, in a parallel cohort of adult offspringwhich had been subjected togestational chronodisruption, we investigated cardiac morphologicalchanges which might derive from altered gene expression in the fetalheart.

2. Material and methods

2.1. Animals

The protocolswere approved by the Local Bioethics ReviewCommit-tees from both, Faculty of Medicine, Universidad de Chile (CBA#0234)and Universidad Austral de Chile (CB#20/10). All fetal and adulthearts used in the present study were obtained and either preservedor processed as described below, from larger cohorts raised in ourprevious report Mendez et al. [21]. Briefly, animal handling and carewere performed following the NIH Guide for Animal ExperimentationCare recommendations. Virgin, female Sprague–Dawley rats (3 monthsold) were maintained in a 12:12 light/dark cycle (light on at 0700),under controlled temperature (18–20 °C), with food and water adlibitum. Timed-pregnant female rats were obtained after mating (thepresence of spermatozoa in the smear of the vaginal contents wasconsidered to be day 0 of pregnancy). During pregnancy, maternalweight, food and water intake were quantified and documented daily.

2.2. Experimental procedures

From day 10 of pregnancy, rats were randomly divided in twogroups (n = 10 per group): control (LD; 12:12 light/dark cycle, lighton at 0700) and gestational chronodisruption by exposure to constantlight (LL; 24-h under ‘lights on’ condition). For RNA isolation from thefetal heart, dams were weighed on day 18 of gestation (term is approxi-mately 21 days; n = 5 per group). The dams were euthanized with anoverdose of sodiumthiopental (150 mg/kg) at 2000 h (i.e., 1 h after lightsoff), the pregnant uterus was exposed via a mid-line incision and theanesthetized pups were euthanized by spinal transection. All fetuseswere weighed and their hearts were dissected out under sterile condi-tions. The fetal hearts were removed and immediately subjected toRNA isolation. The rationale to disrupt the normal LD cycle from day10 to 18 of gestationwas as follows: (1) to avoid interferingwithmater-nal reproductive capacity [20]; (2) to rule out effects on early organo-genesis; (3) to make sure that the fetal circadian master clock is notyet functional, thus allowingus to begin characterizingmaternal controlof the fetal circadian system [23]; and (4) to reduce retinal damageinduced by prolonged exposure to constant light [24], whichmay entailaltered maternal behavior and/or stress responses.

For longitudinal study of the adult heart morphology and geneexpression, the remaining pregnant females were allowed to deliver(n = 5per group), when themothers and their offspringwere immedi-ately returned to a control LD photoperiod. Infants were weaned at21 days old, with the males being held to be studied at 90 days of age.

These males for long-term protocols were maintained in LD cycle undercontrolled temperature (18–20 °C), with food and water ad libitum.

2.3. Microarray analysis

For RNA isolation, heartswere pooled from every litter and thereforeRNA samples do not represent individual fetuses. Thus, any given poolconsisted of 8 fetal hearts coming from one litter; with n = 5 poolsfor the LD and LL conditions; respectively. It should be stressed thatRNA samples are often pooled in a microarray experiment to reducethe effects of biological variation; with the rationale being that differ-ences due to subject-to-subject variation will be minimized, makingsubstantive features easier to find. In a seminal paper, Kendziorski andcolleagues showed that microarray pooled designs do not performworse than non-pooled designs [25].

RNA was extracted using the ‘SV Total RNA Isolation System’

(Promega Corporation, Madison, WI) according to the manufacturer'sinstructions, as previously described [21]. Sample processing andmicro-array hybridization were performed by an external dedicated CoreFacility, the ‘KFB — Center of Excellence for Fluorescent Bioanalytics’(Regensburg, Germany; www.kfb-regensburg.de). The hybridizationand scanning steps of microarray analysis were carried out exactly aspreviously described [26], using Affymetrix Rat Gene 1.1 ST GeneChiparrays (containing about 700,000 probe sets representing 28,000 ratgenes).

All array data were normalized using Robust Multi-array Average(RMA) [27] with the Expression Console software (Affymetrix, SantaClara, CA). RMA utilizes the probe set annotation provided by Affymetrixto identify genes directly from the CEL files. Genes that were significantlyup or down regulated were identified using Significance Analysis ofMicroarrays (SAM) [28]. SAM assigns a score to each gene on the basisof a change in gene expression relative to the standard deviation of re-peated measurements. For genes with scores greater than an adjustablethreshold, SAMuses permutations of the repeatedmeasurements to esti-mate the percentage of genes identified by chance — the false discoveryrate (FDR). Analysis parameters were set to 800 permutations, whileDelta was set to 0.576 to result in FDR ≤10%.

2.4. Functional analysis of differentially expressed genes

We applied two analytical approaches for functional genomics. First,the functional analysis of differentially expressed genes was performedby assessing the statistical significance of enrichment of different geneontologies (GO) [29] and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways [30], using DAVID functional analysis tool (Databasefor Annotation, Visualization, and Integrated Discovery) [31]. Analysisof KEGG pathways enrichment usingDAVIDwas assessed by calculatingthe p-value (as derived from a modified Fisher Exact test; EASE Score).DAVID p-values were corrected to be more conservative in orderto lower family-wise FDR, using different standard statistics formultiplecomparison corrections: Bonferroni, Benjamini and FDR. Enriched GeneOntologies with FDR-values less than 0.05 were taken into account.Second, to identify functional connections between deregulated tran-scripts, both network and pathway analyses of the genes filtered bymi-croarray were performed as previously described by Jovov et al. [32],using Ingenuity Pathways Analysis (IPA;www.ingenuity.com, IngenuitySystems, Mountain View, CA). The significance of networks was calcu-lated by integrated Ingenuity algorithms. IPA calculates a p-valuebased on the right-tailed Fisher's exact test for each canonical pathway,which is a measure of the likelihood that the association between asubset of genes from the whole experimental data set and a relatedfunction/pathway is due to random association. Relevant pathwayswith p-values less than 0.05 were taken into account. In addition, IPAcompares the direction of change for the differentially expressedgenes with expectations based on the literature (cured in the IngenuityKnowledge Base) to predict an integrated direction of change for each

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function, using the z-score algorithm. It is designed to reduce the chancethat random data will generate significant predictions. z-scores ≥2,indicate that the function is significantly increased and z-scores ≤−2,indicate that the function is significantly decreased. Hence, the z-score in-dicates how much each ontology is over-represented (positive z-score)or under-represented (negative z-score) in a gene list (further detailson the z-score algorithm are given in Bouchard-Mercier et al.) [33].

2.5. Analysis of microRNA (miRNA) validated targets

We were interested in determining whether certain microRNAs(miRNAs; small non-coding regulatory RNA molecules), might beinvolved in regulating genes responsive to gestational chrono-disruption. To this end, we used miRWalk Database (available athttp://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/) [34] to per-form bioinformatics analysis of up-regulated miRNAs and theirvalidated target sequences among genes down-regulated by gesta-tional chronodisruption.

2.6. Reverse transcription coupled to quantitative PCR (RT-qPCR)

Quantitative PCRwasused to validate selected deregulated genes. Tothis end, relevant qPCR primers were either taken from the literature ordesigned from the coding sequence of each gene of interest, generallyoverlapping two consecutive exons (PRIMER3 software; http://frodo.wi.mit.edu/cgi-bin/primer3). The different primer pairs were alignedagainst the rat genome using BLASTN 2.2.27 to check their specificity(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Primer sequences are providedas supplemental information (Supplemental Table 1). cDNA synthesisand qPCR reactions were performed using 18S-rRNA as housekeepinggene, exactly as previously described by us [35]. In the AffymetrixRat Gene 1.1 ST GeneChip, 18S-rRNA is identified as LOC310926;which displayed a Standard Deviation =0.0543, as calculated fromthe RMA normalized data for all 5 LD and 5 LL chips. Therefore, 18S-rRNAwas chosen as a robust housekeeping gene for the present micro-array data set. For qPCR, samples were run in triplicate and after thefinal amplification cycle, a melting curve analysis was performed oneach sample to ensure that a single product was obtained; whereasagarose gel electrophoresis confirmed that the single PCR product wasof the expected size. All amplicons were sequenced to confirm appro-priate targeting of the genes of interest. The efficiency for the PCRprimers used in the present study ranged between 90–110%. Relativeamounts of all mRNAs were calculated by the comparative Ct methodusing the Eq. (2)−ΔΔCt [36], and significant differences were assessedby Student's t-test (P b 0.05).

2.7. Analysis of morphology and transcription of disease markers in theadult heart

For morphological studies, male rats (90 days of age), which hadbeen gestated under LL or LD conditions, were euthanized with a sodi-um thiopental overdose (150 mg/kg; n = 5 per treatment) at 1400 h(i.e., 7 h after lights on). The hearts were dissected out, weighed andfixed in 4% paraformaldehyde during 24 h. Tissue blocks were embed-ded in paraffin (Histosec; Merck KGaA, Darmstadt, Germany) and5 μm-thick sections were obtained for staining with Hematoxylin-Eosin or Masson-Trichrome. Histological analysis included: cardiomyo-cyte area, cardiomyocyte nuclear diameter, left ventricle thicknessand ventricular cavity size. For cardiomyocyte area and diametermeasurements, 30 nuclei per field were counted over 15 fields under40× magnification, using an Olympus BX51 microscope coupled to animage capture system equipped with Image Pro Plus v6.3 software(Media Cybernetics Inc., Rockville, MD). All morphological comparisonswere performed manually, with the researcher blinded to treatment.

In addition, RNA samples were obtained from the heart of adultoffspring which had been gestated under LL and LD conditions, as

described above (‘SV Total RNA Isolation System’; n = 5 per treat-ment). To this end, animals were euthanized with an overdose of sodi-um thiopental (150 mg/kg) at 2300 h (i.e., 4 h after lights off). Thetranscriptional rate of a set of selected transcripts was assessed bymeans of RT-qPCR in the adult LL versus LD RNA samples, exactly as de-scribed in Section 2.6 above; but using HPRT1 instead of 18S-rRNA ashousekeeping gene due to its better efficiency and robustness at this de-velopmental stage (see Supplemental Table 1 for details). All selectedtranscripts were either potential or establishedmarkers of cardiovascu-lar disease; namely: Hsd3b1, Star, Cyp11a1, Hmga1, Cited1, Lrrc10, Mdk,Adra1a, Slc2a1 and Kcnip2.

2.8. Statistical analysis

FDR-values, z-scores and P-values were calculated as appropriate byintegrated algorithms of DAVID, IPA and miRWalk software systems.RT-qPCR data for microarray validation purposes (fetal hearts) andgene expression analysis (adult hearts) were expressed asMean ± SEM.The values of 2^ − ΔΔCT for each gene measured were analyzed byStudent's t-test and results were considered significant when P b 0.05.For fetal hearts, correlation between fold change values by microarrayversus RT-qPCR was performed with best-fit linear regression to deter-mine the r2 coefficient. For adult heart morphology, Mean ± SEM ofmeasurements taken from histological sections were analyzed byStudent's t-test for n = 5 animals per treatment. Statistical differenceswere accepted when P b 0.05. All statistical analyses were performedusing GraphPad Prism version 5.01 for Windows (GraphPad SoftwareInc., San Diego, CA).

3. Results

3.1. Effects of gestational chronodisruption on pregnancy outcome

Pregnant dams kept in constant light (LL) did not display any behav-ioral sign of stress, as we found no hair loss, stereotyped movements oraltered water/food intake. Furthermore, at 18 days of gestation LL damspresented similar plasma corticosterone levels (as area under curve in24-h) relative to LD mothers, displaying a circadian rhythm with onlya slight phase delay of peak plasma corticosterone in the 24-h (for de-tails, see Mendez et al. [21]). Overall LL and LD maternal weight gainalong days 10 to 18 of pregnancy and maternal body weight at day 18of gestationwere also similar (again, seeMendez et al. [21]). All dams car-ried fetuses evenly distributed in each uterine horn, with no signs of fetalreabsorption. There were no differences in litter size (LL = 14 ± 1,n = 10 vs. LD = 15 ± 1, n = 10). It is important to stress that thefetal hearts used in the present study come from the same animalsreported for analysis of the fetal adrenal in Mendez et al. [21]. Finally, inthe cohort of animals which was allowed to deliver for longitudinalstudy of the adult heart, maternal exposure to constant light did nothave a significant effect on birth weight (LL = 6.79 ± 0.03, n = 76 vs.LD = 6.59 ± 0.07, n = 71).

3.2. Validation of microarray data by means of RT-qPCR

We used the same RNA samples from the microarray analysis toperform RT-qPCR validation assays. A FDR threshold of 10% predictsthat about 1 out of 10 transcripts differentially expressed in microarraymight not be validated by qPCR. This figure agrees well with the resultsobtained for the 13 mRNAs analyzed so far; with 11 of them displayingdifferential expression in both microarray and RT-qPCR. Indeed, asshown in Fig. 1A, there was a highly significant correlation betweenmicroarray and RT-qPCR (r2 = 0.9845). Thus, mRNAs encoding forcardiovascular disease markers (Mdk, Slc2a1 and Adra1a), individualcomponents of the steroidogenic pathway (Hsd3b1, Star and Cyp11a1)and molecules displaying nuclear function/localization (Hmga1, Cited1and Lrrc10); were all efficiently validated by means of RT-qPCR

-3 -2 -1 1 2 3

-3

-2

-1

1

2

3y = 0.8437x-0.1781

R 2= 0.9845

qPCR

Mic

roar

ray

Steroidogenesis Nuclear location Cardiac disease

Hsd3b1

0.0000

0.5000

1.0000

1.5000

*

Star

0.0000

0.0020

0.0040

0.0060

0.0080

*

Cyp11a1

0.0000

0.5000

1.0000

1.5000

2.0000

2.5000

*

Mdk

Adra1a

Slc2a1

0.0000

0.2000

0.4000

0.6000

0.8000

*

Hmga1

0.0000

5.0000

10.0000

15.0000

20.0000

*

Cited1

Lrrc10

LD LL 0.0000

1.0000

2.0000

3.0000

4.0000

5.0000

*

LD LL 0.0000

0.0100

0.0200

0.0300

0.0400

0.0500

*

0.0000

5.0000

10.0000

15.0000

20.0000

*

LD LL 0.0000

0.0200

0.0400

0.0600

0.0800

*

A

B

Fig. 1.Microarray validation by RT-qPCR in the heart of rat fetuses subjected to gestational chronodisruption. (A) Correlation between expression changes bymicroarray vs. RT-qPCRwithbest-fit linear regression line. (B) Relative expression ofMdk, Adra1a, Kcnip2, Hsd3b1, Star, Cyp11a1, Hmga1, Cited1 and Lrrc10mRNA by RT-qPCR in the heart of fetuses gestated under LLrelative to LD conditions. Transcriptional rate was assessed in the same RNA samples used for microarray hybridization. Y axis units are 2^–ΔΔCT for qPCR. Values are Mean ± SEM(black bars: LD condition; white bars: LL condition). *: different from LD; P b 0.05, Student's t-test for n = 5. Note that 2^–ΔΔCT calculates relative expression levels (using the internalcontrol 18S rRNA as reference) and it displays a wide range (1 × 10−4–2 × 101); depending on each gene's actual level of expression.

4 H.A. Galdames et al. / Journal of Molecular and Cellular Cardiology 66 (2014) 1–11

(Fig. 1B). The mRNAs encoding for Kv channel-interacting protein 2(Kcnip2; Fig. 4E) and hemoglobin zeta (Hbz; data not shown) werealso validated by RT-qPCR. Collectively, these qPCR results confirm themicroarray technically.

3.3. Identification of genes deregulated by gestational chronodisruption

Comparison of global transcription in the fetal heart from motherskept under LL with those gestated in LD conditions revealed 383

Table 2Top down-regulated genes in the heart of fetuses gestated under LL condition.

N° Genesymbol

Gene name Fold change q-value

1 Hsd3b1 Hydroxy-delta-5-steroid dehydrogenase,3 beta-steroid delta-isomerase 1

6.7 4.5

2 Cyp21a1 Cytochrome P450, family 21, subfamily a,polypeptide 1

6.2 7.4

3 Cyp11a1 Cytochrome P450, family 11, subfamily a,polypeptide 1

5.5 3.2

4 Star Steroidogenic acute regulatory protein 5.4 9.55 Cyp11b1 Cytochrome P450, family 11, subfamily b,

polypeptide 14.9 6.7

6 Asb11 Ankyrin repeat and SOCS box-containing 11 2.2 0.07 Rasgrp1 RAS guanyl releasing protein 1

(calcium and DAG-regulated)2.1 0.0

8 Mgp Matrix Gla protein 1.9 0.09 Zbtb16 Zinc finger and BTB domain containing 16 1.8 3.210 Myom2 Myomesin 2 1.8 2.211 Bdh1 3-hydroxybutyrate dehydrogenase, type 1 1.8 0.012 Gipr Gastric inhibitory polypeptide receptor 1.8 5.013 Sult1a1 Sulfotransferase family, cytosolic, 1A,

phenol-preferring, member 11.7 1.7

14 Kcnip2 Kv channel-interacting protein 2 1.7 0.015 Pla2g5 Phospholipase A2, group V 1.7 0.016 Adra1a Adrenergic, alpha-1A-, receptor 1.7 0.017 Lonp1 Lon peptidase 1, mitochondrial 1.7 2.218 Cox8b Cytochrome c oxidase, subunit VIIIb 1.6 0.019 Fkbp2 FK506 binding protein 2 1.6 8.020 Car12 Carbonic anyhydrase 12 1.5 4.2

5H.A. Galdames et al. / Journal of Molecular and Cellular Cardiology 66 (2014) 1–11

differentially expressed transcripts, accounting for almost 1.5% of thewhole rat transcriptome. The majority of the differential genes wereoverexpressed (280 out of 383 genes; i.e., 73.1%), while less differentialgenes were down-regulated (103 out of 383 genes; i.e., 26.9%) in thefetal heart from LL relative to LD condition. A full list of differentiallyexpressed genes is provided as supplemental information (SupplementalTable 2). The top 20 up-regulated genes are shown in Table 1, with thetop six genes displaying a consistent Fold Change of at least 1.8-fold. In-terestingly, three of them are plasma carrier proteins (Hbz, Afp andSerpina6). Among all 280 overexpressed transcripts, there were severalestablished markers of cardiovascular disease, including Slc2a1, Apex1andMdk. Potential markers of cardiovascular disease, which were signif-icantly overexpressed in the LL fetal heart, included Cited1 and Hmga1mRNAs, as well as the microRNAs Mir218-1 and Mir501. On the otherhand, the top 20 down-regulated genes are shown in Table 2, with achange range of 4.9- to 6.7-fold for the top five genes. Besides themarkeddown regulation of this subset of transcripts, it is striking that all of thembelong to the canonical steroidogenic synthesis pathway (Hsd3b1,Cyp21a1, Cyp11a1, Star and Cyp11b1). Among all 103 down-regulatedtranscripts, there were several establishedmarkers of cardiovascular dis-ease, including Adra1a, Gas6 and Mgp. Several other differentiallyexpressed genes of interest are mentioned below (see Section 3.4 andalso Discussion).

3.4. Pathways and gene ontology (GO) processes modulated bygestational chronodisruption

The AssociatedNetwork Functions identified for genes deregulated bychronodisruption are shown in Table 3 (IPA analysis; maximum networksize: 140 genes/proteins and 25 networks). The top Associated NetworkFunctions were related with endocrine system, developmental andother processes. For each of theseNetworks, gestational chronodisruptionhad a marked effect on the fetal cardiac gene expression level of most ofthe molecules associated (Table 3). It is worth mentioning that the top 1Associated Network Function (Cancer, Drug Metabolism, EndocrineSystem Development and Function; Table 3) contains 6 out of the top20 up-regulated genes displayed in Table 1 (Hbz, Afp, Hist2h4, Arnt2,Luc7l3 and Cdh3), while it also contains 5 out of the top 20 down-regulated genes displayed in Table 2 (Cyp11a1, Star, Mgp, Girp andAdra1a). Likewise, the top 2 Associated Network Function (Endocrine

Table 1Top up-regulated genes in the heart of fetuses gestated under LL condition.

N° Genesymbol

Gene name Foldchange

q-value

1 Hbz Hemoglobin, zeta 2.2 3.22 Afp Alpha-fetoprotein 2.2 5.33 Serpina6 Serine (or cysteine) peptidase inhibitor, clade A,

member 62.1 2.1

4 Mir218-1 microRNA mir-218-1 1.9 5.35 Upk3b Uroplakin 3B 1.8 2.16 Lrrn1 Leucine rich repeat neuronal 1 1.8 1.47 Akr7a3 Aldo-keto reductase family 7, member A3

(aflatoxin aldehyde reductase)1.6 5.8

8 Mogat2 Monoacylglycerol O-acyltransferase 2 1.6 0.09 Hist2h4 Histone cluster 2, H4 1.6 2.110 Clk1 CDC-like kinase 1 1.6 5.311 Rnpc3 RNA-binding region (RNP1, RRM) containing 3 1.6 4.512 Arnt2 Aryl hydrocarbon receptor nuclear translocator 2 1.5 0.013 Zdhhc12 Zinc finger, DHHC-type containing 12 1.5 1.414 P2rx2 Purinergic receptor P2X, ligand-gated ion

channel, 21.5 0.0

15 Lrrn4 Leucine rich repeat neuronal 4 1.5 1.416 Crip Cysteine-rich intestinal protein 1.5 5.017 Fah Fumarylacetoacetate hydrolase 1.5 0.018 Cpa2 Carboxypeptidase A2 (pancreatic) 1.5 0.019 Luc7l3 LUC7-like 3 (S, cerevisiae) 1.5 7.420 Cdh3 Cadherin 3 1.5 0.0

System Development and Function, Molecular Transport, Small MoleculeBiochemistry; Table 3) contains 6 out of the top 20 up-regulated genesdisplayed in Table 1 (Afp, Mir218, Clk1, P2rx2, Fah and Cpa2), while italso contains 7 out of the top 20 down-regulated genes displayed inTable 2 (Cyp11a1, Star,Myom2, Sult1a1, Pla2g5, Lonp1 and Cox8b).

The interactome for all components from top 1 Associated NetworkFunction (Cancer, Drug Metabolism, Endocrine System Developmentand Function) is depicted in Fig. 2. Next, we increased the stringency ofthis interactomeby setting amaximumnetwork size of 25 genes/proteinsand 10 networks, which refined the top 1 Associated Network to‘Endocrine System Development and Function’ (inset in Fig. 2), includ-ing the strongly repressed steroidogenic genes Cyp21a2, Cyp11a1,Hsd3b2 and Star. Indeed, the stronger P-value for IPA inferred CanonicalPathways was ‘C21-Steroid Hormone Metabolism’ (P = 0.000585).These IPA results agreewith those obtained usingDAVID gene ontology,which featured ‘C21-steroid hormone metabolic process’ as the mostsignificant term (see Fig. 3A).

The analysis of the whole microarray data set using DAVID, pointedout several gene ontology (GO) categories displaying strong P-values.Other than steroid and general hormone metabolism and biosynthesis,the principal GO categories outlined by this analysis are related toDNA replication, response to organic substance and negative regulationof cell proliferation (Fig. 3A). Regarding DNA function and integrity,although the top P-value was linked to DNA replication, significancewas also displayed by DNA base excision repair, mismatch repairand homologous recombination (data not shown). Other fetal cardiacGO terms markedly altered by gestational chronodisruption were(Glycosylphosphatidylinositol)-anchor biosynthesis and Spliceosome(data not shown).

Regarding Pathological Processes (‘Toxic Functions’ identified by IPA),the identified significant terms for Cardiac included: Hypertrophy,Stenosis andNecrosis/Cell Death (Fig. 3B). A number of geneswerehighlyrepresented in both Biological and Toxic Functions, including: Casq2,Ckm, Gata5, Mb, Nrg1, Trim63 and Srl, among others. Of note, the lattergene symbol stands for Sarcalumenin, which is a validated target of themicroRNA miR-218-1 (see Section 3.5 and also Discussion).

Finally, IPA analyses for significant enrichment of canonical path-ways and also for predicted abnormalities, are shown as supplemental

Table 3Functional networks including transcripts differentially expressed in the heart of fetuses gestated under LL condition.

ID Molecules in Associated Network1 Associated Network Functions Score2 LL-deregulatedtranscripts

1 CKM, NUP62, SGMS,1 SOD2, PLG, GIPR, VASP, ARNT2, TCIRG1, RCE1, C11orf9, PPP1R3C, SORBS1, NRAP, TP53,MPO, RND2, FZD2, HBZ, LUC7L3, LGALS3BP, ROBO1, MN1, FMOD, RGS16, DPT, IRF6, VANGL2, AFP, SLIT2,APEX1, ADRA1A, DZIP1, WNT5A, JAK3, NRG1, GAS6, ACSL1, STAR, Ctla2a, NFIL3, MCM10, TTR, CDH3, GLRX,FABP4, Aph1a, SKP2, Hist2h4, HSD3B2, CYP21A2, HMGA1,MAPK13, POU5F1, PRRX2, GSN, PRNP,MAP1LC3A,Gnas,MDK, RASGRP1, JAG1, ZFP64, FBLN2, DOK1,MCM2, CD9,MGP, BCAT1, SUMO2, SLC2A1, TRIM63,NOC2L,SIGMAR1, RHOQ, CRIP1, GPIHBP1, SLC52A2, PRR5, FBLN1, RAC2, CNN1, CDON, CYP11A1, GORASP1, CXCR7,mir-500, DCX, ITGB1BP2, USP11, CITED1, 26 s proteasome, actin, Akt, AMPK, Ap1, calpain, Cg, CREM, cyclin A,ERK, ERK1/2, FSH, Cbp (family), GNAS, growth hormone, hCG, HDL-cholesterol, histone h3, IgG, Igm, IL12(complex), Jnk, Lh, Mapk, NFkB (complex), P38MAPK, PDGF BB, PI3K (complex), PP2A, proinsulin, Rac, Ras, RNApolymerase II, Sos, Tgf beta

Cancer, drug metabolism, endocrinesystem development and function

123 91

2 ERCC2, BCAT2, ANPEP, FAH, SULT1A1, Cox8b, PPP1R3C, CORO7, NME3, B9D1, SLFN5, SLFN12L, Ccl6, IGF2BP1,ETV4, CDC45, P2RX2, MCM7, ORAI1, OLFML2B, CAMK1G, AFP, mir-218, SLC7A7, TGM1, NEIL2, LONP1, GAS6,HPGD, STAR, MYOM2, ZNF608, CMTM3, NDRG4, HIST1H2BL, PIAS3, SELENBP1, HSD3B2, ENPEP, HMGA1,MAPK13, NNAT, JAG1, PLAGL2, GATA5, CPA2, CPA1, FBLN2, CD163, FRMD4B, MUTYH, KLHL21, CA8, PLA2G5,APOBEC3B, FBLN1, CYP11A1, CXCR7, CLK1, COL6A3, EMR1, ERCC1, hemoglobin, miR-193a-5p miRNAs, MT1L,RBPJL, Tni, TP53I3, XPA, histone h4

Endocrine system development andfunction, molecular transport, smallmolecule biochemistry

66 61

1 The list includes all Molecules in Associated Network as per the Ingenuity Knowledge Base, whereas terms in bold displayed statistically significant changes in the presentmicroarrayexperiments for the LL condition.

2 Score definition. Ingenuity Pathway Analysis computes a score for each network according to the fit of that network to the user-defined set of Focus Genes. The score is derived from aP-value and indicates the likelihood of the Focus Genes in a network being found together due to random chance. A score of 2 indicates that there is a 1 in 100 chance that the Focus Genesare together in a network due to random chance. Therefore, scores of 2 or higher have at least a 99% confidence of not being generated by random chance alone.

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information (Supplemental Table 3 and Supplemental Table 4;respectively).

3.5. microRNAs (miRNAs) upregulated by gestational chronodisruption

Amechanism by which gene expression can be regulated is throughmicroRNAs (miRNAs); a class of small noncoding RNAs, that regulategene expression primarily through post-transcriptional repressionby promoting mRNA degradation in a sequence-specific manner [37].The microarray analysis filtered two miRNAs which were deregulatedby LL in the heart: miR-218-1 is included in Table 1 and also in Network2 in Table 3;whilemiR-501 is included inNetwork 1 in Table 3. In the LLfetal heart, miR-218-1 and miR-501 were overexpressed by 1.9- and1.3-fold; respectively. Therefore, we aimed to identify genes known tobe their targets among the list of 103 genes down-regulated by gesta-tional chronodisruption. As shown in Table 4, our analysis identified10 validated targets for miR-218-1 (Dpt, Gnas, Slfn5, Srl, Gipr, Xirp2,Ppp1r3c, Acsl1, Hpgd, Nav3 and Nfil3; P b 0.05) and one validated targetmRNA for miR501 (Nfil3; P b 0.001).

3.6. Long-term effects of gestational chronodisruption on morphology andtranscription of disease markers in the adult heart

The weight of the heart from gestation under LL and LD conditionswas not different in 90 days old male rats (LL: 1.957 ± 0.0553 g vs.LD: 1.801 ± 0.0819 g). However, gestational chronodisruption alteredthe morphology of the left ventricle, which featured a thicker wall anda smaller cavity area (LL: 2.51 ± 0.02 mm vs. LD: 2.24 ± 0.02 mm,Fig. 4A; and LL: 12.40 ± 0.08 mm2 vs. LD: 16.82 ± 0.41 mm2, Fig. 4B;respectively, P b 0.05). Regarding the left ventricle's cardiomyocytes,there were significant differences in both, their calculated area (LL:546.5 ± 26.5 μm2 vs. LD: 480.4 ± 11.5 μm2; Fig. 4C; P b 0.05) andnuclear diameter (LL: 13.5 ± 0.27 μm vs. LD: 12.0 ± 0.51 μm; Fig. 4D;P b 0.05). Given that we found no changes in the geometry of theright ventricle (data not shown), these combined data agree withadult onset of asymmetric cardiac pathology, such as left ventriclehypertrophy. However, it is possibly a mild condition, given that thecardiomyocytes' size increase was rather moderate and no signs offibrosis or perivascular hyperplasia were detected. Representativestained sections of the adult LL versus LD heart are provided as supple-mental information (Supplemental Fig. 1).

In attempting to determine the molecular basis of this hypertrophicphenotype, we sought to examine the transcriptional rate of selectedcardiovascular disease markers in the adult heart. The biomarkers cho-sen for this analysis were those identified by microarray and validatedby qPCR in the LL fetal heart. No significant differences inmRNA expres-sion level for LL relative to LD adult hearts were found for Hsd3b1, Star,Cyp11a1, Hmga1, Cited1, Lrrc10, Mdk, Adra1a nor Slc2a1. However, themRNA encoding for Kv channel-interacting protein 2 (Kcnip2) displayeda persistently reduced expression level in LL relative to LD heart, asshown by microarray (fold-change: −1.70; Table 2) and RT-qPCR(fold-change: −1.62; Fig. 4E) in fetal samples versus RT-qPCR (foldchange: −1.64 with P = 0.004; Student's t-test for n = 5; Fig. 4F) inadult samples.

4. Discussion

Through whole transcriptome profiling and systematic use offunctional genomics tools, here we show for the first time that the fetalcardiac transcriptome is altered by gestational chronodisruption. Further-more, we provide evidence for transcriptional deregulation of severalestablished and potential markers of cardiovascular disease; which mayhelp to explain the left ventricle hypertrophy found in the adult offspringwhich had been gestated under constant light. Quantitatively, theeffects of gestational chronodisruption on the fetal heart involved 383deregulated transcripts (1.5% of the whole rat transcriptome). However,not all genes are expressed in the heart at any given time; for instance,it has been estimated that only 9407 transcripts are expressed in thesheep fetal heart [38]. Thus, the 280 up-regulated plus 103 down-regulated transcripts found in the LL fetal heart might represent a highlysignificant fraction of the active cardiac transcriptome.

Given that the mRNAs exhibiting the highest increase of expressionlevel under gestational constant light encode for plasma carrier proteins(HBZ, AFP and SERPINA6), it is tempting to speculate that increasedplasma transport of metabolites and hormones is a component of thefetal systemic response to chronodisruption. In particular, the Serpina6gene encodes an alpha-globulin protein which is the major transportprotein for glucocorticoids in the blood of most vertebrates [39]. Onthe other hand, the top five down-regulated transcripts in LL fetalheart displayed a high fold change range, all of them belonging to thecanonical steroidogenic synthesis pathway (Hsd3b1, Cyp21a1, Star andCyp11a1/b1). Indeed, RT-qPCR validation indicated that transcriptionof Hsd3b1, Star and Cyp21a1 was repressed by 86.8- , 24.5- and 12.8-

Fig. 2. Interactome of a pathway significantly enriched in genes differentially expressed in the fetal rat heart under LL condition. This particular set of genes was grouped in the IngenuityTop Network 1 ‘Cancer, Drug Metabolism, Endocrine System Development and Function’. Inset: a sub-net from this interactome, accounting for ‘Endocrine System Development andFunction’, is depicted. Red: significantly up-regulated gene; Green: significantly down-regulated gene; Gray: non-significant differential expression.

7H.A. Galdames et al. / Journal of Molecular and Cellular Cardiology 66 (2014) 1–11

fold, respectively; suggesting that the encoded proteins might be closeto undetectable in LL relative to control LD gestational photoperiod.These combined results are in agreement with the predicted

inactivation of themaster transcription factorNR5A1 (IPA algorithmpa-rameters: P = 0.001 and z-score =−1.9); which is known to contrib-ute to the integrated steroidogenic transcriptional profile. Also in

A) Gene Ontology (GO; DAVID)

B) Pathological Processes (IPA)

0 2

GO:0042446

GO:0008610

GO:0006694

GO:0010033

GO:0006700

GO:0008202

GO:0010817

GO:0008207

GO:0042445

GO:0034754

4 6 8

Regulation of hormone levels

Cellular hormone metabolic process

Hormone metabolic process

Steroid metabolic process

C21-steroid hormone metabolic process

C21-steroid hormone biosynthetic process

Response to organic substance

Steroid biosynthetic process

Lipid biosynthetic process

Hormone biosynthetic process

Threshold

-Log (FDR)

0 1 2 3 4

Liver Cirrhosis

Cardiac Hypertrophy

Cardiac Stenosis

Renal Degeneration

Cardiac Necrosis/Cell Death

Cardiac Pulmonary Embolism

Threshold

-Log (p-value)

Fig. 3. Identification of overrepresented pathways and networks by functional genomics analyses. (A) KEGG pathways significantly enriched by DAVID analysis of genes differentiallyexpressed in the fetal rat heart under LL condition (filtered for FDR b0.05). (B) Ingenuity Pathway Analysis (IPA) software was set to pinpoint Pathological Processes predictably affectedby the interaction of the whole set of differentially expressed genes in the fetal rat heart subjected to LL condition. In (A) and (B) the threshold (blue) line marks P = 0.05. (For interpre-tation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 4Up-regulated microRNAs and down-regulated target genes identified in the heart offetuses gestated under LL condition.

Up-regulatedMicroRNA 1

Hits P-value Down-regulated genes

miR-218-1* 7 P b 0.05 Dpt, Gnas, Slfn5, Srl, Gipr, Xirp2, Ppp1r3cmiR-218-1 4 P b 0.05 Acsl1, Hpgd, Nav3, Nfil3miR-218-2* 2 P b 0.05 Srl, Ppp1r3cmiR-501-5P 1 P b 0,001 Nfil3

1 Only mature miRNAs are shown. The designation miR-218-1 and miR-218-2 representdistinct precursor sequences and genomic loci that express identical mature sequences.miR-218-1 and miR-218-1* originate from the same predicted precursor. However, therelative abundances clearly indicate miR-218-1 is the predominantly expressed miRNA,whilst miR-218-1* comes from the opposite arm of the precursor. For miR-501-5P, thereare not sufficient data to determine which sequence is the predominant one, so ‘-5p’ isused only to indicate that this miRNA comes from the 5′ arm. Nomenclature definitionstaken from: http://www.mirbase.org/help/nomenclature.shtml.

8 H.A. Galdames et al. / Journal of Molecular and Cellular Cardiology 66 (2014) 1–11

keeping with the present results, it has been recently shown that thefetal sheep heart expresses 9 genes from the steroidogenic pathway[38]. In terms of potential consequences of depressed cardiac steroido-genesis, myocardial local pathways of aldosterone and corticosteronesynthesis have been reported in the adult rat heart; whichmay promotepost-infarction ventricular fibrosis and hypertrophy during progressionto heart failure [40,41]. It is worth mentioning that we have reportedthat gestational constant light leads to disruption of the fetal ratadrenal's circadian clock, accompanied by blunted corticosterone pro-duction [21]. The latter effectmay be explained at least in part by the ob-served decrease in response to ACTH in the fetal LL adrenals. Hence,a compelling experiment would be to measure fetal cardiac responseto ACTH and local corticosterone production. Although themechanismsregulating local cardiac steroidogenesis are by no means as wellestablished as in the adrenal gland, it is interesting that we observedalteration of fetal local steroidogenesis under LL conditions in thesetwo dissimilar tissues.

*

2-

Ctx

10-4

Fetus (E18)3.0

2.0

1.0

0.0

Kcnip2 expression in fetal and adult heart

0.0

0.5

1.0

1.5

2.0

*

2-C

t

Adult (P90)E F

1.5

2.0

2.5

3.0

*

LV

wal

l th

ickn

ess

(mm

)

5

10

15

20

*

LV

cav

ity

area

(m

m2 )

LD LL LD LL

LD LL LD LL

200

300

400

500

600

700

*

LV

car

dio

myo

cyte

s ar

ea(

m2)

6

9

12

15

18

*

LV

Car

dio

myo

cyte

s n

ucl

ear

dia

met

er (

m)

A B

C D

Adult heart morphologyΔ

Δ

ΔΔ

Fig. 4. Effects of gestational chronodisruption on adult cardiac morphology and fetal andadult transcription of the diseasemarker Kcnip2 in the rat. Mean ± SEMofmeasurementstaken from heart histological sections of 90 days old (P90) rats which had been gestatedunder LL (white bars) relative to LD (black bars) conditions, as follows: (A) LV wall thick-ness, (B) LV cavity area, (C) LV cardiomyocytes area, and (D) LV cardiomyocytes nucleardiameter. *: different from LD; P b 0.05, Student's t-test for n = 5. LV, left ventricle.(E) Relative expression of Kv channel-interacting protein 2 (Kcnip2) mRNA by RT-qPCRin the heart of E18 fetuses gestated under LL versus LD conditions. (F) Relative expressionof Kcnip2mRNA by RT-qPCR in the heart of P90 adults gestated under LL versus LD condi-tions. See Legend in Fig. 1 for further details on RT-qPCR analysis and symbols.

9H.A. Galdames et al. / Journal of Molecular and Cellular Cardiology 66 (2014) 1–11

An established pathological mechanism brought about by gestationalchronodisruptionwas vascular calcification; which is very aggressive, oc-curring even in patients with osteoporosis [42,43]. Two calcification-related genes down-regulated in the LL fetal heart were Gas6 (1.3-fold)and Mgp (1.9-fold). Human polymorphisms increasing GAS6 activityraise the risk of stroke, while GAS6 inhibits vascular calcification at leastin vitro. In turn, a genetic disease named Keutel syndrome involvesmutations of Mgp and is correlated with vascular calcification; whilethe phenotype of MGP-deficient mouse features full ossification of the

aortic wall and its major collaterals [42,43]. Transcription of several addi-tional markers of cardiovascular disease was deregulated by gestationalchronodisruption in the fetal heart. Mdk (up-regulated; fold changeby qPCR: 1.9) participates in inflammatory reactions, cell growth, cellsurvival, migration and angiogenesis. In adults, MDK appears to be help-ful for cardiac injury treatment [44]. Another important transcript up-regulated in the LL fetal heart was Cited1 (fold change by qPCR: 1.7), atranscriptional coactivator and epigenetic modifier expressed in theembryonic heart, which strongly activates TGFβ-induced transcription[45]. The Slc2a1 mRNA (encoding for glucose transporter 1; GLUT1)was up-regulated under developmental chronodisruption (fold changeby qPCR: 1.6). Heart Slc2a1 gene expression is highest during fetal life,gradually decreasing thereafter. SLC2A1 is mainly responsible for basalcardiomyocyte glucose uptake [46]. The Adra1a mRNA encoding foradrenergic receptor alpha 1A was down-regulated under LL conditions(fold change by qPCR: 1.8). Cardiac activation of alpha 1 adrenergic re-ceptors is involved in developmental growth, proliferation, hypertrophy,increased contractility, as well as changes in electrophysiological proper-ties and metabolism. In pathological conditions, such as under pressureoverload or myocardial infarction, ADRA1Amay promote cardiomyocytesurvival to protect from pathological cardiac remodeling and decompen-sation/heart failure [47]. Lrrc10 mRNA encodes for a cardiac-specificfactor that was down-regulated by LL (fold change by qPCR: 1.5). Lrrc10is exclusively expressed in the precardiac region (E7.5 mice) starting inearly embryos and continuing throughout development of the adultheart, with a marked elevation in expression upon birth [48]. A develop-mental role of Lrrc10with a tight spatiotemporal expression pattern hasbeen reported [48].

Besides cardiac steroid hormone synthesis, several other gene ontolo-gy categories were overrepresented, such as DNA metabolism, functionandmaintenance. HMG (highmobility group) are themost abundant nu-clear proteins after histones, and despite members of the HMGA subfam-ily they do not have intrinsic transcriptional activity, they operateas permissive ‘architectural’ transcription factors [49,50]. In particular,HMGA1 is highly and ubiquitously expressed during embryonic develop-ment [49]. Given that HMGA1 grants accessibility of DNAwithin chroma-tin, it affects transcription, replication, recombination, chromosomalrearrangements and repair [50]. Therefore, the fact that Hmga1 mRNAexpression was increased in the fetal heart subjected to gestationalchronodisruption (fold change by qPCR: 2.1), may contribute to theoverrepresentation of gene ontology terms related to DNA integrity.Interestingly, MYC is an upstream transcriptional regulator for Hmga1which was predicted to be potentially activated (IPA algorithm parame-ters: P = 0.006 and z-score = +1.7).

Another potential mechanism for gestational chronodisruptionto regulate fetal cardiac gene expression could be through miRNA.In the heart gestated under constant light, miR-218-1 and miR-501were overexpressed by 1.9- and 1.3-fold, respectively. miR-218-1 wasincluded in the Functional Network ‘Endocrine System Developmentand Function, Molecular Transport and Small Molecule Biochemistry’;while miR-501 was included in the Functional Network ‘Cancer, DrugMetabolism and Endocrine System Development and Function’. Ouranalysis identified 10 validated targets for miR-218-1 and one validatedtargetmRNA formiR-501. Interestingly, the nuclear factor, interleukin 3regulated (Nfil3) gene seems to be under negative control of bothmiRNAs, miR-218-1 and miR-501. Nfil3 is a transcription factor thatbinds to the promoter region of interleukin-3 which is restricted to ac-tivated T cells, natural killer cells, andmast cell lines in adult individuals[51]. Regarding the remaining transcripts potentially targeted for degra-dation by increased miR-218-1 under gestational chronodisruption,two of them have been recently reported as relevant for cardiac devel-opment and function. Sarcalumenin (Srl) has been shown to playa role in age-related cardiac dysfunction due to a decrease in SERCA2aexpression and/or activity, which is relevant since impaired SERCA2aactivity is a hallmark of cardiac dysfunction in aging population [52].In turn, Xin repeat-containing protein 2 (Xirp2 or Xinβ) was reported

10 H.A. Galdames et al. / Journal of Molecular and Cellular Cardiology 66 (2014) 1–11

to initiate the molecular mechanisms leading to maturation of interca-lated disks during postnatal heart growth [53].

The identification of 42 transcripts displaying a 1.5-fold or greaterchange in fetal cardiac expression level, with about 25% of them beingestablished markers of cardiovascular disease, prompted us to examineany long term effect of developmental chronodisruption on cardiomyo-cyte or even whole heart morphology. Actually, in 90 days old ratswhich had been gestated under constant light, we observed an alteredleft ventricle phenotype; namely, increased cardiomyocyte nuclearand cell diameter as well as increased wall thickness, accompanied byreduced cavity area. This alteredmorphology is consistentwithmorbid-ity andmortality associatedwith increased ventricular pressure, systolicstress, interstitial fibrosis and asymmetric left ventricular hypertrophy[54]. It is conceivable that the present paradigm of developmentalchronodisruptionmay contribute to the observed abnormal adult cardi-acmorphology, through early onset of persistently altered transcriptionof cardiovascular disease markers in the heart. In fact, when we investi-gated this possibility for 10 selected transcripts in the LL versus LD heartof fetal and adult offspring, a lasting and significant decrease for Kcnip2mRNA was found (fold change by qPCR: 1.62 and 1.64; respectively).KCNIP2 is a calcium-binding protein acting as a subunit of the voltage-gated potassium Kv4 channel complex, which is crucial for the conduc-tion system to regulate calcium-dependent A-type currents. Studiesin humans and mice have linked mutation of the Kcnip2 gene witharrhythmogenesis, which can be life threatening [55]. Moreover, thereis solid evidence accounting for a significant decrease of Kcnip2 gene ex-pression not only in heart failure, but also in hypertrophy, particularly inthe left ventricle wall [56]. Indeed, by inducing Kcnip2 overexpressionthrough gene transfer into banded rat hearts (which featured a pro-nounced left ventricular hypertrophy), these authors found a significantattenuation of the cardiac hypertrophic response. Therefore, in the con-text of the present investigation, it was of special interest to find a mo-lecular link between derangement of the circadian system and Kcnip2gene expression. Notably, in an elegant study, Jeyaraj and colleagues re-ported that cardiac Kcnip2 gene expression is under tight control of thekrüppel-like factor 15 (Klf15),which is a bonafide clock-controlled gene[57]. Ongoing experiments are aimed to verify whether the long termeffects of gestational chronodisruption on adult heart morphologymay be explained, at least in part, by sustained down-regulation ofKcnip2 gene expression. To this end, both the epigenetic status of theKcnip2 gene promoter and the ensuing mRNA and protein expressionlevels are under study at different developmental stages.

How does the alteration of a circadian cue for the pregnant mothermodify the transcription rate of 383 genes in the fetal heart? The syn-chronization of the circadian system has been exhaustively investigatedin adult mammals, but little is known about the fetal circadian system[58]. Emerging evidence indicates that the suprachiasmatic nucleus(adult's master clock) does not display circadian oscillations in thefetus. Nevertheless, rhythmic clock gene expression has been shown inother fetal organs, already displaying marked day/night differences at18 days of gestation in the rat [23]. Concerning long-lasting effects ofchronodisruption, several reports provide compelling evidence for epi-genetic modifications in the promoter region of clockwork genes in dif-ferent experimental settings; either through histone acetylation or genepromoter hypermethylation [59–62]. Given that canonical clock genes(together with a myriad of clock-controlled transcription factors) drivetranscription of a large fraction of the genome [58], these recentfindingsbring about the possibility that such a programming mechanism mayoperate in fetal cardiomyocytes submitted to chronodisruption.

In conclusion, a relevant fraction of the fetal cardiac transcriptomeresponds to chronodisruption along pregnancy; which in translationmight have far-reaching consequences for the offspring of pregnantshift working women. The diversity and complexity of the cardiac genenetworks altered by gestational chronodisruption (including severalestablished cardiovascular diseasemarkers), togetherwith the persistenttranscriptional down-regulation of Kcnip2, suggest enduring molecular

changes which may shape the observed adult LL offspring's left ventriclehypertrophy.

Disclosures

None declared.

Funding

This work was supported by grants 1110220 from FONDECYT(to HGR) and ACT1116 from CONICYT (to CT-F and HGR), Chile.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.yjmcc.2013.10.020.

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