Foetal nicotine exposure causes PKCε gene repression by promoter methylation in rat hearts

Lawrence, Jennifer; Chen, Man; Xiong, Fuxia; Xiao, Daliao; Zhang, Haitao; Buchholz, John N.; Zhang, Li

doi:10.1093/cvr/cvq270

Cited by 56 publications

(109 citation statements)

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“…Our recent studies demonstrated that fetal hypoxia increased CpG methylation of the Sp1 binding sites at rat PKCε gene promoter, which contributed to PKCε gene repression in the developing heart (29). Subsequently, our studies also demonstrated a key role of the early growth response factor-1 (Egr-1) binding site in the regulation of PKCε promoter activity in fetal rat hearts (18). Yet, it remains elusive whether heightened methylation of the Egr-1 binding site contributes to sex dimorphism of hypoxia-induced programming of PKCε gene repression in the developing heart.…”

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confidence: 59%

“…RNA was extracted from hearts using TRIzol protocol (Invitrogen, Carlsbad, CA). PKCε mRNA abundance was determined by real-time RT-PCR using Icycler Thermal cycler (BioRad) with the primers of 5=-GCGAAGCCCCTAAGACAAT-3= (forward) and 5=-CACCCCAGATGAAATCCCTAC-3= (reverse) (18). Real-time RT-PCR was performed in a final volume of 25 l. Each PCR reaction mixture consisted of 600 nM of primers, 33 units of M-MLV reverse transcriptase (Promega, Madison, WI), and iQ SYBR Green Supermix (Bio-Rad) containing 0.625 unit Taq polymerase; 400 M each of dATP, dCTP, dGTP, and dTTP; 100 mM KCl; 16.6 mM ammonium sulfate; 40 mM Tris·HCl; 6 mM MgSO 4; SYBR Green I; 20 nM fluorescine; and stabilizers.…”

Section: Methodsmentioning

confidence: 99%

“…DNA was isolated from hearts using a GenElute Mammalian Genomic DNA Mini-Prep kit (Sigma), denatured with 2 N NaOH at 42°C for 15 min, and treated with sodium bisulfite at 55°C for 16 h, as previously described (18). DNA was purified with a Wizard DNA clean up system (Promega) and resuspended in 120 l of H2O.…”

Section: Methodsmentioning

confidence: 99%

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Promoter methylation of Egr-1 site contributes to fetal hypoxia-mediated PKCε gene repression in the developing heart

Chen

Xiong

Zhang

2013

American Journal of Physiology-Regulatory, Integrative and Comp

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Chen M, Xiong F, Zhang L. Promoter methylation of Egr-1 site contributes to fetal hypoxia-mediated PKCε gene repression in the developing heart. Am J Physiol Regul Integr Comp Physiol 304: R683-R689, 2013. First published February 20, 2013 doi:10.1152/ajpregu.00461.2012.-Fetal hypoxia causes protein kinase Cε (PKCε) gene repression in the heart resulting in heightened ischemic injury in male offspring in a sex-dependent manner. The present study tested the hypothesis that heightened methylation of the early growth response factor-1 (Egr-1) binding site at PKCε gene promoter contributes to sex dimorphism of hypoxia-induced programming of PKCε gene repression in the developing heart. Pregnant rats were divided into normoxic and hypoxic (10.5% O 2 from day 15 to 21 of gestation) groups. Hypoxia selectively decreased PKCε mRNA and protein abundance in the heart of male, but not female, near-term (21 days) fetuses. Methylation of the CpG site at the Egr-1 binding site of PKCε promoter was significantly increased in the male hearts by hypoxia, resulting in decreased Egr-1 binding affinity and reduced Egr-1 binding to the PKCε promoter. Nuclear Egr-1 levels were not affected by hypoxia. There was significantly higher abundance of estrogen receptor ␣ (ER␣) and ␤ (ER␤) isoforms in female than in male fetal hearts, which were not significantly altered by hypoxia. Both ER␣ and ER␤ bind to the Egr-1 binding site with significant greater levels in the female fetal hearts. The increased methylation with reduced Egr-1 binding and PKCε gene repression persisted in 3-mo-old adult male hearts in a sexdependent manner. The results indicate a key role for heightened methylation of the Egr-1 binding site in hypoxia-mediated programming of PKCε gene repression in the developing heart and suggest a novel protective mechanism of ER by binding to the Egr-1 binding site in epigenetic regulation of PKCε gene expression patterns in the early developmental stage. hypoxia; heart; PKCε; DNA methylation; Egr-1 LARGE EPIDEMIOLOGICAL STUDIES have shown that adverse environment in utero causes increased risk of developing endocrine and cardiovascular disorders such as diabetes, hypertension, and ischemic heart disease in adult life (4,5,9,11,21,32). Hypoxia during gestation is one of the most common insults to the fetal development and is thought to be associated with fetal intrauterine growth restriction and increased risk of cardiovascular dysfunction in offspring (3,12,22,25,35). Recent studies in rats have found that maternal hypoxia results in developmental programming of heightened heart vulnerability to ischemia and reperfusion injury in male offspring in a sex-dependent manner (3,22). Among other mechanisms, it has been demonstrated that repression of protein kinase Cε (PKCε) gene in the developing heart is a congruent mechanism for programming of increased heart susceptibility to ischemia and reperfusion injury in offspring (2,19,24,35).DNA methylation is a chief mechanism for epigenetic modification of gene expression patterns that are...

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confidence: 59%

Section: Methodsmentioning

confidence: 99%

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Promoter methylation of Egr-1 site contributes to fetal hypoxia-mediated PKCε gene repression in the developing heart

Chen

Xiong

Zhang

2013

American Journal of Physiology-Regulatory, Integrative and Comp

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“…It has been demonstrated that nicotine downregulates miR-133 and miR-590 by the nicotinic acetylcholine receptor, α7-nAChR, subsequently activates the transforming growth factor (TGF)-β1 receptor and increases the synthesis of TGF-β1 protein, resulting in structural alterations of increased collagen and reduced elastin content in vascular media (34). Additionally, maternal nicotine administration caused programming of protein kinase Cε gene repression through promoter methylation, simultaneously activating the sympathetic nervous system in the fetal heart, which compromised cardiovascular homeostasis (35).…”

Section: Discussionmentioning

confidence: 99%

Prenatal nicotine exposure induces gender-associated left ventricular-arterial uncoupling in adult offspring

Wang

Ouyang

Huang

et al. 2012

Molecular Medicine Reports

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Abstract. Alterations in vascular or myocardial structure and function have been demonstrated in offspring subjected to prenatal nicotine exposure (PNE), however, limited data are available on how these changes interact. The present study assessed the hypothesis that prenatal nicotine exposure induced gender-specific alterations of left ventricular-arterial coupling indices in adult offspring. Female Sprague-Dawley rats were exposed to either nicotine (8 mg/kg/day) or saline via subcutaneous osmotic mini-pumps throughout gestation. Male and female offspring, aged 12 months, underwent non-invasive echocardiography and invasive left ventricular cannulation. Left ventricular-arterial coupling was analysed as the ratio of effective arterial elastance (Ea) to ventricular end-systolic elastance (Ees). The left ventricular myocardium and aorta were stained with hematoxylin and eosin and the myocardial cell cross-sectional area was calculated. Simultaneously, the ratio of medium thickness to internal diameter in the aorta and mesenteric artery was determined. The fibrosis component of left ventricle myocardium was analyzed by Sirius-red staining and further confirmed by hydroxyproline determination. The elastic properties of the aortic wall were analyzed by van Gieson staining. PNE caused significant increases in pulse pressure (56.36±7.41 vs. 50.16±4.94 mmHg; P<0.05) and left ventricular meridional wall stress (78.25±9.12 vs. 69.64±7.58 kdyne/cm 2 ; P<0.05) in male offspring compared with the control. Conversely, no similar effect was observed in female offspring. An elevated augmentation index was noted in male and female pups. Additionally, Ea/Ees was reduced in PNE males compared with control males, due to a disproportionate increase in Ees vs. Ea whereas in females, Ea/Ees did not differ significantly due to tandem increase in Ea and Ees. In addition, collagen cross-linking was markedly higher in male offspring, whereas it was unaltered in females compared with their respective controls. Fragmentation of the elastic network in the aorta and the increased ratio of medial thickness to internal diameter in the mesenteric artery were more evident in male offspring when compared with female offspring. PNE caused combined ventricular-arterial stiffening in male and female offspring, with lower Ea/Ees in males, while Ea/Ees was preserved in females. Enhanced collagen cross-linking in the myocardium, underdeveloped elastic fibers in the aorta and remodeled resistance vessels were associated with pathological ventricular arterial mismatching. The results of the present study indicated that male offspring were more susceptible to the development of ventricular and arterial dysfunction in response to PNE compared with female offspring.

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“…These changes to DNA methylation may have functional consequences, with one of these studies identifying changes in gene expression associated with these methylation changes . There is also some evidence from a rat model for the capacity 88 DNA METHYLATION CHANGES FOLLOWING NICOTINE EXPOSURE for prenatal nicotine exposure (from gestational day 4) to impact on both DNA methylation and expression of protein kinase C epsilon (PKCε) -a gene which encodes for a protein associated with cardiac injury in the fetal heart (Lawrence et al, 2010;Murriel and Mochly-Rosen, 2003). Whether an in utero exposure to nicotine restricted to early gestation also has the potential to affect DNA methylation is unknown.…”

Section: Introductionmentioning

confidence: 99%

A study of the consequences of early gestational ethanol and/or nicotine exposure in the mouse

Yamada¹

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The in utero environment is a critical determinant of the immediate and future health of the developing fetus. In Australia, two of the most commonly used drugs during pregnancy are alcohol and nicotine. Prolonged periods of gestational exposure to alcohol and nicotine, separately, have been associated with a range of adverse outcomes in the offspring, in both humans and experimental animal models. The consequences of ethanol and nicotine exposure restricted to early gestation on resultant offspring are poorly characterised, and few animal models exist to build greater understanding. Moreover, the immediate molecular changes associated with exposures to ethanol and/or nicotine restricted to early gestation has yet to be described.This thesis utilised mouse models of early gestational ethanol and/or nicotine exposure to explore the impact of each of these exposures on offspring growth, as well as their associated transcriptional and epigenetic changes. In each model, pregnant C57BL/6 dams were provided with ad libitum access to solutions of 10% ethanol and/or 100 µg/ml nicotine from fertilisation (0.5 dpc) to midgestation (8.5 dpc). The exposure window utilised in each of the models is developmentally equivalent to the first three to four weeks of a human pregnancy.Previously, our laboratory identified reduced growth, microcephaly, and gene expression changes in the liver and hippocampus of the postnatal offspring of the model of prenatal ethanol exposure utilised in this thesis. In contrast, at mid-gestation (9.5 dpc), there was no evidence of developmental delay (somite number), reduced growth (crown-rump length), or microcephaly.Further, a genome-wide gene expression microarray (targeting 30 855 genes) and a screen of the expression of 641 microRNAs, identified only one gene (Bcl11b) as differentially expressed in male 9.5 dpc whole embryos following the ethanol exposure. These data suggest that there is a delay between the ethanol exposure and the onset of adverse phenotypes -arguing that the embryo is somehow able to form a memory of the ethanol exposure. The absence of widespread transcriptional changes suggests that changes in embryonic mRNA and microRNA expression are unlikely to be major contributors to the memory of the exposure.A early gestational model of nicotine exposure was newly established in this thesis. This nicotine exposure resulted in an average serum cotinine (metabolite of nicotine) concentration similar to that reported in humans who smoke 10 to 15 cigarettes per day. This exposure was sufficient to induce a reduction in body size both at mid-gestation and postnatally, but not at late gestation. Furthermore, the increased expression of Zscan10 in the whole male 9.5 dpc embryo was identified and validated across multiple cohorts. Zscan10 encodes for a transcription factor expressed throughout development, with a proposed role in the regulation of progenitor cells. The increased expression of Zscan10 in the nicotine-exposed embryos was associated with a reduction in DNA methylation ...

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Foetal nicotine exposure causes PKCε gene repression by promoter methylation in rat hearts

Abstract: this study demonstrates that prolonged nicotine exposure increases the sympathetic neurotransmitter release in the foetal heart and causes programming of PKCε gene repression through promoter methylation, linking maternal smoking to pathophysiological consequences in the offspring heart.

Cited by 56 publications

References 45 publications

Promoter methylation of Egr-1 site contributes to fetal hypoxia-mediated PKCε gene repression in the developing heart

Promoter methylation of Egr-1 site contributes to fetal hypoxia-mediated PKCε gene repression in the developing heart

Prenatal nicotine exposure induces gender-associated left ventricular-arterial uncoupling in adult offspring

A study of the consequences of early gestational ethanol and/or nicotine exposure in the mouse

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