Foetal nicotine exposure causes PKCε gene repression by promoter methylation in rat hearts (original) (raw)

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Department of Physiology and Pharmacology

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Center for Perinatal Biology, Loma Linda University School of Medicine

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Loma Linda, CA 92350

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USA

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Department of Physiology and Pharmacology

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Center for Perinatal Biology, Loma Linda University School of Medicine

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Loma Linda, CA 92350

,

USA

Search for other works by this author on:

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Department of Physiology and Pharmacology

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Center for Perinatal Biology, Loma Linda University School of Medicine

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Loma Linda, CA 92350

,

USA

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Department of Physiology and Pharmacology

,

Center for Perinatal Biology, Loma Linda University School of Medicine

,

Loma Linda, CA 92350

,

USA

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,

Department of Physiology and Pharmacology

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Center for Perinatal Biology, Loma Linda University School of Medicine

,

Loma Linda, CA 92350

,

USA

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Department of Physiology and Pharmacology

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Center for Perinatal Biology, Loma Linda University School of Medicine

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Loma Linda, CA 92350

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USA

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Department of Physiology and Pharmacology

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Center for Perinatal Biology, Loma Linda University School of Medicine

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Loma Linda, CA 92350

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USA

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Jennifer Lawrence, Man Chen, Fuxia Xiong, Daliao Xiao, Haitao Zhang, John N. Buchholz, Lubo Zhang, Foetal nicotine exposure causes PKCε gene repression by promoter methylation in rat hearts, Cardiovascular Research, Volume 89, Issue 1, 1 January 2011, Pages 89–97, https://doi.org/10.1093/cvr/cvq270
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Abstract

Aims

Foetal nicotine exposure results in decreased protein kinase C epsilon (PKCε) expression and increased cardiac vulnerability to ischaemia and reperfusion injury in adult rat offspring. The present study tested the hypothesis that maternal nicotine administration causes increased promoter methylation of the PKCε gene resulting in PKCε repression in the heart.

Methods and results

Nicotine treatment of pregnant rats starting at day 4 of gestation increased the methylation of the Egr-1 binding site at the PKCε gene promoter and decreased PKCε protein and mRNA abundance in near-term foetal hearts. Methylation of the Egr-1 binding site reduced Egr-1 binding to the PKCε promoter in the heart. Site-specific deletion of the Egr-1 binding site significantly decreased PKCε promoter activity. The effects of nicotine were sustained in the heart of adult offspring. Ex vivo studies found no direct effect of nicotine on PKCε gene expression. However, maternal nicotine administration increased norepinephrine content in the foetal heart. Treatment of isolated foetal hearts with norepinephrine resulted in the same effects of increased methylation of the Egr-1 binding site and PKCε gene repression in the heart. 5-Aza-2′-deoxycytidine inhibited the norepinephrine-induced increase in methylation of the Egr-1 binding site and restored Egr-1 binding and PKCε gene expression to the control levels.

Conclusion

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.

1. Introduction

Maternal cigarette smoking is a major worldwide health concern and has been associated with adverse pregnancy outcomes for the mother, her foetus, and the newborn. The adverse consequences have been well identified in epidemiological studies, including intrauterine growth restriction, sudden infant death syndrome, and cardiovascular disease in offspring.1–3 As one of the major toxic components in cigarette smoking, nicotine is likely to be a major contributor to the development of cardiovascular disorders. Indeed, our previous studies demonstrated that perinatal nicotine exposure altered vascular function in adult offspring and decreased the ability of the heart to recover from acute ischaemia and reperfusion injury in rats.4–6

These findings are consistent with a large body of evidence of epidemiological and animal studies suggesting a clear link between adverse intrauterine environment and an increased risk of ischaemic heart disease in later adult life.7–9 Our previous studies demonstrated that foetal nicotine exposure resulted in a decrease in protein kinase C epsilon (PKCε) protein expression in the heart of adult offspring,5 suggesting a possible mechanism in nicotine-mediated foetal programming of increased cardiac vulnerability to ischaemic injury in offspring. Indeed, the causal role of reduced PKCε expression in foetal programming of increased heart susceptibility to ischaemia and reperfusion injury in offspring has been demonstrated previously.10–12 These findings are consistent with other studies demonstrating that PKCε plays a pivotal role of cardioprotection in heart ischaemia and reperfusion injury.13–16

The finding of decreased PKCε protein expression in the heart of adult offspring that had been exposed to nicotine before birth5 suggests that an epigenetic mechanism may explain PKCε gene repression in the heart. Indeed, recent studies suggest that epigenetic modification of gene expression patterns plays a key role in foetal programming of cardiovascular dysfunction.9,17 DNA methylation is a chief mechanism for epigenetic modification of gene expression patterns and occurs at cytosines in the dinucleotide sequence of CpG.18 Although transcriptional regulation by DNA methylation is often observed in CpG islands located around the promoter region via the sequence-nonspecific and methylation-specific binding of inhibiting methyl-CpG binding proteins,19,20 DNA methylation of sequence-specific transcription factor binding sites can alter gene expression through changes in the binding affinity of transcription factors by altering the major groove structure of DNA to which the DNA binding proteins bind.21–23 Herein, we present evidence that maternal nicotine administration causes an increase in methylation of the early growth response protein 1 (Egr-1) binding site at the proximal promoter region of PKCε gene, resulting in PKCε gene repression in the foetal and offspring heart. Furthermore, we demonstrate that the effect of nicotine is mediated by the increased sympathetic neurotransmitter norepinephrine content in the foetal heart.

2. Methods

An expanded Methods section is available in the Supplementary material online.

2.1 Experimental animals

Pregnant Sprague Dawley rats were randomly divided into two groups: (i) saline control and (ii) nicotine administration through an osmotic mini-pump at 4 µg/kg/min from day 4 to 21 of gestation, as described previously.4 Hearts were isolated from near-term (21 days) foetuses and 3 months old offspring. To study the direct effect of nicotine on the foetal heart, hearts were isolated from day 17 foetal rats and cultured at 37°C in 95% air/5% CO224 in the absence or presence of nicotine and norepinephrine, respectively, for 48 h. All procedures and protocols were approved by the Institutional Animal Care and Use Committee of Loma Linda University and followed the guidelines by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

2.2 Cell culture

Rat embryonic heart-derived myogenic cell line H9c2 cells were grown and subcultured and experiments were performed at 70–80% confluent. Cells were treated with 10 µmol/L norepinephrine in the absence or presence of 5-aza-2′-deoxycytidine, prazosin, or propranolol, respectively, for 48 h.

2.3 Western blot analysis

Protein abundance of PKCε, PKCδ, and Egr-1 in the hearts and H9c2 cells was measured with western blot analysis, as described previously.24

2.4 Real-time RT–PCR

mRNA abundance of PKCε and PKCδ in the hearts and H9c2 cells was determined by real-time RT–PCR, as described previously.24

2.5 Quantitative methylation-specific PCR

DNA collected from the hearts and H9c2 cells was treated with sodium bisulfite at 55°C for 16 h, and was used as a template for real-time fluorogenic methylation-specific PCR (MSP) using primers to amplify promoter binding sites containing possible methylation sites at rat PKCε promoter.24

2.6 Electrophoretic mobility shift assay (EMSA)

Nuclear extracts were collected from the hearts, and electrophoretic mobility shift assay (EMSA) was performed using the oligonucleotide probes with either CpG or mCpG in the Egr-1 binding sites (−1008) at rat PKCε promoter region, as described previously.24

2.7 Chromatin immunoprecipitation (ChIP)

Chromatin extracts were prepared from the hearts and H9c2 cells, and chromatin immunoprecipitation (ChIP) assays were performed for the Egr-1 binding sites (−1008) at rat PKCε promoter in DNA sequences pulled down by an Egr-1 antibody, as described previously.24

2.8 Reporter gene assay

A 1941 bp fragment of rat PKC promoter region spanning −1941 to −1 bp relative to the transcriptional start site of the PKCε was amplified by PCR and inserted into the luciferase reporter gene plasmid, pGL3 (Promega) to yield the full-length promoter–reporter plasmid denoted as pPKCε1941. Two 5′-truncation mutants (pPKCε1163 and pPKCε826) as well as one site-specific Egr-1 deletion mutation [pPKCε(-Egr-11008)] were constructed. Cell transfection was performed using H9c2 cells. After 48 h, firefly and Renilla reniformis luciferase activities in cell extracts were measured in a luminometer using a dual-luciferase reporter assay system.24,25

2.9 High-performance liquid chromatography (HPLC)

Foetal hearts were homogenized and norepinephrine extracted according to the method described previously.26 Norepinephrine content was measured by a high-performance liquid chromatography (HPLC) system with an electrochemical detection, as described previously.26

2.10 Statistical analysis

Data are expressed as mean ± SEM. Statistical significance (P < 0.05) was determined by analysis of variance followed by Neuman–Keuls post hoc test or Student's _t_-test, where appropriate.

3. Results

3.1 Nicotine suppresses PKCε expression in the foetal heart

Maternal nicotine administration resulted in significant decreases in both protein and mRNA levels of PKCε in foetal rat hearts. In contrast, PKCδ protein expression was not affected (Figure 1).

3.2 Nicotine increases methylation of the Egr-1 binding site at the PKCε promoter

Out of the eight CpG dinucleotide-containing transcription factor binding sites identified at rat PKCε gene promoter,24 methylation levels were significantly increased at the putative Egr-1 (−1008) and MTF-1 (−603) binding sites in the hearts of foetuses with maternal nicotine administration, when compared with the control (Figure 2A). Nicotine did not significantly change the methylation status of Stra13 (−1723), PPARG (−1688), E2F (−1621), SP1 (−346 and −268), and MTF-1 (−168).

3.3 Involvement of the Egr-1 binding site in PKCε promoter activation

Given the previous findings of the lack of the MTF-1 binding to the putative MTF-1 site at −603,25 our further investigation was focused on the Egr-1 binding site. Incubation of nuclear extracts from foetal rat hearts with the double-stranded oligonucleotide probes encompassing the putative Egr-1 binding site resulted in the appearance of a major DNA–protein complex, which was further super-shifted by an Egr-1 antibody (Figure 3A). The involvement of the Egr-1 binding site in the regulation of PKCε promoter activity was further determined with a reporter gene assay in the rat embryonic ventricular myocyte cell line H9c2. The level of basal full-length promoter activity obtained with pPKCε1941 was approximately four-fold greater than that obtained with the vector alone in the high-glucose medium. Whereas 5′ deletion of the promoter at −1163 (pPKCε1163) had no significant effect on the promoter activity, the promoter activity fell by 37% with a further deletion from −1163 to −826 (pPKCε826) that contains the Egr-1 binding site at −1008 (Figure 3B). Additionally, a site-specific deletion of the Egr-1 binding site at −1008 caused a 26% decrease in the PKCε promoter activity (Figure 3B), indicating a strong stimulatory role of Egr-1 binding to its binding site in regulating PKCε promoter activity.

3.4 Methylation of the Egr-1 binding site inhibits Egr-1 binding

To determine whether methylation of the Egr-1 site inhibits Egr-1 binding, the binding affinity of Egr-1 to the methylated and unmethylated oligonucleotide probes containing the Egr-1 binding site was determined by competitive EMSA performed in pooled nuclear extracts from foetal hearts with the increasing ratio of unlabelled/labelled oligonucleotides encompassing the Egr-1 site. As shown in Supplementary material online, Figure S1, methylation of the Egr-1 binding site significantly decreased the Egr-1 binding affinity to it (slope: −27.9 ± 5.4 vs. −50.4 ± 7.2, P < 0.05). To determine whether nicotine affects Egr-1 binding to the unmethylated binding site, the binding affinity of the nuclear extracts to the unmethylated oligonucleotide probes containing the Egr-1 site was also measured. Data showed that nicotine had no significant effect on the binding of nuclear extracts to the unmethylated Egr-1 binding site in foetal hearts (Supplementary material online, Figure S1). To further determine whether nicotine-mediated increase in methylation of the Egr-1 binding site inhibits Egr-1 binding to the PKCε promoter in vivo in the context of intact chromatin, ChIP assays were performed. Figure 2B shows that foetal nicotine exposure caused a marked decrease of 78% in Egr-1 binding to the PKCε promoter in the heart.

3.5 Lack of effect of nicotine on Egr-1 protein abundance

To determine whether the nicotine-induced decrease in Egr-1 binding was in part due to decreased Egr-1 protein abundance, western analyses were performed using an Egr-1 antibody. There was no significant difference in nuclear Egr-1 protein abundance either in foetal hearts between control and maternal nicotine treatment groups or in the hearts of both male and female offspring between the control and prenatally nicotine-treated animals (Supplementary material online, Figure S2).

3.6 Direct effect of norepinephrine, but not nicotine, on PKCε expression in the foetal heart

To determine whether the effect of maternal nicotine administration on the foetal heart was caused by the direct effect of nicotine, PKCε and PKCδ protein expression was measured in foetal rat hearts in ex vivo treatment with 1 or 10 µM nicotine for 48 h. As shown in Figure 4A, nicotine had no direct effect on either PKCε or PKCδ protein expression in the foetal heart. Given that nicotine stimulates sympathetic neurotransmitter release, we determined the effect of foetal nicotine exposure on norepinephrine content in the foetal heart. Figure 4B shows a significant increase in norepinephrine content in the hearts of foetuses with maternal nicotine treatment. Consistent with the findings of foetal nicotine exposure, the direct treatment of foetal hearts with norepinephrine for 48 h resulted in a significant decrease in PKCε, but not PKCδ, protein and mRNA abundance (Figure 4C). Furthermore, norepinephrine caused a similar extent of increase in methylation of the Egr-1 binding site at the PKCε promoter in the foetal heart (Figure 4D).

3.7 Inhibition of DNA methylation restores PKCε expression

To determine further the causal role of Egr-1 binding site methylation in the downregulation of PKCε expression, rat embryonic ventricular myocyte cell line H9c2 cells were treated with norepinephrine in the absence or presence of a DNA methylation inhibitor 5-aza-2′-deoxycytidine. Consistent with the findings in foetal hearts, the norepinephrine treatment caused a significant decrease in both mRNA and protein abundance of PKCε in H9c2 cells, which was inhibited by α1 adrenoceptor antagonist prazosin, but not by β adrenoceptor antagonist propranolol (Figure 5A). 5-Aza-2′-deoxycytidine inhibited the norepinephrine-induced increase in methylation of the Egr-1 binding site, reversed the decreased Egr-1 binding to the PKCε promoter (Figure 5B), and restored PKCε mRNA and protein expression to the control levels (Figure 5C).

3.8 Effect of foetal nicotine exposure is sustained in the hearts of adult offspring

Consistent with the previous finding that maternal nicotine administration resulted in a significant decrease in PKCε protein abundance in the hearts of both male and female offspring in rats,5 foetal nicotine exposure resulted in a significant decrease in PKCε mRNA levels in the hearts of male and female offspring (Figure 6). Consistently, the nicotine-induced increase in methylation of the Egr-1 binding site at the PKCε promoter was sustained in the adult hearts, resulting in decreased Egr-1 binding to the promoter as determined by ChIP assays (Figure 6). Similar to the findings in the foetal heart, competitive EMSA revealed that maternal nicotine administration did not alter the Egr-1 binding affinity to the unmethylated binding site in nuclear extracts from the hearts of both male and female offspring, but the binding affinity of the nuclear extracts to the methylated Egr-1 binding sequence was significantly reduced in both males (slope: −30.5 ± 5.5 vs. −61.7 ± 10.4, P < 0.05) and females (slope: −23.4 ± 6.7 vs. −52.8 ± 7.2, P < 0.05) (Supplementary material online, Figure S1).

4. Discussion

The present study demonstrates in a rat model that maternal nicotine administration causes PKCε gene repression in the foetal heart through an increase in methylation of the Egr-1 binding site at the PKCε gene promoter and decreased Egr-1 binding to the promoter. These altered promoter methylation and gene expression patterns are sustained in the heart of adult offspring. Additionally, the study demonstrates that nicotine has no direct effect but rather stimulates the release of sympathetic neurotransmitter norepinephrine in the foetal heart, resulting in PKCε gene repression.

Figure 1

Effect of in utero nicotine exposure on PKCε and PKCδ protein and mRNA abundance in foetal hearts. Pregnant rats were treated with saline (control) or nicotine, and hearts were obtained from near-term foetuses. Data are mean ± SEM, *P < 0.05 vs. control (n = 5).

Figure 2

Effect of in utero nicotine exposure on CpG methylation of the PKCε promoter in foetal hearts. Pregnant rats were treated with saline (control) or nicotine, and hearts were obtained from near-term foetuses. (A) DNA was isolated and methylation levels were determined by methylation-specific real-time PCR. (B) Egr-1 binding to the PKCε promoter at −1008 Egr-1 binding site was determined by ChIP assays. Data are means ± SEM. *P< 0.05 vs. control (n = 5).

The finding of increased methylation of the Egr-1 binding site at the PKCε gene promoter in foetal hearts caused by maternal nicotine treatment suggests an epigenetic mechanism in programming of PKCε gene repression in the heart. Although methylation of the putative MTF-1 site at −603 was also increased, the previous study failed to demonstrate the binding of MTF-1 to this site in the antibody supershift experiments, suggesting the lack of its effect in regulating PKCε promoter activity.25 In contrast, the finding that an antiserum to Egr-1 caused a supershift of the protein–DNA complex, resulting from the binding of nuclear extracts from the foetal heart with the double-stranded oligonucleotide probes containing the putative Egr-1 element at the PKCε promoter at −1008, demonstrates a consensus Egr-1 binding site in the PKCε promoter in rat hearts. Although the previous study showed that an extended 5′-deletions of the PKCε promoter from −1163 to −444, which contains the Egr-1 binding site at −1008, as well as multiple other binding sites, had no significant effect on the promoter activity,25 the more confined deletion from −1163 to −826 in the present study reveals a significant decrease in the PKCε promoter activity. The functional significance of the Egr-1 binding site in regulating PKCε gene activity is further demonstrated by a site-specific deletion of the Egr-1 element at −1008. In the present study, we found that methylation levels of the Egr-1 binding site at the PKCε promoter were low in control foetal hearts, suggesting an active and important role of Egr-1 in regulating PKCε gene activity. The relatively higher methylation levels of Egr-1 as well as other binding sites found in the previous study25 were possibly due to the inefficient DNA bisulfite treatment. In the present study, instead of using a heat block, the bisulfite treatment was performed using an iCycler Thermal cycler with a 4-cycle program of 95°C for 5 min and 55°C for 4 h to maintain the DNA denatured and a stable temperature of the reaction system. The other possible cause is the developmental difference in promoter methylation patterns. When compared with the present study of foetal hearts, the previous study was conducted in three months old rats. The finding that Egr-1 protein abundance in foetal hearts was not significantly affected by maternal nicotine treatment, together with the finding that nicotine had no significant effect on the Egr-1 binding affinity to the unmethylated binding site, suggest a primary role of increased methylation of the Egr-1 binding site in nicotine-mediated PKCε gene repression in foetal hearts. Indeed, there was a significant decrease in the Egr-1 binding affinity to the methylated Egr-1 binding element when compared with the unmethylated sequence. This resulted in a significant decrease in Egr-1 binding to the PKCε promoter in vivo in the context of intact chromatin in foetal hearts, as demonstrated by ChIP assays. Similarly, methylation of the Egr-1 binding site in muscle p57kip2 promoter repressed its expression by directly interfering of methyl groups with the binding of the transcription factor.27

In the present study, the dose rate of nicotine administration (4 µg/kg/min) results in blood nicotine concentrations similar to those found in humans who smoke or use nicotine gum and patch.1,28 Nicotine readily crosses the placenta into the foetal circulation and foetal nicotine concentrations are generally 15% higher than maternal levels.29 Although this suggests a possible direct effect of nicotine on the foetal heart, the finding that nicotine had no significant effect on either PKCε or PKCδ protein abundance in isolated foetal hearts indicates a lack of the direct effect of nicotine on PKCε expression, and raises a possibility of a mediator(s) in the nicotine's effect. Indeed, foetal nicotine exposure significantly increased norepinephrine content in the heart, consisting with the previous findings in humans that maternal nicotine consumption resulted in an increase in foetal heart rate through a catecholamine response.28 Similarly, maternal nicotine treatment resulted in increased norepinephrine levels in foetal rat brains.30 The levels of norepinephrine content in the foetal heart determined in the present study are similar to those found in human foetal hearts,31 and, as expected, somewhat lower than those in adult rat hearts.32 The finding that the treatment of isolated foetal heart with norepinephrine in an ex vivo culture system produced the same result as that obtained with maternal nicotine administration, i.e. the decreased PKCε but not PKCδ protein and mRNA abundance in the foetal heart, indicates norepinephrine as the mediator of the nicotine's effect in foetal hearts. This is further supported by the finding that norepinephrine caused the same pattern of increased methylation of the Egr-1 binding site at the PKCε promoter in the foetal heart, as that seen in the maternal nicotine treatment.

We have recently demonstrated in a rat model that maternal hypoxia causes an increase in methylation in non-CpG island and sequence-specific SP1 binding sites and decreased SP1 binding and the PKCε promoter activity in the foetal heart.12 Interestingly, maternal hypoxia caused an increase in methylation at both the SP1 and Egr-1 binding sites in the foetal heart, yet the direct effect of hypoxia on H9c2 cells resulted in increased methylation only at the SP1 binding sites.12 These findings are intriguing and suggest a lack of the direct effect of hypoxia on methylation at the Egr-1 binding site. Given that foetal hypoxia increases sympathetic activity as well,33,34 it is possible that the increased Egr-1 methylation at the PKCε promoter in foetal hearts seen in the rat model of maternal hypoxia12 was mediated in part by an increase in sympathetic neurotransmitter norepinephrine content in the heart in response to hypoxic stress. Whereas these studies demonstrate the different patterns of promoter methylation between maternal hypoxia and nicotine treatments, they reveal a possible common mechanism of norepinephrine-mediated increase in methylation of the Egr-1 binding site at the PKCε promoter in foetal hearts.

Figure 3

The role of the Egr-1 binding site in the PKCε promoter. (A) Nuclear extracts (N/E) from foetal hearts were incubated with double-stranded oligonucleotide probes containing the PKCε gene consensus Egr-1 binding motif in the absence or presence of Egr-1 and SP1 antibodies. (B) A 1941 bp fragment of rat PKC promoter region spanning −1941 to −1 bp relative to the transcriptional start site of the PKCε was amplified by PCR and inserted into pGL3 to yield the full-length promoter–reporter plasmid denoted as pPKCε1941. Two 5′-truncation mutants (pPKCε1163 and pPKCε826) and one site-specific deletion mutation (pPKCε(-Egr-11008)) were constructed. Constructs were then transfected to H9c2 cells. Firefly and Renilla reniformis luciferase activities were measured after 48 h in a luminometer using a dual-luciferase reporter assay system. Data are means ± SEM, *P< 0.05 vs. pPKCε1941 (n = 5).

Figure 4

Effect of nicotine and norepinephrine on Egr-1 methylation and PKCε/PKCδ expression in isolated foetal hearts. (A, C, and D) Hearts were isolated from 17-day foetal rats and were incubated in the absence (control) or presence of nicotine or norepinephrine for 48 h. PKCε and PKCδ protein and mRNA abundance (A and C) and methylation of Egr-1 and MTF-1 (−168) binding sites (D) were determined. (B) Pregnant rats were treated with saline (control) or nicotine, and hearts were obtained from near-term foetuses. Norepinephrine content was measured by HPLC. Data are mean ± SEM. *P < 0.05 vs. control (n = 5).

Figure 5

Reversal effect of 5-aza-2′-deoxycytidine on norepinephrine-induced Egr-1 methylation and PKCε gene repression in H9c2 cells. (A) PKCε protein and mRNA abundance was determined in the absence (control) or presence of norepinephrine (NE), NE plus prazosin, and NE plus propranolol. (B) Egr-1 methylation and Egr-1 binding to the PKCε promoter in the context of intact chromatin were determined in the absence (control) or presence of NE and NE plus 5-aza-2′-deoxycytidine (5-Aza-2′-dC). (C) PKCε protein and mRNA abundance was determined in the absence (control) or presence of NE and NE plus 5-Aza-2′-dC. Data are mean ± SEM. *P < 0.05 vs. control (n = 5–12).

Figure 6

Effect of in utero nicotine exposure on Egr-1 methylation and PKCε expression in adult offspring. Pregnant rats were treated with saline (control) or nicotine, and hearts were isolated from 3 months old male and female offspring. PKCε mRNA abundance, methylation of the Egr-1 and MTF-1 (−168) binding sites, and Egr-1 binding to the PKCε promoter in the context of intact chromatin were determined by real-time RT–PCR, quantitative methylation-specific PCR and ChIP assay, respectively. Data are mean ± SEM. *P < 0.05 vs. control (n = 5).

The causal effect of increased methylation in norepinephrine-induced PKCε gene repression was further demonstrated in H9c2 cells with a DNA methylation inhibitor 5-aza-2′-deoxycytidine.12,35–39 H9c2 cells have been widely used in a variety of myocardiocyte studies, including those investigating apoptosis, differentiation, and ischaemia and reperfusion injury. In the present study, the norepinephrine-induced decrease in PKCε expression observed in foetal hearts mirrored that found in H9c2 cells, suggesting a congruent underlying mechanism and providing a comparable model of H9c2 cells in the study of PKCε gene regulation. This is supported by a recent study of hypoxia on PKCε gene repression in the heart.12 5-Aza-2′-deoxycytidine inhibited norepinephrine-induced downregulation of PKCε expression and restored the mRNA and protein abundance to the control levels. This is caused by inhibition of the norepinephrine-induced methylation of Egr-1 binding site resulting in recovered Egr-1 binding to the PKCε promoter in the intact chromatin. These findings are in agreement with the previous studies demonstrating that 5-aza-2′-deoxycytidine inhibited promoter methylation and restored PKCε mRNA and protein expression in the presence of a stressor in H9c2 cells12 and foetal rat hearts.24 Although it is not clear at present what mechanisms mediate the norepinephrine-induced promoter methylation in cardiomyocytes, recent studies have suggested a link between prolonged oxidative stress and aberrant DNA methylation patterns.40–42 The present study demonstrates that norepinephrine-induced PKCε gene repression is mediated by α1-adrenoceptors. Given the findings that norepinephrine increases reactive oxygen species production in cardiomyocytes through α1-adrenoceptors,43–46 and that cardiomyocytes are major producers of reactive oxygen species due to their high metabolic demand, it is plausible to suggest that increased oxidative stress is a mechanism in the norepinephrine-induced methylation and repression of PKCε gene in foetal hearts.

The finding that the same pattern of increased Egr-1 site methylation and decreased PKCε gene transcription induced by foetal nicotine exposure was sustained in adult hearts is consistent with the previous findings of decreased PKCε protein abundance in the hearts of adult offspring that had prenatal nicotine exposure,5 demonstrating a mechanism of epigenetic programming of PKCε gene repression in the heart. Consistent with the findings in foetal hearts, prenatal nicotine exposure had no significant effects on nuclear Egr-1 protein abundance or the Egr-1 binding affinity to the unmethylated binding site, but methylation of the Egr-1 binding site significantly decreased the Egr-1 binding affinity and reduced Egr-1 binding to the PKCε gene promoter in adult hearts. PKCε plays a pivotal role of cardioprotection in heart ischaemia and reperfusion injury.13–15 The study in a PKCε knock-out mouse model has demonstrated that PKCε expression is not required for cardiac function under normal physiological conditions, but PKCε activation is necessary for acute cardioprotection during cardiac ischaemia and reperfusion.16 The causal role of reduced PKCε gene expression in programming of increased heart vulnerability to ischaemia and reperfusion injury in adult offspring has been demonstrated by selective activation and/or inhibition of PKCε in the heart.10–12

In contrast to the previous findings that prenatal hypoxia and cocaine treatments caused sex-dependent programming of PKCε gene repression and increased heart vulnerability to ischaemia and reperfusion injury in adult male offspring,10,12,25,47 the foetal nicotine exposure resulted in sex-independent programming of PKCε gene repression and heart vulnerability in both male and female offspring.5 These findings suggest a sex dichotomy of PKCε gene expression patterns in the heart and reinforce a key role for PKCε in programming of heart vulnerability to ischaemia and reperfusion injury in adult offspring. The sex dichotomy in foetal programming of adult disease has been demonstrated in several animal models.48,49 Although it is plausible to speculate a primary role of sex hormones developed postnatally for the sex dichotomy seen in foetal programming of adult disease, our recent studies demonstrated that foetal hypoxia and cocaine exposure caused sex-dependent changes in PKCε gene expression pattern in the hearts of foetuses and neonates before the sexual maturity.12,25 This is possibly due to the greater expression of oestrogen receptor α and β isoforms in the hearts of female foetuses, which bind to the SP1 binding sites at the PKCε promoter and protect them from methylation in response to foetal stresses.12 The interaction between oestrogen receptors and SP1 binding sites has been well documented.12,50 In contrast, the lack of sex difference in methylation of the Egr-1 binding site and PKCε gene expression patterns in the hearts observed in the present study is possibly due to a lack of the protection of oestrogen receptors on the Egr-1 binding site at the PKCε promoter.

Together with the previous studies, the present investigation provides evidence of differential promoter methylation patterns in subtle epigenetic modifications of PKCε gene repression in the developing heart caused by adverse intrauterine environment, supporting the concept that foetal programming may result in highly specific altered methylation patterns in a gene promoter and gene expression patterns in adult offspring. Several animal models of foetal insults, including hypoxia, cocaine, and nicotine, have demonstrated PKCε gene repression in the heart and increased heart vulnerability to ischaemia and reperfusion injury in offspring, suggesting a common mechanism of PKCε in foetal programming of heart disease in later adult life. Although the caution should be observed in extrapolating the findings of animal studies directly to the humans, several lines of evidence suggest a possible clinical significance of these studies. Ischaemic heart disease is the leading cause of death in the USA, and PKCε is a key player in cardioprotection in acute heart ischaemia and reperfusion injury. Additionally, maternal cigarette smoking and use of nicotine gum and patch are a major stress to the developing foetuses, and large epidemiological studies indicate a link between in utero adverse stimuli during gestation and an increased risk of ischaemic heart disease in the adulthood.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.

Funding

This work was supported in part by the National Institutes of Health [Grant nos HL82779 (L.Z.), HL83966 (L.Z.)].

References

1

Fetal nicotine or cocaine exposure: which one is worse?

,

J Pharmacol Exp Ther

,

1998

, vol.

285

(pg.

931

-

945

)

2

Increased blood pressure in neonates and infants whose mothers smoked during pregnancy

,

J Pediatr

,

1996

, vol.

128

(pg.

806

-

812

)

3

et al.

Maternal cigarette smoking during pregnancy, low birth weight and subsequent blood pressure in early childhood

,

Early Hum Dev

,

2000

, vol.

57

(pg.

137

-

147

)

4

Fetal and neonatal nicotine exposure differentially regulates vascular contractility in adult male and female offspring

,

J Pharmacol Exp Ther

,

2007

, vol.

320

(pg.

654

-

661

)

5

Prenatal nicotine exposure increases heart susceptibility to ischemia/reperfusion injury in adult offspring

,

J Pharmacol Exp Ther

,

2008

, vol.

324

(pg.

331

-

341

)

6

Prenatal gender-related nicotine exposure increases blood pressure response to angiotensin II in adult offspring

,

Hypertension

,

2008

, vol.

51

(pg.

1239

-

1247

)

7

Infant mortality, childhood nutrition, and ischaemic heart disease in England and Wales

,

Lancet

,

1986

, vol.

1

(pg.

1077

-

1081

)

8

et al.

Developmental plasticity and human health

,

Nature

,

2004

, vol.

430

(pg.

419

-

421

)

9

Effect of in utero and early-life conditions on adult health and disease

,

N Engl J Med

,

2008

, vol.

359

(pg.

61

-

73

)

10

Prenatal hypoxia causes a sex-dependent increase in heart susceptibility to ischemia and reperfusion injury in adult male offspring: Role of PKCε

,

J Pharmacol Exp Ther

,

2009

, vol.

330

(pg.

624

-

632

)

11

Prenatal cocaine exposure abolished ischemic preconditioning-induced protection in adult male rat hearts: role of PKCε

,

Am J Physiol Heart Circ Physiol

,

2009

, vol.

296

(pg.

H1566

-

H1576

)

12

Chronic hypoxia induces epigenetic programming of PKCε gene repression in fetal rat heart

,

Circ Res

,

2010

[Epub ahead of print, 10 June]

13

et al.

Opposing cardioprotective actions and parallel hypertrophic effects of delta PKC and epsilon PKC

,

Proc Natl Acad Sci USA

,

2001

, vol.

98

(pg.

11114

-

11119

)

14

Functional proteomic analysis of protein kinase C epsilon signaling complexes in the normal heart and during cardioprotection

,

Circ Res

,

2001

, vol.

88

(pg.

59

-

62

)

15

Opposing roles of delta and epsilonPKC in cardiac ischemia and reperfusion: targeting the apoptotic machinery

,

Arch Biochem Biophys

,

2003

, vol.

420

(pg.

246

-

254

)

16

Preservation of base-line hemodynamic function and loss of inducible cardioprotection in adult mice lacking protein kinase C epsilon

,

J Biol Chem

,

2004

, vol.

279

(pg.

3596

-

3604

)

17

Epigenetic modification of the renin-angiotensin system in the fetal programming of hypertension

,

Circ Res

,

2007

, vol.

100

(pg.

520

-

526

)

18

The role of DNA methylation in mammalian epigenetics

,

Science

,

2001

, vol.

293

(pg.

1068

-

1070

)

19

Cancer epigenetics comes of age

,

Nat Genet

,

1999

, vol.

21

(pg.

163

-

167

)

20

Methyl CpG-binding proteins and transcriptional repression

,

Bioessays

,

2001

, vol.

23

(pg.

1131

-

1137

)

21

CpG methylation as a mechanism for the regulation of E2F activity

,

Proc Natl Acad Sci USA

,

2000

, vol.

97

(pg.

6481

-

6486

)

22

et al.

Methylation of adjacent CpG sites affects Sp1/Sp3 binding and activity in the p21Cip1 promoter

,

Mol Cell Biol

,

2003

, vol.

23

(pg.

4056

-

4065

)

23

Methylation adjacent to negatively regulating AP-1 site reactivates TrkA gene expression during cancer progression

,

Oncogene

,

2005

, vol.

24

(pg.

5108

-

5118

)

24

Direct effect of cocaine on epigenetic regulation of PKCepsilon gene repression in the fetal rat heart

,

J Mol Cell Cardiol

,

2009

, vol.

47

(pg.

504

-

511

)

25

Fetal exposure to cocaine causes programming of prkce gene repression in the left ventricle of adult rat offspring

,

Biol Reprod

,

2009

, vol.

80

(pg.

440

-

448

)

26

Chronic nicotine administration does not affect peripheral vascular reactivity in the rat

,

J Pharmacol Exp Ther

,

1994

, vol.

271

(pg.

1135

-

1142

)

27

Regulation of p57KIP2 during muscle differentiation: role of Egr1, Sp1 and DNA hypomethylation

,

J Mol Biol

,

2008

, vol.

380

(pg.

265

-

277

)

28

The maternal and fetal physiologic effects of nicotine

,

Semin Perinatol

,

1996

, vol.

20

(pg.

115

-

126

)

29

Fetal toxicology of environmental tobacco smoke

,

Curr Opin Pediatr

,

1995

, vol.

7

(pg.

128

-

131

)

30

Alterations of brain tissue in fetal rats exposed to nicotine in utero: possible involvement of nitric oxide and catecholamines

,

Neurotoxicol Teratol

,

2004

, vol.

26

(pg.

103

-

112

)

31

Catecholamines in tissues of the human fetus

,

Pediatrics

,

1961

, vol.

27

(pg.

904

-

911

)

32

et al.

Cardiac norepinephrine transporter protein expression is inversely correlated to chamber norepinephrine content

,

Am J Physiol Regul Integr Comp Physiol

,

2008

, vol.

295

(pg.

R857

-

R863

)

33

Purinergic contribution to circulatory, metabolic, and adrenergic responses to acute hypoxemia in fetal sheep

,

Am J Physiol Regul Integr Comp Physiol

,

2001

, vol.

280

(pg.

R678

-

R685

)

34

Fetal sheep endocrine responses to sustained hypoxemic stress after chronic fetal placental embolization

,

Am J Physiol

,

1997

, vol.

272

(pg.

E817

-

E823

)

35

DNA methylation controls the inducibility of the mouse metallothionein-I gene in lymphoid cells

,

Cancer Cell

,

1980

, vol.

25

(pg.

233

-

240

)

36

et al.

Comparison of biological effects of non-nucleoside DNA methylation inhibitors versus 5-aza-2′-deoxycytidine

,

Mol Cancer Ther

,

2005

, vol.

4

(pg.

1515

-

1520

)

37

et al.

Histone H3-lysine 9 methylation is associated with aberrant gene silencing in cancer cells is rapidly reversed by 5-aza-2′-deoxycytidine

,

Cancer Res

,

2002

, vol.

62

(pg.

6456

-

6461

)

38

Inhibition of DNA methylation by 5-aza-2′-deoxycytidine suppresses the growth of human tumor cell lines

,

Cancer Res

,

1998

, vol.

58

(pg.

95

-

101

)

39

Procainamide is a specific inhibitor of DNA methyltransferase 1

,

J Biol Chem

,

2005

, vol.

280

(pg.

40749

-

40756

)

40

et al.

Epigenetic changes induced by reactive oxygen species in hepatocellular carcinoma: methylation of the E-cadherin promoter

,

Gastroenterology

,

2008

, vol.

135

(pg.

2128

-

2140

)

2140 e2121–2128

41

Oxidative stress, DNA methylation and carcinogenesis

,

Cancer Lett

,

2008

, vol.

266

(pg.

6

-

11

)

42

Downregulation of catalase by reactive oxygen species via hypermethylation of CpG island II on the catalase promoter

,

FEBS Lett

,

2010

, vol.

584

(pg.

2427

-

2432

)

43

Norepinephrine induces apoptosis in neonatal rat cardiomyocytes through a reactive oxygen species-TNF alpha-caspase signaling pathway

,

Cardiovasc Res

,

2004

, vol.

62

(pg.

558

-

567

)

44

et al.

Alpha-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes is mediated via thioredoxin-1-sensitive oxidative modification of thiols on Ras

,

Circulation

,

2005

, vol.

111

(pg.

1192

-

1198

)

45

et al.

Reactive oxygen species mediate alpha-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes

,

J Mol Cell Cardiol

,

2001

, vol.

33

(pg.

131

-

139

)

46

Role of reactive oxygen species and NAD(P)H oxidase in alpha(1)-adrenoceptor signaling in adult rat cardiac myocytes

,

Am J Physiol Cell Physiol

,

2002

, vol.

282

(pg.

C926

-

C934

)

47

Prenatal cocaine exposure increases heart susceptibility to ischemia/reperfusion injury in adult male but not female rats

,

J Physiol

,

2005

, vol.

565

(pg.

149

-

158

)

48

et al.

Intrauterine undernutrition—renal and vascular origin of hypertension

,

Cardiovasc Res

,

2003

, vol.

60

(pg.

228

-

234

)

49

Developmental origins of the metabolic syndrome: prediction, plasticity, and programming

,

Physiol Rev

,

2005

, vol.

85

(pg.

571

-

633

)

50

et al.

Mechanisms of estrogen action

,

Physiol Rev

,

2001

, vol.

81

(pg.

1535

-

1565

)

Author notes

†

These two authors contribute equally to the work.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2010. For permissions please email: journals.permissions@oup.com.

Topic:

• nicotine
• norepinephrine
• pregnancy
• adult
• binding sites
• fetal heart
• genes
• methylation
• mothers
• repression
• rna, messenger
• heart
• rats
• fetal tobacco exposure
• offspring

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