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. Author manuscript; available in PMC: 2022 Oct 1.
Published in final edited form as: Reprod Toxicol. 2021 Sep 15;105:198–210. doi: 10.1016/j.reprotox.2021.09.007

Inhibition of DNA methylation in newborns reprograms ischemia-sensitive biomarkers resulting in development of a heart ischemia-sensitive phenotype late in life

Yanyan Zhang 1, Meizi Yang 1,2, Yong Li 1, Bailin Liu 1, Lubo Zhang 1, Daliao Xiao 1
PMCID: PMC8511209  NIHMSID: NIHMS1741354  PMID: 34536542

Abstract

Adverse environmental stress exposure at critical perinatal stages can alter cardiovascular development, which could persist into adulthood and develop a cardiovascular dysfunctional phenotype late in life. However, the underlying molecular mechanisms remain largely unknown. The present study provided a direct evidence that DNA methylation is a key epigenetic mechanism contributing to the developmental origins of adult cardiovascular disease. We hypothesized that DNA hypomethylation at neonatal stage alters gene expression patterns in the heart, leading to development of a cardiac ischemia-sensitive phenotype late in life. To test this hypothesis, a DNA methylation inhibitor 5-Aza-2-deoxycytidine (5-Aza) was administered in newborn rats from postnatal day 1 to 3. Cardiac function and related key genes were measured in 2-week- and 2-month-old animals, respectively. 5-Aza treatment induced an age- and sex-dependent inhibition of global and gene-specific DNA methylation levels in left ventricles, resulting in a long-lasting growth restriction but an asymmetry increase in the heart-to-body weight ratio. In addition, treatment with 5-Aza enhanced ischemia and reperfusion-induced cardiac dysfunction and injury in adults as compared with the saline controls, which was associated with up-regulations of miRNA-181a and angiotensin II receptor type 1 & 2 gene expressions, but down-regulations of PKCε, Atg5, and GSK3β gene expressions in left ventricles. In conclusion, our results provide compelling evidence that neonatal DNA methylation deficiency is a key mechanism contributing to differentially reprogram cardiac gene expression patterns, leading to development of a heart ischemia-sensitive phenotype late in life.

Keywords: Neonatal DNA methylation deficiency, reprogramming of ischemic sensitive gene, angiotensin II receptor (ATR), miRNA-181a, development of heart ischemia-sensitive phenotype

1. Introduction

Developmental origins of adult cardiovascular disease is one of the major health public concerns in modern life. Numerous epidemiologic and animal studies have suggested that adult cardiovascular disease has its origins in early development of fetal and neonatal periods [1-6]. The risk factors from adverse environmental exposures during perinatal stage can affect the fetal and neonatal development, which may persist into adult life and increases the risk of cardiovascular disease in adulthood. Early studies have shown that malnutrition during perinatal stage is one of the major risk factors for coronary heart disease in adulthood. The data suggested that under-nutrition during pregnancy caused fetal growth restriction, resulting in an increased risk for coronary heart disease in adulthood [7,8]. Since then, growing evidence has demonstrated that adverse environmental exposures such as gestational diabetes, maternal smoking/nicotine and hypoxia exposure at critical stages of development can lead to developmental programming of adult cardiovascular disease [4-6,9,10]. Our recent studies have also demonstrated that adult offspring exposed to nicotine, hypoxia or inflammation factor during fetal and neonatal stage develop a phenotype of cardiovascular dysfunction [9-11]. However, the molecular mechanisms remain largely unknown.

Growing evidence has shown that epigenetic regulation plays a key role in developmental origins of adult cardiovascular disease [5,9,12-14]. Epigenetics refers to heritable programming that alters gene expression without changing the DNA sequence [15]. There are three canonical epigenetic regulatory pathways including DNA methylation, histone modifications and noncoding RNA signaling. Epigenetic regulation plays an essential role in differentiation and maintenance of cell fate, and is thus required for normal development and health, but epigenetic regulation is also responsible for the setting of programmed disease [16]. Epigenetic alterations involve changes in phenotype without changes in genotype and are reversible, and as such are promising targets for devising preventive and therapeutic approaches for cardiovascular diseases [17-19].

Of the epigenetic mechanisms, DNA methylation has emerged as a primary mechanism involved in the development origins of adult cardiovascular disease [20,21]. Our recent studies have also demonstrated that nicotine exposure during fetal and neonatal developmental stage differentially reprograms cardiovascular genes such as angiotensin II receptors (ATRs) expression patterns associated with changes of DNA methylation level, resulting in development of a cardiac ischemia-sensitive phenotype and hypertensive phenotype in adult life [9,22]. These studies suggest that DNA methylation is a key epigenetic linker between perinatal stresses and developmental programming of adult cardiovascular disease. To improve our better understanding of the cause-effect of DNA methylation in the development of cardiac dysfunctional phenotype, in this proof-of-principle study we used a newborn rat model and a standard DNA methylation inhibitor, 5-Aza-2-deoxycytidine (5-Aza) as a stressor to see whether inhibition of DNA methylation at neonatal stage may directly contribute to the development of cardiac ischemic-sensitive phenotype later in life. Specifically, we treated the new born rats with 5-Aza at early age (postnatal day 1-3) to induce DNA methylation deficiency. Then we examined the DNA methylation levels of cardiac tissues at late age (from postnatal day 14 to 2-month-old age), and concomitantly we measured the specific pro-ischemic gene expression patterns in the cardiac tissues. Finally, we investigated the effect of DNA methylation inhibition on ischemia/reperfusion (I/R)-induced cardiac infarction and function at the age of 2 months old. Our findings provide the direct evidence that alternation of DNA methylation at early life affects ischemic-sensitive gene expression patterns in the heart, which will persist into late life, leading to development of cardiac ischemic-sensitive phenotype later in life.

2. Material and Methods

2.1. Experimental Animals

Time-dated pregnant Sprague-Dawley rats were purchased from Charles River Laboratories (Portage, MI). Animals were allowed to give birth and newborn rats of both sexes were randomized to receive saline (control) or 5-aza-2-deoxycytidine (5-Aza, 1 mg/kg, Sigma-Aldrich) via intraperitoneal (i.p.) injection consecutively for 3 days from postnatal day 1 to 3 (P1-3) [13]. The rationale for the 5-Aza dosage used in this study was based on previous study [13] that 5-Aza (1mg/kg, i.p.) treatment in newborn rats induced an effective inhibition of DNA methylation. In addition, this dosage was also in the therapeutic range used in children with relapsed or refractory acute leukemia [23]. Animals were randomly assigned to four groups including saline-treated males, 5-Aza treated males, saline-treated females, and 5-Aza-treated females, each group including 5-7 rats from different litters. Animals were kept in a 12-h light/dark cycle and provided ad libitum access to normal rat chow and filtered water. The experiments were conducted at the age of 2 weeks (2w) and 2 months (2m) old. The rationale is that 2 weeks old age of rat is in a neonatal stage and heart developmental stage. On the other hand, 2 months old age of rat is in a sexually mature (adult) stage and heart near fully developed stage. The rats were euthanized with infusion of 5% isoflurane followed by removal of hearts after experiments. All of the procedures and protocols in present study 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. Echocardiography measurement

Cardiac functions of the rats (2 months old) from each group were measured by echocardiography (GE Healthcare, USA). Echocardiography cardiac function assessment were conducted at 3 time points for each rat including Baseline (7 days before I/R), 1 day after I/R, and 7 days after I/R. Briefly, offspring were anesthetized with inhalation of 4% isoflurane and placed on a pre-warmed (37°C) work surface. The rats were shaved in the thorax area and placed in the left lateral decubitus position, then applied a layer of acoustic-coupling gel to the chest. M-mode recording of the left ventricle was obtained at the level of the mitral valve in the parasternal view using two-dimensional (2D) echocardiographic guidance in both short and long axis views. Cardiac function and heart dimensions were evaluated by 2D echocardiography on the anesthetized (2% isoflurane) rat. M-mode tracing was used to measure functional parameters such as LV end-diastolic dimension (LVEDD), LV end-systolic dimension (LVESD), LV end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV) were calculated using the above primary measurements and accompanying software. The percentage of LV ejection fraction (EF) was calculated as (LVEDV-LVESV)/LVEDV x 100% and the percentage of LV fractional shortening (FS) was calculated as (LVEDD-LVESD)/LVEDD x 100%. Echocardiography data were recorded and analyzed blindly to the different treatments.

2.3. Heart ischemic-reperfusion (I/R) model and measurement of myocardial infarct size

Two-month-old rats were subjected to heart ischemia-reperfusion procedure in vivo as described previously [9]. Briefly, rats were anesthetized with isoflurane mixed with oxygen by inhalation and placed on the RoVent Jr. Small Animal Ventilator (Kent Scientific). The respiratory parameters of the mechanical ventilator were adjusted by the body weight of rats. The ischemia was induced by a ligation on left anterior descending artery (LAD) with a 6-0 PROLENE® suture (Ethicon, USA) for 45 mins, afterwards removed the suture to achieve reperfusion. After surgery, rats were recuperated in a single cage for 20 mins before they returned to their house cages.

For measurement of cardiac infarct size, the whole hearts were collected and their LV tissues were isolated and cut into 5 slices on ice, after 24 hours of I/R. Then, LV slices were incubated in 2% triphenyl tetrazolium chloride (TTC, Sigma-Aldrich, USA) solution for 10 minutes at 37°C away from light and immersed in formalin (Thermo Scientific, USA) over night. Each slice was photographed and the areas of myocardial infraction size were analyzed by Image J (National Institutes of Health, USA). The size of myocardial infarct was expressed as the ratio of myocardial infarct size to whole LV area, as previously described [10].

2.4. DNMT Activity Assay and 5-Methylcytosine DNA ELISA

DNA methyltransferease (DNMT) activity assay was performed using the EpiQuik DNMT activity Assay Kit (Epigentek) as described previously [24]. Briefly, nuclear extracts isolated from left ventricles of 2 weeks or 2 months old rats were incubated with S-adenosylmethionine and a universal proprietary DNMT substrate in the DNMT assay buffer at 37°C for 2 h. The blank contained only S-adenosylmethionine and substrate without nuclear extracts, while the positive control contained S-adenosylmethionine and substrate with the purified DNMT enzyme preparation containing both maintenance and de novo DNMTs, supplied in the kit. After the incubation, the capture antibody and detection antibody were added in sequence, followed by incubation with developing solution for 10 min at room temperature. Signal was measured by a dual wavelength microplate reader at 450/655 nm. The DNMT activity [optical density (OD)/h/mg] was calculated by DNMT activity = (sample OD – blank OD)/[protein amount (20 μg) × 2 h] × 1000.

Genomic DNA was isolated from left ventricles of 2 weeks or 2 months old rats, respectively, and global DNA methylation was determined by measuring 5-methylcytosine (5-mC) using a 5-mC DNA ELISA Kit (Zymo Research) as described previously [25]. Briefly, 100 ng of genomic DNA and standard controls provided by the kit were denatured and used to coat the plate wells with 5-mC coating buffer. After incubation at 37°C for 1 h, the wells were washed with 5-mC ELISA buffer. Then, an antibody mix consisting of anti-5-mC and a secondary antibody was added to each well and incubated at 37°C for 1h. After the antibody mix was washed with the 5-mC ELISA buffer 3 times, a HRP developer was added to each well and incubated at room temperature for 30 min. The absorbance at 405 nm was measured using an ELISA plate reader. The percent 5-mC was calculated using the logarithmic second-order regression equation of the standard curve that was constructed with negative control and positive controls in the same experiment.

2.5. Quantitative methylation-specific PCR (MSP)

CpG methylation at rat AT1aR and AT2R gene promoter was determined as previously described [26,27]. Briefly, genomic DNA was bisulfite-converted using a BisulFlash DNA modification kit (Epigentek, Farmingdale, NY, USA). The bisulfite-treated DNA was used as a template for PCR using specific primers designed to amplify the regions of interest with unmethylated CpG dinucleotides or methylated CpG dinucleotides (CmG), respectively [9]. GAPDH was used as an internal reference gene. Real-time methylation-specific PCR was performed using the iQ SYBR Green Supermix with a CFX Connect Real-Time PCR Detection System (Bio-Rad). Data were presented as the percent of methylation at the region of interest (methylated CpG/methylated CpG + unmethylated CpG × 100%).

2.6. qRT-PCR analysis

Total RNA was extracted from left ventricle of 2 weeks or 2 months old rats using the TRIzol reagent as instructed by the manufacturer and reverse transcribed to cDNA with the miScript II RT kit (Qiagen, USA) for miRNA analysis or with the Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, USA) for mRNA analysis, respectively. For measurement of miR-181a levels, Real-time PCR was performed with miScript SYBR Green PCR kit and miScript Universal Primer plus specific primer assays for miR-181a and SNORD61. All reagents were from Qiagen. For measurement of mRNA levels, the AT1aR, AT1bR and AT2R mRNA expression were measured in a CFX Connect Real-Time PCR Detection System (Bio-Rad) by using iQ SYBR Green Supermix (Bio-Rad) according to the manufacturer’s instructions. The primers used were: AT1aR, 5′-ggagaggattcgtggcttgag-3′ (reverse) and 5′-ctttctgggagggttgtgtgat-3′ (forward); AT1bR, 5′- atgtctccagtcccctctca-3′ (reverse) and 5′-tgacctcccatctccttttg-3′ (forward); and AT2R, 5′-caatctggctgtggctgactt-3′ (reverse) and 5′-tgcacatcacaggtccaaaga-3′ (forward) [22]. Data were analyzed using the threshold cycle (Ct) relative quantification method (ΔΔCt) [28]. Expression for each gene was normalized to SNORD61 or GAPDH and expressed as a percentage of control.

2.7. Western blot analysis

Left ventricle tissues from 2 weeks or 2 months old rats were harvested and homogenized in ice-cold lysis buffer with protease inhibitors (cOmplete Mini Protease Inhibitor Cocktail, Roche). The homogenates were then centrifuged at 13,400g for 15 min (4°C) and the supernatants were collected. Total protein concentration was determined using a BCA kit and the same amount of protein was loaded. Protein extracts were separated on 10% SDS polyacrylamide gels and transferred to Nitrocellulose (NC) membranes (Bio-rad, USA). The membranes were blocked in Tris-buffered saline with Tween-20 (TBST) containing 5% non-fat milk and incubated with primary antibodies at 4°C overnight. Antibodies and dilutions were as follows: polyclonal anti-PKCε (1:4000; Santa Cruz Biotechnology), anti-Atg5 (1:2000; Cell Signaling Technology), anti-GSK3β (1:2000; Cell Signaling Technology), anti-AT1R (1:1000; Abcam), anti-AT2R (1:4000; Abcam) and monoclonal anti-GAPDH (1:4000; MilliporeSigma). After extensive washing in TBST, membranes were probed with goat anti-rabbit or goat antimouse IgG-HRP (1:10000; MilliporeSigma) and then washed in TBST. Immunoreactive brands were identified by enhanced chemiluminescence (ECL, ThermoFisher Scientific) and recorded by photographic films (MidSci, USA). GAPDH was used to normalize loading of all samples.

2.8. Statistical analysis

All data are expressed as the mean ± SEM obtained from the number (n) of experimental animals given. Difference between the groups was compared by Student’s t-test or analysis of variance (ANOVA) using the Graph-Pad Prism software (GraphPad Software Version 8, San Diego, CA, USA) or SPSS software 19 (SAS, NC) wherever appropriate. For all comparisons, P-values less than 0.05 indicated statistical significance.

3. Results

3.1. Effects of neonatal 5-Aza treatment on body and heart weight

As shown in Fig. 1, 5-Aza significantly reduced body weights in both male and female rats at the age of 2 weeks (Fig. 1A) and 2 months (Fig. 1B). Neonatal 5-Aza treatment had no effect on heart weights of 2-week-old (Fig. 1C) but decreased the heart weights of 2-month-old male and female rats (Fig. 1D). In addition, the 5-Aza treatment significantly increased the heart to body weight ratio in 2 weeks (Fig. 1E) and 2 months (Fig. 1F) old male but not female rats.

Figure 1. Effects of neonatal 5-Aza treatment on physiological parameters of 2 weeks and 2 months old rats.

Figure 1.

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Body weight (A and B), heart weight (C and D) and the heart to body weight ratio (E and F) in Control and 5-Aza groups at the ages of 2 weeks (2w) and 2 months (2m) old male and female rats. Data are means ± SEM. n=5-8. *P<0.05, 5-Aza versus saline control.

3.2. Neonatal 5-Aza treatment impairs heart function and increases heart susceptibility to ischemic injury in 2-month-old rats

Cardiac function at the baseline and post ischemia/reperfusion (I/R) was measured by echocardiography. As shown in Fig. 2 for the 2-month-old male rats, 5-Aza treatment had no effect on cardiac EF, LVESD, ESV values but decreases in FS, LVEDD, EDV, SV and CO as compared to the saline controls at baseline. However, 5-Aza treatment decreased the EF, FS, SV and CO values at 24 hours and 7th day after I/R procedure in male rats. In contrast to the effect of 5-Aza in male rats, there were no significant differences of the baseline heart function between the saline and 5-Aza treatment groups in the 2-month-old female (Fig. 3). However, neonatal 5-Aza treatment decreased the values of EF, FS, SV and CO at 24 hours and 7th day after I/R procedure in the female rats (Fig. 3).

Figure 2. Effects of neonatal 5-Aza treatment on heart function in 2 months old male rats.

Figure 2.

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The male rats at 2-month-old age from saline control and 5-Aza treated groups were subjected to 45 min of heart ischemia followed by reperfusion. Echocardiographic analysis was obtained from different time periods including baseline, 1 day after I/R, and 7 days after I/R. A: ejection fraction (EF %); B: fractional shortening (FS %); C: left ventricular end-systolic dimension (LVESD); D: end systolic volume (ESV); E: left ventricular end-diastolic dimension (LVEDD); F: end diastolic volume (EDV); G: stroke volume (SV); H: cardiac output (CO). n=6 for each group. *P<0.05, 5-Aza versus saline control in each time point.

Figure 3. Effects of neonatal 5-Aza treatment on heart function in 2-month old female rats.

Figure 3.

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The female rats at 2-month-old age from saline control and 5-Aza treated groups were subjected to 45 min of heart ischemia followed by reperfusion. Echocardiographic analysis was obtained from different time periods including baseline, 1 day after I/R, and 7 days after I/R. A: ejection fraction (EF %); B: fractional shortening (FS %); C: left ventricular end-systolic dimension (LVESD); D: end systolic volume (ESV); E: left ventricular end-diastolic dimension (LVEDD); F: end diastolic volume (EDV); G: stroke volume (SV); H: cardiac output (CO). n=7 for each group. *P<0.05, 5-Aza versus saline control in each time point.

As shown in Fig. 4, neonatal 5-Aza treatment induced a significant increase in myocardial infarct size as compared with the saline control group under I/R procedure in both 2-month-old male and female rats. These data indicate that neonatal 5-Aza treatment has a profound effect on heart function and I/R-induced heart injury in male and female adulthood.

Figure 4. Effects of neonatal 5-Aza treatment on ischemia-reperfusion (I/R)-induced heart infarction size.

Figure 4.

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A: Representative images of 2% TTC staining heart sections in 2-month-old rats subjected to ischemia/reperfusion (I/R). The red portion shows the nonischemic, normal region; the white portion shows the infarcted region. B: Quantified data showing infarct size (IA) as a percentage of total left ventricle (LV) area 24 hours after reperfusion in each group. n=6-7/group. *P<0.05, 5-Aza versus saline control.

3.3. Neonatal 5-Aza treatment inhibits DNMT activity and decreases global DNA methylation in the left ventricle

To see whether the alteration of DNA methylation pattern in early development persists into later life, newborn rats were treated with 5-Aza (1 mg/kg) via i.p. injection daily from postnatal day 1 to 3 (P1-3). As shown in Fig. 5A-B, 5-Aza inhibited total DNMT activities in left ventricles of both male and female rats at the age of 2 weeks and 2 months, respectively. Furthermore, as shown in Fig. 5C-D, compared to the saline control, quantitative 5-mC ELISA revealed that 5-Aza treatment significantly reduced global DNA methylation levels in left ventricles, suggesting that 5-Aza treatment effectively altered the normal DNA methylation patterns and induced global hypomethylation in the developing heart in both male and female rats.

Figure 5. Effects of neonatal 5-Aza treatment on DNMT activity, DNMT3A protein expression and global DNA methylation in left ventricles.

Figure 5.

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A and B: DNMT activity determined with a DNMT activity assay kit in 2 weeks (2w) and 2 months (2m) old rats of both male and female (n=5-7 for each). C and D: Global DNA methylation levels determined with a 5-mC DNA ELISA Kit (n=5-7 for each). Data are means ± SEM. *P<0.05, 5-Aza versus saline control.

3.4. Neonatal 5-Aza treatment down-regulates specific CpG methylation pattern of AT1R/AT2R promoter in left ventricles

Quantitative methylation-specific PCR (MSP) was used to identify the DNA methylation status of AT1R and AT2R gene promoter region in left ventricles. From rat AT1aR gene bank, we have identified, at least five transcription factors binding sites located in CpG locus at rat AT1aR gene promoter region [22]. As shown in Figure 6A, 5-Aza treatment differentially decreased the methylation levels of CpG locus at the binding site of −484 but not −809, −725, −150 and −96 in AT1aR promoter region in left ventricles of both 2-week-old male and female rats as compared with the saline control. However, to our surprised, there was no significant differences of the methylation levels at AT1aR gene promoter region between the 5-Aza-treated and saline control animals at the age of 2 months (Fig. 6C). In contrast to the effect of 5-Aza on AT1aR gene, 5-Aza selectively decreased methylation levels of CpG locus at the binding site of −52 and +11 in AT2R promoter region as compared with the control in the 2-week- and 2-month-old rat left ventricles of both sexes (Figure 6B and D).

Figure 6. Effects of neonatal 5-Aza treatment on DNA methylation of AT1aR and AT2R promoter region in left ventricles.

Figure 6.

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A: Methylation levels of CpG−809, CpG−725, CpG−484, CpG−150 and CpG−96 sites at AT1aR promoter region in the left ventricle tissues isolated from 2 weeks (2w) old rats of both control and 5-Aza treated groups. B: Methylation levels of CpG−52 and CpG+11 at AT2R promoter region in the left ventricle tissues isolated from 2 weeks (2w) old rats of both control and 5-Aza treated groups. C: Methylation levels of CpG−809, CpG−725, CpG−484, CpG−150 and CpG−96 at AT1aR promoter region in the left ventricle tissues isolated from 2 months (2m) old rats of both control and 5-Aza treated groups. D: Methylation levels of CpG−52 and CpG+11 at AT2R promoter region in the left ventricle tissues isolated from 2 months (2m) old rats of both control and 5-Aza treated groups. n=5-7/group. *P<0.05, 5-Aza versus saline control.

3.5. Neonatal 5-Aza treatment increases cardiac angiotensin II receptor (ATR) mRNA and protein expression

As shown in Figure 7, 5-Aza significantly enhanced mRNA levels of AT1aR (Fig. 7A) and AT2R (Fig. 7B) in left ventricles (LVs) as compared to the saline control group in the 2-week-old rats of both sexes. Similarly, 5-Aza also significantly enhanced the mRNA levels of AT1aR (Fig. 7E) and AT2R (Fig. 7F) in LVs as compared to the saline control group in the 2-month-old rats of both sexes. The mRNA levels of AT1bR did not change in any experimental groups (Fig 7A and E). The protein levels of AT1R and AT2R in the left ventricle tissues were determined by western blot analysis. As shown in Figure 7C-D and G-H, neonatal 5-Aza treatment significantly increased the protein levels of both AT1R and AT2R in LV tissues as compared with the saline control group in the 2-week- and 2-month-old rats of both sexes.

Figure 7. Effects of neonatal 5-Aza treatment on AT1R and AT2R mRNA and proteins expression in left ventricles.

Figure 7.

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A and B: Bar plot summarizing quantitative real-time PCR data for AT1aR, AT1bR and AT2R transcript levels in left ventricle tissues isolated from 2 weeks (2w) old rats of both control and 5-Aza treated groups, in which the relative mRNA levels in the 5-Aza treated group are expressed to the fold of control group (n=6). C and D: Representative Western blots of AT1R, AT2R and GAPDH in left ventricle tissues isolated from 2 weeks (2w) old rats of both control and 5-Aza treated groups, and corresponding densitometry summary data (n=5). E and F: Bar plot summarizing quantitative real-time PCR data for AT1aR, AT1bR and AT2R transcript levels in left ventricle tissues isolated from 2 months (2m) old rats of both control and 5-Aza treated groups, in which the relative mRNA levels in the 5-Aza treated group are expressed to the fold of control group (n=5). G and H: Representative Western blots of AT1R, AT2R and GAPDH in left ventricle tissues isolated from 2 months (2m) old rats of both control and 5-Aza treated groups, and corresponding densitometry summary data (n=5). *P<0.05, 5-Aza versus saline control.

3.6. Neonatal 5-Aza treatment enhances miR-181a expression in left ventricles

Previous studies have shown that miR-181a plays a key role in the setting of chronic heart failure, hypertrophy and myocardial infarction [9,29,30]. Our previous study has demonstrated that fetal and neonatal nicotine exposure enhances cardiac ischemic injury and heart dysfunction associated with miR-181a over-expression in left ventricle in adult offspring [9]. To determine whether neonatal 5-Aza-mediated heart function is related to alteration of miR-181a expression in left ventricles, we examined the miR-181a levels of LV tissues in the 2-week- and 2-month-old rats. As shown in Fig. 8, 5-Aza significantly upregulated the levels of miR-181a expression in LV tissues isolated from both male and female rats at the age of 2 weeks (Fig. 8A) and 2 months (Fig. 8B).

Figure 8. Effects of neonatal 5-Aza treatment on miR-181a levels in left ventricles.

Figure 8.

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A: Bar plot summarizing quantitative real-time PCR data for miR-181a levels in left ventricle tissues isolated from 2 weeks (2w) old rats of both control and 5-Aza treated groups, in which the relative miR-181a levels in the 5-Aza treated group are expressed to the fold of control group (n=6). B: Bar plot summarizing quantitative real-time PCR data for miR-181a levels in left ventricle tissues isolated from 2 months (2m) old rats of both control and 5-Aza treated groups, in which the relative miR-181a levels in the 5-Aza treated group are expressed to the fold of control group (n=5). SNORD61 was served as an internal control for the PCR data analysis. *P<0.05, 5-Aza versus saline control.

3.7. Neonatal 5-Aza treatment alters the expression of cardiac ischemia-sensitive biomarkers in left ventricle

To determine whether DNA demethylation-induced cardiac ischemic injury and dysfunction are associated with alterations of the key cardiac ischemic-sensitive biomarkers, we examined the protein abundances of protein kinase C epsilon (PKCε), autophagy related 5 (ATG5) and glycogen synthase kinase-3β (GSK3β) of left ventricles isolated from rats in response to the neonatal DNA methylation inhibition. As shown in Figure 9, 5-Aza treatment significantly decreased the cardiac protein abundance of PKCε in male rats at the age of both 2 weeks (Fig. 9A) and 2 months (Fig. 9D) old. However, in the female rats, 5-Aza treatment had no effect on the cardiac protein abundance of PKCε at the age of 2 weeks old (Fig. 9A) but attenuated it at the age of 2 months old (Fig. 9D). Neonatal 5-Aza attenuated the protein abundance of ATG5 in the left ventricle from both male and female at the age of both 2 weeks old (Fig. 9B) and 2 months old (Fig. 9E). In addition, neonatal 5-Aza treatment had no effect on the cardiac protein abundance of GSK3β gene at the age of 2 weeks old (Fig. 9C) but attenuated the protein abundance at the age of 2 months old (Fig. 9F) of both male and female rats as compared with the saline controls.

Figure 9. Effects of neonatal 5-Aza treatment on PKCε, ATG5 and GSK3β protein expression in left ventricles.

Figure 9.

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A, B and C: Immunoreactive bands corresponding to PKCε, ATG5, GSK3β and GAPDH in left ventricle tissues isolated from 2 weeks old rats of both control and 5-Aza treated groups, and the mean data for PKCε, ATG5 and GSK3β protein levels were expressed as a ratio to GAPDH (n=5 for each group). D, E and F: Immunoreactive bands corresponding to PKCε, ATG5, GSK3β and GAPDH in left ventricle tissues isolated from 2 months old rats of both control and 5-Aza treated groups, and the mean data for PKCε, ATG5 and GSK3β protein levels were expressed as a ratio to GAPDH (n=5 for each group). *P<0.05, 5-Aza versus saline control.

4. Discussion

Epidemiological and animal studies have shown that adverse environmental stresses exposure in early life could increase the risk of cardiovascular disease later in life, which is associated with alteration of DNA methylation profile [5,9,20,31]. In the present study, we provide evidence of proof of concept that DNA methylation deficiency in the early development plays a causal role in programming of heart ischemic-sensitive phenotype in the adult life. Specifically, the major findings in this study are: 1) 5-Aza treatment in newborn rats inhibited cardiac DNMT activity and DNA methylation levels and this inhibition persisted into adulthood; 2) Neonatal inhibition of DNA methylation produced a growth restriction and sex-dependent asymmetric increase in the heart/body weight ratio in male but not in female rats; 3) Neonatal 5-Aza treatment selectively decreased methylation levels of specific CpG sites at AT1R and AT2R promoter regions and enhanced their expression levels in the LV; 4) Neonatal 5-Aza treatment enhanced cardiac miRNA-181a expression and cardiac developmental/ischemia-sensitive biomarkers (PKCε, Atg5 & GSK3β gene) expression patterns; 5) Finally, neonatal 5-Aza treatment enhanced I/R-induced injury and increased cardiac dysfunction in adulthood. These findings suggest that DNA methylation deficiency in early life may serve as a biomarker as well as epigenetic molecular linker for the development of cardiac ischemic-sensitive phenotype in adult life.

Short-term exposure of adverse environmental stress during a critical window of an individual’s development can alter organogenesis leading to long-term phenotypic consequences later in life [4, 32-35]. Growing studies and evidence suggest that epigenetic regulation may be one of the key molecular linkers between the early life adverse environmental stress exposure and the development of long-term chronic disease later in life [32]. In the present study, we exposed newborn rats to 5-Aza-2-deoxycytidine, a standard DNA methylation inhibitor for three days from postnatal day 1 to 3. Our present data showed that 5-Aza administration in newborns significantly inhibited cardiac DNMT activities and global DNA methylation levels in 2-week-old male and female rats. These findings are consistent with previous reports that 5-Aza exposure caused a rapid reduction at both global and gene specific methylation levels in vitro in cell lines [36, 37], as well as in vivo in animal models [13, 24]. Furthermore, our present study also found that this inhibitory effect of 5-Aza on DNA methylation in left ventricles was still observed in 2-month-old rats. Similarly, it has been shown that the effect of 5-Aza at DNA methylation level can persist after the removal of 5-Aza and be retained for two generations in Daphnia [4,38]. Our current findings with some of others further support the hypothesis that organism can maintain an epigenetic memory of the exposure, which will lead to a permanent altered epigenetic state and result in long-term effects on gene expression and function.

Numerous epidemiological and animal studies have shown that adverse environmental stress exposures during pre- and post-natal stage impairs organ development and body growth later in life [39,40]. In the present study, we found that treatment with 5-Aza during the postnatal period (day 1 to 3) decreased the body weight of both male and female rats at the age of 2 weeks and 2 months old. Similarly, our previous study has also shown that neonatal 5-Aza treatment triggers asymmetric growth restriction with an asymmetric increase in the brain to body weight ratio in both male and female rats at 14 days and 3 months of age [13]. In addition, previous study has shown that 5-Aza treatment significantly improves hyperoxia-induced weight loss in neonatal rats [41]. These observations suggest that alteration of DNA methylation in early life is an important epigenetic regulator that may directly contribute to the development of growth restriction phenotype. Our current findings that neonatal 5-Aza treatment significantly increased the heart to body weight ratio in male but not in female group, suggest that DNA methylation deficiency in early life causes a gender-dependent development of asymmetric cardiac hypertrophy in male but not female animals. Based on the present data that 5-Aza administration caused growth retardation, we expect that, in addition to alteration of heart development, neonatal DNA demethylation could also impair other organs development. This opens a wide door for our future studies.

The echocardiography data showed that neonatal 5-Aza treatment not only attenuated cardiac function (EF, FS, SV and CO) after in vivo I/R procedure but also impaired cardiac function before I/R procedure in 2-month-old male rats. These findings suggest a key role of DNA methylation in the setting of heart function at both normal physiologic condition and ischemic stress-induced pathologic condition. Interestingly, in 2-month-old female rats, we found that neonatal 5-Aza treatment had no effect on pre-ischemic baseline values of cardiac function but enhanced I/R-induced cardiac dysfunction, which suggests that neonatal DNA methylation deficiency does not impair the female heart function at a resting physiologic condition but alters the heart function when it encounters an ischemic stress challenge later in life. The enhanced I/R-induced heart dysfunction is regulated by the enhanced I/R-induced LV infarction in both male and female rats in response to 5-Aza stimulation. The sex difference of heart functional response to adverse environmental exposure is often observed in different animal models. Our previous studies have shown a gender-dependent alterations of vascular reactivity, blood pressure, and heart function in male but not female offspring in response to maternal nicotine exposure [9, 42, 43]. Although the mechanisms underlying neonatal 5-Aza or perinatal nicotine exposure-mediated gender differences of cardiac dysfunction are not fully understood, sex hormones have been reported to be the major intrinsic regulators that may contribute to the gender different response to the adverse environmental stress exposures [44]. Previous study has demonstrated that estrogen has a significant role in regulation of Dnmt expression and DNA methylation level [45]. However, our current findings that neonatal 5-Aza treatment inhibited cardiac Dnmt activity and reduced cardiac DNA methylation level in both male and female rats without a significant sex difference, suggest that sex hormones (such as estrogen) may regulate DNA methylation-mediated down-stream signaling pathways, which are contributing to the sex difference of heart dysfunctional phenotype per se. This finding opens a wide door for us to further investigate whether and how estrogen plays a direct role in protection of female rats to development of cardiac dysfunctional phenotype in response to the neonatal DNA methylation deficiency.

Growing evidence has shown that excessive angiotensin II signaling system activation triggers the pathological process of cardiovascular diseases [29,46-48]. In this study, we found that neonatal 5-Aza exposure increased angiotensin II receptor, AT1R and AT2R gene expression of left ventricles in both 2-week and 2-month-old rats, suggesting that 5-Aza-induced cardiac dysfunction may be associated with over-expression of the ATR. Indeed, when we examined the CpG methylation status in AT1aR and AT2R promoter, we found that 5-Aza exposure selectively decreased the methylation levels at −484 CpG locus of AT1aR promoter, as well as the CpG−52 and CpG+11 binding sites of AT2R promoter, suggesting that hypomethylation of the selective CpG locus directly contributes to the over-expression of AT1R and AT2R gene of left ventricles in response to neonatal 5-Aza exposure. However, our findings that 5-Aza treatment significantly increased cardiac AT1R and AT2R gene expression but without changes of the selected CpG methylation levels of AT1aR gene in the 2-month-old rats. This suggests that 5-Aza may inhibit the methylation levels at other CpG sites of the AT1aR promoter region. On the other hands, 5-Aza may alter the DNA methylation levels in the other target gene or signaling pathway, which in turn indirectly regulate ATR gene expression [9].

In response to perinatal stress, alterations of the key ischemia-sensitive gene expressions are contributed to the development of the heart ischemia-sensitive phenotype [11]. MiRNA-181a is one of these genes that is associated with chronic heart failure, infarction fibrosis, hypertrophy and myocardial infarction [9,29,30]. Our previous study has demonstrated that fetal and neonatal nicotine exposure enhances cardiac ischemic injury and heart dysfunction associated with miR-181a over-expression in left ventricle tissues in adult male offspring [9]. In the present study, we also found that neonatal 5-Aza exposure enhanced cardiac miR-181a expression later in life. These results suggest that DNA hypomethylation could up-regulate miR-181a expression, leading to development of heart ischemia-sensitive phenotype. PKCε has a significant impact in the regulation of heart hypertrophy and plays a vital role in cardio-protection in the setting of heart ischemia and reperfusion injury [49]. Consistent with this, our current data that neonatal 5-Aza exposure decreased cardiac PKCε protein expression in adult, suggest that the PKCε gene repression may be one of the common mechanisms underlying pre- and early post-natal insults-mediated cardiac ischemic injury. Autophagy is an important mechanism that can be dramatically elevated as a protective response when cells encounter environmental stresses [50]. We found neonatal 5-Aza exposure downregulated key autophagy-related protein ATG5 and GSK3β expression in adult, which may also partially lead to the development of heart ischemia-sensitive phenotype in adult. It is well-known that DNA hypermethylation will down-regulate gene expression. Our present findings that inhibition of DNA methylation by 5-Aza down-regulated these ischemia-sensitive genes (PKCε, ATG5 and GSK3β) expression, suggests that 5-Aza may not directly target these genes. We speculate that neonatal 5-Aza treatment causes DNA demethylation at ATR promoter or miR-181a promoter, leading to up-regulations of ATR and miR-181a genes in the developing heart. The upregulated ATR or miR-181a will be reprogrammed into adulthood and down-regulate the ischemia-sensitive genes (such as PKCε, ATG5 and GSK3β), resulting in development of heart ischemia-sensitive phenotype later in life.

In conclusion, our data demonstrate that neonatal DNA methylation deficiency upregulates cardiac ATR expression and reprograms ischemia-sensitive biomarkers during heart development, contributing to the development of heart ischemia-sensitive phenotype and dysfunction in adult life. Our present findings provide direct evidence that DNA demethylation may be an important epigenetic linker between adverse environmental stress exposure during early life and programming of cardiac ischemic disease later in life. Our findings suggest that DNA methylation deficiency could serve as an early life biomarker to predict the risk of cardiac ischemic disease later in life. Furthermore, the findings of the detrimental effects of neonatal DNA demethylation on heart development have a great therapeutic potential in targeting DNA methylation of heart ischemia-sensitive genes during early development to prevent heart dysfunction and cardiovascular diseases in adulthood. In addition, it is well known that 5-Aza is one of the most common used drugs for cancer treatment through inhibition of DNA methylation. It has been reported that 5-Aza is used for treatment of neonatal patients with acute leukemia [23, 51]. Although 5-aza has a therapeutic effect for cancer through inhibition of DNA methylation, our present findings suggest that neonatal exposure of 5-aza could induce side toxicological effect and increase the risk of cardiac disease later in life.

Highlights.

  • Neonatal 5-Aza exposure inhibits cardiac DNA methylation persisting into adulthood.

  • Neonatal DNA demethylation produces a growth restriction and heart hypertrophy.

  • DNA demethylation enhances cardiac ATR and miR-181a expression.

  • DNA demethylation alters cardiac ischemia-sensitive biomarkers (PKCε, Atg5 & GSK3β).

  • Neonatal DNA demethylation programs of a heart ischemic-sensitive phenotype.

Acknowledgments

This work was supported by National Institutes of Health Grants HL135623 (DX), DA041492 (DX), and HD088039 (DX). This project was partially supported by funds provided by The Regents of the University of California, Research Grants Program Office, Tobacco Related Disease Research Program (TRDRP) grant # T29IR0437 (DX) and T30FT0936 (BL). The funders had no role in experimental design, data collection and analysis, decision to publish, or preparation of the manuscript.

Footnotes

Conflict of interest

No conflicts of interest, financial or otherwise, are declared by the author(s).

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