Previous study shows aldehyde dehydrogenase 2 (ALDH2) plays a critical role in myocardial protection against ischemia, and down-regulation of ALDH2 expression is associated with exacerbated myocardial ischemia injury [
In the light of the epigenetic theory [
Adult male Sprague-Dawley rats weighing 240–250 g were purchased from Shanghai Animal Administration Center. MI was produced by left anterior descending (LAD) artery ligation as described previously [
Experimental rats were randomized into 4 groups, which included one Sham group and three MI experiment groups. Each MI group contained 5 successfully LAD ligations with reduced ejection fraction (<40%) and myocardial tissue was harvested at 1 week, 2 weeks, and 3 weeks after MI, respectively.
Genomic DNA was extracted from the infarction border using a QIA amp DNA Mini Kit according to manufacturer’s instructions (Qiagen, Hilden, Germany), and myocardial tissue in the same region of Sham group was also obtained for DNA extraction. The concentration and purity of the DNA were determined by absorbances at 260 and 280 nm by NanoDropTM 1000 spectrophotometer (Thermo Scientific, Wilmington, USA).
Sodium bisulfite modification for the extracted DNA was performed using an EZ DNA Methylation Kit
CpG and CpG islands of ALDH2 core promoter BSP were determined with online software (
Primer sequences, product length, and CpG units used for BSP.
Primer |
Forward primer (5′-3′) | Reverse primer (5′-3′) | Product |
CpG unit |
---|---|---|---|---|
M1 | GTGAGTTGGGTAGGGATGGA | CRTCTTCCCCTACCCATATAACC | 101 | 9 |
M2 | GGGATAAAGAGGATTGTTTAGGATA | ATACCAACCCCCAACCAAC | 139 | 19 |
M3 | GTTGGTTGGGGGTTGGTAT | CCRTATCTCTACCTCCCATTAATAACC | 177 | 20 |
M4 | GAGTAATYGGYGATTGTAGTTTTGTAGA | ATCCCCATATTCTACAAACTCCATCTC | 105 | 10 |
A 20
Sequencing reaction were performed in reaction mixture including 2
Final results were evaluated in ABI3130XL sequencer.
The Sequenom MassARRAY platform was used for the quantitative methylation analysis of the upstream sequence of ALDH2 gene promoter. The methylation status of a detected pattern was then analyzed using Epityper software version 1.0 (Sequenom, San Diego, CA, USA). The promoter regions of the upstream sequence were chosen according to the website:
Primer sequences, product length, and CpG units used for MassARRAY.
Primer |
Forward primer (5′-3′)1 | Reverse primer (5′-3′)2 | Product |
CpG unit |
---|---|---|---|---|
SQ1 | TTAAGGATTTGTTTGTATTTAATTGG | CAAACAATACCACAATTTATATCTTTTA | 416 | 6 |
SQ2 | AGATTTGGAGAGATGGTTTAGTGGT | ACCCATTTCCTACAAAAAAATATCC | 498 | 10 |
SQ3 | TTAAAAGATATAAATTGGGGTTGGG | ACCAACAAAACCCCTCTAAATAAAC | 349 | 9 |
The procedures and reaction system were reported before [
Upstream sequence of ALDH2 core promoter in detail.
AGACCTGGAGAGATGGCTCAGTGGTTAAGAGCAC |
||
0 | ||
CAGGTTCACAACCATTTGTAATGAGATCTGATGCCCTCCTCTGGTATGTCTGAAGACAGCTACAGTGTACTTAT | ||
ATATAACAATAAATAAAATTAAAAAAAAAAAAAGCAGTAGACATCC |
||
1 | ||
ATTAGTAAGTCAAAAGACACAAACTGGGGTTGGGGATTTAGCTCAGTGGTAGAG |
||
2 3 | ||
|
||
4 5 | ||
GTGGCATTGTTTGTAATCACACAAGATTGGAAGCCACCTTAAATGTCACCCACAGAACTTTCTTTAAATGGG | ||
AATGCACAGGTAG |
||
6 7 |
The valid CpG sites are written in bold, while invalid CpG sites are written in italic. Valid CpG sites are numbered.
Western blot was carried out as described previously [
Total RNA was extracted from the infarction border zone using reverse transcription and real-time PCR was performed according to the manufacturer’s protocol of PrimeScriptTM RT reagent kit and SYBR Premix Ex TaqTM kit (TaKaRa). Real-time quantification was applied in Bio-Rad IQ5 real-time PCR machine. Relative expression of ALDH2 gene was calculated by using 2−ΔΔCt method. Primers are the same as described previously [
Global methylation level of myocardial cell was examined with Methylated DNA quantification kit (purchased from Epigentek). All steps were followed according to the protocol of kit.
Adult male Sprague-Dawley rats were administered intraperitoneal DNA methyltransferase (DNMT) inhibitor, Decitabine (DAC), by 1 mg/kg once a day for 7 days. Myocardial infarction model procedure was performed on half of rats intervened by DAC which is DAC+MI group, while the group with only DAC intervention is DAC group. Heart tissues of both groups were harvested seven days after myocardial infarction model procedure. Global DNA methylation level and ALDH2 upstream target sequence methylation level were examined with Methylated DNA quantification Kit and MassARRAY, respectively, and expressions of related proteins were determined with Western blot.
Data were analyzed using GraphPad Prism (version 5.0; GraphPad Software Inc., San Diego, CA, US) and SPSS (version 15.0; SPSS Inc., Chicago, IL, US). ANOVA with post-hoc test was performed to compare the methylation levels between MI and Sham groups and between different MI groups.
The transcription factor binding elements were predicted using the known DNA-binding profiles (JASPAR,
ALDH2 protein expression result and real-time PCR result are shown in Figures
The sequence of CpG sites in ALDH2 core promoter was shown in Figure
BSP analysis of DNA methylation level of CpG sites in ALDH2 core promoter.
M1
CpG number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
---|---|---|---|---|---|---|---|---|---|
A1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
M2
CpG number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
A1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
M3
CpG number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
A1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
M4
CpG number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
---|---|---|---|---|---|---|---|---|---|---|
A1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
A5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
B5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
C5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Sh5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
(a), (b), (c), and (d) represent four pairs of BSP primers, respectively, which were numbered in Table
(A): for 1st week MI group.
(B): for 2nd week MI group.
(C): for 3rd week MI group and SH for Sham group.
ALDH2 core promoter sequence. (a) ALDH2 core promoter sequence. (b) CpG distribution of ALDH2 core promoter was analyzed in Methyl Primer Express; red pillar stands for CpG sites and bar underlying represents CpG island (criteria: up to 200 bp, and GC percentage: up to 50%).
Upstream sequence of ALDH2 core promoter. (a) The chart is the analysis result of Methyl Primer Express; red pillars stand for CpG sites, and bar underlying highlights CpG high density region; (b) another analysis in the same region; blue dots represent valid CpG sites while red dots are invalid which cannot be detected in MassARRAY. Valid dots were numbered.
Result and analysis chart of Western blot and real-time PCR. (a) Protein level of ALDH2 was determined by Western blot. (b) mRNA of ALDH2 was determined by real-time PCR.
To maintain the homogenous characteristic of tissue between BSP and MassARRAY, tissue samples from the same myocardial region were used to examine the methylation level of upstream sequence of ALDH2 core promoter. A 500 to 600 bp length region of DNA sequence was targeted, which existed in upstream direction of ALDH2 promoter and was 763 bp distant from rat ALDH2 core promoter. Valid CpG sites were numbered and methylation level was detected in this region with SQ2 primer.
The mean methylation levels of seven CpG sites varied across different CpG units, ranging from 21.2% to 69.0% (Figure
Methylation level in the upstream of ALDH2 core promoter. CpG numbers are described in Table
Further MassARRAY evaluation and verification were performed with SQ1 and SQ3 primers, which contained part of valid CpG sites of SQ2 primer, respectively (Table
Two more pairs of primer for verification of upstream sequence. (a) SQ1 Primer and SQ1 MassARRAY result. (b) SQ3 primer MassARRAY result. Both (a) and (b) are performed according to Table
SQ3 primer results were similar among MI groups (Figure
Taken together, hypermethylation was evidenced at CpG1, CpG2, and CpG7 in infarction border zone. It indicated that SOX10 (SRY-related HMG-box 10, SRY for sex determining region Y) was the most possible TF binding to target sequence (Table
Predicted transcription factors by JASPAR database.
Model ID | Model name | Score | Relative score | Start | End | Strand | Predicted site sequence |
---|---|---|---|---|---|---|---|
MA0152.1 | NFATC2 | 6.724 | 0.829025039347648 | 26 | 32 | 1 | TCTTCCT |
MA0442.1 | SOX10 | 5.846 | 0.863982829885209 | 31 | 36 | 1 | CTCTGT |
MA0099.2 | JUN::FOS | 5.636 | 0.816470638357913 | 36 | 42 | 1 | TGAATTA |
MA0040.1 | Foxq1 | 11.209 | 0.889149675048185 | 182 | 192 | 1 | CATTGTTTGTA |
MA0442.1 | SOX10 | 8.625 | 0.987356608195436 | 182 | 187 | 1 | CATTGT |
MA0442.1 | SOX10 | 4.805 | 0.817767607423551 | 186 | 191 | 1 | GTTTGT |
MA0038.1 | Gfi1 | 7.464 | 0.846275818186275 | 190 | 199 | 1 | GTAATCACAC |
MA0116.1 | Zfp423 | 6.657 | 0.801580348722667 | 210 | 224 | 1 | GCCACCTTAAATGTC |
MA0160.1 | NR4A2 | 8.782 | 0.899753978671343 | 219 | 226 | 1 | AATGTCAC |
MA0442.1 | SOX10 | 6.636 | 0.899054900725468 | 235 | 240 | 1 | CTTTCT |
MA0038.1 | Gfi1 | 6.129 | 0.806171416304315 | 279 | 288 | 1 | TAAACCAGTG |
Putative sites were predicted with the setting of ALDH2 promoter upstream sequence, and predicted scores were
DNA global methylation level of myocardial cells from the infarction border was significantly reduced after intervention of DAC (Figure
Global methylation level of myocardial cell. (a) Global methylation level of myocardial cell decreased after the intervention of DAC (
MassARRAY analysis was performed in target upstream sequence with primer SQ1 for DAC group and DAC+MI group. Western blot of related proteins, including DNMT1 and ALDH2, was performed.
DAC intervention reversed hypermethylation of target upstream sequence in MI (Figure
DNA methylation level of target upstream sequence and related proteins expression. (a) MassARRAY analysis result of target upstream sequence for DAC group and DAC + MI group. Sham group and MI-1-week group are chosen as comparative groups (
Our study demonstrates that DNA methylation might contribute to the upstream regulation of ALDH2 after myocardial infarction, suggesting that there is an association between ALDH2 promoter hypermethylation and myocardial infarction (The location of ALDH2 in
It has been substantially shown that DNA methylation plays a pivotal role in the regulation of DNA transcription level [
We hypothesized that DNA methylation may actively participate in the pathogenesis of myocardial infarction and is related to ALDH2 decrease after MI. Till now, there is no report exploring the association between ALDH2 gene methylation and myocardial infarction. In this study, DNA methylation was evaluated in the present study with BSP and MassARRAY. Core promoter sequence of rat ALDH2 gene was analyzed and possible CpG sites were mapped in BSP examination. BSP analysis showed that DNA methylation did not affect ALDH2 core promoter in the setting of myocardial infarction. To overcome the limitation of BSP technique [
Our first MassARRAY experiment with SQ2 suggested there were slightly significant differences in MI groups (Figure
In our study, BSP was performed to examine DNA methylation level of ALDH2 core promoter, which turned out to be a negative result. However, MassARRAY was performed in this region as well, while the preliminary PCR result of MassARRAY was invalid because of extensively high production of dimers, and regulation of PCR temperature or consequences of primers had no effect on eliminating dimers, so MassARRAY final result is far from ideal. It is suggested that the appendix of Primer is combined with specific DNA sequence in ALDH2 core promoter to trigger high production of dimer, while appendix of Primer is constant and essential for mass spectrum in MassARRAY, and ALDH2 core promoter cannot be changed. The preferential method was supposed to choose BSP instead of MassARRAY, and this result also indicated the limitation of MassARRAY in some specific genes; moreover, combination of MassARRAY and BSP may be a rational procedure for detecting DNA methylation level of specific genes, especially in ALDH2 promoter of
Global methylation status in this study was detected to get a comprehensive evaluation of myocardial DNA methylation level. Though study in cancers suggested that hypomethylation of ALDH2 belongs to part of a global DNA methylation change [
Further experiment was carried out to determine the potential mechanism for hypermethylation. Target sequence, which is upstream sequence of ALDH2 core promoter, possesses a baseline methylation status, and DNMT 1 can influence the maintenance of DNA methylation [
The underlying mechanism for the above change could be that hypoxia induced the increase of DNMT1, at least in its enzyme expression, and that DNMT1 subsequently enhanced DNA methylation status of target sequence; finally, ALDH2 was downregulated (Figure
Potential mechanism for alternation of DNA methylation level in ALDH2 core promoter upstream sequence.
Besides methylation changes of ALDH2 after myocardial infarction, there might be a series of genes, which would also face methylation changes in the setting of MI. Breitling et al. reported that F2RL3 gene hypermethylation was associated with the incidence of MI [
As a key enzyme against myocardial ischemia injury, ALDH2 is also regulated under some other potential mechanisms which could induce the declination of its expression or enzyme activity. In our previous study, ALDH2 was proved to protect myocardial cells against ischemia-reperfusion injury through regulation of autophagy via AMPK- and Akt-mTOR signaling [
There are still some limitations in our study. We only observed that MI was associated with aberrant hypermethylation of CpG sites; future studies are needed to prove the mechanism how myocardial ischemia injury could affect DNA methylation level in target sequence. This procedure may involve specific gene, such as NKCC1 gene as reported [
In summary, the present study suggests DNA methylation has an effect on the upstream sequence of ALDH2 promoter, which is possibly associated with the decrease of ALDH2 expression after myocardial infarction. These findings could be a potential epigenetic explanation for ALDH2 decrease after myocardial infarction or ischemia injury.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by a grant to Aijun Sun from the program for New Century Excellent Talents (NCET-12-0125).