Secondary Logo

Share this article on:

Plasma nuclear and mitochondrial DNA levels in acute myocardial infarction patients

Wang, Lei*; Xie, Liang*; Zhang, Qigao; Cai, Xiaomin; Tang, Yi; Wang, Lijun; Hang, Tao; Liu, Jing; Gong, Jianbin

doi: 10.1097/MCA.0000000000000231
Original Research

Objective Plasma nuclear and mitochondrial DNA (mtDNA) levels are altered in many diseases. However, it is not known whether they are also altered in acute myocardial infarction (AMI). In the present study, we examined plasma nuclear and mtDNA levels in the patients with AMI before and after a percutaneous coronary intervention (PCI) to explore their potential as biomarkers.

Methods and results Plasma nuclear and mtDNA levels were measured by quantitative PCR in 25 AMI patients, 25 non-myocardial infarction (MI) control participants (with MI risk), and 20 healthy individuals during the study period. The concentrations of nuclear and mtDNA were significantly higher in the AMI group on hospital day 1 than that in the non-MI controls (nuclear: 0.4948±0.0830 vs. 0.2047±0.0222 ng/μl, P<0.05; mitochondrial: 3.754±0.384 vs. 1.851±0.3483 ng/μl, P<0.05) and healthy individuals (nuclear: 0.4948±0.0830 vs. 0.1683±0.0254 ng/μl, P=0.001; mitochondrial: 3.754±0.384 vs. 0.1517±0.0924 ng/μl, P<0.05) and decreased shortly after PCI.

Conclusion Both plasma nuclear and mtDNA levels are elevated in AMI patients, but return to normal levels immediately after PCI, suggesting that they are potentially novel biomarkers for AMI.

Department of Cardiology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China

* Lei Wang and Liang Xie contributed equally to the writing of this article and share first authorship.

Correspondence to Jianbin Gong, MD, Department of Cardiology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu 210009, China Tel/fax: +86 25 80860022; e-mail: agong62@126.com

This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially. http://creativecommons.org/licenses/by-nc-nd/4.0/.

Received October 22, 2014

Received in revised form January 10, 2015

Accepted January 15, 2015

Back to Top | Article Outline

Introduction

Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide 1. Partial or complete epicardial coronary artery occlusion from plaques vulnerable to rupture or erosion is the most common cause of MI 2. MI may be a minor event in a lifelong chronic disease and may even go undetected, but it may also be a major catastrophic event leading to sudden death or severe hemodynamic deterioration. The term MI reflects cell death of cardiomyocyte caused by ischemia, which is the result of a perfusion imbalance between supply and demand. Because of tissue damage and necrosis of cardiac cells, danger signals, such as extracellular matrix breakdown products, mitochondrial DNA (mtDNA), heat shock proteins, and high mobility box 1, are released 3.

Over the years, the use of more specific and sensitive biomarkers of myocardial necrosis has improved the detection of MI. For example, lactate dehydrogenase was shown to be better than glutamine-oxaloacetic transaminase and replaced it in the diagnosis of MI, and later, creatine kinase (CK) and the MB fraction of CK, that is, CKMB activity and CKMB mass were used instead for the diagnosis 4. Nevertheless, more specific and sensitive biomarkers to detect MI are required.

Mitochondria are double-membrane organelles in the cytoplasm of eukaryotic cells and contain their own DNA. They carry out oxidative phosphorylation to produce ATP that is required for many cellular activities as energy 5. Recent studies have shown that mtDNA is released as damage-associated molecular patterns into circulation after aseptic trauma and its levels correlate with the incidence of distant organ failure and death 6–8. mtDNA that escapes from autophagy cell autonomously has been shown to act through toll-like receptor 9 to induce inflammation and cardiomyocyte injury 9.

mtDNA could be part of plasma cell-free DNA, which includes both mtDNA and nuclear DNA 10. A recent study shows that plasma nuclear DNA concentrations increase proportionately to the complications arising from acute coronary syndrome 11. Moreover, another study has shown that acute myocardial infarction (AMI) can lead to an increase in circulating mtDNA 12. However, the changes in circulating nuclear and mtDNA levels have not been examined simultaneously in AMI.

We hypothesized that nuclear and mtDNA contents are increased after AMI and decreased by a percutaneous coronary intervention (PCI). In a clinical setting, mtDNA or nuclear DNA may serve as a prognostic and/or a diagnostic biomarker in AMI.

Back to Top | Article Outline

Methods

Ethics statement

The present study protocol was approved by Jinling Hospital’s Institutional Review Committee on Human Research. All experimental procedures and written informed consent obtained from all donors were reviewed and approved by the Local Ethics Committee.

Back to Top | Article Outline

Enrollment of patients and normal controls

A total of 25 AMI patients were enrolled from Jinling Hospital between January 2014 and April 2014. AMI was diagnosed according to the European Society of Cardiology (ESC), the American College of Cardiology, and American Heart Association (ACC/AHA) redefined guidelines. Exclusion criteria were as follows: surgery, trauma, previous transmural infarction, cardiogenic shock, severe liver disease, renal failure, underlying neoplasm, hematologic disorders that affect platelet count or function, and fever or infectious conditions upon study entry. Twenty-five control participants without acute coronary syndrome, whose routine coronary angiography was negative, served as non-MI controls. Twenty healthy volunteers served as an internal control. This study was approved by the local ethics committees. All participants signed an informed consent.

Back to Top | Article Outline

Plasma preparation

In the study, 5 ml of whole blood samples were drawn at hospital days 1 (within 8 h of admission) and 3 (2 days after PCI), transferred into EDTA-coated blood collection tubes, and processed within 2 h after venipuncture. Briefly, whole blood was centrifuged at 500 g at room temperature for 10 min, and the supernatant was transferred to a fresh tube and centrifuged at 700 g at 4°C for 5 min. Then, the supernatant (240 μl) obtained from the whole blood (5 ml) was collected carefully using a pipette without touching the pellet or the bottom of the tube. The supernatant obtained was further centrifuged at 15 000 g at 4°C for 10 min, and the resulting supernatant (200 μl) was collected carefully. The plasma samples were stored at −80°C and were used for DNA isolation within 4 months of storage.

Back to Top | Article Outline

DNA isolation from plasma

Plasma DNA was isolated from plasma using the QIAamp DNA Blood Mini Kit (#51104; Qiagen, Valencia, California, USA) following the manufacturer’s manual 13. In brief, samples were thawed on ice and were then mixed briefly by vortex. Then, we incubated the plasma samples with lysis buffer and proteinase K at 56°C for 10 min. At the final step of isolation, DNA was eluted with 150 μl of nuclease-free deionized and distilled H2O, followed by a quantitative real-time PCR assay.

Back to Top | Article Outline

Plasma DNA quantification by qPCR

mtDNA and nuclear DNA were quantified by real-time PCR using the Lightcycler 96 sequence detection system (Roche, Mannheim, Germany) with the following primers: human NADH dehydrogenase 1 gene (mtDNA): forward 5′-ATACCCATGGCCAACCTCCT-3′, reverse 5′-GGGCCTTTGCGTAGTTGTAT-3′ 6,14; human b-globin (nuclear DNA): forward 5′-GTGCACCTGACTCCTGAGGAGA-3′, reverse 5′-CCTTGATACCAACCTGCCCAG-3′ 15. The thermal profile for mtDNA quantitative real-time PCR was as follows: denaturation at 95°C for 10 min, followed by 40 cycles of 10 s at 95°C, 10 s at 58°C, and 10 s at 72°C. In each application, samples were analyzed in duplicate and the mean was used in the subsequent analysis. The concentration of the standards was quantified spectrophotometrically (Nano Drop 2000; Thermo Fischer, Wilmington, Delaware, USA). The standard curve is shown in Fig. 1. The unknown samples were compared with the standard curve. Plasma DNA concentrations were expressed as ng/μl.

Fig. 1

Fig. 1

Back to Top | Article Outline

Statistical analysis

Statistical analyses were carried out using the statistical package for social sciences (SPSS Inc., Chicago, Illinois, USA). Results are shown as the mean±SEM. Paired data were evaluated using a Student’s t-test. A one-way analysis of variance with the Bonferroni post-hoc test was used for multiple comparisons. The χ 2-test was used to assess the association between two categorical variables. P value less than 0.05 was considered statistically significant.

Back to Top | Article Outline

Results

The AMI and control groups were similar in baseline clinical characteristics

The baseline characteristics of the two groups are listed in Table 1. Twenty-five adults (four women and 21 men, aged 44–81 years) with AMI were evaluated. The mean age of the AMI patients was 59.3±13.4 years and that of the controls was 64.5±12.0 years (P=0.165). According to the ECG criteria, 14 patients showed signs of inferior/posterior wall infarction and 11 patients showed signs of anterior wall infarction. There were no significant differences in other vascular risk factors, including hypertension, diabetes mellitus, dyslipidemia, and smoking status, between the two groups.

Table 1

Table 1

Back to Top | Article Outline

Plasma nuclear and mtDNA in AMI and control groups

The levels of plasma nuclear and mtDNA in patients with AMI, non-MI controls, and healthy volunteers are shown in Figs 2 and 3. The concentrations of nuclear and mtDNA were significantly higher in the AMI group on hospital day 1 than that in the non-MI controls (nuclear: 0.4948±0.0830 vs. 0.2047±0.0222 ng/μl, P<0.05; mitochondrial: 3.754±0.384 vs. 1.851±0.3483 ng/μl, P<0.05) and healthy individuals (nuclear: 0.4948±0.0830 vs. 0.1683±0.0254 ng/μl, P=0.001; mitochondrial: 3.754±0.384 vs. 0.1517±0.0924 ng/μl, P<0.05). There was no significant difference in the concentration of plasma nuclear DNA between the non-MI controls and the healthy individuals (0.2047±0.0222 vs. 0.1683±0.0254 ng/μl, P>0.05). Plasma levels of nuclear DNA were 0.4948±0.0830 ng/μl in the patients with AMI on hospital day 1 and 0.2709±0.0386 ng/μl on hospital day 3 (P<0.05). Levels of plasma mtDNA were 3.754±0.384 ng/μl in the patients with AMI on hospital day 1 and 2.112±0.213 ng/μl on hospital day 3 (P<0.05).

Fig. 2

Fig. 2

Fig. 3

Fig. 3

Back to Top | Article Outline

Discussion

We evaluated circulating nuclear and mtDNA levels in the patients with AMI. This is the first study that has shown marked increases in circulating nuclear and mtDNA levels in the patients with AMI compared with non-AMI patients and decreases after PCI. Our work has confirmed and extended previous studies showing that a high level of mtDNA or cell-free DNA may be accompanied by myocardial damage in patients 3,5,10,12,16–18.

To our knowledge, plasma DNA has been studied in a wide range of human diseases from cancer to diabetes, as well as in development, aging, and exercise 19–24. The prognostic and diagnostic utility of plasma DNA has been proven in some critical conditions 25. Increase in plasma DNA appears to be common in the diseases involving cell death, including infections, cancers with metastasis, hepatitis, irreversible cardiac failure, severe respiratory insufficiency, and thrombophlebitis 26. The majority of plasma DNA is derived from apoptotic or necrotic cells 24. The cellular origin of plasma DNA seems to be different in various pathologic conditions, but remains uncertain in most cases. Moreover, mtDNA acts as a damage-associated molecular pattern 27 that can promote molecular processes, leading to inflammatory responses and organ injuries 6,13,28,29. Inflammation can affect the release of DNA from cells undergoing apoptosis or necrosis, although the nature of this effect may vary depending on the inflammatory stimulus and local cellular events 30. Furthermore, inflammatory responses and recruitment of neutrophils in AMI are more pronounced than that in chronic heart failure 31. Therefore, it is possible that mtDNA released from necrotic cells or escaped from autophagy may induce a danger signal, leading to inflammatory responses in AMI.

Currently, cardiac troponin T and troponin I are the best biomarkers for the diagnosis of AMI because of its cardiac specificity and sensitivity 32. However, measurable amounts of troponin proteins are usually not released from the damaged myocardium before 4–8 h after the onset of symptoms, making an early biomarker-based diagnosis of AMI rather difficult. By contrast, mtDNA arises ahead of time (about 1 h after chest pain) in the plasma 12, potentially making it a novel biomarker for early diagnosis of AMI. However, the specificity and sensitivity of mtDNA in the diagnosis of AMI need to be further investigated.

Our study has several limitations. First, the levels of plasma nuclear and mtDNA may be affected by age and pre-existing diseases 33. Second, the number of cases in the study was small and the follow-up period was also short. Large-scale prospective studies are warranted to evaluate the diagnostic and prognostic utility of plasma DNA for AMI.

In summary, plasma nuclear and mtDNA levels increase after AMI and peak rapidly. Further studies are essential to show the specificity and sensitivity of mtDNA in the diagnosis of AMI. It is likely that plasma nuclear and mtDNA may serve as biomarkers and should be tested routinely in the future.

Back to Top | Article Outline

Conclusion

Concentrations of plasma nuclear and mtDNA in patients with AMI were significantly higher than those in the non-MI controls and healthy participants and decreased shortly after PCI. Plasma nuclear and mtDNA levels may be a novel biomarker for AMI.

Back to Top | Article Outline

Acknowledgements

This work was supported by a grant from the National Natural Science Foundation of China (No. 81400238).

The authors thank Xiaomin Cai for blood collection and Liang Xie for providing technical assistance in the sample analyses.

Back to Top | Article Outline

Conflicts of interest

There are no conflicts of interest.

Back to Top | Article Outline

References

1. White HD, Chew DP. Acute myocardial infarction. Lancet 2008; 372:570–584.
2. Libby P. Current concepts of the pathogenesis of the acute coronary syndromes. Circulation 2001; 104:365–372.
3. De Haan JJ, Smeets MB, Pasterkamp G, Arslan F. Danger signals in the initiation of the inflammatory response after myocardial infarction. Mediators Inflamm 2013; 2013:206089.
4. Thygesen K, Alpert JS, White HD. Joint ESC/ACCF/AHA/WHF Task Force for the Redefinition of Myocardial Infarction. Universal definition of myocardial infarction. J Am Coll Cardiol 2007; 50:2173–2195.
5. Malik AN, Czajka A. Is mitochondrial DNA content a potential biomarker of mitochondrial dysfunction? Mitochondrion 2013; 13:481–492.
6. Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, et al.. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 2010; 464:104–107.
7. Simmons JD, Lee YL, Mulekar S, Kuck JL, Brevard SB, Gonzalez RP, et al.. Elevated levels of plasma mitochondrial DNA DAMPs are linked to clinical outcome in severely injured human subjects. Ann Surg 2013; 258:591–596; discussion 596–598.
8. Nakahira K, Kyung SY, Rogers AJ, Gazourian L, Youn S, Massaro AF, et al.. Circulating mitochondrial DNA in patients in the ICU as a marker of mortality: derivation and validation. PLoS Med 2013; 10:e1001577.
9. Oka T, Hikoso S, Yamaguchi O, Taneike M, Takeda T, Tamai T, et al.. Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure. Nature 2012; 485:251–255.
10. Chen C, Xu J, Huang F. Recent players in the field of acute myocardial infarction biomarkers: circulating cell-free DNA or microRNAs? Int J Cardiol 2013; 168:2956–2957.
11. Rainer TH, Lam NY, Man CY, Chiu RW, Woo KS, Lo YM. Plasma beta-globin DNA as a prognostic marker in chest pain patients. Clin Chim Acta 2006; 368:110–113.
12. Bliksoen M, Mariero LH, Ohm IK, Haugen F, Yndestad A, Solheim S, et al.. Increased circulating mitochondrial DNA after myocardial infarction. Int J Cardiol 2012; 158:132–134.
13. Nakahira K, Haspel JA, Rathinam VA, Lee SJ, Dolinay T, Lam HC, et al.. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat Immunol 2011; 12:222–230.
14. McGill MR, Sharpe MR, Williams CD, Taha M, Curry SC, Jaeschke H. The mechanism underlying acetaminophen-induced hepatotoxicity in humans and mice involves mitochondrial damage and nuclear DNA fragmentation. J Clin Invest 2012; 122:1574–1583.
15. Moreira VG, Prieto B, Rodriguez JS, Alvarez FV. Usefulness of cell-free plasma DNA, procalcitonin and C-reactive protein as markers of infection in febrile patients. Ann Clin Biochem 2010; 47 (Pt 3):253–258.
16. Ide T, Tsutsui H, Hayashidani S, Kang D, Suematsu N, Nakamura K, et al.. Mitochondrial DNA damage and dysfunction associated with oxidative stress in failing hearts after myocardial infarction. Circ Res 2001; 88:529–535.
17. Chang CP, Chia RH, Wu TL, Tsao KC, Sun CF, Wu JT. Elevated cell-free serum DNA detected in patients with myocardial infarction. Clin Chim Acta 2003; 327:95–101.
18. Jing RR, Wang HM, Cui M, Fang MK, Qiu XJ, Wu XH, et al.. A sensitive method to quantify human cell-free circulating DNA in blood: relevance to myocardial infarction screening. Clin Biochem 2011; 44:1074–1079.
19. Mehra N, Penning M, Maas J, van Daal N, Giles RH, Voest EE. Circulating mitochondrial nucleic acids have prognostic value for survival in patients with advanced prostate cancer. Clin Cancer Res 2007; 13 (Pt 1):421–426.
20. Davidson SM, Yellon DM. Mitochondrial DNA damage, oxidative stress, and atherosclerosis: where there is smoke there is not always fire. Circulation 2013; 128:681–683.
21. Kohler C, Radpour R, Barekati Z, Asadollahi R, Bitzer J, Wight E, et al.. Levels of plasma circulating cell free nuclear and mitochondrial DNA as potential biomarkers for breast tumors. Mol Cancer 2009; 8:105.
22. Ellinger J, Muller SC, Wernert N, von Ruecker A, Bastian PJ. Mitochondrial DNA in serum of patients with prostate cancer: a predictor of biochemical recurrence after prostatectomy. BJU Int 2008; 102:628–632.
23. Bugger H, Abel ED. Mitochondria in the diabetic heart. Cardiovasc Res 2010; 88:229–240.
24. Budnik LT, Kloth S, Baur X, Preisser AM, Schwarzenbach H. Circulating mitochondrial DNA as biomarker linking environmental chemical exposure to early preclinical lesions elevation of mtDNA in human serum after exposure to carcinogenic halo-alkane-based pesticides. PLoS One 2013; 8:e64413.
25. Butt AN, Swaminathan R. Overview of circulating nucleic acids in plasma/serum. Ann N Y Acad Sci 2008; 1137:236–242.
26. Fournie GJ, Martres F, Pourrat JP, Alary C, Rumeau M. Plasma DNA as cell death marker in elderly patients. Gerontology 1993; 39:215–221.
27. Krysko DV, Agostinis P, Krysko O, Garg AD, Bachert C, Lambrecht BN, Vandenabeele P. Emerging role of damage-associated molecular patterns derived from mitochondria in inflammation. Trends Immunol 2011; 32:157–164.
28. Sursal T, Stearns-Kurosawa DJ, Itagaki K, Oh SY, Sun S, Kurosawa S, Hauser CJ. Plasma bacterial and mitochondrial DNA distinguish bacterial sepsis from sterile systemic inflammatory response syndrome and quantify inflammatory tissue injury in nonhuman primates. Shock 2013; 39:55–62.
29. Collins LV, Hajizadeh S, Holme E, Jonsson IM, Tarkowski A. Endogenously oxidized mitochondrial DNA induces in vivo and in vitro inflammatory responses. J Leukoc Biol 2004; 75:995–1000.
30. Jiang N, Pisetsky DS. The effect of inflammation on the generation of plasma DNA from dead and dying cells in the peritoneum. J Leukoc Biol 2005; 77:296–302.
31. Konstantinidis K, Kitsis RN. Cardiovascular biology: escaped DNA inflames the heart. Nature 2012; 485:179–180.
32. Penttila I, Penttila K, Rantanen T. Laboratory diagnosis of patients with acute chest pain. Clin Chem Lab Med 2000; 38:187–197.
33. Rainer TH, Lam NY. Circulating nucleic acids and critical illness. Ann N Y Acad Sci 2006; 1075:271–277.
Keywords:

acute myocardial infarction; mitochondrial DNA; nuclear DNA

Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.