Variation of perioperative plasma mitochondrial DNA correlate with peak inflammatory cytokines caused by cardiac surgery with cardiopulmonary bypass
© Qin et al. 2015
Received: 15 April 2015
Accepted: 19 June 2015
Published: 24 June 2015
Cardiac surgery with cardiopulmonary bypass (CPB) may cause inflammatory responses, which can deteriorate the outcomes. Inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6,–8 and -10, can act as both the effector and the predictor for post-operative inflammatory responses. Plasma mitochondrial DNA (mtDNA) was found as a pro-inflammatory agent recently, which was released when cells were insulted.
In the present study, we included 38 patients undergoing coronary artery bypass graft (CABG) to analyze their perioperative plasma mtDNA and levels of inflammatory cytokines. Blood samples were collected before aortic cross-clamping (T1), at the end of CPB (T2), 6 h post-CPB (T3), 12 h post-CPB (T4), and 24 h post-CPB (T5). Rt-PCR and specific ELISA kits were used to quantify the plasma mtDNA and inflammatory cytokines, respectively. Bivariate correlations analysis was used to check the correlations between plasma mtDNA and inflammatory cytokines respectively.
Results shown that plasma mtDNA elevated significantly at T2 and peaked at T4. Furthermore, plasma TNF-α, IL-6 and IL-8 levels significantly increased at T2 and peaked at T3 while IL-10 elevated and peaked at T2. Bivariate correlations analysis showed that the peak plasma mtDNA were positively correlated with the peak TNF-α (r = 0.677, P < 0.001), the peak IL-6 (r = 0.706, P < 0.001), the peak IL-8 (r = 0.584, P < 0.001) and the peak IL-10 (r = 0.565, P < 0.001).
We found that plasma mtDNA might play a key role in CPB-induced post-operative inflammatory responses.
KeywordCardiopulmonary bypass Mitochondrial DNA Inflammatory cytokine
The first case of extracorporeal circulation (ECC) was reported by Gibbon JH in 1954 to successfully save a patient with atrial septal defect (ASD) . In 1955, Kirklin et al. combined the ECC with the oxygenator in intra-cardiac surgery and since then, the technique of cardiopulmonary bypass (CPB) was developed to be the key process in modern cardiothoracic surgery . While the CPB helping to bypass circulation out of the heart and lung during the operation, the abnormal status of circulation and the ischemia/reperfusion injury (I/R injury) may cause inflammatory responses . It is well known that most of postoperative complications are related to the systemic inflammatory response syndrome (SIRS) and many studies demonstrated that inflammatory mediators, like tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8 and IL-10, increased significantly after cardiac surgery with CPB [4–7]. Although many anti-inflammatory agents and coated circuits were investigated to reduce the inflammatory responses after CPB, we still need a more efficient way to eliminate the inflammatory responses and then protect the secondary damage to the heart and other organs [8, 9].
Human mitochondrial DNA (mtDNA) consist of 16569 nucleotide bases and take responsible for encoding 13 polypeptides of the electron transport chain, 22 transfer RNAs, and 2 ribosomal RNAs . mtDNA was released into circulation and trigger inflammatory responses when cells were dealing with harmful insults . With the pro-inflammatory CpG motif, mtDNA acts as a damage-associated molecular patterns (DAMPs) . It is documented that mtDNA played a pro-inflammatory role in several diseases. Plasma mtDNA was elevated remarkably in trauma patients, comparing to volunteers . Recently, a study based on multiple cohorts showed that mtDNA can improve risk prediction and there is a tight relationship between elevated plasma mtDNA level and 28-day mortality .
Given the pro-inflammatory features of mtDNA and series of inflammatory responses after CPB, we hypothesized that mtDNA may act as a pro-inflammatory factor after cardiac surgery with CPB and play a key role in post-CPB inflammatory responses with other inflammatory factors, such as TNF-α, IL-6, IL-8 and IL-10.
Thirty-eight patients were included from January 2014 to August 2014, who were admitted to the Department of Cardiovascular Surgery, West China Hospital, requiring coronary artery bypass graft (CABG). Medical histories of endocarditis, diabetes, hypertension, neurological diseases, psychiatric diseases, infectious diseases, and post-surgical acute renal failure and low cardiac output syndrome were designed as the excluding criteria. All patients had normal function of kidney, liver and lung prior to surgery. Informed consents were signed by every patients. CABG with CPB and postoperative standard care in cardiac intensive care unit (CICU) were performed successfully for all patients. The study was conducted following the Declaration of Helsinki and registered in the research committee at the Sichuan University.
Blood samples collection
Blood samples were collected in EDTA-coated blood collection tube before aortic cross-clamping (T1), at the end of CPB (T2), 6 h post-CPB (T3), 12 h post-CPB (T4), 24 h post-CPB (T5). The whole blood was centrifuged at 1000 rpm/min for 15 min at 4° and subsequently supernatant was collected as plasma. Samples of plasma were stored in -80° freezer and ready for rt-PCR and ELISA.
DNA isolation and rt-PCR for mtDNA
The whole plasma DNA was isolated from plasma using the DNeasy Blood and Tissue Kit (#69504, Qiagen). Briefly, 50 μL plasma samples were added to50 μL phosphate buffered saline (PBS) and then centrifuged at 16000 g for 15 min at 4°. 90 μL of supernatant were kept for the next procedures. The rest procedures were performed according to the manufacture’s protocol. At the last step, 200 μL elution buffer were added to resolve the plasma DNA.
Plasma mtDNA levels were measured by SYBR-green dye-based rt-PCR assay using a PRISM 7300 sequence detection system. The primer sequences were human NADH dehydrogenase 1 gene (mtDNA): forward CGAGCAGTAGCCCAAACAAT, reverse TGTGATAAGGGTGGAGAGGTT. Plasmid DNA with complementary DNA sequence for human mtDNA was obtained from ORIGENE (SC101172, USA). Concentration of plasma mtDNA were converted to copy number via a DNA copy number calculator (http://cels.uri.edu/gsc/cndna.html; University of Rhode Island Genomics and Sequencing Center). Plasmid DNA were diluted in 10-fold serial dilutions and measured as standard curve .
Measurement of cytokines
Plasma TNF-α, IL-6, IL-8 and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA) kit (Solarbio, China). All procedures were followed standard protocols (included in the ELISA kits). Spectrophotometry was used to detect the intensity of the transmitted light. Data was expressed in picogram per mL.
All descriptive data were shown as mean ± standard error of the mean (SEM). Multiple comparisons were analyzed by one-way ANOVA followed by Bonferroni’s test. The Pearson correlation coefficient test were performed. P < 0.05 was considered to statistically significant.
N = 38
49.4 ± 12.7
24 (63.2 %)
23.2 ± 3.2
7 (18.4 %)
9 (23.7 %)
22 (57.9 %)
5 (13.2 %)
8 (21.1 %)
14 (36.8 %)
11 (28.9 %)
Aortic Cross-clamping Time (min)
68 ± 21
CPB Time (min)
96 ± 27
Serum Creatinine (μmol/L)
0.6 ± 0.2
130.3 ± 11.7
Total Cholesterol (mg/dL)
198.2 ± 35.9
1.1 ± 0.2
2.9 ± 0.6
1.5 ± 0.4
140.1 ± 34.8
39.4 ± 3.9
Changes of perioperative plasma mtDNA levels
Changes of perioperative cytokines levels
Correlations between plasma mtDNA and cytokines
Our study demonstrated that plasma mtDNA was released during cardiac surgery with CPB. We found the peak value of plasma mtDNA was shown up at 12 h post-CPB through five time points tracking analysis. Meanwhile, we confirmed releasing patterns of inflammatory cytokines, such as TNF-α, IL-6, IL-8 and IL-10, after cardiac surgery with CPB. We revealed a significantly and positively correlation between the peak plasma mtDNA and peak cytokines levels.
CPB-induced systemic inflammatory response syndrome was affected by many factors, such as the contact between blood and foreign surface of the extracorporeal circuits, hypothermia, reduction of pulmonary blood flow, direct surgical damage to the heart and endotoxemia . It is studied that excessive inflammatory responses might delay the recovery and attenuate hospital outcomes . As many studies reported, post-operative inflammatory responses were almost happened in every cardiac surgery with CPB, characterized with elevated inflammatory cytokines [6, 17]. In addition, levels of TNF-α, IL-6, IL-8 and IL-10, well-known inflammatory cytokines, represented the severity of inflammatory responses. Dr. Mei YQ et al found that concentrations of plasma IL-6 and IL-8 might be able to evaluate the severity of SIRS after CABG and establish the prognosis . In the present study, we measured inflammatory cytokine levels after CABG with CPB. We found that all these inflammatory cytokines increased significantly after surgery with CPB, which apparently demonstrated that post-operative inflammatory responses occurred. Except for IL-10, which peaking at the end of CPB, all other cytokines peaked at 6 h post-CPB and levels of IL-8 dropped dramatically after peak time while others decreased gradually. Given that CPB-induced I/R condition may cause inflammatory responses , we suggest that IL-10 may act as the factor indicating direct damage-caused inflammatory responses while TNF-α, IL-6, IL-8 may represent I/R injury-induced inflammatory responses.
It is well studied that mtDNA was released after trauma surgery and the release of mtDNA happened in a cell necrosis-independent way. Additionally, the study also revealed that levels of plasma mtDNA were positively correlation with the invasiveness and the complexation of surgery . As a pro-inflammatory agent, mtDNA was studied in many different fields and was confirmed that it can cause inflammatory responses [21–23]. Dr. Zhang Q et al. revealed that circulating mtDNA, a kind of mitochondrial damage-associated molecular patterns, can cause inflammatory responses and create a sepsis-like state . In our study, after CABG with CPB, we found that plasma mtDNA levels elevated after the end of CPB and climbed to the peak value at 12 h post-CPB. And then it decreased gradually but still higher than the baseline level. It is reported that mtDNA was continuously released for at least five days after surgery . As mentioned above, CPB, act as an I/R condition, can cause inflammatory responses. Combing with our result, plasma mtDNA elevation might indicate I/R injury-induced inflammatory responses. After the end of CPB, post-ischemia reperfusion caused secondary damage to the heart and late-peaked plasma mtDNA may indicate this secondary I/R injury-induced damage.
It is studied that plasma mtDNA can be a promising predictor for ICU stay and 28-day mortality . In our study, the bivariate correlations analysis demonstrated a surprising positively correlation between the peak plasma mtDNA and the peak inflammatory cytokines. It is well known that excessive production of inflammatory cytokines may be a predictor for outcomes in different diseases and treatments [24, 25]. Considering that positively correlation between plasma mtDNA and inflammatory cytokines, we suggested that mtDNA might be involved in the progression of post-operative inflammatory responses.
Our study found that release of mtDNA after cardiac surgery with CPB and positively correlation between plasma mtDNA and inflammatory cytokines, suggesting that mtDNA may play an important role in inflammatory responses after CPB. Our data strongly recommended a new insightful point at attenuating post-operative inflammation, which need further studies to illustrate more details about mtDNA-relative mechanism and effects. Additionally, considering that peak time of plasma mtDNA was 12 h post-CPB, later than other inflammatory cytokines, we need further studies to figure out the interaction between mtDNA and other inflammatory cytokines.
- Gibbon Jr JH. Application of a mechanical heart and lung apparatus to cardiac surgery. Minn Med. 1954;37(3):171–85. passim.PubMedGoogle Scholar
- Kirklin JW, Dushane JW, Patrick RT, Donald DE, Hetzel PS, Harshbarger HG, et al. Intracardiac surgery with the aid of a mechanical pump-oxygenator system (gibbon type): report of eight cases. Proc Staff Meet Mayo Clin. 1955;30(10):201–6.PubMedGoogle Scholar
- Zhang Z, Wu Y, Zhao Y, Xiao X, Liu J, Zhou X. Dynamic changes in HMGB1 levels correlate with inflammatory responses during cardiopulmonary bypass. Exp Ther Med. 2013;5(5):1523–7. doi:https://doi.org/10.3892/etm.2013.1026.PubMedPubMed CentralGoogle Scholar
- Caputo M, Mokhtari A, Miceli A, Ghorbel MT, Angelini GD, Parry AJ, et al. Controlled reoxygenation during cardiopulmonary bypass decreases markers of organ damage, inflammation, and oxidative stress in single-ventricle patients undergoing pediatric heart surgery. J Thorac Cardiovasc Surg. 2014;148(3):792–801. doi:https://doi.org/10.1016/j.jtcvs.2014.06.001. 3; discussion 0–1.View ArticlePubMedGoogle Scholar
- Samankatiwat P, Samartzis I, Lertsithichai P, Stefanou D, Punjabi PP, Taylor KM, et al. Leucocyte depletion in cardiopulmonary bypass: a comparison of four strategies. Perfusion. 2003;18(2):95–105.View ArticlePubMedGoogle Scholar
- Serrano Jr CV, Souza JA, Lopes NH, Fernandes JL, Nicolau JC, Blotta MH, et al. Reduced expression of systemic proinflammatory and myocardial biomarkers after off-pump versus on-pump coronary artery bypass surgery: a prospective randomized study. J Crit Care. 2010;25(2):305–12. doi:https://doi.org/10.1016/j.jcrc.2009.06.009.View ArticlePubMedGoogle Scholar
- Mahle WT, Matthews E, Kanter KR, Kogon BE, Hamrick SE, Strickland MJ. Inflammatory response after neonatal cardiac surgery and its relationship to clinical outcomes. Ann Thorac Surg. 2014;97(3):950–6. doi:https://doi.org/10.1016/j.athoracsur.2013.10.069.View ArticlePubMedGoogle Scholar
- De Amorim CG, Malbouisson LM, da Silva Jr FC, Fiorelli AI, Murakami CK, Carmona MJ. Leukocyte depletion during CPB: effects on inflammation and lung function. Inflammation. 2014;37(1):196–204. doi:https://doi.org/10.1007/s10753-013-9730-z.View ArticlePubMedGoogle Scholar
- Paparella D, Scrascia G, Rotunno C, Marraudino N, Guida P, De Palo M, et al. A biocompatible cardiopulmonary bypass strategy to reduce hemostatic and inflammatory alterations: a randomized controlled trial. J Cardiothorac Vasc Anesth. 2012;26(4):557–62. doi:https://doi.org/10.1053/j.jvca.2012.04.010.View ArticlePubMedGoogle Scholar
- Wallace DC. Mitochondrial DNA, in aging and disease. Sci Am. 1997;277(2):40–7.View ArticlePubMedGoogle Scholar
- Mittra I, Nair NK, Mishra PK. Nucleic acids in circulation: are they harmful to the host? J Biosci. 2012;37(2):301–12.View ArticlePubMedGoogle Scholar
- Sun S, Sursal T, Adibnia Y, Zhao C, Zheng Y, Li H, et al. Mitochondrial DAMPs increase endothelial permeability through neutrophil dependent and independent pathways. PLoS One. 2013;8(3), e59989. doi:https://doi.org/10.1371/journal.pone.0059989.View ArticlePubMedPubMed CentralGoogle Scholar
- 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(7285):104–7. doi:https://doi.org/10.1038/nature08780.View ArticlePubMedPubMed CentralGoogle Scholar
- 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(12):e1001577. doi:https://doi.org/10.1371/journal.pmed.1001577. discussion e.View ArticlePubMedPubMed CentralGoogle Scholar
- Kapitein B, van Saet A-W, Golab HD, de Hoog M, de Wildt S, Tibboel D, et al. Does Pharmacotherapy Influence the Inflammatory Responses During Cardiopulmonary Bypass in Children? J Cardiovasc Pharmacol. 2014;64(2):191–7. doi:https://doi.org/10.1097/fjc.0000000000000098.View ArticlePubMedGoogle Scholar
- Onorati F, Rubino AS, Nucera S, Foti D, Sica V, Santini F, et al. Off-pump coronary artery bypass surgery versus standard linear or pulsatile cardiopulmonary bypass: endothelial activation and inflammatory response. Eur J Cardiothorac Surg. 2010;37(4):897–904. doi:https://doi.org/10.1016/j.ejcts.2009.11.010.View ArticlePubMedGoogle Scholar
- Berger MM, Delodder F, Liaudet L, Tozzi P, Schlaepfer J, Chiolero RL, et al. Three short perioperative infusions of n-3 PUFAs reduce systemic inflammation induced by cardiopulmonary bypass surgery: a randomized controlled trial. Am J Clin Nutr. 2013;97(2):246–54. doi:https://doi.org/10.3945/ajcn.112.046573.View ArticlePubMedGoogle Scholar
- Mei YQ, Ji Q, Liu H, Wang X, Feng J, Long C, et al. Study on the relationship of APACHE III and levels of cytokines in patients with systemic inflammatory response syndrome after coronary artery bypass grafting. Biol Pharm Bull. 2007;30(3):410–4.View ArticlePubMedGoogle Scholar
- Bourbon A, Vionnet M, Leprince P, Vaissier E, Copeland J, McDonagh P, et al. The effect of methylprednisolone treatment on the cardiopulmonary bypass-induced systemic inflammatory response. Eur J Cardiothorac Surg. 2004;26(5):932–8. doi:https://doi.org/10.1016/j.ejcts.2004.07.044.View ArticlePubMedGoogle Scholar
- McIlroy DJ, Bigland M, White AE, Hardy BM, Lott N, Smith DW, et al. Cell necrosis-independent sustained mitochondrial and nuclear DNA release following trauma surgery. The J Trauma Acute Care Surg. 2015;78(2):282–8. doi:https://doi.org/10.1097/TA.0000000000000519.View ArticlePubMedGoogle Scholar
- Cao H, Ye H, Sun Z, Shen X, Song Z, Wu X, et al. Circulatory mitochondrial DNA is a pro-inflammatory agent in maintenance hemodialysis patients. PLoS One. 2014;9(12), e113179. doi:https://doi.org/10.1371/journal.pone.0113179.View ArticlePubMedPubMed CentralGoogle Scholar
- Wilkins HM, Carl SM, Weber SG, Ramanujan SA, Festoff BW, Linseman DA, et al. Mitochondrial lysates induce inflammation and Alzheimer’s disease-relevant changes in microglial and neuronal cells. J Alzheimer’s Dis: JAD. 2015;45(1):305–18. doi:https://doi.org/10.3233/JAD-142334.PubMedPubMed CentralGoogle Scholar
- Kim YS, Kwak JW, Lee KE, Cho HS, Lim SJ, Kim KS, et al. Can mitochondrial dysfunction be a predictive factor for oxidative stress in patients with obstructive sleep apnea? Antioxid Redox Signal. 2014;21(9):1285–8. doi:https://doi.org/10.1089/ars.2014.5955.View ArticlePubMedPubMed CentralGoogle Scholar
- Drapalova J, Kopecky P, Bartlova M, Lacinova Z, Novak D, Maruna P, et al. The influence of deep hypothermia on inflammatory status, tissue hypoxia and endocrine function of adipose tissue during cardiac surgery. Cryobiology. 2014;68(2):269–75. doi:https://doi.org/10.1016/j.cryobiol.2014.02.007.View ArticlePubMedGoogle Scholar
- Liao C, Yu Z, Guo W, Liu Q, Wu Y, Li Y, et al. Prognostic value of circulating inflammatory factors in non-small cell lung cancer: a systematic review and meta-analysis. Cancer Biomark. 2014;14(6):469–81. doi:https://doi.org/10.3233/CBM-140423.PubMedGoogle Scholar
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