Mitochondrial DNA Copy Number Is Increased in Plasma and Associated with Postoperative Complications After Coronary Artery Bypass Grafting

Mitochondrial DNA Copy Number Is Increased in Plasma and Associated with Postoperative Complications After Coronary Artery Bypass Grafting | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mitochondrial DNA Copy Number Is Increased in Plasma and Associated with Postoperative Complications After Coronary Artery Bypass Grafting Elena A. Kuzheleva, Alla A. Garganeeva, Alexei A. Sleptsov, Olga V. Tukish, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7999546/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract To determine the mtDNA-copy number in the blood of patients before and within 10 days after coronary artery bypass grafting (CABG), and to assess the association between mtDNA-copy number and the postoperative complications was aims of this study. Methods. This single-center, prospective, non-blinded study enrolled 26patients who underwent CABG, with a median age of 67 years (IQR: 58-71). Blood plasma and peripheral blood mononuclear cells (PBMCs) were collected from patients before CABG and at 3, 5, 7, and 10 days post-CABG. Mitochondrial DNA copy number was measured using digital PCR. Results. In plasma, the median mtDNA-copy number was 5.12(3.35-7.44) million copies per mL before CABG, 2.65 (1.88-3.04) on 3rd day, 4.04 (2.28-7.00) on 5th day, 6.50 (3.47-11.31), on 7th day, and 10 (7.2-13.4) on 10th day. The median mtDNA-copy number in PBMCs was 79 (63.5-95.2) copies per cell before CABG and did not significantly change post-CABG (р>0.05). Patients with complications had a significantly increased plasma mtDNA-copy number (198%; interquartile range: 101; 321%) compared with patients without complications (11%; interquartile range: -36; 127%; p = 0.008). Conclusions. Increases mtDNA-copy number by 10th day post-CABG is associated with postoperative complications. The mtDNA-copy number in PBMCs did not significantly change after CABG. Clinical Trial registration: The study protocol was registered at ClinicalTrials.gov: NCT05770349. Registration date 02/21/2023. Coronary artery bypass grafting CABG heart failure with reduced ejection fraction HFrEF heart failure with mildly reduced ejection fraction HFmrEF mitochondrial DNA copy number mtDNA-copy number Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Heart failure (HF) remains a global health challenge and represents the final stage of the cardiovascular continuum. The role of mitochondria in the HF pathogenesis is an area of ongoing investigation. Studying mitochondrial function in HF patients with coronary artery disease (CAD) is particularly important, as both conditions can impair mitochondrial function [1, 2]. It should be noted that 90% of the energy used by the cardiomyocyte contraction-relaxation cycle comes from the de novo synthesis of adenosine triphosphate in mitochondria [3, 4]. A key characteristic of mitochondria that distinguishes them from other intracellular organelles is their self-contained DNA (mtDNA) [5]. When released extracellularly, mtDNA acts as a mitochondrial damage-associated molecular patterns (mtDAMPs), triggering inflammatory responses [6]. Previous research has shown that mtDNA-copy number increases in peripheral blood during cardiac surgeries, especially during coronary artery bypass grafting (CABG) with cardiopulmonary bypass and returns to baseline levels within 24–72 hours post-surgery [7, 8]. Furthermore, elevated pre-CABG mtDNA-copy number has been linked to the development of post-operative atrial fibrillation [6, 9], which is thought to be due to the pro-inflammatory effects of mtDNA. Studies on post-CABG mtDNA-copy number changes have been limited to the first 24–72 hours and it has not been studied how the mtDNA-copy number changes in the following days after CABG which is the aim of our study. The development of complications in the postoperative period after on-pump CABG is primarily related to inflammation. Such complications include atrial fibrillation, post-pericardiotomy syndrome, and infection. These conditions promote the active release of mitochondrial DNA (mtDNA) from cells and an increase in mtDNA-copy number in plasma. At the same time, increased levels of mtDAMPs lead to the progression of these complications and worsening of the postoperative course after CABG [10]. Confirmation of an association between increased plasma mtDNA-copy number and complications after heart surgery may provide a rationale for a more focused study of this parameter. The mechanisms of the release of mtDNA from cells into the extracellular compartment have not been fully elucidated now. In passive mechanisms, mtDNA is believed to be released into the extracellular space following cellular apoptosis or necrosis [11]. Passive release of mtDNA can include an increase in the number of mtDNA-copy by mechanical damage to cells due to CABG surgery. Another mechanism of the release of mtDNA from cells may be active release as part of extracellular vesicles or as a result of release in the form of neutrophils extracellular traps [12]. It is likely that such a mechanism may be involved in changing the mtDNA-copies number in plasma at later stages after CABG and may be associated with the development of post-operative complications after CABG. In addition to the increase in the mtDNA-copy number, indirect evidence of ongoing damage to mitochondria may be an increase in Cytochrome C. Cytochrome C is one of the components of the mitochondrial respiratory chain, which enters the blood when these organelles are damaged [13]. The aim of this study was to determine the changes in blood mtDNA-copy number in patients undergoing on-pump CABG, from pre-operative baseline to day 10 after CABG, and to assess its association with post-operative complications. 2. Materials and Methods Study design This study enrolled 26 patients undergoing CABG, with a median age of 67 years (interquartile range: 58–71 years). This was a single-center, prospective, non-blinded cohort study (Protocol registered at ClinicalTrials.gov: NCT05770349). Inclusion criteria were: presence of HF with left ventricular ejection fraction (LVEF) < 50%; atherosclerotic plaques of 70% or more in at least two major coronary arteries; decision by the cardiology team to perform on-pump CABG; signed informed consent; and availability of biomaterial samples. Exclusion criteria included: refusal of revascularization or participation; need for additional cardiac surgical interventions other than CABG; active oncological diseases; presence of implanted devices; severe renal dysfunction (estimated glomerular filtration rate CKD-EPI < 30 ml/min/1.73 m2); infiltrative heart diseases; acute infections or exacerbations of chronic somatic diseases; severe chronic obstructive pulmonary disease; bronchial asthma; type 1 or type 2 diabetes mellitus; and anemia. All patients provided voluntary written informed consent for the use of blood and plasma samples. The Ethics Committee of the Research Institute of Cardiology of Tomsk National Research Medical Center approved this study (Approval No. 241 of March 9, 2023), which was conducted in accordance with the Declaration of Helsinki (2013). Blood plasma and peripheral blood mononuclear cells (PBMCs) were collected from patients at baseline (pre-CABG) and on days 1, 3, 5, 7, and 10 after CABG. The research was carried out using the equipment of the Center for Collective Use "Medical Genomics" of Cardiology Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences, Tomsk, Russia. DNA preparation To obtain DNA from PBMC, the whole blood was washed twice with ACK Lysing Buffer, prepared according to the protocol outlined by AAT Bioquest [14]. A D-Blood kit (Biolabmix) was used to extract DNA from 300 µL of blood. The quantity and quality of the extracted DNA samples were assessed using the Nanodrop-8000 (Thermo) spectrophotometer. DNA degradation was evaluated via 1% agarose gel electrophoresis. DNA samples were diluted to a working concentration of 20 ng/µL using 1X TE buffer with 0.1 mM EDTA (SibEnzyme). For digestion, 20 ng of DNA was treated with 1 unit of HinfI enzyme (Thermo) and 1X Tango buffer (Thermo) for 1 hour at 37°C. Subsequently, 1 ng of digested DNA per 20 µL of reaction mixture was used for digital PCR. Due to the low quality, low reproducibility and high dispersion of the results of measuring the amount of mtDNA in blood plasma using direct droplet digital PCR technique by adding 1 µL of blood plasma to the reaction mixture, it was decided to use cfDNA isolation and purification in this study. Blood plasma was collected using GBM scf-DNA vacuum tubes (GRADBIOMED) specifically designed for cell-free DNA analysis. A MagPure Circulating DNA Kit (Magen Biotech) was used to extract cfDNA from 300 µL of plasma following the manufacturer’s protocol. cfDNA samples were resuspended in 50 µL of elution buffer. cfDNA quantification was performed fluorometrically using the Qubit dsDNA HS Assay Kit (Thermo) on a Qubit 3 fluorometer (Thermo). Then, the cfDNA samples were diluted 200-fold, and 1 µL of the diluted cfDNA was used for mtDNA quantification by digital PCR. MT-DNA measurement by Digital PCR This study employed primers and probes, developed and patented by our research group, for mtDNA quantification using digital PCR. These primers were designed to quantify mtDNA by measuring the copy number of the MT-RNR2 gene, using the ACTB gene as a reference. The patent for these primers was registered under the number RU2755663C1 [15]. The 20 µl standard ddPCR reaction mixture contained two pairs of primers for MT-RNR2 and ACTB at a final concentration of 500 nM, 250 nM of each sample, and 1 µL of prepared DNA. All manipulations were performed using consumables with low adhesion. 2X Digital PCR Mastermix for Probes (RainSure) was used as a master mix, and Generation Oil for Probes (RainSure) was used to generate droplets. Further preparation of droplet digital PCR was performed according to the protocol of the manufacturer of the QX200 Droplet Digital PCR System (BioRad). The amplification program on the compatible CFX96 Touch Real-Time PCR Detection System (BioRad) was used according to the Bio-Rad manufacturer's recommendations with modifications, namely, with the ramp rate set to 1.5°C/sec and an additional storage step of + 4°C in the refrigerator overnight to stabilize the droplets and increase the yield of the number of read droplets. The results were analyzed using QuantaSoft software version 1.7.4. Quantification of mtDNA in PBMC was performed per cell, i.e., the number of MT-RNR2 copies in 20 µl / the number of ACTB copies in 20 µl × 2 (diploid chromosome set). The amount of mtDNA was calculated per 1 ml of blood plasma, namely the obtained result of the number of MT-RNR2 copies in 20 µl of the reaction mixture x 200 (dilution factor) x 50 µl (eluate volume) and all this is divided by 300 µl of the used plasma and multiplied by 1000. Measurement of cytochrome C concentration in blood The concentration of cytochrome C (ng/ml) in the blood serum was assessed by the ELISA method using a kit from CusaBio (USA) using the equipment of the Center for Collective Use "Medical Genomics" of the Tomsk National Research Medical Center. This analysis was performed before CABG and 3 and 10 days after CABG. Definition of postoperative complications Postoperative complications included infectious complications such as surgical site wound infections, pneumonia, urinary tract infections, and deep sternal wound infections; acute kidney injury, defined as an acute decline in kidney function leading to a rise in serum creatinine and/or a fall in urine output [16]; post-pericardiotomy syndrome, diagnosed if patients exhibited at least two of the following criteria: fever without an alternative cause, pleuritic chest pain, friction rub, new or worsening pleural effusion, and new or worsening pericardial effusion [17]; acute myocardial infarction, diagnosed according to the Fourth Universal Definition of Myocardial Infarction [18]; imaging-confirmed stroke; and newly diagnosed atrial fibrillation. Statistical analysis Data were analyzed using IBM SPSS Statistics version 21. For assessment of normal distribution, the Shapiro-Wilk test was used. Given the small sample size and lack of normal distribution for most features, nonparametric methods were used for statistical data analysis. Continuous variables were presented as median (Q25-Q75). Categorical data are presented as counts (percentages). The Mann-Whitney U test was used to compare continuous variables between independent groups, while the Wilcoxon signed-rank test was used for paired samples. Spearman’s rank correlation coefficient was used to assess correlations. The χ² test or Fisher’s exact test (for small sample sizes) was used to compare categorical variables. The change in mtDNA-copy number from pre-operation to 10 days after CABG was calculated by Formula 1. $$\:\left(\frac{"mtDNAcopynumberat10daysafterCABG"-"Pre-operationmtDNAcopynumber"}{"Pre-operationmtDNAcopynumber"}\right)*100\%$$ Formula 1 – Formula used for calculation the median percentage change in mtDNA-copy number. The change in cytochrome C concentration from pre-operation to 10 days after CABG was calculated by Formula 2. $$\:\left(\frac{"CytochromeCconcentrationat10daysafterCABG"-"Pre-operationCytochromeCconcentration"}{"Pre-operationCytochromeCconcentration"}\right)*100\%$$ Formula 2 – Formula used for calculation the median percentage change in cytochrome C concentration. The relationship between this change in mtDNA-copy number and postoperative complications was assessed using receiver operating characteristic (ROC) curve analysis (area under the curve [AUC]). A p. value < 0.05 was considered statistically significant. 3. Results This study enrolled 26 patients with CAD who underwent CABG. The median age was 67 (58–71) years, and 92.3% of the patients were male. Detailed patient demographics are provided in Supplementary data in Table S1 . Plasma mtDNA-copy number before surgery and at 3, 5, 7, and 10 days after surgery is shown in Table 1 and Fig. 1 . Table 1 Plasma mtDNA levels before and after coronary artery bypass grafting Parameter MtDNA-copy number per 1 ml of blood plasma (10 6 /ml) p.value (Wilcoxon Test) Median (Q25-Q75) Background 3rd day 5th day 7th day 10th day Background 5.12 (3.35–7.44) - 0.005 0.852 0.024 0.001 3rd day 2.65 (1.88–3.04) - - 0.017 0.005 0.005 5th day 4.04 (2.28-7.00) - - - 0.005 < 0.001 7th day 6.50 (3.47–11.31) - - - - 0.067 10th day 10 (7.2–13.4) - - - - - Blood was sampled pre-operatively (before CABG), and at 3, 5, 7, 10 days after CABG. MtDNA was measured by digital PCR and expressed as million copies/mL. Data are presented as box plots, showing median values, 25th and 75th percentiles, and minimum and maximum values. P values for paired comparisons using the Wilcoxon signed-rank test are as follows. The results showed a post-operative increase in plasma mtDNA-copy number, from a median of 2.65 million copies/mL (interquartile range: (1.88–3.04)) at 72 hours post-surgery to a median of 10 million copies/mL (interquartile range: (7.2–13.4)) at 10 days post-CABG (p = 0.005). Additionally, the 10-day post-operative plasma mtDNA-copy number was significantly higher than the pre-operative level (p = 0.001). We also quantified mtDNA-copy number per PBMC (results are shown in Table 2 and Fig. 2 ). Table 2 MtDNA-copy number per PBMC before CABG and at 3, 5, 7, and 10 days after CABG Parameter MtDNA-copy number per cell in PBMCs p.value (Wilcoxon Test) Median (Q25-Q75) Background 3rd day 5th day 7th day 10th day Background 79 (63.5–95.2) - 0.689 0.689 0.867 0.346 3rd day 65 (58–88) - - 0.401 0.475 0.123 5th day 81 (59.5-107.5) - - - 0.556 0.198 7th day 82 (67–94) - - - - 0.411 10th day 86 (70–114) - - - - - PBMC samples were collected preoperatively (before CABG), and at 3, 5, 7, 10 days after CABG. MtDNA-copy number was quantified by digital PCR and expressed as copies per cell. Data are presented as box plots with medians, 25th and 75th percentiles, and minimum and maximum values. P values for paired comparisons using the Wilcoxon signed-rank test are as follows. The mtDNA-copy number in PBMCs remained stable throughout the perioperative period, with no significant changes observed (p > 0.05). Additionally, we assessed the correlation between mtDNA-copy number in PBMCs and several clinical parameters. No significant correlations were observed with body mass index (r = 0.053, p = 0.800), waist circumference (r = 0.370, p = 0.130), or blood glucose (r = -0.261, p = 0.199). The median percentage change in mtDNA-copy number calculated by formula (1) was 116% (interquartile range: 9.6–267%). This value did not correlate with patient age (r = -0.166, p = 0.449), NT-proBNP concentration (r = 0.162, p = 0.521), LVEF (r = 0.233, p = 0.296), on-pump time (r = 0.027, p = 0.907), or aortic cross-clamp time (r = 0.408, p = 0.159). Furthermore, mtDNA-copy number was not significantly different between men and women (p = 0.743). Considering the increase in plasma mtDNA-copy number alongside a stable mtDNA-copy number in PBMCs, we hypothesized that the increased plasma mtDNA was related to further cell damage postoperatively. Therefore, we analyzed the relationship between this parameter and postoperative complications during this hospitalization. Indeed, patients with any complication (infection, acute kidney injury, post-pericardiotomy syndrome, acute myocardial infarction, stroke, atrial fibrillation) (n = 17) had a significantly higher median plasma mtDNA-copy number change (198%; interquartile range: 101; 321%) compared to patients without complications (n = 9) (11%; interquartile range: -36; 127%; p = 0.008) (Fig. 3 , Table 3 ). However, there were no significant differences in the mtDNA-copy number change for individual complication categories: infection (p = 0.520), acute kidney injury (p = 0.129), post-pericardiotomy syndrome (p = 0.806), acute myocardial infarction (p = 0.583), stroke (p = 0.392), and atrial fibrillation (p = 0.463). In addition, we identified a group of patients with serious complications such as deep sternal wound infection, stroke, or myocardial infarction (n = 3). Plasma mtDNA-copy number change in this group ranged from − 44 to 453%. We did not obtain statistically significant results in this group due to the large scatter of data and the small number of patients with serious complications (p = 0.263). Whereas statistically significant differences for the mtDNA-copy number change remained between patients with less severe complications and patients without complications (р=0.009). The frequency of postoperative complications is presented in Supplementary data in Table 2 . Comparison of clinical data for patients with and without complications after on-pump CABG is presented in Table S3 of the supplementary material. The groups did not statistically significantly differ in the main clinical parameters. Table 3 MtDNA-copy number changes in patients with and without complications after CABG Parameters Without complications (n = 9) With complications (n = 17) р MtDNA-copy number per 1 ml of blood plasma before CABG, (106/ml) 5.05 (3.2–7.9) 5.2 (3.1–6.7) 0.718 MtDNA-copy number per 1 ml of blood plasma at 10 days after CABG, (106/ml) 7.25 (6-11.4) 11.6 (8-14.6) 0.147 MtDNA-copy number change, % 11 ((-36) − 127) 198 (101–321) 0.008 Group 1 represents patients without any complications (n = 9), and Group 2 represents patients with complications (n = 17), including infection, acute kidney injury, postpericardiotomy syndrome, acute myocardial infarction, stroke, and atrial fibrillation. The p-value for the comparison using the Wilcoxon signed-rank test is 0.008. Data are presented as box plots showing median values, 25th and 75th percentiles, and minimum and maximum values. 95% confidence interval for Mean MtDNA-copy number change in Group 1: 95%CI [-28; 125], in Group 2: 95%CI [130; 298]. The median plasma mtDNA-copy number was not significantly different between patients with complications (5.05 million copies/mL; interquartile range: 3.2–7.9) and those without complications (5.2 million copies/mL; interquartile range: 3.1–6.7; p = 0.718). Receiver operating characteristic (ROC) curve analysis showed that a 30% or greater increase from the pre-operative value in plasma mtDNA-copy number was associated with the development of postoperative complications, with a sensitivity of 0.88 and specificity of 0.67 (Fig. 4 ). The curve was generated based on patients experiencing postoperative complications (including infection, acute kidney injury, postpericardiotomy syndrome, acute myocardial infarction, stroke, and atrial fibrillation). A 30% increase in plasma mtDNA-copy number was associated with postoperative complications, with a sensitivity of 0.88, a specificity of 0.67, and an area under the ROC curve (AUC) of 0.824 (95% confidence interval [CI]: 0.653–0.994; p = 0.008). To confirm that patients with postoperative complications have more pronounced mitochondrial destruction, we analyzed the concentration of cytochrome C before CABG and on days 3 and 10 after CABG (Table 4 ). Table 4 Cytochrome C concentration changes in patients with and without complications after CABG Parameters Without complications (n = 9) With complications (n = 17) р Cytochrome С before CABG, ng/mL 25.1 (18.2; 32.6) 24.8 (14; 31.7) 0.779 Cytochrome С at 3 days after CABG, ng/mL 32.4 (25.8; 39) 32.5 (22.3; 49) 0.968 Cytochrome С at 10 days after CABG, ng/mL 27.4 (18.8; 32.1) 34.2 (25.6; 39.3) 0.282 Changes of Cytochrome C, % 4.1 (-34; 54) 31.7 (6.3; 92.8) 0.039 Thus, the concentration of cytochrome C before CABG did not differ between the groups with and without complications. By day 3 after surgery we recorded an increase in the concentration of cytochrome C in both groups (p = 0.968 when comparing groups 1 and 2). Thereafter, the concentration of cytochrome C remained high in the group with complications and decreased in the group without complications at 10 days after CABG. We calculated the change in cytochrome C concentration from the preoperative period to day 10 after surgery. In the group without complications the change in cytochrome C concentration was 4.1 (-34; 54) %, in the group with complications 31.7 (6.3; 92.8) %, the differences were statistically significant (p = 0.039). 4. Discussion This study demonstrated, for the first time, the dynamics of plasma mtDNA-copy number within 10 days after CABG with cardiopulmonary bypass. We observed that on day 3 post-operation, plasma mtDNA-copy number decreased slightly compared to pre-operative levels, consistent with existing literature [8]. Subsequently, there was a statistically significant increase in plasma mtDNA-copy number within 10 days after surgery. According to literature, an increase in the MtDNA-copy number in blood plasma during the perioperative period may be associated with both mechanical trauma to cells and the use of artificial circulation in cardiac surgery as well as reperfusion. However, after surgery, the MtDNA-copy numbers should return to normal within 24 to 72 hours [7, 8, 19]. Given the design of our study, we were able to observe an increase in the amount of plasma mtDNA 10 days after on-pump cardiac surgery. Given that mechanical cell damage and use of on-pump circulation does explain this change, we hypothesize that ongoing inflammation-induced cell damage after cardiac surgery leads to release of mtDNA, which in turn enhances inflammatory processes [6]. Mitochondrial DNA, the organelle's genome, has been widely studied as a danger signal. It is a molecule that gives an alarm signal to the organism, activating an inflammatory program in target cells [12]. Mitochondrial DNA, which exits mitochondria, is a potent activator of cyclic GMP–AMP synthase, resulting in stimulation of the subsequent synthesis of cytokines, such as interferon-β1, IL-6, and tumour necrosis factor [10]. Progressive inflammation, in turn, can trigger the development and progression of complications after on-pump CABG, primarily atrial fibrillation and post-pericardiotomy syndrome, which significantly worsen the course of the postoperative period and increase the time of hospitalization for patients [20, 21]. We found that the greatest increase in plasma mtDNA-copy number occurred in patients who developed any postoperative complication, including infections, acute kidney injury, post-pericardiotomy syndrome, acute myocardial infarction, stroke, and atrial fibrillation. The plasma mtDNA-copy number at 10 days post-surgery was not significantly associated with post-operative complications. These findings are consistent with previous research, which showed that a greater than two-fold increase in plasma mtDNA-copy number within 2 days after CABG is linked to a higher risk of complications such as atrial fibrillation and infections [6]. This may be due to the adverse effects of mtDNA-associated damage-associated molecular patterns (mtDNA DAMPs) [22]. Other studies have reported an association between pre-operative blood mtDNA-copy number and the development of atrial fibrillation [6, 9]. We did not observe this in our cohort, possibly due to the inclusion of patients with severe heart failure, which may have led to elevated pre-operative plasma mtDNA-copy number. Another likely explanation is the small number of patients who developed atrial fibrillation (n = 6), which may limit statistical power. We showed that a 30% or more increase in plasma mtDNA-copy number was associated with the development of postoperative complications, with a sensitivity of 88% and a specificity of 67% (ROC analysis). This indirectly confirms the existence of a pathological link between them. To confirm this connection, we studied an additional marker - cytochrome C, which enters the blood when mitochondria are destroyed. Indeed, the change in the concentration of cytochrome C 10 days after CABG was associated with the development of postoperative complications. Previous studies have shown that the level of cytochrome C increases by the patients after cardiac arrest, and to a greater extent in those patients who died, which proves its connection with damage to cardiomyocytes [23]. The connection of cytochrome C concentration with mitochondrial damage has also been proven in experimental studies [13]. In turn, another study showed no change in the concentration of cytochrome C 6 hours after CABG [24]. However, we could not find literature that would study the change in the concentration of cytochrome C 10 days after CABG. In our group of patients with complications after CABG, an increase in the concentration of cytochrome C was observed by day 3 after surgery, which remained high until day 10. Whereas in the group of patients without complications, a decrease in the concentration of cytochrome C was recorded from day 3 to day 10. The obtained data, together with the increase in the mtDNA-copy number in plasma, indicate ongoing damage to cells and mitochondria in the postoperative period in patients with complications. The complication rate in our study was 65.4%, which was quite high compared to the literature even when compared with the more severe cohort of patients included in the STICH study [25], where the complication rate was 31. However, the STICH trial did not account the development of post-pericardiotomy syndrome, which is one of the most common complications in our group. In addition, if we single out only serious complications such as deep chest infection, stroke, and myocardial infarction, the frequency of such complications was low and amounted to 11.5% in our group. The remaining cases of complications were associated with less serious events. We believe that the development of complications was not related to the specific surgical procedure itself, since the main characteristics of the cardiac surgery did not differ between patients with complications and without complications. We associate the high rate of complications with the initial severity of theclinical condition of our patients, who had reduced left ventricular ejection fraction and more than 80% of whom had a history of myocardial infarction. Every fourth patient had experienced heart failure decompensations within a year prior to surgery. As is known, there have been increasing studies exploring the correlation between mitochondrial DNA copy number abnormalities (mtDNA-copy number) and cardiovascular disease. However, their findings have been contradictory. Studies analyzing the relationship between mtDNA-copy number and complications after cardiac surgery [6–8] were limited to a 72-hour period, during which time changes in mtDNA-copy number could be caused by mechanical trauma, which could affect the measured indicator. Some of these studies also included a small number of patients. Despite the inclusion of a large numbers of patients in other studies, they studied only preoperative values of [9], which is not enough to show how this parameter changes after on-pump CABG. We have an advantage in that we study mtDNA-copy in a series and have been able to evaluate changes in this parameter up to 10 days after on-pump CABG. Thus, our data is novel and can inspire larger studies in this area. Thus, our results highlight an increase in plasma mtDNA-copy number that is not associated with mechanical trauma or on-pump circulation. This suggests that reducing plasma mtDNA-copy number may reduce inflammation and the risk of complications. Mitochondria are being increasingly considered as a target for new drugs for cardiovascular diseases [19], and our study adds new data to this concept. In our study, the mtDNA-copy number in in PBMCs did not change before surgery or on days 3, 5, 7, and 10 after CABG. According to literature data, a decrease in mtDNA-copy number in PBMCs is associated with metabolic disorders [26], however, in our study, no statistically significant correlations were found between this indicator and the level of glycemia, body mass index, or waist circumference. Low levels of mtDNA-copy number in PBMCs in the cohort studied may indicate a high baseline cardiovascular risk in patients with HF and CAD [27–29]. Furthermore, the dynamics of mtDNA-copy number in PBMCs has not been investigated previously in patients after CABG. Thus, we have shown for the first time that the mtDNA-copy number in PBMCs does not change significantly after CABG, which may indirectly indicate a decrease in the reparative activity of mtDNA in PBMCs in our group of patients [30]. Study Limitations. The main limitation of this study was the small sample size. However, the number of patients included was sufficient to demonstrate the dynamics of mtDNA-copy number in peripheral blood and identify statistically significant associations between this biomarker and the development of postoperative complications. It should be noted that most of the of complications in our patients were minor, while deep sternal wound infections, strokes, and myocardial infarctions were recorded in only three patients. As a result, we cannot distinguish between minor and severe complications. This may be the subject of further research. Another problem with a small number of patients is that 90% are men. This fact should be taken into consideration when extrapolating findings to the general population undergoing on-pump CABG. Another limitation of our study was the inability to calculate the sample size before the study began. This was because, to our knowledge, this was the first study analyzing the dynamics of mtDNA-copy number over 10 days after on-pump CABG and studying the association between changes in mtDNA-copy numbers and postoperative complications. 5. Conclusions Plasma mtDNA-copy number is decreased to pre-operative levels 3 days after CABG. Subsequently, the plasma mtDNA-copy number increases in peripheral blood by 10 days after surgery. A 30% or greater increase in plasma mtDNA-copy number at 10 days post-CABG, compared to pre-operative levels, is associated with the development of postoperative complications, which mostly concerns less serious events. The mtDNA-copy number in PBMCs does not change after on-pump CABG. Abbreviations mtDNA Mitochondrial DNA CABG Coronary artery bypass grafting PBMCs Peripheral blood mononuclear cells PCR Polymerase chain reaction HF Heart failure CAD Coronary artery disease mtDAMPs Mitochondrial damage-associated molecular patterns LVEF Left ventricular ejection fraction AUC Area under the curve ROC Receiver operating characteristic Declarations Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Table S1. Baseline demographic and clinical characteristics of patients before CABG; Table S2. Complications after CABG surgery in the study cohort; Table S3. Baseline demographic and clinical characteristics of patients with and without complications. Author Contributions: GAA and KEA designed the research study, analyzed the data. SAA performed the research number of copy of mtDNA. KEA, TOV and DBB wrote the manuscript. KMU and SEE provided help and advice on data collection and analysis. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. Funding: The study was supported by the grant from the Russian Science Foundation No. 23-75-00009, https://rscf.ru/project/23-75-00009/ Institutional Review Board Statement: The study protocol was approved by the local ethical committee of the Cardiology Research Institute, Tomsk National Research Medical Center (protocol No. 241 of March 9, 2023), and all of the participants provided signed informed consent. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study Data Availability: The datasets used during the current study are available from the corresponding author on reasonable request. Conflicts of Interest: The authors declare no conflicts of interest References Atici AE, Crother TR, Noval Rivas M. Mitochondrial quality control in health and cardiovascular diseases. 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Method for measuring number of copies of human mitochondrial DNA by digital polymerase chain reaction (Patent No. RU2755663C1). Additional info: application 2021-04-07, granted 2021-09-20 Stevens, Paul E. et al. KDIGO 2024 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease. Kidney International, Volume 105, Issue 4, S117 - S314 Imazio M, Brucato A, Ferrazzi P, Spodick DH, Adler Y. Postpericardiotomy syndrome: a proposal for diagnostic criteria. J Cardiovasc Med (Hagerstown). 2013 May;14(5):351-3. doi: 10.2459/JCM.0b013e328353807d. Fourth universal definition of myocardial infarction (2018). Rev Esp Cardiol (Engl Ed). 2019 Jan;72(1):72. English, Spanish. doi: 10.1016/j.rec.2018.11.011 Paillard M, Abdellatif M, Andreadou I, Bär C, Bertrand L, Brundel BJJM, et al. Mitochondrial targets in ischaemic heart disease and heart failure, and their potential for a more efficient clinical translation. 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Russian. doi: 10.18087/cardio.2023.7.n2229 Paunel-Görgülü A, Wacker M, El Aita M, Hassan S, Schlachtenberger G, Deppe A, Choi YH, Kuhn E, Mehler TO, Wahlers T. fDNA correlates with endothelial damage after cardiac surgery with prolonged cardiopulmonary bypass and amplifies NETosis in an intracellular TLR9-independent manner. Sci Rep. 2017 Dec 12;7(1):17421. doi: 10.1038/s41598-017-17561-1 Cocchi MN, Andersen LW, Rittenberger J, Abella B, Gaieski D, Peberdy MA, et al. Abstract 19510: cytochrome c levels in post-cardiac arrest patients. Circulation 2015; 132(132):A19510. Andersen LW, Liu X, Montissol S, Holmberg MJ, Fabian-Jessing BK, Donnino MW; Center for Resuscitation Science Research Group. Cytochrome c in patients undergoing coronary artery bypass grafting: A post hoc analysis of a randomized trial. J Crit Care. 2017 Dec;42:248-254. doi: 10.1016/j.jcrc.2017.08.006. Wrobel K, Stevens SR, Jones RH, Selzman CH, Lamy A, Beaver TMet al.Influence of Baseline Characteristics, Operative Conduct, and Postoperative Course on 30-Day Outcomes of Coronary Artery Bypass Grafting Among Patients With Left Ventricular Dysfunction: Results From the Surgical Treatment for Ischemic Heart Failure (STICH) Trial. Circulation. 2015 Aug 25;132(8):720-30. doi: 10.1161/CIRCULATIONAHA.114.014932 Agius R, Pace NP, Fava S. Reduced leukocyte mitochondrial copy number in metabolic syndrome and metabolically healthy obesity. Front Endocrinol (Lausanne). 2022 Jul 25;13:886957. doi: 10.3389/fendo.2022.886957 Ashar FN, Zhang Y, Longchamps RJ, Lane J, Moes A, Grove ML, Mychaleckyj JC, Taylor KD, Coresh J, Rotter JI, Boerwinkle E, Pankratz N, Guallar E, Arking DE. Association of Mitochondrial DNA Copy Number With Cardiovascular Disease. JAMA Cardiol. 2017 Nov 1;2(11):1247-1255. doi: 10.1001/jamacardio.2017.3683 Koller A, Fazzini F, Lamina C, Rantner B, Kollerits B, Stadler M, Klein-Weigel P, Fraedrich G, Kronenberg F. Mitochondrial DNA copy number is associated with all-cause mortality and cardiovascular events in patients with peripheral arterial disease. J Intern Med. 2020 May;287(5):569-579. doi: 10.1111/joim.13027 Li X, Liu X, Chen X, Wang Y, Wu S, Li F, Su Y, Chen L, Xiao J, Ma J, Qin P. Leukocyte mitochondrial DNA copy number and cardiovascular disease: A systematic review and meta-analysis of cohort studies. iScience. 2024 Jul 17;27(9):110522. doi: 10.1016/j.isci.2024.110522 Chan SW, Chevalier S, Aprikian A, Chen JZ. Simultaneous quantification of mitochondrial DNA damage and copy number in circulating blood: a sensitive approach to systemic oxidative stress. Biomed Res Int. 2013;2013:157547. doi: 10.1155/2013/157547 Additional Declarations No competing interests reported. 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10:42:37","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":108102,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7999546/v1/daeb8d4cc512a84feea9fd28.html"},{"id":97689101,"identity":"b60ac99c-be68-48bd-8eec-b9753e576532","added_by":"auto","created_at":"2025-12-08 10:42:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":457537,"visible":true,"origin":"","legend":"\u003cp\u003ePlasma mtDNA-copy number in patients undergoing coronary artery bypass grafting (n = 26) with cardiopulmonary bypass and cardioplegia.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7999546/v1/290e04a5a05cfcce2da0c2ae.png"},{"id":97689099,"identity":"073cd350-b858-4d0f-b549-ccfde50d90b0","added_by":"auto","created_at":"2025-12-08 10:42:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":435763,"visible":true,"origin":"","legend":"\u003cp\u003eMtDNA-copy number per cell in PBMCs from patients undergoing coronary artery bypass grafting (n = 26) with cardiopulmonary bypass and cardioplegia.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7999546/v1/44d636a042ea2aae3482bae5.png"},{"id":97892573,"identity":"cf2b66b4-c7eb-4295-a9c4-66d1bd1645c5","added_by":"auto","created_at":"2025-12-10 15:15:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":352273,"visible":true,"origin":"","legend":"\u003cp\u003ePercent change in plasma mtDNA-copy number from pre-operative levels to 10 days post-CABG, in patients with and without post-operative complications.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7999546/v1/d9fb90e5a98661ae742c5ead.png"},{"id":97892637,"identity":"a179e4fd-de4a-408f-a5db-fa108975f2cc","added_by":"auto","created_at":"2025-12-10 15:16:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":41206,"visible":true,"origin":"","legend":"\u003cp\u003eReceiver operating characteristic (ROC) curve for predicting postoperative complications based on the change in plasma mtDNA-copy number.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7999546/v1/ea0971e62306d0a2611dfdac.png"},{"id":106396164,"identity":"fd59c310-1058-4226-be3b-595bec3c19cf","added_by":"auto","created_at":"2026-04-08 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Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eHeart failure (HF) remains a global health challenge and represents the final stage of the cardiovascular continuum. The role of mitochondria in the HF pathogenesis is an area of ongoing investigation. Studying mitochondrial function in HF patients with coronary artery disease (CAD) is particularly important, as both conditions can impair mitochondrial function [1, 2]. It should be noted that 90% of the energy used by the cardiomyocyte contraction-relaxation cycle comes from the de novo synthesis of adenosine triphosphate in mitochondria [3, 4].\u003c/p\u003e\u003cp\u003eA key characteristic of mitochondria that distinguishes them from other intracellular organelles is their self-contained DNA (mtDNA) [5]. When released extracellularly, mtDNA acts as a mitochondrial damage-associated molecular patterns (mtDAMPs), triggering inflammatory responses [6]. Previous research has shown that mtDNA-copy number increases in peripheral blood during cardiac surgeries, especially during coronary artery bypass grafting (CABG) with cardiopulmonary bypass and returns to baseline levels within 24\u0026ndash;72 hours post-surgery [7, 8]. Furthermore, elevated pre-CABG mtDNA-copy number has been linked to the development of post-operative atrial fibrillation [6, 9], which is thought to be due to the pro-inflammatory effects of mtDNA. Studies on post-CABG mtDNA-copy number changes have been limited to the first 24\u0026ndash;72 hours and it has not been studied how the mtDNA-copy number changes in the following days after CABG which is the aim of our study.\u003c/p\u003e\u003cp\u003eThe development of complications in the postoperative period after on-pump CABG is primarily related to inflammation. Such complications include atrial fibrillation, post-pericardiotomy syndrome, and infection. These conditions promote the active release of mitochondrial DNA (mtDNA) from cells and an increase in mtDNA-copy number in plasma. At the same time, increased levels of mtDAMPs lead to the progression of these complications and worsening of the postoperative course after CABG [10]. Confirmation of an association between increased plasma mtDNA-copy number and complications after heart surgery may provide a rationale for a more focused study of this parameter.\u003c/p\u003e\u003cp\u003eThe mechanisms of the release of mtDNA from cells into the extracellular compartment have not been fully elucidated now. In passive mechanisms, mtDNA is believed to be released into the extracellular space following cellular apoptosis or necrosis [11]. Passive release of mtDNA can include an increase in the number of mtDNA-copy by mechanical damage to cells due to CABG surgery. Another mechanism of the release of mtDNA from cells may be active release as part of extracellular vesicles or as a result of release in the form of neutrophils extracellular traps [12]. It is likely that such a mechanism may be involved in changing the mtDNA-copies number in plasma at later stages after CABG and may be associated with the development of post-operative complications after CABG.\u003c/p\u003e\u003cp\u003eIn addition to the increase in the mtDNA-copy number, indirect evidence of ongoing damage to mitochondria may be an increase in Cytochrome C. Cytochrome C is one of the components of the mitochondrial respiratory chain, which enters the blood when these organelles are damaged [13].\u003c/p\u003e\u003cp\u003eThe aim of this study was to determine the changes in blood mtDNA-copy number in patients undergoing on-pump CABG, from pre-operative baseline to day 10 after CABG, and to assess its association with post-operative complications.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eStudy design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study enrolled 26 patients undergoing CABG, with a median age of 67 years (interquartile range: 58\u0026ndash;71 years). This was a single-center, prospective, non-blinded cohort study (Protocol registered at ClinicalTrials.gov: NCT05770349). Inclusion criteria were: presence of HF with left ventricular ejection fraction (LVEF)\u0026thinsp;\u0026lt;\u0026thinsp;50%; atherosclerotic plaques of 70% or more in at least two major coronary arteries; decision by the cardiology team to perform on-pump CABG; signed informed consent; and availability of biomaterial samples. Exclusion criteria included: refusal of revascularization or participation; need for additional cardiac surgical interventions other than CABG; active oncological diseases; presence of implanted devices; severe renal dysfunction (estimated glomerular filtration rate CKD-EPI\u0026thinsp;\u0026lt;\u0026thinsp;30 ml/min/1.73 m2); infiltrative heart diseases; acute infections or exacerbations of chronic somatic diseases; severe chronic obstructive pulmonary disease; bronchial asthma; type 1 or type 2 diabetes mellitus; and anemia. All patients provided voluntary written informed consent for the use of blood and plasma samples. The Ethics Committee of the Research Institute of Cardiology of Tomsk National Research Medical Center approved this study (Approval No. 241 of March 9, 2023), which was conducted in accordance with the Declaration of Helsinki (2013). Blood plasma and peripheral blood mononuclear cells (PBMCs) were collected from patients at baseline (pre-CABG) and on days 1, 3, 5, 7, and 10 after CABG. The research was carried out using the equipment of the Center for Collective Use \"Medical Genomics\" of Cardiology Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences, Tomsk, Russia.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA preparation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo obtain DNA from PBMC, the whole blood was washed twice with ACK Lysing Buffer, prepared according to the protocol outlined by AAT Bioquest [14]. A D-Blood kit (Biolabmix) was used to extract DNA from 300 \u0026micro;L of blood. The quantity and quality of the extracted DNA samples were assessed using the Nanodrop-8000 (Thermo) spectrophotometer. DNA degradation was evaluated via 1% agarose gel electrophoresis. DNA samples were diluted to a working concentration of 20 ng/\u0026micro;L using 1X TE buffer with 0.1 mM EDTA (SibEnzyme). For digestion, 20 ng of DNA was treated with 1 unit of HinfI enzyme (Thermo) and 1X Tango buffer (Thermo) for 1 hour at 37\u0026deg;C. Subsequently, 1 ng of digested DNA per 20 \u0026micro;L of reaction mixture was used for digital PCR.\u003c/p\u003e\u003cp\u003eDue to the low quality, low reproducibility and high dispersion of the results of measuring the amount of mtDNA in blood plasma using direct droplet digital PCR technique by adding 1 \u0026micro;L of blood plasma to the reaction mixture, it was decided to use cfDNA isolation and purification in this study.\u003c/p\u003e\u003cp\u003eBlood plasma was collected using GBM scf-DNA vacuum tubes (GRADBIOMED) specifically designed for cell-free DNA analysis. A MagPure Circulating DNA Kit (Magen Biotech) was used to extract cfDNA from 300 \u0026micro;L of plasma following the manufacturer\u0026rsquo;s protocol. cfDNA samples were resuspended in 50 \u0026micro;L of elution buffer. cfDNA quantification was performed fluorometrically using the Qubit dsDNA HS Assay Kit (Thermo) on a Qubit 3 fluorometer (Thermo). Then, the cfDNA samples were diluted 200-fold, and 1 \u0026micro;L of the diluted cfDNA was used for mtDNA quantification by digital PCR.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMT-DNA measurement by Digital PCR\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study employed primers and probes, developed and patented by our research group, for mtDNA quantification using digital PCR. These primers were designed to quantify mtDNA by measuring the copy number of the MT-RNR2 gene, using the ACTB gene as a reference. The patent for these primers was registered under the number RU2755663C1 [15].\u003c/p\u003e\u003cp\u003eThe 20 \u0026micro;l standard ddPCR reaction mixture contained two pairs of primers for MT-RNR2 and ACTB at a final concentration of 500 nM, 250 nM of each sample, and 1 \u0026micro;L of prepared DNA. All manipulations were performed using consumables with low adhesion. 2X Digital PCR Mastermix for Probes (RainSure) was used as a master mix, and Generation Oil for Probes (RainSure) was used to generate droplets. Further preparation of droplet digital PCR was performed according to the protocol of the manufacturer of the QX200 Droplet Digital PCR System (BioRad).\u003c/p\u003e\u003cp\u003e The amplification program on the compatible CFX96 Touch Real-Time PCR Detection System (BioRad) was used according to the Bio-Rad manufacturer's recommendations with modifications, namely, with the ramp rate set to 1.5\u0026deg;C/sec and an additional storage step of +\u0026thinsp;4\u0026deg;C in the refrigerator overnight to stabilize the droplets and increase the yield of the number of read droplets. The results were analyzed using QuantaSoft software version 1.7.4.\u003c/p\u003e\u003cp\u003eQuantification of mtDNA in PBMC was performed per cell, i.e., the number of MT-RNR2 copies in 20 \u0026micro;l / the number of ACTB copies in 20 \u0026micro;l \u0026times; 2 (diploid chromosome set). The amount of mtDNA was calculated per 1 ml of blood plasma, namely the obtained result of the number of MT-RNR2 copies in 20 \u0026micro;l of the reaction mixture x 200 (dilution factor) x 50 \u0026micro;l (eluate volume) and all this is divided by 300 \u0026micro;l of the used plasma and multiplied by 1000.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMeasurement of cytochrome C concentration in blood\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe concentration of cytochrome C (ng/ml) in the blood serum was assessed by the ELISA method using a kit from CusaBio (USA) using the equipment of the Center for Collective Use \"Medical Genomics\" of the Tomsk National Research Medical Center. This analysis was performed before CABG and 3 and 10 days after CABG.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDefinition of postoperative complications\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePostoperative complications included infectious complications such as surgical site wound infections, pneumonia, urinary tract infections, and deep sternal wound infections; acute kidney injury, defined as an acute decline in kidney function leading to a rise in serum creatinine and/or a fall in urine output [16]; post-pericardiotomy syndrome, diagnosed if patients exhibited at least two of the following criteria: fever without an alternative cause, pleuritic chest pain, friction rub, new or worsening pleural effusion, and new or worsening pericardial effusion [17]; acute myocardial infarction, diagnosed according to the Fourth Universal Definition of Myocardial Infarction [18]; imaging-confirmed stroke; and newly diagnosed atrial fibrillation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eData were analyzed using IBM SPSS Statistics version 21. For assessment of normal distribution, the Shapiro-Wilk test was used. Given the small sample size and lack of normal distribution for most features, nonparametric methods were used for statistical data analysis. Continuous variables were presented as median (Q25-Q75). Categorical data are presented as counts (percentages). The Mann-Whitney U test was used to compare continuous variables between independent groups, while the Wilcoxon signed-rank test was used for paired samples. Spearman\u0026rsquo;s rank correlation coefficient was used to assess correlations. The χ\u0026sup2; test or Fisher\u0026rsquo;s exact test (for small sample sizes) was used to compare categorical variables. The change in mtDNA-copy number from pre-operation to 10 days after CABG was calculated by Formula 1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\left(\\frac{\u0026quot;mtDNAcopynumberat10daysafterCABG\u0026quot;-\u0026quot;Pre-operationmtDNAcopynumber\u0026quot;}{\u0026quot;Pre-operationmtDNAcopynumber\u0026quot;}\\right)*100\\%$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFormula 1 \u0026ndash; Formula used for calculation the median percentage change in mtDNA-copy number.\u003c/p\u003e\u003cp\u003eThe change in cytochrome C concentration from pre-operation to 10 days after CABG was calculated by Formula 2.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\left(\\frac{\u0026quot;CytochromeCconcentrationat10daysafterCABG\u0026quot;-\u0026quot;Pre-operationCytochromeCconcentration\u0026quot;}{\u0026quot;Pre-operationCytochromeCconcentration\u0026quot;}\\right)*100\\%$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFormula 2 \u0026ndash; Formula used for calculation the median percentage change in cytochrome C concentration.\u003c/p\u003e\u003cp\u003eThe relationship between this change in mtDNA-copy number and postoperative complications was assessed using receiver operating characteristic (ROC) curve analysis (area under the curve [AUC]). A p. value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThis study enrolled 26 patients with CAD who underwent CABG. The median age was 67 (58\u0026ndash;71) years, and 92.3% of the patients were male. Detailed patient demographics are provided in Supplementary data in Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003ePlasma mtDNA-copy number before surgery and at 3, 5, 7, and 10 days after surgery is shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePlasma mtDNA levels before and after coronary artery bypass grafting\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eMtDNA-copy number per 1 ml of blood plasma (10\u003csup\u003e6\u003c/sup\u003e/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003ep.value (Wilcoxon Test)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e(Q25-Q75)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBackground\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e3rd day\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e5th day\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e7th day\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e10th day\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBackground\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e(3.35\u0026ndash;7.44)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.005\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.852\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.024\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3rd day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e(1.88\u0026ndash;3.04)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.017\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.005\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.005\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5th day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e(2.28-7.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.005\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7th day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e(3.47\u0026ndash;11.31)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.067\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10th day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e(7.2\u0026ndash;13.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cem\u003eBlood was sampled pre-operatively (before CABG), and at 3, 5, 7, 10 days after CABG. MtDNA was measured by digital PCR and expressed as million copies/mL. Data are presented as box plots, showing median values, 25th and 75th percentiles, and minimum and maximum values. P values for paired comparisons using the Wilcoxon signed-rank test are as follows.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eThe results showed a post-operative increase in plasma mtDNA-copy number, from a median of 2.65 million copies/mL (interquartile range: (1.88\u0026ndash;3.04)) at 72 hours post-surgery to a median of 10 million copies/mL (interquartile range: (7.2\u0026ndash;13.4)) at 10 days post-CABG (p\u0026thinsp;=\u0026thinsp;0.005). Additionally, the 10-day post-operative plasma mtDNA-copy number was significantly higher than the pre-operative level (p\u0026thinsp;=\u0026thinsp;0.001). We also quantified mtDNA-copy number per PBMC (results are shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMtDNA-copy number per PBMC before CABG and at 3, 5, 7, and 10 days after CABG\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eMtDNA-copy number per cell in PBMCs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003ep.value (Wilcoxon Test)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e(Q25-Q75)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBackground\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e3rd day\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e5th day\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e7th day\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e10th day\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBackground\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(63.5\u0026ndash;95.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.689\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.689\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.867\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.346\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3rd day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(58\u0026ndash;88)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.401\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.475\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.123\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5th day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(59.5-107.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.556\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.198\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7th day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(67\u0026ndash;94)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.411\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10th day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(70\u0026ndash;114)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cem\u003ePBMC samples were collected preoperatively (before CABG), and at 3, 5, 7, 10 days after CABG. MtDNA-copy number was quantified by digital PCR and expressed as copies per cell. Data are presented as box plots with medians, 25th and 75th percentiles, and minimum and maximum values. P values for paired comparisons using the Wilcoxon signed-rank test are as follows.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eThe mtDNA-copy number in PBMCs remained stable throughout the perioperative period, with no significant changes observed (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Additionally, we assessed the correlation between mtDNA-copy number in PBMCs and several clinical parameters. No significant correlations were observed with body mass index (r\u0026thinsp;=\u0026thinsp;0.053, p\u0026thinsp;=\u0026thinsp;0.800), waist circumference (r\u0026thinsp;=\u0026thinsp;0.370, p\u0026thinsp;=\u0026thinsp;0.130), or blood glucose (r = -0.261, p\u0026thinsp;=\u0026thinsp;0.199).\u003c/p\u003e\n \u003cp\u003eThe median percentage change in mtDNA-copy number calculated by formula (1) was 116% (interquartile range: 9.6\u0026ndash;267%). This value did not correlate with patient age (r = -0.166, p\u0026thinsp;=\u0026thinsp;0.449), NT-proBNP concentration (r\u0026thinsp;=\u0026thinsp;0.162, p\u0026thinsp;=\u0026thinsp;0.521), LVEF (r\u0026thinsp;=\u0026thinsp;0.233, p\u0026thinsp;=\u0026thinsp;0.296), on-pump time (r\u0026thinsp;=\u0026thinsp;0.027, p\u0026thinsp;=\u0026thinsp;0.907), or aortic cross-clamp time (r\u0026thinsp;=\u0026thinsp;0.408, p\u0026thinsp;=\u0026thinsp;0.159). Furthermore, mtDNA-copy number was not significantly different between men and women (p\u0026thinsp;=\u0026thinsp;0.743).\u003c/p\u003e\n \u003cp\u003eConsidering the increase in plasma mtDNA-copy number alongside a stable mtDNA-copy number in PBMCs, we hypothesized that the increased plasma mtDNA was related to further cell damage postoperatively. Therefore, we analyzed the relationship between this parameter and postoperative complications during this hospitalization. Indeed, patients with any complication (infection, acute kidney injury, post-pericardiotomy syndrome, acute myocardial infarction, stroke, atrial fibrillation) (n\u0026thinsp;=\u0026thinsp;17) had a significantly higher median plasma mtDNA-copy number change (198%; interquartile range: 101; 321%) compared to patients without complications (n\u0026thinsp;=\u0026thinsp;9) (11%; interquartile range: -36; 127%; p\u0026thinsp;=\u0026thinsp;0.008) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). However, there were no significant differences in the mtDNA-copy number change for individual complication categories: infection (p\u0026thinsp;=\u0026thinsp;0.520), acute kidney injury (p\u0026thinsp;=\u0026thinsp;0.129), post-pericardiotomy syndrome (p\u0026thinsp;=\u0026thinsp;0.806), acute myocardial infarction (p\u0026thinsp;=\u0026thinsp;0.583), stroke (p\u0026thinsp;=\u0026thinsp;0.392), and atrial fibrillation (p\u0026thinsp;=\u0026thinsp;0.463). In addition, we identified a group of patients with serious complications such as deep sternal wound infection, stroke, or myocardial infarction (n\u0026thinsp;=\u0026thinsp;3). Plasma mtDNA-copy number change in this group ranged from \u0026minus;\u0026thinsp;44 to 453%. We did not obtain statistically significant results in this group due to the large scatter of data and the small number of patients with serious complications (p\u0026thinsp;=\u0026thinsp;0.263). Whereas statistically significant differences for the mtDNA-copy number change remained between patients with less severe complications and patients without complications (р=0.009). The frequency of postoperative complications is presented in Supplementary data in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Comparison of clinical data for patients with and without complications after on-pump CABG is presented in Table S3 of the supplementary material. The groups did not statistically significantly differ in the main clinical parameters.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMtDNA-copy number changes in patients with and without complications after CABG\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWithout complications\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWith complications\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;17)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eр\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMtDNA-copy number per 1 ml of blood plasma before CABG, (106/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.05 (3.2\u0026ndash;7.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.2 (3.1\u0026ndash;6.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.718\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMtDNA-copy number per 1 ml of blood plasma at 10 days after CABG, (106/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.25 (6-11.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.6 (8-14.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.147\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMtDNA-copy number change, %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11 ((-36) \u0026minus;\u0026thinsp;127)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e198 (101\u0026ndash;321)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cem\u003eGroup 1 represents patients without any complications (n\u0026thinsp;=\u0026thinsp;9), and Group 2 represents patients with complications (n\u0026thinsp;=\u0026thinsp;17), including infection, acute kidney injury, postpericardiotomy syndrome, acute myocardial infarction, stroke, and atrial fibrillation. The p-value for the comparison using the Wilcoxon signed-rank test is 0.008. Data are presented as box plots showing median values, 25th and 75th percentiles, and minimum and maximum values. 95% confidence interval for Mean MtDNA-copy number change in Group 1: 95%CI [-28; 125], in Group 2: 95%CI [130; 298].\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eThe median plasma mtDNA-copy number was not significantly different between patients with complications (5.05 million copies/mL; interquartile range: 3.2\u0026ndash;7.9) and those without complications (5.2 million copies/mL; interquartile range: 3.1\u0026ndash;6.7; p\u0026thinsp;=\u0026thinsp;0.718). Receiver operating characteristic (ROC) curve analysis showed that a 30% or greater increase from the pre-operative value in plasma mtDNA-copy number was associated with the development of postoperative complications, with a sensitivity of 0.88 and specificity of 0.67 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003e\u003cem\u003eThe curve was generated based on patients experiencing postoperative complications (including infection, acute kidney injury, postpericardiotomy syndrome, acute myocardial infarction, stroke, and atrial fibrillation). A 30% increase in plasma mtDNA-copy number was associated with postoperative complications, with a sensitivity of 0.88, a specificity of 0.67, and an area under the ROC curve (AUC) of 0.824 (95% confidence interval [CI]: 0.653\u0026ndash;0.994; p\u0026thinsp;=\u0026thinsp;0.008).\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eTo confirm that patients with postoperative complications have more pronounced mitochondrial destruction, we analyzed the concentration of cytochrome C before CABG and on days 3 and 10 after CABG (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCytochrome C concentration changes in patients with and without complications after CABG\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWithout\u003c/p\u003e\n \u003cp\u003ecomplications (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWith complications\u003c/p\u003e\n \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;17)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eр\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCytochrome С before CABG, ng/mL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.1 (18.2; 32.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24.8 (14; 31.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.779\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCytochrome С at 3 days after CABG, ng/mL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.4 (25.8; 39)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.5 (22.3; 49)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.968\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCytochrome С at 10 days after CABG, ng/mL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.4 (18.8; 32.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.2 (25.6; 39.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.282\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eChanges of Cytochrome C, %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.1 (-34; 54)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.7 (6.3; 92.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.039\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\u003cbr\u003e\u003c/div\u003e\n\u003cp\u003eThus, the concentration of cytochrome C before CABG did not differ between the groups with and without complications. By day 3 after surgery we recorded an increase in the concentration of cytochrome C in both groups (p\u0026thinsp;=\u0026thinsp;0.968 when comparing groups 1 and 2). Thereafter, the concentration of cytochrome C remained high in the group with complications and decreased in the group without complications at 10 days after CABG. We calculated the change in cytochrome C concentration from the preoperative period to day 10 after surgery. In the group without complications the change in cytochrome C concentration was 4.1 (-34; 54) %, in the group with complications 31.7 (6.3; 92.8) %, the differences were statistically significant (p\u0026thinsp;=\u0026thinsp;0.039).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis study demonstrated, for the first time, the dynamics of plasma mtDNA-copy number within 10 days after CABG with cardiopulmonary bypass. We observed that on day 3 post-operation, plasma mtDNA-copy number decreased slightly compared to pre-operative levels, consistent with existing literature [8]. Subsequently, there was a statistically significant increase in plasma mtDNA-copy number within 10 days after surgery.\u003c/p\u003e\u003cp\u003eAccording to literature, an increase in the MtDNA-copy number in blood plasma during the perioperative period may be associated with both mechanical trauma to cells and the use of artificial circulation in cardiac surgery as well as reperfusion. However, after surgery, the MtDNA-copy numbers should return to normal within 24 to 72 hours [7, 8, 19].\u003c/p\u003e\u003cp\u003eGiven the design of our study, we were able to observe an increase in the amount of plasma mtDNA 10 days after on-pump cardiac surgery. Given that mechanical cell damage and use of on-pump circulation does explain this change, we hypothesize that ongoing inflammation-induced cell damage after cardiac surgery leads to release of mtDNA, which in turn enhances inflammatory processes [6]. Mitochondrial DNA, the organelle's genome, has been widely studied as a danger signal. It is a molecule that gives an alarm signal to the organism, activating an inflammatory program in target cells [12]. Mitochondrial DNA, which exits mitochondria, is a potent activator of cyclic GMP\u0026ndash;AMP synthase, resulting in stimulation of the subsequent synthesis of cytokines, such as interferon-β1, IL-6, and tumour necrosis factor [10].\u003c/p\u003e\u003cp\u003eProgressive inflammation, in turn, can trigger the development and progression of complications after on-pump CABG, primarily atrial fibrillation and post-pericardiotomy syndrome, which significantly worsen the course of the postoperative period and increase the time of hospitalization for patients [20, 21].\u003c/p\u003e\u003cp\u003eWe found that the greatest increase in plasma mtDNA-copy number occurred in patients who developed any postoperative complication, including infections, acute kidney injury, post-pericardiotomy syndrome, acute myocardial infarction, stroke, and atrial fibrillation. The plasma mtDNA-copy number at 10 days post-surgery was not significantly associated with post-operative complications. These findings are consistent with previous research, which showed that a greater than two-fold increase in plasma mtDNA-copy number within 2 days after CABG is linked to a higher risk of complications such as atrial fibrillation and infections [6]. This may be due to the adverse effects of mtDNA-associated damage-associated molecular patterns (mtDNA DAMPs) [22].\u003c/p\u003e\u003cp\u003eOther studies have reported an association between pre-operative blood mtDNA-copy number and the development of atrial fibrillation [6, 9]. We did not observe this in our cohort, possibly due to the inclusion of patients with severe heart failure, which may have led to elevated pre-operative plasma mtDNA-copy number. Another likely explanation is the small number of patients who developed atrial fibrillation (n\u0026thinsp;=\u0026thinsp;6), which may limit statistical power.\u003c/p\u003e\u003cp\u003eWe showed that a 30% or more increase in plasma mtDNA-copy number was associated with the development of postoperative complications, with a sensitivity of 88% and a specificity of 67% (ROC analysis). This indirectly confirms the existence of a pathological link between them.\u003c/p\u003e\u003cp\u003eTo confirm this connection, we studied an additional marker - cytochrome C, which enters the blood when mitochondria are destroyed. Indeed, the change in the concentration of cytochrome C 10 days after CABG was associated with the development of postoperative complications. Previous studies have shown that the level of cytochrome C increases by the patients after cardiac arrest, and to a greater extent in those patients who died, which proves its connection with damage to cardiomyocytes [23]. The connection of cytochrome C concentration with mitochondrial damage has also been proven in experimental studies [13]. In turn, another study showed no change in the concentration of cytochrome C 6 hours after CABG [24]. However, we could not find literature that would study the change in the concentration of cytochrome C 10 days after CABG. In our group of patients with complications after CABG, an increase in the concentration of cytochrome C was observed by day 3 after surgery, which remained high until day 10. Whereas in the group of patients without complications, a decrease in the concentration of cytochrome C was recorded from day 3 to day 10. The obtained data, together with the increase in the mtDNA-copy number in plasma, indicate ongoing damage to cells and mitochondria in the postoperative period in patients with complications.\u003c/p\u003e\u003cp\u003eThe complication rate in our study was 65.4%, which was quite high compared to the literature even when compared with the more severe cohort of patients included in the STICH study [25], where the complication rate was 31. However, the STICH trial did not account the development of post-pericardiotomy syndrome, which is one of the most common complications in our group. In addition, if we single out only serious complications such as deep chest infection, stroke, and myocardial infarction, the frequency of such complications was low and amounted to 11.5% in our group. The remaining cases of complications were associated with less serious events. We believe that the development of complications was not related to the specific surgical procedure itself, since the main characteristics of the cardiac surgery did not differ between patients with complications and without complications.\u003c/p\u003e\u003cp\u003eWe associate the high rate of complications with the initial severity of theclinical condition of our patients, who had reduced left ventricular ejection fraction and more than 80% of whom had a history of myocardial infarction. Every fourth patient had experienced heart failure decompensations within a year prior to surgery.\u003c/p\u003e\u003cp\u003eAs is known, there have been increasing studies exploring the correlation between mitochondrial DNA copy number abnormalities (mtDNA-copy number) and cardiovascular disease. However, their findings have been contradictory. Studies analyzing the relationship between mtDNA-copy number and complications after cardiac surgery [6\u0026ndash;8] were limited to a 72-hour period, during which time changes in mtDNA-copy number could be caused by mechanical trauma, which could affect the measured indicator. Some of these studies also included a small number of patients. Despite the inclusion of a large numbers of patients in other studies, they studied only preoperative values of [9], which is not enough to show how this parameter changes after on-pump CABG. We have an advantage in that we study mtDNA-copy in a series and have been able to evaluate changes in this parameter up to 10 days after on-pump CABG. Thus, our data is novel and can inspire larger studies in this area.\u003c/p\u003e\u003cp\u003eThus, our results highlight an increase in plasma mtDNA-copy number that is not associated with mechanical trauma or on-pump circulation. This suggests that reducing plasma mtDNA-copy number may reduce inflammation and the risk of complications. Mitochondria are being increasingly considered as a target for new drugs for cardiovascular diseases [19], and our study adds new data to this concept.\u003c/p\u003e\u003cp\u003eIn our study, the mtDNA-copy number in in PBMCs did not change before surgery or on days 3, 5, 7, and 10 after CABG. According to literature data, a decrease in mtDNA-copy number in PBMCs is associated with metabolic disorders [26], however, in our study, no statistically significant correlations were found between this indicator and the level of glycemia, body mass index, or waist circumference. Low levels of mtDNA-copy number in PBMCs in the cohort studied may indicate a high baseline cardiovascular risk in patients with HF and CAD [27\u0026ndash;29]. Furthermore, the dynamics of mtDNA-copy number in PBMCs has not been investigated previously in patients after CABG. Thus, we have shown for the first time that the mtDNA-copy number in PBMCs does not change significantly after CABG, which may indirectly indicate a decrease in the reparative activity of mtDNA in PBMCs in our group of patients [30].\u003c/p\u003e\u003cp\u003e\u003cb\u003eStudy Limitations.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe main limitation of this study was the small sample size. However, the number of patients included was sufficient to demonstrate the dynamics of mtDNA-copy number in peripheral blood and identify statistically significant associations between this biomarker and the development of postoperative complications. It should be noted that most of the of complications in our patients were minor, while deep sternal wound infections, strokes, and myocardial infarctions were recorded in only three patients. As a result, we cannot distinguish between minor and severe complications. This may be the subject of further research.\u003c/p\u003e\u003cp\u003eAnother problem with a small number of patients is that 90% are men. This fact should be taken into consideration when extrapolating findings to the general population undergoing on-pump CABG.\u003c/p\u003e\u003cp\u003eAnother limitation of our study was the inability to calculate the sample size before the study began. This was because, to our knowledge, this was the first study analyzing the dynamics of mtDNA-copy number over 10 days after on-pump CABG and studying the association between changes in mtDNA-copy numbers and postoperative complications.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ePlasma mtDNA-copy number is decreased to pre-operative levels 3 days after CABG. Subsequently, the plasma mtDNA-copy number increases in peripheral blood by 10 days after surgery. A 30% or greater increase in plasma mtDNA-copy number at 10 days post-CABG, compared to pre-operative levels, is associated with the development of postoperative complications, which mostly concerns less serious events. The mtDNA-copy number in PBMCs does not change after on-pump CABG.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emtDNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMitochondrial DNA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCABG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCoronary artery bypass grafting\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBMCs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePeripheral blood mononuclear cells\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePolymerase chain reaction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHeart failure\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCAD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCoronary artery disease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emtDAMPs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMitochondrial damage-associated molecular patterns\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLVEF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLeft ventricular ejection fraction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAUC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eArea under the curve\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eROC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eReceiver operating characteristic\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials:\u003c/strong\u003e The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Table S1. Baseline demographic and clinical characteristics of patients before CABG; Table S2. Complications after CABG surgery in the study cohort; Table S3. Baseline demographic and clinical characteristics of patients with and without complications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e GAA and KEA designed the research study, analyzed the data. SAA performed the research number of copy of mtDNA. KEA, TOV and DBB wrote the manuscript. KMU and SEE provided help and advice on data collection and analysis. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThe study was supported by the grant from the Russian Science Foundation No. 23-75-00009, https://rscf.ru/project/23-75-00009/\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u003c/strong\u003e The study protocol was approved by the local ethical committee of the Cardiology Research Institute, Tomsk National Research Medical Center (protocol No. 241 of March 9, 2023), and all of the participants provided signed informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eInformed consent was obtained from all subjects involved in the study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e The datasets used during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflicts of interest\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAtici AE, Crother TR, Noval Rivas M. 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Biomed Res Int. 2013;2013:157547. doi: 10.1155/2013/157547\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Coronary artery bypass grafting, CABG, heart failure with reduced ejection fraction, HFrEF, heart failure with mildly reduced ejection fraction, HFmrEF, mitochondrial DNA copy number, mtDNA-copy number","lastPublishedDoi":"10.21203/rs.3.rs-7999546/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7999546/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo determine the mtDNA-copy number in the blood of patients before and within 10 days after coronary artery bypass grafting (CABG), and to assess the association between mtDNA-copy number and the postoperative complications was aims of this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods.\u003c/strong\u003e This single-center, prospective, non-blinded study enrolled 26patients who underwent CABG, with a median age of 67 years (IQR: 58-71). Blood plasma and peripheral blood mononuclear cells (PBMCs) were collected from patients before CABG and at 3, 5, 7, and 10 days post-CABG. Mitochondrial DNA copy number was measured using digital PCR.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults.\u003c/strong\u003e In plasma, the median mtDNA-copy number was 5.12(3.35-7.44) million copies per mL before CABG, 2.65 (1.88-3.04) on 3rd day, 4.04 (2.28-7.00) on 5th day, 6.50 (3.47-11.31), on 7th day, and 10 (7.2-13.4) on 10th day. The median mtDNA-copy number in PBMCs was 79 (63.5-95.2) copies per cell before CABG and did not significantly change post-CABG (р\u0026gt;0.05). Patients with complications had a significantly increased plasma mtDNA-copy number (198%; interquartile range: 101; 321%) compared with patients without complications (11%; interquartile range: -36; 127%; p = 0.008).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions.\u003c/strong\u003e Increases mtDNA-copy number by 10th day post-CABG is associated with postoperative complications. The mtDNA-copy number in PBMCs did not significantly change after CABG.\u003c/p\u003e\n\u003cp\u003eClinical Trial registration: The study protocol was registered at ClinicalTrials.gov: NCT05770349. Registration date 02/21/2023.\u003c/p\u003e","manuscriptTitle":"Mitochondrial DNA Copy Number Is Increased in Plasma and Associated with Postoperative Complications After Coronary Artery Bypass Grafting","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 10:42:32","doi":"10.21203/rs.3.rs-7999546/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e63cca5d-129f-4e42-bf7c-0cdf112ddbe6","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-08T07:58:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-08 10:42:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7999546","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7999546","identity":"rs-7999546","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}