Atrial Dyssynchrony Measured by Strain Echocardiography as a Marker of Proarrhythmic Remodeling and Oxidative Stress in Cardiac Surgery Patients

Research Article Atrial Dyssynchrony Measured by Strain Echocardiography as a Marker of Proarrhythmic Remodeling and Oxidative Stress in Cardiac Surgery Patients Francisco J. Sánchez , 1,2 Valeria A. Gonzalez, 3 Martin Farrando, 2 Ariel O. Baigorria Jayat, 2 Margarita Segovia-Roldan , 4 Laura García-Mendívil , 4 Laura Ordovás , 4,5 Natalia J. Prado , 6 Esther Pueyo , 4,7 and Emiliano R. Diez 1,6 1 Department of Morphophysiology, School of Medicine, National University of Cuyo, Centro Universitario, Mendoza 5500, Argentina 2 Department of Cardiovascular Surgery, Clinic of Cuyo, Mendoza 5500, Argentina 3 Department of Cardiology, Lagomaggiore Hospital, Mendoza 5500, Argentina 4 Biomedical Signal Interpretation and Computational Simulation (BSICoS), Aragon Institute of Engineering Research (I3A), University of Zaragoza Instituto de Investigación Sanitaria (IIS), Zaragoza 50018, Spain 5 Aragon Agency for Research and Development (ARAID), Zaragoza 50018, Spain 6 Institute of Experimental Medicine and Biology of Cuyo (IMBECU)-CONICET, Mendoza 5500, Argentina 7 Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Zaragoza 50018, Spain Correspondence should be addressed to Emiliano R. Diez; [email protected] Received 22 September 2020; Revised 12 November 2020; Accepted 20 November 2020; Published 30 December 2020 Academic Editor: Laura Sartiani Copyright © 2020 Francisco J. Sánchez et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Aging leads to structural and electrophysiological changes that increase the risk of postoperative atrial arrhythmias; however, noninvasive preoperative markers of atrial proarrhythmic conditions are still needed. This study is aimed at assessing whether interatrial dyssynchrony determined using two-dimensional speckle tracking echocardiography relates to proarrhythmic structural and functional remodeling. A cohort of 45 patients in sinus rhythm referred for cardiac surgery was evaluated by echocardiography and surface electrocardiogram the day before the intervention. Transmembrane potential, connexin, and potassium channel distribution, inflammatory, and nitrooxidative markers were measured from right atrial tissue obtained from patients. A difference greater than 40 milliseconds between right and left atrial free wall contraction confirmed the presence of interatrial dyssynchrony in 21 patients. No difference in relation with age, previous diseases, and 2-dimensional echocardiographic findings as well as average values of global longitudinal right and left atrial strain were found between synchronic and dyssynchronic patients. Postoperative atrial fibrillation incidence increased from 8.3% in the synchronic group to 33.3% in the dyssynchronic ones. P wave duration showed no difference between groups. Action potentials from dyssynchronous patients decreased in amplitude, maximal rate of depolarization, and hyperpolarized. Duration at 30% of repolarization increased, being markedly shorter at 90% of repolarization. Only the dyssynchronous group showed early and delayed afterdepolarizations. Atrial tissue of dyssynchronous patients displayed lateralization of connexin 40 and increased connexin 43 expression and accumulation of tumor necrosis factor-αin the intercalated disc. Tumor necrosis factor-αdid not colocalize, however, with lateralized connexin 40. Nitroxidative marks and K ATP channels increased perivascularly and in myocytes. Our results demonstrate that, as compared to a traditional surface electrocardiogram, the novel noninvasive echocardiographic evaluation of interatrial dyssynchrony provides a better identification of nonaged-related proarrhythmic atrial remodeling with increased susceptibility to postoperative atrial fibrillation. Hindawi Oxidative Medicine and Cellular Longevity Volume 2020, Article ID 8895078, 14 pages https://doi.org/10.1155/2020/8895078 1. Introduction Arrhythmias usually complicate cardiovascular surgery, and aging is the main risk factor. Atrial fibrillation is the most frequent sustained arrhythmia with a peak of appearance between the second and fifth days of the postoperative stay [1]. Its incidence progressively increases from 18% in sexagenarians to 50% in octogenarians [2]. Arrhythmic events are usually self-limited, and treatment frequently restores sinus rhythm. However, postoperative atrial fibrillation (POAF) prolongs hospital stay and increases the risk of stroke and mortality [1]. Atrial aging is an elusive prooxidative and proarrhythmic condition. Oxidative stress, hyperadrenergic states, and inflammation contribute to age-related atrial remodeling, but these tissue alterations are challenging to identify noninvasively [3, 4]. In this context, atrial enlargement, stiffness, and conduction blockade are known risk factors, but they are present in only a few patients. Therefore, the mechanisms involved in the onset and perpetuation of POAF are difficult to foresee [5]. Additionally, surgery per se facilitates arrhythmias due to ischemia-reperfusion injury, which increases the preexisting oxidative stress state. The lack of tools to estimate the proarrhythmic substrate evidences the absence of preventive interventions. Structure and function of the beating heart can reveal atrial hidden oxidation and inflammation. Signs of tissue remodeling, before dilatation, arise from atrial dynamic cyclic changes. New echocardiographic techniques like strain and strain rate represent the magnitude and rate of myocardial deformation. Strain can reflect distensibility and atrial contractility [6]. Nonmyocytic cells and extracellular matrix mainly affect distensibility [7]. Cardiomyocyte structure and intercellular communication determine contractile function. Each atrial segment follows a trajectory during the cardiac cycle representative of the tissue physiology [8]. Electrical remodeling involves alterations in both myocytes and nonmyocytic cells, which manifest as contractile dyssynchrony in the echocardiogram, in a similar way as reported for the ventricles [9]. Gap junctions in the heart provide low resistance pathways for propagating the action potential across the myocardium, contributing to electrical coupling and signal propagation [10]. Alteration of cardiomyocyte gap junctions and their main components, connexins (Cx), has been suggested to contribute to the formation of arrhythmias, including atrial fibrillation [11–13]. Accumulating evidence also suggests that inflammation and oxidative stress are involved in atrial remodeling. Detection of protein 3-nitrotyrosine is regarded as a marker of nitrooxidative stress and is observed especially in inflammatory processes. The reaction of peroxynitrite with tyrosine leads to the formation of 3nitrotyrosine and promotes protein, lipid, and DNA damage [14, 15]. ATP-regulated potassium channels (K ATP ) are wellcharacterized metabolic and oxidative sensors in ischemia/reperfusion arrhythmias and here postulated as an interesting substrate of POAF [16]. This study is aimed at assessing whether interatrial dyssynchrony, determined by using two-dimensional speckle tracking echocardiography, relates to proarrhythmic structural and functional remodeling of the atria and whether this increases the susceptibility for POAF. 2. Materials and Methods 2.1. Subjects and Ethical Considerations. Patients with coronary artery disease, aortic stenosis, or the combination of both pathologies, scheduled for surgery at the Department of Cardiac Surgery (Clinic of Cuyo, Mendoza, Argentina), were prospectively enrolled between January 2018 and March 2020. Coronary disease and aortic valve stenosis severity were defined according to current ESC Guidelines to determine surgery indication [17, 18]. All subjects provided written informed consent under the research protocol approved by the Ethics Committee of the National University of Cuyo (Exp-Cuy: 22959/2017). Clinical data including age, gender, and history of previous myocardial infarction and heart failure was collected. The presence of preoperative atrial fibrillation was determined according to previous electrocardiographic reports or diagnosis in medical history. Information regarding the following cardiovascular risk factors was collected: hypertension, dyslipidemia (low-density lipoprotein cholesterol above 100mg/dL or the use of lipid-lowering drugs), smoking (current or any smoking habit in the past ten years), and diabetes mellitus (previous diagnosis of diabetes mellitus or glycated hemoglobin greater than 6.5%). The preoperative use of medications was also documented. 2.2. Inclusion and Exclusion Criteria. Patients over 18 years of age in sinus rhythm with an indication of cardiovascular surgery that gave written informed consent were included in the study. Exclusion criteria were as follows: indication of mitral or tricuspid valve repair or replacement, being older than 80 years, history of previous atrial fibrillation, presence of moderate valvular disease or valvular prosthesis, history of congenital cardiac abnormalities or cardiac tumors, emergency surgery, inability to provide informed consent, and a not entirely detectable left and right atrial profile from the apical four-chamber view during preoperative echocardiography. 2.3. Preoperative Electrocardiogram. Before surgery, patients underwent a 12-lead electrocardiogram (ECG) using the Synchronous ECG software V1.3.5. Measurement in milliseconds of the P wave and the PR segment was performed with the software caliper. According to the current classification, these patients were evaluated for the presence of an interatrial conduction disturbance called Bayes syndrome [19]. There are two major categories for this syndrome: complete and incomplete, both based on P wave duration and morphology in the 12-lead ECG. 2.4. Echocardiography and Atrial Strain. All patients were imaged in a left lateral decubitus position using ESAOTE ultrasound system equipment (MyLab30Gold Cardiovascular) with a 2-4 MHz/PA240 probe. Two-dimensional speckle tracking strain imaging was performed from the apical position by an experienced technician. The average frame rate for analysis was 60-80 frames/s. During a single breath-hold, 2 Oxidative Medicine and Cellular Longevity three consecutive cardiac cycles were stored digitally for offline analysis in the four-, two-, and three-chamber view. The entire right and left atriums were carefully visualized to prevent walls’dropout. Measurements focused on evaluating the indexed volume of the left atrium and the area of the right atrium. In the parasternal long-axis or short-axis view, M-mode of the left ventricle chamber was measured for diameter and wall thickness. In the apical 4-chamber view, the left ventricle ejection fraction was determined using the Simpson measurement. Mitral and tricuspid inflows were recorded at the tip of the valve leaflets. The peak velocities of early and late diastolic filling waves (E wave and A wave) and the E/A velocity ratio were measured. The e velocity was obtained by tissue Doppler averaging the lateral and septal mitral annulus values and the e wave of the tricuspid lateral wall. E/e’values were obtained from both ventricles. Strain and strain rate datasets were analyzed using a wall motion tracking software (ESAOTE MyLab). In apical views (4-chamber, 3-chamber, and 2-chamber), left atrial endocardial boundaries were manually measured at the end-diastolic phase. Right atria were only registered in the 4-chamber view. The values of the reservoir, conduit, and atrial contraction strain were recorded according to the EACVI/ASE/Industry Task Force to standardize deformation imaging [20]. The values of strain rate for the reservoir phase, the conduit, and contraction phases were also recorded (Figure 1). The same was also done in 4 chambers for the right atrium. The left atrium was subsequently divided into basal, medial, and roof atrial segments. This determined a total of 15 segments for the left atria when the 3 apical views were added. The right atrium was evaluated similarly, but only the lateral segments were taken into account, adding 3 more segments, thus making a total of 18 segments. Septal segments were considered as left atria. Deformation rate time (DRT) was recorded from the beginning of the P wave in the ECG to the maximum deflection of the atrial contraction in the strain rate for each of the segments, as indicated in Figures 1(a) and 1(e). To determine interatrial synchrony, an adaptation of atrial strain was performed to evaluate the right and left atrial walls at the same time. This new evaluation was called “Omega”(ω) because of the form the strain takes in the images. Here, the maximal time difference between the strain rate contraction peaks of both right and left atrial lateral walls was recorded (Figure 2). Blinded analysis of the echocardiograms was performed by two independent researchers (FJS and VAG). 2.5. Atrial Transmembrane Potential. Right atrium samples taken in the operating room were transferred within 15 to 20 minutes to the laboratory using a cold oxygenated Ringer-type solution with albumin. Upon arrival, all samples were dissected into smaller pieces, pinned at the bottom of a perfusion chamber with the endocardial surface facing up, and continuously superfused with a modified Krebs–Henseleit solution containing (in mM): 121 NaCl, 25 NaHCO 3 , 1.2 Na 2 HPO 4 , 5 KCl, 2 CaCl 2 , 1.2 MgSO 4 , and 11 glucose. Once equilibrated with 5% CO 2 in O 2 at 36:5±0:5 ° C, the pH of the solution was 7:4±0:02. The membrane potential was recorded with flexibly mounted glass microelectrodes from subendocardial trabecular atrial cells. Microelectrodes were filled with 3 mM KCl and had resistances of 10–15MΩ. After 20 to 30 minutes of stabilization, we continuously obtained epicardial transmembrane potential using a custom-made microelectrode amplifier. The signals were digitized with an analog-to-digital converter (NI PCI-6221; National Instruments, Austin, Texas) and recorded using LabView SignalExpress 2.5 (National Instruments, Austin, Texas). The following properties of the transmembrane potentials were quantified: action potential amplitude, resting potential, maximum upstroke velocity (ΔV/Δtmax), and action potential duration at 30 and 90% of repolarization. Arrhythmic events and arrhythmogenic triggers were evaluated by visual supervision of the traces blindly regarding the patient’s characteristics. 2.6. Structural, Inflammatory, and Nitrooxidative Evaluation by Fluorescent Immunohistochemistry. Part of each atrial sample was fixed in a 4% paraformaldehyde solution for 1 h at 4 ° C before embedding in paraffin blocks. Fivemicrometer-thick tissue sections were stained using the following primary antibodies or label: rabbit polyclonal antiCx40 (Cx40 H-116, Santa Cruz, sc-28658, dilution 1:300), rabbit polyclonal anti-Cx43 (Cx43, Abcam, ab11370, dilution 1: 1000), rabbit polyclonal anti-Kir6.1 (Kir6.1, Thermo Fisher, PA5-48354, dilution 1 :500), mouse monoclonal anti-TNFα(TNFα52B83, Santa Cruz, sc-52746, dilution 1: 300), mouse monoclonal antinitrotyrosine (3-nitrotyrosine, Santa Cruz, sc-32757, dilution 1 :200), and mouse monoclonal anti-SERCA2a (ab2817 Abcam, dilution 1: 1000). Wheat germ agglutinin (WGA) conjugated to Alexa Fluor 555 against the extracellular matrix (W32464 Thermo Fisher, dilution 1 :500) and fluorescently conjugated F-actin against the intracellular content (ab112124 Abcam, dilution 1: 300). The secondary antibodies were anti-rabbit conjugated with Alexa Fluor 633 and anti-mouse labeled with the Alexa Fluor 488 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA, dilution 1 :500). Images were acquired with a confocal microscope Zeiss LSM 880 and processed with the Zen Blue 2.5 software (Carl Zeiss Microscopy GmbH, 2018). The maximum intensity projection of 20 to 40 z stacks was used to analyze the lateralization of connexins and the integrated optical density (IOD). Lateralization was measured with an open access automated program designed by our group called MARTA [21]. This software generates cell masks, contours individual cells, and splits the cells into 4 rectangles to estimate the lateral-to-total ratio of connexins. The IOD quantified the area (>3 pixels connected) multiplied by the average intensity using the software ImageProPlus 4.5, 2001 (Media Cybernetics, Inc., Rockville, MD, USA). Values of IOD measured for 18 pictures per immunofluorescence channel were grouped after the blinded analysis was completed by two independent researchers (NJP and ERD), and the results were assigned to the corresponding groups. The values of IOD are relatively expressed to the level measured in the synchronic group. 3Oxidative Medicine and Cellular Longevity 2.7. Postoperative Atrial Fibrillation Detection. Continuous ECG monitoring after surgery was performed in the cardiovascular intensive care unit for 48–96 hours to detect any new onset of atrial fibrillation. Arrhythmic events of at least 1min length were considered POAF when assessed by a monitoring system or using a 12-lead ECG in case of a symptomatic episode that required intervention. 2.8. Statistical Analysis. Qualitative variables are expressed as number and percentage. Quantitative variables are expressed as mean ± standard deviation (SD) if they are normally distributed and median with a range if they are not normally distributed. For qualitative data, the chi-square test is used. For nonnormally distributed data, the Mann–Whitney tests are used; for normally distributed data, the Student t-test is used. The receiver-operating characteristic (ROC) curve analysis is performed to determine the cutoffvalue of atrial variables as predictors of POAF. The area under the curve and 95% confidence interval (CI) are used to determine the parameters’incremental diagnostic value. Log-rank (Mantel Cox) test is used to assess the incidence of POAF. A pvalue below 0.05 was chosen as an indicative of statistical significance. The GraphPad Prism version 9.0.0, 2020 (GraphPad Software, San Diego, CA, USA, http://www.graphpad.com) was used for statistical analysis. 3. Results 3.1. Postoperative Atrial Fibrillation, Patient Characteristics, and Classification regarding Interatrial Synchrony. A total of 45 patients were included in the study. Forty-one underwent coronary artery bypass surgery, three aortic valve replacement, and one combined surgery. The mean age was 67:4±7:7years, and 69% were male. No patient was under treatment with digitalis or antiarrhythmics. This population was divided into synchronic and dyssynchronic patients based on the difference between the activation of the lateral right and left atrial walls determined from the echocardiogram (Omega). This was used as a factor related to the incidence of POAF. A time difference greater than 40 milliseconds had a sensitivity of 88.9% and a specificity of 55.6% for identifying the atrial fibrillation events, with an area under the curve of 0.728 (95% CI 0.575-0.850) (Figure 3(a)). Dyssynchronic patients suffered from a higher incidence of POAF. The median time of atrial fibrillation onset after surgery was 2.5 days (Figure 3(b)). All patients recovered sinus rhythm prior to discharge from hospital with the use of intravenous amiodarone. No electrical cardioversion was required. The receiver-operating characteristic curve (ROC) of atrial echocardiographic measurements had the following areas under the curve and cutoffvalues: right atrial area 0.796 (95% CI 0.650-0.902), cutoff17.6cm 2 ; left atrial volume index 0.742 (95% CI 0.590-0.861), cutoff29.86 mL/m 2 ; left atria contraction strain 0.645 (95% CI 0.488-0.782), cutoff 15.4%; left atrial reservoir strain 0.636 (95% CI 0.4790.774), cutoff30.7%. After adjusting the right atrial area, left atrial volume index, left atrial reservoir strain, and left atria contraction strain for the cutoffvalues, according to current guidelines, only left atrial volume index remained as predictors of postoperative atrial fibrillation (area under the curve: 0.681 95% CI 0.525-0.812), but the area under the curve was lower than the one obtained with the interatrial Conduit strain (𝜀CD) Contractile strain (𝜀CD) SR contractile (SRct) SR conduit (SRcd) Reservoir +30 +2 –10 0 –2 400 800 1200 1600 (ms) R-R gating DRT Atrial strain (%)Atrial strain (1/s) SR reservoir (SRr) strain (𝜀R) (a) (b) 𝜀R 𝜀CD 33 22 11 0 0200 400 882 ms 600 800 𝜀CT (c) 3 1.5 –1.5 –3 0 0200 400 882 ms 600 800 SRr SRcd SRct (d) 400 120 ms 600 800 DRT (e) B Right atria Left atria M R (f) Figure 1: Two-dimensional atrial strain curves. (a) Atrial strain and strain rate curves and determination of atrial mechanics according to an R-R wave-timed analysis. Deformation rate time (DRT) is determined from the beginning of the P wave in the electrocardiogram to the maximum deflection of the strain rate contractile phase (STct). (b) Four-chamber view of speckle tracking left atrial strain. (c) Atrial strain curves. The white, dashed, and dotted line shows the average strain curve for the six segments of the atria in that view. (d) Atrial strain rate curves. (e) Determination of DRT in a segment of left atria. The measurement agreed with the scheme in (a). (f) Schematic four-chamber view of how DRT can show the activation in time of different segments from both atria. B: base segment; M: medium segment; R: roof segment. 4 Oxidative Medicine and Cellular Longevity dyssynchrony [22–24]. These results supported dyssynchrony as the grouping variable for the comparisons throughout the study. Patients in the synchronic and dyssynchronic groups did not show differences in age, type of surgery, comorbidity, risk factors, or treatment received before surgery (Table 1). The average pump time of the patients was 118 ± 39 minutes, and the clamping time was 75 ± 24 minutes, with no significant differences between groups. During the study, there was only one death from a patient in the synchronic group. 3.2. Atrial-Related ECG. P wave duration in the ECG was 125 ± 15 ms and 118 ± 12 ms in the synchronic and dyssynchronic groups, respectively (p=0:128 by t-test). PR interval was longer in the synchronic group than in the dyssynchronic group (179 ± 32 ms vs. 160 ± 25 ms, respectively; p=0:044 by Mann–Whitney test). No significant differences in Bayes syndrome incidence were found between groups (p=0:985). 3.3. Echocardiography and Atrial Strain. Regarding general data from the echocardiograms, the mean ejection fraction of the left ventricle was 56:83 ± 15:36%. The mean indexed volume of the left atrium was 30:78 ± 9:04 mL/m 2 , and the area of the right atrium was 16:20 ± 3:36 cm 2 . The E/e’ratio of the left ventricle showed a value of 8:6±3:5, which indicated end-diastole pressures at the normal upper limit in most patients (Table 2). There was no other significant difference between the groups in echocardiographic findings, except for the left ventricular index mass. The analysis of the atrial speckle tracking strain showed no significant differences between groups in the deformation rate times corresponding to the activation of any of the 18 atrial segments (Table 3). The rest of the usual strain analysis also showed no differences between groups (Table 4). 3.4. Action Potentials and Proarrhythmic Triggers. Action potentials from dyssynchronous patients decreased by 3:9 ±1:9mV in amplitude and by 66 ± 13 V/s in the maximal upstroke velocity (Figures 4(b) and 4(c)). During the beginning of the upstroke of the action potential, we observed that a segment of the ascendent activation had a little nudge, like a foot that delayed the maximal upstroke occurrence. The resting membrane potential was hyperpolarized by 3:3±1:3mV in the atrial tissue samples from dyssynchronic patients. The action potential duration at 30% of repolarization increased by 4:0±0:7ms but markedly shortened when measured at 90% of repolarization by 107 ± 12 ms. Only action potentials from synchronic patients displayed a plateau and slow repolarization typical of a standard atrial action potential. Early and delayed afterdepolarizations were only observed in the dyssynchronic group. 3.5. Atrial Remodeling, Inflammation, and Nitrooxidative Stress. Cx40 lateralization increased by 9.81% (95% CI of difference 7.71-10.93) in samples from dyssynchronic patients with respect to synchronic ones (Figure 5). (a) +2 –2 400 800 1200 1600 (ms) Atrial SR (1/s) Synchrony time between lateral walls (ms) a (b) 56 ms 30 ms Right atria Right atria Left atria Left atria Dyssynchronic Synchronic (c) Dyssynchronic 1200 1500 1800 2100 56 ms –30 ms 2400 600 800 1000 1200 1400 Synchronic (d) Figure 2: (a) Longitudinal strain echocardiography in a four-chamber view with the Omega (ω) adaptation for the analysis technique of time activation between lateral walls. Yellow and blue dots show the measured segment of the lateral wall. (b) Measurement technique to determine differences between the activation time of two segments at the lateral atrial walls, from one maximal deflection strain rate contraction of a curve to the same point in the other curve. (c) Schematic visualization in a four-chamber view with an example of the determination of time difference in activation between the lateral wall of the right and left atria. (d) Example of strain rate from right and left atrial walls in synchronic and dyssynchronic patients. 5Oxidative Medicine and Cellular Longevity 0 0 20 40 60 80 100 20 40 60 80 100 RAA Interatrial dyssynchrony LA𝜀R LA𝜀CT LAVi 100 - specificity Sensitivity (a) 0246810 0 20 40 60 80 100 Days after surgery Postoperativeatrialfibrillation(%) Dissynchronic Synchronic p = 0.0416 (b) Figure 3: Receiver-operating characteristic curves (ROC) for atrial fibrillation incidence after cardiac surgery. (a) ROC curves for right atrial area (RAA), interatrial dyssynchrony, left atrial reservoir strain (LAεR), left atria contraction strain (LAεCT), and left atrial volume index (LAVi). (b) Postoperative atrial fibrillation incidence in patients with synchronic and dyssynchronic activation of the right and left atrial lateral walls. Table 1: Demographics and medication of patients before surgery. Variables Dyssynchronous (n=21) Synchronous (n=24)pvalue Demographics Age in years (n)68 ± 9 67 ± 7 0.262 Patient sex (male) 15 (71.4) 16 (66.6) 0.731 Type of surgery: coronary 20 (95.2) 21 (87.5) 0.559 Coronary disease 20 (95.2) 23 (95.8) 0.923 Diabetes mellitus 13 (61.9) 10 (41.6) 0.175 Hypertension 20 (95.2) 22 (91.6) 0.632 Dyslipidemia 19 (90.5) 20 (83.3) 0.482 Dilated cardiomyopathy 4 (19.0) 1 (4.2) 0.113 Chronic pulmonary obstructive disease 1 (4.8) 3 (12.5) 0.363 Chronic renal failure 3 (14.3) 1 (4.2) 0.234 Obesity 10 (47.6) 8 (33.3) 0.329 Stroke 0 1 (4.2) 0.344 Heart failure in last month 3 (14.3) 5 (20.8) 0.567 Smoker 2 (9.5) 4 (16.6) 0.482 Presurgical medications Aspirin use 21 (100) 24 (100) 1 βBlocker use 15 (71.4) 17 (70.8) 0.965 Statins 20 (95.2) 24 (100) 0.280 ACEI or ARA 15 (71.5) 14 (58.3) 0.360 Spironolactone or eplerenone 5 (23.8) 2 (8.3) 0.153 Furosemide 3 (14.3) 2 (8.3) 0.526 Data are expressed as number and (%), unless otherwise indicated. pvalues are calculated by Kruskal-Wallis test or chi-square test. ACEI: angiotensinconverting enzyme inhibitors; ARA: angiotensin II, type 1 receptor antagonists. 6 Oxidative Medicine and Cellular Longevity This lateralization was not colocalized with the increased signal of tumor necrosis factor-α(TNFα) seen in the dyssynchronic group (Figure 6). The TNFαwas mainly colocalized with Cx40 close to the intercalated discs. Nitrotyrosine is a relatively stable marker of nitroxidative stress that is formed by peroxynitrite interaction with tyrosine. The nitrotyrosine signal was clearly higher in the dyssynchronic group, especially around the blood vessels. However, it was diffusely observed in the tissue and the microvasculature (Figure 7). Cx43 increased in intercalated discs and also lateralized 14.6% (95% CI of difference 10.81-16.56) more in dyssynchronic patients (Figure 5), but both changes were unrelated to nitrotyrosine marks. The expression of K ATP channels assessed by the detection of its pore-forming unit Kir6.1 increased in atrial myocytes of dyssynchronic samples (Figure 8). K ATP channels usually aggregated at a vascular level in images of both synchronic and dyssynchronic patients. The action potential shortening and the hyperpolarization observed with the microelectrodes agrees with increased expression of this channel. 4. Discussion Our study found that interatrial dyssynchrony assessed by echocardiography was associated with electrical and structural atrial remodeling and the incidence of postoperative atrial fibrillation. The innovative measurement interatrial dyssynchrony was suitable to estimate noninvasively nitrooxidative and inflammatory substrate, as well as, an electrophysiological arrhythmogenic remodeling. Table 2: Comparative echocardiographic measurements in both groups. Echocardiographic variables Dyssynchronous Synchronous pvalue Atrial index volume (mL/m 2 ) 32.51 ±8.27 29.45 ±9.76 0.534 Right atrial area (cm 2 ) 17.07 ±3.54 15.57 ±3.13 0.694 Fey Simpson (%) 54.71 ±15.36 59.21 ±15.47 0.887 Mitral E wave velocity (cm/sec) 68.14 ±20.32 71.83 ±16.82 0.216 Mitral A wave velocity (cm/sec) 85.33 ±21.44 76.96 ±18.4 0.568 E/e ratio LV 8.83 ±3.83 8.53 ±3.44 0.542 E/e ratio RV 4.42 ±1.37 4.38 ±1.08 0.404 LV index mass (g/m 2 ) 138.29 ±52.97 108.33 ±33.61 0.021 Data are express as mean and SD. pvalues are analyzed by t-test. LA: left atria; RA: right atria; LVEF: left ventricular ejection fraction; LV: left ventricle; RV: right ventricle. Table 3: Deformation rate time per segment in both groups. Deformation rate time Dyssynchronous Synchronous pvalue Left atria Base septum segment 138.95 ±44.43 142.46 ±39.48 0.780 Medium septum segment 146.57 ±40.89 136.54 ±30.11 0.350 Roof septum segment 157.76 ±47.46 134.46 ±30.11 0.078 Base lateral segment 168.38 ±45.32 150.79 ±41.49 0.181 Medium lateral segment 169.48 ±41.77 149.67 ±42.23 0.122 Roof lateral segment 161.67 ±33.42 156.83 ±41.26 0.671 Base anterior segment 151.71 ±43.26 155.67 ±34.38 0.734 Medium anterior segment 154.05 ±41.87 154.54 ±35.02 0.966 Roof anterior segment 157.05 ±41.48 151.67 ±49.59 0.697 Base inferior segment 159.52 ±48.36 140.04 ±30.32 0.108 Medium inferior segment 154.24 ±51.01 144.42 ±28.44 0.422 Roof inferior segment 161.43 ±63.04 150.08 ±33.65 0.447 Base inferior lateral segment 153.29 ±49.10 145.38 ±35.49 0.535 Medium inferior lateral segment 150.90 ±44.43 148.04 ±31.73 0.803 Roof inferior lateral segment 133.62 ±45.81 151.54 ±27.04 0.112 Right atria Base lateral segment 138.29 ±62.95 135.17 ±42.17 0.844 Medium lateral segment 131.24 ±57.68 141.71 ±35.19 0.460 Roof lateral segment 138.81 ±62.89 143.88 ±35.68 0.737 Data are expressed as mean and SD. pvalues for analysis by unpaired t-test. 7Oxidative Medicine and Cellular Longevity 4.1. Atrial Dyssynchrony and Postoperative Atrial Fibrillation. Interatrial dyssynchrony is not a common risk factor for POAF. This is one of the first reports of a preoperative indicator of proarrhythmic tissue remodeling. Reservoir and contractile strain of the left atria have been previously related to the development of paroxysmal and chronic atrial fibrillation [25–27]. In this study, both were below the reference values agreed in the clinical guidelines [22]. This indicates more rigid atria, with less pump function, but without adifference between the two groups. One of the reasons for this absence of difference could be the way in which the patients were classified. We did not separate the groups according to the incidence of atrial fibrillation but to the presence of dyssynchrony determined by echocardiogram. Another reason for our patients having a better pump and reservoir function is that we did not include patients with mitral or tricuspid valve disease known for having larger atrial volumes, more rigid atria, and a clear tendency to develop atrial fibrillation. Also, one of our exclusion criteria was a previous history of atrial fibrillation. We found that interatrial dyssynchrony was a better predictor of postoperative atrial fibrillation than other atrial measurements. Atrial structural and functional remodeling has many causal factors such as oxidative stress, inflammation, fibrosis, and the normal process of aging. Previous studies have shown that left and right atrial enlargement predicts postoperative atrial fibrillation [28, 29]. All our patients had left atrial size below the cutoffvalues for atrial enlargement. Moreover, the atrial size was preserved in the dyssynchronic patients despite the higher left ventricle index mass and the slightly reduced conduit strain. Left ventricular hypertrophy is associated with higher end-diastolic left ventricular pressures, explaining the difference in the conduit left atrial strain, but other markers of elevated end-diastolic pressures like E/e’relation showed no differences. Right atrial size had no significant difference between groups but was slightly bigger in the dyssynchronic group. Evaluating the activation of the different segments of the atria did not show differences between the two groups. This supports the standardization guidelines that recommend the longitudinal analysis of the atria as a whole [22]. Additionally, segment by segment analysis takes a longer time and requires a more experienced operator. Nevertheless, several studies have already shown that intra-atrial dyssynchrony is related to atrial fibrillation development [30, 31]. So, this analysis may be a useful tool for the prediction of arrhythmias. The omega approach, presented here for the first time, is an adaptation of the technique that not only introduces the evaluation of the right atria but it also reduces the time for analysis. This facilitates the interatrial dyssynchrony evaluation with significant potential for arrhythmogenic substrate identification in the preoperative setting. Structural remodeling produces changes in atrial function. These changes can be accelerated in many disease processes, like hypertension, diabetes mellitus, and ischemic heart disease. These changes can reflect modifications in structural proteins that produce atrial electrical remodeling, which, in turn, could be the cause for longer times of atrial mechanical activation. The results of our study support that atrial dyssynchronic activation is related to both electrical and structural changes, without an apparent influence of other risk factors. 4.2. Atrial Dyssynchrony, Action Potentials, Connexins, and K ATP Channels. Dyssynchronic patients presented abnormalities in the upstroke of the action potentials. In multicellular preparations, both Na + channels and connexins contribute to phase 0 of the action potentials. The foot and the delay in the upstroke observed in dyssynchronic patients have been previously associated with connexin alterations (see Figure 3(a)) [32]. Connexin expression, conductance, and location can influence the initial part of the electrical activation [33]. The lateralization of Cx40 and Cx43 shown in Figure 5 could support these findings at the beginning of the action potentials. Still, the concurrent increase in Cx43 at the intercalated discs goes against this idea, but agrees with previous reports that reduce the link with action potential morphology [34]. The reduced amplitude and maximal rate of depolarization indicate an impairment in the sodium currents. Therefore, both sodium channels and connexins can Table 4: Comparative atrial strain measurements in both groups. Atrial strain variables Dyssynchronous Synchronous pvalue Reservoir LA strain (%) 25.34 ±8.7 31.30 ±10.99 0.433 Conduit LA strain (%) 11.62 ±5.08 15.61 ±6.99 0.063 Contractile LA strain (%) 14.58 ±5.8 16.37 ±5.57 0.963 Strain rate LA reservoir (1/s) 1.16 ±0.51 1.44 ±0.58 0.867 Strain rate LA conduit (1/s) -1.13 ±0.60 1.63 ±1.45 0.419 Strain rate LA contraction (1/s) -1.01 ±0.61 -1.15 ±0.66 0.623 Reservoir RA strain (%) 46.81 ±21.29 52.50 ±19.13 0.623 Conduit RA strain (%) 20.62 ±12.59 25.56 ±14.26 0.787 Contractile RA strain (%) 17.92 ±13.19 26.73 ±14.69 0.598 Strain rate RA reservoir (1/s) 1.96 ±0.83 2.04 ±0.59 0.336 Strain rate RA conduit (1/s) -1.52 ±1.02 -2.10 ±0.86 0.605 Strain rate RA contraction (1/s) -1.22 ±0.74 -1.68 ±1.31 0.080 Data are expressed as mean and SD. pvalue values for analysis of t-test. LA: left atria; RA: right atria. 8 Oxidative Medicine and Cellular Longevity contribute to interatrial dyssynchrony because they are the major determinants of electrical impulse propagation between cardiomyocytes. K ATP channels could explain the action potential shortening and the hyperpolarization, and these features can worsen under stressful situations like ischemia and reperfusion. Our results bring a new potential substrate to the complex electrophysiology of the atrial tissue. The subunit Kir6.1 is an inward rectifier current mainly expressed in the vasculature [16]. Here, we report the expression in human atrial cardiomyocytes of dyssynchronic patients. The activation of these channels reduces the refractory period, thus facilitating reentrant currents that could lead to postoperative atrial fibrillation. K ATP channels are particularly sensitive to the metabolic and oxidative cellular conditions [35]. In the context of cardiovascular surgery, their properties make them attractive candidates for potential preventive antiarrhythmic therapies. The afterdepolarizations observed in the dyssynchronic group could be related to alterations in calcium handling and leaks from the sarcoplasmic reticulum through the ryanodine receptors. These alterations have already been described as associated with a more oxidized substrate in patients undergoing coronary artery bypass grafting [36]. Dyssynchrony can promote stretching of the cells during the repolarization phase of shortened action potentials, leading to mechanoelectric feedback capable of early afterdepolarization induction [37]. Transmembrane potential (mV) Time (ms) 0 100 200 300 400 500 Synchronic Dyssynchronic –80 –60 –40 –20 0 20 40 (a) 0 20 40 100 120 Amplitude (mV) 0 100 200 300 400 𝛥V/𝛥tmax (V/s) –80 –60 –40 –20 0 Resting potential (mV) * Dyssynchronic Synchronic Dyssynchronic Synchronic Dyssynchronic Synchronic Dyssynchronic Synchronic 0 5 10 15 20 25 Duration at 30% (ms) 0 100 200 300 400 Duration at 90% (ms) ⁎⁎ ⁎ ⁎ (b) Transmembrane potential (mV) –80 –60 –40 –20 0 20 40 (c) Figure 4: Action potentials and arrhythmic triggers in interatrial dyssynchronic patients. (a) Averaged action potentials from three different patients from each group; (b) quantification of action potential amplitude, resting membrane potential, maximum upstroke velocity (ΔV/Δtmax), and the action potential duration at 30 and 90% of the repolarization from both groups. ∗p<0:01 by t-test; (c) arrhythmogenic triggers in 2 seconds traces from two dyssynchronic patients. The blue arrows mark delayed afterdepolarizations, which are very frequent in dyssynchronous patients. Green arrowheads indicate triggered events from early afterdepolarizations. 9Oxidative Medicine and Cellular Longevity