Two-hundred eighteen patients undergoing elective on pump CABG between August 2016 and February 2018 were enrolled in this study. All patients were informed and gave their written consent to participate. The study was approved by the University Hospital ethics committee (IRB approval: 143/17-sc 6.10.2017). Patients with valve pathologies, reoperations, emergency indications or pulmonary hypertension of any origin were excluded.
All patients received a routine preoperative echocardiographic examination (Philips Epiq 7, Philips Health System, Hamburg, Germany). Measurements and analysis were performed by trained echocardiography technician, blinded to patients. We measured left and right atrial end-systolic areas, systolic pulmonary artery pressure (sPAP), TAPSE, left ventricular ejection fraction (LVEF), according to echocardiographic guidelines for assessment of right heart function (10). We divided the patients in two groups: group 1 with TAPSE values ≥ 20 and group 2 with TAPSE value < 20 mm and compared experimental force values from the right atrial auricle as well as all baseline clinical characteristics among both groups. All data including clinical findings and preoperative medication were recorded pseudonymously in a departmental database. Prior to induction of anesthesia, preoperative routine blood samples were drawn from all patients and immediately sent to the laboratory to be stored at -80 °C.
Preoperative clinical chemistry included creatinine, albumin, glomerular filtration rate (GFR) and N-terminal pro brain natriuretric peptid (NT-proBNP). We performed enzyme-linked immunosorbant assay (ELISA) for the following markers of fibrosis and inflammation, taken from serum blood samples of the patients. All ELISAs were conducted with the ELISA reader Tecan Infinity Pro (Tecan Group Ltd., Switzerland): Galectin, Transforming Growth Factor ß (TGFß), N Acyl- Symmetric Dimethylarginine (N-Acyl-SDMA), Arginine, Asymmetric Dimethylarginine (ADMA) (Quantikine Elisa, DGD150, R&D Systems, Bio-Techne, Wiesbaden, Germany) and Pentraxin (Pentraxin 3 Human ELISA, RD191477200R, BioVendor, Czech Repuplic).
Experimental set-up:
We measured the calcium-induced force of right atrial skinned human fibers as previously described (8, 9, 10). We resected the RAA for venous cannulation of extracorporal circulation. This RAA tissue was used for experimental calcium-induced force measurements. Briefly, the intraoperatively resected tissue was transported in ice-cold oxygenated cardioplegic solution, containing BDM (Sigma Aldrich Chemie GmbH, Steinheim, Germany). For the skinning procedure, the trabeculae were excised, immersed in Triton-X solution (Sigma Aldrich Chemie GmbH, Steinheim, Germany) and processed for sarcolemma removal
The trabeculae were then cut in to small bundles for the experimental cycle. The experimental set-up has been previously described (8, 9, 10). Briefly, the bundles were fixed between two forceps. A force transducer is connected to one of the forceps for recording any calcium-induced length changes. The bundles were then immersed increasing calcium concentrations. The calcium concentration is displayed as logarithmic calcium concentration (pCa), which is a negative decadic logarithm. We started with the lowest calcium concentration at pCa 7.0 and increasing at 6.5, 6.0, 5.75, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.75, 4.52.
Statistical Analysis
Demographic variables, comorbidities, blood tests, ventricular function, operative data, 30-day, and mid-term outcomes in patients undergoing elective CABG were compared between patients with TAPSE ≤ 20 vs. ≥ TAPSE. Continuous and categorical variables were compared using t-test and Fisher’s exact test, respectively. Multivariable logistic regression models were constructed to identify factors associated with reduced right heart function (TAPSE ≤ 20 mm). Candidate variables include age, sex, Euro Score II, diabetes mellitus (DM), preoperative AF, glycated haemoglobin (HbA1c), NTPro-BNP), GFR, albumin, ADMA, Pentraxin-3, right end-systolic area, sPAP, TAPSE, left atrial diameter, LVEF, cardiopulmonary bypass (CPB) time, incision-suture time, ventilation time, prolonged ventilation.
Overall survival estimates were obtained by Kaplan-Meier method. The groups were compared with a log-rank test. Cox proportional hazards regression was used to discriminate risk factors associated with time of death. Model assumptions, including proportional hazards assumption, linearity, and normality were evaluated. All estimates are provided with 95% confidence interval (CI). Multiple imputation, analysis and pooling of the results for missing continuous variables were performed reporting mean value of the study cohort for that parameter (missing: 62 [27.3%] for FEV1, 30[13,2%] for NT Pro BNP 29[12,8%] for CRP, 9[4,0%] for GFR, 9[4,0%] for HbA1c, 9[4,0%] for Albumin, 60[26,4%] for Galectin, 50[22,0%] for TGFß1, 53[23,3%] for N Acyl-SDMA, 60[26.4%] for arginine,56[24.7%] for ADMA, 49[21,6%] for Pentraxin-3, 29[12,8%] for RA area, 29[12,8%] for LA area,17[7,5%] for LVEF, 63[27,8%] for sPAP and a dummy variable was used to indicate replacement of missing data. Appropriate models were selected with appropriate candidate variables included with respect to relevance to right heart failure and missing completely at random assumption. Five imputed datasets were used for efficiency and reproducibility of the results. Cox regression using complete cases, single mean and multiple imputation were performed and compared with patients without missing data. Models including only patients without missing data yielded no significant change in overall results.
Candidate variables considered for the multivariable analyses were those detected by univariate models as having a p < 0.05 or suggestive trend toward association (p = 0.05–0.20) predictive of abnormal TAPSE and mortality; retention of variables in the multivariable model was set at p < 0.05. To discriminate independent risk factors, multivariable modeling was performed with methods of stepwise selection, with entry/stay criteria of 0.1/0.1 and exclusion of highly intercorrelated, redundant explanatory variables with r values set at > 0.70, with TAPSE groups and candidate variables all competing for entry into a final model. The final model included all independent risk factors for reduced right heart function and mortality meeting these criteria. Multivariable linear regression was used to adjust for significant independent variables for reduced right heart function (TAPSE ≤ 20 mm) and multivariable logistic regression was used to adjust for significant variables for POAF. All estimates are provided with 95% confidence intervals (CI). Probability p-values were considered as statistically significant when p < 0.05. All analyses were performed using SPSS 25 (IBM; Amonk, New York, USA).
Clinical Outcomes
An endpoint of all-cause mortality was considered to be the primary endpoint. Death notification was confirmed by medical record (evidence of life or death).