Ethical Statement
This study was approved by the Institutional Review Board of the Beth Israel Deaconess Medical Center. All methods were carried out in accordance with relevant guidelines and regulations. The study commenced in August 2019 and completed data collection in August 2021. Data involving human research participants was de-identified and performed in accordance with the Declaration of Helsinki. Furthermore, all animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at BIDMC and adhered to the National Institutes of Health guidelines for the care and use of laboratory animals under the Animal Welfare Act and followed ARRIVE guidelines.
Human Study Design
Inclusion and Exclusion Criteria
Patients undergoing elective heart surgery (coronary artery bypass graft surgery and aortic valve replacement surgery) that required use of CPB were prospectively recruited in this study over a span of three years from August 2018 to August 2021. Patients under the age of 18 or patients undergoing emergency heart surgeries were excluded from the final patient cohort. All patients provided written informed consent prior to enrollment in the study.
Study Variables
Data was manually extracted for 27 relevant variables. Key demographic variables included age, gender, and BMI. Patient comorbidities included hypertension, diabetes mellitus, asthma, hypothyroidism, anti-phospholipid syndrome, and history of heart failure, coronary artery disease, transient ischemic attack, and renal failure. Intra-operative variables such as bypass time and clamp time were also collected. Post-operative outcomes of interest included diastolic dysfunction, atrial fibrillation, cardiac arrest, reintubation, supplemental oxygen used, renal failure (2.5x increase in serum creatinine from baseline or the need for dialysis), and hospital length of stay. Myocardial blush grade (MBG) is used as an angiographic assessment of myocardial reperfusion.
Additionally, all patients underwent a pre-defined echocardiography protocol based on the 2016 American Society of Echocardiography Guidelines for the assessment of diastolic dysfunction. Diastolic dysfunction was defined as two or more abnormal left ventricular filling indices based on e’, E, their ratio (E/e’), LA volume index (LAVI), and peak tricuspid regurgitation jet velocity.
Sample collection
Ten mL of blood was collected through a radial arterial catheter just before induction of patients. Pre-operative right atrial tissue was harvested after median sternotomy and before administration of antegrade cardioplegia. Serum was separated from blood after collection by centrifuging blood for 15 minutes at 2000 G. Post-operative right atrial tissue sample was harvested from between the purse strings during removal of the venous cannula. Samples (50g and 100g) were snap frozen in liquid nitrogen and subsequently stored in a secure -80oC freezer.
Western Blotting
Tissue lysates were prepared through homogenization of atrial tissue with
commercially prepared RIPA buffer. For electrophoresis, 30μg of protein was loaded and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking, membranes were incubated with CD39 (Invitrogen, PA5-80587), CD73 (cell signaling, 13160S), adenosine receptor 2A (ADORA2A) (abcam, ab3461), adenosine receptor 2B (ADORA2B) (abcam, ab40002, cytochrome C (cell signaling, 4280T), cyclooxygenase IV (COX IV) (cell signaling, 4850T), superoxide dismutase 1 (SOD1) (cell signaling, 4266T).
HRP-linked secondary antibody and chemiluminescent substrates were used to visualize signal strength of membranes using Chemi Doc. Image J (National Institutes of Health, Bethesda, MD) was used to perform densitometry analysis for quantification of signal intensity. Western blot images were quantified and normalized using densitometry of housekeeping proteins glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and/or betaactin.
Immunohistochemistry & Serum Assays
Immunohistochemical staining was performed on formalin fixed, paraffin embedded human atrial tissue sections. After serum blocking, the slides were stained for fibrosis with Direct Picosirius stain after slide section (Sigma, Cat # 365548). P2X7 distribution in atrial tissue was also performed on sectioned tissues (Invitrogen, Cat # PA5-28020) Serum adenosine deaminase activity was measured using Sigma Aldrich ADA colorimetric assay (EPI023). Brain-natriuretic peptide (BNP) levels were quantified using (Sigma-Aldrich, RAB0386).
Human and Mouse Serum Cytokine
Chemokine 65-Plex Discovery AssayTM (Eve Technologies, Calgary, Canada), a cytokine array (based on Luminex's xMAP technology, Austin, TX), allows for the quantification of 65 inflammatory cytokines, chemokines, and growth factors (endothelial growth factor/eotaxin/fibroblast growth factor–2/ Fit-3L/fractalkine/granulocyte colony-stimulating factor/granulocyte-macrophage colony-stimulating factor [GM-CSF]/GRO/interferon [IFN]α2/IFNγ/interleukin [IL]-1α/IL-1β/IL-1ra/IL-2/IL-3/IL-4/IL-5/IL-6/IL-7/IL-8/IL-9/IL-10/IL-12(p40)/IL-12(p70)/IL-13/IL-15/IL-17/IP-10/monocyte chemotactic protein [MCP]-1/MCP-3/macrophage-derived chemokine/macrophage inflammatory protein [MIP]-1α/MIP-1β/platelet-derived growth factor[PDGF]-AA/PDGF-AB+BB/regulated on activation, normal T expressed and secreted/sCD40K/sIL 2Rα/tumor growth factor α/tumor necrosis factor [TNF]α/TNFβ/vascular endothelial growth factor/6Ckine/B-cell attracting chemokine 1/cutaneous T cell attracting chemokine/epithelial neutrophil activating peptide 78/eotaxin-2/eotaxin-3/I-309/IL-16/IL-20/IL-21/IL-23/IL-28a/IL-33/leukemia inhibitory factor/MCP-2/MCP-4/MIP-1d/stromal cell-derived factor-1 α+β/SCF/TARC/TPO/TRAIL/TSLP). Assay sensitivities range from 0.1 to 96.5 pg/mL. 10 samples of each group of human serum (male and female) and mouse serum (control, high fat diet, ovariectomized, high fat diet and ovariectomized) serum was sent to the Eve technologies which analyzed and returned fluorescent intensity readings for each sample.
Angiogenesis Antibody Array
Analysis of proangiogenic cytokine synthesis was performed using an antibody array (ab134000; Abcam). The assay was performed in accordance with the manufacturer’s instructions using serum totally 100 μg/ml of total protein per sample from a total group involving 16 samples. Membranes were visualized using the ChemiDoc Imaging System (Bio-Rad, Hercules, USA).
Mouse Study Design
40 Female C57BL/6 mice were obtained from Charles River Labs (CRL, Wilmington, MA). Six-week-old mice were housed in box cages, maintained on a 12:12-h light-dark cycle, and fed for 24 weeks either a normal diet (LFD: D09100304; 10% of total calories from fat, which consists of 5.5% of total calories from soybean oil and 4.5% kcal from lard) or High-Fat Diet (60% of total calories from fat, which consists of 5.5% from soybean oil and 54.5% from lard) purchased from Research Diets (D09100310i, New Brunswick, NJ). At 12 weeks, twenty mice underwent ovariectomy (OVX) under sterile conditions at CRL and monitored appropriately. Ten of these mice were continually fed a high-fat diet (HFD). There were four groups in total, each consisting of 10 mice: a control (wild type), HFD, OVX and OVX+HFD group. At 22 weeks they were transferred to our care at Beth Israel Deaconess Medical Center (Boston, MA)
At week 24, animals were anesthetized with isoflurane at 2.0%, weighed, assessed for cardiac function (ejection fraction, left ventricular systolic volume, left ventricular diastolic volume, fractional shortening) by trained echocardiographers using Vevo 2100 (Fujifilm VisualSonics, Inc), and then euthanized. Whole blood was collected by terminal cardiac puncture, placed in heparin containing tubes, and centrifuged for plasma separation. Following euthanasia, cardiac tissue was rapidly excised and processed. The samples were snap frozen in liquid nitrogen and stored in a -80 oC freezer. Tissue lysates were prepared through homogenization of whole cardiac tissue in RIPA buffer.
Western Blotting
Tissue lysates were prepared through homogenization of atrial tissue with commercially prepared RIPA buffer. 15ug of protein was loaded for electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking, membranes were incubated with CD 73 (cell signaling 13160), and CD 39 (cell signaling 14481). Forkhead box O3A (FOXO3A) (abcam ab47285), Manganese Superoxide Dismutase (MnSOD) (abcam ab68155), B-cell lymphoma-extra-large (Bcl-XL) (abcam ab32370), Adenosine Deaminase (ADA) (cell signaling 65184s), Leptin (Invitrogen, PA1-051).
HRP-linked secondary antibody and chemiluminescent substrates were used to visualize signal strength of membranes using Chemi Doc. Image J 3.0 (National Institutes of Health, Bethesda, MD) was used to perform densitometry analysis for quantification of signal intensity. Western blot images were quantified and normalized using densitometry of housekeeping proteins glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and/or beta Actin.
Assay
Mouse serum was extracted from the blood of the forty mice as detailed prior. Extracellular adenosine levels were determined enzymatically with the help of a fluorometric adenosine assay (abcam, Cat# ab211094) performed according to the manufacturer's instructions.
Statistical Analysis
Data analysis was performed using SPSS (Version 27, IBM Corporation) and/or Prism GraphPad 9.0 (GraphPad Software, Inc., La Jolla, CA, USA). A descriptive analysis of all demographic variables including features of hospital stay was conducted. Shapiro-Wilk test for normality (p > 0.05) was utilized to assess all continuous demographic and echocardiographic variables to satisfy the assumption of normality between genders. For normally distributed data, mean ± standard deviation was used to describe central tendencies while median (interquartile range) was used to describe central tendencies for skewed data. Independent sample t-test was used to compare means between male and female patients. To assess differences in dichotomous variables, Pearson’s chi-squared test was utilized. A p-value less than 0.05 was considered statistically significant.
For mice and cytokine data, independent sample t-test for two independent groups was used. Results of western blotting, immunohistochemistry and quantitative assays were compared between pre- and post-CPB patients using an independent sample t-test unless stated otherwise. Scatter plots were created to represent individual data points.