The aim of the present study is to investigate intra-operative changes in markers of myocardial injury during ischemia and reperfusion, comparing three methods of myocardial protection; St. Thomas' cold crystalloid cardioplegia, Calafiore warm blood cardioplegia, or modified del Nido cold blood cardioplegia in routine coronary artery bypass grafting procedures.

Thirty consecutive patients undergoing elective primary coronary artery bypass grafting will be enrolled in this study. Patients were excluded from this study if they had suffered from previous myocardial infarction, had reduced ejection fraction or were undergoing combined or repeated surgery. Patients will be randomly assigned to undergo surgery using one of three methods for myocardial protection: Group 1: St. Thomas' cold crystalloid cardioplegia (consisting of 750 ml of normal saline solution, 110 - 147 mmol/L of sodium chloride, . 16 - 20 mmol/L of potassium chloride, 16-32 mmol/L of magnesium chloride, 1.2-2.4 mmol/L of Calcium Chloride, 10 mmol/L of Sodium Bicarbonate). This solution will be administered as a 1-liter bolus at 3-4 degrees C following application of the aortic cross-clamp, and repeated as a 500 ml bolus if cross-clamp time extended beyond 30 minutes. Group 2: Calafiore intermittent antegrade warm blood cardioplegia (consisting of normothermic pump blood, potassium chloride 2 mEq/ml and magnesium sulfate 50%). This cardioplegic solution will be administered at 37 degrees C following application of the aortic cross-clamp and repeated after completion of each distal coronary anastomosis. • Group 3: Modified del Nido intermittent antegrade cold blood cardioplegic solution (consisting of 750 ml of lactated Ringer solution, 200 ml of pump blood, 13 ml of sodium bicarbonate 8.4%, 16 ml of mannitol 20%, 4 ml of magnesium sulfate 50%, 13 ml of lidocaine 2% and 13 ml of potassium chloride 2 mEq/ml). This solution will be administered as a 1-liter bolus at 3-4 degrees C following application of the aortic cross-clamp, and repeated as a 500 ml bolus if cross-clamp time extended beyond 60 minutes.

Collection of blood samples and myocardial biopsies:

Peripheral blood samples will be collected immediately prior to surgery, 15 minutes after removal of cross-clamp, and at 6 hours and 24 hours postoperatively. Myocardial biopsies (4-14 mg wet weight) will be taken using a Tru-cut needle from the apex of the left ventricle in every patient as follows: Biopsy 1: "Resting specimen", immediately after beginning of extracorporeal circulation. Biopsy 2: "Ischemic specimen", 30 minutes after application of the aortic cross-clamp. Biopsy 3: "Reperfusion specimen", 20 minutes following the removal of aortic cross-clamp. Each specimen will be immediately snap-frozen in liquid nitrogen (-185 degrees C) until processing.

Extracellular vesicles-derived miRNA (miR-499, miR-208b, miR-133a, EV-miR-1) will be measured, and release kinetics of EV-miRNAs will be determined and compared between the three groups, including correlation between EV-miRNA and cross-clamp time.

Complement system activation (sC5b-9, C5a, C3a) will be determined and compared between the three groups, including whether blood cardioplegia reduces complement activation and the relation between cardioplegia temperature and complement activation.

Neutrophil extracellular Traps (dsDNA) will be determined and compared between the three groups, including the effect of hypothermia on NETs formation.

Inclusion Criteria:

• All consecutive patients undergoing elective primary coronary artery bypass grafting will be enrolled in this study.

Exclusion Criteria:

• Patients will be excluded from this study if they had suffered from previous myocardial infarction, had reduced ejection fraction or were undergoing combined or repeated surgery

Data will be uploaded on (figshare) data repository site

Information will be made available in March 2027 and will be available permanently

open to all