Supporting Methods Study Patients and Tissue Sample Collection

One hundred twenty-four patients who underwent elective coronary artery bypass grafting (CABG) at the Karolinska University Hospital, Solna from 2002 to 2005 were included in the STAGE study. Exclusion criteria were severe diseases (e.g., cancer, kidney disease, and chronic systemic inflammatory diseases) other than coronary artery disease (CAD). The number of surgeons collecting tissue samples during the CABG was restricted to four. A strict protocol was used to standardize the collection procedure, including a specific order for obtaining each tissue sample in relation to the start of surgery. Anesthesia was standardized to keep systolic blood pressure <150 mm Hg throughout the operation. The time of extraction of each biopsy, deviations from the protocol, and no routine events were noted. A total of five tissue samples were obtained. Skeletal muscle (~0.5 g) was taken from the medial border of the apical rectus abdominus muscle close to the incision in sternum. Visceral fat (~1 g) was taken from the mediastinal tissue anterior to the pericardium and great vessels. The internal mammary artery (IMA) was dissected from the inside of the left chest wall, and 1 cm of the distal part of the artery was collected. Aortic wall samples were obtained from the whole punch used to create the proximal vein graft anastomoses at the aortic root during the operation. Liver tissue (3 mm diameter, 0.05 g) was taken from the very inferior border of the left liver lobe at the end of the operation. This part of the liver was easily accessed after the peritoneum was opened through a small incision in the diaphragm a few centimeters below the xiphoid process. The incisions in the liver and diaphragm were sutured after removal of the biopsy. All tissue samples were taken without use of cautery, and none of the patients suffered from bleeding at the biopsy sites or other complications related to the tissue sampling. The tissue samples were rinsed with RNAlater (Qiagen) to remove blood and immediately (~10 seconds) put into vials