Ascending Aorta

A schematic overview of the myocyte isolation procedure is shown in Figure 2. An expanded description of the procedure, accompanied with images and videos, and complete materials list is available in the Online Data Supplement, alongside full details of additional methods applied in this study (Appendix A-ix). All animal work was undertaken in accordance with Singapore National Advisory Committee for Laboratory Animal Research guidelines. Relevant national and institutional guidelines and regulations must be consulted before commencement of all animal work.
Buffers and media were prepared as detailed in Appendix D. EDTA, perfusion, and collagenase buffers were apportioned into sterile 10 mL syringes, and sterile 27 G hypodermic needles were attached (Online Figure IA).
C57/BL6J mice aged 8 to 12 weeks were anesthetized, and the chest was opened to expose the heart. Descending aorta was cut, and the heart was immediately flushed by injection of 7 mL EDTA buffer into the right ventricle. Ascending aorta was clamped using Reynolds forceps, and the heart was transferred to a 60-mm dish containing fresh EDTA buffer. Digestion was achieved by sequential injection of 10 mL EDTA buffer, 3 mL perfusion buffer, and 30 to 50 mL collagenase buffer into the left ventricle (LV). Constituent chambers (atria, LV, and right ventricle) were then separated and gently pulled into 1-mm pieces using forceps. Cellular dissociation was completed by gentle trituration, and enzyme activity was inhibited by addition of 5 mL stop buffer.
Cell suspension was passed through a 100-μm filter, and cells underwent 4 sequential rounds of gravity settling, using 3 intermediate calcium reintroduction buffers to gradually restore calcium concentration to physiological levels. The cell pellet in each round was enriched with myocytes and ultimately formed a highly pure myocyte fraction, whereas the supernatant from each round was combined to produce a fraction containing nonmyocyte cardiac populations.
CM yields and percentage of viable rod-shaped cells were quantified using a hemocytometer. Where required, the CMs were resuspended in prewarmed plating media and plated at an applicationdependent density, onto laminin (5 μg/mL) precoated tissue culture plastic or glass coverslips, in a humidified tissue culture incubator (37°C, 5% CO₂). After 1 hour, and every 48 hours thereafter, media was changed to fresh, prewarmed culture media.
The cardiac nonmyocyte fraction was collected by centrifugation (300g, 5 minutes), resuspended in fibroblast growth media, and plated on tissue-culture treated plastic, area ≈ 23 cm² (0.5× 12-well plate) per LV, in a humidified tissue culture incubator. Media was changed after 24 hours and every 48 hours thereafter.

For participants in the COPDGene trial, analysis of the lung parenchyma and airways was performed on volumetric CT scans of the chest obtained without the administration of contrast material. Parenchymal analysis was performed with the use of the Slicer software package (www.Slicer.org), and airway analysis was performed with the use of Volumetric Information Display and Analysis (VIDA) Pulmonary Workstation 2 software (www.vidadiagnostics.com). Emphysema was defined by a CT attenuation value of less than –950 Houns-field units on inspiratory scans, and gas trapping was defined by a CT attenuation of less than –856 Hounsfield units on expiratory scans. We assessed airway disease by measuring the wall-area percent ([the bronchial wall area ÷ total cross-sectional area of the wall and lumen] × 100), using the average of six fourth-generation airways, as reported previously.^{19 (link)}Vascular measurements in the COPDGene and ECLIPSE cohorts were performed on baseline CT scans by an investigator who was unaware of the participants’ clinical characteristics. Measurements were made from axial CT images with the use of inspiratory acquisitions with digital imaging and communications in medicine (DICOM) software (OsiriX DICOM Viewer, version 4.0, 32-bit; www.osirix-viewer.com). The interpreter measured the diameter of the main pulmonary artery at the level of its bifurcation and measured the diameter of the ascending aorta in its maximum dimension using the same images, as shown in Figure 1.