An in vitro model of coronary artery disease and the changes in intracellular calcium regulation during its progression.

Abstract

Ca2+ is a ubiquitous second messenger and is tightly regulated in coronary smooth muscle (CSM) cells. In coronary artery disease (CAD) Ca2+ regulation will change as well as the CSM phenotype. CSM will dedifferentiate from a contractile to a synthetic/proliferative phenotype, which will lead to neointimal thickening and CAD progression. We studied these changes by inducing CAD progression in vitro via an organ culture model. Healthy coronary rings were harvested from lean Ossabaw miniature swine, isolated under sterile conditions, and stored at 37°C in low glucose Dulbecco's Modified Eagle's Medium (DMEM) + 1% penicillin/streptomycin for up to 11 days. On the day of collection (day 0), tissue was used for histology and fura‐2 fluorescence Ca2+imaging experiments. The same was performed on days 4, 7 and 11 to assess any changes over this time period. Histology supported our hypothesis of CSM dedifferentiation by demonstrating neointimal thickening, increased collagen deposition, and loss of structural organization in the tunica media. During fura‐2 experiments, cells were exposed to 80 mM K+ to assess voltage‐gated Ca2+ channel (VGCC) activity and 5 mM caffeine to assess ryanodine receptor (RyR)‐sensitive sarcoplasmic reticulum (SR) Ca2+ store release. A significant decrease in VGCC activity was consistent with CAD progression and CSM dedifferentiation (p<0.05). There was a trend toward increased SR Ca2+ release in cells from days 4 and 7 followed by a significant decrease by day 11 (p<0.05). This in vitro model of CAD progression mimics the same trends we observed from our in vivo models of CAD.