A study in the American Heart Association Journal of Arteriosclerosis, Thrombosis, and Vascular Biology has found that acetylcholine (ACh) treatment is effective in protecting human umbilical vein endothelial cells (HUVECs) from hypoxia/reoxygenation (H/R) injury by reducing interactions between mitochondria and the endoplasmic reticulum (ER). The goal of this study was to investigate the role of ER-mitochondria calcium crosstalk in epithelial cell recovery from H/R damage, a common result of chronic heart failure and other conditions involving a dysfunctional circulatory system. The study also seeks to define the protective effects of ACh, which has been documented to have antioxidative and anti-inflammatory effects in cardiomyocytes, but has yet to have records on how it affects calcium overload (a main factor in H/R damage) in endothelial cells.

To begin the study, the researchers decided to arrange a time course displaying how calcium levels in oxygen-deprived endothelial cells typically responded to reoxygenation, without any presence of acetylcholine. They used the calcium indicator Cal-520^® to track calcium flux for up to 24 hours after reoxygenation of their HUVECs, which had been subjected to 8 hours of hypoxia. Through this initial day-long interval of reperfusion, the researchers determined the peak difference in calcium level to be at 2 hours after the start of reoxygenation. This 2 hour time point was noted and used in subsequent calcium monitoring experiments under this study as the point of highest calcium level and potential overload. The researchers would then proceed to compare this information to calcium level data acquired with HUVECs under the treatment of acetylcholine, and continue exploring calcium dynamics in relation to mitochondria-ER interplay.

To acquire their (2 hour) baseline indicating when calcium levels were highest upon reoxygenation, the study had to utilize a calcium dye that would remain emitting a detectable fluorescence even 24 hours after application. With that requirement known, the selected Cal-520^® is one of the best calcium dyes for such situations. Unlike other more popular dyes like Fluo-4, which generates a signal that fades away relatively quickly, Cal-520^® is enhanced for significantly improved intracellular retention qualities that make it especially suitable for long-duration calcium detection and imaging.