Extracellular low pH affects circadian rhythm expression in human primary fibroblasts

Abstract

Circadian rhythm is a fundamental biological system involved in the regulation of various physiological functions. However, little is known about a nature or function of circadian clock in human primary cells. In the present study, we have applied in vitro real time circadian rhythm monitoring to study human clock properties using primary skin fibroblasts. Among factors that affect human physiology, slightly lower extracellular pH was chosen to test its effects on circadian rhythm expression. We established human primary fibroblast cultures obtained from three healthy subjects, stably delivered a circadian reporter gene Bmal1-luciferase, and recorded circadian rhythms in the culture medium at pH 7.2 and 6.7. At pH 7.2, robust and sustained circadian rhythms were observed with average period length 24.47±0.03h. Such rhythms were also found at pH 6.7; however, period length was significantly shortened to 22.60±0.20, amplitude was increased, and damping rate was decreased. The effect of exposure to low pH on the period length was reversible. The shortened period was unlikely caused by factors affecting cell viability because cell morphology and MTT assay showed no significant difference between the two conditions. In summary, our results showed that the circadian rhythm expression is affected at pH 6.7 in human primary fibroblasts without affecting cell viability.