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 ± 0.03 h. 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.

Figure 1

Altered circadian rhythm expression at pH 6.7 in human primary skin fibroblasts. Fibroblasts were cultured in culture medium either at pH 7.2 or pH 6.7, and circadian rhythms were recorded for six days. (A) Left. Robust and sustained Bmal1 promoter driven bioluminescence rhythms were observed at both pH 7.2 and pH 6.7. Right. At pH 6.7 the period length was significantly shortened. (B) The damping rate was shortened and amplitude was increased at pH 6.7. (C) Circadian rhythms were recorded at pH 7.2 or pH 6.7 and, then, rhythms were monitored again at the alternate pHs. Irrespective of orders used for different pH treatment, incubation at pH 6.7 consistently shortened the period lengths. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Values are means ± S.E.M. N=3 subjects.

Figure 2

Cell morphology or cell viability was not significantly affected by exposure to the low pH. Cell morphology and MTT assay were examined six days after the initiation of rhythm recordings. Similar cell morphology was observed at both pH 6.7 and pH 7.2 (upper). The MTT assay showed no significant difference in the absorbance that indicates that amount of metabolic activity between pH 6.7 and pH 7.2 conditions was the same. Values are means ± S.E.M. N=3 subjects.