Effect of 1a, 25(OH)2D3 on circadian clock gene expression in cardiac myocytes

Abstract

Objective To explore the effect of 1a, 25(OH)2D3 on circadian clock gene expressions in cardiac myocytes. Methods Cultured cardiac myocytes isolated from 7-day-old Sprague-Dawley(SD) rats were identified by immunofluorescence.The medium including 1a, 25(OH)2D3 (final concentrations were 0 nmol/L, 1 nmol/L, 10 nmol/L, 50 nmol/L and 100 nmol/L) were added to primary myocardial cells to culture for 2 h and then total RNA was extracted.Real-time polymerase chain reaction (RT-PCR) was applied to analyze myocardial cells circadian clock gene (Bmal1, Per2, Rev-erba) transcript levels to determine optimum concentration of 1a, 25(OH)2D3.Then, the primary myocardial cells cultured for 72 h were divided into 3 groups: the control group was of serum-free culture medium; serum shock group was of DMEM containing 50%volume fraction of horse serum cultured 2 h; 1a, 25(OH)2D3 treatment group receiving 1a, 25(OH)2D3 at optimal concentration cultured 2 h. The cells were collected at 7 time points (0 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h) and then total RNA was extracted.RT-PCR was applied to analyze circadian clock gene (Bmal1, Per2, Rev-erba) transcript levels in the myocardial cells. Results In the presence of 50 nmol/L 1 a, 25(OH)2D3, the Bmal1 mRNA expression showed the highest level, but the Per2 and Rev-erba mRNA expression levels were minimum.Compared with the control group, both 1a, 25(OH)2D3 treatment group and serum shock group caused day-cycle rhythmic oscillation in circadian clock genes(Bmal1, Per2, Rev-erba) in the cardiac myocytes.And the expressions pattern of Bmal1 and Per2 genes were in the opposite phase.While Bmal1 gene expression appeared at peak at 12 h, Per2 gene expression appeared in a trough.Expression of Rev-erba gene trend began to rise at 8 h, and the highest expression level appeared at 12-16 h. Conclusions 1a, 25(OH)2D3 can affect Bmal1, Per2 and Rev-erba mRNA expressions of circadian clock genes in the cardiac myocytes. Key words: 1a, 25(OH)2D3; Cardiac myocytes; Circadian clock; Circadian clock gene