Abstract 14036: Identification of Rho-associated Kinase 2 (ROCK2) as a Key Molecule Generating the Intrinsic Circadian Rhythm of Vascular Contractility
The occurrence of coronary artery events is known to have a circadian variation, with a peak during the early morning. The circadian changes in vascular contractility may underlie this diurnal occurrence of events. However, the peripheral circadian control of vascular contractility remains largely unknown. This study elucidated the mechanism of the vascular intrinsic clock that generates the circadian oscillation of vascular contractility. We identified ROCK2 as a key regulatory molecule, which receives a cue from the biological clock, thereby generating the circadian rhythm of contractility. The dexamethasone pulse treatment was used to induce a circadian oscillation in cultured porcine coronary artery smooth muscle cells. Under this condition, the response of myosin light chain (MLC) phosphorylation to thrombin and endothelin-1 exhibited a circadian change, with a 24-hr cycle, from 0.85 at a nadir to 1.05 PO4 mol/MLC mol at the peak. The inhibition of ROCK2, but not MLCK, PKC or ZIPK, by either pharmacological inhibitors or siRNA, abolished this oscillation. The contractile response of the cells, as indicated by a decrease in the planar cell surface area, also changed with MLC phosphorylation. Thrombin-induced phosphorylation of MYPT1 at Thr853 also exhibited circadian oscillation, thus suggesting that ROCK2 activity also oscillates. A pull-down assay revealed no changes in the level of GTP-bound RhoA, while the expression of ROCK2 protein exhibited the circadian oscillation with a similar phase as was seen with MLC phosphorylation. The level of rock2 mRNA also oscillated. RORE is the only regulatory element for the known clock genes, which was preserved in the mammalian rock2 promoters. The forced expression of RORα and RORγ, but not RORβ, increased the rock2 promoter activity more than 5-fold that of the control. The knockdown of RORα abolished the circadian rhythm of the ROCK2 expression. In the aortas of mice kept under a 12-hr dark-light cycle, the ROCK2 expression exhibited a circadian oscillation in vivo, with a peak during the early morning. The present study thus provides the first evidence that the ROR-regulated circadian rhythm of ROCK2 expression plays a pivotal role in generating the circadian oscillation of vascular contractility.
- © 2010 by American Heart Association, Inc.