The circadian clock regulates KCNH2 (hERG) promoter activity through daily temperature rhythms.

Abstract

Background: KCNH2 encodes Kv11.1 channel proteins that conduct the rapidly activating delayed-rectifier K+ current (IKr), which is critical for cardiac repolarization. KCNH2 encodes two functional isoforms, Kv11.1a and Kv11.1b, via alternative transcription start sites. Kv11.1a is the principal determinant of cardiac IKr and ventricular repolarization. The circadian clock, a transcriptional-translational feedback loop that cycles with a period of ~24 hours and drives the circadian expression of many genes, including Kcnh2 in the mouse heart. Because daily body temperature rhythms provide a systemic signal that synchronizes cardiac circadian clocks, we tested whether physiological temperature cycles drive the circadian promoter activity of the cloned human KCNH2 (hKCNH2) promoter. Hypothesis: hKCNH2 is a direct transcriptional target of the circadian clock, with temperature driving its promoter activity through BMAL1:CLOCK acting at a conserved tandem E-box. Methods: We cloned the conserved proximal promoter of KCNH2 (-1631 bp upstream of Kv11.1a exon 1) to generate hKCNH2 promoter luciferase reporter constructs. Constructs were transfected into C2C12 myotubes and synchronized by serum shock (static 37{degrees}C) or temperature cycling (36.5-38.5{degrees}C). Bioluminescence was recorded and assessed for period, phase, and amplitude. BMAL1:CLOCK dependence was tested via dominant-negative CLOCK{Delta}19 co-expression. Results: Temperature cycling did not exhibit the rapid damping characteristic of serum-shock-synchronized oscillations, consistent with continuous entrainment by an external zeitgeber rather than a free-running oscillator. Deletion analysis identified a conserved tandem E-box required for oscillation under both serum shock and temperature cycling, and for BMAL1:CLOCK-dependent transactivation (1.75 {+/-} 0.21 vs. 0.86 {+/-} 0.06 RLU, p = 0.0038). CLOCK{Delta}19 reduced hKCNH2 promoter amplitude under temperature cycling without altering period. Conclusion: The circadian clock regulates KCNH2 promoter activity through daily temperature rhythms.