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

BACKGROUND

KCNH2 encodes Kv11.1 channel proteins that conduct the rapidly activating delayed-rectifier K+ current (I Kr ), 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 I Kr 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 (h KCNH2 ) promoter.

HYPOTHESIS: h KCNH2 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 h KCNH2 promoter luciferase reporter constructs. Constructs were transfected into C2C12 myotubes and synchronized by serum shock (static 37°C) or temperature cycling (36.5-38.5°C). Bioluminescence was recorded and assessed for period, phase, and amplitude. BMAL1:CLOCK dependence was tested via dominant-negative CLOCKΔ19 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). CLOCKΔ19 reduced h KCNH2 promoter amplitude under temperature cycling without altering period.

CONCLUSION

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