Identification of conserved cis-regulatory elements in the GJA1 promoter

Background: GJA1 encodes Connexin 43 (Cx43), the gap junction protein essential for ventricular conduction. We previously found that GJA1 mRNA exhibits circadian oscillations in male and female mouse ventricular heart tissue. Hypothesis: We hypothesize that conserved cis regulatory elements in the human GJA1 promoter region modulate circadian amplitude and phase of GJA1 promoter activity. Methods: We constructed a luciferase promoter reporter vector containing the human (h) GJA1 promoter sequence (6987 bp; (pGL3 basic vector + proximal promoter)) and assessed its circadian activity using real-time bioluminescence monitoring (LumiCycle, Actimetrics) in C2C12 myoblasts. Cells were transiently transfected and monitored continuously for 7-10 days with 10-minute sampling intervals. Circadian parameters were quantified using established analytical methods. Two targeted deletion promoter constructs with the following deletions upstream of the transcription start site (ΔhGJA1 (-10 to -393bp; mutant 1 (M1)); ΔhGJA1 (-444 to -1973bp; mutant 2 (M2))) were generated to map functional regulatory regions. Statistical significance was determined using ANOVA with Bonferroni post-hoc correction. Data is reported as mean ± SD. Results: The full-length hGJA1 (n=22), M1 (n=23), and M2 (n=18) constructs demonstrated circadian bioluminescence patterns and had periods of 24.1 ± 0.3 hrs, 24.5 ± 0.3 hrs, and 23.9 ± 0.1 hrs respectively. There was a significant increase in the amplitude of M1 compared to the full-length construct (62.9 ± 13.9 counts/sec vs 3.0 ± 1.5 counts/sec; 20-fold increase, p< 0.0001), but no difference was observed between M2 and the full-length construct (1.8 ± 0.5 counts/sec vs 3.0 ± 1.5 counts/sec). The phase of the full-length hGJA1 and M2 (3.0 ± 3.4 hrs vs 2.4 ± 2.9 hrs) were not different, but there was a significant difference between the full-length construct and M1 (3.0 ± 3.4 hrs vs 9.4 ± 2.8 hrs, p< 0.0001). The increase in amplitude and phase shift of M1 compared to the full-length construct suggest that this deleted region contains repressor elements. Conclusions: Human GJA1 promoter reporter constructs exhibit robust circadian bioluminescence patterns in C2C12 cells. Our data reveal a novel regulatory model in which the hGJA1 promoter harbors endogenous repressor elements that constrain the amplitude of rhythmic expression. Within the conserved M1 promoter region, we identified these repressive elements. Collectively, these findings uncover previously unknown mechanisms underlying circadian regulation of hGJA1. Funding Sources: This work was supported by National Heart, Lung, and Blood Institute grants R01HL153042, R01HL141343, and R01HL172813. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.