Circadian disruption induces sex-dependent aortic dysfunction

Vascular dysfunction, especially aortic stiffness, is a known predictor and risk for vascular disease. Disruptions in circadian rhythms are known to increase aortic stiffness. We recently reported that alteration of the feeding-fasting cycle or chronic mistimed feeding increases aortic stiffness in male but not female adult mice. However, it is unknown whether mistimed feeding, acute or chronic, interferes with the aortic molecular clock. Thus, we hypothesized that circadian disruption through acute or chronic mistimed feeding interferes with the aortic molecular clock in a sex-dependent manner. To test this hypothesis, we first utilized male and female Per2Luc+/- mice, which allow for luminescent detection of the core clock protein PER2, to assess rhythmicity of the molecular clock. Mice were assigned to one of three feeding groups: ad libitum, acute mistimed feeding (6 days of light-period time-restricted feeding, or iTRF), or chronic mistimed feeding (10 weeks of iTRF). At the termination of the protocol, aortae were harvested and cultured in a LumiCycle luminometer to assess PER2::LUC rhythm via cosinor analyses (GraphPad Prism). In males, the phase of the aortic rhythm revealed that chronic iTRF significantly shifted the phase of the aortic molecular clock compared to ad libitum feeding (zeitgeber time or ZT of first peak: ad libitum, 26.1±0.7 hr, n=7; acute iTRF, 28.1±1.9 hr, n=4; chronic iTRF, 21.6±1.3 hr, n=5, where ZT 0 refers to the prior lights-on time; one-way ANOVA, p=0.04 for ad libitum vs. chronic iTRF). The acute iTRF phase was similar to the phase in ad libitum fed males (p=0.49). In females, however, the phase of the aortic rhythm was shifted in both the acute and chronic mistimed feeding groups (ZT of first peak: ad libitum, 35.7±0.4 hr, n=5; acute iTRF, 34.0±0.5 hr, n=5; chronic iTRF, 33.6±0.4 hr, n=4, one-way ANOVA, p=0.04 for ad libitum vs. acute iTRF; p=0.02 for ad libitum vs. chronic iTRF). These results indicate that the aortic clock rhythmicity is phase shifted by mistimed feeding in a sex-dependent manner. Bmal1 is a core circadian gene and global deletion of Bmal1 leads to sex differences at the whole animal level. Further, sex differences in endothelial function are well-known. Thus, we hypothesized that endothelial cell-specific Bmal1 knockout mice would exhibit aortic stiffness in a sex-dependent manner. We generated an inducible endothelium-specific Bmal1 KO mouse model (ieBmal1-/-). Briefly, male and female ieBmal1-/- mice and WT Cre+ littermates were administered tamoxifen to induce Bmal1 deletion. After 8 weeks, pulse wave velocity (PWV) was measured by ultrasound (VEVO 3100). Our data revealed that male ieBmal1-/- mice displayed significantly increased PWV compared to WT (WT: 2.0±0.3 m/s, n=5; ieBmal1-/-: 5.6±0.7 m/s, n=7, unpaired t-test, p=0.003), indicating that genetic disruption of the endothelial clock promotes aortic stiffness in male mice. However, female ieBmal1-/- and WT mice had similar PWV (p=0.49). These data indicate that aortic clock homeostasis is integral for vascular function, especially in males. These results further suggest that therapies targeting the circadian system in males would be beneficial for treatment of vascular disease. 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.