Single-cell transcriptomics reveals cell-type-specific circadian rhythms and their disruption by acute misalignment...

The circadian molecular clock is a 24-hour cellular timekeeper that influences many features of cardiovascular function. Disruption of the circadian clock via misalignment with the light-dark cycle is associated with a higher incidence of cardiovascular disease and raises cardiovascular risk factors in humans. Nonetheless, the cell-type-specific molecular basis for how misalignment affects the vasculature remains poorly understood. To address this, we performed single-cell RNA-sequencing from whole mouse aorta at ZT0, ZT6, ZT12, and ZT18 under aligned and acutely misaligned (6-hour phase advance) light-dark cycles in both male and female mice. Leveraging Bayesian variational inference, we estimated posterior waveforms for 141,752 cells across four major cell types and identified hundreds of cycling genes in vascular smooth muscle cells (SMCs) and fibroblasts. Pathway and transcription factor enrichment analyses revealed coordinated circadian activity in cholesterol biosynthesis, smooth muscle contraction, and extracellular matrix organization. Notably, SMC genes implicated in phenotypic switching showed coordinated temporal patterns, with genes promoting switching peaking at dusk and genes restraining switching peaking at dawn. Comparing males and females, we found that female SMCs are broadly more rhythmic, with higher amplitudes and nearly twice as many cycling genes after controlling for cell counts and library sizes-a sex difference that was cell-type-specific and not observed in fibroblasts. After acute misalignment, cycling genes showed reduced amplitudes, and peak times showed limited adaptation to the new light-dark cycle. Given that the central clock is known to adapt near-completely during this timeframe, these observations suggest internal misalignment between central and peripheral rhythms. Moreover, altered relative timing of core clock genes within cells indicates that misalignment is created at the intracellular level as well. In SMCs, gene expression patterns were consistent with proteostatic stress, including broad downregulation of protein chaperones and stress-response genes, which cells appear to cope with by upregulating protein degradation pathways. In parallel, in vivo vascular phenotyping showed increased vascular permeability in both sexes, reduced urinary nitrate in males, and increased microvascular thrombus formation in males following acute misalignment. This atlas provides a resource for understanding how circadian misalignment disrupts vascular homeostasis and may contribute to cardiovascular disease risk.