Introduction: Mice at room temperature are cold-stressed, with elevated metabolic rate and increased sympathetic input to the heart. Thermoneutrality reduces sympathetic input to the heart. Here, we examined circadian rhythmicity of the cardio-metabolic transcriptome in male mice under room temperature and thermoneutrality. Hypothesis: Thermoneutrality increases rhythmic gene expression in cardio-metabolic tissues. Methods: Male SV129s/J mice (n=53) were housed at room temperature (25 ± 1°C) in a 12-hour/light/12-hour dark cycle with ad libitum access to food and water. They were assigned to one of two temperature conditions: continued room temperature (n=26) or thermoneutrality (30 ± 1°C, n=27) for two weeks. At the end of day 14, mice were released into free-running, constant-dark conditions. Hearts, livers, and diaphragms were collected on day 15 across the circadian cycle at 12 time points over 48 hours (circadian time; CT 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, and 62 hours aligned to the corresponding zeitgeber time; n = 2–3 mice/time point/housing condition). RNA sequencing was performed on the Illumina platform. DESeq2 normalized expression values were used for differential expression analysis. Gene ontology (GO) and KEGG pathways were generated using ClusterProfiler (RStudio). DiffCircadian (RStudio) was used to identify rhythmically expressed genes at FDR-corrected q< 0.05. Results: Thermoneutrality increased REGs compared with room temperature. Specifically, the heart showed four times more REGs in thermoneutrality (room temperature: 1410; thermoneutrality: 5282), the liver showed a similar number of REGs (room temperature: 4041; thermoneutrality: 4150), and the diaphragm showed 1.5 times more REGs in thermoneutrality (room temperature: 795; thermoneutrality: 1153). Interestingly, under thermoneutrality, REGs showed two distinct peaks, more prominently in the heart and liver at activity and rest transitions. Thermoneutrality also increased the shared REGs between three tissues (room temperature: 118; thermoneutrality: 232). Thermoneutrality-specific REGs KEGG pathway enriched protein processing, MAPK signaling, and glycerophospholipid metabolism. Room temperature -specific REGs enriched mineral absorption, lipid metabolism, and various signaling pathways. Surprisingly, core clock genes and accessory clock genes showed robust rhythmicity in both room temperature and thermoneutrality with the same mesor, amplitude, and acrophase. Conclusion: Thermoneutrality increases circadian rhythmicity of the transcriptome in cardiometabolic tissues without altering core clock gene expression patterns. Funding: This study was supported by the National Heart, Lung, and Blood Institute (grant no. R01HL172813, to Dr Brian Delisle and Elizabeth Schroder); and by the Pathway to Independence Grant, the Diabetes and Obesity Research Priority Area, and the Barnstable Brown Diabetes and Obesity Center, University of Kentucky (to Abhilash Prabhat). 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.
Prabhat et al. (Fri,) studied this question.