Housing mice in thermoneutrality causes peripheral tissue-specific changes in the number, identity, and phase of circadian-expressed mRNA transcripts

Laboratory mice are typically housed at room temperature (20-25°C), well below their thermoneutral zone (about 30°C), imposing chronic mild cold stress that increases metabolic rate by 30-40%. Because circadian and metabolic physiology are highly temperature-sensitive, we tested whether this housing condition reshapes rhythmic gene expression in tissues central to cardiovascular and metabolic health. We hypothesized that mild cold stress suppresses circadian gene expression in peripheral tissues. We performed RNA sequencing on hearts, livers, and diaphragms collected every 4 hours over 48 hours in constant darkness from male mice to identify transcripts with approximately 24-hour rhythms. Thermoneutral housing reorganized the circadian transcriptome tissue-specifically. Out of ∼13000 filtered transcripts, the proportion of rhythmic transcripts (q<0.05) in the heart nearly quadrupled, from 11% (1410) at room temperature to 42% (5282) at thermoneutrality, while the diaphragm rose modestly (6% to 9%; 792 vs. 1153). The liver's overall proportion was unchanged (36% vs. 37%; 4041 vs. 4150), yet more than 30% of rhythmic transcripts were unique between housing conditions. Almost 80% of rhythmic genes in the heart under thermoneutrality show acrophase near the subjective activity/rest transition. Gene ontology analysis revealed coordinated changes in the temporal organization of metabolic pathways in the heart and liver but less pronounced in the diaphragm. These changes occurred without detectable changes in core clock expression, implicating regulators acting downstream of, or parallel to, the clock itself. Because room temperature housing is standard across biomedical research, our findings show that this overlooked variable can distort the circadian transcriptome of tissues central to cardiovascular and metabolic disease, with implications for how mouse studies model human physiology.