Housing Mice in Thermoneutrality Causes Tissue-specific Changes in Number, Identity, and Phase of Circadian-expressed mRNA Transcripts

Abstract

Most laboratory mice are housed at room temperature (20-25{degrees}C), which exposes them to chronic mild cold stress because it is below their thermoneutral temperature (30{degrees}C). 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 produced tissue-specific changes in the number, identity, and timing of rhythmic transcripts without altering the expression of core circadian clock genes. In the heart, the number of rhythmic transcripts increased fourfold, whereas the diaphragm showed a 1.5-fold increase. In the liver, the overall number of rhythmic transcripts showed little change, but their identity changed by 30%. Gene Ontology analysis revealed coordinated changes in the temporal organization of metabolic pathways in the heart and liver. Together, these findings demonstrate that ambient housing temperature is a major determinant of tissue-specific circadian gene expression, altering the abundance, identity, and timing of rhythmic transcripts independently of the core circadian clock. SignificanceScientists typically house laboratory mice at room temperature, below their thermoneutrality, forcing them to increase their metabolic rate to maintain core body temperature. Since ambient temperature plays an important role in metabolism, cold stress could disrupt circadian gene expression. Comparing mice housed at room temperature with a warmer, thermoneutral temperature, we found that housing temperature causes tissue-specific differences in rhythmically expressed genes in the heart, liver, and diaphragm, without altering core clock genes. The heart was especially sensitive, with rhythmic genes peaking at the transition between subjective light and dark cycles, increasing fourfold. These results identify ambient housing temperature as an underrecognized variable that biases circadian gene expression in cardio-metabolic tissues, affecting interpretation of preclinical studies of metabolism and disease.