Entrainment of Circadian Rhythms to Temperature Reveals Amplitude Deficits in Fibroblasts from Patients with Bipolar Disorder and Possible Links to Calcium Channels

Abstract

Bipolar disorder (BD) is characterized by recurrent mood episodes, and circadian rhythm disturbances. Past studies have identified calcium channel genes as risk loci for BD. CACNA1C encodes an L-type calcium channel (LTCC) involved in the entrainment of circadian rhythms to light. Another calcium channel, i.e., the ryanodine receptor (RYR), is involved in -circadian phase delays. It is unknown whether variants in CACNA1C or other calcium channels contribute to the circadian phenotype in BD. We hypothesized that, by using temperature cycles, we could model circadian entrainment in fibroblasts from BD patients and controls to interrogate the circadian functions of LTCCs. Using Per2-luc, a bioluminescent reporter, we verified that cells entrain to temperature rhythms in vitro. Under constant temperature conditions, the LTCC antagonist verapamil shortened the circadian period, and the RYR antagonist dantrolene lengthened the period. However, neither drug affected temperature entrainment. Fibroblasts from BD patients and controls also entrained to temperature. In cells from BD patients, the rhythm amplitude was lower under entrained, but not constant, conditions. Temperature entrainment was otherwise similar between BD and control cells. However, the CACNA1C genotype among BD cells predicted the degree to which cells entrained. We conclude that assessment of rhythms under entrained conditions reveals additional rhythm abnormalities in BD that are not observable under constant temperature conditions.

Keywords: Bipolar disorder; Calcium channel; Circadian rhythm; Gene expression.

Fig. 1

Temperature cycles entrain rhythms in NIH3T3 cells. a The period is shortened by temperature cycles that approximate the duration of daily light/dark cycles. Compared to cells examined under constant temperature conditions (n = 33), cells run under temperature cycles (h at 35°C:h at 37.5°C) of 10: 14 (n = 3), 12: 12 (n = 12), or 14: 10 (n = 14) showed shorter periods of Per2-luc expression that converged upon the period of the temperature cycle. Representative traces are shown for rhythms entrained to 10: 14 (b), 12: 12 (c), and 14: 10 (d) temperature cycles (red) compared to cells from the same plate run in parallel under constant 35°C conditions (black). The white bar indicates a temperature of 35°C, and the gray bars indicate a temperature of 37.5°C (applies only to the samples measured under temperature cycles). * Significant (post hoc t test, p < 0.05) difference compared to controls. ** Significant difference (post hoc t test, p < 0.05) between 12: 12 and 14: 10 cycles.

Fig. 2

Temperature cycles entrain rhythms in immortalized mouse hippocampal neurons. a Rhythms in mouse neurons. Compared to neuronal cultures examined under constant temperature conditions (n = 4), cells run under a temperature cycle (h at 35°C:h at 37.5°C) of 12: 12 (n = 4) showed shorter periods of Per2-luc expression that converged upon the period of the temperature cycle. Representative traces are shown for rhythms entrained to 12: 12 temperature cycles (b) (red) compared to neurons run in parallel under constant 35°C conditions (black). The white bar indicates a temperature of 35°C, and the gray bar indicates a temperature of 37.5°C (applies only to the samples measured under temperature cycles). * Significant (p < 0.05) difference compared to controls as determined by a two-tailed t test.

Fig. 3

Calcium channel antagonists alter the circadian period in NIH3T3 cells. a Under constant conditions, the LTCC antagonist verapamil shortens the circadian period at 1 (n = 3) and 10 µM (n = 11) in NIH3T3^P2L cells compared to vehicle-treated controls (n = 22). b Representative traces demonstrating the period-shortening effect (under constant conditions) of verapamil 10 µM (red) compared to vehicle-treated cells (black) in NIH3T3^P2L cells. c Under 12: 12 temperature-entrained conditions, verapamil at 10 µM has no significant effect on period (p = 0.11, n = 9/group). d Under constant conditions, the RYR antagonist dantrolene lengthens the circadian period in a concentration-dependent manner at 1 (n = 4) and 10 µM (n = 12) in NIH3T3^P2L cells compared to vehicle-treated controls (n = 12). e Representative traces demonstrating the period-lengthening effect of dantrolene at 10 µM (blue) compared to vehicle-treated cells (black) in NIH3T3^P2L cells. f Under 12: 12 temperature-entrained conditions, dantrolene at 10 µM has no significant effect on period (n = 6 per group). For each panel, a positive period change indicates a lengthening of period by the drug. Box plots indicate minimum/maximum/mean values and SEM. * Significant (p < 0.05) difference compared to controls as determined by one-way ANOVA.

Fig. 4

Entrained circadian rhythms in subjects with BD and controls. a Entrained period length of human fibroblasts run under varying temperature cycles. b The rhythm amplitude under constant temperature conditions does not differ in fibroblasts from BD cases and controls, whereas under 12: 12 temperature cycle entrained conditions the amplitude is lower in cells from BD patients. The results shown reflect samples collected under constant conditions from controls (n = 10) and BD patients (n = 56) and samples analyzed under temperature-entrained conditions (BD, n = 22; controls, n = 7). The latter cohort fully includes the former. Two-way ANOVA revealed the main effect of temperature (p = 0.001), i.e., and a trend towards a bipolar × temperature interaction (p = 0.07). A post hoc t test comparing BD and control samples under temperature-entrained conditions revealed a significant difference (p = 0.01) (indicated by *). c Representative baseline subtracted, raw data traces of Per2-luc expression rhythms in fibroblast cultures under temperature entrainment from a control (black) and a BD patient (red), illustrating the higher rhythm amplitude in controls. White vertical bars indicate the 12-h periods of 35°C, and gray bars indicate the 12-h periods of 37.5°C. d Under entrained conditions, there was no significant difference in period in cells from controls vs. BD. However, there was a significant difference in period by genotype. BD cells harboring a CACNA1C rs4765913 BD risk allele showed a reduced ability to entrain to 12: 12 temperature cycles compared to cells homozygous for the more common CACNA1C T allele. * p < 0.05 in a post hoc t test. e Representative baseline subtracted, raw data traces of Per2-luc expression rhythms in fibroblast cultures from a CACNA1C risk allele carrier (black) and sample homozygous for the CACNA1C common allele (red), illustrating the longer period of the risk allele carrier under entrained conditions. White bars indicate the 12-h periods of 35°C, and gray bars indicate the 12-h periods of 37.5°C.