Calcium channel genes associated with bipolar disorder modulate lithium's amplification of circadian rhythms

Abstract

Bipolar disorder (BD) is associated with mood episodes and low amplitude circadian rhythms. Previously, we demonstrated that fibroblasts grown from BD patients show weaker amplification of circadian rhythms by lithium compared to control cells. Since calcium signals impact upon the circadian clock, and L-type calcium channels (LTCC) have emerged as genetic risk factors for BD, we examined whether loss of function in LTCCs accounts for the attenuated response to lithium in BD cells. We used fluorescent dyes to measure Ca(2+) changes in BD and control fibroblasts after lithium treatment, and bioluminescent reporters to measure Per2::luc rhythms in fibroblasts from BD patients, human controls, and mice while pharmacologically or genetically manipulating calcium channels. Longitudinal expression of LTCC genes (CACNA1C, CACNA1D and CACNB3) was then measured over 12-24 h in BD and control cells. Our results indicate that independently of LTCCs, lithium stimulated intracellular Ca(2+) less effectively in BD vs. control fibroblasts. In longitudinal studies, pharmacological inhibition of LTCCs or knockdown of CACNA1A, CACNA1C, CACNA1D and CACNB3 altered circadian rhythm amplitude. Diltiazem and knockdown of CACNA1C or CACNA1D eliminated lithium's ability to amplify rhythms. Knockdown of CACNA1A or CACNB3 altered baseline rhythms, but did not affect rhythm amplification by lithium. In human fibroblasts, CACNA1C genotype predicted the amplitude response to lithium, and the expression profiles of CACNA1C, CACNA1D and CACNB3 were altered in BD vs.

Controls: We conclude that in cells from BD patients, calcium signaling is abnormal, and that LTCCs underlie the failure of lithium to amplify circadian rhythms.

Keywords: Bipolar disorder; Calcium; Cells; Circadian rhythms; Gene; Lithium.

Figure 1

The intracellular Ca²⁺ response to acute lithium is attenuated in BD fibroblasts. A) Normalized increase in Ca²⁺ fluorescence observed in cells from controls (N=9) and BD patients (N=10). The Ca²⁺ increase following lithium in controls was significantly higher than in the BD group. Experiment was run four times. A two-tailed T-test comparing normalized increase in intracellular Ca²⁺ in BD and control cells indicated a significant (p<0.05) group difference. B) Representative traces showing the absolute increase in Ca²⁺ fluorescence for a control and BD cell line. Black arrow indicates the time of lithium application.

Figure 2

Blocking Ca²⁺ channels attenuates lithium's amplification of circadian rhythms. Circadian rhythms of mouse NIH3T3^P2L fibroblasts (A) respond to lithium in a manner similar to human cells: increasing amplitude (B), and lengthening period (C). The Ca²⁺ channel antagonist diltiazem decreases rhythm amplitude in NIH3T3^P2L cells (D) and blocks the rhythm amplifying effect of lithium (E). Similar effects of diltiazem on rhythm amplitude are observed in parallel cell cultures from human controls (n=4 / group). Because BD patients had more variable amplitude responses to lithium, they were not considered for this experiment (F). Values are means ± SEM; *p < 0.05 using ANOVA with post-hoc T-tests.

Figure 3

Effects of calcium channel knockdown on rhythms in NIH3T3^P2L cells. Knockdown of CACNA1C (A-C) caused increases in amplitude at baseline, and reversed lithium's effect on rhythms, causing an amplitude decrease. CACNA1C knockdown alone had no effect on period, but attenuated the period lengthening action of lithium. CACNA1D (D-F) also caused increases in amplitude at baseline, and blocked lithium's ability to increase amplitude. Knockdown of CACNA1D shortened period, but had no effect on the period lengthening action of lithium. CACNA1A (G-I) caused increases in amplitude at baseline, but had no effect on lithium's ability to increase amplitude. CACNA1A knockdown alone had no effect on period, but attenuated the period lengthening action of lithium. Knockdown of CACNB3 (J-L) decreased baseline amplitude, but had no effect on lithium's amplification of rhythms. CACNB3 knockdown had no statistically significant impact on period at baseline or after lithium. Sample sizes are as follows: CACNA1C N= 12-15/ group, CACNA1D N= 3-6/ group, CACNA1A N= 4-6/ group, CACNB3 N= 9/ group. Normalized amplitude values for negative controls ± lithium were combined across experiments (total N=25 siRNA alone, N=31 siRNA+lithium). Values are means ± SEM; Analyses performed by one-way ANOVA, *p < 0.05 in post-hoc tests.

Figure 4

Lithium's amplification of circadian rhythms depends on CACNA1C genotype. Per2∷luc circadian rhythms were recorded in parallel at baseline and during treatment with 1 mM lithium in 55 human cell lines (32 BD, 23 control). Rhythms from two representative cell lines are shown ± lithium (A) one homozygous for the common G allele and (B) one carrier of the BD-associated C allele at CACNA1C rs4765913. Raw data and best fit damped sine curves are shown for each. (C) Lithium increased amplitude differentially according to genotype [two-way ANOVA indicates effect of drug (p<0.001) and drug × genotype interaction (p<0.05)]. N=38 GG genotype, N=17 GC/CC genotypes, values are means ± SEM; *p < 0.05 by post-hoc T-test.

Figure 5

Gene expression profiles using fibroblasts from BD patients (n=4) and controls (N=3) A) Expression profiles and best fit curves indicate that expression of CACNA1C is rhythmic over 24 hr, but more so in controls compared to BD cells. Phase in 24 hr (A) and 12 hr (B-E) experiments may differ due to minor technical differences between experiments. To allow direct comparison to other genes, B) the 12 hr profile of CACNA1C is shown. C) CACNA1D expression over 12 hr was higher in BD compared to controls. D) Amplitude of CACNB3 expression over 12 hr was lower in BD compared to controls. E) PER1 and CLOCK were used as phase markers after combining BD and control samples. As expected, expression of these genes varied over time with opposite phases.