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Na+/Ca2+ exchanger mediates cold Ca2+ signaling conserved for temperature-compensated circadian rhythms

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Science Advances  30 Apr 2021:
Vol. 7, no. 18, eabe8132
DOI: 10.1126/sciadv.abe8132
  • Fig. 1 CaMKII and NCX activities are essential for temperature compensation.

    (A) Effects of chemical inhibitors on Q10 of bioluminescence rhythms in Rat-1–Bmal1-luc cells. To normalize experiment-to-experiment variations, ΔQ10 values compared to the vehicle (DMSO) control were used for comparison of the all screening data. The waveforms and the other parameters are shown in figs. S1 to S3. (B) Representative bioluminescence rhythms of Rat-1–Bmal1-luc cells in the presence of KN-93 (left) or KB-R7943 (right) at 32° or 37°C. (C) Dose-dependent effect of KN-93 or KB-R7943 on Q10. ★P < 0.05 compared to DMSO (Dunnett’s test). (D) Dose- and temperature-dependent effect of KN-93, KB-R7943, or SEA0400 on period length at 32° or 37°C. ★P < 0.05 compared to DMSO (Dunnett’s test). (E) Effect of KN-92, KN-93, or SEA0400 on ΔQ10 value. We used a concentration of 10 μM consistently in the first screening (A), and two compounds, KN-93 and KB-R7943, met our criteria. Then, we performed reproducibility test and dose dependency test for the two compounds with several control compounds (B to E). Representative data (B) or the means with SEM from three independent samples (A and C to E) are shown.

  • Fig. 2 Temperature compensation is compromised by CaMKII or NCX inhibitor.

    (A) Period length of Rat-1–Bmal1-luc cells in the presence of KN-93 or KB-R7943 at 32°, 33°, 34°, 35°, 36°, or 37°C. ★P < 0.05 compared to DMSO (Student’s t test). (B) Temperature-dependent effect of KN-93 or KB-R7943 on the period length. The means with SEM from three independent samples (A and B) are shown.

  • Fig. 3 NCX-dependent Ca2+-CaMKII signaling is a key determinant of the state of circadian oscillator.

    (A) Effects of various ion channel modulators on the amplitude of Rat-1–Bmal1-luc cells. Bioluminescence rhythm data are shown in fig. S2. (B) Effects of NCX inhibitors on intracellular Ca2+ levels. The Ca2+ level changes by the drug were measured by using Fluo-4 in NIH3T3 cells. ★P < 0.05 compared to DMSO (Dunnett’s test). (C) Effects of the NCX inhibitors on intracellular CaMKII levels in NIH3T3 cells. After 1-day treatment with the inhibitor or DMSO, phosphorylation activity of the cell lysate was measured with syntide-2. ★P < 0.05 compared to DMSO (Student’s t test). (D) Effects of Ca2+-CaMKII signaling inhibitors on amplitude of the rhythms in Rat-1–Bmal1-luc cells. ★P < 0.05 compared to DMSO (Dunnett’s test). The level of DMSO control was set to 100% (A to D). TFP, trifluoperazine. (E) Reversible effects of NCX inhibitors on bioluminescence rhythm of Rat-1–Bmal1-luc cells or Rat-1–Per2-luc cells. (F) Effects of pulse inhibition of CaMKII or NCX on phase of the oscillator. Two-hour treatment of KN-93 (left) or KB-R7943 (middle) or 1-hour treatment of SEA0400 (right) was applied to Rat-1–Bmal1-luc cells at various circadian time (CT). CT12 was defined as trough level of Bmal1-luc rhythm. Because the treatment of KN-93 at CT24 resulted in the disappearance of the cellular rhythm, the extent of the phase shift was not determined. Data shown are means with SEM from three (A, B, D, and F) or eight (C) independent samples. All experimental data in this figure were obtained from cells cultured at 37°C. nd, not determined.

  • Fig. 4 Hypothermic activation of NCX-dependent Ca2+-CaMKII signaling.

    (A) Effects of temperature on amplitude of cellular rhythm in Rat-1–Bmal1-luc cells. (B) Temperature- and dose-dependent effects of KN-93 on amplitude of cellular rhythm in Rat-1–Bmal1-luc cells. (C) Hypothermic Ca2+ response in NIH3T3 cells. The mean value at 37°C is set to 100. ★P < 0.05 compared to 37°C (Dunnett’s test). Right panels are representative images of intracellular Ca2+ levels in NIH3T3 cells at 37° or 25°C. (D) KB-R7943 or SEA0400 blocks hypothermic Ca2+ response in NIH3T3 cells. Initial value of each cell at 37°C was set to 100%. ★P < 0.5 × 10−7 compared to DMSO (Student’s t test). The Ca2+ imaging analysis was started from 37°C down to 25°C (C and D). (E) NCX mediates hypothermic CaMKII activation in NIH3T3 cells. The mean value of DMSO at 37°C is set to 100%. ★P < 0.05 (Student’s t test). ns, not significant. (F) Ca2+ ionophore up-regulates clock gene Per1, Per2, and Dec1. Thirty-six hours after the rhythm induction with dexamethasone, 10 μM (final concentration) ionomycin, A23187, or the same volume of DMSO was applied to Rat-1–Bmal1-luc cells. One hour after the treatment, the cells were harvested to detect clock gene mRNA levels. The mean value of pretreatment is set to 100%. ★P < 0.05 compared to DMSO (Student’s t test). (G) Hypothermic response of clock genes in Rat-1–Bmal1-luc cells. (H) NCX and CaMKII mediate hypothermic up-regulation of Per1 and Per2 in Rat-1–Bmal1-luc cells. The mean value at 37°C is set to 100%. ★P < 0.05 and ★★P < 0.005 compared to DMSO-treated cells at 27°C (Student’s t test). The cells were harvested to detect clock gene mRNA levels at indicated time points (G) or 5 days (H) after rhythm induction by dexamethasone. Representative data [panels of (C)] or means with SEM from 3 (A, B, F, and G), 8 (E), 9 (H), or 20 (C and D) independent samples are shown.

  • Fig. 5 Mechanism of temperature compensation.

    (A) Effect of temperature on phosphorylation activity of purified CaMKII. Phosphorylation activity of rat CaMKII against CLOCK peptide was measured by autoradiography. The level of phosphorylated CLOCK at 20°C was set to 100%. (B) Effect of CaMKII overexpression on bioluminescence rhythm by Bmal1-luc in NIH3T3 cells. (C) Effect of CaMKII overexpression on period length and amplitude of cellular rhythm. ★P < 1.0 × 10−6 (Student’s t test with Bonferroni correction). (D) Mathematical simulation of effect of CLOCK-BMAL1 activation on period length and amplitude of Bmal1 expression rhythms. (E) Circadian Ca2+ oscillator regulates TTFL to generate temperature-compensated overt rhythms in mammalian circadian clock. (F) Effect of temperature on Ca2+ oscillation in SCN. (G) Hypothermia increases trough and peak levels of Ca2+ oscillation in SCN. The fluorescence levels of GCaMP6s were divided by those of mRubby for normalization of effect of temperature on the fluorescence indicator. Representative data [top panels of (A), (B) and (F)] or means with SEM (A, C, and G) from three (A), four (B and C), or seven (F and G) independent samples are shown.

  • Fig. 6 Cold-responsive phosphorylation signaling conserved in animals and plants.

    (A) Body temperature of mice at normal (23°C) or cold (4°C) temperature. Surface body temperature was measured by infrared thermography. Representative images are shown in the left panel. Core body temperature of mice was measured by implantable device in peritoneal cavity (fig. S7). (B) Hypothermic activation of cellular phosphorylation activity against syntide-2 in the ear or tail of Mus musculus, the head of D. melanogaster, or the shoot of A. thaliana. The phosphorylation activity at normal temperature was set to 100%. ★P < 0.01 and ★★P < 1.0 × 10−5 compared to nontreated samples (Student’s t test). Data are means with SEM from three independent samples (A and B).

  • Fig. 7 Conserved roles of Ca2+ signaling in circadian clockwork.

    (A) Wheel-running rhythm of NCX mutant mice. LD, light-dark; DD, constant dark.(B) Period length of wheel-running rhythm of the NCX mutant mice. Animal number of wild type (WT), NCX2+/−, or NCX2+/− NCX3−/− is 6, 10, or 7, respectively. ★P < 0.05 compared to WT (Student’s t test). (C) Aberrant pattern of morning and evening activity rhythms in NCX2+/− NCX3−/− mice. (D) Locomotor activity rhythm of calx mutants of D. melanogaster. (E) Relative FFT power of locomotor activity rhythm of calx mutants of D. melanogaster. Animal number of WT, calxA, or calxB is 15, 16, or 14, respectively. ★P < 0.5 × 10−4 compared to WT (Student’s t test). (F) Effect of Ca2+ depletion on gene expression rhythm by CCA1::LUC reporter in A. thaliana. (G) Effect of Ca2+ depletion on period length of gene expression rhythm by CCA1::LUC reporter in A. thaliana. (H) Effect of Ca2+ depletion on Q10 of gene expression rhythm by CCA1::LUC reporter in A. thaliana. The sample number of each group is 20. ★P < 1.0 × 10−7 (Student’s t test). (I) Effect of knockout of yrbG on gene expression rhythm by PkaiBC::luxAB in Synechococcus elongatus PCC 7942 at 25° or 30°C. (J) Effect of knockout of yrbG on period length of gene expression rhythm at 25° or 30°C. ★P < 0.05 and ★★P < 0.0005. (K) Effect of knockout of yrbG on Q10 of gene expression rhythm at 25° or 30°C. *P < 0.005. The sample number of each group is 4 (J and K). Data shown are representative (A, C, D, F, and I) or means with SEM (B, E, G, H, J, and K).

  • Fig. 8 Involvement of ancient Ca2+ signaling for temperature-compensated circadian rhythms.

    Clock genes involved in the TTFLs evolved independently after divergence of each lineage. In animals, fungi, and plants, common multifunctional kinases, such as casein kinase I (CKI), CKII, glycogen synthase kinase 3 (GSK3), or Ca2+-dependent kinase (CaMK), are involved in posttranslational regulation of clock gene products. In cyanobacteria, posttranslational oscillator by KaiA/KaiB/KaiC drives the TTFL. NCX, a highly conserved molecule among three domains of life, is a common circadian timekeeping element in the eukaryotes and prokaryotes, and its original function is regulation of Ca2+ homeostasis.

Supplementary Materials

  • Supplementary Materials

    Na+/Ca2+ exchanger mediates cold Ca2+ signaling conserved for temperature-compensated circadian rhythms

    Naohiro Kon, Hsin-tzu Wang, Yoshiaki S. Kato, Kyouhei Uemoto, Naohiro Kawamoto, Koji Kawasaki, Ryosuke Enoki, Gen Kurosawa, Tatsuto Nakane, Yasunori Sugiyama, Hideaki Tagashira, Motomu Endo, Hideo Iwasaki, Takahiro Iwamoto, Kazuhiko Kume, Yoshitaka Fukada

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