Cardiomyocyte Circadian Oscillations Are Cell-Autonomous, Amplified by β-Adrenergic Signaling, and Synchronized in Cardiac Ventricle Tissue

<div><p>Circadian clocks impact vital cardiac parameters such as blood pressure and heart rate, and adverse cardiac events such as myocardial infarction and sudden cardiac death. In mammals, the central circadian pacemaker, located in the suprachiasmatic nucleus of the hypothalamus, synchronizes cellular circadian clocks in the heart and many other tissues throughout the body. Cardiac ventricle explants maintain autonomous contractions and robust circadian oscillations of clock gene expression in culture. In the present study, we examined the relationship between intrinsic myocardial function and circadian rhythms in cultures from mouse heart. We cultured ventricular explants or dispersed cardiomyocytes from neonatal mice expressing a PER2::LUC bioluminescent reporter of circadian clock gene expression. We found that isoproterenol, a β-adrenoceptor agonist known to increase heart rate and contractility, also amplifies PER2 circadian rhythms in ventricular explants. We found robust, cell-autonomous PER2 circadian rhythms in dispersed cardiomyocytes. Single-cell rhythms were initially synchronized in ventricular explants but desynchronized in dispersed cells. In addition, we developed a method for long-term, simultaneous monitoring of clock gene expression, contraction rate, and basal intracellular Ca²⁺ level in cardiomyocytes using PER2::LUC in combination with GCaMP3, a genetically encoded fluorescent Ca²⁺ reporter. In contrast to robust PER2 circadian rhythms in cardiomyocytes, we detected no rhythms in contraction rate and only weak rhythms in basal Ca²⁺ level. In summary, we found that PER2 circadian rhythms of cardiomyocytes are cell-autonomous, amplified by adrenergic signaling, and synchronized by intercellular communication in ventricle explants, but we detected no robust circadian rhythms in contraction rate or basal Ca²⁺.</p></div

Simultaneous monitoring of basal Ca²⁺ and PER2 levels in dispersed cardiomyocytes.

<p>(A) Representative PER2::LUC (left) and GCaMP3 (right) images of a single cardiomyocyte in dispersed culture. (B) Patterns of Ca²⁺ (GCaMP3, black) and PER2 (PER2::LUC, red) of the cardiomyocyte shown in a recorded over 7d.</p

The SCN oscillator network is responsible for stable rhythms in <i>Clock</i>^<i>-/-</i> SCN neurons.

<p>(A) Raster plots of mPer2^Luc bioluminescence intensity of individual dispersed wild-type (left, n = 246) and <i>Clock</i>^<i>-/-</i> (right, n = 161) SCN cells. Data are presented as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005882#pgen.1005882.g001" target="_blank">Fig 1C</a>. (B) mPer2^Luc bioluminescence rhythms of two representative rhythmic individual dispersed SCN neurons from wild type (left) and <i>Clock</i>^<i>-/-</i> mice (right). (C) Circadian period, amplitude, and sine wave goodness-of-fit of cellular mPer2^Luc rhythms, and the percentage of rhythmic neurons in dispersed SCN cultures from wild type (white), <i>Clock</i>^<i>-/-</i> (black), and <i>Bmal1</i>^<i>-/-</i> (patterned) mice. <i>Bmal1</i>^<i>-/-</i> data are from Ko et al [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005882#pgen.1005882.ref018" target="_blank">18</a>]. Data are shown as mean ± SEM; ***<i>p</i>≤0.001 (student’s t-test); or as percentage of cells that are significantly rhythmic; WT: n (rhythmic/total) = 234/255; <i>Clock</i>^<i>-/-</i>: n = 138/208; <i>Bmal1</i>^<i>-/-</i>: n = 30/243.</p

Peripheral organs of <i>Clock</i>^<i>-/-</i> mice can exhibit circadian rhythms <i>in vitro</i>.

<p>(A) Two representative mPer2^Luc bioluminescence rhythms of rhythmic organotypic liver (black), lung (blue), kidney (red), and adrenal (green) slice cultures from wild type (left) and <i>Clock</i>^<i>-/-</i> (right) mice. After ~7 culture days, samples were treated with 10 μM forskolin (arrow). Y-axis scales are adjusted to amplitudes for better visualization of data. (B) Circadian mPer2^Luc rhythm period, amplitude, damping constant (days to reach 1/e of initial amplitude), and phase of first peak after forskolin treatment, and % of slices from wild type (unfilled bars) and <i>Clock</i>^<i>-/-</i> mice (filled bars) that were significantly rhythmic after forskolin treatment (culture days 8–14). Data are shown as mean ± SEM; *<i>p</i>≤0.05, **<i>p</i>≤0.01, ***<i>p</i>≤0.001 (student’s t-test); or % of slices rhythmic; n = 8.</p

NPAS2 Compensates for Loss of CLOCK in Peripheral Circadian Oscillators

<div><p>Heterodimers of CLOCK and BMAL1 are the major transcriptional activators of the mammalian circadian clock. Because the paralog NPAS2 can substitute for CLOCK in the suprachiasmatic nucleus (SCN), the master circadian pacemaker, CLOCK-deficient mice maintain circadian rhythms in behavior and in tissues <i>in vivo</i>. However, when isolated from the SCN, CLOCK-deficient peripheral tissues are reportedly arrhythmic, suggesting a fundamental difference in circadian clock function between SCN and peripheral tissues. Surprisingly, however, using luminometry and single-cell bioluminescence imaging of PER2 expression, we now find that CLOCK-deficient dispersed SCN neurons and peripheral cells exhibit similarly stable, autonomous circadian rhythms <i>in vitro</i>. In CLOCK-deficient fibroblasts, knockdown of <i>Npas2</i> leads to arrhythmicity, suggesting that NPAS2 can compensate for loss of CLOCK in peripheral cells as well as in SCN. Our data overturn the notion of an SCN-specific role for NPAS2 in the molecular circadian clock, and instead indicate that, at the cellular level, the core loops of SCN neuron and peripheral cell circadian clocks are fundamentally similar.</p></div

Knockdown of <i>Npas2</i> expression suppresses circadian rhythms in <i>Clock</i>^<i>-/-</i> fibroblasts.

<p>(A) Fibroblasts dispersed from wild type and <i>Clock</i>^<i>-/-</i> mice were treated with lentiviral vectors carrying an <i>Npas2</i>-KD or scrambled DNA sequence, as well as a GFP reporter. Simultaneous fluorescence and bioluminescence images of a representative field show GFP expression marking transfected cells (left, green) and mPer2^Luc bioluminescence from both transfected and untransfected cells (middle, red). The overlay shows that circadian rhythms could be measured from both transfected (filled arrowheads) and untransfected (unfilled arrowheads) fibroblasts in the same culture dish. (B) Percentage of wild type (left) and <i>Clock</i>^<i>-/-</i> (right) fibroblasts treated with <i>Npas2</i>-KD lentiviruses (top) or scrambled lentiviruses (bottom) that were significantly rhythmic. ***<i>p</i>≤0.001 (Fisher’s exact test); number of cells are given in parentheses. (C) mPer2^Luc rhythms of representative individual dispersed untransfected (top) and transfected (bottom) <i>Clock</i>^<i>-/-</i> fibroblasts from the same culture dish.</p

Dispersed <i>Clock</i>^<i>-/-</i> fibroblasts show circadian rhythms comparable to those of dispersed <i>Clock</i>^<i>-/-</i> SCN neurons.

<p>(A) Raster plots of bioluminescence intensity of individual dispersed wild-type (left, n = 320) and <i>Clock</i>^<i>-/-</i> (right, n = 121) SCN cells. Data are presented as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005882#pgen.1005882.g001" target="_blank">Fig 1C</a>. (B) Images of mPer2^Luc expression of representative rhythmic wild-type (left) and <i>Clock</i>^<i>-/-</i> (right) fibroblasts. (C) Two representative mPer2^Luc bioluminescence rhythms of individual rhythmic wild-type (left) and <i>Clock</i>^<i>-/-</i> (right) fibroblasts. (D) Circadian period, amplitude, and goodness of fit of mPer2^Luc rhythms, and % of cells that were significantly rhythmic, for individual wild type (left) and <i>Clock</i>^<i>-/-</i> (right) fibroblasts. Data are shown as mean ± SEM; *<i>p</i>≤0.05, ***<i>p</i>≤0.001 (Student’s t-test); or % of cells rhythmic; ***<i>p</i>≤0.001 (Fisher’s exact test); WT: n (rhythmic/total) = 320/321; <i>Clock</i>^<i>-/-</i>: n = 121/163.</p

SNPs with psychiatric illness associations and their location relative to the most proximal clock gene.

<p>The SNPs proximal to PER1 and RORC were determined to lie outside of these genes. The SNP proximal to NR1D2 was indeterminate. The remaining SNPs were determined to be located with the corresponding clock gene.</p

Overlap among clock genes, genes associated with BD spectrum illnesses by GWAS, and lithium-responsive genes.

<p>For each set of genes, the % associated with BD spectrum illnesses by GWAS (blue), the % found to be lithium-responsive (red), and the overlap between these (purple) is shown for 18 core clock genes (A), 342 clock modulator genes (C) and 136 pervasively rhythmic clock controlled genes (CCGs) (E) compared to their respective randomly selected controls (B and D), CCGs that are less pervasively rhythmic (F), and genes with no evidence for rhythmicity (G). Gray indicates % of genes with no relationship to either BD or lithium response.</p

Analysis of cell periods.

<p>(A) Histogram of cell periods (mean peak-to-peak times). (B) Raster plot showing two cells with clearly different periods. In the raster plot, time of day is plotted left to right and successive days down the page, such that vertically adjacent points are 24 h apart. Each row is extended to 48 h, duplicating data in the next row, so that patterns crossing midnight can be appreciated. Thick bars designate times when the luminescence for a cell was above the mean for each row. Cell #66 with period 25.5 h is plotted in red; cell #68 with period 22.5 h is plotted in blue. Due to different circadian periods, the two cells' phase relationship changes over time. (C) Standard deviation in period over the population of cells as a function of the number of cycles used for period determination. Here period for each cell is calculated as the mean of peak-to-peak times over the indicated number of cycles. This curve is expected to decrease to the true value in proportion to one over the square root of the number of cycles used. The dashed line shows the ANOVA prediction of the true value of the standard deviation in period among the fibroblasts. Note that if all cells had the same intrinsic period, and variability of observed period was only due to stochastic fluctuations, then we would expect this graph to approach zero, rather than having a positive horizontal asymptote.</p