The Clock Mechanism Influences Neurobiology and Adaptations to Heart Failure in Clock∆19/∆19 Mice With Implications for Circadian Medicine

Abstract

In this study we investigated the role of the circadian mechanism on cognition-relevant brain regions and neurobiological impairments associated with heart failure (HF), using murine models. We found that the circadian mechanism is an important regulator of healthy cognitive system neurobiology. Normal Clock^∆19/∆19 mice had neurons with smaller apical dendrite trees in the medial prefrontal cortex (mPFC), and hippocampus, showed impaired visual-spatial memory, and exhibited lower cerebrovascular myogenic tone, versus wild types (WT). We then used the left anterior descending coronary artery ligation model to investigate adaptations in response to HF. Intriguingly, adaptations to neuron morphology, memory, and cerebrovascular tone occurred in differing magnitude and direction between Clock^∆19/∆19 and WT mice, ultimately converging in HF. To investigate this dichotomous response, we performed microarrays and found genes crucial for growth and stress pathways that were altered in Clock^∆19/∆19 mPFC and hippocampus. Thus these data demonstrate for the first time that (i) the circadian mechanism plays a role in neuron morphology and function; (ii) there are changes in neuron morphology and function in HF; (iii) CLOCK influences neurobiological gene adaptations to HF at a cellular level. These findings have clinical relevance as patients with HF often present with concurrent neurocognitive impairments. There is no cure for HF, and new understanding is needed to reduce morbidity and improve the quality of life for HF patients.

Figure 1

Murine Clock^∆19/∆19 and WT models. (a) Wheel running actigraphy characterizing Clock^∆19/∆19 (left) and WT (middle) mice under normal diurnal conditions (12 hour light:12 hour dark) for 10 days, and circadian (constant darkness, DD) conditions for 10 days. Quantification of period under DD (right) showing that the CLOCK mutation extends the circadian period from 23.9 hours to ~27.6 hours, as anticipated (n = 4 mice/group, P < 0.05). (b) Actigraphy of HF mice, showing that Clock^∆19/∆19 (left) and WT (middle) mice, and period under DD (right) maintain their respective phenotypes, and are significantly different from each other (n = 4 mice/group, P < 0.05). (c) Exemplar photomicrographs of one traced neuron are shown. A low-magnification view is shown in the left panel, with the neuron of interest indicated by the red arrow and a scale bar of 500 µm. A high-magnification view of the area enclosed by the red box is shown in the middle panel, with a scale bar of 100 µm, and a tracing of a layer 2/3 neuron on the right, with a scale bar of 100 µm. (d) Representative neuron tracings illustrating the smaller mPFC apical dendrite tree size in Clock^∆19/∆19 versus WT mice. *Indicates P < 0.05 by Bonferroni post-hoc analysis.

Figure 2

Apical dendrite morphology differs in Clock^∆19/∆19 vs. WT mice, baseline and HF. (a) Apical dendrite morphology was analyzed for mPFC layer 2/3 and hippocampus CA1 pyramidal neurons using a modified three-dimensional Sholl analysis. This analysis measured the length of apical dendrite between concentric spheres radiating outward from the soma. (a) Normal Clock^∆19/∆19 mice have less apical dendrite length compared to WT mice in the mPFC (left, P = 0.0001) and the hippocampus (right, P = 0.001). (b) WT mice with HF exhibit decreased apical dendrite length in the mPFC (left, P = 0.003), but no change in apical dendrite length in hippocampus (right), as compared to non-HF WT controls. In contrast, (c) Clock^∆19/∆19 mice with HF exhibit increased apical dendrite length in the mPFC (left, P = 0.03), and increased apical dendrite length in the hippocampus (right, P = 0.01), versus Clock^∆19/∆19 controls. (d) Thus HF is associated with changes in apical dendrite length, and the direction and magnitude of change is different in Clock^∆19/∆19 HF versus WT HF mice, in mPFC (left) and hippocampus (right) neurons. For mPFC: n = 4 mice per baseline group, n = 5 mice per HF group. For hippocampus: n = 4 mice per baseline group, n = 4 mice per HF group. Four neurons were traced and averaged for each mouse, and data are shown as mean ± SEM. *Indicates P < 0.05 by Bonferroni post-hoc analysis.

Figure 3

Basal dendrite morphology, baseline and HF. Basal dendrite morphology was analyzed for mPFC layer 2/3 and hippocampus CA1 pyramidal neurons using a modified three-dimensional Sholl analysis. This analysis measured the length of basal dendrite between concentric spheres radiating outward from the soma. (a) Normal Clock^∆19/∆19 mice exhibit similar basal dendrite length compared to WT mice in the mPFC (left) and the hippocampus (right). (b) WT HF mice exhibit increased basal dendrite length in the mPFC (left, P = 0.0007), and in the hippocampus CA1neurons (right, P = 0.049), versus WT controls. In contrast, (c) Clock^∆19/∆19 HF mice exhibit no difference in basal dendrite length in the mPFC (left) and increased basal dendrite length in the hippocampus neurons (right, P = 0.001), versus Clock^∆19/∆19 controls. (d) Thus HF is associated with changes to basal dendrite length that differ in magnitude and direction for mPFC neurons, but are similar for hippocampus neurons, between Clock^∆19/∆19 HF and WT HF mice. For mPFC: n = 4 mice per baseline group, n = 5 mice per HF group. For hippocampus: n = 4 mice per baseline group, n = 4 mice per HF group. Four neurons were traced and averaged for each mouse, and data are shown as mean ± SEM. *Indicates P < 0.05 by Bonferroni post-hoc analysis.

Figure 4

Visual-spatial memory differs in Clock^∆19/∆19 and WT mice. (a) At baseline, Clock^∆19/∆19 mice show impaired short term OiP memory (5 minutes) compared to WTs (n = 23 Clock^∆19/∆19, n = 27 WT). (b) At 1 week post-myocardial infarction, OiP performance is impaired in both Clock^∆19/∆19 mice and WT mice at both immediate (45 seconds) and 5-minute retention delays (n = 16 Clock^∆19/∆19, n = 17 WT). (c) In the 8-week HF mice, OiP performance is impaired in Clock^∆19/∆19 vs. WT mice at immediate (45 second) delays, and is for both Clock^∆19/∆19 and WT mice at 5-minute retention delays (n = 16 Clock^∆19/∆19, n = 17 WT). Object oddity discrimination is similar at (d) baseline (n = 5/group), (e) 1 week post-myocardial infarction (n = 6/group), and in the (f) 8 week HF mice (n = 16 WT, n = 17 Clock^∆19/∆19), as the oddity preference significantly differed from 0.33 (chance performance). However, Clock^∆19/∆19 mice performed worse than WT at 1 week post-myocardial infarction *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

Pressure myography of posterior cerebral arteries, neurobiology of HF. (a) At baseline, Clock^∆19/∆19 PCA have lower myogenic tone versus WT PCA at all transmural pressures >60 mmHg. In the HF mice, and compared to baseline, (b) WT PCA exhibit increased myogenic tone at transmural pressures of 40 mmHg and 60 mmHg, whereas (c) Clock^∆19/∆19 PCA have increased myogenic tone at all transmural pressures tested>20mmHg. (d) PCA of all groups respond similarly to phenylephrine, suggesting similar capability to respond. However, as compared to WT PCA, the Clock^∆19/∆19 PCA exhibit reduced myogenic tone at baseline, and (e) a greater delta change in myogenic tone in response to HF (e), supporting the notion that the circadian mechanism can influence responses in cerebrovasculature. (f) Summary of the different responses of WT PCA versus Clock^∆19/∆19 PCA. All PCA were collected during the middle of the animals’ wake period (Zeitgeber time (ZT19)). *P < 0.05, n = 6 WT PCA, n = 8 WT HF PCA, n = 7 Clock^∆19/∆19 PCA, n = 7 Clock^∆19/∆19 HF PCA. (g) The circadian mechanism is an important regulator of healthy cognitive system neurobiology. Neurobiological adaptations to HF differ in magnitude and direction in Clock^∆19/∆19 versus WT mice, including neuron morphology, visual-spatial memory and cerebrovascular myogenic tone, leading to convergence of end stage measures. These findings highlight the need to better understand how the circadian mechanism affects neurobiological adaptations to HF, a leading cause of morbidity and mortality worldwide. Large black double-sided arrow denotes comparison of normal Clock^∆19/∆19 and WT mice. Open white single sided arrows denote comparison of each genotype at baseline and in HF.

Figure 6

Gene expression in Clock^∆19/∆19 versus WT mPFC and hippocampus, and adaptations in response to HF. (a) Gene expression was assessed in Clock^Δ19/Δ19 and WT mPFC and hippocampus at baseline (BL), after MI, and in HF, by using microarrays and bioinformatics analyses. (b) Key Gene Ontology (GO) pathways identified for genes with ≥ 1.3-fold difference in Clock^Δ19/Δ19 versus WT mice, in the mPFC (dark boxes) or hippocampus (light boxes). (c) Baseline conditions. Circadian mechanism and output genes that differ in expression (≥ 1.3-fold change) in the mPFC (top) or hippocampus (bottom) for Clock^Δ19/Δ19 versus WT mice. (d) Response to MI. Genes that differ in expression (≥ 1.3-fold change) in the mPFC of Clock^Δ19/Δ19 versus WT mice. (e) Response to HF. Clock^Δ19/Δ19 versus WT genes that exhibit dichotomous expression at baseline, and then converge in HF, mPFC (top) or hippocampus (bottom). For images in Fig. 6, RFU  =  relative fluorescence units, *P < 0.05 by Student’s t-test, further information on genes shown is provided in Table 2, and all gene shown as well as additional identified genes are further detailed in Supplementary Table S1.