The circadian clock maintains cardiac function by regulating mitochondrial metabolism in mice

Abstract

Cardiac function is highly dependent on oxidative energy, which is produced by mitochondrial respiration. Defects in mitochondrial function are associated with both structural and functional abnormalities in the heart. Here, we show that heart-specific ablation of the circadian clock gene Bmal1 results in cardiac mitochondrial defects that include morphological changes and functional abnormalities, such as reduced enzymatic activities within the respiratory complex. Mice without cardiac Bmal1 function show a significant decrease in the expression of genes associated with the fatty acid oxidative pathway, the tricarboxylic acid cycle, and the mitochondrial respiratory chain in the heart and develop severe progressive heart failure with age. Importantly, similar changes in gene expression related to mitochondrial oxidative metabolism are also observed in C57BL/6J mice subjected to chronic reversal of the light-dark cycle; thus, they show disrupted circadian rhythmicity. These findings indicate that the circadian clock system plays an important role in regulating mitochondrial metabolism and thereby maintains cardiac function.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. H-Bmal1 −/− mice develop progressive congestive heart failure with age.

(A) Representative gross morphology of hearts from 12-week-old control and H-Bmal1−/− mice. (B) Ratios of heart weight to body weight (HW/BW) at 12 weeks of age (n = 6 per group). (C) Low-power views after H&E staining of transverse sections from control and Bmal1−/− hearts at 12 weeks of age. (D) LV lateral wall thickness determined in histological images used in (C) (n = 5 per group). (E) Echocardiographic analysis in 12-week-old control and H-Bmal1−/− animals (n = 8–9 per group). LV internal diameter at diastole (LVIDd) and at systole (LVIDs) and fractional shortening (FS) are shown in bar graph format. (F) Transcript expression levels of ANP and BNP in control and H-Bmal1−/− mice at 12 weeks of age (n = 6 per group). (G) Kaplan-Meier survival curves of control and H-Bmal1−/− animals (n = 31 per group). (H) Low-power views (top panels, scale bar: 1 mm) and high-magnification views (bottom panels, scale bar: 25 µm) of Masson's trichrome staining of heart sections from 33-week-old control and H-Bmal1−/− mice. (I) High-magnification views of H&E staining of lung (top panels) and liver (bottom panels) sections from the same animals used in (H). Scale bar: 50 µm. Data are the mean ± SEM. *P<0.05, **P<0.01, unpaired two-tailed Student's t-test.

Figure 2. Gene expression profiles of cardiac energy metabolism in H-Bmal1 −/− mice.

(A) Heat-map representations of several classes of metabolic genes detected in the expression analysis of Bmal1−/− hearts from 12-week-old animals. (B-D) Relative expression levels of representative genes regulating (B) fatty acid metabolism, (C) glycolysis/TCA cycle, and (D) ETC/OXPHOS are shown (n = 6 per group). Data are the mean ± SEM. *P<0.05, **P<0.01, unpaired two-tailed Student's t-test (B-D).

Figure 3. Mitochondrial abnormalities in the hearts of 12-week-old H-Bmal1 −/− mice.

(A) A heat-map representing the expression profiles of genes regulating mitochondrial structure and function in Bmal1−/− hearts. (B) Relative expression levels of genes associated with mitochondrial biogenesis, dynamics, and membrane potential in the heart tissue of control and H-Bmal1−/− animals (n = 6 per group). (C) Representative electron micrographs of sections taken from the left ventricular muscle from control and H-Bmal1−/− mice at two different magnifications. (D) Mitochondrial protein concentration in control and Bmal1−/− hearts (n = 8 per group). (E) Mitochondrial DNA to nuclear DNA ratio in control and Bmal1−/− hearts (n = 6 per group). (F-G) Enzymatic activities of (F) complex I and (G) complex IV in control and Bmal1−/− hearts (n = 8 per group). The activities of the mitochondrial respiratory enzymes are expressed either per milligram of tissue used for mitochondrial isolation (top panels) or per microgram of mitochondrial protein (bottom panels). (H) NAD+ and NADH concentrations in control and Bmal1−/− hearts (n = 6 per group). Data are the mean ± SEM. *P<0.05, **P<0.01, unpaired two-tailed Student's t-test.

Figure 4. Expression profiles of genes associated with pathologic ventricular remodeling in Bmal1 −/− hearts.

(A-D) Heat-map representations of genes involved in (A) the response to oxidative stress, (B) programmed cell death, (C) inflammation, and (D) fibrosis in hearts from 12-week-old Bmal1−/− mice.

Figure 5. Circadian desynchronization reduces cardiac function in C57BL/6J mice with drug-induced cardiomyopathy.

(A) A light-dark (LD) cycle regimen used to examine the effects of a variable LD schedule on cardiac function. C57BL/6J mice were either maintained on a constant LD schedule (fixed LD cycle group) or were subjected to a 12-h phase shift in LD cycle every 3 days (disrupted LD cycle group). To compare the effects of the LD schedule between animals with healthy hearts and those with cardiomyopathy, either normal saline (NS) or phenylephrine (PE) was continuously infused via an osmotic pump in each LD cycle group. (B) Locomotor activity records of NS-infused (top panel) and PE-infused (bottom panel) mice. Two representative records from animals subjected to the fixed (left column) and disrupted (right column) LD cycle are shown. Activity counts are indicated by the vertical black marks. The records are double-plotted such that 48 h are shown for each horizontal trace. The blue shaded and unshaded areas indicate the dark and light period, respectively. The duration of NS or PE infusion is indicated by the vertical line at the right margin. (C) The ratios of heart weight to body weight (HW/BW) were increased by the PE infusion but were not influenced by the disruption in LD cycle (n = 5–8 per group). (D) The effects of PE, disrupted LD cycle, or both on ventricular function were evaluated using echocardiographic measurements (n = 5–8 per group). LVIDd, LVIDs, and FS are shown in bar graph format. Data are the mean ± SEM. *P<0.05, **P<0.01, two-way ANOVA.

Figure 6. Circadian desynchronization not only disrupts rhythms but also reduces the expression levels of clock and metabolic genes in the heart of C57BL/6J mice with PE-induced cardiomyopathy.

(A-C) Relative expression levels of genes regulating (A) clock machinery as well as (B) glucose and (C) lipid metabolism in heart. All heart tissues used were from PE-infused animals subjected to either a fixed or a disrupted LD cycle as described in Figure 5A (n = 4 per group per time point). To provide a 24-h overall mean expression level, the data over a 24-h time period in each group were also averaged and are expressed using a bar graph format. Data are the mean ± SEM. *P<0.05, **P<0.01, unpaired two-tailed Student's t-test.

Figure 7. Circadian desynchronization impairs mitochondrial function in the hearts of C57BL/6J mice with PE-induced cardiomyopathy.

(A-B) Relative expression levels of genes regulating (A) mitochondrial structure or (B) mitochondrial oxidative metabolism in heart. All heart tissues used are from PE-infused animals subjected to either a fixed or a disrupted LD cycle as described in Figure 5A (n = 4 per group per time point). To provide a 24-h overall mean expression level, the data over a 24-h time period in each group were also averaged and are expressed in bar graph format. (C) Enzymatic activity of complex I in PE-infused animals exposed to a fixed or disrupted LD cycle (n = 4–5 per group). The complex I activity is expressed per milligram of tissue used for mitochondrial isolation. (D) The relative number of mitochondria in the left ventricular muscle was counted using electron microscope images. Representative images are shown. Data are the mean ± SEM. *P<0.05, **P<0.01, unpaired two-tailed Student's t-test.