The circadian clock remains intact, but with dampened hormonal output in heart failure

Abstract

Background: Circadian (24-h) rhythms are important regulators in physiology and disease, but systemic disease may disrupt circadian rhythmicity. Heart failure (HF) is a systemic disease affecting hormonal regulation. We investigate whether HF affects the rhythmic expression of melatonin and cortisol, main endocrine products of the central clock, and cardiac-specific troponin in patients. We corroborate the functionality of the peripheral clock directly in the organs of translational models, inaccessible in human participants.

Methods: We included 46 HF patients (71.7% male, median age of 60 years, NYHA class II (32.6%) or III (67.4%), ischemic cardiomyopathy (43.5%), comorbidities: diabetes 21.7%, atrial fibrillation 30.4%), and 24 matched controls. Blood was collected at seven time-points during a 24-h period (totalling 320 HF and 167 control samples) for melatonin, cortisol, and cardiac troponin T (cTnT) measurements after which circadian rhythms were assessed through cosinor analyses, both on the individual and the group level. Next, we analysed peripheral circadian clock functionality using cosinor analysis in male animal HF models: nocturnal mice and diurnal zebrafish, based on expression of core clock genes in heart, kidneys, and liver, every 4 h during a 24-h period in a light/darkness synchronised environment.

Findings: Melatonin and cortisol concentrations followed a physiological 24-h pattern in both patients and controls. For melatonin, acrophase occurred during the night for both groups, with significantly decreased amplitude (median 5.2 vs 8.8, P = 0.0001) and circadian variation ([maximum]/[minimum]) in heart failure patients. For cortisol, mesor showed a significant increase for HF patients (mean 331.9 vs 275.1, P = 0.017) with a difference of 56.8 (95% CI 10.3-103.3) again resulting in a relatively lower variation: median 3.9 vs 6.3 (P = 0.0058). A nocturnal blood pressure dip was absent in 77.8% of HF patients. Clock gene expression profiles (Bmal, Clock, Per, Cry) were similar and with expected phase relations in animal HF models and controls, demonstrating preserved peripheral clock functionality in HF. Furthermore, oscillations in diurnal zebrafish were expectedly in opposite phases to those of nocturnal mice. Concordantly, cTnT concentrations in HF patients revealed significant circadian oscillations.

Interpretation: Central clock output is dampened in HF patients while the molecular peripheral clock, as confirmed in animal models, remains intact. This emphasises the importance of taking timing into account in research and therapy for HF, setting the stage for another dimension of diagnostic, prognostic and therapeutic approaches.

Funding: Hartstichting.

Keywords: Circadian rhythms; Cortisol; Human heart failure; Melatonin; Mouse; Zebrafish.

Fig. 1

24-hour hormonal output is intact in HFrEF patients. (a) Parameters used for quantitative analysis of the 24-h melatonin and cortisol variation. 24-hour (b) melatonin (pmol/L), (c) cortisol (nmol/L) and (d) cTnT (ng/L) concentration. Each dot (red = patients, blue = controls) represents individual concentration at a given time-point. Lines indicate median values of all patients (red; N = 44–46) and controls (blue; N = 23–24). Horizontal bar indicates day (=white) and night (=black). cTnT = cardiac troponin T; HFrEF = heart failure with reduced ejection fraction.

Fig. 2

24-hour expression of core clock genes in murine organs. 24-hour (a) Bmal1, (b) Clock, (c) Per1, and (d) Cry2 expression levels in murine heart, and (e, g) Bmal1 and (f, h) Per1 in (e, f) kidneys and (g, h) liver between sham and HF group (N = 3–5 mice/group/ZT; mean ± SEM), normalised against sham ZT1. Statistical significance of cosinor rhythmicity of delta Ct values per group is indicated at the top left graph corner: ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. Horizontal bar indicates lights-on (=white; ZT0) and lights-off (=black; ZT12) period. Bmal1 = Brain and Muscle ARNT-Like 1; Clock = Circadian Locomotor Output Cycles Kaput; Cry2 = Cryptochrome Circadian Regulator 2; HF = heart failure; Per1 = Period Circadian Regulator 1; ZT = zeitgeber time.

Fig. 3

24-hour expression profiles of core clock genes in zebrafish organs. 24-hour (a) arntl1b, (b) clocka, (c) per3, and (d) cry1ba expression levels in zebrafish heart, and (e) arntl1b and (f) per3 in kidneys between control and HF ((a–d) N = 9, (e) N = 3, (f) N = 6 zebrafish/group/ZT; mean ± SEM), normalised against control ZT0. Statistical significance of cosinor rhythmicity of delta Ct values per group is indicated at the top left graph corner: ∗P < 0.05, ∗∗∗∗P < 0.0001. Horizontal bar indicates lights-on (=white; ZT0) and lights-off (=black; ZT14) period. arntl1b = Aryl Hydrocarbon Receptor Nuclear Translocator Like 1 b. Other abbreviations as in Fig. 2.

Central Illustration

In HF, molecular clocks of heart, kidney and liver are preserved, with expression comparable to that of healthy controls. Conversely, HF negatively affects the central clock output: 24-h concentrations of melatonin and cortisol are dampened in HF, when compared to healthy controls. The prevalence of non-dipping BP and HR patterns in the HF population is high, correlating with lower nocturnal melatonin levels and higher nocturnal cortisol levels. HF patients suffer from poor subjective sleep quality and impaired activity. Altogether, these findings open various opportunities for developing new therapeutic (clock) interventions in order to improve the long-term quality of life and survival of patients suffering from HF, and warrant further investigations (e.g. melatonin supplementation, prognosis/prevention strategies based on non-dipping BP and HR, behavioural interventions for restoring subjective sleep quality and physical activity: light and exercise therapy, chronopharmacology). BMAL, Brain and Muscle ARNT-Like; BP, blood pressure; CLOCK, Circadian Locomotor Output Cycles Kaput; CRY, Cryptochrome Circadian Regulator; HF, heart failure; HR, heart rate; PER, Period Circadian Regulator; SCN, suprachiasmatic nucleus