Regulation Of Peripheral Cardiac Clock Gene Expression By Chronic β-adrenergic Stimulation

Background:Ventricular arrhythmias in heart failure exhibit distinct circadian rhythm. Elevated sympathetic tone is the main pathophysiologic mechanism of chronic heart failure and arrhythmias. Cardiac resynchronization therapy (CRT), as the standard therapy of chronic heart failure, improves cardiac function and meanwhile restores sympathovagal balance and reduces the occurrence of ventricular arrhythmias. Emerging evidence has shown the relationship between aberrant clock genes and dysregulated ion channels, thus leading to lethal arrhythmias. The association of autonomic nerve activity with peripheral cardiac clock genes was not well elucidated.Objective:To explore the regulation of peripheral cardiac clock genes by chronic β-adrenergic stimulation.Methods:1. We used isoproterenol (Iso)-induced cardiac hypertrophy in mice and collected heart tissues every four hours. Real-time PCR was applied to detect the expression of clock and calcium-regulated genes. Western blot was used to measure the protein expression.2. Holter was monitored in CRT patients before implantation and at follow-up. Venous blood was collected at 06:00,11:00,18:00 and 23:00 on the same day of holter monitoring. Plasma cortisol and catecholamines were measured, and clock genes in peripheral blood or mononuclear cells were determined. Pearson analysis was done between clock gene expression and heart rate variability or cardiac function index.3. H9c2 cells were cultured and stimulated by Iso for 24 hours. Clock genes were detected by PCR. Then, Iso, CGP-20712A (a selective β1-AR blocker) or ICI118551 (a selective β2-AR blocker) was used to stimulate NIH3T3 cells and luciferase activities were measured by luciferase reporter assays.Results:1. Expression of clock genes in hypertrophic hearts was altered. The peak expression of BMAL1 at 07:00 was down-regulated and peak expression of PER1, PER2 and CRY1 at 19:00 was up-regulated in hypertropic hearts. For calcium-regulated genes, expression of ASPH was significantly reduced at 23:00 and RYR expression was markedly elevated at 15:00-23:00. Protein levels of Bmall and Junctin (encoded by ASPH) were consistently decreased.2. Two groups of CRT patients were included. One group was new implanted (12 patients, all responders) and the other was super-responder group (16 patients). There were 10 and 16 patients with dilated cardiomyopathy in the two groups, respectively. Circadian rhythm of plasma cortisol was apparently similar before or after the implantation. Heart rate variability was improved in CRT responders or super-responders. Expression of PER1 showed obvious circadian rhythm with peak at 06:00 and trough at 18:00. Amplitude of PERI expression was associated with high frequency value and left ventricular ejection fraction.3. After stimulation of Iso in H9c2 cells, circadian phase of BMAL1 expression was significantly advanced and the peak expression of PERI and PER2 was increased compared with serum shock group. For NIH3T3 cells, luciferase’activities of BMAL1, PER1 and CRY1 were elevated in Iso (200μM) group, inhibited in ICI 118551 (100μM) group, but not affected in CGP-20712A (100μM) compared to control group. Pretreatment of ICI 118551 completely blocked Iso action. Iso increased activities of BMAL1 and PER1 in the context of pretreatment of CGP-20712A.Conclusions:1. Chronic β-adrenergic stimulation altered peripheral cardiac clock gene and calcium-regulated gene expression.2. Cardiac resynchronization therapy restored clock gene expression in peripheral blood of heart failure patients. Amplitude of circadian PER1 expression may be a marker of improvement of heart rate variability or cardiac function in super-responders.3. Isoproterenol probably changed peripheral clock gene expression by β2-adrenoceptor.