Sustained Hemodynamic Stress Disrupts Normal Circadian Rhythms in Calcineurin-Dependent Signaling and Protein Phosphorylation in the Heart

Abstract

Rationale: Despite overwhelming evidence of the importance of circadian rhythms in cardiovascular health and disease, little is known regarding the circadian regulation of intracellular signaling pathways controlling cardiac function and remodeling.Objective: To assess circadian changes in processes dependent on the protein phosphatase calcineurin, relative to changes in phosphorylation of cardiac proteins, in normal, hypertrophic, and failing hearts. Methods and Results:We found evidence of large circadian oscillations in calcineurin-dependent activities in the left ventricle of healthy C57BL/6 mice. Calcineurin-dependent transcript levels and nuclear occupancy of the NFAT (nuclear factor of activated T cells) regularly fluctuated as much as 20-fold over the course of a day, peaking in the morning when mice enter a period of rest. Phosphorylation of the protein phosphatase 1 inhibitor 1 (I-1), a direct calcineurin substrate, and phospholamban, an indirect target, oscillated directly out of phase with calcineurin-dependent signaling. Using a surgical model of cardiac pressure overload, we found that although calcineurin-dependent activities were markedly elevated, the circadian pattern of activation was maintained, whereas, oscillations in phospholamban and I-1 phosphorylation were lost. Changes in the expression of fetal gene markers of heart failure did not mirror the rhythm in calcineurin/NFAT activation, suggesting that these may not be direct transcriptional target genes. Cardiac function in mice subjected to pressure overload was significantly lower in the morning than in the evening when assessed by echocardiography. Key Words: calcineurin Ⅲ circadian rhythms Ⅲ heart failure Ⅲ RCAN1/MCIP1 C ircadian rhythms are self-sustaining, 24-hour cycles in molecular, biochemical, and behavioral parameters that help an organism prepare for anticipated changes in physiological demand. Many important cardiovascular factors, including metabolism, heart rate, blood pressure, and hormone release, oscillate over a 24-hour period. 1 In humans, the incidence of adverse cardiac events, such as myocardial infarction, ventricular tachycardia, and death from ischemic heart disease, vary according to the time of day. 2 Despite overwhelming evidence of the importance of circadian rhythms in cardiovascular health and disease, little is known regarding the circadian regulation of intracellular signaling pathways in the heart. The molecular basis of the circadian clock consists of cellautonomous, positive and negative transcriptional and posttranscriptional feedback loops. 3 The "master clock," located in the suprachiasmatic nucleus within the hypothalamus, influences the phase of independent molecular clocks found in peripheral organs, including the heart. Many cells and tissues also display circadian fluctuations in cytoplasmic Ca 2ϩ levels, although the source of these Ca 2ϩ oscillations and their relationship to the transcriptional clock mechanism is not fully understood. 4 Dysregulation of Ca 2ϩ handling is a hallmark of hear...

Calcineurin and its regulator, RCAN1, confer time-of-day changes in susceptibility of the heart to ischemia/reperfusion

“…Real-time PCR was carried out in a Roche 480 Lightcycler. Primers sequences were as reported previously [40]. Transcript levels were normalized to 18s rRNA.…”

“…Our laboratory recently demonstrated a strong circadian oscillation in protein and transcript levels of Rcan1.4 in the hearts of normal, healthy mice indicating a circadian pattern of activation of the cardioprotective calcineurin/ Rcan1.4 feedback loop [40]. The peaks in both transcript and protein levels occurred in the early morning coinciding with the transition to rest and the time of day when the murine heart is reported to be most resistant to damage from I/R [3].…”

Calcineurin signaling in the heart: The importance of time and place

“…The cardiovascular system displays significant circadian rhythms in many physiological processes and continual disruption of normal circadian rhythms predisposes humans to cardiovascular disease [270–274]. In a healthy rodent heart there is a circadian pattern of calcineurin activity that peaks at the end of the animal's active period and is at its lowest at the end of its rest period [275]. As much as a 20-fold change can be seen in nuclear translocation of NFAT, binding of NFAT to chromatin, and expression of the calcineurin/NFAT controlled gene, Rcan1.4 , over the course of twenty-four hours.…”

“…Therefore, although both sustained activation of calcineurin [4] and its complete absence [45] have pathological consequences, a diurnal pattern of calcineurin activation is compatible with heart health. The peak in calcineurin activity immediately precedes a time of day when the heart is less susceptible to damage from I/R [275, 276]. These diurnal changes in susceptibility appear to be mediated by changes in RCAN1 levels, as Rcan1 −/− mice no longer display this temporal window of protection [89].…”

Cardiomyocyte-Specific BMAL1 Plays Critical Roles in Metabolism, Signaling, and Maintenance of Contractile Function of the Heart

“…However, it is becoming increasingly clear that cardiovascular components exhibit time-of-day-dependent oscillations in their responsiveness to extrinsic factors, which in turn likely influence outcomes following pathologic events ( e.g. , myocardial infarction) (Collins and Rodrigo 2010; Durgan, et al 2010; Durgan, et al 2006; Durgan, et al 2011b; Durgan and Young 2010; Sachan, et al 2011). Currently, the relative contribution of extrinsic versus intrinsic factors towards rhythms in cardiovascular function and dysfunction remains undefined.…”