These studies provide several important new insights into the dynamics and consequences of calcineurin signaling in the heart. First, we present evidence that there is a circadian rhythm in calcineurin activity in a normal, healthy mouse heart. This assertion is supported by five different assessments of calcineurin activity: RCAN1.4 protein levels, Rcan1.4 mRNA levels, NFATc1 nuclear translocation, NFATc1 occupancy of the Rcan1.4 promoter, and phosphorylation levels of the calcineurin substrate I-1Ser67. Second, although activation of calcineurin has been viewed primarily as a stress response driving pathological hypertrophic remodeling, these daily increases in calcineurin activity are not associated with transcriptional changes indicative of pathological remodeling. Third, calcineurin activity oscillates out of phase with phosphorylation of proteins that promote cardiac contractility. Fourth, cardiac function in failing hearts shows diurnal variation. Finally, in failing hearts, calcineurin activity is elevated above normal peak activity throughout the day. Although a pronounced circadian oscillation in calcineurin-dependent activities persists, the amplitude of circadian variation declines correlating to a decline in cardiac function. Circadian rhythmicity in the phosphorylation of the regulatory proteins I-1 and PLB is lost in hypertrophic and failing hearts. Based on our findings we propose that circadian oscillations provide temporal separation of kinase and phosphatase activities that help to regulate changes in cardiac function in response to physiological demand and that this temporal relationship is disrupted when the heart is placed under sustained hemodynamic load.
In nocturnal animals, the light to dark transition at CT12 is anticipated by a rapid increase in physical activity, blood pressure, and β-adrenergic drive,24
all factors shown to increase calcineurin activity in cardiomyocytes. We therefore expected Rcan1.4
expression to increase in parallel with these physiological parameters at dusk. Instead, there was a gradual increase in Rcan1.4
expression throughout the course of the night suggesting a progressive activation of calcineurin that peaked at dawn when β-adrenergic drive is minimal in a mouse. depicts a model of how calcineurin activity could crosstalk with cardiac function via dephosphorylation of I-1, thus promoting reversal of phosphorylation-driven increases in cardiac contractility as discussed in the introduction. The findings we present here document that in a normal heart, circadian control provides temporal separation of these opposing processes as schematized in .
Figure 8 The model in (A) depicts interacting pathways through which PKA, PP1 and calcineurin (Cn) can influence calcium handling and contractility. Please refer to text for details. Solid arrows indicate direct interactions. Dashed arrows indicate multi-step (more ...)
The peak in NFAT activity and Rcan1.4
transcription at dawn (CT0) implies that calcineurin activity and cytoplasmic Ca2+
levels are also maximal at this time of day. Conversely, Ca2+
levels are likely lowest at dusk (CT12) when these indicators of calcineurin activity are lowest. Oscillating out of phase with calcineurin-dependent activities is phosphorylation of I-1 and PLB in response to increased β-adrenergic drive as the animal wakes. Phosphorylation of PLB, releases inhibition of SERCA2 increasing the rate of Ca2+
uptake, consequently lowering resting cytoplasmic Ca2+
levels, and potentially helping to maintain calcineurin in an inactive state. Eventually, cardiomyocytes become desensitized to prolonged β-adrenergic stimulation leading to a decrease in I-1 and PLB phosphorylation by PKA. Restored inhibition of SERCA2 by PLB would cause a gradual rise in cytoplasmic Ca2+
consistent with a gradual increase in calcineurin activity during the second half of the night. We postulate that the peak in calcineurin activity at the dark to light transition (CT0) helps maintain PLB and other key proteins in an unphosphorylated state as the mouse transitions to a time of lower cardiac demand. Conversely, a trough in calcineurin activity around dusk (CT12) would allow maximal β-adrenergic responses when the animal enters its waking hours. Consistent with this, fractional shortening of sTAC mice was higher in the evening than in the morning () potentially reflecting circadian differences in the adrenergic response of the unanaesthetized mice to handling. Likewise, a recent study demonstrated circadian differences in the response of isolated adult cardiomyocytes to β-adrenergic stimulation.25
Although in this study we used changes in Rcan1.4
transcript levels primarily as an indication of changes in calcineurin activity, the RCAN1 protein itself likely contributes to shaping the dynamics of calcineurin-dependent signaling. It is interesting to note that cardiac damage from ischemia-reperfusion (I/R) is greater in mice lacking Rcan1
than in wild type hearts.26
Furthermore, I/R damage in wild type mice has been shown to be greater when the procedure is performed at CT12 than when I/R is performed at CT0.27
This time of greater susceptibility corresponds to the time when we find RCAN1.4 protein levels in the heart are lowest.
An important short-coming of the current study is our inability to monitor changes in I-1Thr35
phosphorylation and the as yet incomplete understanding of the cumulative effect of changes in I-1 phosphorylation at other sites including Ser67
. Taking these limitations into account, the model in suggests that daily oscillations in Ca2+
handling and calcineurin activity form interdependent positive and negative feedback loops typical of circadian rhythms that result in a separation between times of day when kinase activities predominate and times of day when phosphatase activities predominate. Clearly this simple model does not take into account many additional factors that can influence calcineurin activity, PP1 activity, and cardiac contractility. For instance, the density of β-adrenergic receptors, adenylyl cyclase activity, and phosphodiesterase activity have all been shown to undergo circadian cycling both in neurons and the heart.28
Sustained hemodynamic stress elicits increases in both calcineurin activity and β-adrenergic drive. The model in suggests that these are opposing processes. Our data indicates that in hypertrophy and failure the normal temporal separation of calcineurin activity and phosphorylation of contractile proteins is disrupted. PLB phosphorylation was elevated throughout the day even during the animal’s sedentary period. In contrast, I-1Ser67
phosphorylation was completely lost, consistent with increased calcineurin activity. A corresponding loss of I-1Thr35
phosphorylation would release PP1 inhibition and result in uncoupling of circadian changes in calcineurin activity from regulation of PLB phosphorylation (). Initially, release of I-1 inhibition would be compensatory, helping to reverse hyper-phosphorylation of regulatory proteins, however, sustained loss of a circadian pattern of PP1 inhibition could ultimately contribute to declining cardiac function. Consistent with our observations, patients with heart failure demonstrate an increase in PP1 activity coincident with a decrease in I-1 phosphorylation.29, 30
The present studies provide a deeper understanding of the dynamics of calcineurin regulation in the heart and draw attention to the need to control for normal, underlying circadian changes in the activity of intracellular signaling pathways. In a human heart, it is likely that calcineurin activity cycles with a reverse phase compared to a mouse heart although as yet this is not known. In humans there is disproportionate ischemic activity, arrhythmic activity and acute cardiovascular events in the first few hours after waking.31
Interestingly this would correspond to the time of day when calcineurin activity would be lowest allowing maximal β-adrenergic responsiveness in a normal heart. Elevation of the trough in calcineurin activity in heart failure would compromise the ability to respond appropriately. There is a growing appreciation for the potential therapeutic benefit of timing drug delivery to correlate with maximal biological need.32
Our findings highlight the importance of remaining mindful of inherent circadian oscillations in the cardiovascular system during both the study and treatment of heart disease.
Novelty and Significance
What is known?
Circadian rhythms are important for maintaining cardiovascular health.
Activation of the protein phosphatase calcineurin is known to promote cardiac hypertrophy and heart failure.
What new information does this article contribute?
In healthy hearts, there is an apparent circadian rhythm in calcineurin activity that oscillates out of phase with phosphorylation of proteins, such as phospholamban (PLB) and inhibitor I (I-1), that regulate cardiac contractility.
In heart failure, calcineurin activity increases but continues to cycle, whereas cycling of PLB and I-1 phosphorylation is lost.
Despite overwhelming evidence that circadian rhythms are important in cardiovascular health and disease, little is known regarding circadian regulation of intracellular signaling pathways that control cardiac function and remodeling. Activation of calcineurin is known to promote pathological cardiac remodeling. Here we present evidence that there is a circadian rhythm in calcineurin activity in normal, healthy hearts that is not associated with transcriptional changes indicative of pathological remodeling. Calcineurin activity oscillates out of phase with phosphorylation of proteins, such as PLB and I-1 that promote cardiac contractility. In heart failure, calcineurin activity increases but continues to cycle, whereas cycling of PLB and I-1 phosphorylation is lost. We propose a model in which daily oscillations in Ca2+ handling and calcineurin activity form interdependent positive and negative feedback loops typical of circadian rhythms that result in separation between times of day when kinase activities predominate and times of day when phosphatase activities predominate. These studies provide a deeper understanding of the dynamics of calcineurin regulation in the heart and draw attention to the importance of normal, underlying circadian changes in regulating the activity of intercellular signaling pathways.