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Hill and Beverly A. Rothermel Sikder, Victoria Copeland, Misook Oh, Erik Bush, John M. Shelton, James A. Bibb, Joseph A. Nita Sachan, Asim Dey, David Rotter, D. Bennett Grinsfelder, Pavan K. Battiprolu, Devanjan Calcineurin-Dependent Signaling and Protein Phosphorylation in the Heart Sustained Hemodynamic Stress Disrupts Normal Circadian Rhythms in Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 2011 American Heart Association, Inc. All rights reserved. is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research doi: 10.1161/CIRCRESAHA.110.235309 2011;108:437-445; originally published online January 13, 2011;Circ Res. http://circres.ahajournals.org/content/108/4/437 World Wide Web at: The online version of this article, along with updated information and services, is located on the http://circres.ahajournals.org/content/suppl/2011/01/13/CIRCRESAHA.110.235309.DC1.html Data Supplement (unedited) at: http://circres.ahajournals.org//subscriptions/ is online at: Circulation Research Information about subscribing to Subscriptions: http://www.lww.com/reprints Information about reprints can be found online at: Reprints: document. Permissions and Rights Question and Answer about this process is available in the located, click Request Permissions in the middle column of the Web page under Services. Further information Editorial Office. Once the online version of the published article for which permission is being requested is can be obtained via RightsLink, a service of the Copyright Clearance Center, not theCirculation Researchin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions: by guest on April 17, 2013http://circres.ahajournals.org/Downloaded from Sustained Hemodynamic Stress Disrupts Normal Circadian Rhythms in Calcineurin-Dependent Signaling and Protein Phosphorylation in the Heart Nita Sachan,* Asim Dey,* David Rotter, D. Bennett Grinsfelder, Pavan K. Battiprolu, Devanjan Sikder, Victoria Copeland, Misook Oh, Erik Bush, John M. Shelton, James A. Bibb, Joseph A. Hill, Beverly A. Rothermel 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. Conclusions: Normal, opposing circadian oscillations in calcineurin-dependent activities and phosphorylation of proteins that regulate contractility are disrupted in heart failure. (Circ Res. 2011;108:437-445.) Key Words: calcineurin � circadian rhythms � heart failure � RCAN1/MCIP1 Circadian rhythms are self-sustaining, 24-hour cycles inmolecular, biochemical, and behavioral parameters that help an organism prepare for anticipated changes in physio- logical demand. Many important cardiovascular factors, in- cluding 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 cell- autonomous, positive and negative transcriptional and posttran- scriptional 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 Ca2� levels, although the source of these Ca2� oscillations and their relationship to the transcriptional clock mechanism is not fully understood.4 Dysregulation of Ca2� handling is a hallmark of heart disease. Several Ca2�-responsive signaling pathways, including the pro- tein phosphatase calcineurin, have been causally linked to the progression of heart failure.5 Sustained activation of calcineurin Original received October 30, 2008; resubmission received October 26, 2010; revised resubmission received December 27, 2010; accepted January 3, 2011. In November 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.2 days. From the Departments of Internal Medicine (N.S., A.D., D.R., D.B.G., P.K.B., M.O., J.M.S., J.A.H., B.A.R.), Molecular Biology (J.A.H.), and Psychiatry, University of Texas Southwestern Medical Center (J.A.B.), Dallas; Sanford-Burnham Medical Research Institute (D.S.), Orlando, FL; GlycoFi (V.C.), Lebanon, NH; and Thermo Fisher Scientific (E.B.), Lafayette, CO. *Both authors contributed equally to this work. Correspondence to Beverly A. Rothermel, PhD, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-8573. E-mail beverly.rothermel@utsouthWestern.edu © 2011 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.110.235309 437 by guest on April 17, 2013http://circres.ahajournals.org/Downloaded from is sufficient to drive pathological hypertrophic remodeling of the myocardium with subsequent heart failure and premature death.6 Calcineurin is activated by an elevation in intracellular Ca2� and binding of a Ca2�/calmodulin complex. In a healthy heart, calcineurin is thought to be inactive and unresponsive to high- amplitude, high-frequency waves of Ca2� that drive contraction. Calcineurin is activated in response to stress presumably when either diastolic resting Ca2� or Ca2� in subcellular domains exceed a required threshold. Calcineurin has numerous substrates including the tran- scription factor NFAT (nuclear factor of activated T cells) through which calcineurin influences long-term changes in gene expression associated with pathological cardiac remod- eling.6 When NFAT is dephosphorylated, it translocates to the nucleus where it binds to and activates calcineurin- responsive genes. Among target genes is the exon 4 isoform of the Rcan1.4 (regulator of calcineurin 1), previously called MCIP1, DSCR1, or calcipressin. RCAN1 proteins are potent inhibitors of calcineurin activity.7 Expression of the mouse Rcan1.4 gene is extremely responsive to changes in cal- cineurin activity in vivo8; thus, altered Rcan1.4 transcript levels have been used as a sensitive indicator of changes in calcineurin activity in the heart and other tissues. Both Ca2� handling and cardiac myocyte contractility are regulated by changes in phosphorylation of key proteins.9 �-adrenergic stimulation activates the cAMP-dependent protein kinase (PKA), which increases cardiac output by phosphorylat- ing a number of proteins including phospholamban (PLB). This releases inhibition of the sarcoplasmic reticulum Ca2� ATPase (SERCA)2, thereby enhancing relaxation rate and contractility. PLB and other regulatory proteins are dephosphorylated by the protein phosphatase 1 (PP1). Phosphorylation of the PP1 inhib- itor-1 (I-1) by PKA at threonine 35 (I-1Thr35) prolongs �-adrenergic responses by inhibiting PP1, thus slowing dephos- phorylation of PLB.10 Changes in I-1 levels and/or phosphory- lation have been implicated in human heart failure and chronic atrial fibrillation.9,11 I-1 can be phosphorylated at other sites including Ser67.12 The in vivo consequence of phosphorylation at these various sites remains controversial, however, calcineurin can dephosphorylate both I-1Ser67 and I-1Thr35.12,13 In vitro and in vivo studies suggest that calcineurin activity can promote de- phosphorylation of PLB via regulation of I-1.14,15 Given the need for the heart to adapt to daily changes in cardiac demand and the potentially antagonistic roles of PKA and calcineurin, we asked whether calcineurin activity and/or PLB phosphorylation change over the course of 24 hours in a healthy heart. We found circadian oscillations in both these parameters that were directly out of phase with each other. We then tested what happens to these rhythms when both �-adrenergic activity and calcineurin activity increase in the pressure stressed myocardium. Methods An expanded Methods section is available in the Online Data Supple- ment at http://circres.ahajournals.org and provides expanded details for in situ hybridization, immunohistochemistry, chromatin immunopre- cipitation, quantitative RT-PCR, and Western blot analysis. Animal Procedures Male C57BL/6 mice were housed and fed under standard laboratory conditions with a strict 12:12 hour light:dark cycle with lights turning on at 6:00 AM, circadian time 0 (CT0), and off at 6:00 PM (CT12). For pressure-overload experiments, mice were subjected to thoracic aortic constriction (TAC) or severe (s)TAC for 3 weeks as described previously.16 Mice were shifted to constant darkness at the end of the normal light cycle for 24 hours before harvesting. Hearts were removed and the ventricles flash frozen within 30 seconds of euthanasia to preserve phosphorylation. A minimum of 3 animals was analyzed for each time point. A VisualSonics Vevo 770 imaging system was used to assess cardiac function in unanesthetized animals. The �MHC-RCAN1 mice were described previously,17 and wild-type littermates were used as controls. Surgically implanted miniosmotic pumps (Alzet, Palo Alto, CA) were used to deliver cyclosporine at a rate of 50 �g per hour per 25 kg of body weight. Results Changes in RCAN1.4 Protein and mRNA Levels Display Circadian Rhythmicity Biochemical assays of calcineurin activity are limited to mea- suring the potential activity of the entire cellular pool of calcineurin, rather than the fraction of the pool that was active in vivo. We therefore used multiple indirect methods to assess calcineurin activity. Initially, we quantified changes in both protein and mRNA levels of the Rcan1.4 gene, a direct target of calcineurin/NFAT. Male C57BL/6 mice were entrained to a 12:12 light:dark cycle then shifted to constant darkness at circadian time 12 (CT12) the day before samples were harvested for analysis. RCAN1.4 protein levels were highest in the heart at the beginning of the day (CT1 to CT3) and lowest at the end of the day (CT11 to CT13) (Figure 1A). In comparison, there were no significant changes in either the level of the exon 1 isoform of RCAN1 (RCAN1.1) or tubulin. A similar circadian pattern in RCAN1.4 protein levels was found in the hearts of 129/Sv and C3H/He inbred lines demonstrating that the oscillations were not strain dependent (data not shown). These findings are consistent with genome-wide microarray analysis identifying Rcan1 as having a circadian pattern of mRNA expression in the mouse heart.18 We found a 20-fold oscillation in Rcan1.4 mRNA levels with a peak at CT23 to CT1 and a trough at CT11 (Figure 1B) Non-standard Abbreviations and Acronyms ANF atrial natriuretic factor CT circadian time Glut4 high-affinity glucose transporter GSK glycogen synthase kinase I-1 protein phosphatase 1 inhibitor-1 I/R ischemia/reperfusion MHC myosin heavy chain NFAT nuclear factor of activated T cells Per2 period 2 PKA cAMP-dependent protein kinase PLB phospholamban PP1 protein phosphatase 1 RCAN1 regulator of calcineurin 1 SERCA sarcoplasmic reticulum Ca2� ATPase sTAC severe transverse aortic constriction TAC transverse aortic constriction 438 Circulation Research February 18, 2011 by guest on April 17, 2013http://circres.ahajournals.org/Downloaded from directly preceding the peak and trough in RCAN1.4 protein levels. In contrast, there were no significant circadian changes in the transcript levels of either Rcan1.1 or I-1 (Figure 1C and 1D). Thus, circadian regulation of Rcan1 expression is unique to the Rcan1.4 isoform and controlled at the level of transcript abun- dance. Transcription of the circadian clock gene Period 2 (Per2) oscillated with 24-hour periodicity (Figure 1E) verifying the presence of a functional clock in these samples. Calcineurin-Dependent Signaling Is Most Active in a Mouse Heart as the Animal Enters a Period of Decreased Physical Activity Immunohistochemical analysis for NFATc1 in the left ven- tricle revealed nuclear staining at 6:00 AM (CT0) (Figure 2A) but not at 6:00 PM (CT12) (Figure 2B). Although only a modest number of nuclei stained positive for NFATc1 even at the peak of activity, these positive nuclei were always Figure 1. Rcan1.4 mRNA and protein levels oscillate with 24-hour periodic- ity in the hearts of healthy mice. Sam- ples were pooled from 3 mice for each time point. A, Immunoblot analysis of RCAN1.4, RCAN1.1, and tubulin are shown. B through E, Real-time RT-PCR for Rcan1.4 (B), Rcan1.1 (C), I-1 (D), and Per-2 (E) mRNA levels were normalized to 18S rRNA. Trough values for each gene were set at a value of 1 (n�3 each time point in B through E). Figure 2. More NFATc1 is located in the nucleus and bound to chromatin at CT0 than at CT12. Left ventricular free-wall harvested at 6:00 AM (CT0) (A, C, and D) and 6 PM (CT12) (B) were stained with a FITC-labeled NFATc1 antibody (yellow/green) and propidium iodide (PI) (red). Cardiac myocytes have a high level of autofluorescence because of the abundance of mitochondrial flavins and fla- voproteins, which emit in a broad band overlapping the FITC-NFATc1 signal. In image C, the gain on the green channel has been turned down to obtain a clear outline of the nuclear PI signal. In D, the intensity of the green overlay has been restored, so that the autofluorescence of the sarcomere now obscures the myocyte-localized NFATc1-positive nuclei marked with arrows. A nonmyocyte nucleus is marked with an asterisk (*) and is not obscured. NFATc1 occupancy of the RCAN1.4 promoter was determined by chromatin immunoprecipitation from ventricular lysates using either preimmune IgG or NFATc1 antibodies (E) (n�4 each time point). In situ hybridization was carried out using an Rcan1.4-specific probe on the left ventricular wall harvested at 6:00 AM (CT12). Antisense probe (F) and sense probe (G) are shown. White bars denote 20 �m (A, B, F, and G) or 10 �m (C and D). Sachan et al Calcineurin Circadian Rhythms in the Heart 439 by guest on April 17, 2013http://circres.ahajournals.org/Downloaded from embedded within sarcomere-positive cells and never ob- served in nonmyocyte nuclei (Figure 2C and 2D). NFAT binding to the Rcan1.4 promoter was assessed by chromatin immunoprecipitation studies. A six-fold increase in NFAT occupancy of the Rcan1.4 promoter was detected at CT0 compared with at CT12 (Figure 2E), verifying that the circadian expression pattern of Rcan1.4 was driven by changes in NFAT nuclear translocation. An in situ hybridization specific for Rcan1.4 indicated that transcription was elevated uniformly across the wall of the myocardium (Figure 2F and 2G). Peak Rcan1.4 transcript levels were blunted in the hearts of mice with cardiomyocyte-specific expression of a transgene encoding RCAN1 to inhibit calcineurin (Figure 3A) or treated with the calcineurin inhibitor cyclosporine (Figure 3B). Taken as a whole, these results suggest that activation of the calcineurin/ NFAT signaling pathway occurs throughout the left ventricular myocardium and is greatest when the animal is entering its rest phase and cardiac demand decreases. Phosphorylation of I-1 and PLB Oscillates Out of Phase With Calcineurin Activity Immunoblot analyses were conducted to assess phosphoryla- tion of the calcineurin substrate I-1 at Thr35 and Ser67. Unfortunately, we were not able to detect phosphorylation of Thr35 in either heart extracts or forskolin-treated cells trans- fected with an I-1 expression construct (data not shown). There were, however, pronounced circadian changes in Ser67 phosphorylation. Phospho–I-1Ser67 was lowest in the morning (CT1 to CT8), increased notably at CT11 as the animals became active, and peaked at CT14 directly opposed to circadian changes in calcineurin activity (Figure 3C and 3D). In contrast, total I-1 protein (Figure 3C) and transcript levels (Figure 1D) did not change. Cardiac contractility and �-adrenergic drive are both higher at night in the hearts of nocturnal rodents.19,20 Anti- bodies specific for phospho-PLBSer16 showed a peak at CT14, coincident with the peak in I-1Ser67 phosphorylation (Figure 3C). The change in phosphorylation was even more pro- nounced when protein extracts were run such that PLB was maintained as a pentameric complex. Phospho-PLBSer16 and phospho-PLBThr17 were both elevated at CT14 compared with CT2 (Figure 4A, 4B, 4D, and 4E). This was evident in the slower electrophoretic migration of total PLB complexes from CT14 lysates (Figure 4C). Thus, the overall phosphor- ylation state of PLB was elevated during the period when calcineurin-dependent activities were lowest. Although we were not able to detect phosphorylation of I-1 at Thr35 we predict that phosphorylation of this site by PKA should parallel PLBSer16 phosphorylation. The kinase responsi- ble for I-1Ser67 phosphorylation in the heart has not yet been identified definitively. To test whether I-1Ser67 is phosphorylated Figure 3. Calcineurin-dependent tran- scription oscillates out of phase with protein phosphorylation. Rcan1.4 tran- script levels were quantified in the hearts of wild-type (WT) and �MHC-Rcan1 (TG) mice (A) or wild-type mice receiving either vehicle (V) or cyclosporine A (CsA) via a mini-osmotic pump (B) (n�4 each). Ventricular lysates collected from 3 dif- ferent wild-type hearts at the times indi- cated were pooled and probed for phospho–I-1Ser67, total I-1, phospho- PLBSer16, total PLB, phospho- GSK3Ser9, and total ERK1/2 (C). Signals from 3 experiments were quantified by densitometry (D and E). Figure 4. Increased PLB and I-1 phosphorylation occurs dur- ing times of increased adrenergic activity. Ventricular lysates isolated at CT2 and CT14 were probed for phospho-PLBSer16 (2 �g of protein per lane) (A), phospho-PLBThr17 (0.5 �g of protein) (B), or total PLB (0.5 �g of protein) (C) per lane. Phospho-PLBSer16 and phospho-PLBThr17 were quantified by densitometry (n�6 each) (D and E). In F and G, wild-type mice were injected with 8 mg/kg body weight of isoproterenol at CT10. Hearts were harvested at the indicated time points after injection. 440 Circ