Nuclear factors of activated T cells (NFATs) are Ca2+-sensitive transcription factors that have been implicated in hypertrophy, heart failure (HF), and arrhythmias. Cytosolic NFAT is activated by dephosphorylation by the Ca2+-sensitive phosphatase calcineurin, resulting in translocation to the nucleus, which is opposed by kinase activity, rephosphorylation, and nuclear export. Four different NFAT isoforms are expressed in the heart. The activation and regulation of NFAT in adult cardiac myocytes, which may depend on the NFAT isoform and cell type, are not fully understood. This study compared basal localization, import, and export of NFATc1 and NFATc3 in adult atrial and ventricular myocytes to identify isoform- and tissue-specific regulatory mechanisms of NFAT activation under physiological conditions and in HF. NFAT-green fluorescent protein fusion proteins and NFAT immunocytochemistry were used to analyze NFAT regulation in adult cat and rabbit myocytes. NFATc1 displayed basal nuclear localization in atrial and ventricular myocytes, an effect that was attenuated by reducing intracellular Ca2+ concentration and inhibiting calcineurin, and enhanced by the inhibition of nuclear export. In contrast, NFATc3 was localized to the cytoplasm but could be driven to the nucleus by angiotensin II and endothelin-1 stimulation in atrial, but not ventricular, cells. Inhibition of nuclear export (by leptomycin B) facilitated nuclear localization in both cell types. Ventricular myocytes from HF rabbits showed increased basal nuclear localization of endogenous NFATc3 and reduced responsiveness of NFAT translocation to phenylephrine stimulation. In control myocytes, Ca2+ overload, leading to spontaneous Ca2+ waves, induced substantial translocation of NFATc3 to the nucleus. We conclude that the activation of NFAT in adult cardiomyocytes is isoform and tissue specific and is tightly controlled by nuclear export. NFAT is activated in myocytes from HF animals and may be secondary to Ca2+ overload.
- nuclear factor of activated T cells
- intracellular Ca2+ concentration
- nuclear translocation
during pathological remodeling associated with hypertension, hypertrophy, heart failure (HF), and arrhythmias, the cardiovascular system experiences changes in gene transcription and protein expression. Nuclear factor of activated T cells (NFAT) transcription factors play a key role in cellular remodeling, integrating intracellular Ca2+ signals and gene transcription. NFAT is controlled by phosphorylation [e.g., by the kinases glycogen synthase kinase (GSK)3β, p38, and JNK], and its phosphorylation status regulates its translocation into the nucleus. Intracellular Ca2+ signals stimulate the Ca2+/calmodulin-dependent phosphatase calcineurin (CaN), which dephosphorylates NFAT, causing translocation to the nucleus, where it regulates gene transcription (4, 43, 59). Rephosphorylation of NFAT causes NFAT nuclear export and relieves its transcriptional effect. Cardiac myocytes display large rhythmic changes in intracellular Ca2+ concentration ([Ca2+]i) with every heart beat (5). This raises the question of how NFAT is activated in a Ca2+-dependent fashion either normally or during pathological situations (5, 35, 57). Several mechanisms have been hypothesized to selectively activate CaN and NFAT dephosphorylation [e.g., Ca2+ fluxes through voltage-gated L-type Ca channels and Ca2+-induced Ca2+ release (CICR) from ryanodine receptor (RyR) sarcoplasmic reticulum (SR) Ca2+-release channels, Ca2+ entry via T-type Ca2+ channels, store-operated Ca2+ entry, and inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release; for a review, see Ref. 58]. However, none of these pathways has been demonstrated experimentally in conclusive and unequivocal ways. Nonetheless, several recent reports have shed new light on two Ca2+ signaling mechanisms that appear to be involved in the activation of NFAT in diseased cardiomyocytes: first, under conditions of tachycardia, the high frequency of action potentials can cause a net intracellular Ca2+ gain that increases [Ca2+]i and can activate the CaN/NFAT pathway (30). Two more recent studies (42, 61) have revealed a potential link between NFAT activation and remodeling of ion channels [L-type Ca2+ channels and voltage-gated K+ channels (transient outward K+ current)] in canine myocytes in response to high-frequency pacing. Second, chronic activation of Gq protein-coupled receptors by neurohumoral agonists [e.g., angiotensin (ANG) II and endothelin (ET)-1] may represent an alternative mechanism for NFAT activation observed during hypertrophy (32, 36). Gq proteins stimulate phospholipase C, which generates the second messengers IP3 and diacylglycerol. IP3 promotes Ca2+ release from intracellular stores by activating specific Ca2+-release channels of the SR (IP3 receptors). IP3-mediated Ca2+ release could either act as a Ca2+ source independent from RyR-mediated Ca2+ release or modulate RyR-dependent CICR, resulting in arrhythmogenic Ca2+ release and elevated systolic [Ca2+]i (63). The application of ET-1 has been shown to generate cytoplasmic Ca2+ signals that affect the nucleus and activate NFAT, thereby inducing hypertrophy in ventricular myocytes (23). In addition, IP3-dependent Ca2+ signaling also occurs locally at the nuclear envelope and affects the nuclear Ca2+ concentration, which, in turn, controls many cellular functions in cardiac cells (22, 27), including the regulation of transcription factors (60).
The mammalian heart expresses four different NFAT isoforms (NFATc1, NFATc2, NFATc3, and NFATc4), which are regulated by [Ca2+]i via CaN (54). Open questions regarding our understanding of NFAT regulation in adult cardiac myocytes are: Is there a common mechanism that activates all NFAT isoforms in cardiac cells, or are there isoform-specific or even tissue-specific (atrium vs. ventricle) differences? Is NFAT solely regulated via the balance of cytosolic phosphatase (CaN) and kinase activity, or are regulated nuclear export pathways and nuclear kinases involved in NFAT activation/deactivation?
The present study analyzed basal and agonist-induced (ET-1 and ANG II) activation of NFATc1 and NFATc3 in quiescent myocytes (measured as nuclear localization of NFAT) using NFAT-green fluorescent protein (GFP) fusion proteins in conjunction with confocal microscopy. We found isoform- and tissue-specific differences in the activation of NFATc1 and NFATc3 in adult cardiac myocytes as well as increased basal activity of NFATc3 in myocytes from failing hearts.
MATERIALS AND METHODS
Isolation, Cell Culture, and Viral Transduction of Cardiac Myocytes
Atrial and ventricular myocytes were isolated from cat or rabbit hearts as previously described (26, 50). The procedure for cell isolation was approved by the Institutional Animal Care and Use Committees. Briefly, atrial and ventricular myocytes were isolated from cat or rabbit hearts using animals of either sex. Animals were anesthetized with thiopental sodium (35 mg/kg ip). After a thoracotomy, the hearts were quickly excised, mounted on a Langendorff apparatus, and retrogradely perfused via the aorta. After an initial washing step with an oxygenated Ca2+-free solution [which contained (in mM) 137 NaCl, 5.4 KCl, 1.0 MgCl2, 12 NaHCO3, 0.6 NaH2PO4, and 11 glucose], the heart was perfused with oxygenated HEPES-buffered saline solution (HBSS) containing 36 μM Ca2+ and collagenase at 37°C (0.06% collagenase type II, Worthington Biochemical, Freehold, NJ) to obtain single myocytes. Isolated cells were adapted to the final Ca2+ concentration of the medium (1.8 mM) over a period of 2 h and plated on sterile, laminin-coated glass coverslips. Myocytes were cultured using serum-free medium 199, which was supplemented with 25 μg/ml gentamycin and 25 μg/ml kanamycin (all from Mediatech, Hernon, VA). One day after isolation, cells were infected with adenoviruses encoding for NFATc1-GFP and NFATc3-GFP, and experiments were performed 24 h (ventricular myocytes) or 48 h (atrial myocytes) after infections. In addition, ventricular myocytes were isolated from rabbits with nonischemic HF induced by combined aortic insufficiency and stenosis (38, 39). This HF model is characterized by the combination of the gradual development of hypertrophy and HF (over 4–9 mo, as monitored by serial echocardiography), depressed systolic function, and arrhythmogenesis in an animal that has human-like Ca2+ handling and cellular electrophysiological properties. The echocardiographic index of severe left ventricular dysfunction is a left ventricular end-systolic dimension of >1.4 cm (>40% increase). This model has been well characterized on structural, biochemical, molecular, Ca2+ handling, and electrophysiological levels (14, 15, 38, 40, 41, 49).
Solutions and Chemicals
During all experiments, cells were bathed in an extracellular solution (HBSS), which contained (in mmol/l) 135 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES (pH 7.3 with NaOH). Agonists or antagonists were prepared in HBSS for acute application or added to the medium for longer incubations. Unless otherwise stated, all chemicals, agonists, or inhibitors were purchased from Sigma (St. Louis, MO) or Tocris (Ellisville, MO).
Measurements of NFAT-GFP.
To analyze the subcellular localization of NFAT-GFP with confocal microscopy (Biorad Radiance 2000/MP) in virally transduced myocytes, we measured the mean fluorescence of a region of interest (ROI) covering the nucleus (NFATnuc) and a cytoplasmic ROI (NFATcyt) of the same size. The subcellular distribution of NFAT was then quantified as the ratio of NFATnuc to NFATcyt. In some experiments, where agonists or antagonists were applied acutely, this ratio was normalized to the ratio measured from the same cell before the stimulus application. GFP was excited with an argon ion laser (wavelength: 488 nm), and emitted fluorescence was collected at 500–520 nm.
Intracellular Ca2+ measurements.
Intracellular Ca2+ transients from single myocytes evoked by electrical field stimulation (0.5 Hz) or [Ca2+]i signals evoked by Ca2+ overload were measured with rhod-2 and confocal microscopy in the line-scan mode (3 ms/line, pixel size: 1.5 μm). Briefly, cells were loaded for 20 min at room temperature with 5 μM rhod-2 AM and 5 μM Pluronic F-127 in HBSS (Pluronic stock solution: 0.2 g/ml DMSO). After the removal of excessive dye, rhod-2 was excited at 543 nm (green He-Ne laser), and emitted fluorescence was recorded at a wavelength of ≥570 nm. Changes in rhod-2 fluorescence were normalized to the level of fluorescence before stimulus application. Rhod-2 was chosen as the Ca2+-sensitive dye because it allows for measurements of [Ca2+]i in the presence of GFP (46).
The preparation and immunofluorescence staining of rabbit ventricular myocytes were carried out as previously described (46). Myocytes were plated on laminin-coated coverslips and washed with solution A (450 mM NaCl, 20 mM phosphate buffer, pH 7.2). Cells were fixed for 30 min using 4% (wt/vol) paraformaldehyde in 200 mM phosphate buffer (pH 7.4). Excessive paraformaldehyde was washed out using solution A, and cells were permeabilized using solution B (0.3% Triton X-100, 450 mM NaCl, and 20 mM phosphate buffer; pH 7.2) supplemented with 1% goat serum. Cells were then incubated with goat polyclonal antibody C-20 against NFATc3 (sc-1152, Santa Cruz Biotechnology, Santa Cruz, CA) using a 1:300 dilution. For visualization, an Alexa fluor488 donkey anti-goat secondary antibody (A11055, Invitrogen, Carlsbad, CA) was used at a 1:500 dilution. Fluoromount G was used as a mounting medium.
Data Analysis and Presentation
Data are presented as individual observations or as means ± SE and were analyzed using a Student's t-test. n represents the number of individual cells, and differences were considered significant at P < 0.05.
Localization of NFAT in Resting Myocytes Is Isoform Specific
The subcellular distribution of NFAT-GFP in resting (extracellular Ca2+ concentration: 2 mmol/l) adult cat myocytes was analyzed with confocal microscopy 24–48 h after infections. NFATc1-GFP was predominantly localized to the nucleus of atrial and ventricular cells (Fig. 1, A,a and A,c). Average NFATc1nuc-to-NFATc1cyt ratios were 1.71 ± 0.31 (n = 31) for atrial myocytes and 2.50 ± 0.12 (n = 22) for ventricular myocytes. In contrast, NFATc3-GFP displayed cytoplasmic localization under basal conditions, as indicated by lower average NFATc3nuc-to-NFATc3cyt ratios of 0.54 ± 0.05 (n = 17) and 0.51 ± 0.04 (n = 34) for atrial myocytes (Fig. 1A,b) and ventricular (Fig. 1A,d) myocytes, respectively.
Adult myocytes in culture may be subject to morphological and functional changes that are characterized by the loss of t-tubules and insensitivity to electrical excitability (20, 33, 34, 55) and may affect Ca2+ handling. To test for functional changes of excitation-contraction coupling and CICR, we measured action potential-evoked intracellular Ca2+ transients from myocytes expressing NFATc1-GFP using the Ca2+-sensitive dye rhod-2. Cultured myocytes responded to electrical field stimulation (0.5 Hz) with Ca2+ transients that were typical for atrial cells (Fig. 1B, top) or ventricular cells (Fig. 1B, bottom), indicating no significant functional changes due to time in culture.
Pharmacological Manipulation of NFATc1 in Cardiac Myocytes
If the nuclear localization of NFATc1 is due to basal Ca2+-dependent CaN activity, then reduction of [Ca2+]i or inhibition of CaN with cyclosporin A (CsA) would be expected to shift NFATc1 toward the cytoplasm (i.e., decrease the NFATnuc-to-NFATcyt ratio). Indeed, the acute application of CsA (1 μmol/l) in Ca2+-free HBSS for 60 min resulted in the redistribution of NFATc1 to the cytoplasm in atrial cells (Fig. 2, A,c and A,d), which was quantified as a decrease in the NFATnuc-to-NFATcyt ratio by up to 50% of the initial level (summary data shown in Fig. 2A,e); however, NFATc1 retained the preferential nuclear localization.
The observation that nuclear localization of NFAT is reversible indicates the involvement of a nuclear export process for NFAT. It has been suggested that the nuclear export of NFAT involves the transport protein Crm1 (exportin 1), which can be inhibited by leptomycin B (LB) (28). The application of LB for 60 min resulted in an even higher NFATnuc-to-NFATcyt ratio for NFATc1 (Fig. 2, A,a, A,b, and A,e). This indicates that in the basal state there is some dynamic balance of NFATc1 import (driven by CaN activity) and export via a Crm1-dependent pathway (as opposed to a maximal nuclear concentration).
These observations were not restricted to atrial tissue, since LB also increased the steady state ratio by a factor of ∼1.8 (Fig. 2, B,a and B,b), and CaN inhibition (and reduction of [Ca]i) caused nearly a 50% reduction in the ratio (Fig. 2, B,c and B,d) in cat ventricular myocytes.
Kinase activity and rephosphorylation of NFAT can also influence NFAT localization in ventricular myocytes (4, 9, 53). When we inhibited the cellular kinases GSK3β with 1 μmol/l alsterpaullone or JNK2 using 1 μmol/l SP-600125 in ventricular cells by incubations overnight, there was a substantial nuclear accumulation of NFATc1, as indicated by an increase in the NFATnuc-to-NFATcyt ratio from 2.10 ± 0.08 (control, n = 34) to 4.17 ± 0.32 (SP-600125, n = 20) and 5.28 ± 0.36 (alsterpaullone, n = 24).
These data suggest that the CaN/NFATc1 pathway has a high basal activity in resting atrial myocytes and even more so in ventricular myocytes. Nonetheless, nuclear kinase activity and export processes can shift the nuclear/cytosolic localization of NFATc1 in adult cardiac myocytes.
Activation of NFATc3 by Neurohumoral Stimuli
The NFATc3 isoform has been shown to play an important role in hypertrophy and HF-related remodeling in the cardiovascular system (11, 19, 48, 61). In contrast to NFATc1, we found NFATc3 to be localized to the cytoplasm of resting atrial and ventricular myocytes (cf. Fig. 1A) under basal conditions. We tested whether the hypertrophy-related extracellular agonists ANG II or ET-1 were capable of inducing the translocation of NFATc3 to the nucleus. These agonists activate the Gq protein/IP3 pathway and liberate Ca2+ from the SR via IP3 receptor Ca2+-release channels (44, 63), a pathway that has been linked to the activation of transcription factors in myocytes (22, 60). Atrial myocytes expressing NFATc3-GFP were incubated overnight in medium containing 2 μmol/l ANG II or 100 nmol/l ET-1. Both agonists induced the nuclear translocation of NFATc3, which was quantified as a two- to threefold increase in the NFATnuc-to-NFATcyt ratio (representative images are shown in Fig. 3A; summary data are shown in Fig. 3B). The degree of agonist-induced translocation was comparable with nuclear accumulation induced by LB (Fig. 3B). The combination of agonist stimulation and block of nuclear export further enhanced the nuclear accumulation of NFAT (4- to 5-fold increase of the NFATnuc-to-NFATcyt ratio compared with control). Shorter incubation times (up to 4 h) did not result in detectable changes in the subcellular distribution of NFATc3 (data not shown).
In striking contrast to atrial myocytes, stimulation with ANG II and ET-1 failed to induce the nuclear accumulation of NFATc3 in ventricular myocyes (Fig. 4, A and B). However, the inhibition of nuclear export (40 nmol/l LB) resulted in the substantial nuclear accumulation of NFATc3-GFP in time-matched parallel cultures (Fig. 4B). Combined incubation with ANG II and LB did not induce further nuclear accumulation of ventricular NFATc3, in contrast to observations in atrial cells (compare Fig. 3B).
Furthermore, the inhibition of cellular kinases (with alsterpaullone or SP-600125), an experimental intervention that induced the nuclear accumulation of NFATc1 in ventricular cells (see above), did not induce nuclear localization of NFATc3 in ventricular myocytes [NFATnuc-to-NFATcyt ratios: control, 0.66 ± 0.04 (n = 51); alsterpaullone, 0.65 ± 0.06 (n = 12); and SP-600125, 0.63 ± 0.07 (n = 13)].
Another notable observation was a strong fluorescence signal of NFATc3-GFP around the nucleus (Fig. 4C; see also Fig. 1A,a for atrial cells). As shown for ventricular cells at two different magnifications, NFATc3 did accumulate around the nucleus in nonstimulated cells (Fig. 4C). This finding might support the hypothesis that NFATc3 is regulated locally by IP3-dependent nuclear Ca2+ signals (see the discussion) (27).
Our data suggest that the activation and regulation of NFATc3 are different in atrial cells compared with ventricular cells. The activation of atrial (but not ventricular) IP3 pathways by hypertrophic, neurohumoral agonists appears to be capable of inducing the nuclear translocation of NFATc3. Comparable with the NFATc1 isoform above, nuclear NFATc3 accumulation is also limited by dynamic nuclear export mechanisms under basal conditions.
HF and Ca2+ Overload Induce Nuclear Localization of NFATc3
HF is characterized by profound changes in Ca2+ handling (38) and gene transcription (7). Since the activation of CaN depends on the sustained elevation of [Ca2+]i (59), a mechanism that results in Ca2+ overload would favor the activation of NFAT transcription factors under pathological conditions. NFATc3, in particular, is active during pathological situations, including atrial and ventricular fibrillation or the presence of hypertrophic extracellular agonists (23, 42, 61). We analyzed the nuclear localization of endogenous NFATc3 in ventricular cells from a chronic rabbit HF model (38) using immunocytochemistry (NFATc3-specific antibody). In ventricular myocytes from HF rabbits, nuclear localization of NFATc3 was enhanced compared with myocytes from normal rabbits [NFATnuc-to-NFATcyt ratios: 0.49 ± 0.014 (n = 37) vs. 0.40 ± 0.013 (n = 22), P < 0.002; Fig. 5A]. The application of phenylephrine (100 μmol/l) resulted in further nuclear accumulation of NFATc3 in ventricular cells from control rabbits but failed to facilitate nuclear localization in myocytes from HF animals (Fig. 5B). Similar to cat ventricular myocytes, ET-1 and ANG II, however, failed to induce translocation in rabbit ventricular cells (data not shown). Phenylephrine application in the presence of CsA prevented the agonist-induced nuclear translocation in control cells and reduced the NFATnuc-to-NFATcyt ratio below control levels in HF myocytes. These data suggest that the Ca2+/CaN pathway may be basally activated in ventricular myocytes from HF animals.
Cardiac myocytes from failing hearts develop irreversible changes in Ca2+ homeostasis due to altered function and/or the expression of several Ca2+-handling proteins, among them, the Na+/Ca2+ exchanger (NCX). In HF rabbits, alterations in [Ca2+]i contribute to systolic dysfunction and arrhythmogenesis (39, 40) and may also contribute to altered transcriptional regulation (7). To test whether diastolic [Ca2+]i elevation (e.g., at a high heart rate in HF) activates NFATc3 in ventricular myocytes from normal rabbits, we induced Ca2+ overload in myocytes expressing NFATc3-GFP by Na+,K+-ATPase inhibition (K+-free solution), which elevates the intracellular Na+ concentration and, in turn, [Ca2+]i [via NCX, leading to SR Ca2+ overload (18)]. Figure 6A,a shows the sustained elevation of [Ca2+]i in K+-free solution, which caused the translocation of NFATc3 to the nucleus (over 2 h) to a similar extent as LB in normal HBSS (Fig. 6A,b). This NFATc3 translocation was prevented by either blocking CaN with CsA or removing extracellular Ca2+ (Fig. 6A,b) and further enhanced by 40 nM LB. The further analysis of [Ca2+]i signals under Ca2+ overload conditions using higher temporal resolution (confocal line scan mode) indicated that these cells developed spontaneous Ca2+ release in the form of spontaneous Ca2+ waves and a net increase in diastolic [Ca2+]i (see Fig. 6B,a). In contrast, control cells did not show comparable changes in [Ca2+]i (control myocytes; Fig. 6B,b).
These data indicate that the nuclear localization of NFATc3 is regulated by a dynamic balance between import and export rates. Under normal conditions, a net nuclear export rate prevents the nuclear localization of NFATc3. A pathological Ca2+ signal (e.g., Ca2+ waves and elevated diastolic [Ca2+]i, as in Fig. 6) changes this balance to a net nuclear import, thereby stabilizing the nuclear localization of NFATc3 in cardiomyocytes.
Transcription factors of the NFAT family are involved in the pathological remodeling of cardiac myocytes (42, 61). The mammalian heart expresses four different Ca2+-sensitive isoforms of NFAT (NFATc1, NFATc2, NFATc3, and NFATc4) (54). Although Ca2+-dependent activation and regulation of NFAT have been investigated in detail in many cell types (10, 21, 59), it is not fully understood how NFAT is regulated in adult cardiac myocytes, which experience large changes in [Ca2+]i during every heart beat (5, 35, 57). Several Ca2+-dependent signals for the activation of NFAT have been proposed for neonatal and adult cardiac tissue, including contributions of extracellular Ca2+ (i.e., Ca2+ influx through L-type voltage-operated Ca2+ channels) (24, 42, 56) or local spatially restricted Ca2+ signals, such as nuclear Ca2+ and IP3-mediated Ca2+ release (27). The activation of NFAT in adult myocytes has been observed during high-frequency pacing (42, 61) or the application of neurohumoral stimuli (ET-1) that activate the IP3 pathway (23). In addition, recent evidence has suggested an upstream regulatory function of Ca2+/calmodulin-dependent kinase II (CaMKII) during NFAT activation by means of CaN phosphorylation (31, 61); however, the details of this type of regulation remain to be substantiated. The studies mentioned above have also indicated that the activation of NFAT is tissue specific and may also be regulated through pathways other than the Ca2+-dependent activation of CaN alone.
The present study focused on the analysis of basal and agonist-induced activation of NFAT (measured as nuclear translocation of NFAT) in adult myocytes. To visualize and analyze the subcellular localization of NFAT, we used NFAT-GFP fusion proteins (NFATc1 and NFATc3 isoforms) and quantified the subcellular localization of NFAT as the NFATnuc-to-NFATcyt ratio. NFAT-GFP fusion proteins are widely used to study NFAT in living cells because they behave similarly to endogenous proteins (13, 17, 25, 31, 46, 51).
We found that the NFATc1 isoform, but not the NFATc3 isoform, displayed nuclear localization in resting myocytes. To our best knowledge, this is the first study to compare these isoforms in adult cardiac myocytes. Consistent with the notion of the high activity of NFATc1 compared with other isoforms, a recent study on skeletal muscle cells has described the robust nuclear translocation of NFATc1 but only the transient nuclear localization of NFATc3 in response to electrical stimulation. In those cells, as well as in endothelial cells, it was found that NFATc3, but not NFATc1, was tightly controlled by nuclear export processes (46, 51).
The molecular mechanism for the basal nuclear localization of NFATc1 is not well understood. Transcriptional activity and the localization of NFAT in the nucleus are determined by binding DNA and other accessory transcription factors in the nucleus (52, 59). This mechanism would provide another layer of (isoform-specific) regulation of NFAT in the nucleus. Cardiac NFATc1 controls valve formation and morphogenesis of the heart, and disruption of this isoform results in lethal defects (12). Thus, the basal activity of NFATc1 in cardiac myocytes is likely to be required to maintain the differentiated phenotype of adult myocytes. This idea is supported by a more recent study that analyzed the role of different NFAT isoforms for the differentiation of skeletal muscle fibers. The study (8) demonstrated that the activation of a single NFAT isoform or the concerted activation of up to four NFAT isoforms controls the differentiation of skeletal muscle fibers into slow or fast fibers. However, it remains to be determined how exactly specific patterns of NFAT isoforms are activated differentially. It is conceivable that NFAT isoforms reside in distinct cytosolic domains and restricted Ca2+ signals activate CaN locally, that the spatiotemporal organization of the Ca2+ signal (e.g., steady-state elevation vs. oscillatory changes) is responsible for the activation of a specific isoform, or that different NFAT isoforms have different levels of sensitivity for dephosphorylation by CaN.
Another striking result of the present study is the specific activation of NFATc3 by the neurohumoral agonists ANG II and ET-1 in atrial, but not ventricular, myocytes from the cat. Our laboratory has previously demonstrated that the density of IP3 receptors is higher in atrial than ventricular myocytes (16) and that IP3 signaling strongly influences Ca2+ handling, excitation-contraction coupling, and arrhythmogenesis in atrial myocytes (29, 62, 63). While the more pronounced IP3-dependent Ca2+ release in atrial cells would support the hypothesis that this specific source of Ca2+ preferentially activates NFAT, further investigation will be required to clarify whether this difference in IP3 receptor signaling indeed represents the molecular mechanism responsible for the cellular differences in NFAT regulation or if there are other mechanisms involved, such as different expression levels of membrane surface receptors for these agonists.
We also observed a concentrated localization of NFATc3 around the nucleus. This observation supports the notion that cardiac NFAT and other Ca2+-dependent transcription factors are regulated by local, nuclear Ca2+ signals, independent from normal “beat-to-beat” Ca2+ (22, 27, 60). In support of this idea are the observations that the nuclear envelope is well equipped with IP3 receptors (3) and that IP3 can release Ca2+ from the cytosolic side of the nuclear envelope as well as into the nucleoplasm via IP3 receptors located in the inner membrane of the nuclear envelope (62). Thus, IP3-mediated Ca2+ release can act locally around the nucleus and affect nuclear envelope [Ca2+] and the nuclear Ca2+ concentration (62, 63), which could represent the basic elements of a mechanism for NFAT activation independent from global cytoplasmic Ca2+ signals. Thus, the perinuclear region could reflect a local reserve of NFAT that is poised for shuttling in and out of the nucleus when local [Ca2+]i is elevated.
In ventricular myocytes from HF rabbits, basal nuclear localization of NFATc3 was increased compared with control cells (Fig. 5A). HF myocytes are characterized by increased expression of IP3 receptors and increased diastolic Ca2+ release (1) as well as calmodulin/CaMKII-dependent nuclear export of HDAC5 (another transcriptional regulator) (7).
The higher circulating levels of neurohumoral factors in HF, which trigger IP3 signaling (e.g., ET-1 and ANG II), may contribute to the enhanced IP3-dependent NFAT nuclear import seen here and may reinforce the HF phenotype. In HF, the intracellular Na+ concentration is increased (2, 6, 37) and can increase [Ca2+]i and spontaneous SR Ca2+ release (especially at higher heart rates). This could also contribute to enhanced CaN activity and nuclear translocation of NFATc3-GFP in HF, as seen here in Ca2+-overloaded control ventricular myocytes (Fig. 6).
The fact that there are substantial basal levels of nuclear NFATc3 (and especially NFATc1) that are acutely increased by block of nuclear export implies that there is a significant rate of basal CaN-dependent import and export of NFAT that results in the steady-state distribution. Moreover, this may poise this system for dynamic manipulation by cytosolic or perinuclear Ca2+ signals.
We conclude that the regulation of NFAT in adult cardiac myocytes is isoform specific and differs among atrial and ventricular tissue. Nuclear localization of NFAT is regulated by both the Ca2+ signal, which activates CaN, but also by nuclear NFAT kinases and the nuclear export machinery.
This work was supported by grants National Heart, Lung, and Blood Institute Grants HL-62231 and HL-80101 (to L. A. Blatter), HL-089617 (to K. Banach), HL-30077 and HL-80101 (to D. M. Bers), HL-62927 (to J. D. Molkentin), and HL-46929 and HL-73966 (to S. M. Pogwizd) and by American Heart Association Grant 0820080Z (to A. Rinne).
No conflicts of interest are declared by the author(s).
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