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European Journal of Heart Failure 2004 6(1):3-9; doi:10.1016/j.ejheart.2003.07.007
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© 2004 European Society of Cardiology

Increased regulatory activity of the calcineurin/NFAT pathway in human heart failure

Holger Diedrichsa, Mei Chia, Birgit Boelcka, Uwe Mehlhormb and Robert H.G. Schwingera,*

a Laboratory of Muscle Research and Molecular Cardiology University of Cologne, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany
b Department of Cardiothoracic Surgery University of Cologne, Cologne, Germany

* Corresponding author. Tel.: +49-221-478-6205; fax: +49-221-478-6550. E-mail address: robert.schwinger{at}medizin.uni-koeln.de


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Background: Cardiac hypertrophy may initiate progression to a compromised cardiac function. While the clinical consequences of hypertrophy are well understood, only little is known about the underlying molecular pathways. As reported from animal experiments, the Ca2+-calmodulin activated phosphatase calcineurin and its downstream transcriptional effector NFAT have been implicated as transducers of the hypertrophic response.

Methods and results: To study whether the calcineurin pathway is activated in human heart failure, we investigated samples of human left ventricular myocardium from patients with dilated (idiopathic) cardiomyopathy (DCM, NYHA IV, n=8) in comparison with non-failing controls (NF, n=8). We not only analyzed the pathway by measuring the calcineurin activity, but also by determination of the protein expression of the calcineurin B subunit and additional key markers of the calcineurin signaling cascade (NFAT-3, GATA-4). Calcineurin enzymatic activity was increased by 80% in human dilated cardiomyopathy compared with non-failing human hearts (135.424±11.69 and 83.484±1.81 nmol Pi/min per µl). This was in line with increased protein expression of calcineurin B in DCM (71.18+9.11 vs. 46.41±11.23 densitometric units (DU)/µg protein). In order to verify the activated calcineurin pathway as described in animal models, we compared the protein expression of NFAT-3 in homogenates within nuclear extracts. In nuclear extracts the protein level of NFAT-3 was increased in dilated cardiomyopathy compared with non-failing myocardium (104.01±8.85 vs. 71.47±8.79 DU/µg protein). In contrast, in homogenates the expression of NFAT-3 was higher in the non-failing tissue indicating subcellular redistribution (19.56±3.36 vs. 25.84±3.16 DU/µg protein). The protein expression of GATA-4 was increased in DCM (43.14±2.89 vs. 29.87±2.17 DU/µg protein).

Conclusions: In human heart failure (DCM) the calcineurin signaling pathway is activated not only by an increased activity of calcineurin and expression of GATA-4, but also by the shift from dephosphorylated NFAT-3 to the nucleus indicating subcellular redistribution and regulatory activation.

Key Words: Calcineurin • NFAT-3 • GATA-4 • Cardiomyopathy • Hypertrophy • Heart failure

Received May 26, 2003; Revised July 1, 2003; Accepted July 8, 2003


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Cardiac hypertrophy occurs in response to a variety of stimuli mostly as an adaptive mechanism. While it is typically viewed as a compensatory response, sustained hypertrophy leads to irreversible cardiomyopathy and increased incidence of arrhythmia and sudden cardiac death [1,2].

Several intracellular signaling pathways have been implicated in the induction of cardiac hypertrophy. Among these, the Ca2+-calmodulin activated phosphatase calcineurin and its downstream transcriptional effector nuclear factor of activated T-cells (NFAT) have been implicated as critical transducers of the hypertrophic response that uniquely link alterations in intracellular calcium handling in a myocyte to cardiac hypertrophy [3]. Once activated, calcineurin directly dephosphorylates members of the NFAT transcription factor family (NFAT-3) in the cytoplasm, resulting in their nuclear translocation and the activation of hypertrophic genes by interacting specifically with the cardiac-restricted zinc finger protein GATA-4 [3–5].

The initial description of the calcineurin/NFAT pathway as hypertrophic transducer involved transgenic overexpression of each factor in the heart, which promoted a dramatic myocardial hypertrophy with progression to heart failure [3]. Furthermore, pharmacological studies in animal models for hypertrophy with calcineurin inhibitors like cyclosporine A and FK506 showed equivocal results [6–12]. Recently, rodent models with genetically suppressed calcineurin function by overexpressing the myocyte-enriched calcineurin-interacting protein (MCIP1) renewed the hypothesis of the important role of calcineurin in the development of hypertrophy and heart failure [13].

However, it is still unclear whether the calcineurin/NFAT-3 pathway is involved in the development of human heart failure [14]. Presently, there is little information regarding the calcineurin expression and activity without studying NFAT levels. Furthermore, these studies give inconsistent results [14,16–18].

To study the role of the calcineurin signaling pathway in human heart failure, we investigated the calcineurin expression and activity as well as the levels of NFAT-3 and GATA-4. As NFAT may be translocated to the nucleus by dephosphorylation in heart failure, we compared the protein expression of NFAT-3 in homogenates with nuclear extracts. Furthermore, we solely examined heart tissue from patients with the diagnosis of dilated cardiomyopathy (ischemic cardiomyopathy or other reasons were excluded).


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Patients
Left ventricular myocardium was obtained from terminally failing human hearts of patients after cardiectomy during cardiac transplantation (five men, three women; age 47±4 years; left ventricular end diastolic volume 280±29 ml; ejection fraction 25±4.9%). Patients gave written informed consent prior to operation. The preoperative diagnosis was dilated cardiomyopathy (DCM) in all patients. Detailed data about the patients with heart failure is shown in Table 1. As control, non-failing human myocardium was obtained from eight donors who were brain dead as a result of traumatic injury. Echocardiography showed normal ejection fraction and in patient history there was no evidence of cardiovascular medication (seven men, one woman; age 34±10 years). These non-failing hearts were not used for transplantation because of technical reasons (e.g. death of recipient, logistic problems and fever shortly before explantation). Drugs used for general anesthesia were flunitrazepam, fentanyl and pancuronium bromide with isoflurane. The investigation conforms with the principles outlined in the Declaration of Helsinki and was approved by the local ethics committee.


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  		Table 1 Detailed data about the patients with heart failure (n=8)

 
2.2. Protein preparation (homogenates)
Protein extracts were prepared by homogenization of frozen human myocardial tissue. Myocardial tissue was stored at –80 °C immediately after explantation. Between 0.5 and 1 g myocardium from the free left ventricular wall were broken with a hammer and reduced to powder in liquid nitrogen (Microdismembrator, U-Braun International, Melsungen, Germany), then transferred to a glass–teflon homogenizer and thawed on ice in three volumes of chilled preparation buffer (300 mM Saccharose reinst DAB 10, 1 mM PMSF, 20 mM Pipes, 10 mM EDTA, 50 mM NaH2PO4, pH 7.4) as previously described [19,20]. Samples were minced with three strokes of 30 s with an ultra turrax (Janke und Kunkel, IKA Werke, Staufen, Germany) at a constant temperature of 4 °C. The obtained homogenate was further diluted with the same volume of a storage buffer (400 mM Saccharose reinst DAB 10, 5 mM HEPES, 5 mM Tris, 10 mM EDTA, 50 mM NaH2PO4, pH 7.2), and frozen in liquid nitrogen and stored at –80 °C. Protein concentrations were determined by Bradford's assay. The homogenates were diluted until the optimal protein concentration (2500 µg/ml), which was suitable for Western blotting, were obtained.

As we observed under fluorescence microscopy in the homogenates, the nuclei are mostly intact. Thus, when measuring the expression of a certain protein in the homogenates, we determine the expression of this protein mostly in the cytoplasm without its expression in the nucleus!

2.3. Extraction of DNA-binding proteins from myocardial tissues (preparation of nuclear extracts)
As described by Deryckere and Gannon [21], between 300 and 400 mg of tissue, frozen in liquid nitrogen, were broken in liquid nitrogen with hammer, then transferred to a Microdismembrator (U-Braun International, Melsungen, Germany) under agitation at 1600 rev./min for 2 min and reduced to powder. All the following steps were carried out on ice and centrifugations at 0 °C. The thawed powder was homogenized in 5 ml of solution A [0.6% P-40 (NP40), 150 mM NaCl, 10 mM HEPES (pH 7.9), 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF)]. After centrifugation the supernatant was incubated for 5 min on ice and then centrifuged for 5 min at 4800 rev./min. The pelleted nuclei were resuspended in 300 µl of solution B [255 glycerol, 20 mM HEPES (pH 7.9), 420 mM NaCl, 1.2 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol (DTT), 0.5 mM PMSF, 2 mM benzamidine, 5 µg/ml of each of these three protease inhibitors: pepstatin, leupeptin, and aprotinin] and incubated on ice for 20 min for high-salt extraction. The lysed nuclei were transferred to a Eppendorf tube and cellular debris were pelleted by a 15 s centrifugation. The supernatant containing the DNA-binding proteins was then frozen in liquid nitrogen and stored at –80 °C.

2.4. Western blot analysis
In order to detect the protein expression immunoblots were performed as previously described [19]. Preparations were thawed on ice and suspended with buffer (Tris/HCl 0.05 mol/1; glycerol 10%; sodium dodecyl sulfate 2%; 2-mercaptoethanol 5%, Bromophenol Blue 0.05%). The samples were subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE) (Hoefer SE 600, Hoefer Scientific Instruments, San Francisco, CA, USA) and transferred to PVDF membrane (Bio-Rad, Bio-Rad laboratories, Hercules, CA, USA) (Hoefer Transphor unit)). Immunoblotting was performed using rabbit anti-Calcineurin B (UpstateTM) polyclonal, rabbit anti-GATA-4 polyclonal and Goat anti-NFAT3 polyclonal antibodies (Santa Cruz Biotechnology), at dilutions of 1:2000, 1:300, 1:250 and 1:500, respectively. For detection of antibody binding an enhanced chemiluminescence assay (ECL Kit, Amersham, Amersham-Life Science, Buckinghamshire, England) was used and exposed to an X-ray film (Amersham) according to the intensity of the signal. Protein expression was quantified after scanning of films into a personal computer and analysis of densitometric volume of bands with a commercially available computer program (ImageQuant, Molecular Dynamics Sunnyvale, USA, Germany).

Quantitative control of the protein of each slot was performed by Western blot analysis of calsequestrin, the marker protein of human myocardium.

2.5. Calcineurin activity assay
We used Promega's non-radioactive Ser/Thr phosphatase assay system for measuring the calcineurin (PPase-2B) activity. PPase-2B can phosphorylate a number of smaller substrates including p-nitrophenylphosphate (PNPP), we used PNPP activity assay to measure the activity of PPase-2B in crude extracts. The assay was carried out on 96-well plates. Calmodulin and NiCl2 were used for evidence that the phosphatase activity is only attributable to PPase-2B (and not PPase-2A, etc.). The solution A (20 mM PNPP, 0.5 mg/ml acetylated BSA, 50 mM Tris pH 7.4) and solution B (20 mM PNPP, 0.5 mg/ml acetylated BSA, 50 mM Tris pH 7.4 1 mM NiCl2, 10 µg/ml calmodulin) reaction mixtures were prepared immediately before use. Homogenate which was prepared as previously described was diluted in enzyme dilution buffer (0.5 mg/ml acetylated BSA, 50 mM Tris pH 7.4) in 1:20 ratio, the final concentration was 125 µg protein/ml. Then the solution B (95 µl/well) was dispensed into separate wells for the homogenate samples and the same volume of solution A for controls. This prepared plate was prewarmed at 30 °C for 5–10 min. Then 5 µl diluted homogenate samples and control enzyme dilution buffer were added to the separated wells, samples and control were run in triplicate. After being incubated for exactly 15 min at 30 °C, absorbance at 410 nm was read. In order to reduce measurement errors by interfering substances in the background (e.g. free phosphate) we assayed the crude extracts for linearity between phosphatase activity and protein concentration and used optimized protein concentration for the final enzymatic assay.

2.6. Statistical analysis
All data are presented as mean±S.E.M. Differences among groups were compared by an unpaired t-test. Significance was assigned to a value of P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Calcineurin activity
The calcineurin activity was measured by using human heart tissue homogenates. The assay was done on a 96-well plate with all samples in the same plate. Calcineurin activity was calculated as phosphate released in presence of calmodulin and Ni2+ minus phosphate released without calmodulin and Ni2+. We investigated calcineurin phosphatase activity in eight patients with dilated cardiomyopathy and eight donor hearts. In patients with dilated cardiomyopathy, calcineurin activity was significantly increased (P<0.05) compared with non-failing (135.424±11.69 and 83.484±1.81 nmol Pi/min per µl, respectively; Fig. 1a).


Figure 1
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  		Fig. 1 (a) Assessment of calcineurin phosphatase activity. Calcineurin activity was calculated as phosphate released in the presence of calmodulin and Ni2+ minus phosphate released without calmodulin and Ni2+. Calcineurin activity in dilated cardiomyopathy (n=9) was significantly increased compared with non-failing hearts (n=8). (b,c) Western blot analysis of calcineurin expression in homogenates of human left ventricular myocardium (DCM: n=9, NF: n=8). The expression of calcineurin was significantly increased in failing myocardium (b). In (c) the expression of calsequestrin shows no difference between non-failing and failing human myocardium.

 
3.2. Protein expression of calcineurin
Western blot analysis was performed to investigate calcineurin B (CnB) protein expression in myocardial homogenates. Using a specific antibody for the regulatory subunit, signals of CnB protein expression was significantly (P<0.05) increased in dilated cardiomyopathy (DCM 71.18±9.11 densitometric units/µg protein) compared with non-failing (NF: 46.41±11.23 densitometric units/µg protein, Fig. 1b).

3.3. Translocation of the nuclear factor of activated T cells (NFAT-3)
To evaluate the effect of calcineurin dephosphorylation the protein expression of NFAT-3 in homogenates and in nuclear extracts were compared. Signals of NFAT-3 protein expression in homogenates were significantly (P=0.006) decreased in DCM (19.56±3.36 densitometric units/µg protein) compared with non-failing (NF: 25.84±3.16 densitometric units/µg protein; Fig. 2a). In contrast, NFAT-3 expression in nuclear extracts was significantly (P<0.05) increased in DCM (104.01±8.85 densitometric units/µg protein) compared with non-failing (71.47±8.79 densitometric units/µg protein; Fig. 2b).


Figure 2
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  		Fig. 2 (a) Western blot analysis of NFAT-3 expression in homogenates of human left ventricular myocardium (DCM: n=9, NF: n=8). Intracellular NFAT-3 was reduced in failing myocardium. (b) Western blot of calsequestrin without difference in protein expression between NF and DCM. (c) Western blot analysis of NFAT-3 expression in nuclei of human left ventricular myocardium (DCM: n=9, NF: n=8). Expression of nuclear translocated NFAT-3 was enhanced in failing myocardium.

 
3.4. Protein expression of the cardiac-restricted zinc finger transcription factor GATA-4
The expression of GATA-4 in the nuclear extract was significantly (P<0.05) increased in dilated cardiomyopathy (DCM) (291.78±11.5 densitometric units/µg protein) compared with non-failing (220.65±12.13 densitometric units/µg protein; Fig. 3a). Protein expression of GATA-4 in homogenates was also significantly (P=0.004) increased in DCM (DCM 43.14±2.89 vs. NF: 29.87±2.17 densitometric units/µg protein; Fig. 3b).


Figure 3
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  		Fig. 3 (a) Western blot analysis of GATA-4 expression in homogenates of human left ventricular myocardium (DCM: n=9, NF: n=8). GATA-4 was significantly increased in human failing myocardium. (b,c) Western blot analysis of GATA-4 expression in nuclei of human left ventricular myocardium (DCM: n=9, NF: n=8). Expression of GATA-4 was significantly enhanced in nuclear extracts of failing myocardium (c). (b) shows an equal expression of calsequestrin in NF and DCM.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In animal models the Ca2+-calmodulin activated phosphatase calcineurin and its downstream transcriptional effector NFAT-3 have been implicated as transducers of the hypertrophic response [3,22,23]. In addition, myocardial hypertrophy may precede cardiac failure at least under certain conditions. To date, there are mostly controversial data available on calcineurin activity in the end staged failing human heart [14–17]. One reason for variable results could be the use of human heart tissue of mixed etiologies, e.g. ischemic and hypertrophic. The present study gives conclusive evidence that there is a significant activation of calcineurin in the failing human heart due to dilated cardiomyopathy. Haq et al. reported a higher calcineurin activity in the failing heart due mainly to an increased expression [17]. As in most of the previous studies this group focused on the protein expression of calcineurin A (isoform β), the binding site of calmodulin. Thus, Ca2+ levels in human failing myocardium are increased and calcineurin requires Ca+ for its enzymatic activity in the present study we measured the expression of calcineurin B, the binding site of Ca2+ enabling calmodulin to bind to calcineurin A [24]. Our results are consistent with the hypothesis that calcineurin activity in the human failing heart is increased at least partly due to an upregulation of the Ca-binding subunit calcineurin B.

It is still a problem whether calcineurin activity and expression measured in tissue homogenates reflect the situation in vivo. From animal models it is known that activated calcineurin dephosphorylates the nuclear transcription factor NFAT-3 resulting in NFAT-3 translocation to the nucleus and activation of transcription factor GATA-4 [3]. Currently, a few studies could show the dephosphorylation of NFAT-3 in failing and/or hypertrophied human myocardium [17], but there is no study to investigate the subcellular redistribution and nuclear translocation of NFAT-3 in the human myocardium. In order to verify the previously described downstream effectors of the calcineurin pathway we compared the expression of NFAT-3 and GATA-4 in homogenates (cytoplasm) with nuclear extracts. In nuclear extracts the protein level of NFAT-3 was increased in dilated cardiomyopathy compared with non-failing myocardium. In contrast, in homogenates the expression of NFAT-3 was higher in the non-failing tissue. This indirect measurement of NFAT-3 translocation to the nucleus may be evidence for an activated calcineurin pathway with NFAT-3 as a downstream mediator in human DCM in vivo. A limitation of our measurements could be that in the homogenates not all nuclei may be still intact and in the nuclear extracts may be a contamination with cytosolic components. Nevertheless we think that these contaminations are small and do not affect the overall results. Besides the close relation between the subcellular redistribution of NFAT-3 and the increased expression and activation of calcineurin in human end-staged heart failure, could be influenced by morphologic variations in the disease process from non-failing to hypertrophic and failing myocardium.

NFAT-3 interacts with high affinity and specificity with the cardiac-restricted zinc finger protein GATA-4 [4]. GATA-4 turns on myocardial genes during hypertrophy. In previous studies, the function of GATA-4 was mostly investigated in hypertrophic myocardium. Hautala et al. reported that GATA-4 DNA binding activity is rapidly upregulated in response to pressure overload in rats. Nevertheless, the mRNA levels of GATA-4 were not increased [25]. In the present study we found an elevated protein expression of GATA 4 in DCM compared with non-failing in homogenates and nuclear extracts. This is consistent with GATA-4 as a downstream part of the activated calcineurin/NFAT pathway. In summary, the human failing heart (DCM) is characterized by a significant activation of the calcineurin cascade. This could be shown in human myocardium not only by an increased calcineurin activity but also by changes in localization and expression of two downstream effectors of the calcineurin signaling pathway indicating subcellular redistribution and regulatory activity.


   Acknowledgements
 
This study was supported by the Else Kröner-Fresenius-Stiftung and Köln Fortune (to Holger Diedrichs and Robert H.G. Schwinger). We are grateful for the technical assistance of Katja Rössler and Sabine Danneschewski.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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