Targeted Proteolysis Sustains Calcineurin Activation
http://www.100md.com
循环学杂志 2005年第3期
the Department of Medicine (N.B., J.B., C.H., O.R.) and Institute of Clinical Biochemistry and Pathobiochemistry (K.S.)
University of Wuerzburg, Wuerzburg, Germany
University Department of Medicine, Manchester Royal Infirmary, Manchester, UK (L.N.).
Abstract
Background— Calcineurin (CnA) is important in the regulation of myocardial hypertrophy. We demonstrated that targeted proteolysis of the CnA autoinhibitory domain under pathological myocardial workload leads to increased CnA activity in human myocardium. Here, we investigated the proteolytic mechanism leading to activation of CnA.
Methods and Results— In patients with diseased myocardium, we found strong nuclear translocation of CnA. In contrast, in normal human myocardium, there was a cytosolic distribution of CnA. Stimulation of rat cardiomyocytes with angiotensin (Ang) II increased calpain activity significantly (433±11%; P<0.01; n=6) and caused proteolysis of the autoinhibitory domain of CnA. Inhibition of calpain by a membrane-permeable calpain inhibitor prevented proteolysis. We identified the cleavage site of calpain in the human CnA sequence at amino acid 424. CnA activity was increased after Ang II stimulation (310±29%; P<0.01; n=6) and remained high after removal of Ang II (214±17%; P<0.01; n=6). Addition of a calpain inhibitor to the medium decreased CnA activity (110±19%; P=NS; n=6) after removal of Ang II. Ang II stimulation of cardiomyocytes also translocated CnA into the nucleus as demonstrated by immunohistochemical staining and transfection assays with GFP-tagged CnA. Calpain inhibition and therefore suppression of calpain-mediated proteolysis of CnA enabled CnA exit from the nucleus.
Conclusions— Ang II stimulation of cardiomyocytes increased calpain activity, leading to proteolysis of the autoinhibitory domain of CnA. This causes an increase in CnA activity and results in nuclear translocation of CnA. Loss of the autoinhibitory domain renders CnA constitutively nuclear and active, even after removal of the hypertrophic stimulus.
Key Words: calcium ; myocardium ; hypertrophy ; molecular biology
Introduction
The calcineurin (CnA)/NF-ATc signaling cascade is of major importance in the development of cardiac hypertrophy.1 Activation of the Ca2+-calmodulin–dependent phosphatase CnA leads to dephosphorylation of the nuclear transcription factor NF-ATc (nuclear factor of activated T cells), enabling its nuclear translocation and resulting in induction of genes typical of cardiac hypertrophy.2 Lines of transgenic mice confirmed the functional significance of this pathway for the development of myocardial hypertrophy.1,3,4
According to the current understanding of activation of the CnA/NF-ATc pathway, sustained high Ca2+ levels and binding of calmodulin result in structural changes and subsequent displacement of the C-terminal autoinhibitory domain from the catalytic subunit.2,5 Studies in other cell types have also demonstrated that NF-ATc remains nuclear only in response to prolonged, low-amplitude Ca2+ signals and is insensitive to transient, high-amplitude Ca2+ alterations.6 However, we7 recently found evidence that targeted proteolysis of the CnA autoinhibitory domain also causes an increase in CnA activity in human myocardium. This is in line with a mouse model of transgene overexpression of a constitutively active form of CnA. This form of CnA lacks the autoinhibitory domain and leads to myocardial hypertrophy.1
In this study, we investigated whether targeted proteolysis of CnA in the myocardium occurs in vivo and whether this mechanism can regulate CnA activity under pathophysiological circumstances.
Methods
CnA Protein Expression
Nuclear protein extracts were prepared as described previously8 with minor modifications. Protein concentrations were determined by a Bradford assay.9 Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with anti-CnA antibodies: anti-CnA polyclonal rabbit anti-mouse antibody against the C-terminal domain (Upstate; 1:300; epitop sequence corresponding to amino acids 500 to 521) and anti-CnA polyclonal goat anti-mouse antibody against the N-terminal domain (StressGen; 1:100; epitop sequence corresponding to amino acids 264 to 283). Antibodies were described earlier.7 Further processing was according to standard procedures.9 Protein was visualized with an ECL detection system (Amersham).
Preparation of Neonatal Rat Cardiomyocytes
Neonatal rat cardiomyocytes of Wistar rats were isolated as described previously.10 Briefly, hearts were removed from 1- to 3-day-old rats, and ventricular tissue was digested in calcium- and bicarbonate-free Hanks’ medium with HEPES with 1.5 mg/mL trypsin and 10 μg/mL DNase. Cells were resuspended in minimum essential medium/5% FCS; fibroblasts were removed by preplating for 1 hour and were used for cell cultures. After preplating, the supernatant (cardiomyocytes) was recovered, and cells were plated in minimum essential medium on 6-well plates at a density of 1x106 cells per well. Medium for cardiomyocytes contained 5-bromo-2'-deoxyuridine (0.1 mmol/L) to suppress fibroblast growth. Fibroblast contamination of cardiomyocyte cultures was between 4% and 7% as regularly determined by immunohistochemical staining for troponin T.
Stimulation of Cell Cultures
Forty-eight hours after preparation, cells were stimulated with 10 nmol/L angiotensin II (Ang II). Cells were harvested 24 hours after stimulation. The protocol was described previously.10 Specifically, time points for investigation were 0, 12, and 24 hours. Stimulation of cells was according to the following protocol: (1) Cells were left unstimulated for 12 and 24 hours; (2) cells were stimulated for 12 and 24 hours with Ang II; (3) cells were stimulated with Ang II for 12 hours, and then Ang II was removed for the next 12 hours; (4) cells were stimulated with Ang II for 12 hours, and then Ang II was removed for the next 12 hours (During the complete 24 hours, calpain inhibitor was in the medium); and (5) cells were stimulated with Ang II for 12 hours, and then Ang II was removed for the next 12 hours. Calpain inhibitor was added to the medium after removal of Ang II, ie, from 12 to 24 hours.
Immunostainings
The patient population was described earlier.7 For immunocytochemistry of human samples, 5-μm sections from frozen tissue were fixed in 2% paraformaldehyde in PBS (15 minutes at 4°C), permeabilized in 0.1% Triton X-100 (15 minutes at 21°C), and blocked in PBS with 10% goat serum (3 hours at 21°C). The primary antibodies used in fluorescence staining are described above. Secondary antibodies were Cy-3–labeled sheep anti-rabbit IgG or Cy2-conjugated mouse anti-goat IgG (Jackson Laboratories). Primary cell culture of neonatal cardiomyocytes was performed as described above. Cells were cultured on slides for 48 hours in medium containing 0.5% FCS and for another 24 hours in serum-free medium. They were fixed for 30 minutes with 2% paraformaldehyde at room temperature and then rinsed twice in PBS. Samples were blocked and permeabilized in PBS containing 1% BSA, 0.5% Triton X-100, and 0.1% Tween 20 for 30 minutes at 20°C and then were incubated at 4°C overnight in a PBS/0.1% BSA solution containing the primary antibodies. For staining, the above-mentioned antibodies were used.
Proteolytic Fragmentation, Immunoprecipitation, and Sequencing of Calcineurin
Cleavage of CnA by μ-calpain was performed as described earlier.11 Briefly, lysates of cardiomyocytes were incubated with 0.1 U μ-calpain for 30 minutes at 25°C in a medium containing 50 mmol/L Tris (pH 7.4), 0.5 mmol/L EDTA, 1 mmol/L CaCl2, and 10 mmol/L DTT. Immunoprecipitations were made using a standard protocol. Tissues were lysed in precipitation assay buffer (1x PBS, 1% IGEPAL CA-630, Sigma; 0.5% sodium deoxycholate, Sigma/Complete EDTA-free protease inhibitor mixture, Roche Diagnostics). After an initial preclearing step of 1 hour at 4°C, antigens were coupled to the following antibody (5 μg purified antibodies): anti-CnA N-terminal polyclonal goat anti-mouse (StressGen). Protein-antibody complexes were precipitated with a mix of 50 μL protein A and 50 μL protein G–sepharose beads for 1 hour at 4°C. After 4 washing steps with precipitation assay buffer and 1 with 50 mmol/L Tris, pH 8.0, 100 μL sample buffer was added to the washed beads, and 30 μL of each sample was loaded and separated in an 8% SDS-PAGE. Immunodetection of proteins on membrane was done with the ECL system according to the manufacturer’s protocol (Amersham Pharmacia Biotech). For sequencing of the 48-kDa CnA fragment, immunoprecipitation was performed as described. After in-gel digestion with trypsin, the fragments were analyzed by MALDI-TOF/mass spectrometry (TOPLAB).
Determination of CnA/NF-ATc Transcriptional Activity and CnA Phosphatase Activity
The protocol for transfection was described elsewhere.10 Neonatal rat cardiomyocytes were transfected with the LipofectAMINE PLUS system (Life Technologies). Cells were incubated for 3 hours at 37°C. For cotransfection experiments, human embryonic kidney (HEK-293) cells instead of cardiomyocytes were used because of higher transfection efficiencies. Luciferase activity was determined according to the manufacturer’s protocol (Promega). NF-ATc reporter plasmid consisted of the Il-2 promoter followed by luciferase. For cloning the constitutively active form of CnA, the pSp6 backbone (Invitrogen) with human CnA; cDNA as insert was digested with PstI and XbaI. The protruding ends were filled in with T4 DNA polymerase and ligated. For determination of subcellular CnA localization, GFP-tagged CnA was transfected into cardiomyocytes. GFP was fused with full-length CnA; cDNA at the 5' end. For inhibition of CnA/NF-ATc interaction, a synthetic peptide (50 μg/mL; VIVIT; Calbiochem) was added to the medium.
For determination of CnA phosphatase activity, a commercial kit (CnA kit assay, Biomol) was used on the basis of a specific CnA phosphosubstrate. Free PO4 was indicated by a malachite green dye. The kit was described elsewhere.7
Statistical Analysis
All data are presented as mean±SEM. Statistical analysis was performed with a Student t test. Significance was assigned a value of P<0.05.
Results
Proteolysis and Localization of Calcineurin in Human Myocardium
To identify the exact cleavage site of calpain in the CnA sequence and to investigate whether truncated CnA exits in vivo, lysates of hypertrophied human myocardium were immunoprecipitated with an antibody against the N-terminal half of CnA. After immunoprecipitation, CnA was separated in an SDS gel (10%), and the 48-kDa lane was isolated. The 48-kDa fragment of CnA was digested with trypsin in a gel, and the tryptic peptides were loaded onto a MALDI-TOF (Figure 1A). The C-terminal residues (425 to 521 residues) of CnA were not found in the digested peptides. These findings suggest that in human hypertrophied myocardium, calpain cleaved off the C-terminal region of CnA and produced a 1- to 424-residue truncated CnA (Figure 1B and 1C). From aligned sequence analysis, we found 100% sequence similarity between CnA and CnA; in the region around the putative cleavage site (Figure 1D). Therefore, μ-calpain may cleave both CnA isoforms.
We further investigated subcellular CnA localization on histological sections of human normal hearts (n=4) and diseased human myocardium from patients with coronary artery disease (CAD; n=3), hypertrophic cardiomyopathy (HCM; n=9), and aortic stenosis (AS; n=7). Patients with HCM and AS had maintained systolic function but marked left ventricular hypertrophy with diastolic dysfunction. Patients with CAD had systolic heart failure. In normal heart, there was homogeneous cytosolic distribution of CnA. In all patient groups, there was a marked increase in nuclear localization of CnA. About 70% of cardiomyocytes in the diseased heart displayed an increase in nuclear CnA with no difference between the AS, HCM, or CAD group (Figure 1E).
Calpain Activity
To investigate whether calpain is the protease responsible for the cleavage of CnA, recombinant CnA was incubated with recombinant μ-calpain (0.1 U at room temperature). Cleavage of CnA by calpain was time dependent. Calpain-mediated proteolysis of CnA revealed a 48-kDa fragment. Addition of calpain inhibitor III suppressed cleavage of CnA (Figure 2A).
To investigate whether calpain-mediated cleavage of CnA was dependent on calmodulin, we examined proteolysis of CnA by μ-calpain in the absence or presence of calmodulin. Truncation of CnA by μ-calpain was not dependent on calmodulin. Using an antibody against the C-terminal end of CnA, we detected similar 59-kDa (the catalytic subunit) and 11-kDa (the autoinhibitory domain) fragments when the phosphatase was incubated with μ-calpain in the presence and absence of calmodulin (Figure 2B).
Calpain activity was investigated by incubation of cardiomyocytes with a specific synthetic membrane-permeable fluorescent calpain substrate (10 μm; Boc-Leu-Met-CMAC; Molecular Probes). Activity of calpain in cardiomyocytes was assessed after 24 hours of stimulation with Ang II or after 24 hours in unstimulated cardiomyocytes. Calpain activity was calculated as area under the curve. Calpain activity in unstimulated cells was set at 100%. Ang II stimulation (100 nm) for 24 hours increased calpain activity 4.3-fold (433±11%; n=6; P<0.01) in neonatal cardiomyocytes (Figure 2C and 2D). Increased calpain activity in cardiomyocytes resulted in proteolysis of CnA, whereas in unstimulated cells, calpain activity was not increased. Therefore, no truncated CnA could be detected in unstimulated cells (Figure 2C). The increase in calpain activity is probably due to an increase in calpain protein expression after 24 hours of stimulation, as demonstrated in Figure 2E.
CnA in Nuclear Extracts
To assess nuclear translocation of CnA, nuclear extracts of cardiomyocytes were investigated. An antibody directed against the N-terminal half of CnA detected 2 bands (59 and 48 kDa) in nuclear extracts of Ang II–stimulated (24 hour) cardiomyocytes. If calpain inhibitor III was added during the complete time of Ang II stimulation (24 hours), the same antibody recognized only 1 band (59 kDa) in nuclear extracts (Figure 3A), indicating that in this experiment calpain-mediated cleavage of CnA was prevented and only "full-length" CnA was translocated to the nucleus. In nuclear extracts of unstimulated (24 hours) cardiomyocytes, no full-length CnA could be detected.
Repetition of the same experiment with antibodies directed against the C-terminal end of CnA (the autoinhibitory domain) confirmed these results. In nuclear extracts of cardiomyocytes stimulated with Ang II for 24 hours, there was only one 59-kDa band. Addition of calpain inhibitor III for 24 hours yielded only 1 band as well because the 48-kDa N-terminal truncated product of CnA was not recognized by this antibody (Figure 3A). In Ang II–stimulated cardiomyocytes, 24±4% (n=6; P<0.01) of the total nuclear amount of CnA consisted of the truncated CnA isoform, lacking the autoinhibitory domain (Figure 3B).
In Ang II–stimulated cardiomyocytes, the smaller 11-kDa C-terminal fragment containing the autoinhibitory domain was detectable in the cytosol but not in nuclear extracts (Figure 3C).
To rule out contamination of the cytosolic and nuclear extracts, membrane (caveolin), cytosolic (GAPDH), and nuclear (histone) markers were used (Figure 3D).
Nuclear Calcineurin Translocation
Using an antibody directed against the N-terminal (catalytic) part of CnA, we found a cytosolic distribution of CnA in unstimulated cardiomyocytes. After stimulation with Ang II for 12 or 24 hours, CnA was translocated into the nucleus. After removal of Ang II from the medium for 12 additional hours, CnA remained in the nucleus in part. Addition of calpain inhibitor III (during 12 hours of stimulation and subsequently 12 hours without stimulation) led to removal of CnA from the nucleus back into the cytosol, indicating inhibition of proteolytic degradation of CnA. When the calpain inhibitor was added only after removal of Ang II, CnA remained nuclear. In this case, CnA was possibly truncated before addition of the calpain inhibitor (Figure 4).
To confirm the results, GFP-tagged CnA was transiently transfected into cardiomyocytes (Figure 5A). CnA that was fused to GFP at the N-terminal end was in the cytosol in unstimulated cells. After Ang II stimulation, the N-terminal GFP-CnA fusion protein was translocated to the nucleus. After removal of the stimulus from the medium after 12 hours, a part of GFP-CnA was still nuclear. However, if the calpain inhibitor III was added to the medium during the same experiment, GFP-CnA was exported from the nucleus again after removal of the stimulus. Addition of calpain inhibitor only after removal of Ang II did not prevent proteolysis of CnA. GFP-CnA remained nuclear (Figure 5A).
To further delineate the role of the autoinhibitory domain for nuclear translocation, full-length GFP-CnA and the 48-kDa GFP-CnA fragment (lacking the autoinhibitory domain) were transfected into cardiomyocytes. The 48-kDa fragment was translocated into the nucleus even without Ang II stimulation. Stimulation and removal of the stimulus did not alter the localization. The full-length CnA was in the cytosol in control cells, was translocated to the nucleus after stimulation, and was exported from the nucleus after removal of the stimulus when a calpain inhibitor was present (Figure 5B).
Western blotting for GFP of nuclear extracts of unstimulated cardiomyocytes demonstrated that only the 48-kDa GFP-CnA was translocated to the nucleus. The full-length GFP-CnA was not detectable in nuclear extracts in unstimulated cardiomyocytes (Figure 5C).
CnA Transcriptional and Phosphatase Activity
CnA activity was assessed with NF-ATc reporter assays. Cardiomyocytes transiently transfected with pNF-AT-Luci were left unstimulated for 24 hours or stimulated with Ang II (100 nmol/L) for 24 hours. Luciferase activity of cells transfected with pNF-AT-Luci and left unstimulated for 24 hours was taken as 100%. Stimulation of cardiomyocytes with Ang II resulted in a 3-fold increase (301±16%; n=6; P<0.01) in CnA activity as measured by luciferase activity. Stimulation of cells with Ang II plus calpain inhibitor III resulted in a 3.2-fold increase (321±9%; n=6; P<0.01) in CnA activity (Figure 6A).
The major result could be demonstrated after removal of the hypertrophic stimulus (Ang II) from the medium: after stimulation of cells with Ang II for 12 hours and removal of Ang II for an additional 12 hours, CnA/NF-ATc transcriptional activity remained elevated compared with control values (214±17%; n=6; P<0.01). Addition of calpain inhibitor III and therefore calpain inhibition caused a decrease in CnA transcriptional activity after removal of Ang II (111±9%; n=6; P<0.01) back to background levels. When the calpain inhibitor (10 μmol/L) was added from 12 to 24 hours, after 12 hours of Ang II stimulation, transcriptional activity remained elevated compared with control cells (209±10%; n=6; P<0.01).
To investigate the possibility that the autoinhibitory domain affects CnA/NF-ATc interaction, HEK-293 cells were transfected with a constitutively active form of CnA (CnA). Cells were cotransfected with a NF-AT reporter plasmid. Stimulation of cell cultures with Ang II resulted in a 3-fold increase (301±16%; n=6; P<0.01) in CnA activity as measured by luciferase activity. Transfection of cells with CnA increased CnA/NF-ATc transcriptional activity significantly (367±16%; n=6; P<0.01). However, inhibition of CnA/NF-ATc interaction by the inhibitory peptide VIVIT resulted in a significant decrease in transcriptional activity (121±9%; n=6; P<0.01) in CnA/NF-ATc activity (Figure 6B). Therefore, CnA/NF-ATc interaction seems not to be affected by proteolysis of the autoinhibitory domain.
To investigate CnA phosphatase activity (Figure 6C), we determined the amount of PO4 set free from a specific CnA phosphosubstrate. Ang II stimulation for 24 hours increased CnA phosphatase activity significantly. The average increase in CnA phosphatase activity after Ang II stimulation was 2.1-fold (0.021±0.002 nmol PO4 in whole-cell extracts, 0.016±0.002 nmol PO4 in nuclear extracts; n=6; P<0.01). Calpain inhibitors had no influence in this experiment. Similar to transcriptional activity, CnA phosphatase activity remained elevated in whole-cell extracts (0.016±0.0017 nmol PO4) and nuclear extracts (0.012±0.0018 nmol PO4) after removal of Ang II after 12 hours compared with the values in unstimulated cells (n=6; P<0.01). In the presence of a calpain inhibitor for 24 hours, CnA phosphatase activity decreased after removal of Ang II in whole cells (0.011±0.0013 nmol PO4; n=6; P<0.01) and nuclear extracts (0.007±0.0009 nmol PO4; n=6; P<0.01). When the calpain inhibitor was added only after 12 hours of Ang II stimulation, CnA phosphatase activity remained elevated in whole-cell extracts (0.017±0.004 nmol PO4; n=6; P=NS) and in the nucleus (0.011±0.002 nmol PO4; n=6; P=NS).
CnA expression in whole-cell and nuclear extracts was investigated by use of an antibody against the catalytic domain (Figure 6D). After stimulation of cardiomyocytes for 24 hours with Ang II, CnA was presumably truncated. Therefore, 2 CnA bands were located. Removal of Ang II after 12 hours of stimulation displayed the same results. Addition of a calpain inhibitor for 24 hours in this situation prevented proteolysis. If the calpain inhibitor was added only after 12 hours, proteolysis could not be prevented. In nuclear extracts, there was predominantly the smaller, presumably truncated CnA fragment after removal of Ang II after 12 hours. The part of the CnA pool that remained intact may have been relocated to the cytosol in its majority. Calpain inhibition during 12 hours of Ang II stimulation and subsequent removal of Ang II suppressed proteolysis of CnA; therefore, CnA export from the nucleus was still possible, and only traces of CnA were detectable in the nucleus. When a calpain inhibitor was added after removal of Ang II, proteolysis could not be prevented. The smaller CnA fragment remained nuclear.
Discussion
Experiments in cultured cardiomyocytes and transgenic mice established CnA, the calcium-calmodulin–dependent phosphate (formerly PP2B, now PP3), as a central player in the signal transduction pathways that culminates in cardiomyocyte hypertrophy.3,12 Because the CnA/NF-ATc signaling cascade was first described in the immunosystem, the precise regulation of CnA activity has been investigated intensively in lymphocytes.
The catalytic center of CnA is blocked by the autoinhibitory domain. The mechanism of CnA activation involves binding of calcium and calmodulin, resulting in a displacement of the C-terminal autoinhibitory domain.2 Once activated, the phosphatase CnA dephosphorylates NF-ATc, promoting its nuclear import. It has also been reported that in lymphocytes sustained high concentrations of Ca2+, but not transient pulses, are required to maintain NF-ATc transcription factors in the nucleus, where they participate in Ca2+-dependent induction of genes required for lymphocyte activation and proliferation.5
In resting cells, NF-ATc proteins reside in the cytoplasm. Their nuclear localization signal is masked by phosphorylation. In stimulated cells, CnA dephosphorylates the "masking" residues, exposing the nuclear localization signal and initiating nuclear import. If CnA activity declines, NF-ATc proteins are exported from the nucleus, and NF-ATc–dependent transcription stops.13,14 Nuclear export is accompanied by rephosphorylation of the NF-ATc regulatory domain.15 When it has moved to the nucleus by activated CnA, NF-ATc binds DNA through its Rel homology domain and transactivates genes responsible for myocardial hypertrophy. However, despite its nuclear localization, NF-ATc alone could not form effective transcriptional complexes. Additional activated CnA is necessary to maintain the transcriptional activity of NF-ATc, not by promoting its import but by suppressing its export.16 It appears that suppression of the NF-ATc nuclear export process through CnA binding is a prerequisite to the formation of effective NF-ATc transcriptional complexes.
In a myocardial context, this means that more CnA that is in the nucleus longer results in pronounced transcriptional activity. This mechanism has been used in a model of transgene overexpression of a constitutively active form of CnA, lacking the autoinhibitory domain. These mice develop severe myocardial hypertrophy.1
In a study from our own group,7 we found data that this mechanism, deletion of the autoinhibitory domain, not only is a transgene approach but also occurs in vivo as a pathophysiological mechanism to activate CnA in humans with cardiac hypertrophy. Therefore, in this study, we investigated whether there is proteolytic activation of CnA in the myocardium in vivo.
It has been shown previously that the Ca2+-dependent protease calpain is able to cleave CnA, thereby increasing phosphatase activity.11 Additionally, in a very recent publication,17 cleavage of CnA by calpain has been demonstrated in cell cultures of neuronal cells. Proteolysis of CnA revealed 3 cleavage sites of calpain. Two truncated fragments were constitutively active. In our own experiments, we found that calpain-induced proteolysis of CnA occurs at only 1 site (residue 424). The remaining fragment is also constitutively active. Isolation and sequencing of this truncated product from hypertrophied human myocardium confirms our hypothesis that this mechanism of CnA activation occurs in vivo.
Of the 3 CnA isoforms (CnA, CnA;, and CnA), only CnA and CnA; are expressed in the myocardium. Previous studies4 have demonstrated that CnA and CnA; are expressed at equal protein levels, whereas total CnA activity was only 20% in CnA;-null mice.4 CnA activity is probably regulated on several levels. However, the primary amino acid sequences of CnA and CnA; are 100% homologous in the area around the putative cleavage site, suggesting that calpain-cleavage of CnA is common to both isoforms of CnA.
The influence of calpain-induced proteolysis on the CnA/NF-ATc pathway was demonstrated recently on another level: Proteolysis of the endogenous CnA inhibitor cain/cabin by μ-calpain increased CnA activity.18 Therefore, calpain-mediated proteolysis emerges as a powerful activator of the CnA/NF-ATc pathway. Additionally, targeted proteolysis of specific signaling molecules like protein kinase C19 and of G-proteins20 by calpain could selectively increase the effect of signaling cascades. The major cardiac calpain isoforms are μ-calpain (calpain 1) and m-calpain (calpain 2), named according to Ca2+ concentrations for maximum in vitro activity. It is not clear whether the calpain isoforms in the myocardium display specific function or whether there is redundancy. In our experiments, we used μ-calpain. We found an increase in μ-calpain protein expression in stimulated cardiomyocytes.
Here, we could also demonstrate that CnA is translocated to the nucleus in most cardiomyocytes in the diseased human myocardium. To confirm this observation in an experimental setup, cardiomyocytes were stimulated with Ang II, causing an increase in calpain activity. Subsequently, CnA was translocated to the nucleus and remained nuclear after removal of the hypertrophic stimulus. In nuclear extracts from stimulated cardiomyocytes, we found that up to 30% of CnA is made up of the truncated, constitutively active form. Our observation of nuclear translocation of CnA confirms results from earlier reports that also describe nuclear import of CnA in activated cells.16,21,22 The small 11-kDa fragment, containing the autoinhibitory domain, was not detected in nuclear extracts. Therefore, persistent inhibition of CnA activity by the autoinhibitory domain after its proteolytic truncation, although possible, is unlikely. In vivo, there may be further degradation of this fragment by other proteases.
The nuclear translocation of the constitutively activated CnA increased transcriptional activity even after removal of the hypertrophic stimulus. It is important in this context that the catalytic activity of CnA has been demonstrated to not necessarily be a prerequisite for nuclear import.16 A synthetic peptide (VIVIT)23 that blocked the CnA/NF-ATc interaction inhibited transcriptional activity of the truncated form of CnA (CnA). Therefore, CnA/NF-ATc interaction seems not to be affected by the loss of the autoinhibitory domain. From these data, the persistent nuclear localization of the truncated CnA may account for the lasting increase in transcriptional activity.
We propose here a model of dual activation of CnA. The conventional mechanism is activation by sustained high calcium levels that led to a conformational change and displacement of the autoinhibitory domain away from the catalytic domain as described above. Activated by this mechanism, CnA can shuttle across the nuclear membrane. An additional new mechanism is proteolysis of the CnA autoinhibitory domain by calpain, leaving CnA constitutively active and possibly constitutively nuclear (Figure 7). Once activated this way, CnA activation is not reversible, and CnA, together with NF-ATc, causes a persistent induction of hypertrophic genes.
In summary, we show here that Ang II stimulation of cardiomyocytes increased calpain activity, which led to proteolysis of the autoinhibitory domain of CnA. This caused an increase in CnA activity and resulted in nuclear translocation of CnA. Cleavage of the autoinhibitory domain leaves CnA constitutively active and nuclear, even after removal of the hypertrophic stimulus. We isolated the truncated 48-kDa CnA product lacking the autoinhibitory domain from human hypertrophied myocardium. This demonstrates that proteolysis of the C-terminus of CnA occurs in vivo in human hypertrophied myocardium. We conclude that targeted proteolysis of CnA could represent an additional mechanism to control CnA activity.
Acknowledgments
This work was supported by grants to Dr Ritter from the German Research Foundation (DFG Ri 1085/3-1) and the Interdisciplinary Centre for Clinical Research Wuerzburg (IZKF E-251).
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Aramburu J, Yaffe MB, Lopez-Rodriguez C, Cantley LC, Hogan PG, Rao A. Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A. Science. 1999; 285: 2129–2133.(Natalie Burkard, MSc; Jan)
University of Wuerzburg, Wuerzburg, Germany
University Department of Medicine, Manchester Royal Infirmary, Manchester, UK (L.N.).
Abstract
Background— Calcineurin (CnA) is important in the regulation of myocardial hypertrophy. We demonstrated that targeted proteolysis of the CnA autoinhibitory domain under pathological myocardial workload leads to increased CnA activity in human myocardium. Here, we investigated the proteolytic mechanism leading to activation of CnA.
Methods and Results— In patients with diseased myocardium, we found strong nuclear translocation of CnA. In contrast, in normal human myocardium, there was a cytosolic distribution of CnA. Stimulation of rat cardiomyocytes with angiotensin (Ang) II increased calpain activity significantly (433±11%; P<0.01; n=6) and caused proteolysis of the autoinhibitory domain of CnA. Inhibition of calpain by a membrane-permeable calpain inhibitor prevented proteolysis. We identified the cleavage site of calpain in the human CnA sequence at amino acid 424. CnA activity was increased after Ang II stimulation (310±29%; P<0.01; n=6) and remained high after removal of Ang II (214±17%; P<0.01; n=6). Addition of a calpain inhibitor to the medium decreased CnA activity (110±19%; P=NS; n=6) after removal of Ang II. Ang II stimulation of cardiomyocytes also translocated CnA into the nucleus as demonstrated by immunohistochemical staining and transfection assays with GFP-tagged CnA. Calpain inhibition and therefore suppression of calpain-mediated proteolysis of CnA enabled CnA exit from the nucleus.
Conclusions— Ang II stimulation of cardiomyocytes increased calpain activity, leading to proteolysis of the autoinhibitory domain of CnA. This causes an increase in CnA activity and results in nuclear translocation of CnA. Loss of the autoinhibitory domain renders CnA constitutively nuclear and active, even after removal of the hypertrophic stimulus.
Key Words: calcium ; myocardium ; hypertrophy ; molecular biology
Introduction
The calcineurin (CnA)/NF-ATc signaling cascade is of major importance in the development of cardiac hypertrophy.1 Activation of the Ca2+-calmodulin–dependent phosphatase CnA leads to dephosphorylation of the nuclear transcription factor NF-ATc (nuclear factor of activated T cells), enabling its nuclear translocation and resulting in induction of genes typical of cardiac hypertrophy.2 Lines of transgenic mice confirmed the functional significance of this pathway for the development of myocardial hypertrophy.1,3,4
According to the current understanding of activation of the CnA/NF-ATc pathway, sustained high Ca2+ levels and binding of calmodulin result in structural changes and subsequent displacement of the C-terminal autoinhibitory domain from the catalytic subunit.2,5 Studies in other cell types have also demonstrated that NF-ATc remains nuclear only in response to prolonged, low-amplitude Ca2+ signals and is insensitive to transient, high-amplitude Ca2+ alterations.6 However, we7 recently found evidence that targeted proteolysis of the CnA autoinhibitory domain also causes an increase in CnA activity in human myocardium. This is in line with a mouse model of transgene overexpression of a constitutively active form of CnA. This form of CnA lacks the autoinhibitory domain and leads to myocardial hypertrophy.1
In this study, we investigated whether targeted proteolysis of CnA in the myocardium occurs in vivo and whether this mechanism can regulate CnA activity under pathophysiological circumstances.
Methods
CnA Protein Expression
Nuclear protein extracts were prepared as described previously8 with minor modifications. Protein concentrations were determined by a Bradford assay.9 Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with anti-CnA antibodies: anti-CnA polyclonal rabbit anti-mouse antibody against the C-terminal domain (Upstate; 1:300; epitop sequence corresponding to amino acids 500 to 521) and anti-CnA polyclonal goat anti-mouse antibody against the N-terminal domain (StressGen; 1:100; epitop sequence corresponding to amino acids 264 to 283). Antibodies were described earlier.7 Further processing was according to standard procedures.9 Protein was visualized with an ECL detection system (Amersham).
Preparation of Neonatal Rat Cardiomyocytes
Neonatal rat cardiomyocytes of Wistar rats were isolated as described previously.10 Briefly, hearts were removed from 1- to 3-day-old rats, and ventricular tissue was digested in calcium- and bicarbonate-free Hanks’ medium with HEPES with 1.5 mg/mL trypsin and 10 μg/mL DNase. Cells were resuspended in minimum essential medium/5% FCS; fibroblasts were removed by preplating for 1 hour and were used for cell cultures. After preplating, the supernatant (cardiomyocytes) was recovered, and cells were plated in minimum essential medium on 6-well plates at a density of 1x106 cells per well. Medium for cardiomyocytes contained 5-bromo-2'-deoxyuridine (0.1 mmol/L) to suppress fibroblast growth. Fibroblast contamination of cardiomyocyte cultures was between 4% and 7% as regularly determined by immunohistochemical staining for troponin T.
Stimulation of Cell Cultures
Forty-eight hours after preparation, cells were stimulated with 10 nmol/L angiotensin II (Ang II). Cells were harvested 24 hours after stimulation. The protocol was described previously.10 Specifically, time points for investigation were 0, 12, and 24 hours. Stimulation of cells was according to the following protocol: (1) Cells were left unstimulated for 12 and 24 hours; (2) cells were stimulated for 12 and 24 hours with Ang II; (3) cells were stimulated with Ang II for 12 hours, and then Ang II was removed for the next 12 hours; (4) cells were stimulated with Ang II for 12 hours, and then Ang II was removed for the next 12 hours (During the complete 24 hours, calpain inhibitor was in the medium); and (5) cells were stimulated with Ang II for 12 hours, and then Ang II was removed for the next 12 hours. Calpain inhibitor was added to the medium after removal of Ang II, ie, from 12 to 24 hours.
Immunostainings
The patient population was described earlier.7 For immunocytochemistry of human samples, 5-μm sections from frozen tissue were fixed in 2% paraformaldehyde in PBS (15 minutes at 4°C), permeabilized in 0.1% Triton X-100 (15 minutes at 21°C), and blocked in PBS with 10% goat serum (3 hours at 21°C). The primary antibodies used in fluorescence staining are described above. Secondary antibodies were Cy-3–labeled sheep anti-rabbit IgG or Cy2-conjugated mouse anti-goat IgG (Jackson Laboratories). Primary cell culture of neonatal cardiomyocytes was performed as described above. Cells were cultured on slides for 48 hours in medium containing 0.5% FCS and for another 24 hours in serum-free medium. They were fixed for 30 minutes with 2% paraformaldehyde at room temperature and then rinsed twice in PBS. Samples were blocked and permeabilized in PBS containing 1% BSA, 0.5% Triton X-100, and 0.1% Tween 20 for 30 minutes at 20°C and then were incubated at 4°C overnight in a PBS/0.1% BSA solution containing the primary antibodies. For staining, the above-mentioned antibodies were used.
Proteolytic Fragmentation, Immunoprecipitation, and Sequencing of Calcineurin
Cleavage of CnA by μ-calpain was performed as described earlier.11 Briefly, lysates of cardiomyocytes were incubated with 0.1 U μ-calpain for 30 minutes at 25°C in a medium containing 50 mmol/L Tris (pH 7.4), 0.5 mmol/L EDTA, 1 mmol/L CaCl2, and 10 mmol/L DTT. Immunoprecipitations were made using a standard protocol. Tissues were lysed in precipitation assay buffer (1x PBS, 1% IGEPAL CA-630, Sigma; 0.5% sodium deoxycholate, Sigma/Complete EDTA-free protease inhibitor mixture, Roche Diagnostics). After an initial preclearing step of 1 hour at 4°C, antigens were coupled to the following antibody (5 μg purified antibodies): anti-CnA N-terminal polyclonal goat anti-mouse (StressGen). Protein-antibody complexes were precipitated with a mix of 50 μL protein A and 50 μL protein G–sepharose beads for 1 hour at 4°C. After 4 washing steps with precipitation assay buffer and 1 with 50 mmol/L Tris, pH 8.0, 100 μL sample buffer was added to the washed beads, and 30 μL of each sample was loaded and separated in an 8% SDS-PAGE. Immunodetection of proteins on membrane was done with the ECL system according to the manufacturer’s protocol (Amersham Pharmacia Biotech). For sequencing of the 48-kDa CnA fragment, immunoprecipitation was performed as described. After in-gel digestion with trypsin, the fragments were analyzed by MALDI-TOF/mass spectrometry (TOPLAB).
Determination of CnA/NF-ATc Transcriptional Activity and CnA Phosphatase Activity
The protocol for transfection was described elsewhere.10 Neonatal rat cardiomyocytes were transfected with the LipofectAMINE PLUS system (Life Technologies). Cells were incubated for 3 hours at 37°C. For cotransfection experiments, human embryonic kidney (HEK-293) cells instead of cardiomyocytes were used because of higher transfection efficiencies. Luciferase activity was determined according to the manufacturer’s protocol (Promega). NF-ATc reporter plasmid consisted of the Il-2 promoter followed by luciferase. For cloning the constitutively active form of CnA, the pSp6 backbone (Invitrogen) with human CnA; cDNA as insert was digested with PstI and XbaI. The protruding ends were filled in with T4 DNA polymerase and ligated. For determination of subcellular CnA localization, GFP-tagged CnA was transfected into cardiomyocytes. GFP was fused with full-length CnA; cDNA at the 5' end. For inhibition of CnA/NF-ATc interaction, a synthetic peptide (50 μg/mL; VIVIT; Calbiochem) was added to the medium.
For determination of CnA phosphatase activity, a commercial kit (CnA kit assay, Biomol) was used on the basis of a specific CnA phosphosubstrate. Free PO4 was indicated by a malachite green dye. The kit was described elsewhere.7
Statistical Analysis
All data are presented as mean±SEM. Statistical analysis was performed with a Student t test. Significance was assigned a value of P<0.05.
Results
Proteolysis and Localization of Calcineurin in Human Myocardium
To identify the exact cleavage site of calpain in the CnA sequence and to investigate whether truncated CnA exits in vivo, lysates of hypertrophied human myocardium were immunoprecipitated with an antibody against the N-terminal half of CnA. After immunoprecipitation, CnA was separated in an SDS gel (10%), and the 48-kDa lane was isolated. The 48-kDa fragment of CnA was digested with trypsin in a gel, and the tryptic peptides were loaded onto a MALDI-TOF (Figure 1A). The C-terminal residues (425 to 521 residues) of CnA were not found in the digested peptides. These findings suggest that in human hypertrophied myocardium, calpain cleaved off the C-terminal region of CnA and produced a 1- to 424-residue truncated CnA (Figure 1B and 1C). From aligned sequence analysis, we found 100% sequence similarity between CnA and CnA; in the region around the putative cleavage site (Figure 1D). Therefore, μ-calpain may cleave both CnA isoforms.
We further investigated subcellular CnA localization on histological sections of human normal hearts (n=4) and diseased human myocardium from patients with coronary artery disease (CAD; n=3), hypertrophic cardiomyopathy (HCM; n=9), and aortic stenosis (AS; n=7). Patients with HCM and AS had maintained systolic function but marked left ventricular hypertrophy with diastolic dysfunction. Patients with CAD had systolic heart failure. In normal heart, there was homogeneous cytosolic distribution of CnA. In all patient groups, there was a marked increase in nuclear localization of CnA. About 70% of cardiomyocytes in the diseased heart displayed an increase in nuclear CnA with no difference between the AS, HCM, or CAD group (Figure 1E).
Calpain Activity
To investigate whether calpain is the protease responsible for the cleavage of CnA, recombinant CnA was incubated with recombinant μ-calpain (0.1 U at room temperature). Cleavage of CnA by calpain was time dependent. Calpain-mediated proteolysis of CnA revealed a 48-kDa fragment. Addition of calpain inhibitor III suppressed cleavage of CnA (Figure 2A).
To investigate whether calpain-mediated cleavage of CnA was dependent on calmodulin, we examined proteolysis of CnA by μ-calpain in the absence or presence of calmodulin. Truncation of CnA by μ-calpain was not dependent on calmodulin. Using an antibody against the C-terminal end of CnA, we detected similar 59-kDa (the catalytic subunit) and 11-kDa (the autoinhibitory domain) fragments when the phosphatase was incubated with μ-calpain in the presence and absence of calmodulin (Figure 2B).
Calpain activity was investigated by incubation of cardiomyocytes with a specific synthetic membrane-permeable fluorescent calpain substrate (10 μm; Boc-Leu-Met-CMAC; Molecular Probes). Activity of calpain in cardiomyocytes was assessed after 24 hours of stimulation with Ang II or after 24 hours in unstimulated cardiomyocytes. Calpain activity was calculated as area under the curve. Calpain activity in unstimulated cells was set at 100%. Ang II stimulation (100 nm) for 24 hours increased calpain activity 4.3-fold (433±11%; n=6; P<0.01) in neonatal cardiomyocytes (Figure 2C and 2D). Increased calpain activity in cardiomyocytes resulted in proteolysis of CnA, whereas in unstimulated cells, calpain activity was not increased. Therefore, no truncated CnA could be detected in unstimulated cells (Figure 2C). The increase in calpain activity is probably due to an increase in calpain protein expression after 24 hours of stimulation, as demonstrated in Figure 2E.
CnA in Nuclear Extracts
To assess nuclear translocation of CnA, nuclear extracts of cardiomyocytes were investigated. An antibody directed against the N-terminal half of CnA detected 2 bands (59 and 48 kDa) in nuclear extracts of Ang II–stimulated (24 hour) cardiomyocytes. If calpain inhibitor III was added during the complete time of Ang II stimulation (24 hours), the same antibody recognized only 1 band (59 kDa) in nuclear extracts (Figure 3A), indicating that in this experiment calpain-mediated cleavage of CnA was prevented and only "full-length" CnA was translocated to the nucleus. In nuclear extracts of unstimulated (24 hours) cardiomyocytes, no full-length CnA could be detected.
Repetition of the same experiment with antibodies directed against the C-terminal end of CnA (the autoinhibitory domain) confirmed these results. In nuclear extracts of cardiomyocytes stimulated with Ang II for 24 hours, there was only one 59-kDa band. Addition of calpain inhibitor III for 24 hours yielded only 1 band as well because the 48-kDa N-terminal truncated product of CnA was not recognized by this antibody (Figure 3A). In Ang II–stimulated cardiomyocytes, 24±4% (n=6; P<0.01) of the total nuclear amount of CnA consisted of the truncated CnA isoform, lacking the autoinhibitory domain (Figure 3B).
In Ang II–stimulated cardiomyocytes, the smaller 11-kDa C-terminal fragment containing the autoinhibitory domain was detectable in the cytosol but not in nuclear extracts (Figure 3C).
To rule out contamination of the cytosolic and nuclear extracts, membrane (caveolin), cytosolic (GAPDH), and nuclear (histone) markers were used (Figure 3D).
Nuclear Calcineurin Translocation
Using an antibody directed against the N-terminal (catalytic) part of CnA, we found a cytosolic distribution of CnA in unstimulated cardiomyocytes. After stimulation with Ang II for 12 or 24 hours, CnA was translocated into the nucleus. After removal of Ang II from the medium for 12 additional hours, CnA remained in the nucleus in part. Addition of calpain inhibitor III (during 12 hours of stimulation and subsequently 12 hours without stimulation) led to removal of CnA from the nucleus back into the cytosol, indicating inhibition of proteolytic degradation of CnA. When the calpain inhibitor was added only after removal of Ang II, CnA remained nuclear. In this case, CnA was possibly truncated before addition of the calpain inhibitor (Figure 4).
To confirm the results, GFP-tagged CnA was transiently transfected into cardiomyocytes (Figure 5A). CnA that was fused to GFP at the N-terminal end was in the cytosol in unstimulated cells. After Ang II stimulation, the N-terminal GFP-CnA fusion protein was translocated to the nucleus. After removal of the stimulus from the medium after 12 hours, a part of GFP-CnA was still nuclear. However, if the calpain inhibitor III was added to the medium during the same experiment, GFP-CnA was exported from the nucleus again after removal of the stimulus. Addition of calpain inhibitor only after removal of Ang II did not prevent proteolysis of CnA. GFP-CnA remained nuclear (Figure 5A).
To further delineate the role of the autoinhibitory domain for nuclear translocation, full-length GFP-CnA and the 48-kDa GFP-CnA fragment (lacking the autoinhibitory domain) were transfected into cardiomyocytes. The 48-kDa fragment was translocated into the nucleus even without Ang II stimulation. Stimulation and removal of the stimulus did not alter the localization. The full-length CnA was in the cytosol in control cells, was translocated to the nucleus after stimulation, and was exported from the nucleus after removal of the stimulus when a calpain inhibitor was present (Figure 5B).
Western blotting for GFP of nuclear extracts of unstimulated cardiomyocytes demonstrated that only the 48-kDa GFP-CnA was translocated to the nucleus. The full-length GFP-CnA was not detectable in nuclear extracts in unstimulated cardiomyocytes (Figure 5C).
CnA Transcriptional and Phosphatase Activity
CnA activity was assessed with NF-ATc reporter assays. Cardiomyocytes transiently transfected with pNF-AT-Luci were left unstimulated for 24 hours or stimulated with Ang II (100 nmol/L) for 24 hours. Luciferase activity of cells transfected with pNF-AT-Luci and left unstimulated for 24 hours was taken as 100%. Stimulation of cardiomyocytes with Ang II resulted in a 3-fold increase (301±16%; n=6; P<0.01) in CnA activity as measured by luciferase activity. Stimulation of cells with Ang II plus calpain inhibitor III resulted in a 3.2-fold increase (321±9%; n=6; P<0.01) in CnA activity (Figure 6A).
The major result could be demonstrated after removal of the hypertrophic stimulus (Ang II) from the medium: after stimulation of cells with Ang II for 12 hours and removal of Ang II for an additional 12 hours, CnA/NF-ATc transcriptional activity remained elevated compared with control values (214±17%; n=6; P<0.01). Addition of calpain inhibitor III and therefore calpain inhibition caused a decrease in CnA transcriptional activity after removal of Ang II (111±9%; n=6; P<0.01) back to background levels. When the calpain inhibitor (10 μmol/L) was added from 12 to 24 hours, after 12 hours of Ang II stimulation, transcriptional activity remained elevated compared with control cells (209±10%; n=6; P<0.01).
To investigate the possibility that the autoinhibitory domain affects CnA/NF-ATc interaction, HEK-293 cells were transfected with a constitutively active form of CnA (CnA). Cells were cotransfected with a NF-AT reporter plasmid. Stimulation of cell cultures with Ang II resulted in a 3-fold increase (301±16%; n=6; P<0.01) in CnA activity as measured by luciferase activity. Transfection of cells with CnA increased CnA/NF-ATc transcriptional activity significantly (367±16%; n=6; P<0.01). However, inhibition of CnA/NF-ATc interaction by the inhibitory peptide VIVIT resulted in a significant decrease in transcriptional activity (121±9%; n=6; P<0.01) in CnA/NF-ATc activity (Figure 6B). Therefore, CnA/NF-ATc interaction seems not to be affected by proteolysis of the autoinhibitory domain.
To investigate CnA phosphatase activity (Figure 6C), we determined the amount of PO4 set free from a specific CnA phosphosubstrate. Ang II stimulation for 24 hours increased CnA phosphatase activity significantly. The average increase in CnA phosphatase activity after Ang II stimulation was 2.1-fold (0.021±0.002 nmol PO4 in whole-cell extracts, 0.016±0.002 nmol PO4 in nuclear extracts; n=6; P<0.01). Calpain inhibitors had no influence in this experiment. Similar to transcriptional activity, CnA phosphatase activity remained elevated in whole-cell extracts (0.016±0.0017 nmol PO4) and nuclear extracts (0.012±0.0018 nmol PO4) after removal of Ang II after 12 hours compared with the values in unstimulated cells (n=6; P<0.01). In the presence of a calpain inhibitor for 24 hours, CnA phosphatase activity decreased after removal of Ang II in whole cells (0.011±0.0013 nmol PO4; n=6; P<0.01) and nuclear extracts (0.007±0.0009 nmol PO4; n=6; P<0.01). When the calpain inhibitor was added only after 12 hours of Ang II stimulation, CnA phosphatase activity remained elevated in whole-cell extracts (0.017±0.004 nmol PO4; n=6; P=NS) and in the nucleus (0.011±0.002 nmol PO4; n=6; P=NS).
CnA expression in whole-cell and nuclear extracts was investigated by use of an antibody against the catalytic domain (Figure 6D). After stimulation of cardiomyocytes for 24 hours with Ang II, CnA was presumably truncated. Therefore, 2 CnA bands were located. Removal of Ang II after 12 hours of stimulation displayed the same results. Addition of a calpain inhibitor for 24 hours in this situation prevented proteolysis. If the calpain inhibitor was added only after 12 hours, proteolysis could not be prevented. In nuclear extracts, there was predominantly the smaller, presumably truncated CnA fragment after removal of Ang II after 12 hours. The part of the CnA pool that remained intact may have been relocated to the cytosol in its majority. Calpain inhibition during 12 hours of Ang II stimulation and subsequent removal of Ang II suppressed proteolysis of CnA; therefore, CnA export from the nucleus was still possible, and only traces of CnA were detectable in the nucleus. When a calpain inhibitor was added after removal of Ang II, proteolysis could not be prevented. The smaller CnA fragment remained nuclear.
Discussion
Experiments in cultured cardiomyocytes and transgenic mice established CnA, the calcium-calmodulin–dependent phosphate (formerly PP2B, now PP3), as a central player in the signal transduction pathways that culminates in cardiomyocyte hypertrophy.3,12 Because the CnA/NF-ATc signaling cascade was first described in the immunosystem, the precise regulation of CnA activity has been investigated intensively in lymphocytes.
The catalytic center of CnA is blocked by the autoinhibitory domain. The mechanism of CnA activation involves binding of calcium and calmodulin, resulting in a displacement of the C-terminal autoinhibitory domain.2 Once activated, the phosphatase CnA dephosphorylates NF-ATc, promoting its nuclear import. It has also been reported that in lymphocytes sustained high concentrations of Ca2+, but not transient pulses, are required to maintain NF-ATc transcription factors in the nucleus, where they participate in Ca2+-dependent induction of genes required for lymphocyte activation and proliferation.5
In resting cells, NF-ATc proteins reside in the cytoplasm. Their nuclear localization signal is masked by phosphorylation. In stimulated cells, CnA dephosphorylates the "masking" residues, exposing the nuclear localization signal and initiating nuclear import. If CnA activity declines, NF-ATc proteins are exported from the nucleus, and NF-ATc–dependent transcription stops.13,14 Nuclear export is accompanied by rephosphorylation of the NF-ATc regulatory domain.15 When it has moved to the nucleus by activated CnA, NF-ATc binds DNA through its Rel homology domain and transactivates genes responsible for myocardial hypertrophy. However, despite its nuclear localization, NF-ATc alone could not form effective transcriptional complexes. Additional activated CnA is necessary to maintain the transcriptional activity of NF-ATc, not by promoting its import but by suppressing its export.16 It appears that suppression of the NF-ATc nuclear export process through CnA binding is a prerequisite to the formation of effective NF-ATc transcriptional complexes.
In a myocardial context, this means that more CnA that is in the nucleus longer results in pronounced transcriptional activity. This mechanism has been used in a model of transgene overexpression of a constitutively active form of CnA, lacking the autoinhibitory domain. These mice develop severe myocardial hypertrophy.1
In a study from our own group,7 we found data that this mechanism, deletion of the autoinhibitory domain, not only is a transgene approach but also occurs in vivo as a pathophysiological mechanism to activate CnA in humans with cardiac hypertrophy. Therefore, in this study, we investigated whether there is proteolytic activation of CnA in the myocardium in vivo.
It has been shown previously that the Ca2+-dependent protease calpain is able to cleave CnA, thereby increasing phosphatase activity.11 Additionally, in a very recent publication,17 cleavage of CnA by calpain has been demonstrated in cell cultures of neuronal cells. Proteolysis of CnA revealed 3 cleavage sites of calpain. Two truncated fragments were constitutively active. In our own experiments, we found that calpain-induced proteolysis of CnA occurs at only 1 site (residue 424). The remaining fragment is also constitutively active. Isolation and sequencing of this truncated product from hypertrophied human myocardium confirms our hypothesis that this mechanism of CnA activation occurs in vivo.
Of the 3 CnA isoforms (CnA, CnA;, and CnA), only CnA and CnA; are expressed in the myocardium. Previous studies4 have demonstrated that CnA and CnA; are expressed at equal protein levels, whereas total CnA activity was only 20% in CnA;-null mice.4 CnA activity is probably regulated on several levels. However, the primary amino acid sequences of CnA and CnA; are 100% homologous in the area around the putative cleavage site, suggesting that calpain-cleavage of CnA is common to both isoforms of CnA.
The influence of calpain-induced proteolysis on the CnA/NF-ATc pathway was demonstrated recently on another level: Proteolysis of the endogenous CnA inhibitor cain/cabin by μ-calpain increased CnA activity.18 Therefore, calpain-mediated proteolysis emerges as a powerful activator of the CnA/NF-ATc pathway. Additionally, targeted proteolysis of specific signaling molecules like protein kinase C19 and of G-proteins20 by calpain could selectively increase the effect of signaling cascades. The major cardiac calpain isoforms are μ-calpain (calpain 1) and m-calpain (calpain 2), named according to Ca2+ concentrations for maximum in vitro activity. It is not clear whether the calpain isoforms in the myocardium display specific function or whether there is redundancy. In our experiments, we used μ-calpain. We found an increase in μ-calpain protein expression in stimulated cardiomyocytes.
Here, we could also demonstrate that CnA is translocated to the nucleus in most cardiomyocytes in the diseased human myocardium. To confirm this observation in an experimental setup, cardiomyocytes were stimulated with Ang II, causing an increase in calpain activity. Subsequently, CnA was translocated to the nucleus and remained nuclear after removal of the hypertrophic stimulus. In nuclear extracts from stimulated cardiomyocytes, we found that up to 30% of CnA is made up of the truncated, constitutively active form. Our observation of nuclear translocation of CnA confirms results from earlier reports that also describe nuclear import of CnA in activated cells.16,21,22 The small 11-kDa fragment, containing the autoinhibitory domain, was not detected in nuclear extracts. Therefore, persistent inhibition of CnA activity by the autoinhibitory domain after its proteolytic truncation, although possible, is unlikely. In vivo, there may be further degradation of this fragment by other proteases.
The nuclear translocation of the constitutively activated CnA increased transcriptional activity even after removal of the hypertrophic stimulus. It is important in this context that the catalytic activity of CnA has been demonstrated to not necessarily be a prerequisite for nuclear import.16 A synthetic peptide (VIVIT)23 that blocked the CnA/NF-ATc interaction inhibited transcriptional activity of the truncated form of CnA (CnA). Therefore, CnA/NF-ATc interaction seems not to be affected by the loss of the autoinhibitory domain. From these data, the persistent nuclear localization of the truncated CnA may account for the lasting increase in transcriptional activity.
We propose here a model of dual activation of CnA. The conventional mechanism is activation by sustained high calcium levels that led to a conformational change and displacement of the autoinhibitory domain away from the catalytic domain as described above. Activated by this mechanism, CnA can shuttle across the nuclear membrane. An additional new mechanism is proteolysis of the CnA autoinhibitory domain by calpain, leaving CnA constitutively active and possibly constitutively nuclear (Figure 7). Once activated this way, CnA activation is not reversible, and CnA, together with NF-ATc, causes a persistent induction of hypertrophic genes.
In summary, we show here that Ang II stimulation of cardiomyocytes increased calpain activity, which led to proteolysis of the autoinhibitory domain of CnA. This caused an increase in CnA activity and resulted in nuclear translocation of CnA. Cleavage of the autoinhibitory domain leaves CnA constitutively active and nuclear, even after removal of the hypertrophic stimulus. We isolated the truncated 48-kDa CnA product lacking the autoinhibitory domain from human hypertrophied myocardium. This demonstrates that proteolysis of the C-terminus of CnA occurs in vivo in human hypertrophied myocardium. We conclude that targeted proteolysis of CnA could represent an additional mechanism to control CnA activity.
Acknowledgments
This work was supported by grants to Dr Ritter from the German Research Foundation (DFG Ri 1085/3-1) and the Interdisciplinary Centre for Clinical Research Wuerzburg (IZKF E-251).
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