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1 Department of Physiology, Division of Biophysics, Biocentre Oulu, University of Oulu, Finland 2 Department of Pharmacology and Toxicology, Division of Biophysics, Biocentre Oulu, University of Oulu, Finland 3 Department of Physical Sciences, Division of Biophysics, Biocentre Oulu, University of Oulu, Finland 4 Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
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(Received 18 August 2003;
accepted after revision 16 October 2003;
first published online 17 October 2003)
Corresponding author P. Tavi: Karolinska Institutet, 17177 Stockholm, Sweden. Email: pasi.tavi{at}fyfa.ki.se
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Introduction |
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Originally it was shown that transgenic mice with constitutively active calcineurin develop cardiac hypertrophy and express fetal cardiac genes (Molkentin et al. 1998), making the calcineurin–NFAT pathway an attractive candidate for coupling of Ca2+ signals to cardiac gene expression. Moreover, overexpression of an endogenous calcineurin inhibitory protein, modulatory calcineurin-interacting protein-1 (MCIP1), inhibits hypertrophy induced by overexpression of constitutively active calcineurin or chronic administration of ß-adrenoreceptor agonist (Rothermel et al. 2001), indicating that activation of calcineurin is critical for the development of the hypertrophy. However, pharmacological inhibition of calcineurin in a variety of rodent models of heart disease has produced controversial results. Calcineurin inhibitors (CsA or FK506) have been reported to inhibit the load-induced hypertrophy (Shimoyama et al. 1999; Zou et al. 2000) or to have no effect (Ding et al. 1999). CsA has even been found to escalate the development of the mouse cardiomyopathy induced by a myosin heavy chain mutation (Fatkin et al. 2000). Furthermore, calcineurin inhibition is not selective to the pathological hypertrophy, since both CsA (Eto et al. 2000) and MCIP1 (Rothermel et al. 2001) also suppress the favourable hypertrophic adaptation to exercise. One possible explanation for these conflicting results could be that the effect of calcineurin inhibition depends on the hypertrophic model used. For example, lack of MCIP1 in mice heart results in an increased hypertrophic response to overexpression of constitutively active calcineurin, but a reduced hypertrophic response to pressure overload and to adrenergic stimulation (Vega et al. 2003), suggesting that calcineurin may have a different role depending on the stimulus inducing the hypertrophy.
While there is strong evidence from genetic mouse models to support the involvement of calcineurin in the development of various forms of hypertrophy (for reviews see Molkentin, 2000; Wilkins & Molkentin, 2002; Frey & Olson, 2003), very little is known about the normal Ca2+ activation of calcineurin and its immediate functional implications. Because the frequency of Ca2+ transients encodes an adequate stimulus for calcineurin activation in skeletal muscle (Liu et al. 2001; Kubis et al. 2002), we first examined if cardiac calcineurin is activated by pacing-induced [Ca2+]i changes in rat atrium. Secondly, since it was reported that calcineurin inhibitors (McCall et al. 1996; Janssen et al. 2000) and/or activated calcineurin (Bandyopadhyay et al. 2000; Münch et al. 2002) regulate the [Ca2+]i balance in cardiac myocytes, we studied the Ca2+-dependent effects of CsA on the myocyte Ca2+ signalling. Thirdly, the findings that in several cell types the calcineurin–NFAT cascade has been implicated in both activation (Rao et al. 1997; Molkentin et al. 1998; Macian et al. 2000) and suppression of the expression of immediate genes (Su et al. 1996; Bito et al. 1996; Schaefer et al. 1998) led us to examine if the calcineurin-dependent transcriptional pathway shows dual effects with respect to the expression of immediate-early genes.
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Methods |
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The rat atrial appendix preparation was set up and perfused as previously described (Tavi et al. 1999). The Animal Use and Care Committee of the University of Oulu approved the experimental design. Male Sprague-Dawley rats weighing 290–400 g were used. The rats were decapitated, and the hearts were rapidly removed and placed in oxygenated (ca 10°C) Tyrode buffer solution (mM): 113.8 NaCl; 17.6 NaHCO3; 4.7 KCl; 2.0 CaCl2; 1.1 MgSO4; 1.2 KH2PO4; 11.0 glucose; and 10 µIU ml-1 insulin; pH was 7.4 when bubbled with 5% CO2–95% O2 gas. The same solution was used for superfusion of the atrium (2.5 ml min-1) at 37°C. The atria were paced with a field stimulus (1 ms, 50% over threshold) by two platinum electrodes located inside the perfusion chamber. CsA (1 µM, Sigma-Aldrich) was applied to the perfusion medium 30 min before the change of the pacing frequency. For mRNA measurements the atria were preincubated for 50 min with 1 Hz pacing, followed by 30 min pacing at 1, 4, 5, or 6 Hz. The tissue was frozen in liquid nitrogen and stored at –70°C.
[Ca2+]i measurements
For indo-1AM loading, the atria were superfused for 25–40 min at 25–32°C (flow 7 ml min-1) with Tyrode solution (volume 4 ml) containing (mM): 103.8 NaCl; 17.6 NaHCO3; 4.7 KCl; 2.0 CaCl2; 1.1 MgSO4; 1.2 KH2PO4; 10 glucose; 9.1 sodium pyryvate. Indo-1 AM (Molecular Probes, Europe) was dissolved in DMSO with 20% Pluronic. The final concentration of indo-1 AM was 20 µM and the amount of DMSO/Pluronic was 25 µl ml-1 Tyrode solution. The solution was bubbled with 5% CO2–95% O2 (pH 7.4). The Ca2+ measurement setup has been previously described (Tavi et al. 1998, 1999). The Indo-1 emission ratio (405 nm/485 nm) was acquired at 200 Hz with two photomultiplier tubes (Hamamatsu, Japan) and filtered at 100 Hz. Both excitation and emission were guided to and from the tissue with a quartz fibre optic cable (diam. 1 mm). The distance between the tissue and the cable head was constant (
3 mm) and the illuminated area covered 100% of the tissue area facing the cable. Therefore the amount of the Indo-1 molecules within the illuminated area was constant. For calibration of the measured signal, Rmin and Rmax were determined at the end of each experiment. To get Rmin the atria were perfused with Ca2+-free buffer (0 Ca2+, 0.5 Mg2+, 5 mM EGTA) containing 10 µM of the Ca2+ ionophore 4-Br-A23187 (Molecular Probes) until a steady state fluorescence signal was obtained (10–12 min). This was followed by perfusion with high Ca2+ solution (15 mM) with 10 µM Ca2+ ionophore. The atria was then exposed to high frequency stimulation (8–10 Hz), resulting in a tetanic-like contraction within 10 min and a steady-state value of Rmax. To evaluate the degree of loading of the non-cytosolic compartments, 1 mM of Mn2+ was applied at the end of calibration procedure. The residual fluorescence after Mn2+ was 6 ± 2% (n= 6) representing the maximal error caused by non-cytosolic fluorescence. To estimate the non-myocyte fluorescence, we induced Ca2+ release in endothelial cells with 10 µM bradykinin (Field et al. 1994), which caused no detectable change in the fluorescence (n= 4), indicating that either the tissue does not contain endothelial cells or these cells were not loaded with indo-1. The [Ca2+]i was calculated by using Grynkiewicz formalism:
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| (1) |
Isolation of mRNA and quantitative PCR
Total RNA was prepared from rat atria and used as a template for the cDNA first strand synthesized by M-MuLV reverse transcriptase. The quantitative PCR reactions were performed with an ABI 7700 Sequence Detection System using the TaqMan chemistry. The forward and reverse primers for rat b-type natriuretic peptide (BNP) mRNA detection were TGGGCAGAAGATAGACCGGA and ACAACCTCAGCCCGTCACAG, and for c-fos GGCTGAACCCTTTGATGACTTC and GGGCAGTCTCCGAGCCA, respectively. The bifunctional fluorogenic probes for BNP and c-fos were 5'-Fam-CCAAGCGACTGACTGCGCCG-Tamra-3' and 5'-Fam-TGTTTCCGGCATCATCTAGGC-Tamra-3', respectively. The results were normalized to 18S RNA quantified from the same samples as previously described (Majalahti-Palviainen et al. 2000).
Oligonucleotides and EMSA
All oligonucleotides were purchased from Sigma Chemical Co. For electrophoretic mobility shift assay (EMSA), the NFAT binding element located at –927 bp BNP promoter (NFAT-BNP, Molkentin et al. 1998) was used as probe and intact and mutated NFAT binding elements of the interleukin-2 promoter (NFAT-IL-2 and NFATmut-IL-2, respectively, Northrop et al. 1994) were used as unlabelled competitor oligonucleotides (coding strand shown, point mutations in bold and 5'-overhangs in italics): NFAT-BNP, 5'-AGAGCTATCCTTTTGTTTTCCATCCTGGCCC-3'; NFAT-IL-2, 5'-AGAGCGCCCAAAGAGGAAAATTTGTTTCATAGCCC-3' and NFATmut-IL-2: 5'-AGAGCGCCCAAAGCTTAAAATTTGTTTCATAGCCC-3'. The sense and corresponding antisense oligonucleotides were annealed to generate double-stranded oligonucleotides. NFAT-BNP was sticky-end-labelled with [32P]-dCTP by Klenow enzyme. Nuclear extracts from frozen auricular tissue were prepared (Hautala et al. 2001) and protein concentration from each sample was colourimetrically determined (Bio-Rad Laboratories). For each reaction mixture, 12 µg of nuclear protein and 3 µg of poly(dI-dC) were used in a buffer containing 10 mM Hepes (pH 7.9), 1 mM MgCl2, 50 mM KCl, 1 mM DTT, 1 mM EDTA, 10% glycerol, 0.025% NP-40, 0.25 mM PMSF and 1 µg ml-1 each of leupeptin, pepstatin and aprotinin. Reaction mixtures were incubated with a labelled probe for 15 min followed by non-denaturating gel electrophoresis on 5% polyacrylamide gel. Subsequently, gels were dried and exposed in a PhosphorImager screen and analysed with ImageQuant (Molecular Dynamics, Amersham Biosciences, CA, USA). To confirm DNA sequence specificity of the protein–DNA complex formation, competition experiments with 100 M excesses of unlabelled oligonucleotides with intact or mutated NFAT binding sites were performed. Competitor oligonucleotides were added to the reaction mixture 15 min before the labelled probe.
Model for calcineurin Ca2+ activation
We utilized a previously published reaction scheme for Ca2+- and calmodulin-dependent activation of calcineurin (CaN) (Bhalla & Iyengar, 1999), where parameters have been adjusted to better meet the characteristics of cardiac myocytes. The reactions and the corresponding rate constants of the model are shown in Fig. 1A. All the reactions were incorporated into Mathlab (Mathworks, USA) as time-dependent differential equations of concentration and they were solved using the well-stirred assumption, which states that each molecule has equal access to each other in a single compartment. As initial concentrations we have used 6 µM for calmodulin ([CaM]) (Shannon et al. 2000) and 1 µM for calcineurin ([CaN]) (Bhalla & Iyengar, 1999; Crabtree, 1999) while the other concentrations being zero at the beginning. The reaction rates were taken initially from the previous model (Bhalla & Iyengar, 1999) and the equilibrated CaN-activation was calculated at different Ca2+ concentrations, which allowed plotting of the CaN activation against [Ca2+] (Fig. 1B) and data points were fitted to the Hill equation:
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Statistical testing
Results are expressed as mean ±S.E.M. The statistical testing was done with one-way and two-way ANOVA. Pairwise comparisons between different groups were done by Student–Newman-Keuls method (SigmaStat, USA). For comparison between multiple groups, Bonferroni correction was applied. P values smaller than 0.05 were considered statistically significant. The data analysis was done with Origin 6.1 (OriginLab) and equations were solved with Mathlab.
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Results |
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Increase of the pacing frequency in different types of cardiac preparations causes a substantial Ca2+ accumulation, manifested as an elevation of diastolic [Ca2+]i. This has been reported in rat ventricular trebeculae (Layland & Kentish, 1999; Brandes & Bers, 2002) and in isolated cardiac myocytes of mouse (Antoons et al. 2002; Knollmann et al. 2003), rabbit (Chudin et al. 1999) and cat (Wang et al. 2001). Because both the sustained component of the Ca2+ signal, like the diastolic Ca2+ accumulation, and the frequency of Ca2+ transients activate calcineurin (Timmerman et al. 1996; Rusnak & Mertz, 2000; Kubis et al. 2003), we hypothesized that in cardiac myocytes pacing might serve as a physiological stimulus for calcineurin activation. To quantify this, we measured [Ca2+]i levels from atria paced at different frequencies. At 1 Hz atrial myocytes generated Ca2+ transients from a stable diastolic [Ca2+]i of 200 ± 7 nM to a systolic [Ca2+]i of 802 ± 18 nM, resulting in an amplitude of the transients of 600 ± 21 nM and an average [Ca2+]i of 300 ± 8 nM (n= 24). An increase in the pacing frequency rapidly shifted the diastolic Ca2+ to higher levels as shown by the representative recording in Fig. 2A. The diastolic [Ca2+]i was dependent on the frequency of the pacing and increased up to 511 ± 40 nM after 10 min at 6 Hz (n= 6, Fig. 2B). The increase in diastolic [Ca2+]i was accompanied by a corresponding increase in the systolic and average [Ca2+]i, but there was no change in the amplitude of the Ca2+ transients.
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To explore the dynamics of calcineurin activation induced by pacing, we used a mathematical model to simulate calcineurin activation using the measured [Ca2+]i signals as input to the model. A data set for each pacing group was selected where [Ca2+]i was closest to the mean values of systolic, diastolic and average as calculated from six identical experiments. The simulations showed that calcineurin activity is 10% of maximum when the pacing frequency is 1 Hz. This baseline activity is brought about by the diastolic [Ca2+]i since Ca2+ transients at 1 Hz do not cause any accumulative activation of calcineurin in the simulations. By increasing the pacing frequency, the calcineurin activity is increased to 50% at 4 Hz, to 60% at 5 Hz and to 70% at 6 Hz (Fig. 3A). From the simulation with measured [Ca2+]i as an input it cannot be judged how much of the calcineurin activity is caused by diastolic [Ca2+]i increase and how much is due to increase of the frequency of the Ca2+ transients. To study this we modelled artificial data mimicking the normal Ca2+ signals at different frequencies (1, 4, 5 and 6 Hz, Fig. 3B), but with fixed baseline (0.2 or 0.4 µM) and amplitude (0.8 µM). With baseline [Ca2+]i of 0.4 µM, the frequency-dependent calcineurin activity increases from 37% at 1 Hz to 59% at 6 Hz and with baseline [Ca2+]i of 0.2 µM the corresponding values were 13% and 36% (Fig. 3B), indicating that calcineurin is activated by the frequency of cardiac Ca2+ transients.
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Cyclosporin A has been reported to cause a calcineurin-independent suppression of Ca2+ transients with a corresponding negative inotropy in cardiac muscle by acting on the sarcoplasmic reticulum (SR) (Janssen et al. 2000). However, active calcineurin may modulate the Ca2+ release in cardiac myocytes by regulating the ryanodine receptors (RyRs) (Bandyopadhyay et al. 2000) and reduce the SR Ca2+ uptake by dephosphorylating phopholamban, the regulator of cardiac SR Ca2+-ATPase (Münch et al. 2002). Measuring the Ca2+ dependence of the CsA effect can be used to distinguish between the calcineurin-dependent and calcineurin-independent effects of CsA. When we superfused rat atria paced at 1 Hz with 1 µM CsA for 15 min, no statistically significant differences in any of the measured parameters of [Ca2+]i were observed compared to control experiments without CsA (Fig. 4). However, when CsA was applied prior to 6 Hz pacing, [Ca2+]i was decreased. In six experiments the systolic [Ca2+]i (P < 0.05), the average [Ca2+]i (P < 0.01) and the amplitude of the Ca2+ transients (P < 0.05) were all significantly suppressed compared to control. It should be noted that CsA affected the Ca2+ transients in a biphasic manner. After the onset of 6 Hz pacing the amplitude was transiently increased (Fig. 5A), which was then followed by decay to a lower level than in the control (Fig. 5B). This suggests that calcineurin has a role in the maintenance of Ca2+ release in the face of high levels of [Ca2+]i.
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To study the immediate effects of calcineurin activation on gene expression, we measured the mRNA levels of two rapidly inducible genes, BNP and c-fos. First we measured pacing sensitivity of the expressions of these genes. Atrial tissues were subjected to different pacing frequencies (1, 4, 6 and 8 Hz) for 30 min and mRNA levels measured. BNP mRNA level showed near-linear pacing dependence, whereas pacing did not affect the c-fos mRNA level (Fig. 6A). From the frequencies used here, 8 Hz pacing for 30 min gave the maximal BNP mRNA response of 2-fold compared to the baseline expression in 1 Hz. Next we studied the role of calcineurin in these pacing-induced changes in gene expression by exposing atrial tissues to one of the following stimuli for 30 min: 1 Hz pacing, 1 Hz pacing after preincubation (30 min) with 1 µM CsA, 8 Hz pacing, and 8 Hz pacing with preincubation (30 min) with 1 µM CsA. Pacing at 8 Hz induced a significant increase in the BNP mRNA levels (P < 0.05), which was abolished by pre-exposure of CsA (Fig. 6B). It was a surprise to see that while the mRNA levels of c-fos were not sensitive to 8 Hz pacing or CsA alone, pacing at 8 Hz increased the c-fos mRNA levels when the atria were pre-exposed to CsA (n= 6, P < 0.01, Fig. 6C).
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We isolated nuclear fractions from atria exposed to different pacing and/or CsA and from these fractions we measured the NFAT binding activity on the BNP promoter. This step was taken to answer two questions: first, to determine if pacing-induced Ca2+ changes are sufficient to activate calcineurin and resulting NFAT nuclear translocation, and second, to study if cyclosporin A inhibits the NFAT activation. This indirect method was chosen because the fast inactivation of calcineurin after Ca2+ removal (Stemmer & Klee, 1994) might complicate direct activity measurements from tissue samples, while the calcineurin-induced NFAT nuclear accumulation persists for several minutes (Timmerman et al. 1996; Liu et al. 2001; Kubis et al. 2002). The specificity of NFAT complex formation on BNP promoter was confirmed by the competition analysis for nuclear protein binding with NFAT-BNP as a radioactively labelled probe exposed to 100 M excess of unlabelled competitor oligonucleotides of NFATmut-IL-2, NFAT-BNP or NFAT-IL-2 (Fig. 7A). In rat atrium, 8 Hz pacing for 30 min induced a 2-fold increase in NFAT nuclear activation (n= 6, P < 0.01, Fig. 7B). Application of 1 µM of CsA did not change the NFAT activation at 1 Hz pacing but totally inhibited the activation induced by pacing at 8 Hz.
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Discussion |
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Calcineurin activation by pacing
The measurements showed a clear CsA-dependent increase in the nuclear NFAT activity when the frequency of atrial pacing was increased. This is in line with the modelling results that predicted that calcineurin activation depends on the frequency of cardiac Ca2+ transients. Similarly, in skeletal muscle cells NFAT translocation has been shown to be sensitive to the frequency of stimulation and to CsA (Lui et al. 2001; Kubis et al. 2002). Although the NFAT translocation is induced by calcineurin activation, the time courses of these two processes are different. The nuclear import of NFAT requires a minimum stimulation time of 1.5–5 min and is complete after 20–30 min (Liu et al. 2001; Kubis et al. 2002), whereas calcineurin in vitro responds to Ca2+ on a time scale of seconds (Stemmer & Klee, 1994). This was also seen in the present study where the effects of calcineurin inhibition on [Ca2+]i were apparent within seconds after increasing the pacing frequency. In our simulations, the activation of calcineurin was fast enough (
act= 0.7–1.6 s) to respond to a brief rise in [Ca2+]i such as that during the cardiac Ca2+ transient. The kinetics of calcineurin deactivation (deact1.5 s) ensures that activity triggered by a single Ca2+ transient decays if the frequency is low enough. Upon an increase of frequency, the deactivation of calcineurin becomes incomplete before the next transient and calcineurin activation increases. The frequency-dependent calcineurin activation appears to be prominent at frequencies corresponding to the normal rat heart rate (5–8 beats s-1) and thus calcineurin is likely to exhibit a significant level of activity even at rest, with rapid responses to both increase and decreases of heart rate. It is also interesting to note that although pacing produces an increase in the diastolic [Ca2+]i that activates calcineurin effectively, our modelling experiments show that changes in the frequency of Ca2+ transients alone are sufficient to alter calcineurin activity (see Fig. 3B). This supports the idea that calcineurin is part of the normal adaptation mechanism, and as such participates in the maintenance of the normal cardiac phenotype (Eto et al. 2000). Analogously, it has been suggested that in slow type skeletal myocytes, calcineurin maintains the slow phenotype because the continuous activity in these cells upholds the calcineurin activity (Chin et al. 1998).
Suppression of pacing-induced Ca2+ increase by cyclosporin A
In the present study, we found that that all of the effects of CsA on [Ca2+]i were present only with high pacing frequency (see Fig. 4). This indicates that the changes were caused by inhibition of Ca2+-activated calcineurin. In cardiac myocytes, three different calcineurin targets may be involved in Ca2+ regulation. First, the inhibitory regulator of SR Ca2+-ATPase, phospholamban, is inhibited by calcineurin in ventricular myocytes (Münch et al. 2002) and therefore calcineurin inhibition would have a stimulatory effect on the pump. This sort of Ca2+-ATPase stimulation would cause faster Ca2+ transient decay, possibly a reduced diastolic [Ca2+]i and increased SR Ca2+ content resulting in an increased Ca2+ release and subsequently sustained augmentation of Ca2+ transients (Song et al. 2003). This is opposite to what we found to be the effect of CsA during pacing where the endpoint was a significant reduction in Ca2+ transient amplitude. This finding is not surprising since atrial muscle contains much less phospholamban than ventricular muscle (Koss et al. 1995). Second, calcineurin inhibition was recently found to stimulate the L-type calcium current and thereby cause a sustained increase of calcium release in ventricular myocytes (Santana et al. 2002). This mechanism had either a transient or a small effect in our atrial cells where the endpoint was a significant reduction in calcium release. Third, calcineurin may act on the Ca2+ release channels, i.e. the RyRs (Bandyopadhyay et al. 2000) or the IP3 receptors (Cameron et al. 1995). The contribution of IP3 receptors to the normal calcium release is small in rat atrial myocytes (Mackenzie et al. 2002). Calcineurin regulates cardiac ryanodine receptor via FKBP12.6, which de-sensitizes the channel to Ca2+, thereby reducing the Ca2+ leak during diastole (Bandyopadhyay et al. 2000; Marks, 2003). Calcineurin inhibitors make RyRs more sensitive to Ca2+ leading to increased RyR openings at lower levels of Ca2+ manifested as spontaneous Ca2+ release events (McCall et al. 1996; Bandyopadhyay et al. 2000). This type of RyR sensitization leads first to an increase in the Ca2+ release, which in turn leads to a reduction of SR Ca2+ content and Ca2+ release ensuring that the effect is transient (Trafford et al. 2000; Eisner et al. 2000). If this is accompanied by even a small increase in the passive Ca2+ leak like that with ryanodine (Bers et al. 1987) or with CsA (Bandyopadhyay et al. 2000), a sustained decrease of Ca2+ release can be expected. Altogether this is exactly what we found in the present study (see Fig. 4), which supports the idea that RyR-mediated SR Ca2+ release was the main target of CsA.
Effect of calcineurin inhibition on the BNP and c-fos gene expression
Part of the controversy concerning calcineurin signalling in cardiac muscle gene expression and hypertrophy is related to the difficulty in isolating the calcineurin pathway from other hypertrophic pathways. In vivo hypertrophic stimuli consist not only of activation of Ca2+-dependent processes, but also of hormonal, mechanical and neuronal adaptations with corresponding activation of different hypertrophic signalling cascades (for review see Tavi et al. 2001). To study the earliest calcineurin-induced transcriptional events, we measured the effects of altered [Ca2+]i on the expression of the immediate-early genes c-fos and BNP, two rapidly inducible marker genes for load-induced hypertrophy (see, e.g. Tavi et al. 2001). We found that the Ca2+–calcineurin pathway has opposite effects on the expressions of c-fos and BNP. While the BNP expression is part of normal adaptation of heart to increased load (Tavi et al. 2001; Tokola et al. 2001) and a direct target of calcineurin–NFAT-mediated transcription (Molkentin et al. 1998), the c-fos induction is additionally associated with pathological developments such as induction of fetal gene expression by mechanical unloading of the heart (Depre et al. 1998). More importantly, induction of c-fos expression is likely to regulate the impending expression of other genes because it is a part of the heterodimeric transcription factor AP-1. We have shown here that in rat cardiac myocytes, CsA increases the c-fos mRNA levels in a Ca2+-dependent manner. This seems to be a common feature of many cell types since stimulation of c-fos expression by CsA has been demonstrated in T-cell lymphoma cells when exposed to ionomycin (Su et al. 1996) and in murine erythroleukaemia cell line CsA stimulates c-fos expression in a Ca2+-dependent manner in correlation with the inhibition of CaN activity (Schaefer et al. 1998). These phenomena are probably not caused by unspecific actions of CsA since in hippocampal neurones FK506 enhances the c-fos expression after short bouts of electrical stimulation (Bito et al. 1996). These results suggest that the same calcium signal promotes both activation and inhibition of the c-fos expression. To explain a similar phenomenon in hippocampal neurones, Bito et al. (1996) suggested that calcineurin acts indirectly to de-phosphorylate the CaM kinase-phosphorylated cyclic AMP-responsive element binding protein (CREB) transcription factor, thereby suppressing the CREB-induced c-fos expression.
Supporting such a dual role of calcineurin in controlling cardiac gene expression, MCIP1-/- mice have an increased hypertrophic response to constitutively active calcineurin expression but a reduced hypertophic response to pressure overload and to adrenergic stimulation (Vega et al. 2003). If the primary role of calcineurin activation is suppression of some genes, like c-fos, the inhibition of calcineurin would allow these genes to be expressed and would secondarily change the whole pattern of gene expression. This dualism in the calcineurin-dependent immediate-early gene transcription together with the apparent activity of calcineurin, maintained already by moderate heart rates, suggests that calcineurin may orchestrate the transcriptional signals during both normal adaptation and pathological developments. This is not unexpected since the output of calcineurin-dependent transcription has been shown to depend on other accompanying signal cascades (e.g. Rac, Ras or protein kinase C, for review see Crabtree, 2001) also in cardiac myocytes (Bueno et al. 2002; for review see Wilkins & Molkentin, 2002).
Physiological and pathophysiological implications
On the basis of the modelling in the present study it can be predicted that, in addition to the frequency, other factors that modulate the shape or amplitude of cardiac Ca2+ transients and the diastolic [Ca2+]i may have a substantial impact on calcineurin activity. Increased amplitude of the Ca2+ transients, induced by increased SR Ca2+ uptake (for example, by phosphorylation of phospholamban), may actually reduce calcineurin activity by shortening the duration of transients and decreasing the diastolic [Ca2+]i. On the other hand, factors that act to lengthen the Ca2+ transients or augment Ca2+ transients without increasing the rate of Ca2+ removal may elevate calcineurin activity as shown previously with endothelin-1 (Zhu et al. 2000). This mechanism would provide a novel pathway for communication between different hypertrophic pathways.
This study shows that calcineurin influences excitation–contraction coupling in the heart by regulating the Ca2+ release. This extends the spectrum of calcineurin effects in the heart, but also points to the likely consequences of calcineurin inhibition. The vast majority of the reported effects of calcineurin inhibition on the hypertrophy development appear to be due to inhibition of calcineurin-induced transcription activity. Nevertheless, part of the effects can be due to concomitant suppression of cardiac Ca2+ signals, which in turn suppresses many other Ca2+-mediated processes in the myocytes.
The extent to which frequency-dependent calcineurin activation, as described in the present study, contributes to the development of left ventricular hypertrophy and cardiac failure cannot be precisely estimated. However, given that the increased sympathetic outflow with associated tachycardia constitutes one of the major risk factors of chronic heart failure mortality (Poole-Wilson et al. 2003) and calcineurin activity has been reported to increase in patients with cardiac hypertrophy and failure (Haq et al. 2001; Ritter et al. 2002), the drugs that reduce the activation of the sympathetic nervous system like ß-receptor blockers may be effective in control of cardiac growth and remodelling (Packer et al. 1996).
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). C, activation time constants of calcineurin in the model upon steps (from 0.2 µM to 0.4, 0.6 or 0.80 µM [Ca2+]. D, deactivation time constants by corresponding Ca2+ stimuli (as in C).
) baseline [Ca2+], respectively, compared with the simulated CaN activity produced by measured [Ca2+]i changes (•).
) and presence (
