Chronic Elevation of Calmodulin in the Ventricles of Transgenic Mice Increases the Autonomous Activity of Calmodulin-Dependent Protein Kinase II, Which Regulates Atrial Natriuretic Factor Gene Expression

Josep M. Colomer and Anthony R. Means

Department of Pharmacology and Cancer Biology Duke University Medical Center Durham, North Carolina 27710


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although isoforms of Ca2+/calmodulin-dependent protein kinase II (CaMKII) have been implicated in the regulation of gene expression in cultured cells, this issue has yet to be addressed in vivo. We report that the overexpression of calmodulin in ventricular myocytes of transgenic mice results in an increase in the Ca2+/calmodulin-independent activity of endogenous CaMKII. The calmodulin transgene is regulated by a 500-bp fragment of the atrial natriuretic factor (ANF) gene promoter which, based on cell transfection studies, is itself known to be regulated by CaMKII. The increased autonomous activity of CaMKII maintains the activity of the transgene and establishes a positive feedforward loop, which also extends the temporal expression of the endogenous ANF promoter in ventricular myocytes. Both the increased activity of CaMKII and transcriptional activation of ANF are highly selective responses to the chronic overexpression of calmodulin. These results indicate that CaMKII can regulate gene expression in vivo and suggest that this enzyme may represent the Ca2+-dependent target responsible for reactivation of the ANF gene during ventricular hypertrophy.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multisubunit enzyme with a broad substrate specificity. Originally isolated from mammalian brain, CaMKII is encoded by four genes, and isoforms of the enzyme are expressed in all vertebrate cells (1). Evidence suggests that CaMKII may be capable of decoding the frequency of repetitive increases in intracellular Ca2+ (2). Ca2+/CaM binding to a subunit of a CaMKII holoenzyme complex relieves an intrasteric autoinhibition and activates the protein kinase (1). When Ca2+/CaM binds two adjacent subunits, one active molecule phosphorylates the other on a single threonine residue (T286 in CaMK {alpha}) (1). This phosphorylation has two important consequences. First, it markedly increases the affinity of the subunit for Ca2+/CaM, a phenomena called CaM trapping (3). Second, the kinase activity of the subunit is maintained even after the Ca2+ levels have declined (4, 5, 6). The latter property has been called "autonomous" or Ca2+/CaM-independent activity and endows CaMKII with a memory function (5).

Whereas all of the variants of the CaMKII holoenzyme are assembled in the cytoplasm, specific isoforms of the {alpha}, {delta}, and {gamma} types contain nuclear localization sequences and are targeted to the nucleus (7). However, although the membrane and cytoskeletal roles of CaMKII are beginning to be clarified (1), little is known about the nuclear functions of this enzyme. Many of the studies that have addressed nuclear functions of CaMKII have relied on the ability of C terminally truncated, Ca2+/CaM-independent forms of the enzyme to alter transcription when overexpressed in cells along with selected reporter gene constructs. Such studies implicated CaMKII in the phosphorylation of several transcription factors including CREB (cAMP response element binding protein) (8), ATF-1 (activating transcription factor 1) (9), CAAT-enhancer binding protein ß (C/EBPß) (10), and SRF (serum response factor) (11). However, the only direct experimental evidence demonstrating a requirement for nuclear localization of a CaMKII holoenzyme in transcriptional regulation is by Ramirez et al. (12). These authors showed that overexpression of full-length CaMKII {delta}B activated transcription of the atrial natriuretic factor (ANF) gene in cultured neonatal rat ventricular myocytes in response to an {alpha}-adrenergic agonist that increased intracellular Ca2+. The transcriptional response could be inhibited by KN-93, an antagonist of the multifunctional CaM kinases (which include CaMKII), or by blocking nuclear entry of CaMKII.

Gruver et al. (13) generated several lines of transgenic mice that specifically overexpress CaM in the heart. Transcription of the CaM transgene was regulated by the proximal 500-bp fragment of the ANF promoter. Analysis of two lines of mice revealed that whereas the transgene expression was constitutive in atrial myocytes, transcription was terminated soon after birth in ventricular myocytes (13), which is also the fate of the endogenous ANF gene in these cells (14). CaMKII has a much higher KCaM than many other Ca2+/CaM-dependent enzymes (3). We reasoned that if we could identify a line of mice that overexpressed a sufficiently high level of CaM, the CaM might increase the autonomous activity of CaMKII {delta}B as this is the isoform of CaMKII expressed in ventricular myocytes (15). In turn, if the ANF promoter was regulated by CaMKII in vivo, the increased autonomous CaMKII might exert a positive feed-forward effect on the ANF promoter that controlled expression of the CaM transgene and prevent it from being inactivated on schedule. We report that our hypothesis seems valid and, in addition, the autonomous activity of the endogenous CaMKII was sufficient to extend the duration of expression of the endogenous ANF gene in ventricular cells. We propose that CaM and nuclear CaMKII {delta}B may play a role in the reexpression of the ANF gene during cardiac hypertrophy.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of the CaM Transgene in Ventricles of 2295 Mice as a Function of Age
Gruver et al. (13) used DNA constructs regulated by the proximal 500 bp of the human ANF promoter to create eight lines of transgenic mice that specifically overexpressed proteins in the heart. These mice produced either CaM or a mutant form of CaM called CaM-8 that bound Ca2+ normally but neither interacted with nor activated CaM-dependent enzymes due to removal of eight amino acids from the central helix (16). In the four lines of CaM and CaM-8 mice studied by Gruver et al. (13), the ANF transgene was found to be regulated similarly to the endogenous ANF gene. That is, the transgene was constitutively expressed in the atria but transiently expressed in the ventricles as a function of age. Whereas both transgenes and the endogenous ANF gene were expressed in ventricular myocytes during the first postnatal week, expression was extinguished by 21 days of age. Because we were interested in determining the consequences of higher levels of CaM on events in the ventricles, we examined the level of CaM expression in the hearts of newborn mice from the remaining four uncharacterized strains. Whereas the CaM-8 transgene behaved similarly as a function of age regardless of considerable differences in expression at birth, three lines of CaM-overproducing mice, including the 2295 line, exhibited a higher level of CaM at birth and maintained this level for at least 2 weeks postnatally. Southern and quantitative dot blot analysis revealed that the 2295 line expressing CaM and the 4466 line expressing CaM-8 had a similar site of transgene integration and copy number (~10 copies). Therefore, these lines were used in our study. Figure 1Go shows quantification of ventricular CaM in the 2295 line, the 4466 line that expresses CaM-8, and in normal mice as a function of age. These data were collected using a RIA with an antibody that equally recognizes the CaM and CaM-8 proteins. At birth the 2295 and 4466 lines produced 6 times more immunoreactive protein that did nontransgenic mice of the same age. However, by day 4, the levels of immunoreactive protein in the 4466 strain and normal mice were equivalent whereas the 2295 mice continued to demonstrate a 6-fold increase in CaM. Moreover, this increased CaM was maintained for at least 14 postnatal days before beginning to decline. Thus, the 2295 line afforded us the opportunity to evaluate the mechanism responsible for maintaining the high level of CaM past the time at which the ANF-driven transgene and endogenous ANF gene should be silenced in ventricular myocytes. All subsequent studies were carried out in ventricles isolated from the hearts of 14-day-old mice.



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Figure 1. Time Course for the Overexpression of CaM in the Ventricles

CaM levels were monitored by RIA in ventricular homogenates of normal ({circ}), 2295 ({square}), and 4466 (•) mice from birth to 28 days of age. n >= 3 for each time point of each strain. *, P < 0.01 comparing 2295 to normal of the same age; {dagger}, P < 0.01 comparing newborn 4466 to newborn normal.

 
Temporal Elevation of Ventricular CaM Levels Correlates with Increased Autonomous Activity of CaMKII
Our hypothesis proposed that the chronically elevated levels of CaM might result in an increase in the autonomous activity of CaMKII that, in turn, would maintain the activity of the ANF promoter, thus creating a positive feedback loop. As shown in Table 1Go, the total activity (Ca2+ dependent plus Ca2+ independent) of CaMKII did not differ in ventricular extracts prepared from normal, 2295, or 4466 mice at 14 days of age. However, there was a statistically significant increase in the autonomous CaMKII activity (measured in the presence of EGTA) in ventricular extracts of mice overexpressing CaM compared with those prepared from the other two strains of mice. The protein kinase activity was quantified by phosphorylation of the highly selective peptide substrate, autocamtide-II in vitro (17). The isoform of CaMKII expressed in ventricular myocytes is CaMKII {delta}B (15, 18). Autonomy is the result of intersubunit phosphorylation of threonine 287 (T287) in the holoenzyme. To ensure that the protein kinase activity in vitro reflected an increase in the autonomous activity of CaMKII in vivo, we used an antibody to CaMKII that only recognizes the protein when phosphorylated on T287 (19). T287 in {delta}B is equivalent to T286 in the {alpha}-isoform, and the peptide used to raise this antibody is conserved 100% in {delta}B (19). As shown in the top panel of Fig. 2AGo, there is a significant increase in the amount of this immunoreactive epitope in ventricular extracts of 2295 mice relative to normal or 4466 mice. The lower panel of Fig. 2AGo shows results obtained using an antibody that recognizes CaMKII, whether or not it was phosphorylated, and confirms that the total amount of CaMKII does not significantly differ between extracts from the three types of mice. Thus, both protein kinase assays and immunoblots revealed a 2-fold increase in the autonomous activity of CaMKII in mice that overexpress CaM in the ventricles but not change in the total amount of CaMKII.


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Table 1. Effects of overexpression of CaM on CaN and CaMKII in vitro activity and cAMP steady-state levels in the ventricles of 14-day-old mice

 


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Figure 2. Western Blot Analysis of CaMKII and NFAT Phosphorylation in Ventricles of 14-Day-Old Mice Overexpressing CaM

Representative Western blots of three different experiments, with at least three normal and three transgenic mouse ventricular extracts were performed. A, Analysis of CaMKII phosphorylated at T287. Extracts from the indicated mouse ventricles were resolved by SDS-PAGE and probed with an antibody specific for T287 autophosphorylated CaMKII (upper panel). The membrane was stripped and reprobed with an antibody that recognizes CaMKII regardless of the degree of phosphorylation (lower panel). B, Analysis of NFAT mobility. Ventricular extracts were resolved by SDS-PAGE as before and probed with an anti-NFAT antibody (upper panel). The membrane was stripped and reprobed with anti-{alpha}-actinin, a myocyte-specific marker, to check for lane loading (lower panel).

 
As the expression of a transgene shows considerable variability from mouse to mouse, we questioned whether the level of CaM was predictive of the degree of autonomous CaMKII activity in the ventricles of individual mice. Figure 3Go is a plot of autonomous CaMKII activity as a function of CaM levels. The calculated correlation coefficient was r = 0.94, demonstrating that the extent of CaMKII autonomous activity could be predicted from the total CaM content in the ventricle with almost 90% accuracy (r2 = 0.884).



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Figure 3. Positive Correlation between Autonomous CaMKII Activity and CaM Levels in the Ventricles of 14-Day-Old Mice

CaM levels were measured in ventricular extracts by RIA. The same extracts were used to measure autonomous CaMKII activity in the presence of EGTA using autocamtide-II as the substrate. Correlation coefficient r = 0.94; n = 18 mice.

 
Like all cells, the ventricular myocyte contains a number of CaM-dependent enzymes. We wanted to determine whether the elevated CaM level was selectively correlated with an increase in autonomous CaMKII activity or was more globally correlated with changes in the activity of other CaM-dependent systems. Since the cross-sectional diameter, the most accurate prognosticator of cardiomyocyte hypertrophy (20), is increased in the ventricular myocytes of mice overexpressing CaM (16.93 ± 0.83 µm compared with 11.96 ± 0.89 µm and 12.80 ± 0.15 µm in 2295, normal, and 4466 strains, respectively, P <= 0.002), we evaluated two other CaM-dependent signaling pathways that have been linked to hypertrophy. The first is chronic ß-adrenergic stimulation (21). Although the mechanism by which this stimulus induces hypertrophy is not known, the ß-adrenergic receptor is coupled to Gs and thus increases the level of cAMP. The second is the pathway by which the CaM-dependent protein phosphatase 2B (calcineurin) controls the nuclear localization of the transcription factor, nuclear factor of activated T cells (NF-AT), in the cardiomyocyte (22).

The cardiomyocyte contains both a CaMK-regulated isoform of adenylyl cyclase (type III) (23) and a CaM-dependent isoform of cyclic nucleotide phosphodiesterase (24). Since neither of these enzymes exhibit autonomy but the steady-state levels of cAMP reflect the balance of the two enzyme activities, we measured the cAMP levels by RIA. Table 1Go shows that there were no differences in cAMP levels in ventricular extracts prepared from 2295 ventricles compared with those of normal mice.

Activation of calcineurin in ventricular myocytes has been shown to result in dephosphorylation and nuclear translocation of NF-AT (22). Table 1Go shows that overexpression of CaM does not increase the specific activity of calcineurin, which is not surprising as calcineurin also does not show autonomous activity. The levels of calcineurin are not changed in CaM-overexpressing ventricles as assessed by Western blot analysis (data not shown) and total calcineurin activity in vitro (Table 1Go). Therefore, we examined the phosphorylation status of NF-AT using an antibody that recognizes both phosphorylated (the more intensely stained upper band) and dephosphorylated (the more faintly stained lower band) forms of this protein (Fig. 2BGo). The ratio of the two immunoreactive bands is not changed in ventricular extracts prepared from normal and 2295 mice, revealing that overexpression of CaM does not result in a change of the phosphorylation state of NF-AT. Taken together, our results suggest that the overexpression of CaM in ventricular cardiomyocytes selectively increased the autonomous activity of CaMKII relative to changes in the activity of other Ca2+/calmodulin-dependent enzymes implicated in pathways that lead to cardiac hypertrophy.

The Inactivation of ANF Promoter-Dependent Gene Expression Is Delayed in Ventricles of Mice Overexpressing CaM
The fact that increased CaM levels occur in the ventricles of 14-day-old mice of the 2295 strain suggested that the ANF promoter-driven CaM (cCaM) transgene would also remain active at this stage. To test this possibility, we examined the level of the mRNA expressed from the transgene by Northern blot analysis using a probe specific for the 3'-untranslated region of chicken CaM mRNA. As shown in the top panel of Fig. 4Go, cCaM mRNA was not detected in total RNA from normal mouse ventricles (because no transgene is present) or in total RNA isolated from ventricles of the 4466 mice (because transgene expression has been suppressed in the age-dependent manner typical of the endogenous ANF gene). However, cCaM mRNA was clearly present in total RNA isolated from the ventricles of 2295 mice at 14 days of age. Two other CaM-overexpressing mouse lines that express less CaM than 2295 and show different sites of transgene integration, namely 2256 and 2280, showed temporally extended transgene expression in the ventricles relative to the control CaM-8 animals [8 vs. 5 days, respectively (13) and data not shown]. These results suggest that the increased CaM levels correspond to an increase in the autonomous activity of CaMKII which, in turn, may exert a positive feedback on the ANF promoter driving the cCaM transgene to maintain its activity. One corollary to this proposed feedforward loop would be that the endogenous ANF gene should also remain active in the ventricular myocytes from the 2295 mice. As shown in the second panel of Fig. 4Go, the level of endogenous ANF mRNA is increased in 2295 ventricles relative to either normal or 4466 mice of the same age. This experiment was repeated with six mice from each of the three strains, and the blots were quantified by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) analysis. The amount of ANF mRNA in the 2295 samples was increased about 20-fold relative to either of the other lines of mice (P < 0.01 in either comparison).



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Figure 4. Northern Blot Analysis of Various mRNAs in the Ventricles of Normal, 2295, and 4466 Mice

Representative Northern blot analysis of the chicken CaM transgene expression (cCaM), endogenous ANF, skeletal {alpha}-actin (sk {alpha}-actin), vascular smooth muscle {alpha}-actin (vsm {alpha}-actin), ventricular myosin light chain-2 (mlc-2v), and GAPDH in ventricles from normal, 2295, and 4466 mice, as indicated. PC, Positive controls: skeletal muscle for sk {alpha}actin, and the construct for vsm {alpha}-actin for the vsm {alpha}-actin probe. Ten micrograms of total RNA were used for each lane; six mice of each strain were analyzed for every marker. The riboprobes and specific labeled oligonucleotides used to identify each RNA are described in Materials and Methods.

 
As detailed earlier, the ventricles of the 2295 mice are significantly larger than those of either the control or 4466 mice at 14 days of age. The 40% increase in cross-sectional diameter of ventricular myocytes indicates that ventricular hypertrophy accompanies the overexpression of CaM. The ANF gene is reexpressed in ventricles of a variety of animal models of cardiac hypertrophy (25, 26, 27, 28, 29). However, the reexpression of the ANF gene is not a specific event as a number of other genes characteristic of more immature cardiomyocytes are also reexpressed. To examine the specificity of the ANF gene response in the 2295 mice, we also quantified the mRNA encoded by three other genes known to be up-regulated in hypertrophy (30, 31, 32, 33) in the samples of RNA from 2295, 4466, and control mice. As can be seen in Fig. 4Go, neither skeletal muscle {alpha}-actin (sk {alpha}-actin), vascular smooth muscle {alpha}-actin (vsm {alpha}-actin), or ventricular myosin light chain-2 (MLC-2v) mRNAs are increased in the RNA isolated from ventricles of the 14-day-old 2295 mice. Thus, the continued expression of the endogenous ANF gene is a selective response to the overexpression of CaM. As none of the other genes we examined have been shown to be positively regulated by CaMKII, these negative results support our contention that the increase in autonomous CaMKII is responsible for the continued expression of the endogenous ANF gene.

Table 2Go shows that the increase in ANF mRNA results in a 5-fold elevation in the amount of ANF peptide present in ventricular myocytes as well as a 2-fold increase in the circulation. To determine whether this increase in ANF results in physiological changes that could confound our data interpretation, we evaluated both high arterial blood pressure (63.25 ± 6.29 mm Hg; 50.17 ± 5.39 mm Hg; 56.73 ± 3.32 mm Hg in normal, 2295, and 4466 mice, respectively; P = 0.24, not significant) and low arterial blood pressure (26.75 ± 3.31 mm Hg; 23.67 ± 3.58 mm Hg; 22.73 ± 1.22 mm Hg in normal, 2295, and 4466 mice, respectively; P = 0.49, not significant) (28). The absence of a significant change in blood pressure lessens the concern that the small increase in the circulating levels of ANF has physiological consequences. Indeed, even transgenic mice for ANF, which show a chronic 10-fold increase in plasma levels, have no changes in hormones (such as PRA, norepinephrine, phenylephrine, or vasopressin) that could stimulate the heart. Nor do they reveal changes in the expression of the endogenous ANF gene in either the atria or ventricles (34). Therefore, none of these parameters were evaluated in the present study.


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Table 2. Effects of overexpression of CaM in the heart on ANF peptide levels in the ventricles and in plasma of 14-day-old mice

 
During development, ANF expression is restricted to ventricular myocytes that are located in the endomyocardial area of the wall (35), and it is these same cells that reexpress the ANF gene in response to hypertrophy stimuli (36). Since the 500-bp fragment of the ANF promoter and the endogenous ANF gene are coordinately regulated in a temporal manner in ventricles of mice that overexpress CaM, we questioned whether the cellular specificity of the transgene and endogenous gene was similarly coordinated. This question was addressed by in situ hybridization using riboprobes specific for cCaM or ANF mRNA. As shown in Fig. 5BGo, at 14 days of age, ANF expression is restricted to myocytes located in the endomyocardium of the ventricular walls of the hearts from the 2295 strain that overexpress CaM. However, the transgene is expressed in all ventricular myocytes (Fig. 5EGo). Thus, whereas all myocytes that express the endogenous ANF gene also express the transgene, the overexpression of CaM does not induce ANF gene expression in cells that do not normally express the gene. These results also suggest that although the 500-bp fragment of the ANF promoter is sufficient to target cardiomyocyte-specific expression of a transgene, the promoter fragment is missing sequences that restrict expression of the endogenous gene to a subset of ventricular myocytes when CaM is overexpressed.



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Figure 5. Pattern of Endogenous ANF and CaM Transgene Expression in the Heart

In situ hybridization was performed on cryosections of hearts from normal (A, D, and G), 2295 (B, E, and H) and 4466 (C, F, and I) 14-day-old mice (A–F) or newborn mice (G–I). A riboprobe specific for the endogenous ANF (A–C) or the CaM transgene (D–I) was used, and the slides were counterstained with hematoxylin and eosin, as described in Materials and Methods. Representative photomicrographs at low magnification are shown. Three mice from each strain were analyzed.

 
The CaM transgene mRNA is expressed in all ventricular myocytes of 14-day-old mice of the 2295 strain (Fig. 5EGo), whereas no expression of the mutant CaM transgene, CaM-8, was observed at this age (Fig. 5FGo). This result supported our original assertion that overexpression of CaM set up a positive feedback loop that kept the transgene active whereas CaM-8, because it did not lead to increased autonomous activity of CaMKII, was incapable of establishing this loop. This conclusion would be strengthened if expression of the CaM-8 transgene were restricted to those cells that express the endogenous ANF gene, because it would show that a CaM pathway is required for the area-extended expression. To test this possibility, we carried out in situ hybridization analysis on hearts of newborn mice. As shown in Fig. 5HGo, the CaM transgene is expressed in all ventricular myocytes, whereas expression of the CaM-8 transgene is restricted to cells of the ventricular endomyocardium (Fig. 5IGo), and the pattern is very similar to that of the endogenous ANF gene (Fig. 5BGo).

Because of the transgene expression variability, we predicted that the ventricles with initially higher CaM levels would exhibit enhanced expression of the ANF-CaM transgene and the endogenous ANF gene relative to the ones with initially lower levels of CaM because of the positive feedback effect of CaM on the ANF promoter. To test this prediction, we isolated RNA from the ventricles of 14-day-old mice of the 2295 strain, performed Northern analysis using riboprobes specific for the cCaM mRNA or the ANF mRNA, and quantified the amount of RNA with a PhosphorImager. Figure 6Go shows a plot of the level of ANF mRNA as a function of the level of cCaM mRNA. The correlation coefficient is r = 0.728, indicating a good correlation between the expression of the ANF promoter-driven cCaM transgene and the endogenous ANF gene.



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Figure 6. Relationship between CaM Transgene and Endogenous ANF Gene Expression in the Ventricles

ANF and CaM transgene mRNA levels in individual ventricles were quantified from a Northern blot using a PhosphorImager. The values were normalized to GAPDH mRNA levels and expressed as relative units. Correlation analysis shows an r = 0.728; n = 11.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We report that chronic overexpression of CaM in the ventricular myocytes leads to an increase in the degree of autophosphorylation and autonomous activity of endogenous CaMKII in a dose-dependent manner. In turn, the increase in CaMKII activity results in an extended temporal expression of both the ANF promoter-driven CaM transgene and the endogenous ANF gene. Both the activation of CaMKII and the increase in ANF promoter-mediated gene expression are highly selective consequences of the overexpression of CaM. These data are the first to demonstrate a clear association between the degree of autonomous CaMKII activity and regulation of gene expression in vivo in a mammalian system.

Although we cannot directly determine the isoform of CaMKII responsible for the regulation of ANF gene expression, we highly suspect that it is the nuclear form of CaMKII {delta}, CaMKII {delta}B. The CaMKII {delta} gene is expressed in the heart, and the {delta}B-isoform is known to contain a nuclear localization signal (7, 15). Previously, Ramirez et al. (12) used transient transfection to show that overexpression of CaMKII {delta}B in cultured ventricular myocytes isolated from neonatal rats transcriptionally regulates a cotransfected transgene driven by a 638-bp fragment of the rat ANF promoter. The authors showed that the CaMKII effect occurred only with CaMKII isoforms capable of entering the nucleus and required an intact serum response element (SRE) in the ANF promoter. Since CaMKII can phosphorylate the SRF on Ser103 (11) and this modification has been suggested to increase SRF-mediated transcription (37), Ramirez et al. (12) suggested that phosphorylation of SRF might be the mechanism by which CaMKII regulates transcription of the ANF gene. However, we failed to demonstrate an increase in Ser103 phosphorylation of SRF or an increase in the amount of SRF in vivo (data not shown). Thus, it is unlikely that regulation of SRF is the primary mechanism by which CaMKII enhances ANF gene transcription in mouse ventricular myocytes in vivo.

It is not clear why the ANF promoter seems to be selectively targeted by overexpression of CaM and increased activity of CaMKII. Stimulation of the heart by other agents that increase intracellular Ca2+ also leads to increases in ANF gene transcription but the cellular transcriptional response appears to be much less selective. For example, {alpha}1-adrenergic agonists increase intracellular Ca2+ and activate ANF gene expression in cultured ventricular myocytes (38). However, this stimulus also increases transcription of the skeletal {alpha}-actin and mlc-2v promoters (32, 39). Renin, which binds to a Gq-coupled receptor present on the myocyte membrane, activates PLC {gamma}, and increases intracellular Ca2+ (40), also regulates ANF and skeletal {alpha}-actin gene expression (27). These same two genes are up-regulated by overexpression of a Ca2+/CaM-independent form of calcineurin in the heart (22). Interestingly, although all three genes respond to an increase in Ca2+, the promoters contain different regulatory elements (41, 42, 43). It seems possible that multiple Ca2+/CaM-dependent pathways are involved in orchestrating the overall transcriptional response to a rise in intracellular Ca2+ and that activation of CaMKII is only rate limiting for transcription of the ANF gene.

Initially, we questioned the selectivity of the CaMKII effect as other CaM-dependent pathways exist in the myocyte that potentially could alter transcriptional responses. One such pathway involves the Ca2+/CaM-dependent protein phosphatase 2B, or calcineurin. Activation of this enzyme has been implicated in the development of cardiac hypertrophy because it controls the subcellular localization of the transcription factor NFAT (22). Originally identified in T lymphocytes, this regulatory pathway occurs in a number of other cells including myocytes (22). Increased synthesis of calcineurin in response to myocyte stimulation (44) results in dephosphorylation of a subunit of NFAT, which is required for this transcription factor to enter the nucleus (45). We did not find calcineurin activity or levels to be increased by chronic overexpression of CaM, nor did we detect a difference in the degree of phosphorylation of NFAT, which can be readily observed by an altered mobility on polyacrylamide gels (46). This might not be surprising since calcineurin does not exhibit an autonomous state and the myocytes were not subject to stimulation. In addition, calcineurin may be subject to activity-induced inactivation due to oxidation, possibly of its cofactor iron. This latter mechanism, which couples Ca2+/CaM-dependent protein dephosphorylation to the redox state of the cell, provides a way to reversibly desensitize the enzyme (47).

Two other CaM-dependent kinases have been linked to regulation of gene expression in other systems (48). However, one of these enzymes, CaM kinase IV, is not expressed in mouse ventricles based on our in situ hybridization studies (data not shown). The other enzyme, CaM kinase I (CaMKI), is expressed in the heart (49), but a Ca2+/CaM-independent form of CaMKI cannot increase the activity of an ANF promoter-derived reporter gene in ventricular myocytes (50). Thus, CaMKI is an unlikely mediator of ANF gene transcription in vivo.

An alternative pathway by which an elevation of CaM could alter transcription involves a modification of cAMP levels, which would regulate protein kinase A (PKA). PKA is known to phosphorylate a number of transcription factors. The heart contains Ca2+/CaM-dependent and independent isoforms of both cyclic nucleotide phosphodiesterase (24) and adenylyl cyclase (23, 51). However, we did not detect any increase in the steady-state levels of cAMP, suggesting that these signaling pathways may not be chronically modified by the overexpression of CaM. Together, the data support our contention that the increase in autonomous activity of CaMKII is a selective consequence of the elevation of CaM levels and that the increase in the activity of CaMKII is responsible for the increased transcriptional activity of the transgene and endogenous ANF promoters.

Why would an increase in CaM lead to such a selective activation of CaMKII? CaMKII is an unusual CaM-dependent enzyme in several respects. First, when in its inactive state, it has one of the lowest affinities for Ca2+/CaM of any Ca2+-dependent CaM-binding enzyme (Kd {approx}45 nM) (3). Second, at the resting concentration of Ca2+ common to many cells (100–200 nM) CaMKII does not bind CaM (3). Third, when adjacent subunits of the holoenzyme complex bind Ca2+/CaM, one subunit phosphorylates its neighboring subunit on T287 (1). This phosphorylation event increases the affinity of CaM-binding 1000-fold and generates an enzyme that is active even when CaM becomes dissociated (3). Thus, higher concentrations of Ca2+ are required to activate CaMKII than for most other CaM-dependent enzymes, but once activated, the kinase is independent of CaM binding. Increasing the CaM concentration markedly decreases the amount of Ca2+ required for target enzyme activation (52). Therefore, it is possible that increasing the CaM concentration in the myocyte allows CaMKII activation to occur at concentrations of Ca2+ that would normally be insufficient for its activation. Indeed, precedence exists for this suggestion as Wang and Kelly (53) revealed that injection of CaM into postsynaptic neurons decreased pulse-paired facilitation at ambient Ca2+ concentrations. Once activated, CaMKII activity becomes Ca2+/CaM autonomous and inactivation requires dephosphorylation of T287 (1). The continued presence of elevated CaM levels would favor a steady-state increase in autonomous CaMKII activity. Since both the regulatory regions of the CaM transgene and the endogenous ANF gene require the action of CaMKII, a feed-forward loop would be established whereby the increased CaM would activate CaMKII which, in turn, drives the transcription of the transgene.

Whereas the 500 bp fragment of the ANF promoter is sufficient to target expression of the cCaM transgene to cardiomyocytes, it lacks the specificity of the endogenous ANF gene and is expressed in all ventricular cells. Thus, the increase in CaMKII activity might be expected to also occur in all ventricular myocytes. On the other hand, the endogenous ANF gene is only expressed in cells that comprise the endomyocardial wall (35, 36). These are the same cells that express ANF during early development and reexpress ANF in response to hypertrophic stimuli. This suggests that the regulatory elements that are responsible for the regionally restricted expression of the ANF gene must occur upstream of the 500-bp fragment used to drive the transgene. Nevertheless, our results suggest that CaMKII may play a role in regulating the reexpression of the ANF gene that accompanies cardiac hypertrophy. Support for this possibility comes from Kirchhefer et al. (54), who reported an increased CaM kinase activity in human hypertrophied ventricles although the specific CaM kinase was not identified. However, the nuclear isoform of CaMKII {delta}, CaMKII {delta}B, has been shown by Hoch et al. (55) to be up-regulated in human hearts that have dilated ventricles. Our results are the first to demonstrate that increased activity of endogenous CaMKII (presumably CaMKII {delta}B) correlate with increased ANF gene expression in vivo and provide the basis for future experiments aimed at elucidating the Ca2+-dependent signaling pathways involved in the generation of cardiac hypertrophy.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Experimental Animals
Mice were housed in the Duke University Levine Science Research Center Vivarium under a 12-h light, 12-h dark cycle. Food and water were provided ad libitum, and care was given in compliance with NIH guidelines on the use of laboratory and experimental animals. All transgenic mouse strains used in this study were generated by Gruver et al. (13). Animal experiments were carried out in compliance with a protocol approved by the Duke University Animal Care and Use Committee.

RIAs for CaM, cAMP, and ANF
Ventricles were assayed for total soluble protein by the Bradford procedure (Bio-Rad Laboratories, Inc., Hercules, CA) after hearts were harvested from 14-day-old mice (unless otherwise stated) of normal, 2295, or 4466 strains (13). The ventricles were microdissected and homogenized twice with 1-min bursts of a Polytron homogenizer (Brinkmann Instruments, Inc. Westbury, NY) at a setting of 4.5 using a PT-10 generator, in RSB (10 mM Tris pH 7.4, 30 mM NaCl, 3 mM MgCl2) containing 10 mM 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, several proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mg/ml Pefablock, 10 µg/ml aprotinin, 20 µg/ml trypsin inhibitor, 10 µg/ml leupeptin, and 1 µg/ml pepstatin A) and several phosphatase inhibitors (1 µM microcystin, 100 µM Na3VO4, 20 mM NaF). After the samples were centrifuged for 10 min, the soluble fraction was used for the indicated assays.

CaM protein levels were determined in ventricular extracts by RIA, exactly as previously described (56).

cAMP levels in ventricular extracts were assayed using a [8-3H]cAMP assay system (Amersham Pharmacia Biotech, Buckinghamshire, UK). After homogenization of fresh ventricles in RSB buffer containing 10 mM CHAPS and 4 mM EDTA, the extracts were heated for 5 min in a boiling water bath, and centrifuged for 10 min and the supernatant was assayed exactly as indicated by the manufacturer.

ANF peptide levels were determined by RIA both in ventricular extracts and plasma, using the [125I]{alpha}ANP RIA (Amersham Pharmacia Biotech), which recognizes mouse ANF with an efficiency of 95%. To assay tissue-immunoreactive ANF, ventricles were dissected from 14-day-old mice, weighed, and quickly frozen in liquid nitrogen. The tissue extraction was done as described (57). Briefly, the frozen ventricles were boiled in screw cap tubes containing 1 ml of 1 M acetic acid with 0.1 N HCl for 5 min, homogenized with a Polytron tissue homogenizer, and centrifuged for 30 min at 10000 g at 4 C. The supernatant was transferred to 4 ml polypropylene tubes and 2 ml of 2 M Tris-HCl pH 7.4 were added to neutralize. Aliquots of 100 µl were used to assay ANF according to the manufacturer’s instructions. The final ANF amounts were normalized to initial ventricular wet weight. To assay plasma ANF, the manufacturer instructions were followed, including the extraction of ANF with Amprep C8 columns (Amersham Pharmacia Biotech).

Enzyme Assays
Ventricular homogenates were assayed for CaMKII activity in 50 µl of a reaction mixture consisting of 50 mM HEPES pH 7.5, 10 mM MgCl2, 0.5 mM dithiothreitol (DTT), 1 µM CaM, 100 nM microcystin, 50 µM ATP (1500 cpm/pmol [{gamma}-32P]ATP), and 0.1 mM of substrate peptide autocamtide II. Ca2+/calmodulin-dependent kinase activity was determined by including 1 mM CaCl2 in the reaction mixture, while autonomous activity was measured in the presence of 2.5 mM EGTA. The reaction was carried out for 2 min at 30 C and 40-µl aliquots of the reaction mixture were spotted onto P81 phosphocellulose filters (Whatman, Clifton, NJ) as described previously (58).

Inhibitor-1 protein was labeled with [{gamma}-32P]ATP, modified from Stewart et al. (59) as follows. Glutathione-S-transferase (GST)-inhibitor-1 fusion protein (4–6 mg) was incubated with a solution containing 100 µM ATP (200 µCi/ml of [{gamma}-32P]ATP), 1 mM MgCl2, 50 mM HEPES, 100 U of PKA in a final volume of 1 ml at 37 C for 5 h. Aliquots of 2 µl were taken every 30 min to determine [32P] incorporation into GST-inhibitor-1 (2 µl of reaction were added to 600 µg of BSA and 1 ml of 25% TCA, kept on ice for 5 min, centrifuged for 2 min, washed again, and counted). Once saturation was achieved, [{gamma}-32P]ATP was separated from [32P]GST-inhibitor-1 using an Amicon-30 concentrator (Amicon Inc., Beverly, MA). Five hundred microliters of the reaction were added to 1500 µl of 500 mM glycero-phosphate, loaded onto an Amicon-30 concentrator, and centrifuged at 3000 rpm for 1 h. After the concentrator was washed with 2 ml of H2O, [32P]GST-inhibitor-1 protein was eluted by resuspending it in 2 ml of H2O, inverting the concentrator, and centrifuging 10 min at 1000 rpm.

A modification of the method of King et al. (60) was used to assay calcineurin activity. Ventricular homogenates (40 µg) were added to a reaction mixture containing a final concentration of 50 mM HEPES, pH 7.5, 5 mM MgCl2, 0.1 mM MnCl2, 0.5 mM DTT, 0.1 mM EDTA, 10-8 M microcystin-LR, 0.1 mM CaM, 1.5 µM [32P]inhibitor-1 as a substrate, and 0.2 mM CaCl2. Controls without Ca2+ were done in the presence of 1 mM EGTA. The reaction was carried out for 10 min at 37 C and stopped by placing the tubes on ice after adding 100 µl of TCA 25% and 100 µl of 6 mg/ml of BSA. After centrifugation for 5 min at 4 C, 200 µl of the supernatant were counted with 2 ml of scintillation liquid (ultrafluor).

Western Immunoblot Analysis
After homogenizing the ventricles as described before, the ventricular soluble fraction was run on a 6% or a 10% SDS-polyacrylamide gel for NFAT or CaMKII Western blot analysis, respectively. The proteins were transferred to an Immobilon-P membrane (Millipore Corp., Bedford, MA), which then was blocked for 1 h in TBST (25 mM Tris, pH 7.4, 140 mM NaCl, 3 mM KCl, 0.1% Tween-20), containing 5% nonfat dry milk and 0.5% cold water fish skin gelatin (Sigma) as blocker agents. Subsequently, the filter was incubated overnight with the indicated antibody. An anti-NFAT antibody that recognizes all the isoforms of NFAT (sc-1149, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used at a 1:500 dilution or a monoclonal antibody specific for CaMKII phosphorylated at T286 [22B1, a generous gift of Dr. M. K. Kennedy (19)] at a 1:1000 dilution. After 4–6 washes with TBST, the filter was incubated for 1 h in the same buffer with 5% nonfat dry milk and 0.5% gelatin, containing a second antibody labeled with horseradish peroxidase (Amersham Pharmacia Biotech, Arlington Heights, IL). Then the membranes were washed again in TBST, incubated with enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech) as indicated by the manufacturer, and exposed to film (Eastman Kodak Co., Rochester, NY). Afterward, membranes were stripped (according to ECL protocol), and reprobed with another antibody, as indicated in the figures. An anti {alpha}-actinin (A-7811, Sigma) was used at 1:800 dilution or a polyclonal antibody specific for CaMKII [a generous gift of Dr. M. K. Kennedy (19)] at a 1:1000 dilution. The subsequent washes and second antibody incubation were performed as described above.

RNA Analysis
Total RNA from microdissected ventricles was isolated using the Ultraspec RNA kit (Biotex Laboratories, Inc., Houston, TX) and electrophoresed through denaturing 1.5% agarose, 6% formaldehyde gels. RNA was blotted onto Zeta probe membranes and hybridized to riboprobes specific for chicken CaM (cCaM), rat ANF, mouse skeletal muscle {alpha}-actin, or mouse vascular smooth muscle {alpha}-actin, as specified, in a buffer containing 50% formamide, 1.5x SSPE, 1% SDS, 0.5% Blotto, 0.2 mg/ml yeast RNA, 0.5 mg/ml salmon sperm DNA, and [32P]riboprobe. cRNA CaM riboprobe was generated from the 3'-end of the chicken CaM cDNA as described previously (13). cRNA ANF riboprobe was generated from a plasmid (pGEM) containing a 0.6-kb PstI fragment of rat ANF (61), using SP6 RNA polymerase after plasmid linearization with HindIII according to the Promega protocol (Promega Corp., Madison, WI). cRNA skeletal {alpha}-actin riboprobe was generated from a Bluescript plasmid containing 240 bp of the 3'-untranslated region of mouse skeletal muscle {alpha}-actin (generous gift of Dr. R. J. Schwartz), using T3 RNA polymerase. cRNA mouse smooth muscle {alpha}-actin riboprobe was generated from Bluescript plasmid containing 160 bp (DdeI-EcoRI fragment) of the 3'-untranslated region of mouse vascular smooth muscle {alpha}-actin (62), using T3 RNA polymerase. When oligonucleotides were used as probes, the membranes were hybridized in a buffer containing 5xSSC, 20 mM sodium phosphate (pH 7.2), 7% SDS, 1x Denhardt’s, 0.1 mg/ml salmon sperm DNA, and [32P]oligoprobe. The oligonucleotide probe used for myosin light chain-2v, which has been previously described (63), was labeled using T4 polynucleotide kinase. When quantification was required, the membranes were stripped (according to the Bio-Rad Laboratories, Inc. instructions) and reprobed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA, using a 780-bp fragment (PstI-XbaI) of the human GADPH cDNA containing the 5'-UT region and the sequence encoding the first 250 amino acids (64). ANF and MLC-2v mRNA levels, determined by Northern analysis, were normalized to GAPDH mRNA levels after quantification by a PhosphorImager.

In Situ Hybridization
Hearts were rinsed in PBS, frozen in Isopentane at -30 C, and kept in a -80 C freezer. Sections (10 µm) were obtained with a Frigocut cryostat (Leica Corp., Nussloch, Germany) at -20 C and thaw-mounted on RNAse free silylated glass microscope slides (CEL Associates Inc, Houston, TX) and stored at -80 C. Frozen slides were fixed in ice-cold 4% paraformaldehyde in PBS for 10 min, rinsed with DEPCed 2xSSC (SSC: 150 mM NaCl, 15 mM sodium citrate, pH 7.0) and the sections were illuminated with a UV lamp for 5 min to postfix the biological material. After the sections were incubated with the prehybridization buffer (50% formamide, 0.6 M NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, 50 µg/ml salmon sperm DNA, 500 µg/ml yeast total RNA, 50 µg/ml yeast transfer RNA) in a humid oven at 50 C for 1 h, the RNA was hybridized with the hybridization buffer (50% formamide, 0.6 M NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, 10 µg/ml salmon sperm DNA, 50 µg/ml yeast total RNA, 50 µg/ml yeast transfer RNA, 10% dextran sulfate) containing 5000 cpm/µl of a heat-denatured (15 min at 65 C), [35S]RNA probe overnight at 50 C. After hybridization the slides were washed, dehydrated, developed, and counterstained in hematoxylin-eosin as described previously (65). cRNA CaM and cRNA ANF were obtained from the same constructs used for Northern blots, but they were labeled instead with rUTP, according to the Promega Corp. protocol. The specific activity of the [35S]riboprobes was higher than 108 cpm/µg RNA.

Statistical Analysis
ANOVA, Student’s t test, and regression analysis were done using Statview (Abacus Concepts Inc., Berkeley, CA). Statistical significance was accepted when P < 0.05.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. R. J. Schwartz (Baylor College of Medicine, Houston, TX) for providing us with mouse skeletal {alpha}-actin probe and Dr. M. B. Kennedy (Caltech, Pasadena, CA) for kindly sharing her specific antibodies for CaMKII. We also gratefully acknowledge Dr. H. A. Rockman (Duke University) for measuring blood presure in mice.

J. Colomer is a recipient of a Formación de Personel Investigador fellowship from the Spanish Government. This research was supported by NIH Grants HD-07503 and GM-33976 (to A.R.M.).


    FOOTNOTES
 
Address requests for reprints to: Anthony R. Means, Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710. E-mail: tony.means{at}duke.edu

Received for publication February 21, 2000. Revision received April 12, 2000. Accepted for publication April 13, 2000.


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