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Endocrinology Vol. 148, No. 9 4162-4169
Copyright © 2007 by The Endocrine Society

Local Actions of Atrial Natriuretic Peptide Counteract Angiotensin II Stimulated Cardiac Remodeling

Ana Kilic1, Alexander Bubikat1, Birgit Gaßner, Hideo A. Baba and Michaela Kuhn

Institute of Physiology (A.K., A.B., B.G., M.K.), University of Würzburg, 97070 Würzburg, Germany; and Institute of Pathology (H.A.B.), University of Duisburg-Essen, 47057 Duisburg, Germany

Address all correspondence and requests for reprints to: Michaela Kuhn, Physiologisches Institut der Universität Würzburg, Röntgenring 9, D-97070 Würzburg, Germany. E-mail: michaela.kuhn{at}mail.uni-wuerzburg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cardiac hormones atrial and brain natriuretic peptides (NPs) counteract the systemic, hypertensive, and hypervolemic actions of angiotensin II (Ang II) via their guanylyl cyclase-A (GC-A) receptor. In the present study, we took advantage of genetically modified mice with conditional, cardiomyocyte (CM)-restricted disruption of GC-A (CM GC-A knockout mice) to study whether NPs can moderate not only the endocrine but also the cardiac actions of Ang II in vivo. Fluorometric measurements of [Ca2+]i transients in isolated, electrically paced adult CMs showed that atrial NP inhibits the stimulatory effects of Ang II on free cytosolic Ca2+ transients via GC-A. Remarkably, GC-A-deficient CMs exhibited greatly enhanced [Ca2+]i responses to Ang II, which was partly related to increased activation of the Na+/H+-exchanger NHE-1. Chronic administration of Ang II to control and CM GC-A knockout mice (300 ng/kg body weight per minute via osmotic minipumps during 2 wk) provoked significant cardiac hypertrophy, which was markedly exacerbated in the later genotype. This was concomitant to increased cardiac expression of NHE-1 and enhanced activation of the Ca2+/calmodulin-dependent prohypertrophic signal transducers Ca2+/calmodulin-dependent kinase II and calcineurin. On the basis of these results, we conclude that NPs exert direct local, GC-A-mediated myocardial effects to antagonize the [Ca2+]i-dependent hypertrophic growth response to Ang II.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RENIN-ANGIOTENSIN SYSTEM (RAS) plays a crucial role in the endocrine regulation of arterial blood pressure and volume. Angiotensin II (Ang II) is a potent vasoconstrictor; it increases sympathetic tone and exerts indirect antidiuretic and antinatriuretic actions via stimulation of aldosterone and antidiuretic hormone secretion (for review, see Ref. 1). Besides these systemic actions, locally produced Ang II exerts direct trophic actions within the heart, inducing cardiomyocyte (CM) hypertrophy and fibroblast proliferation and thereby causing pathologic cardiac remodeling. Thus, elevated cardiac Ang II levels have been observed in experimental models of hypertensive cardiac hypertrophy and clinically during the development of heart failure (2, 3). Conversely, numerous studies have demonstrated the efficacy of RAS blockade in the treatment of cardiac remodeling and heart failure, independently of the reduction in systemic blood pressure. The hypertensive and cardiac hypertrophic as well as profibrotic actions of Ang II are mediated through the Ang II type 1 (AT1) receptor (4, 5).

The systemic, hypertensive and hypervolemic actions of Ang II are counter-regulated by the cardiac hormones atrial (ANP) and brain (BNP) natriuretic peptides. ANP is mainly produced in the atria and BNP in ventricles, both being released into the bloodstream in response to increased wall stretch (6, 7). They activate the guanylyl cyclase-A (GC-A) receptor to cGMP production in a variety of tissues and thereby ultimately decrease blood pressure and volume (6, 7). Importantly, NPs, via GC-A, reduce both the systemic activity and the effects of the RAS: they inhibit renin release and Ang II-stimulated aldosterone production (8, 9) and antagonize the vasoconstrictor, sympathotonic, and antinatriuretic actions of Ang II (10). Thus, the physiological balance between the endocrine NP/GC-A and Ang II/AT1 systems is critical for the maintenance of blood pressure and volume homeostasis. During chronic hemodynamic overload, the expression levels of ANP and BNP in the cardiac ventricles increase (11). In this situation, NPs may act not only as circulating endocrine factors but also as local antihypertrophic and antifibrotic cardiac factors (12). Hence, it was shown that ANP reduces the trophic actions of Ang II on cultured neonatal CMs and cardiac fibroblasts (13). Also, overexpression of GC-A in CMs exerted antihypertrophic effects in vivo (14). Moreover, CM-restricted deletion of GC-A in mice [CM GC-A knockout (KO)] led to blood pressure-independent cardiac hypertrophy and exacerbated cardiac remodeling in response to increased chronic pressure load (15, 16). These observations indicate a key regulatory role of NPs and GC-A not only in controlling blood pressure and volume but also in locally protecting the heart from abnormal remodeling. However, the molecular mechanisms mediating these protective cardiac effects as well as the cardiac interactions of NPs with the Ang II/AT1 system in vivo are essentially unknown.

To determine whether the local, cardiac NP/GC-A system can counter-regulate the cardiac actions of Ang II in vivo, in the present study, we examined the responses of CM GC-A KO mice (15) to exogenous Ang II treatment. The hypertensive responses to Ang II were not altered. However, Ang II-induced cardiac hypertrophy and fibrosis were markedly enhanced, which was associated with increased cardiac activity of the Ca2+/calmodulin-dependent prohypertrophic pathways Ca2+/calmodulin-dependent kinase II (CaMKII) and calcineurin. Moreover, fluorometric measurements in isolated adult CMs demonstrated that the calcium responses to Ang II were markedly enhanced in GC-A-deficient CMs. We conclude that local, cardiac NP/GC-A signaling moderates the cardiac growth response to Ang II by counter-regulating its stimulatory effects on CM [Ca2+]i levels and on Ca2+-dependent CaMKII and calcineurin activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice with cardiac specific deletion of the ANP receptor
Mice with conditional, CM-restricted deletion of the GC-A receptor (CM GC-A KO mice) and control littermates (floxed GC-A mice, with normal GC-A expression levels) were generated by the Cre/loxP strategy and genotyped as described previously (15). All investigations conform with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication no. 85-23, revised 1996) and were approved by the local animal care committee.

Chronic Ang II infusion
Control and CM GC-A KO mice received Ang II (Sigma, Taufkirchen, Germany) at a dose of 300 ng/kg body weight (BW) per minute during 2 wk. The peptide was dissolved in 0.9% NaCl and 0.01 M acetic acid and then infused subcutaneously via osmotic minipumps (model 2002; Alzet, Colorado City, CO). For comparison, additional mice of both genotypes were only given vehicle. Eight- to 12-wk-old male and female mice were examined (n = 8–10 mice per group).

Hemodynamic measurements and tissue harvesting
Before and during Ang II treatment, arterial blood pressure was measured in conscious mice by tail cuff (Softron, Tokyo, Japan) (15, 17). Mice were killed under urethane anesthesia, the hearts were weighed, and the left ventricles were bisected and frozen in liquid nitrogen (for RNA or protein extraction) and fixed in 4% buffered formaldehyde (for histology).

Histology
For histological analysis, formaldehyde-fixed left ventricles were embedded in paraffin, and 5-µm sections were stained with hematoxylin-eosin, periodic acid Schiff (to discriminate CM cell borders) or 0.1% picrosirius red (for collagen) (15, 17). Photomicrographs of the sections were evaluated using a computer-assisted image analysis system (VIDAS 25; Zeiss, Jena, Germany), with the investigator blinded to the genotypes. The mean cross-sectional CM diameters were calculated by measuring 100 cells with a centrally located nucleus per specimen. Interstitial collagen fractions were obtained by calculating the ratio, in percentage, between the collagen area and the total ventricular area in the corresponding section (15, 17).

Intracellular [Ca2+]i transients
Ventricular myocytes from control (n = 5) and CM GC-A KO (n = 5) hearts were isolated by liberase/trypsin digestion (for procedure, see Protocol PP00000125 from The Alliance for Cellular Signaling), and [Ca2+]i transients were measured in Indo-1-loaded, electrically paced (0.5 Hz) CMs as described previously (17). Excitation was at 365 nm, and the emitted fluorescence was recorded at 405 and 495 nm. The ratio of fluorescence at the two wavelengths was used as an index of the cytosolic Ca2+concentration. Data were collected at 20 Hz, and acquisition and processing were supported by Felix software (Felix version 1.1; Photon Technologies, Seefeld, Germany) (17).

After obtaining basal recordings for 10 min, the acute effects of Ang II were tested by superfusion of CMs with 100 nM Ang II for an additional 10 min. In parallel experiments, the effects of ANP (100 nM; Bachem, Bubendorf, Switzerland) or of the Na+/H+-exchanger NHE-1 inhibitor cariporide (10 µM; Sanofi-Aventis, E Frankfurt am Main, Germany) on baseline vs. Ang II-stimulated [Ca2+]i transients were tested (superfusion of cardiomyocytes with ANP or cariporide during 10 min and then superfusion with Ang II in the presence of ANP or cariporide during the additional 10 min).

Western blot analyses
Left ventricular proteins were solubilized in SDS-sample buffer and separated by 10% PAGE. The primary antibodies were against NHE-1 (Chemicon, Temecula, CA), calcineurin, CaMKII (BD Transduction Laboratories, Lexington, KY), autophosphorylated (active) CaMKII (Santa Cruz Biotechnology, Santa Cruz, CA), and threonine-17-phosphorylated as well as total phospholamban (PLB) (Badrilla, Leeds, UK). Either calsequestrin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Trevigen, Gaithersburg, MD) was used for loading controls (17). The blots were developed using the enhanced chemiluminescence detection system (GE Healthcare, Little Chalfont, UK), and results were quantitated by densitometry (ImageQuant) (17).

Analyses of mRNA
Ventricular mRNA levels of ANP and myocyte-enriched calcineurin interacting protein 1 (MCIP1) were estimated from 20 µg total RNA. A specific ANP cDNA probe as well as a DNA fragment encompassing the 5' exon 4 splice variant of murine MCIP1 (15, 17, 18) were [32P]dCTP labeled. Signals were visualized in a phosphor imager and quantified by ImageQuant software (15). A quantitative analysis of angiotensinogen and AT1 receptor mRNA expression was performed by RT-PCR using the LightCycler Detection System (Roche, Mannheim, Germany) as described previously (19). GAPDH or cyclophilin were used for normalization (19).

Statistics
Results are presented as means ± SEM. Group data were compared using one-way or two-way ANOVA (with genotype and treatment as categories), followed by the multiple comparison Bonferroni’s t test to assess differences. The significance level was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CM GC-A KO mice exhibit exacerbated cardiac remodeling in response to Ang II
Corresponding to our previous study (15), the diastolic and systolic blood pressure levels of vehicle-treated CM GC-A KO mice were not different from their control littermates (floxed GC-A mice with unaltered GC-A expression) (Fig. 1AGo), indicating the preservation of the endocrine effects of ANP. Despite this, these mice exhibit mild increases in cardiac weights (heart weight/BW ratios were 4.6 ± 0.07 in controls and 5.2 ± 0.2 in CM GC-A KO mice) (Fig. 1BGo) together with significant increases in CM diameters (Fig. 2AGo), indicating mild, blood pressure-independent cardiac hypertrophy without cardiac fibrosis (Fig. 2BGo). This mild hypertrophy was accompanied by induction of ventricular ANP mRNA expression (Fig. 1CGo). Of note, the cardiac changes in these young, 2-month-old mice were less pronounced compared with the older mice studied previously (15).


Figure 1
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  		FIG. 1. Effect of chronic Ang II infusion on systolic blood pressure (A), cardiac weights (B), and ventricular ANP mRNA levels (C) of floxed GC-A (controls) and CM GC-A KO mice. C, Representative Northern blot. The four lanes in each group correspond to mRNAs from four separate mice. ANP mRNA levels were normalized to GAPDH and calculated as x-fold respective vehicle-treated controls. The cardiac but not the pressor responses to Ang II were significantly enhanced in CM GC-A KO mice (n = 8–10 per genotype and treatment group; *, P < 0.05 compared with vehicle; #, P < 0.05 compared with controls).

 

Figure 2
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  		FIG. 2. Morphometrical analyses of left ventricular cardiac sections. Under baseline conditions (in vehicle-treated mice), CM diameters were significantly increased in CM GC-A KO compared with control mice (A and B), whereas interstitial collagen contents were not different between genotypes (C and D). Chronic infusion of Ang II provoked significant CM hypertrophy and fibrosis, these responses being enhanced in CM GC-A KO mice (A–D) (n = 8–10 per genotype and treatment group; *, P < 0.05 compared with vehicle; #, P < 0.05 compared with controls).

 
Administration of Ang II (300 ng/kg BW· min) provoked significant increases in arterial blood pressure (increases in systolic blood pressure levels by ~25 mm Hg). This pressor effect of Ang II was not different between genotypes (Fig. 1AGo). In both genotypes, Ang II led to significant increases in the heart weight/BW ratios (Fig. 1BGo), increased ventricular ANP mRNA levels (Fig. 1CGo), CM hypertrophy (as indicated by enlargement of CM diameters) (Fig. 2AGo), and increases in cardiac interstitial collagen content (Fig. 2BGo). Notably, these cardiac responses to Ang II were more pronounced in CM GC-A KO mice than in controls, despite the similar hypertensive reactions. We conclude from these data that a chronic inhibition of the local, cardiac effects of NPs exacerbates the hypertrophic and profibrotic actions of Ang II.

Enhanced effects of Ang II on intracellular Ca2+ transients in GC-A-deficient cardiomyocytes
To elucidate whether altered CM [Ca2+]i responses to Ang II participate in the increased cardiac hypertrophic effects observed in CM GC-A KO mice, we measured [Ca2+]i transients in isolated adult ventricular myocytes from CM GC-A KO and control mice under basal condition and in the presence of Ang II. Fluorometric measurements of [Ca2+]i transients in isolated, electrically paced adult CMs indicated that myocyte diastolic [Ca2+]i levels were not different between genotypes (Fig. 3AGo). However, baseline systolic [Ca2+]i levels and therefore peak amplitude of [Ca2+]i transients were increased in GC-A-deficient myocytes compared with controls (Fig. 3Go, A and B). Superfusion of CMs with Ang II (100 nM) induced acute increases of free systolic [Ca2+]i levels in both genotypes. Interestingly, these [Ca2+]i responses were significantly enhanced in GC-A-deficient myocytes (Fig. 3Go, A and B).


Figure 3
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  		FIG. 3. Effects of Ang II on intracellular Ca2+ transients in electrically paced cardiomyocytes from CM GC-A KO and respective control mice. A, Representative tracings of the Indo-1 ratio in single CMs from control and CM GC-A KO mice under baseline conditions (continuous line) and during superfusion with Ang II (dotted line). B, On average, the peak amplitude of Ca2+ transients (Indo-1 ratio, 405/495 nm, systolic/diastolic) was significantly increased in GC-A-deficient (CM GC-A KO) compared with control myocytes. Superfusion with Ang II increased [Ca2+]i transients in myocytes from both genotypes, the effects being enhanced in GC-A-deficient cells. C, Superfusion with ANP had no effect on baseline Ca2+ transients. Notably, ANP fully prevented the Ca2+ responses to Ang II in control but not in GC-A-deficient myocytes. D, Cariporide did not affect baseline [Ca2+]i transients in controls but reversed the increased [Ca2+]i transients of GC-A-deficient myocytes. Notably cariporide fully prevented the [Ca2+]i responses to Ang II in cells from both genotypes (n = 2 cells from each of 5 mice per genotype; *, P < 0.05 compared with baseline; #, P < 0.05 compared with controls).

 
ANP superfusion had no effect on baseline [Ca2+]i transients (Fig. 3CGo). However, ANP fully prevented the [Ca2+]i responses of control myocytes to Ang II (Fig. 3CGo). This inhibitory effect was abolished in GC-A-deficient myocytes (Fig. 3CGo).

In a recent study, we demonstrated that the increased baseline [Ca2+]i transients of GC-A-deficient myocytes are related to enhanced activity of NHE-1, the main Na+/H+-exchanger in the heart (17). Notably, previous in vitro studies suggested that ANP, via GC-A/cGMP, inhibits (20) whereas Ang II, via AT1 receptors, stimulates sarcolemmal NHE-1 activity (21). To elucidate whether the [Ca2+]i responses of myocytes to Ang II are partly related to NHE-1 activation, we studied the effects of the selective NHE-1 inhibitor cariporide. As shown in Fig. 3DGo, cariporide had no effect on baseline [Ca2+]i transients of control myocytes but reversed the increased [Ca2+]i transients of GC-A-deficient myocytes. Most importantly, cariporide fully prevented the Ca2+ responses to Ang II in both control and GC-A-deficient myocytes.

As shown in Fig. 4Go, under baseline conditions (in vehicle-treated animals), the left ventricular NHE-1 expression levels were not different between control and CM GC-A KO mice, indicating that enhanced activity (not expression) of the exchanger contributes to the increased baseline myocyte [Ca2+]i transients of the later. Notably, chronic treatment of mice with Ang II (300 ng/kg·min, 2 wk) provoked significant increases in left ventricular NHE-1 levels in both control (on average by 1.3-fold) and even more in CM GC-A KO mice (by almost 2-fold) (Fig. 4Go).


Figure 4
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  		FIG. 4. Left ventricular expression levels of NHE-1 in untreated (vehicle) vs. Ang II-treated control and CM GC-A KO mice. Top, Representative Western blots. The two lanes in each group correspond to cardiac proteins from two separate mice. Bottom, Protein levels were normalized to GAPDH and calculated as x-fold respective vehicle-treated controls. Cardiac NHE-1 expression levels were not different between genotypes but were significantly increased by Ang II in control and even more in CM GC-A KO mice (n = 8–10 mice per group; *, P < 0.05).

 
We conclude from these fluorometric and protein data that chronic ablation of ANP/GC-A signaling in CMs increases their [Ca2+]i responses to Ang II, an effect that is at least partly related to increased activity of NHE-1.

Enhanced Ang II-induced cardiac remodeling in CM GC-A KO mice was accompanied by activation of Ca2+/calmodulin-dependent pathways such as CaMKII and calcineurin
Because increases in free cytoplasmic Ca2+ levels can induce myocyte hypertrophy through Ca2+/calmodulin-mediated activation of CaMKII and/or calcineurin signaling (22), we assessed the activity of these pathways. Unfortunately, the traditional biochemical calcineurin and CaMK activity assays rely on homogenized extracts in the presence of exogenously supplied Ca2+ and calmodulin and therefore may approximate total calcineurin or CaMK availability rather than the specific endogenous activity of these enzymes (23). Instead, we estimated their activity by evaluating endogenous cardiac signaling (see below).

The cardiac expression levels of total CaMKII were not different between genotypes and/or treatment groups (Fig. 5AGo). However, the levels of autophosphorylated (active) CaMKII were increased in CM GC-A KO mice already under basal conditions (vehicle treatment) (Fig. 5AGo). In both genotypes, chronic Ang II treatment (300 ng/kg·min, 2 wk) significantly increased cardiac CaMKII autophosphorylation, this effect being markedly enhanced in CM GC-A KO mice. To corroborate these results, we also analyzed the endogenous phosphorylation status of a specific downstream target of CaMKII, the regulatory SR protein PLB (specifically phosphorylated by CaMKII at position threonin-17) (24). As shown in Fig. 5BGo, the phosphorylation of PLB at threonin-17 was increased in hearts from CM GC-A KO mice under baseline conditions. Ang II treatment increased the cardiac levels of phosphorylated PLB in control and, even more, in CM GC-A KO mice (Fig. 5BGo).


Figure 5
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  		FIG. 5. Activation of the cardiac CaMKII signaling pathway by Ang II. Representative Western blots for the left ventricular levels of CaMKII and autophosphorylated CaMKII (A) and of threonine-17 phosphorylated PLB (B). Top, Representative Western blots. Bottom, Levels of phosphorylated CaMKII and PLB were normalized to the total levels of the respective proteins and calculated as x-fold respective vehicle-treated control mice. The levels of autophosphorylated CaMKII and of threonine-17-phosphorylated PLB were significantly increased in CM GC-A KO hearts. Chronic Ang II infusion enhanced the cardiac levels of phosphorylated CaMKII and of phosphorylated PLB in both genotypes. This effect was increased in CM GC-A KO mice (n = 8–10 mice per group; *, P < 0.05).

 
As shown in Fig. 6AGo, the cardiac levels of calcineurin were not different between genotypes and were not affected by Ang II treatment. Because these results reflect total calcineurin availability but not the endogenous activity of this phosphatase, we also measured the mRNA expression of the 5' exon 4 splice variant of MCIP1.4, which is specifically induced by calcineurin/nuclear factor of activated T-cell (NFAT) signaling (18, 25). Hence, the mRNA expression levels of MCIP1.4 have been used in many studies as an indirect index of endogenous cardiac calcineurin activity (17, 18, 25). Northern blot analysis showed no genotype-dependent differences in cardiac MCIP1.4 expression under baseline conditions in vehicle-treated mice (Fig. 6BGo). Ang II treatment caused an approximately 2-fold increase of MCIP1.4 in control mice and a nearly 4-fold induction in CM GC-A KO mice (Fig. 6BGo). Together, these data demonstrate that Ang II led to cardiac CaMKII as well as calcineurin activation, these effects being significantly enhanced in CM GC-A KO mice.


Figure 6
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  		FIG. 6. Activation of the calcineurin/NFAT signaling pathway by Ang II. A, Western blots demonstrated that the cardiac levels of calcineurin were not different between genotypes and were not affected by chronic Ang II infusion. Top, Representative Western blot. Bottom, Protein levels were normalized to the CM-specific protein calsequestrin (CSQ) and calculated as x-fold vs. vehicle-treated control mice. B, Northern blots demonstrated that chronic Ang II infusion enhanced the cardiac levels of MCIP1.4 in both genotypes. This effect was increased in CM GC-A KO mice. Top, Representative Northern blot. Bottom, MCIP1.4 expression levels were normalized to the levels of GAPDH and calculated as x-fold respective vehicle-treated controls (n = 8–10 mice per group; *, P < 0.05).

 
Altered expression of AT1 receptors does not account for the increased effects of Ang II in CM GC-A KO mice
Finally, we found that expression levels of ventricular angiotensinogen and AT1 receptor mRNA (the receptor subtype mediating the effects of Ang II on cardiac remodeling) were similar in control and CM GC-A KO mice (Fig. 7Go).


Figure 7
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  		FIG. 7. The left ventricular mRNA expression levels of angiotensinogen and of the AT1 receptor were not different between genotypes (n = 8 mice per group). m CYC, Mouse cyclophilin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Principal findings
Our study demonstrates that Ang II-induced cardiac hypertrophy and interstitial fibrosis were markedly increased in CM GC-A KO mice. This was concomitant to enhanced myocyte [Ca2+]i responses and greater activity of the Ca2+-dependent prohypertrophic signal transducers CaMKII and calcineurin. Our observations suggest that cardiac NPs and Ang II exert reciprocal effects on CM [Ca2+]i homeostasis and on calcium-dependent hypertrophic signaling pathways such as CaMKII and calcineurin.

Our results are in accordance with previous studies showing that cardiac hypertrophy and fibrosis in mice with global (not CM-specific) GC-A disruption are markedly inhibited by genetic or pharmacological blockade of the AT1 receptor (26). As a corollary, Ang II-induced cardiac remodeling was suppressed in mice overexpressing BNP in the circulation, a member of the NP family that also activates the GC-A receptor, although with less affinity than ANP (27). Our study adds an important piece of information, because the conditional, CM-restricted disruption of the GC-A gene in mice allowed us to specifically dissect the local cardiac from the systemic interactions between the ANP/GC-A and Ang II/AT1 systems. Furthermore, it characterizes the molecular effectors responsible for the crosstalk between these pathways in cardiac remodeling.

Chronic ablation of cardiac GC-A leads to enhanced [Ca2+]i responses to Ang II
Cardiac expression of AT1 receptors was not altered in CM GC-A KO mice, at least at the mRNA level. This suggests that the hyperresponsiveness to Ang II involves modulation of component(s) of the downstream signal transduction cascade from the AT1 receptor to [Ca2+]i handling. Interestingly, separate in vitro studies showed that ANP and Ang II exert opposing, inhibiting vs. stimulating effects on the activity of the sarcolemmal Na+/H+ exchanger (20, 21). Stimulation of NHE-1 can increase CM [Ca2+]i via Na+/Ca2+ exchange, a mechanism that at least partly mediates the hypertrophic effects of Ang II [comprehensively reviewed by Karmazyn and colleagues (28, 29)]. Notably, at least in epithelial cells, ANP directly blocked the stimulatory effects of Ang II on NHE-1 activity and the subsequent increases in intracellular Ca2+ levels (30). The following observations in our study suggest that this reciprocal modulation of NHE-1 also occurs in the heart: 1) ANP, via GC-A, inhibited myocyte [Ca2+]i responses to Ang II; 2) the stimulatory effect of Ang II on [Ca2+]i levels was markedly increased in GC-A-deficient myocytes; 3) inhibition of NHE-1 with cariporide fully abolished the Ca2+-stimulating effects of Ang II in both control and GC-A-deficient myocytes, indicating that these effects are related at least in part to NHE-1 activity; and 4) cardiac expression levels of NHE-1 were enhanced by chronic Ang II administration, and this effect was markedly increased in CM GC-A KO mice. Together, these results suggest that the local, cardiac NP/GC-A system can counter-regulate NHE-1-dependent, stimulatory effects of Ang II on myocytes-free cytosolic [Ca2+]i levels. The exact mechanism for this reciprocal regulation of NHE-1 expression and activity by NPs/Ang II will be our scope in future studies.

Downstream signaling pathways leading to increased hypertrophic responses to Ang II
Several signal transduction pathways have been implicated in Ang II-induced cardiac hypertrophy, many of which are mediated by intracellular calcium signaling (22). The Ca2+/calmodulin-dependent pathways calcineurin and CaMKII are especially effective inducers of cardiac growth. Indeed, our study shows that both pathways, calcineurin and CaMKII, were activated by Ang II treatment, and, in line with the increased cardiomyocyte [Ca2+]i responses to Ang II, both pathways were enhanced in CM GC-A KO mice.

Because isolated cardiomyocytes from CM GC-A KO mice exhibited increased free cytosolic systolic [Ca2+]i transients already under baseline conditions, it seems intriguing that only cardiac CaMKII but not calcineurin activity was increased under baseline conditions (in vehicle-treated CM GC-A KO compared with control mice). It has been suggested that calcineurin is preferentially activated by a sustained, low Ca2+ plateau (31) or by specific subcellular Ca2+ pools (32). Thus, it is conceivable that the increased amplitude of [Ca2+]i transients observed in our genetic mouse model led to a selective activation of CaMKII activity, as the main mechanism contributing to the mild baseline cardiac hypertrophy.

Together with published data, our study indicates that NP/GC-A/cGMP signaling can moderate the activity of different pathways involved in CM growth, such as NHE-1, calcineurin/NFAT, CaMKII (present study), and ERK (27). However, the molecular mechanisms of signaling immediately downstream of cGMP are not completely understood. Recent in vivo and in vitro evidence identifies cGMP-dependent protein kinase I (PKG I) as a major mediator of cGMP signaling in the cardiovascular system (33). Indeed, the overexpression of PKG I in cultured neonatal CMs reduced hypertrophic growth (34). However, neither global nor CM-specific ablation of PKG I affects the development of cardiac hypertrophy under basal or hypertrophy-inducing conditions in vivo (33). Thus, the immediate downstream targets of cGMP in the heart are essentially unknown.

Mechanisms leading to increased fibrotic responses to Ang II
As demonstrated, the hypertrophic responses of CM GC-A KO mice to Ang II were accompanied by pronounced cardiac interstitial fibrosis. The mechanism(s) underlying this effect remains unclear. The GC-A receptors are present on fibroblasts and mediate antiproliferative effects in vitro, but they should not be affected by our strategy of conditional, CM-restricted gene deletion. Thus, increased fibrosis after deletion of myocyte GC-A could result from alterations in the Ang II-stimulated production of profibrotic or antifibrotic factors by myocytes. For instance, Ang II stimulated TGF-ß production and release by adult rat CMs (35). Even more, TGF-ß1 gene expression was significantly increased in the hearts of mice with global (not CM-restricted) GC-A deletion and was reversed by genetic deletion of the AT1 receptor (26). Based on these published observations, we hypothesize that ANP and BNP, via GC-A, might inhibit the stimulatory effects of Ang II on both fibroblast and CM synthesis and/or secretion of profibrotic factors such as TGF-ß1 and thereby moderate cardiac fibrosis.

Conclusion
In some forms of arterial hypertension and as one of the earliest and pathognomonic events in cardiac hypertrophy and insufficiency, the cardiac synthesis and release of ANP and BNP is markedly enhanced, but their GC-A-mediated effects are clearly diminished, indicating a receptor or postreceptor defect (7, 36). Concurrently, the deleterious role of the local RAS-aldosterone system in cardiac remodeling has been demonstrated by many experimental and clinical reports. Our study emphasizes that a disturbance of the delicate systemic and also local, cardiac balance between NPs and the RAS can critically contribute to the progression of cardiac hypertrophy and fibrosis.


   Footnotes
 
This study was supported by Deutsche Forschungsgemeinschaft Sonderforschungsbereich 487-B8 (to M.K.).

Disclosure Statement: A.K., A.B., B.G., H.A.B., and M.K. have nothing to declare.

First Published Online May 17, 2007

1 A.K. and A.B. contributed equally to this work. Back

Abbreviations: Ang II, Angiotensin II; ANP, atrial natriuretic peptide; AT1, angiotensin II type 1; BNP, brain natriuretic peptide; BW, body weight; CaMKII, Ca2+/calmodulin-dependent protein kinase II; CM GC-A KO, cardiomyocyte GC-A knockout; GC-A, guanylyl cyclase-A; MCIP1, myocyte-enriched calcineurin interacting protein 1; NFAT, nuclear factor of activated T-cell; NHE-1, Na+/H+ exchanger type 1; NP, natriuretic peptide; PKG I, cGMP-dependent protein kinase I; PLB, phospholamban; RAS, renin-angiotensin system.

Received February 13, 2007.

Accepted for publication May 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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L. R. Forte Jr.
Atriopeptins: Protection from Myocardial Hypertrophy and Heart Failure
Endocrinology, September 1, 2007; 148(9): 4160 - 4161.
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