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Endocrinology Vol. 148, No. 6 2815-2820
Copyright © 2007 by The Endocrine Society

Calcium-Calmodulin Kinase II Is the Common Factor in Calcium-Dependent Cardiac Expression and Secretion of A- and B-Type Natriuretic Peptides

Jarkko J. Ronkainen, Olli Vuolteenaho and Pasi Tavi

Department of Physiology and Biocenter Oulu, University of Oulu, 90014 Oulu, Finland

Address all correspondence and requests for reprints to: Pasi Tavi, University of Oulu, Department of Physiology and Biocenter Oulu, P.O. Box 5000, University of Oulu, 90014 Oulu, Finland. E-mail: pasi.tavi{at}oulu.fi.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peptides derived from the precursor of A- and B-type natriuretic peptides (ANP and BNP) are powerful clinical markers of cardiac hypertrophy and dysfunction. It is known that many stimuli affecting the intracellular calcium concentration also induce ANP and BNP secretion. It was our intention to study the mechanisms by which calcium regulates the secretion of ANP and BNP. The effects of pacing and calcium-calmodulin kinase II activity on natriuretic peptide secretion were studied in isolated perfused rat atria and cultured rat neonatal cardiomyocytes. In isolated rat atrium pacing induced an increase in diastolic, systolic, and averaged intracellular free calcium concentration and a frequency-dependent increase in the secretion of both ANP and BNP. The molar ratio of the secreted natriuretic peptides (ANP to BNP) remained nearly constant (~1000) at all the pacing frequencies tested (1, 3, 6, and 8 Hz). Calmodulin kinase II inhibitor KN-93 (3 µM) did not affect intracellular free calcium concentration but showed a frequency-dependent inhibitory effect on ANP and BNP secretion without a change in ANP to BNP ratio. In the neonatal cardiomyocytes, KN-93 (3 µM) suppressed the secretion and gene expression of both ANP and BNP. Overexpression of constitutively active (T286D) or nuclear ({delta}B) calcium-calmodulin kinase II induced an increase in ANP and BNP gene expression. The results indicate that the calcium-dependent secretion and gene expression of A- and B-type natriuretic peptides are similarly regulated by calmodulin kinase II-dependent mechanisms. This is a plausible mechanism contributing to exercise-induced natriuretic peptide secretion and the augmented secretion in heart dysfunction due to impaired calcium handling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INCREASED CARDIAC EXPRESSION and secretion of A- and B-type natriuretic peptides (ANP and BNP) is associated with cardiac growth, heart dysfunction, and physical exercise (1, 2). ANP and BNP are useful and powerful clinical tools in diagnosis and guiding the treatment of cardiac failure (3, 4). Cardiac myocytes are the main source of natriuretic peptides (NPs), which they produce and secrete in response to various stimuli (1). A number of neuronal, hormonal, and mechanical stimuli that increase NP production also promote changes in the intracellular free calcium concentration ([Ca2+]i) of the myocytes, implying that the secretion of natriuretic peptides might use calcium-sensitive exocytosis mechanisms which are common in many cell types (5, 6). A suggested link between calcium and ANP secretion is the calcium-calmodulin-dependent protein kinase II (CaMKII) (7, 8). In the heart, calcium-calmodulin-dependent protein kinases are involved in many signaling cascades that promote cardiac hypertrophy and failure (9, 10, 11). Overexpression of either the nuclear ({delta}B) or cytosolic ({delta}C) isoforms of cardiac CaMKII leads to hypertrophy and dilated cardiomyopathy with increased production of ANP (12, 13).

The primary physiological stimulus for CaMKII activation is increased frequency of the intracellular calcium transients (14, 15). Pacing also seems to be a potent physiological stimulus for NP production in the cardiac myocytes. Increased pacing promotes NP secretion in intact tissue preparations as well as cultured neonatal cardiomyocytes (7, 16). A line of evidence suggests a link between calcium activity-dependent CaMKII activation and increased production of ANP; inhibition of CaMKII decreases the pacing-induced secretion of ANP (7). On the other hand, nuclear CaMKII ({delta}B) overexpression activates the ANP promoter constructs as well as increases the number of neonatal cardiomyocytes expressing ANP (8). Although BNP expression and secretion are also stimulated with pacing (16), it is not known whether the secretion of BNP shares the CaMKII-activated mechanism used by ANP. This is possible because ANP has been reported to colocalize with BNP in the same granules in human (17), rat (18), and porcine (19) cardiac myocytes.

The aim of this study was to find out the role of CaMKII in the calcium-induced expression and secretion of A- and B-type natriuretic peptides. We were interested in whether ANP and BNP secretion and gene expression have common or different calcium-dependent regulatory mechanisms in cardiac myocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All experimental protocols were approved by the University of Oulu Animal Care Committee.

Preparation and perfusion of rat atrial appendix
Male Sprague Dawley rats weighing 290–400 g were used. The rats were decapitated, and the hearts were rapidly removed and placed in oxygenated Tyrode buffer solution (millimoles per liter): 113.8 NaCI, 17.6 NaHCO3, 4.7 KCI, 2.0 CaCI2, 1.1 MgSO4, 1.2 KH2PO4, 11.0 glucose, and 10 µIU/ml insulin, pH was 7.4 when bubbled with 5% CO2-95% O2. The same solution was used for superfusion of the atrium (2.5 ml/min) at 37 C. The rat atrial appendix was perfused as described previously (20). The perfusate samples were collected in 4 min (10 ml) fractions. The delay of the perfusate flow from the atria to the fraction collector was approximately 2 min. The atria were paced with a field stimulus (1 msec, 50% over threshold) by two platinum electrodes located inside the perfusion chamber. KN-93 (3 µM, water soluble; Calbiochem, La Jolla, CA) was applied to the perfusion medium 30 min before changing the pacing frequency.

[Ca2+]i measurements
Intracellular calcium signals from perfused rat left atrium were measured with indo-1 (Molecular Probes, Leiden, The Netherlands). The dye loading and calibration of [Ca2+]i were done as previously (21).

Cell culture
Neonatal rat cardiomyocytes were isolated 1–2 d after birth. Ventricles were excised, cut into small pieces, and incubated for 1 h in a solution containing 100 mM NaCl, 10 mM KCl, 1.2 mM KH2PO4, 4.0 mM MgSO4, 50 mM taurine, 20 mM glucose, 10 mM HEPES, 2 mg/ml collagenase type II (Worthington, Freehold, NJ), 2 mg/ml pancreatin (P-3292; Sigma, Buchs, Switzerland), and 1% penicillin-streptomycin. After incubation the detached cells were collected in 15 ml Falcon tubes and centrifuged for 5 min at 160 g. The supernatant and the top layer of the pellet containing damaged cells were discarded, and the isolated cardiomyocytes were plated on 35-mm fibronectin-coated plastic dishes at a density of 750,000 cells/dish. The cells were cultured for 48–72 h to reach confluence in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin before any intervention.

Transfection of neonatal cardiomyocytes
Cells were transiently transfected with Lipofectamine 2000 (10 µl/35 mm plate; Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. After a 5-h transfection period, the Optimem (Invitrogen) transfection medium was replaced with normal culture medium, and the cells were cultured another 48 h before lysis and RNA isolation. To increase CaMKII activity in the cultured cells, an expression vector coding the constitutively active T286D mutant CaMKII{gamma} [pCaMKII-T286D, 8.0 µg/dish (22)] or the overexpression vector of the nuclear CaMKII{delta} splicing variant [pCaMKII{delta}B, 8.0 µg/dish (8)] was used. The expression plasmids were kind gifts from Professor Daniel Goldman (University of Michigan, Ann Arbor, MI) and Professor Joan Brown (University of California, San Diego, San Diego, CA), respectively. In control groups, cells were transfected with an empty cloning vector pUC18 (8.0 µg/dish; Fermentas International, Canada). Transfection efficiency was assessed using the viral vector-driven GFP construct, (pEGFP-N1; CLONTECH Europe, Erembodegen, Belgium). After 48 h approximately 30% of the transfected cells were found to be fluorescent.

Quantitative RT-PCR
Total RNA was isolated from the cultured cardiomyocytes using the GenElute mammalian total RNA miniprep kit (Sigma). After cDNA synthesis (first-strand cDNA synthesis kit; MBI Fermentas), quantitative PCRs were performed with the ABI 7700 sequence detection system (Applied Biosystems, Foster City, CA) using the TaqMan chemistry. The forward and reverse primer and fluorogenic probe sequences used are listed in Table 1Go.


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  		TABLE 1. Oligonucleotide sequences in RT-PCR analysis using TaqMan chemistry

 
Measurement of natriuretic peptide secretion
NT-proANP was assayed directly from the perfusate. BNP was extracted from the perfusate using Sep-Pak C18 cartridges (23). The RIA protocol for NT-proANP has been described previously (23). NT-proANP is secreted in equimolar amounts with ANP. Compared with ANP, NT-proANP is a better marker of proANP-derived peptides because of its higher stability in biological fluids (24). BNP assays were performed with the protocol described previously for ANP (25) using antirat BNP-45 antiserum RIN9080 purchased from Peninsula Laboratories Europe (Merseyside, UK). The sensitivities of the NT-proANP and BNP assays were 0.75 fmol/tube and 1 fmol/tube, respectively. The within- and between-assay coefficients of variation were less than 10% and less than 15%, respectively. The NT-proANP antiserum cross-reacts fully with proANP but does not recognize ANP, BNP, or CNP (cross-reactivity < 0.01%). According to the data supplied by Peninsula Laboratories, the BNP antiserum has 0% cross-reactivity with ANP or fragments of NT-proANP. In our own tests, however, rat BNP antiserum cross-reacted 0.02% with rat ANP.

Samples of medium (0.8 ml) from the cardiomyocyte cultures were acidified with 160 µl of 1 M HCl and extracted with Sep-Pak C18 cartridges using a Gilson Aspec extraction robot. Each cartridge was preconditioned with 2-propanol followed by 0.1% aqueous trifluoroacetic acid. After sample application the cartridge was washed with 5 ml 0.1% aqueous trifluoroacetic acid followed by elution with 1 ml 80% acetonitrile and 0.1% trifluoroacetic acid. The eluates were dried in a SpeedVac centrifuge concentrator for use in RIA.

Statistical testing
Statistical testing was done with one-way or two-way ANOVA followed by Bonferroni test using Origin 7.5 software (OriginLab Corp., Northampton, MA). P < 0.05 was considered statistically significant (***, P < 0.001; **, P < 0.01; *, P < 0.05). All values are expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pacing-induced ANP and BNP secretion
Pacing of the rat atrium resulted in an increase of diastolic and systolic [Ca2+]i as shown in Fig. 1Go. When the atria were subjected to rapid pacing (8 Hz) the secretion of both NT-proANP (Fig. 2AGo) and BNP (Fig. 2BGo) was increased. The onset of increased secretion of both cardiac peptides displayed a 6- to 10-min delay, after which both were activated with a time constant of 11–12 min and reached the maximum secretion rate within 40 min.


Figure 1
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  		FIG. 1. Pacing-induced changes in [Ca2+]i in isolated rat atrium. Representative [Ca2+]i traces at pacing frequencies 1 Hz (left) and 6 Hz (right).

 

Figure 2
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  		FIG. 2. Pacing-induced (8 Hz) NT-proANP secretion (A) and BNP secretion (B) measured from the same samples of the perfusate. Arrow indicates the time of the change of the pacing frequency from 1 to 8 Hz. All values are expressed as mean ± SEM.

 
Assuming that the secretion of both peptides in response to pacing shares the same CaMKII-dependent mechanism, they should also have a similar frequency dependence. To study this we paced atria with different frequencies (1, 3, 6, and 8 Hz) and measured the peptide secretion (Fig. 3Go, A and B). Because both the time course and the extent of secretion of NT-ANP and BNP were almost identical, we calculated the regression coefficient between perfusate NT-proANP and BNP measured from all experiments (Fig. 3Go, A and B). There was a strong linear correlation between the perfusate concentrations of NT-proANP and BNP (r = 0.78, Fig. 3CGo). Furthermore, the molar ratio of NT-proANP/BNP was almost constant regardless of the pacing frequency, ranging from 998 ± 121 at 1 Hz to 1139 ± 123 at 6 Hz (n = 6, not significant) (Fig. 3DGo).


Figure 3
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  		FIG. 3. Cosecretion of NT-proANP and BNP induced by pacing. NT-proANP (A) and BNP secretion (B) at the different pacing frequencies (1, 3, 6, and 8 Hz) in rat atrium (n = 6). Arrows indicate the time of the change of the pacing frequency. C, Correlation of pacing-induced secretion of NT-proANP and BNP. Individual points represent measured NP levels from the same samples of perfusate. Regression line with 95% confidence bands shows correlation coefficient of 0.78. D, Molar ratio of NT-proANP and BNP secretion calculated from A and B. All values are expressed as mean ± SEM.

 
Effect of KN-93 on pacing-induced NT-proANP and BNP secretion
In neonatal cardiac myocytes, pacing-induced ANP secretion can be inhibited by inhibiting CaMKII (7). To test whether CaMKII is involved in pacing-induced natriuretic peptide secretion in rat atrium, we applied CaMKII inhibitor KN-93 (3 µM) before a change in the pacing frequency from 1 to 8 Hz. CaMKII inhibition halved the increase in NT-proANP (Fig. 4Go, A and C) and BNP (Fig. 4Go, B and D) secretion in response to increasing the frequency of pacing from 1 to 8 Hz. It did not, however, have any effect on the secretion of NT-proANP and BNP at 1 Hz or on the delay between the onset of pacing and the induction of peptide secretion. This indicates that ANPs and BNPs share the same CaMKII-dependent secretion mechanism that is recruited by pacing. This is further supported by the fact that KN-93 did not alter the molar ratio of secreted NT-proANP and BNP (Fig. 4EGo).


Figure 4
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  		FIG. 4. Effect of CaMKII inhibition by KN-93 on the pacing-induced secretion of NT-proANP and BNP in rat atrium. Effect of KN-93 (3 µM) on the pacing-induced (1 vs. 8 Hz) NT-proANP (A) and BNP secretion (B). Arrows indicate the time of the change of the pacing frequency. Effect of KN-93 on the steady-state secretion at 1 Hz and 8 Hz of NT-proANP (n = 6) (C) and BNP (n = 6) (D). E, Molar ratio of NT-proANP and BNP secretion at 1 and 6 Hz with and without KN-93. ***, P < 0.001 by two-way ANOVA. All values are expressed as mean ± SEM. ns., Not significant.

 
Effect of KN-93 on the [Ca2+]i and calcium sensitivity of NP secretion
CaMKII can modulate the L-type calcium channels (26), ryanodine receptors (27), phospholamban, and sarcoplasmic reticulum Ca-ATPase (28). Therefore, inhibitors of CaMKII can be expected to modulate [Ca2+]i (29). To find out whether CaMKII influences [Ca2+]i, we measured the pacing-induced calcium changes in the presence of KN-93 (Table 2Go). KN-93 (3 µM) was found to have no significant effect in the atrial preparation on the calcium transient amplitude, diastolic calcium concentration, or time-averaged calcium either at 1 Hz pacing or at 6 Hz.


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  		TABLE 2. Effect of KN-93 on the diastolic, systolic, and average [Ca2+]i at low (1 Hz) and high (6 Hz) pacing frequency in rat atrium

 
Effect of KN-93 on the natriuretic peptide secretion and gene expression in cultured neonatal ventricular cardiomyocytes
The KN-93 effect on natriuretic peptide secretion and gene expression was further studied in cultured rat neonatal ventricular cardiomyocytes. This was done to assess the changes in ANP gene expression, which have been reported to occur on a relatively slow time scale (30). Twenty-four-hour exposure to 3 µM KN-93 resulted in consistent 29 and 50% reduction of NT-proANP and BNP secretion (Fig. 5AGo) as well as 52 and 62% repression of ANP and BNP mRNA levels (Fig. 5BGo), respectively. To control the specificity of the repression, endothelin-1 mRNA levels were measured and found unaffected and no compensatory induction in CaMKII{delta} gene expression after inhibition by KN-93 was found (Fig. 5CGo). Based on this observation, inhibition of CaMKII by KN-93 seems to exert selective inhibition on both the secretion and gene expression of ANPs and BNPs in ventricular cardiomyocytes as well as atrial cells.


Figure 5
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  		FIG. 5. Effect of CaMKII inhibition by KN-93 on secretion and gene expression of NPs in cultured rat neonatal ventricular cardiomyocytes. KN-93 reduces NT-proANP and BNP immunoreactivity in culture medium 29 and 50%, respectively. A, KN-93 represses ANP and BNP gene expression 52 and 62% but not endothelin-1 or CaMKII{delta} (B). In each group n = 6. ***, P < 0.001; **, P < 0.01 by one-way ANOVA. All values are expressed as mean ± SEM.

 
Overexpression of active CaMKII in neonatal cardiomyocytes
To induce a specific and calcium-independent activation of CaMKII, neonatal cardiomyocytes were transfected with a constitutively active CaMKII mutant (T286D) (22). This resulted in 42% increase of ANP (Fig. 6AGo) and 36% increase of BNP (Fig. 6BGo) mRNA levels. Endothelin-1 mRNA, served as a control, was measured and found to be unaffected (data not shown). There was no difference between spontaneous beating rates of the CaMKII{gamma}-T286D and the control groups (1.72 ± 0.29 vs. 1.80 ± 0.48 Hz).


Figure 6
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  		FIG. 6. Expression of constitutively active T286D mutant and nuclear {delta}B-CaMKII in cultured rat neonatal ventricular cardiomyocytes. Activated CaMKII promotes expression of ANP (~42%, n = 6) (A) and BNP (~36%, n = 7) (B). Similarly, nuclear CaMKII{delta}B isoform induces expression of ANP (~37%, n = 6) (C) and BNP (~50%, n = 6) (D). ***, P < 0.001; **, P < 0.01; *, P < 0.05 by one-way ANOVA. All values are expressed as mean ± SEM.

 
Overexpression of the nuclear CaMKII isoform-{delta}B was used to exclude cytosolic targets of CaMKII in regulation of ANP and BNP gene expression. It has been reported earlier that overexpression of CaMKII{delta}B activates the ANP promoter construct and increases the number of cardiomyocytes expressing ANP in cell culture (8). A direct effect on endogenous ANP or BNP gene expression has not been reported. In the neonatal cardiomyocyte culture, overexpression of CaMKII{delta}B resulted in 37% increase in ANP mRNA (Fig. 6CGo) and a 50% increase in BNP mRNA (Fig 6DGo), whereas endothelin-1 mRNA level was not increased (data not shown). Spontaneous beating rates were not significantly different between control and CaMKII{delta}B-overexpressing cardiomyocytes (0.83 ± 0.09 Hz vs. 1.14 ± 0.27 Hz).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that in cardiomyocytes ANPs and BNPs share the same calcium-sensitive secretion mechanism that can be recruited by pacing at physiological frequencies. Inhibition of CaMKII with KN-93 suppresses the secretion of both ANP and BNP in intact perfused rat atrium. In a similar manner, KN-93 inhibits gene expression and secretion of both NPs in neonatal ventricular cardiomyocytes, suggesting that the regulation of NPs by CaMKII is not restricted to either atrial or ventricular myocytes. In addition, specific overexpression of constitutively active or nuclear CaMKII results in a consistent increase in both ANP and BNP gene expression.

In normal physiological conditions most circulating ANP and BNP is of atrial origin (31). However, during the development of various ventricular pathologies, ventricular cells express and secrete increased amounts of NPs (32). The induction of ventricular NP production by remodeling of the muscle makes these peptides good markers of pathological development (32). Although BNP is up-regulated to a larger degree than ANP during ventricular pathogenesis (31), experiments with conscious animals indicate a greater increase in ANP than BNP secretion on an increase in pacing rate (16). This may indicate differential regulation of NP secretion in acute and chronic ventricular load or greater contribution of atrial release of NPs in acute pacing experiments.

The fact that the nuclear isoform of CaMKII is sufficient to activate both ANP and BNP gene expression shows that direct activation of the secretory mechanism by CaMKII is not required for the activation of NP gene expression. Atrial myocytes store a high amount of ANP, and its secretion primarily relies on the use of prestored peptide (31). Consequently, the induction of ANP gene expression requires several hours of continuous stimulation (30). In contrast, the BNP content of atrial myocytes is low, and the de novo synthesis of BNP appears to be required for an increase in the secretion. Because KN-93 inhibits ANP secretion in a much faster time scale than the activation of ANP gene expression is likely to take place, it could be hypothesized that there are two parallel CaMKII-sensitive mechanisms that regulate separately NP gene expression and secretion. Interestingly, CaMKII is the only factor so far reported to regulate the expression and secretion of both ANP and BNP.

In atrial muscle, ANP and BNP have been reported to be colocalized into the same secretory vesicles (17, 18) and are secreted in response to various physiological stimuli including stretch, endothelin-1 and phenylephrine (33). Our data show that during 35 min pacing of rat atria, ANP and BNP are cosecreted in fixed molar proportions with CaMKII-dependent manner. In addition, after 24 h of culture neonatal ventricular cardiomyocytes show similar proportional responses in both expression and secretions upon increased or decreased activity of CaMKII. In these experimental arrangements atrial and ventricular NP productions are not strictly comparable due the fact that in the cell culture, the cumulative 24-h secretion is measured, whereas atrial pacing produces acute NP secretion response. However, our data indicate that both cell types may have the same CaMKII regulated mechanisms for NP expression and secretion. This mechanism would maintain a continuous heart rate-dependent production of ANP and BNP contributing to the basal plasma levels of these peptides as well as increased production upon increased physical load.

NPs, especially BNP, have been established as independent markers of heart dysfunction and predictors of outcome in heart failure (3, 4, 34). Because NPs are such powerful predictors of cardiac pathology, it seems likely that the mechanisms regulating NP expression and secretion include a wide variety of signaling cascades that are also involved in the development of hypertrophy and heart dysfunction in animal models. Among these, calmodulin kinases are known to activate many transcription factors such as myocyte enhancing factor-2, cAMP response element-binding protein (CREB), CREB-binding protein, and serum response factor (9), which are known to control myogenesis and muscle hypertrophy (10, 35). Recently it has been shown that calcium-calmodulin-dependent protein kinase phosphorylates HDAC5 causing its nuclear export (10), potentially activating hypertrophic cardiac gene expression (36). In genetically manipulated mice, both nuclear and cytosolic overexpression of CaMKII produces a hypertrophic phenotype with increased expression of ANP (12, 13).

On the basis of our results CaMKII appears to be a common, direct and independent activator of ANP and BNP expression and secretion. CaMKII is involved in the normal physiological regulation of natriuretic peptide expression and secretion and contributes to NP regulation in the development of cardiac hypertrophy and failure.


   Acknowledgments
 
The authors thank Eero Kouvalainen for help with statistical analysis and Anneli Rautio, Tuula Taskinen, Ulla Weckström, and Mika Ilves for technical help.


   Footnotes
 
First Published Online March 1, 2007

Abbreviations: ANP, A-type NP; BNP, B-type NP; [Ca2+]i, intracellular free calcium concentration; CaMKII, calcium-calmodulin-dependent protein kinase II; NP, natriuretic peptide.

This work was supported by Jenny and Antti Wihuri Foundation, Finnish Foundation of Cardiovascular Research, Emil Aaltonen Foundation, Sigrid Juselius Foundation, and Aarne Koskelo Foundation.

Disclosure Statement: The authors have nothing to disclose.

Received December 14, 2006.

Accepted for publication February 21, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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