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Cardiovascular Research 2007 73(4):678-688; doi:10.1016/j.cardiores.2006.10.003
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Copyright © 2007, European Society of Cardiology

CaMKII-mediated increased lusitropic responses to β-adrenoreceptor stimulation in ANP-receptor deficient mice

Sevdalina Yurukovaa,1, Ana Kilica,1, Katharina Völkera, Kirsten Leineweberb, Nataliya Dybkovac, Lars S. Maierc, Otto-Erich Broddeb and Michaela Kuhna,*

aInstitute of Physiology, University of Würzburg, Röntgenring 9, D-97070 Würzburg, Germany
bDepartment of Pathophysiology and Nephrology, University of Essen School of Medicine, Germany
cDepartment of Cardiology and Pneumology/Heart Center, Georg-August-University of Göttingen, Germany

* Corresponding author. Tel.: +49 931 31 2720; fax: +49 931 31 2741. Email address: michaela.kuhn{at}mail.uni-wuerzburg.de

Received 27 February 2006; revised 23 September 2006; accepted 4 October 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Mice with genetic disruption of the guanylyl cyclase-A (GC-A) receptor for atrial natriuretic peptide (ANP), have chronic arterial hypertension and marked cardiac hypertrophy. Intriguingly, despite pronounced remodeling, cardiac contractile functions and cardiomyocyte Ca2+-handling are preserved and even enhanced. The present study aimed to characterize the specific molecular mechanisms preventing cardiac failure.

Methods and results: Contractile function and expression as well as phosphorylation of regulatory proteins were evaluated in isolated perfused working hearts from wild-type and GC-A KO mice under baseline conditions and during β1-adrenergic stimulation. Cai2+-transients were monitored in Indo-1 loaded isolated adult cardiomyocytes. Cardiac contractile, especially lusitropic responsiveness to β-adrenergic stimulation was significantly increased in GC-A KO mice. This was concomitant to enhanced expression and activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII), increased dual-site phosphorylation of phospholamban (PLB) at Ser16 and Thr17, enhanced amplitude of Cai2+ transients, and accelerated Cai2+ decay. In contrast, the expression of cardiac ryanodine receptors and phosphorylation at Ser2809 and Ser2815 was not altered. Pharmacological inhibition of CaMKII-but not of protein kinase A-mediated PLB phosphorylation totally abolished the increased effects of β-adrenergic stimulation on cardiac contractility and Cai2+-handling. Thus, acceleration of sarcoplasmic reticulum Ca2+-uptake and increased availability of Ca2+ for contraction, both secondary to increased CaMKII-mediated PLB phosphorylation, seem to mediate the augmented responsiveness of GC-A KO hearts to catecholamines.

Conclusion: Our observations show that increased CaMKII activity enhances the contractile relaxation response of hypertrophic GC-A KO hearts to β-adrenergic stimulation and emphasize the critical role of CaMKII-dependent pathways in β1-adrenoreceptor modulation of myocardial Ca2+-homeostasis and contractility.

KEYWORDS Atrial natriuretic peptide; Guanylyl cyclase-A; Cyclic GMP; CaMKII; Calcium cycling; Phospholamban; Heart failure


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiac hypertrophy in response to chronic arterial hypertension is initially adaptive and compensatory, but later often heart failure (CHF) develops. An early and pathognomonic marker in the development of hypertrophy is the increased cardiac expression and release of atrial and B-type natriuretic peptides (ANP and BNP) [1]. These peptides share a common receptor, guanylyl cyclase-A (GC-A), which is expressed mainly in the vasculature and kidneys. Activation of GC-A and subsequent increases in intracellular cGMP initiate vasodilatation, natriuresis, and diuresis, thereby lowering blood pressure and volume [1]. In addition, the GC-A receptor is expressed within the heart itself, in atria and ventricles, and mediates local, autocrine/paracrine effects of ANP and BNP which moderate the cardiac growth response to hypertrophic stimuli as well as cardiac fibrosis [2–4]. Notably, in patients with cardiac hypertrophy and/or CHF elevated levels of circulating ANP and BNP are accompanied by a significant attenuation of their protective vasodilative, natriuretic and cardiac effects, suggesting a downregulation and/or impaired responsiveness of GC-A, which might contribute to the progression of cardiac remodeling [1,5,6].

Mice with a genetic disruption of the GC-A gene (GC-A KO mice) constitute an elegant experimental model to dissect the cardiovascular functions of the ANP/GC-A system and the consequences of its long term inhibition. Their prominent phenotypical features, arterial hypertension, hypervolemia and cardiac hypertrophy, emphasize the important physiological role of the aforementioned endocrine as well as local actions of ANP [7–9]. Remarkably, despite massive hypertrophy, GC-A KO mice do not show alterations in cardiac contractile function until very late stages, when hypertrophy is accompanied by marked interstitial fibrosis and myocyte cytoskeletal changes [10]. Hence, echocardiographic and hemodynamic data of GC-A KO mice showed ventricular enlargement without contractile dysfunction [8] and even with hyperdynamic function [9] until 10–12 months of age. The occurrence of marked chronic hypertrophy with preserved cardiac function constitutes one of the most intriguing aspects of this mouse model, rising relevant questions regarding the specific molecular and biochemical mechanisms delaying cardiac failure. In particular, at difference to other models of cardiac hypertrophy, which are usually accompanied by reduced systolic and increased diastolic Cai2+ levels [11], cardiomyocyte Cai2+-handling in GC-A KO mice is not impaired but even enhanced, with unaltered diastolic and increased systolic free Cai2+-levels and enhanced sarcoplasmic reticulum (SR) Ca2+ storage [12].

The present study aimed to characterize the mechanisms contributing to the preservation of contractile function and Cai2+-handling in hypertrophic GC-A KO hearts. Since attenuation of β1-adrenoreceptor-mediated contractile responses is among the first alterations in patients with CHF [13], our study focused on β-adrenergic responses and signaling pathways. To further characterize the cardiac contractile response of the GC-A KO mice independently of their increased pressure and volume load, we used the isolated perfused "working" heart model to compare left ventricular contractility and the functional and biochemical responses to β-adrenergic stimulation in WT and GC-A KO hearts under identical pre- and after-load conditions. In addition, we evaluated the impact of GC-A ablation on adrenergic modulation of cardiomyocyte free cytosolic Ca2+-transients.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animals
GC-A KO mice were provided by Dr. D.L. Garbers (HHMI, University of Texas Southwestern Medical Center, Dallas) [7]. GC-A KO mice and respective wild type (WT) controls, 6–8 months old, were used. Genotypes were identified by PCR. Blood pressure measurements were in conscious mice using a tail cuff method (Softron, Tokyo) [10,12]. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the local animal care committee.

2.2. Contractile parameters in isolated work-performing heart preparations
Analysis of cardiac function was performed as described previously [10]. In brief, isolated hearts from WT and GC-A KO mice were perfused with Krebs-Henseleit (KH) buffer through the pulmonary vein and left atrium, in an anterograde, fluid-ejecting mode. Fluid ejected from the left ventricle into the aorta and coronary arteries. Distal to the coronary arteries the aorta was cannulated and connected to a hydrostatic fluid column set at a height to yield a mean aortic pressure (afterload) of 50 mmHg. Venous return (preload) and aortic flow were measured using flowmeters (Transonic Systems Inc., Ithaca, USA). Preload was adjusted to 5 ml/min. Coronary flow (calculated as the difference between preload and aortic flow), heart rate, aortic pressure and left intraventricular pressure (LVPmax) were continuously monitored. ±dP/dt (in mmHg/s), as well as the times to peak pressure (T1 99% and 50%) and to relaxation (T2 99% and 50%) were calculated (AMon 2.1 program, Büro Jäckel, Hanau, Germany) [10].

2.3. Experimental protocols
After a 20 min equilibration period with KH buffer, the β1-adrenoreceptor agonist dobutamine (100 nM; Sigma, Deisenhofen, Germany) was infused via the coronary arteries for either 10 (n=6 per genotype) or 60 min (n=14). Control hearts of each genotype were infused with KH-buffer for the same time periods (n=4).

In a second experimental series we tested the effects of inhibition of either Ca2+/calmodulin-dependent protein kinase II (CaMKII, inhibition with 0.5 µM KN93) or cAMP-dependent protein kinase (PKA, inhibition with 100 nM KT-5720) on the responses to dobutamine. KT-5720 (Merck, Darmstadt, Germany), KN93 or its inactive analogue KN92 (both from Sigma) were infused alone for 30 min and were then combined with dobutamine (100 nM) and infused for additional 15 min (n=6 per genotype and treatment). Control hearts were infused with vehicle (DMSO, 0.001% v/v final) for the same time period (DMSO alone for 30 min; then DMSO combined with dobutamine for additional 15 min). All test agents were added to the perfusion solution (KH) entering the heart via the pulmonary vein and left atrium.

At the end of each experiment the atria and right ventricles were removed and the left ventricles were quickly frozen in liquid nitrogen and were then extracted for assay of cAMP and for Western blot analyses.

2.4. Western blot analyses
To determine the protein expression level of PKA, CaMKII, autophosphorylated (active) CaMKII, phospholamban (PLB) and the Ca2+ release channel (ryanodine-receptor, RyR2) as well as the effect of dobutamine on PLB and RyR2 phosphorylation, frozen left ventricles were homogenized and analyzed by Western blot as described previously by our laboratories [12,14]. Samples were either heated to 95 °C in Laemmli buffer for 5 min (kinases, total PLB) or not heated (to preserve CaMKII, PLB and RyR2 phosphorylation), and proteins were size-separated on SDS-PAGE. Antibodies used were against PKA, CaMKII (both BD transduction laboratories, Heidelberg, Germany), autophosphorylated CaMKII (Santa Cruz, Heidelberg, Germany), total PLB (PLB A-1 antibody) and PLB phosphorylated at Ser16 (PLB-PS-16 antibody) or Thr17 (PLB-PT-17) (all Fluorescience Ltd., Leeds, UK). RyR2 expression and phosphorylation were investigated using mouse monoclonal anti-RyR2 (C3-33 clone, Affinity Bioreagents, Golden, CO, USA) and rabbit polyclonal anti-phospho-RyR2 antibodies (anti-RyR-P-2809 and anti-RyR-P-2815, both antibodies kindly provided by Dr. A. Marks, Columbia University, New York) as described in Ref. [14]. An ECL system (Amersham-Pharmacia, Freiburg, Germany) was used for detection and results were quantitated by densitometry (ImageQuant software; Molecular Dynamics, Krefeld, Germany). All immunoreactive signals were normalized to junctin [12] or calsequestrin protein levels [14] and then the ratio to the WT values was calculated.

2.5. Intracellular Ca2+-transients in isolated cardiac myocytes
Ventricular myocytes from WT and GC-A KO hearts were isolated by collagenase digestion and Cai2+-transients were measured in Indo-1 loaded, electrically paced (0.5 Hz) cardiomyocytes using an established protocol [12]. Excitation was at 365 nm, and the emitted fluorescence was recorded at 405 and 495 nm and was used as an index of cytosolic Ca2+ concentration. After 15 min of baseline recording, the acute effects of the β-adrenoreceptor agonist isoproterenol (100 nm; Sigma) in the absence (superfusion with vehicle, 0.001% DMSO) or presence of the CaMKII inhibitory agent KN93 (1 µM) were tested. The amplitude of the Cai2+-transients was calculated as the difference between systolic and diastolic Cai2+-levels [12].

2.6. β-adrenergic receptor density
Whole ventricles were minced and homogenized in 10 volumes ice-cold 5 mM EDTA/5 mMTris–HCl buffer pH 7.4 with an Ultra Turrax homogenizer for 10 s at full speed and twice for 20 s at half-maximum speed in 1 min intervals. The homogenate was diluted to 20 ml with 5 mM EDTA/5 mM Tris–HCl puffer pH 7.4, filtered through 4 layers of cheese-cloth and centrifuged with 10000 g for 20 min at 4 °C. The pellets were resuspended in incubation buffer (10 mM Tris–HCl, 154 mM NaCl containing 0.55 mM ascorbic acid, pH 7.4) to yield a final protein concentration of 0.15 mg/ml. The density of β-adrenoreceptors was assessed in 150 µl aliquots by (–)-[125I]-iodocyanopindolol (ICYP, a specific agonist for β1-adrenoreceptors) binding at six concentrations ranging from 5 to 200 pM for 90 min at 37 °C in a final volume of 250 µl [15]. Non-specific binding of ICYP was defined as binding to membranes that was not displaced by 1 µM of the non-selective β-adrenoceptor antagonist (±)-CGP 12177. Specific binding of ICYP was total binding minus non-specific binding and amounted usually to 70–80% at 50 pM of ICYP [15].

2.7. Tissue cyclic AMP content
Frozen ventricles were homogenized and cAMP was extracted with ice-cold 70% (v/v) ethanol. After centrifugation (3000 g, 5 min, 4 °C) supernatants were dried in a speed vacuum concentrator, resuspended in sodium acetate buffer (50 mM, pH 6.0) and acetylated, and then cAMP contents were quantified by a commercially available radioimmunoassay (Amersham-Pharmacia). The pellets of the ethanol extracts were used for determination of protein content.

2.8. Statistics
Results are presented as means±S.E.M. and, where indicated, values were normalized as % of baseline (functional parameters in working heart experiments) or X-fold vs WT (data from western blot analyses). Student's t test was used for comparison of baseline data. Serial changes in response to β1-adrenoreceptor agonists were tested with a two-way ANOVA (with genotype and treatment as categories). The effects of protein kinase inhibitors on responses to β-adrenergic stimulation were tested with a repeated-measures ANOVA. ANOVA was followed by Bonferroni and Student–Newmann–Keuls post hoc test for multiple comparisons. Results were considered statistically significant at P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Baseline cardiovascular parameters
In agreement with previous observations [7–10,12], GC-A KO mice had higher systolic (139±4.4 vs 107±3.8 mmHg, P<0.05) and diastolic blood pressures (86±5.7 vs 69±3.3 mmHg, P<0.05), and significantly enlarged hearts as compared to WT mice (Table 1). Table 1 also shows that despite this pronounced hypertrophy the baseline contractile parameters of isolated working GC-A KO hearts were similar to WT.


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  		Table 1 Baseline parameters of isolated hearts from WT and GC-A KO mice in the work-performing mode

 
3.2. Enhanced contractile relaxation of GC-A KO hearts in response to dobutamine
Intracoronary infusion of 100 nM dobutamine into isolated WT hearts for 60 min provoked an immediate increase in all contraction and relaxation parameters (Fig. 1). Since the accelerating effects of dobutamine on the time to 50% and to 99% of maximal contraction (T1) and to relaxation (T2) were similar, only the later effects are shown. All responses started immediately after addition of dobutamine, reached their maximum at ~5–10 min and then slowly reversed. These changes were accompanied by stable increases in chronotropy (Fig. 1) and coronary flow (by 44±6%; not shown).


Figure 1
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  		Fig. 1 Time course of the effects of dobutamine (100 nM) on maximal left ventricular systolic pressure (LVPmax), heart rate (HR), maximal rates of contraction (+dP/dt) and relaxation (–dP/dt) and time to contraction (T1 99%) and to relaxation (T2 99%) in isolated working WT (open circles) and GC-A KO (filled circles) hearts. Dobutamine was added at min 1 and infused for 60 min in all panels. Data are expressed as % of baseline (at 0 min). Two-way ANOVA results showed a significant genotype-treatment interaction for –dP/dt, LVPmax, T1 99% and T2 99% (n=14 per genotype, *P<0.01).

 
In isolated GC-A KO hearts, the positive inotropic (LVPmax) and, in particular, the positive lusitropic (–dP/dt) actions of dobutamine were enhanced as compared to WT (Fig. 1). Since the dobutamine-provoked acceleration of the time to contraction (T1 99% and T1 50%) was less pronounced in GC-A KO as compared to WT hearts, the accompanying increases in the maximal rate of contraction (+dP/dt) ultimately were not different between genotypes. Similar to WT, all effects in the GC-A KO group reached their maximum at 5–10 min of dobutamine infusion and then slowly reversed (Fig. 1). The dobutamine-induced increase in heart rate (Fig. 1) and coronary flow (not shown) were similar to WT and stable over the entire experimental period.

Control WT and GC-A KO hearts infused with vehicle (KH buffer) alone exhibited no significant changes in contractile function over a 80 min observation time (data not shown), indicating that the time-dependent attenuation of dobutamine effects does not reflect a spontaneous deterioration of heart function but probably represents desensitization of β-adrenoreceptor signaling during prolonged stimulation.

3.3. Dual-site PLB phosphorylation is increased in GC-A KO hearts whereas RyR2 phosphorylation is unaltered
To investigate the mechanism of the enhanced inotropic and lusitropic effects of dobutamine in GC-A KO hearts, we examined the phosphorylation of the sarcoplasmic reticulum (SR) proteins PLB and RyR2 by Western blot analyses.

The levels of total PLB were not different in GC-A KO as compared to WT ventricles (Fig. 2). The dual-site phosphorylation of PLB at both Ser16 (the PKA site) and Thr17 (the CaMKII site) [16] was already detectable in the isolated hearts under basal conditions (perfusion with vehicle solution during either 10 or 60 min) and was increased in GC-A KO hearts (Fig. 2A). Intracoronary infusion of 100 nM dobutamine markedly enhanced PLB phosphorylation at both sites (Fig. 2B and C). The levels of Ser16- and Thr17-phosphorylated PLB were increased by 1.52±0.11-fold and by 1.7±0.08-fold in WT hearts and by 3.2±0.12-fold and 3.19±0.09 fold in GC-A KO hearts (10 min dobutamine vs vehicle perfusion). The levels of Ser16-and Thr17-phosphorylated PLB were ~2-fold and 2.6-fold greater in GC-A KO as compared to WT hearts treated with dobutamine for 10 or 60 min, respectively (Fig. 2B and C).


Figure 2
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  		Fig. 2 Enhanced dual-site PLB phosphorylation at Ser16 (PLB-PS-16) and Thr17 (PLB-PT-17) in GC-A KO hearts. Protein expression and phosphorylation of PLB (MW ~30 kDa in SDS-PAGE) in isolated WT and GC-A KO hearts perfused with vehicle during 10 min (A) or with 100 nM dobutamine for either 10 (B) or 60 min (C). The levels of total PLB, PLB-PS-16 and PLB-PT-17 expression were normalized to junctin (PLB/junctin, P-PLB/junctin) and calculated as X-fold versus levels in WT (the mean value of the WT group was set as 1). Two-way ANOVA results showed a significant genotype-treatment interaction for PLB-PS-16 and PLB-PT-17 expression (n=6, *P<0.01).

 
During basal conditions (hearts perfused with vehicle during 10 min) the levels of total RyR2 and of RyR2 phosphorylated at the PKA-(Ser2809) and CaMKII-sites (Ser2815) [14] were not different between WT and GC-A KO hearts (Fig. 3A). During β-adrenergic stimulation (dobutamine, 10 min) the phosphorylation of both sites slightly increased, but in contrast to PLB the extent of dual-site RyR2 phosphorylation was similar in WT and GC-A KO hearts (Fig. 3B). The ratio RyR-PS-2809/RyR was increased by 1.4±0.3 fold in WT and by 1.31±0.05 fold in GC-A KO hearts (dobutamine as compared to vehicle); RyR-PS-2815/RyR was increased by 1.6±0.3 fold and by 1.6±0.2 fold, respectively.


Figure 3
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  		Fig. 3 Unaltered dual-site RyR2 phosphorylation at Ser2809 and Ser2815 in GC-A KO hearts. Protein expression and phosphorylation of RyR (MW ~560 kDa) in isolated WT and GC-A KO hearts perfused with vehicle (A) or with 100 nM dobutamine during 10 min (B). The levels of total RyR (n=12 per genotype and condition), RyR-PS-2809 (n=12) and RyR-PS-2815 (n=5–7) were normalized to calsequestrin (RyR/CSQ, P-RyR/CSQ) and calculated as X-fold versus levels in WT (the mean value of WT was set as 1).

 
3.4. Enhanced expression and activation of CaMKII but not PKA in GC-A KO hearts
To study the immediate downstream targets of the β1-adrenoreceptor/cAMP system mediating enhanced dual-site PLB phosphorylation in GC-A KO hearts, the expression level of PKA and CaMKII was studied by Western blot analyses. As shown in Fig. 4A, CaMKII expression and autophosphorylation was significantly increased in the ventricles from GC-A KO as compared to WT hearts. The expression of PKA (C-subunit) was not different between genotypes (Fig. 4A). Infusion of dobutamine (100 nM, 60 min) did not affect ventricular expression levels of protein kinases but significantly increased the levels of autophosphorylated (active) CaMKII in WT (by ~1.7-fold, in comparison to vehicle-perfused hearts) and even more in GC-A KO hearts (by ~2.3-fold) (Fig. 4B).


Figure 4
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  		Fig. 4 Protein expression of PKA (MW ~40 kDa), CaMKII and autophosphorylated (active) CaMKII (MW ~52 kDa) in WT and GC-A KO hearts perfused with vehicle (A) or with dobutamine (B). The relative amounts of PKA and CaMKII expression were normalized to junctin (MW ~25 kDa) and calculated as X-fold versus levels in WT (the mean value of the WT group was set as 1). CaMKII expression and autophosphorylation were significantly enhanced in GC-A KO compared to WT hearts (*P<0.05 vs WT). Two-way ANOVA results showed a significant genotype-treatment interaction for pCaMKII expression (P<0.01; all n=6).

 
3.5. Increased β-adrenoreceptor responses in GC-A KO hearts are mediated by CaMKII
To appraise the role of PKA and CaMKII in mediating the increased lusitropic responses of GC-A KO hearts to dobutamine, we tested the effects of pharmacological inhibition of these kinases. In both genotypes, inhibition of PKA by KT-5720 or CaMKII by KN93 exerted mild inhibitory effects on baseline cardiac contractile parameters, but on average these effects were not statistically significant. KN93 (0.5 µM, 30 min) reduced +dP/dt by 8±3% in WT and by 4±3% in GC-A KO hearts; –dP/dt was decreased by 9±2% and 5±3%, respectively. Similar subtle changes in ±dP/dt were provoked by KT-5720 (100 nM, 30 min), again without differences between genotypes. KT-5720 or KN92, the inactive analogue of KN93, did not affect the contractile relaxation responses to dobutamine or their genotype-dependent differences (Fig. 5A). Also KN93 did not affect the inotropic and lusitropic responses of WT hearts to dobutamine. However, KN93 prevented and even reversed the enhanced inotropism (LVPmax) and lusitropism (–dP/dt, shown in Fig. 5A) of GC-A KO hearts in response to dobutamine.


Figure 5
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  		Fig. 5 A. Peak effects of dobutamine (100 nM) on the maximal rates of relaxation (–dP/dt, mmHg/s) of WT and GC-A KO hearts pretreated with vehicle (0.001% v/v DMSO), the CaMKII inhibitor KN93, its inactive analog KN92, or the PKA inhibitor KT-5720. Data are expressed as % of baseline (pretreatment, as 100%). KN93, but not KN92 or KT-5720, fully prevented the enhanced lusitropism of GC-A KO hearts. B. Effects of dobutamine (100 nM) on PLB phosphorylation at Ser16 and Thr17 in WT and GC-A KO hearts pretreated with KN93, KN92 or KT-5720. The signals obtained with the phosphorylation site-specific antibodies (PLB-PS-16, PLB-PT-17) were normalized to total PLB and calculated as X-fold vs WT. KN93, but not KN92 or KT-5720, prevented the enhanced effects of dobutamine on PLB phosphorylation at Thr17 in GC-A KO ventricles. KT-5720 selectively prevented the enhanced effects of dobutamine on PLB phosphorylation at Ser16 in GC-A KO ventricles (*P<0.05 vs WT; all n=6). ANOVA results showed a significant genotype-treatment interaction for expression levels of PLB-PS-16 and PLB-PT-17 (P<0.05).

 
The inhibitory effects of KN93 on the contractile responses to dobutamine were concomitant with diminished phosphorylation of PLB at Thr17 (Fig. 5B). In particular, pretreatment with KN93 totally abolished the enhanced effects of dobutamine on phosphorylation of PLB at Thr17 in GC-A KO compared to WT hearts but fully preserved the enhanced phosphorylation of PLB at Ser16 (Fig. 5B). Conversely, KT-5720 abolished the enhanced effects of dobutamine on PLB phosphorylation at Ser16 in GC-A KO as compared to WT hearts but fully preserved the enhanced phosphorylation of PLB at Thr17 (Fig. 5B). The inactive analogue KN92 had no effect on the dual-site phosphorylation of PLB in response to dobutamine nor on the increased phosphorylation of this regulatory protein in GC-A KO hearts (Fig. 5B). Thus, inhibition of either PKA or CaMKII selectively prevented the increased effects of β-adrenergic stimulation on PLB phosphorylation at either Ser16 or Thr17 in GC-A KO hearts. However, only the inhibition of CaMKII-mediated PLB phosphorylation at Thr17 prevented the enhanced contractile, i.e. lusitropic responses of GC-A KO hearts to maximal β-adrenoreceptor stimulation.

3.6. CaMKII contributes to the increased Ca2+-transients of GC-A KO cardiomyocytes
PLB, in its unphosphorylated form, serves as a constitutive inhibitor of SERCA2a [16]. Phosphorylation of PLB reverses its inhibition of SERCA2a activity and stimulates SR Ca2+-reuptake [16]. The increased effects of dobutamine on PLB phosphorylation in GC-A KO hearts suggested that enhanced Cai2+-transients augmented the contractile responses to β-adrenoceptor stimulation. To address this possibility, we monitored cytoplasmic Ca2+-transients in isolated Indo-1 loaded myocytes from WT and GC-A KO hearts. In accordance with our previous study [12] GC-A KO myocytes exhibited increased systolic Ca2+-levels already under baseline conditions, resulting in greater baseline peak amplitudes of Ca2+-transients (Table 2, top). The time to 50% decay was not significantly different between genotypes (Table 2, top). Isoproterenol (100 nM) did not affect diastolic Ca2+-levels, but provoked significant increases in systolic Ca2+-levels and in the peak amplitudes of the Ca2+-transients, together with an accelerated Ca2+-decay, in both WT and GC-A KO myocytes. As shown in Table 2, these adrenergic effects were significantly enhanced in GC-A KO myocytes. CaMKII inhibition by KN93 (1 µM, 5 min) attenuated baseline Cai2+-transients and delayed Cai2+-decline only in GC-A KO but not in WT myocytes (Table 2, bottom). Even more, KN93 inhibited the Cai2+-responses to isoproterenol more in GC-A KO than in WT myocytes. In particular, only in GC-A KO myocytes was the stimulatory effect of isoproterenol on Cai2+-decay inhibited by KN93 (WT: the time to 50% decay was shortened by 22% in response to isoproterenol both in the absence and presence of KN93; GC-A KO: the time to 50% decay was shortened in response to isoproterenol by 48% in the absence and only by 30% in the presence of KN93) (Table 2, bottom). Thus, a putative mechanism for the increased baseline and isoproterenol-stimulated SR Ca2+ cycling involves CaMKII-dependent phosphorylation of PLB at Thr17 and subsequent activation of SERCA2a.


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  		Table 2 Effects of isoproterenol (100 nM) and KN93 (1 µM) on Cai2+ transients of isolated cardiomyocytes from WT and GC-A KO mice

 
3.7. GC-A KO hearts exhibit increased density of β-adrenergic receptors and increased cAMP responses to β-adrenoceptor stimulation
Because changes in β-receptor number or affinity can also alter myocardial responses to catecholamines [17], we compared β-adrenoreceptor binding characteristics in ventricular membrane preparations from WT and GC-A KO hearts using ICYP as a specific ligand. As shown in Fig. 6A and C, β-adrenoceptor density in GC-A KO hearts was increased by ~20% in comparison to that of WT hearts. The affinity for the ligand expressed as Kd, however, on average was similar in WT and GC-A KO hearts (Fig. 6B, C). Lastly we investigated whether altered receptor expression leads to increased formation of the second messenger cAMP. As shown in Fig. 7, the cAMP contents of vehicle-perfused WT and GC-A KO hearts were similar. However, the cAMP response to dobutamine infusion (100 nM, 10 min) was significantly increased in the later. These cAMP responses apparently were rather low (~1.5-fold and 1.9-fold cAMP increases in dobutamine-as compared to vehicle-treated WT and GC-A KO hearts, respectively). Please note that these experiments were done in the absence of phosphodiesterase inhibitors and therefore these cAMP contents result from both synthesis and degradation. However, taken together our data indicate that increased β-adrenergic receptor densities in GC-A KO hearts, via enhanced cAMP-mediated activation of PKA, may be involved in the increased phosphorylation of PLB at Ser16 in response to dobutamine.


Figure 6
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  		Fig. 6 The density (A) and affinity (B) of β-adrenergic receptors were determined in cardiac membrane preparations as described in Methods. β-adrenoceptor density is increased in GC-A KO hearts (n=14 per genotype; *P<0.05 vs WT, compared by t test). (C) Saturation curve; Insert: Scatchart plot [B/F] specifically bound [125I]-Iodocyanopindolol binding in fmol/mg protein/free [125I]-Iodocyanopindolol in pmol/l to [B] specifically bound [125I]-Iodocyanopindolol binding in fmol/mg protein (with 20 µg protein per assay, crude membranes). Shown are two representative experiments, with membranes from one GC-A KO (#17) and one WT heart (#25).

 

Figure 7
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  		Fig. 7 Effect of dobutamine (100 nM, 10 min) on cAMP content (in fmol/µg protein) in isolated working WT and GC-A KO hearts. Control hearts were infused with vehicle (KH-buffer) for the same time period. The cAMP-responses to dobutamine are greater in GC-A KO hearts (n=6 per genotype; *P<0.05 vs WT; §P<0.05 vs vehicle). Two-way ANOVA results showed a significant genotype-treatment interaction for cAMP content (P<0.05).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
4.1. Principal findings
In the present study we examined the signaling mechanisms underlying the enhanced contractile relaxation responses to β-adrenoreceptor stimulation of hypertrophic hearts from GC-A-deficient mice. Functional and biochemical measurements in isolated perfused "working" hearts and fluorimetric Ca2+ imaging in isolated myocytes demonstrated that acceleration of SR Ca2+-uptake and increased availability of Ca2+ for contraction, both secondary to increased CaMKII-dependent PLB phosphorylation at Thr17, are major mechanisms for the enhanced inotropic and lusitropic effects. Our observations demonstrate that increased endogenous CaMKII activity can contribute to the preservation of SR Ca2+-cycling and cardiac performance even in the presence of significant cardiac hypertrophy. They also emphasize the involvement of CaMKII-dependent pathways in β1-adrenoreceptor modulation of myocardial contractility.

4.2. Enhanced CaMKII activity mediates increased responses of GC-A KO hearts to β-adrenoreceptor stimulation
Analysis of myocardial contractility revealed similar basal contractile parameters in isolated working WT and hypertrophic GC-A KO hearts. However, the inotropic and especially the lusitropic responses to β-adrenergic stimulation were significantly enhanced in the later. Concomitantly, the stimulatory effects of β-adrenergic agonists on systolic free Cai2+-levels and on the time to Cai2+-decay in isolated myocytes from GC-A KO hearts were increased. This finding indicates that the increased contractile response of GC-A KO hearts to β-adrenergic stimulation involves modulation of components of the signal transduction cascade from the β-adrenergic receptor to Cai2+-handling. Indeed, the density of β-adrenergic receptors was significantly elevated in GC-A KO hearts, leading to increased cardiac cAMP formation after β-adrenoreceptor stimulation. However, these changes were rather small, suggesting that additional modification(s) of components downstream of the β1-adrenoceptor/cAMP system contribute to the increased sensitivity of GC-A KO hearts to catecholamines.

β1-adrenoceptor stimulation in cardiac myocytes concurrently activates two protein kinases regulating SR Ca2+-cycling: activation of PKA phosphorylates PLB at Ser16; and PKA-dependent increases in intracellular Ca2+ stimulate CaMKII, which phosphorylates PLB at Thr17 [13,16]. When PLB is phosphorylated, its inhibitory effect on the SR Ca2+ pump SERCA2a is relieved resulting in increased SR Ca2+-reuptake (reviewed in 16). As shown, the double phosphorylation of PLB was already increased in GC-A KO hearts under baseline conditions. Dobutamine led to marked dual PLB phosphorylation at Ser16 and Thr17 and this effect was significantly increased in GC-A KO as compared to WT hearts, suggesting enhanced activation of both PKA and CaMKII. The following findings indicate that CaMKII mainly mediates the enhanced contractile relaxation of GC-A KO hearts in response to β-adrenergic stimulation: 1) the expression and autophosphorylation (activation) of CaMKII was significantly increased in GC-A KO hearts whereas PKA expression was unaltered; 2) a selective CaMKII inhibitor, KN93, had no significant effect on baseline cardiac contractility but totally abolished the increased effects of β-adrenergic stimulation on PLB-Thr17 phosphorylation, cardiomyocyte Cai2+-handling and cardiac lusitropy in this genotype; and 3) the selective PKA inhibitor KT-5720 prevented the increased effects of β-adrenergic stimulation on PLB-Ser16 phosphorylation in GC-A KO hearts but intriguingly did not affect their enhanced contractile responses to β-adrenoceptor stimulation.

As demonstrated, inhibition of CaMKII with KN93 did not affect the β-adrenoceptor-stimulated PKA-mediated phosphorylation of PLB at Ser16 and the augmentation of this pathway in GC-A KO hearts. Despite this, their enhanced lusitropism was reversed by CaMKII inhibition. We conclude that increased expression of β-adrenoceptors and enhanced cAMP/PKA-signaling might contribute to the increased responses of GC-A KO hearts to dobutamine, but seems to be less critical then CaMKII signalling. Our study is consistent with a study by Wang et al. [18] also demonstrating the critical role of CaMKII in β1-adrenoceptor modulation of myocardial contractility. However, with difference to our study (where CaMKII mediated the immediate, acute effects of β1-adrenoceptor stimulation), in this study with cultured rat cardiomyocytes CaMKII activation was mainly responsible for the long-term, sustained effects on contractility, whereas the short-term β1-adrenoreceptor responses were preferentially mediated by the cAMP/PKA pathway [18]. Our study in GC-A KO mice suggests that an increase in the endogenous cardiac expression and/or activity of CaMKII may not only increase the contribution but also modify the chronology of CaMKII signaling after β-adrenergic stimulation.

4.3. Enhanced cardiac CaMKII activity in GC-A KO hearts is not associated with changes in RyR2 phosphorylation
Another SR protein which is also phosphorylated both by PKA and CaMKII is the Ca2+-release channel, RyR2. RyR2 hyperphosphorylation via PKA [19] or CaMKII [20] can result in diastolic SR Ca2+ leak and depletion of SR Ca2+, ultimately impairing systolic function. As demonstrated, RyR2 expression and phosphorylation at the PKA-(Ser2809) and CaMKII-sites (Ser2815) were not altered in GC-A KO hearts both at baseline and during β-adrenergic stimulation. In line with observations in rat cardiac myocytes [21] RyR-Ser-2809 was already substantially phosphorylated before β-adrenergic stimulation, and the extent of the increase in Ser-2809 phosphorylation after β-adrenergic stimulation was rather small. Similar results were obtained for RyR-Ser-2815. Our observation of more pronounced PLB as compared to RyR phosphorylation in response to dobutamine is in agreement with a study by Ginsburg and Bers [22] which showed that the increased amount of SR Ca2+ release provoked by β-adrenergic stimulation depends primarily on increased ICa trigger and SR Ca2+ load.

Interestingly, in human failing hearts hyperphosphorylation of RyR2 seems to be mainly caused by a decrease in phosphatase bound to the RyR2 channel macromolecular complex, and not by an increase in kinase activity [19]. Since GC-A KO mice did not show signs of heart failure but even improved contractility as well as myocyte Cai2+-transients which are not compatible to SR Cai2+-loss through a possible hyperphosphorylated RyR2, it is unlikely that phosphatases were impaired. We conclude that due to unchanged phosphatases bound to the RyR2, CaMKII exerts its effects in GC-A KO mice mainly through increased PLB phosphorylation without altering RyR2 phosphorylation. Together with recent studies in mice with targeted inhibition of SR CaMKII activity [23], our findings demonstrate that endogenous SR CaMKII activity may improve Cai2+-uptake and cardiac contractility. Only during pathophysiological conditions like heart failure, when increased CaMKII expression is accompanied by decreased expression and/or activity of phosphatases bound to RyR2, RyR hyperphosphorylation and SR Ca2+-leak occur, leading to impaired Ca2+-cycling and contractile dysfunction.

4.4. Mechanisms for increased CaMKII activity in GC-A KO hearts
The mechanism through which chronic GC-A ablation results in increased cardiac CaMKII expression and activity is not yet apparent. Because exogenous synthetic ANP did not inhibit the inotropic and lusitropic responses of isolated working WT mouse hearts as well as the calcium responses of isolated WT cardiomyocytes to β-adrenergic stimulation (own unpublished observations), we propose that the reported molecular changes leading to the increased β-adrenergic responses in GC-A KO hearts derive from long-term absence of the GC-A receptor and not from abolition of direct, acute effects of endogenous ANP. The precise link will be an important issue for our future investigations.

4.5. GC-A KO hearts exhibit delayed contraction
Whereas the effects of dobutamine on maximal systolic contraction force (LVPmax) were greater in GC-A KO as compared to WT hearts, the effects on the time to contraction were diminished in the former. As a result, the stimulatory effects of dobutamine on the maximal rate of left ventricular contraction (+dP/dt, an index of both inotropy and the time to contraction) were not different between genotypes. At present time there is no explanation for this observation. Since it is not concomitant with myocyte Cai2+-cycling, we suspect that the previously reported changes in the cytoskeletal proteins, with increased expression of tubulin and desmin, might lead to cytoskeletal stiffness and retard cardiac contraction in GC-A KO mice [10,24].

4.6. Implications
Together with recent studies in mice with targeted inhibition of SR CaMKII activity [23], our findings demonstrate that CaMKII plays an important role in the regulation of PLB, myocyte Cai2+-cycling and cardiac contractility, especially under conditions of acute cardiovascular stress [23] and catecholamine stimulation ([23] and present study). Hence, increased CaMKII phosphorylation of PLB can be critical to maintain or even rescue cardiac contractile function under pathological conditions such as in the postischemic "stunned" myocardium [25], during recovery from intracellular acidosis [25] or even during development of cardiac hypertrophy (present study). Since excessive CaMKII-mediated phosphorylation of other cardiac targets can be detrimental and may facilitate the occurrence of arrhythmias, apoptosis [26], hypertrophy [12] or heart failure [27], further studies are needed to ascertain what modulates the different CaMK isoforms and the (de)phosphorylation of the different substrates under physiological vs pathophysiological conditions.


   Acknowledgements
 
We thank Anja Beilfuβ for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 487-B8 to Michaela Kuhn). Dr. Lars S. Maier is funded by the Deutsche Forschungsgemeinschaft (DFG) through an Emmy Noether-grant (MA 1982/1-4).


   Notes
 
1 Contributed equally to this manuscript. Back

Time for primary review 27 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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