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Cardiovascular Research 2001 51(4):717-728; doi:10.1016/S0008-6363(01)00346-7
© 2001 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Enhanced protein phosphorylation in hypertensive hypertrophy

Peter Bokníka,*, Ingrid Heinroth-Hoffmannb, Uwe Kirchhefera, Jörg Knappa, Bettina Lincka, Hartmut Lüssa, Thorsten Müllera, Wilhelm Schmitza, Otto-Erich Broddeb and Joachim Neumanna

aInstitut für Pharmakologie und Toxikologie, Universitätsklinikum Münster, Westfälische Wilhelms-Universität, Domagkstraße 12, D-48129 Münster, Germany
bInstitut für Pharmakologie und Toxikologie, Martin-Luther-Universität, Magdeburger Straße 4, D-06097 Halle, Germany

* Corresponding author. Tel.: +49-251-835-5517; fax: +49-251-835-5501 boknik{at}uni-muenster.de

Received 9 January 2001; accepted 4 May 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Chronic pressure overload in spontaneously hypertensive rats (SHR) is accompanied by heart hypertrophy and signs of heart failure. Since there is growing evidence for a possible pathophysiological role of altered protein phosphorylation in heart hypertrophy and failure, we studied here cardiac regulatory phosphoproteins and the kinases and phosphatases which regulate their phosphorylation state. Methods: The experiments were performed in ventricles of SHR (12–13 weeks old) and age-matched normotensive Wistar–Kyoto rats (WKY). Results: Basal as well as isoproterenol (Iso)-stimulated force of contraction (FOC) was markedly decreased in isolated electrically driven papillary muscles of SHR. Iso (3 µmol/l, 10 min) increased FOC by 0.91±0.20 mN in SHR and by 3.88±0.52 mN in WKY, respectively. Ca2+-uptake by sarcoplasmic reticulum (SR) at low ionized Ca2+-concentration was increased in homogenates from SHR. This was not due to altered expression of phospholamban (PLB), SR-Ca2+-ATPase and calsequestrin. However, PLB-phosphorylation at threonine-17 (PLB-PT-17) and the activity of Ca2+/calmodulin dependent protein kinase (Ca2+/Cam-PK) was increased in SHR. In addition, we found an enhanced protein kinase A (PKA)-dependent phosphorylation of the inhibitory subunit of troponin (TnI). In contrast, there was no difference in the activity or expression (protein- and mRNA-level) of protein phosphatases type 1 or type 2A between SHR and WKY. Conclusions: It is suggested that increased Ca2+/Cam-PK-activity with resulting increase of PLB-PT-17 enhanced SR-Ca2+-uptake in SHR and might contribute to the pathophysiological changes in cardiac hypertrophy of SHR.

KEYWORDS Contractile function; Gene expression; Hypertrophy; Protein kinases; Protein phosphorylation; SR (function)


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Spontaneously hypertensive rats (SHR) are a well established model for hypertension related heart hypertrophy and heart failure [1]. SHR manifest a progression from a stable form of hypertrophy with normal cardiac function to chronic heart failure with impaired heart function which corresponds well to the clinical course of patients with hypertension [2].

The SHR heart is characterized, similar to other models of heart hypertrophy and the failing human heart [3], by a decreased inotropic response to β-adrenergic stimulation [4,5]. Although multiple alterations in the function and expression of regulatory proteins in SHR rats were found [6], the subcellular basis for the loss of β-adrenergic responsiveness remains elusive.

Phosphorylation of cardiac regulatory proteins plays an important role in the regulation of cardiac contractility and in mediating contractile effects of β-adrenergic catecholamines. In the intact isolated heart, β-adrenergic stimulation leads to phosphorylation of phospholamban (PLB) on serine-16 by protein kinase A and on threonine-17 by Ca2+/calmodulin dependent protein kinase II [7]. PLB is an intrinsic protein of the sarcoplasmic reticulum (SR) and inhibits the function of SR-Ca2+-ATPase. Stimulation of β-adrenoceptors increases the phosphorylation state of PLB and thereby enhances Ca2+-ATPase activity and augments the rate of SR-Ca2+-uptake. Moreover, SR can release more Ca2+ for the subsequent contraction. These steps contribute to the inotropic and relaxant effects of β-adrenergic stimulation in the heart [8]. In addition, stimulation of β-adrenoceptors increases the phosphorylation state of the inhibitory subunit of troponin (TnI), localized in thin filaments of cardiac contractile apparatus. Both PLB- and TnI-phosphorylation can hasten cardiac relaxation [9].

The two phosphorylation sites of PLB are substrates for type 1 and 2A protein phosphatases (PP) in vitro. PPs of type 1 and type 2A could dephosphorylate phospholamban in membranes from rabbit heart phosphorylated in vitro on serine or threonine [10]. Similarly, type 1 and type 2A PP, separated from human ventricle, dephosphorylate recombinant PLB [11].

There are several lines of evidence that PLB dephosphorylation contributes to impaired inotropic effects of β-adrenergic stimulation and cardiac hypertrophy. The deletion of phosphorylation sites for PKA and Ca2+/Cam-PK II greatly diminished inotropic effect of β-adrenergic stimulation and led to cardiac hypertrophy [12]. Isoproterenol-induced heart hypertrophy was accompanied by an increase of PP-activity and dephosphorylation of PLB at both phosphorylation sites [13].

There is also accumulating evidence for a possible pathophysiological role of increased PP-activity in human heart failure. We have shown that PP-activity is enhanced in failing human hearts [14]. This was accompanied by reduced phosphorylation of cardiac regulatory proteins [15–17]. The reduced phosphorylation of PLB at serine-16 correlated with the reduced Ca2+-sensitivity of SR-Ca2+-ATPase [17].

These data underline the importance of reversible protein phosphorylation for the regulation of cardiac contractility and, in addition, imply its pathophysiological role in heart hypertrophy and failure. We hypothesized that alterations of protein phosphorylation may also play a role in early stages of hypertensive hypertrophy. Therefore, we comparatively studied (i) the expression of Ca2+-regulatory proteins, (ii) the phosphorylation state of phospholamban and the inhibitory subunit of troponin, and (iii) the expression and activity of protein kinases and phosphatases in SHR and age-matched Wistar–Kyoto rats.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animals
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Male SHR of 12–13 weeks of age and age-matched normotensive Wistar–Kyoto (WKY) rats were purchased from Harlan-Winkelmann (Borchen, Germany).

2.2 Contraction experiments
The experiments were performed on electrically driven papillary muscles from the left ventricles [13,18]. The cross-sectional area of papillary muscles amounted to 0.67±0.10 mm2 (n=10) in WKY and to 0.78±0.12 mm2 (n=9) in SHR, respectively. Force of contraction was measured with an inductive force transducer and parameters of contractility were calculated using BEMON-Software (Ingenieurbüro Jäckel, Hanau, Germany).

2.3 Ca2+-uptake measurement
The rate of SR-Ca2+-uptake was measured as described [18,19].

2.4 Total RNA preparation
In order to extract total RNA, a modification of the method described by Chomczynski and Sacchi [20] was employed. Frozen tissue was homogenized in TriStar-ReagentTM (AGS, Heidelberg, Germany) and total RNA was extracted according to the manufacturer's instructions.

2.5 mRNA quantification
The cDNA-probes for rat SERCA2a, rat PLB and rat Gs{alpha}-subunit were obtained as described by Linck [18]. The cDNA-probes for CSQ, TnI and catalytic subunits of PP1 and 2A were constructed by reverse transcription-polymerase chain reaction [21]. The blots were hybridized subsequently with 32P-labeled probes against PP1{alpha}, PP1β, PP1{gamma}, PP2A{alpha} and PP2Aβ, CSQ, TnI, SERCA2a, PLB and Gs{alpha}-subunit. For further hybridization, the membranes were stripped by boiling in 0.1% SDS. Bound radioactivity was visualized and quantified in a PhosphorImagerTM (Molecular Dynamics, Krefeld, Germany).

2.6 Protein quantification
SDS-tissue extracts were prepared as described [19] and 30 or 100 µg (for quantification of PPs and PLB-phosphorylation) of sample protein were loaded per lane. Western blots for PLB, SERCA and CSQ were performed as described [19]. The catalytic subunits of PPs were measured using polyclonal antibodies (UBI, Lake Placid, NY, USA) and 125I-labeled goat-anti-rabbit IgG.

PLB-phosphorylation was assessed using antibodies against PLB-peptide phosphorylated at serine-16 or at threonine-17 (PhosphoProtein Research, Bardsey, UK) [22] and 125I-labeled protein A. The specificity of these antibodies was tested in membrane vesicles from rat ventricles as described [23]. The signals obtained from the phosphorylation site specific PLB-antibodies were normalized to the corresponding values from the A1 PLB-antibody, which recognizes PLB independently of its phosphorylation state.

The phosphorylation state of TnI was measured using the monoclonal antibody 1E11.3 which recognizes TnI phosphorylated at cardiac specific N-terminal sequence [24,25]. The signal was normalized using the phosphorylation-independent antibody 2F6.6. Both primary antibodies were detected by 125I-labeled protein A.

2.7 Protein phosphatase assay
Protein phosphatase activity was determined as described previously [11,13] with 32P-phosphorylase a as substrate.

2.8 Protein kinase assays
Assays on protein kinase A and Ca2+/calmodulin protein kinase II were performed as described previously [13].

2.9 Data analysis
Data shown are means±S.E.M. In Fig. 1 the significant difference from control within SHR and WKY groups was calculated using Student's t-test for paired observations. All statistical comparisons between SHR and WKY were performed using unpaired t-test. A P-value <0.05 was considered significant.


Figure 1
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  		Fig. 1 Effects of isoproterenol (Iso) on parameters of contractility in isolated electrically driven papillary muscles from spontaneously hypertensive (SHR) and Wistar–Kyoto rats (WKY). Panel A depicts changes in force of contraction in mN: for each individual papillary muscle the corresponding pre-drug value has been subtracted from developed force at increasing Iso-concentrations. This basal force of contraction amounted to 4.72±0.36 mN in WKY and to 2.36±0.35 mN in SHR, respectively (P<0.05). In panel B, the first derivative of force (negative deflection) in mN/s is plotted. Panel C shows the duration of isometric relaxation to 10% of peak force in ms. * Depicts significant differences from WKY (P<0.05, unpaired t-test), + denotes first significant difference from pre-drug value (Ctr, P<0.05, paired t-test).

 

   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
Fig. 1 summarizes the data from contraction experiments in isolated electrically driven papillary muscles. The basal force of contraction was markedly decreased in SHR and amounted to 2.36±0.35 mN (n=9) in SHR and to 4.72±0.36 mN in WKY (n=10). The positive inotropic effect of the β-adrenoceptor agonist isoproterenol was noticeably attenuated in SHR (Fig. 1A). In addition, basal as well as isoproterenol-stimulated rate of diastolic relaxation was also diminished (Fig. 1B). However, the EC50-values for the inotropic as well as relaxant effect of isoproterenol did not differ between SHR and WKY. The diastolic dysfunction might be partly due to a prolonged duration of isometric relaxation (Fig. 1C). The time to peak tension was comparable and amounted under basal conditions to 57.1±1.0 ms in WKY (n=10) and to 58.3±1.8 ms in SHR (n=9), respectively.

Next, we determined Ca2+-dependence of Ca2+-uptake in the absence and in the presence of the PLB monoclonal antibody 2D12. This PLB antibody reverses the inhibition of SERCA by PLB at low ionized Ca2+-concentration, giving similar effects as PLB-phosphorylation [19]. Surprisingly, at low ionized Ca2+-concentration (30 mmol/l free Ca2+) Ca2+-uptake was higher in SHR-homogenates (Fig. 2A). Addition of PLB antibody reversed the PLB-inhibition and stimulated Ca2+-uptake to the same maximal level in SHR and WKY ventricles. Fig. 2B plots the fold stimulation of Ca2+-uptake by PLB antibody at different ionized Ca2+-concentrations. At low ionized Ca2+-concentrations stimulation of Ca2+-uptake by the PLB-antibody was markedly higher in WKY-homogenates compared to SHR-homogenates due to greater depression of Ca2+-transport at low ionized Ca2+-concentration. In contrast, no difference in Ca2+-transport rates was observed at high ionized Ca2+-concentrations, at which the extent of antibody stimulation was similar in WKY and SHR homogenates.


Figure 2
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  		Fig. 2 Ca2+-uptake by ventricular homogenates of spontaneously hypertensive (SHR) and Wistar–Kyoto rats (WKY) in the absence (Control) or in the presence of PLB monoclonal antibody 2D12 (+AB) at 30 nmol/l of ionized Ca2+ (A), and fold-stimulation of Ca2+-uptake by the PLB monoclonal antibody 2D12 determined at different ionized Ca2+-concentrations (B). Homogenates were preincubated with PLB monoclonal antibody for 20 min on ice, and ionized Ca2+-concentration was set using 0.05 mmol/l EGTA and different concentrations CaCl2. Ca2+-uptake was assayed by filtration at 8-min incubation. *P<0.05 versus WKY.

 
Next, we addressed whether these differences in contractility and Ca2+-uptake were correlated with altered expression of cardiac regulatory proteins. PLB and SERCA are major determinants of SR-Ca2+-uptake [8]. Calsequestrin (CSQ), a protein located within the SR-lumen, is also tightly linked to the regulation of Ca2+-homeostasis. CSQ-overexpression is accompanied by heart hypertrophy and prolonged relaxation [26]. In addition, we also studied expression of the inhibitory subunit of troponin (TnI), localized in the thin filaments of cardiac contractile apparatus. However, as depicted in representative Northern blots (Fig. 3), there was no difference in the mRNA-expression of SR-proteins and TnI between SHR and WKY. The occurrence of different mRNA-transcripts for PLB due to the distinct polyadenylation sites is a known phenomenon [13,18,27]. However, there was no difference in the relative abundance of PLB-transcripts between SHR and WKY. In agreement with Marcil et al. [28], the mRNA-expression of Gs{alpha}-subunit was similar in SHR and WKY (Fig. 3). Therefore, the data for mRNA-expression were normalized to the corresponding Gs{alpha}-value and are given in Table 1.


Figure 3
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  		Fig. 3 mRNA-expression of Gs{alpha}-subunit (Gs{alpha}), sarcoplasmic Ca2+-ATPase (SERCA), phospholamban (PLB), calsequestrin (CSQ), and the inhibitory subunit of troponin (TnI) in ventricles of spontaneously hypertensive (SHR) and Wistar–Kyoto rats (WKY). A 20-µg amount of total mRNA was electrophoresed, transferred on nylon membrane and hybridized with 32P-labeled cDNA-probes. Radioactive bands were visualized using PhosphorImagerTM.

 

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  		Table 1 mRNA- and protein-expression of cardiac regulatory proteins in ventricles of spontaneously hypertensive (SHR, n=9) and Wistar–Kyoto rats (WKY, n=10)

 
Since the mRNA-levels do not necessarily reflect the expression of respective genes at protein level [27], we also studied protein expression by means of quantitative immunoblotting. In accordance with the data from the mRNA-quantification, the expression of PLB, CSQ, SERCA as well as of TnI at the protein level did not differ between SHR and WKY as demonstrated in Figs. 4 and 5Go and summarized in Table 1.


Figure 4
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  		Fig. 4 Protein-expression of sarcoplasmic Ca2+-ATPase (SERCA) and calsequestrin (CSQ) in ventricles of spontaneously hypertensive (SHR) and Wistar–Kyoto rats (WKY). Samples were electrophoresed, transferred on nitrocellulose membrane and incubated with specific antibodies. Protein binding antibodies were visualized using 125I-labeled protein A. Radioactive bands were visualized using PhosphorImagerTM. On the left, molecular weight markers in kDa are indicated.

 

Figure 5
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  		Fig. 5 Protein expression and phosphorylation of phospholamban (PLB) at serine-16 (PLB-PS-16) and threonine-17 (PLB-PT-17), protein expression and phosphorylation of the inhibitory subunit of troponin (TnI, TnI-P) in ventricles of spontaneously hypertensive (SHR) and Wistar–Kyoto rats (WKY). Samples were electrophoresed, transferred on nitrocellulose membrane and incubated with specific antibodies. Protein binding antibodies were visualized using 125I-labeled protein A or 125I-labeled antimouse IgG and exposed to PhosphorImagerTM-screens. On the left, molecular weight markers in kDa are indicated.

 
Cardiac contractility is tightly controlled by the phosphorylation state of cardiac regulatory proteins. Using phosphorylation specific antibodies [22,24,25] we could address the question of altered protein phosphorylation in SHR. Experiments with the PLB-PS-16 antibody revealed no difference between SHR and WKY (Figs. 5 and 6Go). In contrast, phosphorylation of PLB at threonine-17 was increased in SHR ventricles as demonstrated on representative Western blot using the PLB-PT-17 antibody (Fig. 5). Moreover, by means of the monoclonal antibody 1E11.3 [24,25] we were able also to measure the phosphorylation state of TnI. Similar to the phosphorylation of PLB at threonine-17, phosphorylation of TnI was higher in ventricles of SHR. The results are summarized in Fig. 6.


Figure 6
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  		Fig. 6 Phosphorylation of the inhibitory subunit of troponin (TnI) and phospholamban at serine-16 (PLB-PS-16) and threonine-17 (PLB-PT-17) in ventricles of spontaneously hypertensive (SHR) and Wistar–Kyoto rats (WKY). The phosphorylation state was measured using phosphorylation site specific antibodies. The signals from phosphorylation specific antibodies were referred to the corresponding values from monoclonal antibodies 2F6.6 (TnI) and A-1 (PLB). *P<0.05 versus WKY.

 
Since phosphorylation of at least two substrates was altered in SHR, we investigated the expression and activity of enzymes regulating the phosphorylation state. The main protein phosphatases (PP) dephosphorylating PLB and TnI are PP1 and PP2A [10,11]. Due to the high homology between genes coding for the catalytic subunits, it is not possible to distinguish the isoforms unequivocally at the protein level. Therefore, PCR and Northern blots were initially performed. Using PCR we qualitatively detected catalytic subunits of PP1{alpha}, PP1β, and PP1{gamma} as well as of PP2A{alpha} and PP2Aβ. Moreover, we could use the 32P-labeled PCR-products for mRNA-quantification. As exemplified in Fig. 7, the mRNA-expression of catalytic subunits of PP1{alpha}, PP1β, PP1{gamma} as well as of PP2A{alpha} did not differ between SHR and WKY. In contrast, we could not quantify the mRNA-expression of PP2Aβ as the abundance of mRNA-transcripts was below detection limit. Accordingly, using antibodies raised against the catalytic subunits of PP1{alpha} and PP2A{alpha}/β we did not find any difference in the protein expression between SHR and WKY (Fig. 8). The data on the expression of catalytic subunits of PPs are summarized in Table 2. In addition, we also measured the type 1 and 2A PP-activity using 32P-labeled phosphorylase a. The comparison revealed no difference between SHR and WKY and PP-activity amounted to 1.91±0.15 nmol/min per mg in SHR and to 1.80±0.22 nmol/min per mg in WKY (n=10 each).


Figure 7
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  		Fig. 7 mRNA-expression of catalytic subunits of protein phosphatase 1{alpha} (PP1{alpha}CAT), PP1β, PP1{gamma} and PP2A{alpha} in ventricles of spontaneously hypertensive (SHR) and Wistar–Kyoto rats (WKY). A 20-µg amount of total mRNA was electrophoresed, transferred on nylon membrane and hybridized with 32P-labeled cDNA-probes. Radioactive bands were visualized using PhosphorImagerTM.

 

Figure 8
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  		Fig. 8 Protein-expression of catalytic subunits of protein phosphatase 1{alpha} (PP1{alpha}CAT) and 2A{alpha}β (PP2A{alpha}β) in ventricles of spontaneously hypertensive (SHR) and Wistar–Kyoto rats (WKY). Samples were electrophoresed, transferred on nitrocellulose membrane and incubated with specific antibodies. Protein binding antibodies were visualized using 125I-labeled goat anti-rabbit IgG. Radioactive bands were visualized using PhosphorImagerTM. On the left, molecular weight markers in kDa are indicated.

 

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  		Table 2 Expression of catalytic subunits of protein phosphatases (PP) at mRNA- and protein-levels in ventricles of spontaneously hypertensive (SHR, n=9) and Wistar–Kyoto rats (WKY, n=10)

 
Finally, we also measured the activity of protein kinase A (PKA) and Ca2+/calmodulin dependent protein kinase II (Ca2+/CaM-PK). Whereas PKA activity under basal conditions (absence of exogenous cAMP) did not differ between SHR and WKY, we found a decrease of PKA activity under conditions of maximal stimulation (2 µmol/l cAMP) in SHR-ventricles. As seen in Fig. 9B, Ca2+-independent (presence of 5 mmol/l EGTA) protein kinase activity measured using syntide-2 was comparable in SHR and WKY. However, Ca2+-dependent protein kinase activity under conditions of maximal stimulation (1 mmol/l Ca2+, 10 µg/ml calmodulin) was almost doubled in SHR as compared to WKY. Thus, the difference between stimulated and Ca2+-independent protein kinase activity corresponding to the net activity of Ca2+/CaM-PK was also markedly higher in SHR ventricles (5445±483 vs. 3225±367 nmol/mg per min, respectively).


Figure 9
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  		Fig. 9 Activity of protein kinase A (PKA, A) and Ca2+/calmodulin dependent protein kinase II (Ca2+/CaM-PK, B) in ventricles of spontaneously hypertensive (SHR) and Wistar–Kyoto rats (WKY). (A) PKA-activity was assayed with kemptide as substrate in the absence (–cAMP) or in the presence of 2 µmol/l cAMP (+cAMP). (B) Ca2+/CaM-dependent protein kinase was measured with syntide 2 as substrate in the presence of 1 mmol/l Ca2+ and 10 µg/ml calmodulin (Ca2+/CaM). Ca2+-independent protein kinase activity was determined in the presence of 5 mmol/l EGTA. *P<0.05 versus WKY.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The main new finding of the present study is an increase in the activity of Ca2+/calmodulin dependent protein kinase II (Ca2+/CaM-PK) in ventricles of spontaneously hypertensive rats (SHR). This correlated with an enhanced phosphorylation of phospholamban at threonine-17 (PLB-PT17) and an increased rate of Ca2+-uptake by sarcoplasmic reticulum.

SHR are a well established model of genetic hypertension which also enables investigation of subcellular mechanisms responsible for the development of heart hypertrophy. The aim of the present study was to characterize biochemical alterations in an early stage of hypertensive hypertrophy. We focused on the age of 12–13 weeks, because this time-point is characterized by stable compensated heart hypertrophy [1,2]. In an integrative approach, cardiac contractility, expression of cardiac regulatory proteins and function of cardiac sarcoplasmic reticulum (SR) were compared between SHR and age-matched Wistar–Kyoto rats (WKY). Furthermore, we measured protein phosphorylation and the activity of phosphorylation state regulating enzymes.

We noted a decrease of basal contractile parameters and marked attenuation of β-adrenergic mediated contractile response in papillary muscles from SHR rats [4,5]. This might be partly due to decreased density of β-adrenergic receptors and increased expression of inhibitory G-proteins [29]. These findings also implicate an impairment of SR-Ca2+-transport in SHR. Surprisingly, Ca2+-uptake at low ionized Ca2+-concentration was higher in ventricular homogenates from SHR rats. In contrast, we found no difference in Ca2+-transport rates at high ionized Ca2+-concentrations. Apparently, this difference was not due to alterations of protein expression since the levels of PLB, sarcoplasmic Ca2+-ATPase (SERCA) and calsequestrin (CSQ) were similar in SHR and WKY. Hence, we studied the phosphorylation state of PLB, as this is an important mechanism regulating SR-Ca2+-uptake. PKA and Ca2+/Cam-PK phosphorylate PLB at two different sites: serine-16 and threonine-17 [7]. These phosphorylations relieve inhibition of SERCA by PLB, stimulate SR-Ca2+-uptake and shorten cardiac relaxation [8]. Whereas comparable levels of PKA-mediated PLB-phosphorylation were found, the phosphorylation of PLB at threonine-17 was enhanced in SHR-ventricles. This can explain higher rates of SR-Ca2+-uptake at low ionized Ca2+-concentration, which we observed in vitro.

The above-mentioned findings are, however, somewhat surprising with respect to the observed decrease of contractile parameters and the blunted response to isoproterenol in SHR [4,5]. One would rather expect an increase of basal force of contraction and shortening of relaxation due to enhanced SR-Ca2+-transport. The reason for this discrepancy is not clear. In contrast, there is considerable evidence that PLB dephosphorylation attenuates contractile effects of β-adrenergic stimulation and may play an important role in the development heart hypertrophy [12,13]. However, heart hypertrophy in these models is not induced by pressure overload. Thus, one might speculate that a different etiology may account for this discrepancy.

Next, we focused on the mechanism of increased PLB-phosphorylation. The present data indicate that the enhanced PLB-phosphorylation at threonine-17 is due to increased activity of Ca2+/calmodulin dependent protein kinase in SHR-ventricles. We have reported an increase of Ca2+/CaM-PK-activity also in the hearts of patients suffering from dilated cardiomyopathy [30]. Fittingly, Hoch et al. [31] have found increased expression of {delta}3-isoform of Ca2+/CaM-PK in failing human hearts. Thus, Ca2+/CaM-PK seems to play an important role in heart hypertrophy and failure.

There is accumulating evidence that calmodulin alone or its downstream targets Ca2+/Cam-PK and calcineurin may be crucial regulators of heart hypertrophy. Transgenic overexpression of calmodulin displays heart hypertrophy [32]. Passier et al. [33] have shown that activated Ca2+/CaM-PKs I and IV induce hypertrophic responses in cultured cardiomyocytes. Furthermore, it is also conceivable, that persistent increase of Ca2+-uptake due to enhanced Ca2+/CaM-PK-mediated PLB-phosphorylation leads to SR-Ca2+-overload favoring the development of heart hypertrophy. Interestingly, cardiac specific overexpression of CSQ, which increases SR-Ca2+-stores, leads to heart hypertrophy and failure in transgenic animals [26].

The observed decrease of PKA-activity is in agreement with previous findings of Bhalla et al. [34] and most probably reflects the reduction of type I PKA reported by Prashad [35]. We suggest that the PKA decrease may in part also explain the weak contractile response to isoproterenol in SHR. On the other hand, the decrease of PKA-activity might be relevant also for cardiac growth as there is evidence for inhibition of mitogen-activated protein kinase-pathway by PKA [36].

Based on data from various animal models [13] and on the findings from human failing hearts [14], we initially hypothesized also a possible pathophysiological role of protein phosphatases (PP) in SHR. In contrast however, PPs are apparently not a subject of regulation in the early stage of hypertensive hypertrophy. Our experiments characterizing PPs at various levels provide no indication for any alteration of type 1 and 2A PPs in SHR. On the other hand, this may explain the increase of PLB-PT-17 phosphorylation in SHR in contrast to human heart failure. The increase of PP-activity in human failing hearts [14] apparently overcomes the stimulation of Ca2+/Cam-PK resulting in overall decrease of PLB-phosphorylation at threonine-17 [37].

Another finding, which has to be discussed, is the increased phosphorylation of TnI despite decreased PKA-activity. The phosphorylation specific antibody 1E11.3, which we used, recognizes the phosphorylated N-terminus of TnI-molecule [24,25]. This cardiac specific sequence contains two adjacent serine residues (23 and 24) that can be phosphorylated by either PKA or under some conditions also by protein kinase C (PKC) [38]. We cannot differentiate, whether the increase of TnI-phosphorylation is due to PKA or PKC. Nevertheless, the observed increase in SHR concerns the N-terminus where, independently of the involved kinase (PKA or PKC), the impact of phosphorylation is the same: a decrease of Ca2+-sensitivity of Ca2+-stimulated Mg2+-ATPase without affecting its maximal activity [38,39]. However, the N-terminus seems not to be a primary target for PKC as it is only a weak substrate for PKC in vitro [39]. Thus, we suggest that the increase of TnI-phosphorylation in SHR might be due to PKA. The discrepancy between the increased TnI-phosphorylation and decreased PKA-activity may suggest a local regulation of PKA-activity. One can speculate that the expression of PKA regulatory subunits and herewith the anchoring of catalytic subunits might be altered in SHR [40]. On the other hand, the existence of multiple subcellular pools of cAMP could also explain this discrepancy. The increase of PKA-mediated protein phosphorylation seems to be confined to the cardiac contractile apparatus as the PKA-dependent phosphorylation of PLB is comparable in SHR and WKY. McConnell et al. [41] have reported that isoproterenol led to a higher PKA-dependent phosphorylation of TnI in SHR. This was associated with a rightward shift in the Ca2+-dependence of the actomyosin Mg2+-ATPase-activity as compared to WKY. Thus, it is conceivable that the higher basal TnI-phosphorylation may contribute to lower basal force development observed in our study. The enhanced N-terminal phosphorylation of TnI along with the increased PLB-PT-17 phosphorylation might also counteract the impaired relaxation in SHR. Apparently, these compensatory mechanisms are not sufficient to normalize the duration of cardiac relaxation.

Besides Ser-23 and 24 the cardiac TnI contains multiple phosphorylation sites for PKC, namely Ser-43, Ser-45, Ser-78 and Thr-144 [39]. Their phosphorylation inhibits the Ca2+-stimulated actomyosin Mg2+-ATPase and decreases force of contraction. Interestingly, there is evidence for upregulation of the PKC-cascade in SHR [42]. Thus, an increased PKC-mediated phosphorylation seems to be important not only for the development of heart hypertrophy but might contribute also to the depressed contractility in SHR.

In summary, we have shown that the activity of the Ca2+/calmodulin dependent protein kinase is increased in SHR-ventricles. This leads to enhanced phosphorylation of phospholamban at threonine-17 and stimulation of SR-Ca2+-uptake. These alterations might contribute to the pathophysiological changes in heart hypertrophy. In addition, a lower basal and isoproterenol-stimulated contractility may be partly due to the increased N-terminal phosphorylation of the inhibitory subunit of troponin in SHR.

Time for primary review 26 days.


   Acknowledgements
 
The skilful technical assistance of Cordula Vischedyk is greatly appreciated. This research was supported by the IMF, IZKF A7, SFB556 B1/B2.


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

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