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Cardiovascular Research 2003 58(3):602-610; doi:10.1016/S0008-6363(03)00261-X
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Copyright © 2003, European Society of Cardiology

Trapidil protects ischemic hearts from reperfusion injury by stimulating PKAII activity

Oliver J Sichelschmidta, Claudia Hahnefeldb, Thomas Hohlfelda, Friedrich W Herbergb and Karsten Schröra,*

aInstitut für Pharmakologie und Klinische Pharmakologie, Heinrich-Heine-Universität Düsseldorf, Gebäude 22.21 Universität Strasse 1, 40225 Düsseldorf, Germany
bFachbereich 19, Abteilung Biochemie, Universität Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany

kschroer{at}uni-duesseldorf.de

* Corresponding author. Tel.: +49-211-811-2500; fax: +49-211-811-4781.

Received 16 October 2002; accepted 3 February 2003


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The cardioprotective effects of trapidil on ischemic reperfused (I/R) rabbit hearts were studied. Recently, we had shown that trapidil might activate protein kinase A (PKA). In this study, we examined the exact mode of PKA stimulating activity of trapidil. Finally, we investigated the effect of trapidil on the phosphorylation state of phospholamban (PLB), a major PKA target in the heart and key regulator of Ca2+ sequestration via the sarcoplasmic reticulum Ca2+-ATPase. Methods: Langendorff-hearts of New Zealand White rabbits were perfused at constant volume and subjected to global low-flow ischemia for 2 h, followed by 1 h of reperfusion. Subsequently, hearts were used for Western blot analysis of PLB phosphorylation. Furthermore, three different regulatory subunits and one catalytic subunit of PKA were overexpressed in E. coli. These PKA subunits were purified and used in an in vitro assay system to test the impact of trapidil on PKA activities in the absence and presence of cAMP. Results: I/R resulted in a significant increase in left ventricular end-diastolic pressure and creatine kinase efflux in the hearts. Trapidil (10 µM) prevented these alterations. Using recombinant cAMP-free PKA isoforms, it was found that trapidil specifically stimulated PKAII but only did so in the presence of small amounts of added cAMP. Furthermore, the PKA-dependent 16Ser phosphorylation of PLB was markedly reduced in I/R. Trapidil largely normalized the 16Ser phosphorylation of PLB. Conclusions: The data demonstrate cardioprotective actions of trapidil in I/R and show a PKAII-dependent cAMP sensitizing effect of the compound. They also indicate PKA-dependent PLB phosphorylation as a target, suggesting an improved Ca2+ uptake by the sarcoplasmic reticulum. This action might be involved in the cardioprotective effects of trapidil.

KEYWORDS Ischemia; Protein kinases; Reperfusion; SR (function)


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The triazolopyrimidine trapidil (5-methyl-7-diethylamino-s-triazolo[1,5-{alpha}]pyrimidine) is an antianginal compound with a broad spectrum of pharmacological activities. In the cardiovascular system, these include coronary vasodilation, antiischemic effects in vivo and in vitro, inhibition of vascular smooth muscle cell proliferation, antiplatelet and antiatherosclerotic actions [1–5]. Mechanistically, trapidil was considered a non-selective inhibitor of phosphodiesterases (PDE) [3], eventually resulting in increased cellular cAMP levels and stimulation of cAMP-dependent signaling pathways, specifically protein kinase A (PKA). More recently, we have postulated that trapidil may directly activate PKA since the compound stimulated the purified enzyme in a cell-free system in the absence of added cAMP [6]. Preliminary pharmacological studies further suggested that the compound might exhibit some selectivity for the isoform PKAIIβ [7]. However, neither the exact mechanisms of activation of PKA by trapidil nor its relation to its antiischemic effects have been elucidated. Specifically there is no information about the cellular sites, isoforms and substrates of trapidil-activated PKA, except the demonstration of phosphorylation of vasodilator-stimulated phosphoprotein (VASP) [7].

One possible target of PKA in the ischemic myocardium is the sarcoplasmic reticulum (SR), specifically phospholamban (PLB). PLB is a 52 amino acid SR-membrane-integrated phosphoprotein and the key regulator of SR-Ca2+-ATPase (SERCA2a). PLB forms homopentamers which are in a dynamic equilibrium with PLB monomers in the lipid bilayer. Phosphorylation of PLB drives the equilibrium towards the pentamers [8], thereby reducing the dephosphorylated monomer which in contrast to the pentamer is a potent inhibitor of SERCA [9,10]. Serine-16 (16Ser) and threonine-17 (17Thr) were identified as the residues specifically phosphorylated by PKA and calcium/calmodulin-dependent (CaM) kinase, respectively [11,12]. A third phosphorylation site, serine-10 (10Ser), was also identified. The amino acid is phosphorylated by protein kinase C but this appears not to result in an increase in SERCA activity [13]. Phosphorylation of PLB is markedly reduced in myocardial ischemia [14,15]. As a consequence, intracellular Ca2+ overload to the cytosol due to reduced Ca2+ sequestration into the SR and subsequent tissue injury have been described in cardiomyocytes from hearts that were subjected to I/R [16,17].

In this study we demonstrate for the first time that (i) the action of trapidil is selective for the subtype PKAII, where trapidil probably acts as a cAMP sensitizer and (ii) that I/R-induced reduction in PLB phosphorylation is largely prevented by trapidil, possibly explaining at least parts of the cardioprotective effects of the compound in I/R.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Heart preparations and experimental protocol
The investigation conforms with 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). Langendorff-hearts were prepared from New Zealand White rabbits (1.6–2.4 kg) as previously described [18]. The hearts were perfused at constant flow with Krebs–Henseleit buffer (pH 7.4). The temperature of the hearts was kept constant at 37.0±0.1°C by a surrounding water-jacket. The perfusion buffer was equilibrated with 95% O2+5% CO2 by using a membrane oxygenator (Dideco, Mirandola, Italy).

After recovery from the preparation as seen from a stable contractile function and coronary perfusion pressure (defined as t = 0 min), the hearts were perfused with Krebs–Henseleit buffer at a constant flow rate of 22 ml/min for 210 min or were subjected to 30 min of normal perfusion (22 ml/min) followed by 120 min of low-flow ischemia by reducing the perfusion volume to 1.2 ml/min [18]. Reperfusion was initiated at time 150 min by restoring the normal flow rate of 22 ml/min for another 60 min. The hearts were either untreated or treated with 10 µM trapidil (final concentration) (UCB, Kerpen, Germany) which was added to the Krebs–Henseleit buffer at t = 0 min and was present throughout the experiment. This trapidil concentration was shown in preliminary experiments to have a cardioprotective effect [5], to cause significant stimulation of PKA [6] but not to inhibit PDE [6].

2.2 Functional measurements
The hearts were beating spontaneously. Left ventricular pressure (LVP) was measured by a Statham transducer, connected to a balloon inserted into the cavity of the left ventricle. The filling pressure in all experiments was set between 3 and 5 mmHg. Coronary perfusion pressure (CPP) was measured by a second Statham transducer in the aortic inflow tract. Actively developed left ventricular pressure ({Delta}LVP), left ventricular end-diastolic pressure (LVEDP) and CPP were monitored continuously and recorded on a Mac-Lab-Laboratory computer. Heart rate (HR) was computed from left ventricular pressure signals.

2.3 Biochemical measurements in coronary effluent
Creatine kinase activity was determined in the coronary effluent. Aliquots of the perfusate were taken and the CK activity was measured with a creatine kinase assay kit (Roche Diagnostics, Mannheim, Germany) according to recommendations by the manufacturer.

2.4 Western blotting
At the end of the experiments, left ventricular tissue was homogenized on ice in RIPA buffer containing: NaCl, 150 mM; Tris–HCl (pH 7.5), 50 mM; phenylmethylsulfonylfluoride (PMSF), 10 mM; sodium dodecylsulfate (SDS), 0.1% (w/v); IGEPAL CA-630 (NP-40), 1% (v/v); desoxycholic acid (DOC), 0.5% (w/v); protease inhibitor mix (#P-8340, Sigma, Deisenhofen, Germany), 5 µl per 1 ml of buffer. Lysates were subsequently boiled for 3 min. Aliquots of the lysates were used to determine the protein content with the Bio-Rad kit (#500-0112, Hercules, CA, USA). The lysates were then immediately transferred into an equal volume of 2x Laemmli buffer (sodium phosphate buffer (pH 7.4), 125 mM; dithiothreitol (DTT), 100 mM; glycerol, 20% (v/v); SDS, 4% (w/v); bromphenol blue, 0.002% (w/v)), boiled for 10 min and stored until further processing at –80°C. Samples of the lysate (1 µg protein each) were fractionated on 15% SDS–polyacrylamide gels and transferred to PVDF membranes (Millipore, Eschborn, Germany). Membranes were incubated for 60 min at room temperature in Tris-buffered saline (NaCl, 150 mM; Tris–HCl (pH 7.4), 10 mM; Tween 20, 0.1% (v/v)) containing 5% skim milk and then probed with antibodies to PLB (see below).

The analysis of site-specific phosphorylation of PLB was performed with the polyclonal antibody PS-16 (1:10 000 dilution, Cyclacel, Dundee, Leeds, UK) [19]. The PS-16 antibody only recognizes the monophosphorylated 16Ser-PLB but is unable to detect dual-phosphorylated PLB, i.e. 16Ser/17Thr-PLB [13]. Blots were stripped and reprobed with the monoclonal antibody A1 (1:10 000, Cyclacel, Dundee, Leeds, UK) [20] to detect total PLB and to check for adequate protein loading. Secondary antibodies (anti-rabbit and anti-mouse, respectively) were conjugated to horseradish peroxidase (1:3000, 45 min at room temperature). Proteins were detected by Lumi-Light Western blotting substrate (Roche Molecular Biochemicals, Mannheim, Germany). PLB phosphorylation was quantified by densitometry and is expressed as ratio 16Ser-phosphorylated PLB/total PLB.

2.5 Preparation of recombinant PKA subunits
Murine catalytic subunit (C) {alpha}, bovine regulatory subunit (R) I{alpha}, human RII{alpha} and RIIβ were overexpressed in E. coli BL21.DE3. The C subunit was purified by P11 phosphocellulose chromatography and the type I R subunit by ion-exchange chromatography (DEAE-cellulose) as described previously [21,22]. The type II R subunits were purified by affinity chromatography via cAMP columns (Biolog, Bremen, Germany) as described previously [23] and eluted with cGMP. To obtain cAMP-free RI{alpha} subunit, the protein was unfolded with 8 M urea and refolded as described by Buechler et al. [24]. All R subunits were additionally purified by gel filtration and tested for their ability to inhibit the C subunit stoichiometrically using a spectrophotometric assay.

2.6 PKA activity assay
PKA activity was measured in a cell-free system with a non-radioactive protein kinase assay kit (Calbiochem, San Diego, CA, USA). Isoform-specific measurements were performed using cAMP-free purified human recombinant RII{alpha} and RIIβ subunits as well as cAMP-free purified bovine recombinant RI{alpha} subunit. The holoenzyme was formed by mixing cAMP-free R subunit with murine recombinant C{alpha} in a Mg/ATP buffer containing 5 mM MgCl2 and 1 mM ATP in a 1.2:1 molar ratio (R:C; 1 h, 4°C) using 20 nM C{alpha} subunit. Phosphorylation of a synthetic PKA pseudosubstrate (RFARKGSLRQKNV) by PKA in the presence of trapidil or cAMP or a combination of both was monitored by ELISA according to the manufacturer's instructions. Cyclic AMP was used in a concentration of 2 µM unless otherwise indicated. In this in vitro system as well as in the spectrophotometric PKA assay trapidil was used in a concentration of 100 µM which was previously shown to have cardioprotective effects in I/R (unpublished results) but not to inhibit PDE [5].

2.7 Spectrophotometric PKA assay
This assay was used as another means to determine PKA activity modulation. In brief, holoenzyme was generated by combining cAMP-free R subunit and C subunit (20 nM C{alpha}) in 100 mM MOPS (pH 7), 5 mM DTT in a 1.2:1 molar ratio (R:C;1 h, 4°C). This preformed holoenzyme was incubated for 2 min at room temperature in an assay mix as described by Cook et al. [25] in a total volume of 750 µl. In brief, the assay is based on the quantity turnover of ADP with glycolytic enzymes. The reaction was initiated by adding the heptapeptide kemptide (170 µM) and the activity of the free C subunit was followed for 2 min in the absence or presence of 100 µM trapidil and 2 µM cAMP, respectively, using a Perkin Elmer Lambda Bio UV–Vis spectrophotometer. The compound detected in this assay was NADH at a wavelength of 340 nm.

2.8 Statistics
The data are mean±S.E.M. of n independent experiments. Statistical analysis was performed by one-way ANOVA followed by Bonferroni's multiple comparison test. P values<0.05 were considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Myocardial function
At the beginning of the experiments, HR, CPP, LVEDP, and {Delta}LVP were not significantly different between the several experimental groups. There was a time-dependent increase in CPP and some decrease in HR in all groups of hearts, independent of treatment. Specifically, trapidil did not alter any of these parameters in non-ischemic hearts. However, HR, CPP and {Delta}LVP were substantially reduced at the onset of ischemia. There was an almost complete recovery of {Delta}LVP at the end of reperfusion (Table 1). In contrast, LVEDP was markedly increased during ischemia with an incomplete recovery during reperfusion. Trapidil largely prevented this rise in LVEDP. Thus, at the end of ischemia prior to reperfusion, LVEDP in trapidil-treated I/R hearts was reduced by 59% as compared to untreated I/R hearts. There was also an incomplete recovery of LVEDP in trapidil-treated hearts during reperfusion (Fig. 1). No such improvements were seen, when trapidil was added only during reperfusion (not shown) suggesting that the cardioprotective action of the compound requires its presence during ischemia.


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  		Table 1 Parameters of cardiac and coronary function at the beginning (t = 30 min) and the end (t = 210 min) of the protocol

 

Figure 1
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  		Fig. 1 Left ventricular end-diastolic pressure (LVEDP) of control hearts and I/R hearts with and without treatment with trapidil (10 µM). Triangles mark I/R hearts and squares non-ischemic hearts. Open symbols are trapidil-treated hearts, closed symbols untreated hearts. Data are mean±S.E.M. of 9–14 hearts per group. *P<0.05 (I/R+trapidil vs. I/R).

 
3.2 CK release
Creatine kinase (CK) efflux is an established marker of the loss of myocellular integrity. There was no significant release of CK from non-ischemic hearts. In ischemic hearts, CK release started to increase with the beginning of reperfusion and amounted to 302±57 mU/g·min at the end of reperfusion. This elevated enzyme activity in the cardiac effluent was reduced by 69% by trapidil treatment (Fig. 2). As already seen with LVEDP, there was only a small, insignificant, reduction in CK-overflow, when trapidil was only present during the reperfusion period (not shown).


Figure 2
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  		Fig. 2 Creatine kinase (CK) activity in the coronary effluent of control hearts and I/R hearts with and without treatment with trapidil (10 µM). Triangles mark I/R hearts and squares non-ischemic hearts. Open symbols are trapidil-treated hearts, closed symbols untreated hearts. Data are mean±S.E.M. of 9–14 hearts per group. *P<0.05 (I/R+trapidil vs. I/R).

 
3.3 Modulation of activation of PKA holoenzyme by trapidil
To determine whether trapidil directly activates PKA, cAMP-free holoenzymes (without any free or enzyme-bound cAMP) were analyzed in a cell-free system. To further elucidate a possible isoform specificity of trapidil, holoenzymes containing only the RI{alpha}, RII{alpha} or RIIβ subunits were used (referred to as PKAI{alpha}, PKAII{alpha} and PKAIIβ, respectively). There was no direct effect of 100 µM trapidil on the activity of any of the three holoenzymes in the PKA-activity assay. However, for the two PKAII holoenzymes a significant increase of activity was detected in the preparations containing 2 µM cAMP plus 100 µM trapidil as compared to the preparations containing only 2 µM cAMP in the absence of trapidil. Two µM of cAMP is in the range of endogenous levels in non-ischemic cardiac tissue [26]. This suggests that the activities of PKAII{alpha} and PKAIIβ were significantly increased by trapidil and that this effect required the presence of physiological amounts of cAMP. Similar results were obtained in four independent experiments performed in duplicate (see Fig. 3a for representative data). No difference in phospho-transfer activity was seen with PKAI{alpha}, as confirmed in three independent experiments performed in duplicate (see Fig. 3b for representative data), as well as with the isolated C{alpha} subunit in the presence or absence of trapidil (Fig. 3c), as shown by means of the Cook assay [25]. Two µM cAMP did not fully activate all three holoenzymes. In an independent experiment the concentration-dependent activation of PKA by cAMP was examined (data not shown). Five different cAMP concentrations between 3 nM and 30 µM were assayed. Application of 3 nM cAMP did not induce an activation of PKA holoenzymes. With 30 nM cAMP a first distinct effect on all three isoforms was observed. However, 30 µM cAMP led to a more pronounced activation of PKA than 3 µM cAMP. This indicates that the failure of trapidil to stimulate PKAI{alpha} in the presence of added cAMP cannot be attributed to an already maximum activation of the holoenzyme. Thus, trapidil appears to activate the PKAII-isoform and this effect is probably due to an enhanced binding/activity of cAMP at the regulatory subunit of the enzyme. On average, the stimulation of PKAIIβ appeared to be somewhat more pronounced than that of PKAII{alpha}. The spectrophotometric assay confirmed the data from the PKA activity assay and also detected a more pronounced activation for RIIβ as compared to RII{alpha} (data not shown).


Figure 3
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  		Fig. 3 Activation of protein kinase A (PKA) holoenzyme by 100 µM trapidil (TPD), 2 µM cAMP or a combination of both. PKA activity was measured by phosphorylation of a synthetic PKA pseudosubstrate and monitored by ELISA. (a) Effects of trapidil on the activation of PKAII{alpha} and PKAIIβ. One representative experiment out of four independent experiments with similar results, performed in duplicate, is shown. Experiments were performed by means of the PKA activity assay. (b) Effects of trapidil on the activation of PKAI{alpha}. One representative experiment out of three independent experiments with similar results, performed in duplicate, is shown. Experiments were performed by means of the PKA activity assay. The data are x-fold induction of PKA activity as compared to control and are mean±S.E.M. {dagger}P<0.05 (PKAIIβ+cAMP+TPD vs. PKAIIβ+cAMP); *P<0.05 (PKAII{alpha}+cAMP+TPD vs. PKAII{alpha}+cAMP). (c) Influence of 100 µM trapidil and 2 µM cAMP or a combination of both on the activity of free C{alpha} subunit as measured by means of the Cook assay. Kemptide was used as a substrate of C{alpha}. Data are enzyme activity in µmol/mg protein·min and are mean±S.E.M. of three independent experiments.

 
3.4 PLB phosphorylation
Fig. 4a shows a representative Western blot with antibody PS-16 to identify changes in PKA-dependent phosphorylation of PLB. Total PLB, as identified by the antibody A1, appears to be identical in all groups. In contrast, the PKA-dependent phosphorylation was different. Non-ischemic control hearts exhibited a marked phosphorylation of 16Ser which almost completely disappeared under I/R conditions. The addition of 10 µM trapidil to non-ischemic hearts had no significant effect. However, treatment of I/R hearts with trapidil resulted in an almost complete preservation of the I/R-induced decrease of PLB phosphorylation. Densitometric analysis of the PLB bands validated these findings. The PLB phosphorylation was calculated as ratio of 16Ser-phosphorylated PLB/total PLB. The results show that the 16Ser-phosphorylation rate of PLB was significantly reduced after I/R from 198±32 to 21±7 arbitrary units (AU) (P<0.05). In the presence of 10 µM trapidil, in I/R hearts the phosphorylation was significantly improved, amounting to 129±35 AU at the end of reperfusion (P<0.05) (Fig. 4 b).


Figure 4
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  		Fig. 4 Protein kinase A (PKA)-dependent phosphorylation of phospholamban (PLB) at the end of reperfusion. (a) Representative Western blot showing control hearts and I/R hearts with or without trapidil treatment (10 µM). The PKA-phosphorylated PLB and total PLB are shown from one blot (detection of total PLB after membrane stripping). Each treatment is exemplified by two original protein preparations from different hearts, one is shown for the control group. (b) PLB-phosphorylation expressed as ratio PS-16-phospho-PLB/total PLB. The figure shows control hearts and I/R hearts with or without 10 µM trapidil. Data are mean±S.E.M. of 6–10 hearts per group. {dagger}P<0.05 (I/R+trapidil vs. I/R); *P<0.05 (I/R vs. control).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Confirming an earlier preliminary report [5] we demonstrate here a reduction of I/R injury by 10 µM trapidil, a concentration that is in the range of plasma levels in vivo after oral therapeutic doses [27]. In this study we show for the first time that trapidil specifically activates PKAII and that this is possibly due to a cAMP sensitizing action which is not seen in non-ischemic hearts. Furthermore, we identified PLB, a protein associated with the I/R-influenced Ca2+ homeostasis and a major substrate of PKA in the heart, as a target of the I/R-specific action of trapidil-induced PKA activity.

Four different isoforms of PKA are known. They are distinguished by their regulatory subunits (RI {alpha} and β, RII {alpha} and β) and localized at different sites inside the cell. The type I PKA holoenzyme is predominantly cytoplasmic, whereas the majority of type II PKA is associated with cellular structures and organelles by an interaction with A kinase anchoring proteins (AKAPs) [28,29]. We have previously suggested that trapidil activates PKA in a cAMP-independent manner [6]. This conclusion was based on studies with purified PKA holoenzyme from bovine heart (#P5511, Sigma, Deisenhofen, Germany; contains mainly PKAII{alpha}). However, even this purified PKA might not have been entirely free of enzyme-bound cAMP. Thus, PKA stimulation by trapidil could also indirectly be caused by enhancement of cAMP-mediated PKA activation, for example by an allosteric effect of trapidil on the cAMP binding-sites at the regulatory subunit of the enzyme. In the present study, purified recombinant holoenzymes were available which were free from any cAMP contamination. In these preparations, trapidil was unable to activate PKA but did so after addition of physiological amounts of exogenous cAMP [26]. Thus, the substance rather appears to act as a cAMP sensitizer. Furthermore, this effect was isoform-specific. Only the holoenzymes formed with RII subunits (PKAII{alpha} and PKAIIβ) showed this effect. Hence, only a local PKA activation occurs which is limited to the subcellular sites where PKAII is localized.

One of the prominent features of I/R injury in the heart is the disturbed Ca2+ homeostasis, causing a Ca2+ overload to cardiomyocytes [16,17]. This increase in cytosolic Ca2+ is due to reduced Ca2+ uptake subsequent to inhibition of PKA-dependent phosphorylation of PLB [2,14,15]. Therefore, PLB is a likely candidate for being modified by trapidil-induced modulation of PKA activity in I/R.

In cardiac myocytes the RII subunit is localized to the perinuclear regions and to periodic transverse striations [30]. An association of PKA with cardiac SR vesicles was reported and this PKA isoform was characterized as a type II enzyme [31]. It was concluded that the cardiac SR contains primarily PKAII and that this is probably the enzyme which is responsible for the regulation of Ca2+-transport. No further characterization of the PKAII isoform ({alpha} or β) was performed. Another important observation for a role of PKAII in PLB phosphorylation was made recently by Fink et al. [30]. They demonstrated a reduced PLB phosphorylation and contractile function during isoproterenol stimulation by interrupting the binding of PKAII to AKAPs using a non-selective AKAP competitor peptide (Ht 31), leading to diffuse distribution of PKAII throughout the cytosol. PKA shows a broad range of substrate specificity. Subcellular targeting of PKA via AKAPs leads to compartmentation and has emerged as an important control mechanism by which PKA is able to cause specific phosphorylations of target substrates within the cell [32] (for review, see Ref. [28]). Recent investigations provided evidence for the generation of multiple microdomains with increased concentration of cAMP in cardiac myocytes under β-adrenergic stimulation [33]. These discrete microdomains are localized in the proximity of the region of transverse tubule/junctional SR membrane. Furthermore, these authors [33] demonstrated that such local spots of high cAMP specifically activate a subset of PKA molecules in the T tubule area. Thus, the consequences of cAMP-induced activation of PKA depend on the site of this action and are clearly different in local membrane-bound domains as compared to rather uniform increases in the cytosol, for example after β-adrenergic stimulation subsequent to ischemia-related catecholamine overflow [34,35].

Drago and Colyer [19] introduced a pair of polyclonal antibodies, PS-16 and PT-17 which detect PLB either phosphorylated at 16Ser (PKA-dependent) or at 17Thr (CaM kinase-dependent), respectively. Subsequent characterization demonstrated that the antibody PS-16 interacts with mono-phosphorylated 16Ser-PLB but did not detect dual-phosphorylated 16Ser/17Thr-PLB [13]. Therefore, the changes in the phosphorylation state of PLB found here can be attributed to altered PKA activities.

Trapidil-treated I/R hearts exhibited a significantly improved recovery in PLB phosphorylation as opposed to untreated I/R hearts. This shows that the trapidil-mediated PKA activation is not restricted to cell-free systems but is present in whole organs as well. Potentially, this recovery in PLB phosphorylation allows for improved Ca2+ uptake into the SR, thereby reducing the cytosolic Ca2+ level. Functional consequences of this are a significant reduction in diastolic contracture at the end of ischemia as indicated by the markedly reduced LVEDP at the end of reperfusion as well as a reduced CK leakage from myocardial tissue. In addition, improved Ca2+ uptake is also associated with a lower activity of detrimental Ca2+ activated enzymes (i.e. proteases, hydrolases) [36–38] and reduced mechanical stress for the cardiac myocytes [39]. Although Ca2+ homeostasis is considered to be improved and higher Ca2+ transients at the SR membrane are expected, no significant effect of trapidil on {Delta}LVP was observed. This might be related to the small (–22%) and not significant contractile failure in I/R hearts.

It is interesting that trapidil improved the reduced PLB phosphorylation and, therefore, the reduced PKA activity in I/R but did not stimulate PKA activity above control in non-ischemic hearts. Osada et al. [40] reported a reduction in SERCA content in rats after 30 min of ischemia followed by 30 min of reperfusion. In another publication from the same group a reduction in protein content of SERCA2a by 72% as compared to non-ischemic control hearts was reported in rat hearts subjected to 30 min of global ischemia followed by 60 min of reperfusion. In contrast, the PLB protein content in the I/R hearts was only slightly decreased by 14% as compared to control [41] which is in accordance with our findings. We found an apparently unaltered protein content of PLB in I/R hearts compared to control. This significant higher PLB/SERCA ratio in I/R could have been involved in the I/R-specific action of trapidil.

At this point, it should be noted that there are other potential targets for PKAII in addition to PLB, i.e. substrates that after phosphorylation by PKAII might contribute to the reduced ischemic injury in trapidil-treated hearts. The phosphorylase kinase/glycogen phosphorylase cascade is one of them, eventually providing the ischemic heart with additional substrate for glycolytic ATP production [42,43]. However, in this study we only wanted to show that enhanced PKAII activity by trapidil has functional consequences and have selected reduced PLB-phosphorylation, a generally accepted marker for ischemic injury.

The coronary perfusion pressure shows a time-dependent increase in all experimental groups, independent from ischemia and trapidil application. This is a common feature of all in vitro, buffer-perfused Langendorff-hearts under conditions of constant flow perfusion. This change in CPP has no consequences for myocardial contractile function as confirmed by a missing time-dependent decrease in {Delta}LVP. There were no trapidil-related changes in coronary perfusion pressure in the examined Langendorff-hearts, suggesting that the cardioprotective effects of the compound are independent of changes in coronary perfusion. The occasionally described vasodilation by trapidil at higher concentrations appears to be due to inhibition of PDE, an effect requiring concentrations of ≥300 µM which were not used here [3,44].

In conclusion, trapidil is a potent cardioprotective agent in I/R. The substance selectively increases the activity of PKAII isoforms probably due to a cAMP-sensitizing activity. This property discriminates the substance from catecholamines which induce a rather non-selective activation of PKA via increase in cytosolic cAMP concentration. Furthermore, we demonstrated that PLB is a target of trapidil-mediated PKA activation and that the improved PKA-mediated PLB phosphorylation, possibly via an enhanced Ca2+ uptake, might contribute to cardioprotection in I/R hearts. This might also explain protective effects of trapidil in myocardial ischemia in vivo [45,46].

Time for primary review 21 days.


   Acknowledgements
 
The authors like to thank Dr Stephan Drewianka (Biaffin GmbH & Co. KG) for providing recombinant proteins and Dr A.-A. Weber for helpful discussions and comments. The technical assistance of Daniela Moll and the excellent secretarial support of Erika Lohmann are gratefully acknowledged. The study was supported by the Forschungsgruppe Herz-Kreislauf e.V. (Düsseldorf) and the Deutsche Forschungsgemeinschaft (SFB 612, A6; SFB 394, B4 He 1818/4).


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

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