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Cardiovascular Research 2006 71(2):352-362; doi:10.1016/j.cardiores.2006.02.004
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Copyright © 2006, European Society of Cardiology

Contribution of PI 3-kinase isoforms to angiotensin II- and {alpha}-adrenoceptor-mediated signalling pathways in cardiomyocytes

Sibylle Wenzel*, Yaser Abdallah, Simone Helmig, Claudia Schäfer, Hans Michael Piper and Klaus-Dieter Schlüter

Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, D-35392 Giessen, Germany

* Corresponding author. Tel.: +49 641 99 47 255; fax: +49 641 99 47 219. Email address: sibylle.wenzel{at}physiologie.med.uni-giessen.de

Received 22 July 2005; revised 1 February 2006; accepted 3 February 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective Angiotensin II stimulation increases the formation of reactive oxygen species (ROS), the phosphorylation of p38 mitogen-activated protein kinase (MAPK), and the expression of transforming growth factor beta (TGFβ) in adult cardiomyocytes. The aim of this study was to determine the involvement of PI 3-kinase and to specify the participation of different isoforms in the angiotensin II-induced formation of ROS in comparison to the hypertrophic pathway triggered by {alpha}-adrenoceptor stimulation.

Methods Freshly isolated myocytes were used to examine formation of ROS via H2DCF fluorescence. p38 MAPK phosphorylation, p70S6-kinase phosphorylation, PI 3-kinase, and TGFβ expression were measured by Western blotting. Sense and antisense oligonucleotides were used to down-regulate diverse PI 3-kinase isoforms. Hypertrophy was measured by 14C-phenylalanine incorporation and cell volume.

Results Inhibition of PI 3-kinase by Ly294002 or wortmannin, two inhibitors, decreased formation of ROS, phosphorylation of p38 MAPK, and TGFβ expression. Down-regulation of the p110β isoform by antisense oligonucleotides inhibited the angiotensin II-induced signalling pathway but not the {alpha}-adrenoceptor-mediated hypertrophic growth of cardiomyocytes. In contrast, down-regulation of the p110{alpha} isoform decreased the {alpha}-adrenoceptor-mediated hypertrophic growth of cardiomyocytes but did not affect the angiotensin II-mediated signalling pathway.

Conclusion Thus, our study identifies an involvement of PI 3-kinase in the angiotensin II-induced formation of ROS and provides a biochemical basis for ligand-specific responses for angiotensin II and {alpha}-adrenoceptor stimulation as relates to hypertrophy.

KEYWORDS Reactive oxygen species; p38 MAPK; TGFβ; Antisense oligonucleotides; Hypertrophy


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiac hypertrophy is not only a functionally useful adaptation to an enhanced workload [1], but also one of the most critical clinical complications of cardiovascular disorders [2]. Nevertheless, the cellular mechanisms leading to myocardial hypertrophy are incompletely understood. To date, a number of in vivo animal models have been established to examine the role of an activated renin–angiotensin system in cardiac hypertrophy and heart failure. For example, treatment of stroke-prone spontaneously hypertensive rats with an angiotensin converting enzyme inhibitor (ACE inhibitor) or angiotensin II type1 (AT1 blocker) receptor blocker decreased elevated TGFβexpression [3] and prolonged survival rates, which indicates a dependency between the angiotensin II and TGFβpathways. These in vivo data have been verified in vitro by the use of isolated adult ventricular cardiomyocytes. Our own data demonstrated that angiotensin II is able to increase TGFβexpression in single cardiomyocytes. Formation of radicals by activation of NAD(P)H oxidase and phosphorylation of p38 mitogen-activated protein kinase (MAPK) seem to be key steps in this pathway [4]. Nevertheless, much less is known about the initial steps in this signalling pathway that leads to an activation of NAD(P)H oxidase.

Other cell systems and signalling pathways utilise PI 3-kinase as a signalling molecule for reactive oxygen species (ROS) formation [5]. Furthermore, it is known that angiotensin II directly increases PI 3-kinase activity in embryonic chicken cardiomyocytes [6]. Therefore, we were specifically interested in the question of whether the angiotensin II-dependent formation of ROS is also mediated via induction of the PI 3-kinase pathway in adult ventricular cardiomyocytes. Beyond this, PI 3-kinase participates in many different signalling pathways leading to a multiplicity of end points [7,8]. Important for the aim of this study was the identification of PI 3-kinase as a signalling molecule in another heart-specific signalling pathway that involves coupling between G-proteins and receptors. In adult ventricular cardiomyocytes, stimulation of {alpha}-adrenoceptors with phenylephrine leads to PI 3-kinase activation, p70S6-kinase phosphorylation, and marked hypertrophic growth of cells [9]. Nevertheless, angiotensin II increases protein synthesis in a less efficient way in adult cardiomyocytes in comparison to phenylephrine stimulation [10]. We hypothesised that an isoform-specificity of PI 3-kinase may be responsible for the ligand-specific differences.

The class I PI 3-kinase isoform is the best known isoform [7]. This class can be subdivided into two groups (Ia and Ib) based on structure. All members possess regulatory (p85, p101) and catalytic (p110) subunits [7]. The p85 regulatory subunit of the class Ia isoform is expressed in cardiomyocytes [11]. Less is known about the expression of the p101 regulatory subunit of the class Ib isoform. All members of the class I isoform have a p110 catalytic subunit [7]. This subunit is classified into {alpha}, β, {delta}, and {gamma} isoforms of p110. For this reason, we dealt with the question of whether there is any difference in requirements for diverse p110 catalytic subunits of PI 3-kinase that could be responsible for the different downstream signalling pathways between angiotensin II and {alpha}-adrenoceptor stimulation.

In summary, our study investigated whether the PI 3-kinase pathway is involved in the angiotensin II-mediated formation of radicals and examined the involvement of an idiosyncratic PI 3-kinase isoform in adult ventricular cardiomyocytes.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
All animal studies were performed in accordance with guidelines described in the NIH Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH, publication no. 85-23, revised 1996).

2.1 Cell isolation, short-term cultures
Ventricular cardiomyocytes were isolated from 200 to 250 g male Wistar rats, suspended in basal culture medium, and plated on 60 mm culture dishes as described in detail in Ref. [12]. The culture dishes had been pre-incubated overnight with medium 199 containing 4% (v/v) foetal calf serum (FCS). The basal culture medium (CCT) was modified medium 199 including Earl's salts, 2 mM L-carnitine, 5 mM taurine, 100 IU/ml penicillin and 100 µg/ml streptomycin. To prevent growth of nonmyocytes, media were also supplemented with 10 µM cytosine-β-D-arabinofuranoside (pH 7.4).

Cultures were washed twice with CCT medium after 4 h of plating. As a result of the medium change, broken cells were removed. This resulted in cultures of 93±2% quiescent rod-shaped cells. These cells were stimulated directly.

2.2 SDS-gel electrophoresis
After the particular stimulation, cells were lysed in lysis buffer [composition: 50 mmol/l Tris/HCl, pH 6.7, 2% (w/v) SDS, 2% (v/v) mercaptoethanol, and 1 mmol/l sodium orthovanadate]. Afterwards, nucleic acids were digested with benzonase (Merck, Darmstadt, Germany) [13]. Protein extracts (100 µg) were loaded on a 12.5% (w/v) SDS-gel (acryl amide:bisacryl amide 30:1). After electrophoresis, proteins were transferred onto reinforced nitrocellulose by semi-dry blotting. The sheets were saturated with 2% (w/v) bovine serum albumin and incubated for 2 h with the diverse polyclonal primary antibodies. After washing, the sheets were re-incubated with an alkaline phosphatase-labelled secondary antibody. Finally, bands were visualised by alkaline phosphatase activity using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. p38 MAPK activation as well as p70S6-kinase phosphorylation were determined by a ratio of the amount of phosphorylated protein to the total amount of that protein. Therefore, two blots were performed and two different primary antibodies were used.

2.3 Measurement of reactive oxygen species (ROS)
ROS generation in cells was assessed using the probe 2,7-dichlorofluorescein (DCF) (Molecular Probes). The membrane-permeable diacetate form of the dye (reduced DCF, DCF-DA) was added to the culture medium with a final concentration of 10 µM. Within the cell, esterases cleave the acetate groups on DCFH diacetate, thus trapping the reduced probe (DCFH) intracellularly. ROS in the cells oxidize DCFH, yielding the fluorescent product DCF. Fluorescence intensity was measured in up to ten different cells per preparation, and background was identified as an area without cells. Fluorescence was analysed using a fluorescence microscope combined with a video imaging system (T.I.L.L. Photonics). Cardiomyocytes, cultured on round glass coverslips, were incubated (30 min) in Hepes-buffered medium containing H2DCF-DA.

2.4 RT-PCR
Total RNA from cardiomyocytes was extracted with RNA-Clean (AGS, Heidelberg, Germany) as described by the manufactures. Reverse transcription reactions were performed for 1 h at 37 °C in a final volume of 10 µl RNA, 100 ng oligo(dT) (Boehringer Mannheim, Germany), 1 mM dNTPs (Gibxo-BRL), 8 U RNAse Block (Promega, Mannheim, Germany), and 60 U M-MLV reverse transcriptase (Gibco-BRL). Aliquots (1.5 µl) of the synthesised cDNA were used for polymerase chain reaction in a final volume of 10 µl, containing 10 µl of 10xPCR-Buffer, 5.8 µl of a. b., 3 µl of primer pairs (100 µM), 0.4 µl dNTPs (10 mM), 0.15 µl MgCL2 (50 mM), 0.5 µl 1% W1, and 0.2 µl Taq-polymerase (Gibco-BLR, 5 U/µl). Amplification was performed under the following cycle conditions: 1 min 93 °C, 1 min 60 °C, and 3 min 72 °C. Oligonucleotide primers had the following sequences: β-actin forward: 5'-GAAGTGTGACGTTGACATCCG-3', reverse: 5'-TGCTGATCCACATCTGCTGG-3'; p110{alpha} forward: 5'-GGTGACTGTGTGGGACT TATTG-3', reverse: 5'-ATGTAGTGTGTGGCTGTTGAAC-3'; p110β forward: 5'-TGTGCCCTCTCCAGATTCC-3', reverse: 5'-GACAGTATGCCTCTAGGATGAC-3'; p110{delta} forward: 5'-TTCCACGGCAATGAGATG-3', reverse: 5'-CTTCTCCACGACAGCATAG-3'; p110{gamma} forward: 5'-TCTGGTTCTTGCGAAGTGAG-3', reverse: 5'-GCTGCGTGAAGTCCTGTAG-3'. After amplification, reaction products were separated on a 2% agarose gel, stained with ethidium bromide, and photographed under UV illumination. As a loading control, β-actin was additionally amplified. As a negative control the reaction mixture was run without cDNA. All primers were purchased from Invitrogen.

2.5 Incorporation of 14C-phenylalanine
To measure the rate of protein synthesis, the incorporation of 14C-phenylalanine was determined by exposing cultures to L-14C-phenylalanine (0.1 µCi/ml) for 24 h. Incorporation of radioactivity into acid-insoluble cell mass was determined as described before [14].

2.6 Determination of cell volume
Myocyte growth was determined on phase-contrast micrographs recorded on tape using a CCD-video camera. Cell volume was determined by the following formula: cell volume=(radius)2*{pi}*length. To calculate radius of cardiomyocytes the widest point of the cells was measured and half of that value was used.

2.7 Antisense experiments
In order to down-regulate the expression of the PI 3-kinase isoforms in adult ventricular cardiomyocytes, cells were incubated for 24 h with 10 µg/ml phosphorothioated antisense or sense oligonucleotides in 20-mer lengths that corresponded to the region of the translation initiation site of the proteins. These are: PI 3-kinase p110{alpha} sense: TEF ZEA TGG TCT TGG AEE OFT; antisense: AZE OOT CCA AGA CCA TOF ZOA; PI 3-kinase p110β sense: TZZ EZC AAG TCG ATG OFE OT; antisense: AEO ZEC ATC GAC TTG FOF FA; PI 3-kinase p110{gamma} sense: TFO FZC CGG GAT GCC OZO FT; antisense: AZE FEG GCA TCC CGG FZE ZA; PI 3-kinase p110{delta} sense: AFF EOA ACA TGG CGG OOF OA; antisense: TEZ EEC CGC CAT GTT EOZ ZT. To assess transfection of cardiomoycytes by oligonucleotides, FITC-labelled oligonucleotides were directly added to the medium and fluorescence was measured by 484 nm (Fig. 1). All oligonucleotides were purchased from Invitrogen.


Figure 1
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  		Fig. 1 Verification of the uptake of oligonulceotides (20-mer length) by cardiomyocytes. The uptake of oligonucleotides by cardiomyocytes was controlled by the use of FITC-labelled oligonucleotides. Cells were either cultured under control conditions (C) or trasfected with non-labelled oligonuclotides (oligo, 10 µg/ml) or with FITC-labelled oligonucleotides (oligo-FITC, 10 µg/ml) for 24 h. Afterwards the uptake was visualised by fluorescence microscopy at a wavelength of 484 nm.

 
2.8 Statistics
Data are given as means±s.e.m. from n different culture preparations or single cells. Statistical comparisons were performed by one-way analysis of variance and use of the Student–Newman–Keuls test for post hoc analysis. In cases in which two groups were compared, conventional t-tests were performed. Differences with p<0.05 were regarded as statistically significant. All data analyses were performed using SPSS software, version 11.5.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Influence of PI 3-kinase inhibition on the angiotensin II-induced increase in formation of radicals, phosphorylation of p38 MAPK, and expression of TGFβ
The present study investigated whether PI 3-kinase is involved in angiotensin II-mediated NAD(P)H oxidase activation and downstream mechanisms such as p38 MAPK phosphorylation and TGFβexpression. In order to examine the influence of PI 3-kinase on the aforementioned signalling cascade, we used Ly294002 (Ly, 100 µM) or wortmannin (Wort, 100 nM), two chemically unrelated inhibitors of PI 3-kinase with effects at different sites. Pre-treatment of cardiomyocytes for 15 min with both inhibitors reduced the angiotensin II-induced formation of radicals (Fig. 2A). Inhibition of PI 3-kinase with Ly294002 and wortmannin also blocked the angiotensin II-induced phosphorylation of p38 MAPK (Fig. 2B) and TGFβexpression (Fig. 2C). In all experiments the inhibitors alone had no influence on basal ROS formation, p38 MAPK phosphorylation, or TGFβ expression (Fig. 2A–C).


Figure 2
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  		Fig. 2 Effect of PI 3-kinase inhibition on the angiotensin II-mediated signalling pathway. A) ROS formation: production of radicals was determined via DCF fluorescence. Representative fluorescence images are given. Production of radicals was measured 15 min under untreated test conditions. After pre-incubation with Ly294002 (Ly: 100 µM) or wortmannin (Wort: 100 nM) lasting 15 min, angiotensin II (Ang)-induced formation of radicals was measured for 30 min. Data are means±s.e.m. from n=35 different cells of six different preparations; * = p<0.05 vs. control; # = p<0.05 vs. angiotensin II. B) p38 MAPK phosphorylation: p38 MAPK activation was determined by the ratio of phosphorylated (p38p) to non-phosphorylated p38 MAPK (p38) after an incubation time of 45 min. Cells were stimulated with angiotensin II (Ang: 100 nM), angiotensin II plus Ly294002 (Ly: 100 µM) or angiotensin II plus wortmannin (Wort: 100 nM). Representative Western blots are shown. Data are means±s.e.m. from n=5 cultures; * = p<0.05 vs. control; # = p<0.05 vs. angiotensin II. C) TGFβ expression: TGFβexpression was determined by Western blotting after 24 h of angiotensin II incubation. Cells were stimulated with angiotensin II (Ang: 100 nM), angiotensin II plus Ly294002 (Ly: 100 µM) or angiotensin II plus wortmannin (Wort: 100 nM). TGFβvalues were normalised to β-actin values. Data are means±s.e.m. from n=5 cultures; * = p<0.05 vs. control; # = p<0.05 vs. angiotensin II. Two representative Western blots are additionally shown. In all three cases, the two inhibitors alone had no effect on the examined parameters.

 
3.2 Expression of PI 3-kinase isoforms in cardiomyocytes
Besides investigating the participation of PI 3-kinase in the angiotensin II-induced signalling pathway, this study examined the role of diverse PI 3-kinase isoforms in the aforementioned pathway. Therefore, we focused on the class I PI 3-kinase isoforms, because they are widely distributed and well known. In order to determine whether the four p110 subunit isoforms ({alpha}, β, {gamma}, or {delta}) are expressed in cardiomyocytes, primers for RT-PCR were first created. The products were amplified at a temperature of 60 °C. Specific bands were visualised in the expected amount. As a control, β-actin was additionally examined. Second, the expression of the four isoforms in myocytes was determined with isoform-specific antibodies by SDS-gel electrophoresis and Western blotting. Fig. 3 demonstrates the expression of all four subunits of PI 3-kinase on an mRNA level as well as on a protein level in cardiomyocytes.


Figure 3
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  		Fig. 3 Determination of mRNA and protein expression of the class I PI 3-kinase isoforms (p110{alpha}, β, {gamma}, and {delta}) by RT-PCR or Western blotting. For RT-PCR, products were amplified at a temperature of 60 °C. Protein bands of the four isoforms were visualised with isoform-specific antibodies. As a loading control, β-actin is additionally shown. All four isoforms are expressed in cardiomyocytes (CMC: 1, 2) on the mRNA and protein level. Representative parts of PCR agarose-gels and SDS-gels are shown.

 
3.3 Impact of different PI 3-kinase isoform antisense oligonucleotides on total and isoform-specific PI 3-kinase expression
Sense and antisense oligonucleotides were created against p110{alpha}, p110β, p110{gamma}, and p110{delta} isoforms. In order to test the efficiency of these oligonucleotides, we transfected cells with 10 µg/ml of each oligonucleotide for 24 h and subsequently determined total PI 3-kinase expression with a pan-specific PI 3-kinase antibody. Only antisense oligonucleotides against the isoforms p110{alpha} and p110β were able to down-regulate the total PI 3-kinase expression significantly (by up to 30%). Sense oligonucleotides were used as control and had no effects (Fig. 4A). Antisense oligonucleotides against p110{gamma} and p110{delta}showed no effect on total p110 PI 3-kinase expression within the time period of 24 h of transfection. To exclude unequal protein loading, PI 3-kinase values were normalised to β-actin values as an internal loading control. Significant effects of down-regulation of p110{alpha} and p110β could also be replicated by the use of isoform-specific antibodies (Fig. 4B).


Figure 4
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  		Fig. 4 Down-regulation of PI 3-kinase with antisense oligonucleotides against the diverse PI 3-kinase isoforms. A) On total PI 3-kinase level: quantitative analysis of Western blots are shown. Cells were transfected with sense (S, 10 µg/ml) or antisense (As, 10 µg/ml) oligonucleotides directed against one out of four isoforms as indicated for 24 h. Data are means±s.e.m. using a non-isoform-specific p110 antibody from n=6 cultures; # = p<0.05 vs. the particular sense control. Four representative parts of Western blots for down-regulation of p110{alpha} and p110β are shown. B) On isoform-specific PI 3-kinase protein level: representative parts of Western blots are given on the diverse isoform-specific PI 3-kinase protein level ({alpha}-, β-, {gamma}-, and {delta}-isoform-specific antibody). Cells were transfected with sense (S, 10 µg/ml) or antisense (As, 10 µg/ml) oligonucleotides directed against one out of four isoforms as indicated for 24 h. Afterwards down-regulation by antisense oliognucleotides was visualised with isoform-specific antibodies. Only antisense olionucleotides against p110{alpha} and p110β were able to down-regulate their respective PI 3-kinase isoform. Sense oligonucleotides were used as control. Antisense oligonucleotides against p110{gamma} and p110{delta} had no effect on p110{gamma} or p110{delta}protein level, respectively.

 
3.4 The role of p110β on angiotensin II-induced ROS formation
Using the antisense technique we now had a tool to down-regulate the PI 3-kinase isoforms p110{alpha} and p110β. Transfection of cardiomyocytes with antisense oligonucleotides (10 µg/ml) against PI 3-kinase p110β for 24 h reduced the angiotensin II-induced increase in ROS formation (after 30 min of angiotensin II stimulation) significantly. Sense oligonucleotides were used as control and did not show any effect. In addition, neither antisense oligonucleotides nor sense oligonucleotides against p110{alpha} had any effect on angiotensin II-induced ROS formation (Fig. 5A).


Figure 5
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  		Fig. 5 Effects of down-regulation of p110{alpha} and p110β on the angiotensin II-induced signalling pathway. A) ROS formation: influence of transfection of cells with sense and antisense oligonucleotides against the PI 3-kinase isoform p110β and p110{alpha} on angiotensin II (Ang: 100 nM)-induced ROS formation. Sense oligonucleotides (S) served as control. Cells were transfected with these oligonucleotides (10 µg/ml) for 24 h. Afterwards they were stimulated with angiotensin II. Representative fluorescence images are shown for ROS formation 30 min after angiotensin II stimulation. Only antisense oligonucleotides (As) against p110β were able to block the angiotensin II-induced effect. Data are means±s.e.m. from n=48 different cells of eight different preparations; * = p<0.05 vs. control; # = p<0.05 vs. angiotensin II or vs. angiotensin II plus sense oligonucleotides against p110β. B) p38 MAPK phosphorylation: influence of transfection of cells with sense (S) and antisense oligonucleotides (As) against the PI 3-kinase isoforms p110β and p110{alpha} on angiotensin II (Ang: 100 nM)-induced p38 MAPK phosphorylation. Sense oligonucleotides served as control. Cells were transfected with these oligonucleotides (10 µg/ml) for 24 h. Afterwards, they were stimulated with angiotensin II for 45 min. p38 MAPK activation was determined by the ratio of phosphorylated (p38p) to non-phosphorylated (p38) p38 MAPK. Only antisense oligonucleotides against p110β reduced the angiotensin II-induced p38 MAPK phosphorylation. Four representative Western blots are shown. Data are means±s.e.m. from n=5 cultures; # = p<0.05 vs. angiotensin II plus sense oligonucleotides against p110β. C) TGFβ expression: Influence of transfection of cells with sense (S) and antisense oligonucleotides (As) against the PI 3-kinase isoform p110β and p110{alpha} on angiotensin II (Ang: 100 nM)-induced TGFβ expression. Sense oligonucleotides served as control. Two representative Western blots for the effect of p110{alpha} and p110β are shown. Cells were transfected with these oligonucleotides (10 µg/ml) for 24 h. TGFβ expression was measured 24 h after angiotensin II stimulation and values were normalised to β-actin values. Data are means±s.e.m. from n=6 cultures; * = p<0.05 vs. control; # = p<0.05 vs. angiotensin II or vs. angiotensin II plus sense oligonucleotides against p110β.

 
3.5 The role of p110β on angiotensin II-induced p38 MAPK phosphorylation
To investigate whether the p110β isoform is involved in downstream elements of the angiotensin II-dependent signalling pathway, experiments concerning p38 MAPK phosphorylation were performed. Thus, we transfected cells with antisense oligonucleotides (10 µg/ml) against p110β for 24 h and additionally stimulated with angiotensin II for 45 min. As a control, cells were also transfected with sense oligonucleotides. Only antisense oligonucleotides directed against the p110β isoform were able to reduce the angiotensin II-induced phosphorylation of p38 MAPK. Transfection of cardiomyocytes with antisense or sense oligonucleotides (10 µg/ml) against PI 3-kinase p110{alpha} for 24 h also had no effect on the angiotensin II-induced increase in phosphorylation of p38 MAPK (Fig. 5B).

3.6 The role of p110β on angiotensin II-induced TGFβ expression
In order to examine the influence of down-regulation of p110β on the final step of the angiotensin II-induced signalling pathway, cells were transfected with antisense oligonucleotides (10 µg/ml) against PI 3-kinase p110β for 24 h, and 24 h later TGFβ expression was measured. This procedure reduced TGFβ expression significantly. Sense oligonucleotides were used as control and did not show any effect. Furthermore, neither antisense oligonucleotides nor sense oligonucleotides against p110{alpha} had any effect on angiotensin II-induced TGFβ expression (Fig. 5C).

3.7 The role of p110{alpha} on {alpha}-adrenoceptor-mediated increase in p70S6-kinase phosphorylation
In order to determine which PI 3-kinase isoform is involved {alpha}-adrenoceptor-mediated hypertrophic growth, cells were transfected with sense and antisense oligonucleotides (10 µg/ml) against p110β and p110{alpha} for 24 h, and adrenoceptors were additionally stimulated with norepinephrine (1 µM). As norepinephrine is a agonist of both {alpha}- and β-adrenoceptors, β-adrenoceptor stimulation was blocked simultaneously by using the selective β-adrenoceptor antagonist atenolol (10 µM). {alpha}-Adrenoceptor stimulation for 30 min increased the phosphorylation of p70S6-kinase, a signalling molecule involved in the {alpha}-adrenoceptor-mediated pathway. Transfection of cells with antisense oligonucleotides against p110{alpha} was able to block the {alpha}-adrenoceptor-mediated effect. Sense oligonucleotides for p110{alpha} were used as control and had no effect. Neither sense nor antisense oligonucleotides against p110β were able to reduce the phosphorylation of p70S6-kinase (Fig. 6A).


Figure 6
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  		Fig. 6 Effects of down-regulation of p110{alpha} and p110β on the {alpha}-adrenoceptor-mediated signalling pathway. A) p70S6-kinase phosphorylation: influence of transfection of cells with p110{alpha} and p110βoligonucleotides on the amount of p70S6-kinase phosphorylation in the presence of norepinephrine (Nor, 1 µM) plus atenolol (At, 10 µM) for 30 min. Only transfection of cells with antisense (As) oligonucleotides against p110{alpha} (10 µg/ml) attenuates the increase in p70S6-kinase phosphorylation after {alpha}-adrenoceptor stimulation for 24 h. Sense (S) oligonucleotides against p110{alpha} as well as antisense and sense oligonucleotides against the isoform p110β (10 µg/ml) had no effect. Data are means±s.e.m. from n=5 cultures, * = p<0.05 vs. control; # = p<0.05 vs. norepinephrine plus atenolol or vs. norepinephrine plus atenolol plus sense oligonucleotides against p110{alpha}. B) 14C-Phenylalanine incorporation: Influence of transfection of cells with p110{alpha} and p110β oligonucleotides on norepinephrine (Nor, 1 µM) plus atenolol (At, 10 µM)-induced amount of protein synthesis. Only transfection of cells with antisense (As) oligonucleotides against p110{alpha} (10 µg/ml) attenuates the increase in cell mass after {alpha}-adrenoceptor stimulation for 24 h. Sense (S) oligonucleotides against p110{alpha} as well as antisense and sense oligonucleotides against the isoform p110β (10 µg/ml) had no effect. Data are means±s.e.m. from n=6 cultures, * = p<0.05 vs. control; # = p<0.05 vs. norepinephrine plus atenolol or norepinephrine plus atenolol plus sense oligonucleotides against p110{alpha}. C) Cell volume: influence of norepinephrine (Nor, 1 µM) plus atenolol (At, 10 µM) on cell volume. Transfection of cells with antisense oligonucleotides against p110{alpha} attenuates the increase in cell mass after {alpha}-adrenoceptor stimulation for 24 h. Sense oligonucleotides against p110{alpha} as well as sense and antisense oligonucleotides against p110β had no effect. Data are means±s.e.m. from n=84 different cells of 3 different preparations; * = p<0.05 vs. control, # = p<0.05 vs. norepinephrine plus atenolol alone or vs. norepinephrine plus atenolol plus sense oligonucleotides against p110{alpha}.

 
3.8 The role of p110{alpha} in {alpha}-adrenoceptor-mediated increase in hypertrophic growth
In the same set of experiments, transfection of cells with antisense oligonucleotides (10 µg/ml) against the p110{alpha} isoform attenuated the {alpha}-adrenoceptor-induced hypertrophic growth of cardiomyocytes. The hypertrophic growth was measured first by 14C-phenylalanine incorporation (Fig. 6B) and second by direct measurement of the cell volume (Fig. 6C) of myocytes. Both methods showed a significant increase in hypertrophic growth of cardiomyocytes by {alpha}-adrenoceptor stimulation and a decrease after transfection with antisense oligonucleotides against p110{alpha}. p110{alpha} sense oligonucleotides again had no effect (Fig. 6B, C). Neither sense nor antisense oligonucleotides against p110β had any effect on {alpha}-adrenoceptor-mediated hypertrophic growth (Fig. 6B, C).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Myocardial induction of TGFβ is of particular interest in cardiac biology because this cytokine plays a crucial role in the transition from compensated to de-compensated hypertrophy. In addition, it is known that TGFβ modulates other signalling pathways like that of the β-adrenoceptor [15]. Previous studies demonstrated that the expression of TGFβ is directly mediated by angiotensin II in vivo and in vitro [4]. In order to improve treatment protocols to block the transition of hypertrophy to heart failure, it is important to completely understand the intracellular signalling steps necessary for the angiotensin II-mediated effects on ventricular cardiomyocytes. Earlier studies by our laboratory detected important molecules involved: for example, ROS generated by NAD(P)H oxidase, activation of p38 MAPK, protein kinase C, and the transcription factor AP1 [4]. This study investigated the mechanisms by which angiotensin II activates the formation of ROS through NAD(P)H oxidase in adult ventricular cardiomyocytes with the aim of deciphering the mechanism by which G-protein-coupled intracellular signalling pathways lead to different endpoints while utilizing the same molecules.

Our data clearly implicate PI 3-kinase activation in angiotensin II-induced ROS generation. These data confirm previous experiments on a variety of different cells, including vascular smooth muscle cells [16]. The involvement of PI 3-kinase in the formation of radicals is not consistent for different tissues or cells. There are different isoforms of NAD(P)H oxidases known. The common theory on phagocyte oxygen radical production states that the NAD(P)H oxidase is assembled in the plasma membrane, releasing superoxide and hydrogen peroxide into the extracellular milieu. In contrast, smooth muscle cells, cardiomyocytes, and stem cells, for example, exhibit a type of NAD(P)H oxidase that releases radicals inside the cells. In macrophages (radicals outside), ROS formation is not inhibited by wortmannin, an inhibitor of PI 3-kinase [17]. There seems to be a PI 3-kinase-dependent intracellular release of radicals, whereas the extracellular release is independent of PI 3-kinase [18]. Such a mechanism is consisted with our findings that PI 3-kinase regulates those NAD(P)H oxidases (the smooth muscle types) that release radicals inside the cells, as is the case for cardiomyocytes.

As PI 3-kinase is a multifunctional molecule involved in various signalling pathways, the question arises whether there is any isoform-specificity for the angiotensin II-mediated pathway and the {alpha}-adrenoceptor-mediated pathway, where PI 3-kinase is also involved [13]. In the first pathway, PI 3-kinase activation leads to a ROS-dependent activation of p38 MAPK, and in the second pathway, it is part of a strong hypertrophic growth response that is p38 MAPK independent [9]. In the literature there are some indications from knockout and transgenic studies for the functional specialization of class I isoforms. Knockout of p110{alpha} and p110β results in embryonic death [19,20]. p110{alpha} knockouts demonstrate complete failure of cell proliferation. This isoform has also been implicated in the regulation of cell size, with cardiac-specific expression of a constitutively active or dominant negative form of p110{alpha} resulting in mice with increased or decreased myocyte size, respectively. Other studies demonstrate an involvement of p110{alpha} in the physiological hypertrophy of knockout mice [21]. The isoform p110{gamma} was also demonstrated in knockout studies to have effects, generating mice with a compromised immune system and reduced inflammatory responses. Okkenhaug and Vanhaesebroeck [22] also reported an elegant study with p110{delta} knockout mice, showing impaired B- and T-cell responses and implicating a role for that isoform in immune responses. However, it must be noted that knockout studies can be complicated by alteration of expression of one subunit affecting the expression of others. Moreover, it seems difficult to compare rats and mice concerning some aspects of intracellular signalling [23]. In genetically hypertensive rats, for example, p110{delta} is associated with pathological hypertension and altered arterial hypercontractility [24]. Kessler et al. [11] postulated a role of p110β in diversification of cardiac insulin signalling. Because of these extremes, we used the antisense technique in order to investigate the significance of diverse PI 3-kinase isoforms in our model of cultured adult rat cardiomyocytes. Use of phosphorothioated antisense oligonucleotides is a common and effective way to down-regulate protein expression. Our own studies with radioactively labelled oligonucleotides agree with that from Miller and Das [25] and showed that 1 to 2% (100–200 ng) of the exogenously added oligonucleotides were taken up by cardiac myocytes, with a maximum after 16 h of incubation. The efficiency of transfection of cells with oligonucleotides was additionally checked by FITC-labelled oligonucleotides. This measured small quantity of antisense oligonucleotides directed against one out of four different isoforms expressed in cardiomyocytes is sufficient to significantly down-regulate total PI 3-kinase expression by up to 30%. Transfection of cells with antisense oligonucleotides against the different isoforms then allowed us to directly determine the involvement of the individual isoforms. Our findings indicated that the PI 3-kinase isoform p110β is directly involved in the angiotensin II-mediated signalling pathway, as opposed to the PI 3-kinase isoform p110{alpha}, which is involved in the hypertrophic growth of myocytes. In particular, p110β seems to be associated with G-protein-coupled receptors that consist of β/{gamma} subunits [26]. These data agree with those of Anderson and Jackson [7], who described a preferred involvement of p110{alpha} in cell growth and an involvement of p110β in other aspects of cell signalling.

4.1 Concluding remarks
In summary, our study describes for the first time PI 3-kinase p110β as an important intracellular molecule involved in the angiotensin II-mediated generation of radicals in adult cardiomyocytes. This pathway is one of the critical events in the transition from stable hypertrophy to heart failure. The initial step after stimulation of angiotensin II receptors is the induction of PI 3-kinase activity and of NAD(P)H oxidase. Furthermore, this study helps to clarify the question of why PI 3-kinase, a multifunctional enzyme, leads to hypertrophic growth in the case of {alpha}-adrenoceptor stimulation and to an induction of TGFβexpression the in case of angiotensin II stimulation. We were able to link the p110βisoform of PI 3-kinase to the angiotensin II-dependent pathway and the p110{alpha} isoform of PI 3-kinase to the {alpha}-adrenoceptor-dependent stimulation of cardiac hypertrophy and heart failure.


   Acknowledgements
 
The authors thank Nadine Lorenz and Daniela Schreiber for excellent technical assistance. Financial support of this study by the Deutsche Forschungsgemeinschaft grant Schl324/5-2, is gratefully acknowledged.


   Notes
 
Time for primary review 30 days

* Ajay Shah, King's College, London, served as Guest Editor for this article.


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

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