Effects of cerivastatin on adrenergic pathways, hypertrophic growth and TGFbeta expression in adult ventricular cardiomyocytes

European Journal of Cell Biology 91 (2012) 367–374

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Effects of cerivastatin on adrenergic pathways, hypertrophic growth and TGFbeta expression in adult ventricular cardiomyocytes Hagen Maxeiner b , Yaser Abdallah a , Christoph Rüdiger Wolfram Kuhlmann c , Klaus-Dieter Schlüter a , Sibylle Wenzel a,∗ a

Institute of Physiology, Justus-Liebig-University, Giessen, Germany Department of Anaesthesiology, Intensive Care Medicine, Pain Therapy, University Hospital Giessen and Marburg, Germany c Institute of Physiology and Pathophysiology, Johannes Gutenberg-University, Mainz, Germany b

a r t i c l e

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Article history: Received 13 October 2010 Received in revised form 21 December 2011 Accepted 23 December 2011 Keywords: p38 MAP kinase Rac Isoprenaline Phenylephrine NAD(P)H oxidase

a b s t r a c t The effects of statin treatment in the setting of heart failure have already been shown. Nevertheless, there is little knowledge about its influence on adrenergic pathways in cardiomyocytes. Therefore, this study investigated the impact of cerivastatin on adrenoceptor-mediated signalling pathways in isolated adult ventricular cardiomyocytes. It focused on two endpoints: hypertrophic growth and TGFbeta expression. Cultured cardiomyocytes were used to study rac activation (analysed by its translocation into the membrane fraction), ROS formation (H2 DCF fluorescence) and hypertrophic growth (14 C-phenylalanine incorporation). Alpha- and beta-adrenoceptor stimulation showed significant differences regarding rac activation, ROS formation, and p38 MAP kinase activation. Both alpha- and beta-adrenoceptor stimulation induced TGFbeta expression. Upon activation of alpha-adrenergic signalling – although ROS formation was not influenced by cerivastatin – TGFbeta expression decreased. Following beta stimulation, TGFbeta expression as well as rac and p38 MAP kinase activation were reduced after pre-treatment with cerivastatin. Statin treatment did not show any influence on hypertrophic growth. In summary, this study clearly demonstrates the ability of adrenoceptor stimulation to increase TGFbeta expression. One component of the beneficial effects of statin therapy on heart failure might therefore be due to a dominant reduction and inhibition of TGFbeta, which is involved in many pathophysiological processes in cardiomyocytes. © 2012 Elsevier GmbH. All rights reserved.

Introduction The populations of industrialised nations are undergoing demographic changes. The age structure is shifting to a larger fraction of older people – e.g. due to progress in health care and nutrition. These structural changes have resulted in a growing incidence of people suffering from heart failure. Among many therapeutic approaches statin therapy has been shown to be of great benefit with few side effects in this clinical setting. The beneficial effects have been studied and confirmed on the cellular level in isolated cardiomyocytes. The present study investigates their influence on adrenergic pathways, which are known to lead to cardiac hypertrophy and cardiac failure. The effects of adrenergic stimulation of cardiomyocytes on hypertrophy and contractile function have already been well studied. Activation of the two receptor classes has different effects on isolated adult cardiomyocytes. Alpha-adrenoceptor stimulation strongly induces protein synthesis

∗ Corresponding author at: Physiologisches Institut, Aulweg 129, D-35392 Giessen, Germany. Tel.: +49 641 99 47 255; fax: +49 641 99 47 219. E-mail address: [email protected] (S. Wenzel). 0171-9335/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2011.12.006

but maintains cell function (Anwar et al., 2005). This effect is mediated by PKC isoform ␦, in vivo as well as in vitro (Bowman et al., 1997; Rohde et al., 2000; Ruf et al., 2002; Schafer et al., 2002). Above PKC activation the pro-hypertrophic signalling molecules PI3-kinase, AKT/PKB and the p70S6 kinase are involved (Sadoshima et al., 1995; Boluyt et al., 1997; Schluter et al., 1998). Selective betaadrenergic stimulation of adult cardiomyocytes does not induce protein synthesis in isolated cardiomyocytes but activates nuclear factor of activated T-cells (NFAT) and the calcineurin pathway (Anwar et al., 2005). In contrast to these well-established endpoints and pathways, almost no data exist concerning increased cytokine expression after either alpha- or beta-adrenoceptor stimulation alone. An increased induction of TGFbeta is of particular interest as this cytokine plays a crucial role in the transition from compensated to de-compensated hypertrophy (Boluyt et al., 1995). It is known that in isolated cardiomyocytes TGFbeta modulates other signalling like the beta-adrenoceptor-mediated pathway (Taimor et al., 2001), and induces apoptosis (Schroder et al., 2006), contractile dysfunction (Mufti et al., 2008), and the deposition of extracellular matrix proteins (Wenzel et al., 2010). But only one study by Lai et al. (2009) provides some indication that both alphaand beta-adrenoceptor stimulation increase TGFbeta expression in

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fibroblasts. Zhao et al. (2008) connected TGFbeta expression with ROS production in hypertensive rats. In contrast to these adrenergic pathways, it is well established that angiotensin II (used as a control pathway in this study) indeed increases TGFbeta expression 24 h after stimulation of cardiomyocytes via a radical-dependent pathway (Wenzel et al., 2001). Statins are known to interfere with a variety of signalling pathways in the heart. They are able to inhibit the translocation of the small GTPase rac to the cell membrane and therefore inhibit the activation of NAD(P)H oxidase. Conflicting data exist concerning the involvement of the small GTPase rac or reactive oxygen species (ROS) in the signalling pathways of adrenoceptors in cardiomyocytes. Xiao et al. (2002) found that specific stimulation of alpha1-adrenoceptors leads to a significant increase in oxidative stress related to NAD(P)H oxidase in adult cardiac myocytes. In accordance with that, transgenic mice over-expressing rac1 develop a moderate hypertrophy that can be abolished by the application of N-acetyl cysteine, a radical scavenger (Hassanain et al., 2007). Some data suggest the involvement of rac or ROS in beta-adrenergic signalling in the heart, e.g. the fact that statins inhibit the beta-adrenoceptor-induced increase in apoptosis in a rac-dependent way (Ito et al., 2004). Isoprenaline-induced beta-adrenergic stimulation increases BNP expression in neonatal cardiomyocytes, whereas a dominant negative mutant of rac abolishes these effects (He et al., 2000). Zhang et al. reported an increased generation of ROS and an activation of rac after isoprenaline infusion in rats, however, for these studies the whole heart tissue was analysed (Zhang et al., 2005) and therefore, the target cells of this effect were not defined. Simvastatin inhibits the noradrenaline-induced hypertrophy in neonatal cardiomyocytes, implicating an involvement of rac in this context (Luo et al., 2001). Moreover, rac activation seems to be required for MAP kinase activation and hypertrophy in neonatal cardiomyocytes (Pracyk et al., 1998; Clerk et al., 2001). Whether statins also reduce ROS generation in terminally differentiated cardiomyocytes remains vague. Thus, our study compared the effects of statins on the adrenoceptor-mediated signalling aimed at two endpoints: TGFbeta expression and the hypertrophic growth response of adult ventricular rat cardiomyocytes. As a positive control, the wellestablished angiotensin II-induced signalling pathway in adult cardiomyocytes and its interference with statins was additionally investigated. Methods 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). Cell isolation and 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 previously (Schluter and Schreiber, 2005). The culture dishes had been pre-incubated overnight with CCT medium containing 4% (v/v) foetal calf serum (FCS) and 2% (v/v) penicillin/streptomycin (100 UI/ml). The basal culture medium was modified medium 199 including Earl’s salts, 2 mM l-carnitine and 5 mM taurine. To prevent growth of non-myocytes media were also supplemented with 10 ␮M cytosine-␤-d-arabinofuranoside (pH 7.4). Cultures were washed twice with CCT medium 4 h after plating. The medium change removed broken cells, resulting in a culture of 93 ± 2% quiescent rod-shaped cells. These cells were

stimulated directly with the respective agonists (angiotensin II: 100 nM, phenylephrine: 1 ␮M, isoprenaline: 100 nM) or after pre-incubation with cerivastatin (10 ␮M) for 24 h. For concentration-dependent experiments the following concentrations were additionally used: isoprenaline 1 ␮M and 10 ␮M; phenylephrine 100 nM and 10 ␮M; cerivastatin 100 nM, 1 ␮M, 5 ␮M, and 10 ␮M (Figs. 2, 5, 7 and 8 of additional online supplements). RT-PCR Total RNA from cardiomyocytes, harvested 20 h after stimulation with the respective agonists, was extracted with RNA-Clean (AGS, Heidelberg, Germany) as described by the manufacture. Reverse transcription reactions and RT-PCR were performed as described previously (Wenzel et al., 2006). The detailed sequence for each primer is given in additional online material section. As a negative control, the reaction mixture was run without cDNA. HPRT was used as a housekeeping gene. All primers were purchased by Invitrogen. SDS-gel electrophoresis Protein extracts from adult ventricular cardiomyocytes were prepared for SDS-gel electrophoresis as described before (Schluter et al., 1999). Protein extracts (100 ␮g) were loaded on a 12% (w/v) or 15% (w/v) SDS-polyacrylamide gel (acryl amide:bisacryl amide 30:1). After electrophoresis, proteins were transferred onto a reinforced nitrocellulose membrane by semi-dry blotting. The membranes were saturated with 2% (w/v) bovine serum albumin and incubated for 2 h with a polyclonal first antibody. After washing, the membranes were re-incubated with an alkaline phosphatase-labeled second antibody. Finally, bands were visualised by alkaline phosphatase activity using 5-bromo-4chloro-3-indolyl phosphate and nitro blue tetrazolium. p38 MAP kinase activation was estimated by the ratio of the amount of phosphorylated p38 MAP kinase to the total amount of p38 MAP kinase. Therefore, two blots were performed and two different first antibodies were used. TGFbeta values were normalised to beta-actin values, and rac values to the total amount of protein (nanodrop technique) in the membrane fraction. To properly identify rac protein bands, rac protein was additionally added. The quantitative analysis of bands was performed by using Quantity One® . Detailed informations about the used antibodies are given in additional online material section. Measurement of reactive oxygen species Intracellular ROS generation was assessed using the fluorescent probe 2,7-dichlorofluorescein (DCF). The membrane-permeable diacetate form was added to the culture medium at a final concentration of 10 ␮M and incubated for 30 min. After penetration of the membrane, DFC was reduced (DCFH) and trapped intracellularly. ROS in the cells oxidise DCFH, yielding the fluorescent product DCF. Fluorescence intensity was measured in up to 8 different cells per preparation, and background was identified as an area without cells and subtracted from the signal. Fluorescence was analysed using a fluorescence microscope combined with a video imaging system (T.I.L.L. Photonics). Due to the prior handling process, the initial fluorescence intensity of individual culture dishes differs enormously. Therefore, DCF fluorescence of each examined culture dish was measured under untreated conditions for 15 min (0–15 min). Afterwards, the respective substances were added and fluorescence was measured for another 30 min (15–45 min). The increase in fluorescence intensity per minute during the stimulation time was compared to the change in fluorescence intensity

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radioactivity into acid-insoluble cell mass was determined as described previously (Pinson et al., 1993). Nanodrop technique The nanodrop technique was used to validate the total amount of proteins in the membrane and cytosolic fraction of cells. Two microliters of each sample were used to detect the protein amount at a wavelength of 280 nm. The individual amount of protein determined was used for normalisation of the respective rac values. The average protein amount of the membrane fraction of cells was 450 ␮g/ml and that of the cytosolic fraction 520 ␮g/ml. Statistics

Fig. 1. rac translocation. (A) Influence of angiotensin II (Ang: 100 nM) and adrenoceptor agonists (phenylephrine (PE): 1 ␮M; isoprenaline (ISO): 100 nM) on rac translocation to the cellular membrane (20 min after stimulation). The membrane fraction of cells was obtained using different centrifugation steps. Rac values were normalised to the total protein amount of the membrane fraction. Both angiotensin II and isoprenaline were able to increase rac translocation significantly. Alphaadrenergic stimulation with phenylephrine alone had no effect. Pre-incubation (24 h) of cardiomyoctes with the HMG-CoA reductase inhibitor cerivastatin totally blocked both the angiotensin II- and the isoprenaline-induced effects. Data are means ± s.e.m. from n = 8 cultures, * p < 0.05 vs. control; # p < 0.05 vs. angiotensin II or isoprenaline alone. (B) Representative western blots for the small GTPase rac of the membrane and cytosolic fraction of cells after stimulation with phenylephrine (PE): 1 ␮M; angiotensin II (Ang: 100 nM), and isoprenaline (ISO): 100 nM. Rac protein was additionally visualised as a positiv control of antibody specificity.

per minute during the first 15 min (0–15 min). Additionally, each experimental setup contained two controls (45 min without any treatment, e.g. panel C in Fig. 2). Here again, the first 15 min were compared to the following 30 min. The ratio of the increase in fluorescence intensity per minute between time point 15–45 min and time point 0–15 min of the controls (average of both) was set to 100%. Rac translocation As a measure of rac activation, its translocation to the membrane fraction was investigated. Cardiomyocytes were treated with angiotensin II, phenylephrine or isoprenaline for 10, 20, 30, 40, and 60 min with or without pre-treatment with cerivastatin (concentration–response curve for cerivastatin is given in Fig. 1, additional online supplements). Thereafter, the cultures were washed twice with ice-cold phosphate buffered solution (PBS), scraped off the dishes in lysis buffer (composition: NAHCO3 1 mM; MgCl2 5 mM and PMSF 100 ␮M), mixed vigorously and centrifuged for 5 min at 1000 × g at 4 ◦ C. The supernatant was again centrifuged for 20 min at 16,000 × g at 4 ◦ C. The remaining pellet was used as the membrane fraction and resuspended in 25 ␮l lysis buffer for western blotting. The remaining protein samples were used as described before (Schluter et al., 1999). As a positive control, different amounts of rac protein (rac1, Sigma) were additionally visualised. Incorporation of 14 C-phenylalanine The rate of protein synthesis as a marker of hypertrophic growth was assessed by determination of the incorporation of 14 C-phenylalanine. Therefore, the cultures were exposed to l14 C-phenylalanine (0.1 ␮Ci/ml) for 24 h. The incorporation of

Data are given as means ± S.E.M. from n different culture preparations or different cells, respectively. 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® , V. 17 for Windows® . Results Effect of cerivastatin on beta-adrenoceptor-induced rac translocation Rac, a cytosolic subunit of membrane-bound NAD(P)H oxidase, becomes activated by its translocation to the cell membrane and initiates the generation of ROS. As a measure of activation, the protein amount of rac in the membrane fraction of cardiomyocytes was determined by western blotting. Isoprenaline (100 nM) increased rac translocation to the membrane fraction with a significant maximum after 20 min (ISO: +53 ± 18%, p ≤ 0.05 vs. control, Figs. 1 and 3C of additional online supplements). In contrast, phenylephrine (1 ␮M) did not affect the amount of rac in the cell membrane compartment at all time points examined (10, 20, 30, 45, and 60 min, Figs. 1 and 3B of additional online supplements). As a positive control angiotensin II (100 nM) was additionally examined (Figs. 1 and 3A of additional online supplements). This AT1-receptor stimulation increased rac translocation significantly. Adding cerivastatin (10 ␮M) totally prevented the angiotensin IIinduced and beta-adrenoceptor-induced translocation of rac to the cell membrane (Fig. 1). Therefore, cerivastatin interfered with the angiotensin II- and beta-adrenoceptor-mediated rac translocation. Cerivastatin stimulation alone had no effect on rac translocation. Interestingly in contrast to angiotensin II, neither phenylephrine nor isoprenaline were able to initiate translocation of p67phox, a second important cytosolic subunit of NAD(P)H oxidase, to the cell membrane (Fig. 4 of additional online supplements). Effect of cerivastatin on the alpha-adrenoceptor-dependent ROS formation ROS formation was measured by H2 DCF fluorescence in isolated cardiomyocytes. Cells were incubated for 30 min with angiotensin II at a concentration of 100 nM, which was used as an internal positive control, with phenylephrine at a concentration of 1 ␮M, or with isoprenaline at a concentration of 100 nM (concentration–response curves for ISO and PE are given in Fig. 5 of additional online supplements). In contrast to isoprenaline stimulation, incubation with phenylephrine activated ROS formation (+58 ± 21%, p ≤ 0.05 vs. control, Fig. 2). Cerivastatin blocked only the angiotensin II-induced ROS formation but not ROS formation induced by

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Fig. 2. ROS formation. (A) Influence of angiotensin II (Ang: 100 nM) and adrenoceptors (phenylephrine (PE): 1 ␮M; isoprenaline (ISO): 100 nM) on the generation of ROS. Angiotensin II and phenylephrine increased ROS formation significantly after incubation for 30 min. Isoprenaline had no effect. Pre-incubation with cerivastatin only inhibited the angiotensin II-induced effect but not the phenylephrine-induced effect. Data are means ± s.e.m. from up to 8 different cell of n = 6 cultures, * p < 0.05 vs. control; # p < 0.05 vs. angiotensin II. (B) Representative 2,7 dichlorofluorescein (DCF)-staining (10 ␮M). ROS formation was visualised after angiotensin II and adrenegic stimulation. After stabilisation time for 15 min cells were stimulated with the respective agonist and ROS were measured for 30 min. The brighter the cells appear, the more ROS are present.

alpha-adrenergic stimulation. Cerivastatin treatment alone had no influence on ROS formation. Moreover, to further detect the source of ROS production, ROS production was blocked using different pharmacological inhibitors and oligonucleotides. Pre-incubation of cells with Apocinin (10 ␮M) a selective inhibitor of NAD(P)H oxidase, with NSC23766 (100 ␮M) an inhibitor of rac translocation, and transfection of cells with p22phox sense or antisense oligonucleotides (10 ␮g/ml) did not inhibit phenylephrineinduced ROS production. Only pre-incubation with DPI, an general

inhibitor of flavoenzymes, did slightly reduce PE-induced ROS production (Fig. 6 of additionall online supplements). In contrast, both DPI (Wenzel et al., 2001; Anwar et al., 2005) and NSC23766 as well as p22phox antisense oligonucleotides (Wenzel et al., 2001) were able to block angiotensin II-induced ROS production (Fig. 6 of additional online supplements). Therefore, the source of the alpha-adrenoceptor-dependent ROS production clearly differs from that of the angiotensin II-induced production. These data underline the differences between alpha-adrenoceptor and

Fig. 3. p38 MAP kinase phosphorylation. Influence of angiotensin II (Ang: 100 nM) and adrenoceptor agonists (phenylephrine (PE): 1 ␮M; isoprenaline (ISO): 100 nM) on p38 MAP kinase phosphorylation as an indication of activation. Angiotensin II and isoprenaline increased the amount of phosphorylated p38 MAP kinase after 20 min of incubation. In contrast, single alpha-adrenergic stimulation with phenylephrine was not able to increase activation of p38 MAP kinase. Pre-incubation (24 h) of cardiomyoctes with cerivastatin (10 ␮M) totally abolished both the angiotensin II and the isoprenaline-induced effects. The phosphorylated form of p38 MAP kinase was normalised to the total amount of the enzyme. Data are means ± s.e.m. from n = 5 cultures, * p < 0.05 vs. control; # p < 0.05 vs. angiotensin II or isoprenaline alone.

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Effect of cerivastatin on the beta-adrenoceptor-dependent p38 MAP kinase phosphorylation p38 MAP kinase is involved in the signalling pathways of angiotensin II (Wenzel et al., 2001) and beta-adrenoceptors (Wenzel et al., 2005) but not in alpha-adrenoceptor stimulation (Fig. 7 of additional online supplements), as indicated by the degree of p38 MAP kinase phosphorylation (Figs. 3 and 7 of additional online supplements). Both the angiotensin II- and the isoprenaline-induced p38 MAP kinase phosphorylation were abolished by pre-incubation of cells with cerivastatin (Figs. 3 and 2 of additional online supplements). Therefore although cerivastatin blocked rac activation and p38 MAP kinase phosphorylation in both pathways, ROS generation does not seem to be required in the isoprenaline-dependent activation. Cerivastatin stimulation alone had no influence on the amount of phosphorylated p38 MAP kinase (Fig. 3).

Effect of cerivastatin on the adrenoceptor-dependent TGF-beta mRNA expression Previews studies showed that angiotensin II (100 nM) stimulation of cardiomyocytes is able to increase TGFbeta mRNA expression after 20 h of incubation (Wenzel et al., 2001). We therefore investigated of whether adrenoceptor-induced stimulation is also able to increase TGFbeta mRNA expression. Both adrenergic agonists were able to increase TGFbeta mRNA expression significantly. Pre-incubation with cerivastatin blocked TGFbeta mRNA expression in all cases (Fig. 4).

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TGFbeta mRNA expression (x-fold)

angiotensin II receptor-induced signalling in adult ventricular cardiomyocytes.

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Fig. 4. TGFbeta mRNA expression. Influence of cerivastatin (10 ␮M) on the angiotensin II-induced (Ang: 100 nM) and adrenoceptor-induced (phenylephrine (PE): 1 ␮M; isoprenaline (ISO): 100 nM) increase in TGFbeta mRNA expression after incubation for 20 h. Pre-incubation of cardiomyocytes with cerivastatin (24 h) inhibited the increase in TGFbeta expression induced by all three agonists. Data are means ± s.e.m. from n = 5 cultures, * p < 0.05 vs. control; # p < 0.05 vs. angiotensin II, phenylephrine or isoprenaline alone.

Effect of cerivastatin on the adrenoceptor-dependent TGF-beta protein expression Due to the results on TGFbeta mRNA expression, we now investigated the effect of adrenergic agonists on TGFbeta protein expression (Figs. 5, 6 and 8 of additional online supplements). Incubation with phenylephrine (1 ␮M) increased TGFbeta expression significantly (+41% ± 25%; p ≤ 0.05 vs. control). Isoprenaline (100 nM) had a lesser but also significant effect on TGFbeta expression (+22% ± 15%; p ≤ 0.05 vs. control). The TGFbeta expression was significantly blocked by pre-incubation of cells with cerivastatin (Fig. 5), irrespective of whether they were stimulated with angiotensin II, isoprenaline, or phenylephrine. Cerivastatin stimulation alone had no effect on TGFbeta expression

Fig. 5. TGFbeta protein expression. (A) Influence of cerivastatin (10 ␮M) on the angiotensin II-induced (Ang: 100 nM) and adrenoceptor-induced (phenylephrine (PE): 1 ␮M; isoprenaline (ISO): 100 nM) increase in TGFbeta expression on protein level after incubation for 24 h. Pre-incubation of cardiomyocytes with cerivastatin (24 h) inhibited the increase in TGFbeta expression induced by all three substances. Data are means ± s.e.m. from n = 6 cultures, * p < 0.05 vs. control; # p < 0.05 vs. angiotensin II, phenylephrine or isoprenaline alone. (B) Sections of two representative western blots. The upper blot was stained for TGFbeta, the lower blot was stained for beta-actin and served as an internal loading control.

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Fig. 6. TGFbeta protein expression. (A) Influence of Apocinin (10 ␮M) on the phenylephrine-induced (PE: 1 ␮M) TGFbeta expression and SB202190 (10 ␮M) on the isoprenaline-induced TGFbeta expression on protein level after incubation for 24 h. Data are means ± s.e.m. from n = 5 cultures, * p < 0.05 vs. control; # p < 0.05 vs. isoprenaline alone. (B) Sections of four representative western blots. The upper blot was stained for TGFbeta, the lower blot was stained for beta-actin and served as an internal loading control.

. Furthermore TGFbeta expression was also blocked in the case of isoprenaline with the p38 MAP kinase inhibitor SB 202190 (10 ␮M). In the case of phenylephrine, pre-incubation with the NAD(P)H oxidase inhibitor Apocinin did not prevent TGFbeta expression (Fig. 6).

of cardiomyocytes after either alpha-adrenergic or angiotensin II stimulation (Fig. 7). In both cases, protein synthesis was not affected by cerivastatin. Moreover, cerivastatin alone had no effect on protein synthesis.

No effect of cerivastatin on alpha-adrenoceptor-dependent increase in cardiac hypertrophy-associated parameters

Discussion

Alpha-adrenergic stimulation alone is a strong stimulus for hypertrophic growth of myocytes (Schluter et al., 1999). In adult cardiomyocytes angiotensin II has only a moderate effect on protein synthesis (Ruf et al., 2002), whereas isoprenaline has no direct effect on protein synthesis (Wenzel et al., 2005). Therefore, we investigated the effect of cerivastatin on hypertrophic growth

Fig. 7. Hypertrophic growth. Influence of cerivastatin (10 ␮M) on the angiotensin II-induced (Ang: 100 nM) and adrenoceptor-induced (phenylephrine (PE): 1 ␮M; isoprenaline (ISO): 100 nM) increase in 14 C-phenylalanine incorporation as a parameter of hypertrophic growth after incubation time for 24 h. Alpha-adrenergic stimulation alone is a strong stimulus for hypertrophic growth whereas – in contrast to neonatal cardiomyocytes – angiotensin II only shows moderate effects on hypertrophic growth. Beta-adrenergic stimulation shows no effect in short-time cultures. Pre-incubation with cerivastatin did not influence the effects of these agonists on hypertrophic growth. Data are means ± s.e.m. from n = 5 cultures, * p < 0.05 vs. control.

This study clearly shows for the first time that in addition to angiotensin II, stimulation of either alpha- or beta-adrenoceptors increases TGFbeta expression in isolated adult rat cardiac myocytes and that this effect is blocked by pre-treatment with cerivastatin in all three cases. Angiotensin II directly increases the expression of TGFbeta in vivo and in vitro (Wenzel et al., 2001; Peng et al., 2005). There have already been several reports that adrenergic stimulation in vivo and in vitro indeed is able to modulate TGFbeta expression (Omura et al., 1994; Takahashi et al., 1994; O’Callaghan and Williams, 2002; Briest et al., 2004). For isolated cardiomyocytes, however no data exist about the correlation between adrenergic activity and cytokine expression. Alpha-adrenoceptor stimulation has been shown to be a strong hypertrophic stimulus in isolated cardiomyocytes, whereas angiotensin II is just a moderate stimulus. In contrast, betaadrenoceptor stimulation does not lead to hypertrophy. In the present study we were able to demonstrate that alphaadrenoceptor stimulation does not induce rac translocation and that cerivastatin does not prevent the hypertrophic growth of adult myocytes in response to phenylephrine. This observation is in contrast to results obtained in vascular smooth muscle cells (Nishio et al., 1998) or neonatal cardiomyocytes (Luo et al., 2001; Melchert et al., 2001). However, as described in detail elsewhere, neonatal and terminally differentiated adult cardiomyocytes have totally different signalling pathways and growth responses to various agonists (Schluter and Wenzel, 2008). Transgenic mice constitutively overexpressing active rac in the heart show hypertrophy and dilated cardiomyopathy (Sussman et al., 2000). This may indicate that a constitutively activated rac acts differently compared with a transient activation by receptor stimulation or that species differences are important. As mentioned above, a different picture

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TGFbeta Fig. 8. Overview of the rac-dependent and -independent signalling pathways in adult cardiomyocytes. Illustrated are the angiotensin II- and adrenoceptor-dependent signalling pathways leading to an increased TGFbeta expression in adult cardiomyocytes. Solid arrows give the direct and dotted arrows the indirect influences of cerivastatin treatment in these signalling pathways.

emerges at the level of TGFbeta expression concerning the influence of cerivastatin. In this case, the phenylephrine-induced effect was totally blocked by cerivastatin. Although cerivastatin blocked TGFbeta induction in all three cases (AT1-R, alpha- and betaadrenoceptor stimulation), early post-receptor signalling displays great differences. In neonatal cardiomyocytes statin treatment reduces the phenylephrine-induced hypertrophic growth through inhibition of the Ras homolog gene family members RhoA and cyclin D1 (Morikawa-Futamatsu et al., 2006). Whether RhoA, Ras or cyclin D1 play an important role in the scenario of TGFbeta induction needs to be elucidated. 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 induction of TGFbeta expression in ventricular cardiomyocytes. There is much evidence that the translocation of the small Gprotein rac is directly involved in the formation of radicals by NAD(P)H oxidase (Bokoch and Knaus, 2003). Both Nisimoto et al. (1997) and Koshkin et al. (1996) assume that rac poses as an adapter molecule for the cytosolic subunit p67phox, the target protein influencing the rate-limiting electron-transfer step. In order to participate in the regulation of ROS production, rac needs to translocate to the cell membrane. Besides this redox-inducing effect, rac is known to exert a multiplicity of effects in other signalling pathways. For example, ASK-1, tubulin and PAK are thought to be downstream targets of rac activation (Brown et al., 2006). Moreover, in neonatal cardiomyocytes Muhlhauser et al. (2006) demonstrated a desensitisation of beta-adrenergic signalling via reduced isoprenylation of G-protein gamma-subunits. Thus, our finding that isoprenaline activates rac but does not generate ROS may be due to the inability of beta-adrenoceptors to stimulate co-factors required for NAD(P)H oxidase activation. This assumption was underlined by the inability of isoprenaline to induce p67phox translocation, a necessary requirement for ROS generation by NAD(P)H oxidase. These findings are partially in line with results obtained by Andersson et al. The authors found that betaadrenergic stimulation increases mitchondrial ROS production in

mouse cardiomyocytes (Andersson et al., 2011), but especially concerning adrenergic pathways there are strong differences between mice and rats. Interestingly the highest used concentration of isoprenaline in this study (10 ␮M) was indeed able to induce ROS generation by about 450% (Fig. 5 of additional online supplements). This oxidative burst may induce cell death but not an adequate signalling pathway. Therefore ROS generation as part of a signal transduction after beta-adrenergic stimulation in adult rat cardiomyocytes is of minor relevance. Moreover, an attendance of p38 MAP kinase activation was detected. In accordance with our findings Varon et al. (2008) postulated an activation of p38 MAP kinase downstream of rac activation without an involvement of ROS in vascular cells. Following activation of the alpha-adrenergic-induced signalling pathway, ROS generation, TGFbeta expression, and hypertrophic growth were detected. Among these only TGFbeta expression was diminished by pre-treatment of cardiomyocytes with cerivastatin. Additionally neither pharmacological inhibition of NAD(P)H oxidase by Apocinin nor inhibition of rac activation by NSC23766 nor transfection of cells with p22phox antisense oligonucleotides did reduce PE-induced ROS production, underlining the assumption the alpha-adrenoceptor-induced ROS formation is independent of NAD(P)H oxidase. Indeed some NAD(P)H oxidase isoforms (Sumimoto, 2008; Brandes et al., 2010) and xanthine-oxidase (Adiga and Nair, 2008) may be considered as potential candidates for a rac-independent ROS formation (Lee et al., 2011). In summary, our study clearly demonstrates that cerivastatin differentially affects AT1-R, alpha- and beta-adrenoceptor-induced signalling pathways (Fig. 8). As a common endpoint, cerivastatin affected the induction of TGFbeta, an important cytokine involved in many remodelling processes of the heart.

Conflict of interest The authors state no conflict of interest.

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