© 2002 by European Society of Cardiology
Copyright © 2002, European Society of Cardiology
Impact of HMG CoA reductase inhibition on small GTPases in the heart
Medizinische Klinik und Poliklinik der Universität des Saarlandes, Innere Medizin III, 66421 Homburg/Saar, Germany
ulrich{at}laufs.com
* Corresponding author. Tel.: +49-6841-162-3436; fax: +49-6841-162-3637
Received 9 July 2001; accepted 19 November 2001
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
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Objective: Members of the Rho GTPase family, Rac1 and RhoA have been suggested to be mediators of cardiac hypertrophy in mice. Rho proteins are posttranslationally isoprenylated. In addition to cholesterol-lowering, statins inhibit the isoprenylation of small G proteins. Therefore, it was tested if these drugs inhibit Rac1 and RhoA activity in cardiomyocytes and, thereby, prevent angiotensin II-mediated expression of atrial natriuretic factor (ANF) and myosin light chain (MLC)-2 in the heart. Methods and results: Western and Northern analysis of rat neonatal cardiomyocytes and H9C2 cells showed inhibition of basal and angiotensin-stimulated Rac1 expression, membrane-translocation and activity after statin treatment. Similarly, basal and stimulated RhoA membrane expression was inhibited. Statins concentration- and time-dependently downregulated basal as well as angiotensin-induced expression of ANF by 86±2.3% and 89±1.7%, as well as MLC-2 by 75±4.1% and 84±6%, respectively. Direct inhibition of Rac GTPase by overexpression of the dominant negative mutant RacN17 or by Clostridium sordellii lethal toxin in rat H9C2 cells inhibited ANF expression by 70±4.9% and 78±10%, respectively. Inhibition of RhoA by Clostridium botulinum C3 transferase or the dominant negative mutant RhoN19 reduced ANF mRNA by 19±11% and 23±8%, respectively. To test these findings in vivo, spontaneously hypertensive rats were treated with atorvastatin, leading to a decrease in cardiac Rac1 and RhoA activity as determined by [35S]-GTP
S-binding assays by 61±16% and 72±24%, and downregulation of MLC-2 as well as ANF mRNA expression by 31±16% and 80±24%, respectively. Conclusions: (1) Statins downregulate the activity of small G proteins in cardiomyocytes in culture as well as in vivo. (2) Inhibition of Rac1 and RhoA by statins reduces myocardial expression of ANF and MLC-2. (3) Targeting myocardial Rho GTPases by statins may be a novel treatment strategy to prevent cardiac hypertrophy.
KEYWORDS Myocytes; G-proteins; Gene expression; Lipid metabolism; Hypertrophy
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
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Cardiac hypertrophy is an adaptive response to hemodynamic overload or neurohormonal activation to normalize wall tension and maintain systolic function. Cardiac hypertrophy is associated with an increased risk of heart failure, a major cause of death and disability [1]. On the cellular level, the hypertrophic phenotype is characterized by an increase in cell size and myofibrillar assembly, reactivation of fetal genes (e.g. atrial natriuretic factor, ANF), and increased expression of contractile elements (e.g. myosin light chain-2, MLC-2) [2]. It is thought that many of the same signaling pathways that regulate hypertrophy are involved in the progression to heart failure, thus, pharmacological targeting of key mediators of hypertrophy may prevent the development of cardiac failure.
There is growing evidence that a variety of ligands (e.g. angiotensin II) and mechanical forces known to mediate hypertrophy [3] activate small GTP binding proteins of the Ras and Rho families [4–7]. The Rho subfamily includes the GTPases RhoA, Rac1 and Cdc42. The functions of the small GTPases are not fully understood, but important downstream effects include the regulation of the cytoskeleton and activation of the mitogen-activated protein kinase (MAPK) family in cardiomyocytes [8]. Overexpression of Ras, Rac1 and RhoA in neonatal cardiomyocytes has been shown to provoke hypertrophic target gene expression as a final common response [4,5]. In fibroblasts, a hierarchy of the small GTPases has been described, with Ras able to stimulate Cdc42, which can then stimulate Rac, which in turn activates RhoA [9]. In addition, extensive cross talk between Ras, Rac1 and RhoA has been demonstrated in several cell types [10]. On the other hand, each GTPase mediates specific changes of the cytoskeleton and activates individual downstream targets [9,11]. For example, Ras interacts with protein kinases c-Raf and phosphatidylinositol 3'-kinase (PI3K), Rac1 activates to p21 activated kinases (PAK), and RhoA regulates Rho-Kinase and myosin light chain [4]. Important information on a potentially diverse role of small G proteins in hypertrophy stems from recent experiments with transgenic mice: mice homozygous for the cardiac-targeted V12Hras transgene displayed left ventricular hypertrophy [12]. In contrast, overexpression of RhoA in mouse myocardium showed no evidence of ventricular hypertrophy. Instead, atrial enlargement and a greatly depressed heart rate was observed, leading to contractile failure [13]. Finally, cardiac overexpression of Rac1 in mice resulted in a lethal dilated phenotype or a transient cardiac hypertrophy seen among juvenile mice suggesting Rac1 GTPase as a mediator of dilatation as well as hypertrophy [14]. Therefore, small G proteins may be potential targets for therapeutic intervention.
HMG-CoA reductase inhibitors, or ‘statins’, competitively inhibit mevalonate synthesis and reduce serum cholesterol. At the same time, statins inhibit the synthesis of the isoprenoid intermediates of cholesterol pathway, including farnesylpyrophosphate and geranylgeranylpyrophosphate [15]. Ras and the Rho-family of GTPases are major substrates for isoprenylation. Ras proteins are post-translationally modified by farnesylation, whereas the Rho family is activated by the attachment of geranylgeraniol. This post-translational lipid modification is necessary for the translocation of inactive GTPase from the cytosol to the membrane [9]. Consequently, it has been shown that statins inhibit small GTPase activity in vascular cells [16–20]. Therefore, statins which block geranylgeraniol synthesis, or isoprenoid-transferase inhibitors which prevent the attachment of specific isoprenoids, may inhibit membrane translocation and activity of small G proteins in cardiomyocytes.
We hypothesized that statins may modulate cardiac small GTPase expression and activity and, thereby, may influence surrogate markers of cardiac dysfunction such as ANF and MLC-2. Therefore, the effects of angiotensin and statins were studied in cultured cardiomyocytes and in spontaneously hypertensive rats as a model of pressure-induced hypertrophy.
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2. Methods |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
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2.1. Materials
Angiotensin II, L-mevalonate, and chemicals were purchased from Sigma. GGTI-286 and FTI-276 were obtained from Calbiochem. [32P]dCTP and Hybond N-nylon membranes were obtained from Amersham. [35S]GTP
S was supplied by New England Nuclear. Simvastatin and atorvastatin were gifts of Merck-Sharp-Dohme (Haar, Germany) and Gödecke-Parke-Davis (Freiburg, Germany), respectively. Simvastatin and mevalonate were chemically activated by alkaline hydrolysis [21]. Clostridium botulinum C3 toxin was from List Biological Laboratories, Inc. (Campbell, CA, USA). Clostridium sordellii lethal toxin was kindly provided by K. Aktories (Freiburg, Germany) [22]. RhoN19 and RacN17 were kind gifts from A. Hall (London, UK) [11].
2.2. Cell culture
Cardiomyocytes from neonatal Sprague–Dawley rats were isolated and cultured as described [23]. Plating density was about 3x105 cells/cm2. H9C2 rat heart myoblasts were kindly provided by P. Karczewski (Berlin, Germany). Neonatal cardiomyocytes and H9C2 cells were grown to confluence in Dulbecco's modified Eagle medium supplemented with 10% (v/v) fetal calf serum, glutamine (2 mM) and penicillin (100 IU/ml). Neonatal cardiomyocytes showed spontaneous contractions. Cells were kept in serum-free medium for 24 h before treatment. Cellular viability under all treatment conditions was determined by cell count, morphology, and Trypan blue exclusion. For determination of cellular protein content, cells were counted in a Boyden chamber and total protein concentration was quantitated by a modified Lowry assay.
2.3. Animal treatment
Male, spontaneously hypertensive rats (Hoechst Marion Roussel, Frankfurt/Main, Germany) were randomized to a standard chow supplemented with or without atorvastatin at a dose of 50 mg/kg body weight. Treatment was started at age 18 weeks and was continued for 30 days. Body weights were similar in both groups (350±5 g vs. 357±13 g). Mean serum cholesterol was 89±8 mg/dl in controls and 60±5 mg/dl in the statin group. All animal experiments were conducted in accordance to the German animal protecting law.
2.4. Transfection
H9C2 cells were harvested and resuspended in electroporation medium (Optimem 1, Gibco) at a concentration of 5x107 cells/ml. The following constructs were transfected: insertless vector (pcDNA3) as control, pRK5-myc-Rac1-L61 (constitutively active Rac1 mutant), pRK5-myc-Rac1-N17 (dominant-negative Rac1 mutant) and pcDNA3-myc-N19RhoA (dominant-negative RhoA mutant) [18,24]. Twenty µg plasmid DNA and 200 µl cell suspension were placed in a 0.4-cm cuvette, mixed and incubated for 30 min on ice. After incubation at 37 °C for 30 s, the cuvette was pulsed with 300 V and 500 µF (Electro Cell Manipulator, BioRad). The pulse length was determined by the electroporator based on capacitance, field strength, and resistance of the medium. Upon electroporation the cuvette was incubated at room temperature for an additional 30 min. The cells were plated at tissue culture plates and cultured for 48 h before harvest or treatment with angiotensin II and statins.
2.5. Northern blotting
Northern blotting using [32P]dCTP-labeled, full length ANF cDNA, Rac1 cDNA and MLC-2 cDNA was performed as described previously [25].
2.6. Real-time RT-PCR
Real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR) was performed with the TaqMan system (Prism 7700 Sequence Detection System, PE Biosystems). For MLC-2 the primers were 5'-CGG AAG CTC CAA CGT GTT CT and 5'-TCC TTC TCT TCT CCG TGG GT [26,27]. For 18S the primers were 5'-TTG ATT AAG TCC CTG CCC TTT GT and 5'-CGA TCC GAG GGC CTA ACTA. For quantification, MLC-2 expression was normalized to the expressed housekeeping gene 18S.
2.7. Western blotting
For preparation of membrane and cytosolic proteins, cells were washed with PBS, pelleted and dissolved in x1 reporter lysis buffer (Promega), frozen three times at –80 °C and sonicated. After spinning for 10 min, 3000 rev./min, 4 °C, the supernatant was transferred into ultracentrifuge tubes (Beckmann Quickseal) and spun for 45 min at 25 000 rev./min, 4 °C in a Beckmann NVT 65.2 rotor. The resulting pellet of membrane proteins and the supernatant cytosolic fraction were stored at –80 °C. Total cell lysates, membrane and cytosolic proteins were separated on SDS–PAGE [18]. Immunoblotting was performed using Rac1 and RhoA and phosphorylated (Thr-183/The-185) c-Jun NH2 terminal kinase (JNK) monoclonal antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, 1:250 dilution). Immunodetection was accomplished using a goat anti-rabbit secondary antibody (1:4000 dilution), and an enhanced chemiluminescence kit (Amersham).
2.8. Assay for GTP-binding activity
The Rac1 and RhoA GTP-binding activity was determined by immunoprecipitation of [35S]GTPS-labeled Rac1 and RhoA [18]. Briefly, membrane and cytosolic proteins were isolated, and proteins (20 µg) from control and treated H9C2 cells were incubated for 30 min at 37 °C in a buffer containing [35S]GTPS (20 nmol/l), GTP (2 µmol/l), MgCl2 (5 mmol/l), EGTA (0.1 mmol/l), NaCl (50 mmol/l), creatinine phosphate (4 mmol/l), phosphocreatinine kinase (5 units), ATP (0.1 mmol/l), dithiothreitol (1 mmol/l), leupeptin (100 µg/ml), aprotinin (50 µg/ml), and phenylmethanesulfonyl fluoride (PMSF, 2 mmol/l). The assay was terminated with excess unlabeled GTPS (100 µmol/l). Samples were then resuspended in 100 µl of immunoprecipitation buffer containing Triton-X (1%), SDS (0.1%), NaCl (150 mmol/l), EDTA (5 mmol/l), Tris–HCl (25 mmol/l, pH 7.4), leupeptin (10 µg/ml), aprotinin (10 µg/ml), and PMSF (2 mmol/l). The Rac1 or RhoA antiserum was added to the mixture at a final dilution of 1:75. The samples were allowed to incubate for 4 h with gentle mixing. The antibody-G-protein complexes were then incubated with 50 µl of protein A-sepharose (1 mg/ml, Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 2 h, and the immunoprecipitate was collected by centrifugation at 12 000xg for 10 min. Preliminary studies using Western analysis of the supernatant indicated that Rac1 was completely immunoprecipitated under these conditions. The pellets were washed four times in a buffer containing HEPES (50 mmol/l, pH 7.4), NaF (100 µmol/l), sodium phosphate (50 mmol/l), NaCl (100 mmol/l), Triton X-100 (1%), and SDS (0.1%). The final pellet containing the immunoprecipitated [35S]GTPS-labeled Rac1 proteins was counted in a liquid scintillation counter (LS 1800, Beckman, Munich, Germany). Nonspecific activity was determined in the presence of excess unlabeled GTPS (100 µmol/l).
2.9. Data analysis
Band intensities were analyzed by densitometry. All values are expressed as mean±S.E.M. compared to controls. Paired and unpaired Student's t-tests and ANOVA for multiple comparisons were employed. Differences were considered significant at P<0.05.
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3. Results |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
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3.1. Statins inhibit Rac1 expression and membrane translocation
Rac1 GTPase has been suggested as a mediator of phenotype changes in the heart [14]. Therefore, the effect of statins on Rac1 protein expression in neonatal rat cardiomyocytes was determined by Western blotting. Treatment with angiotensin II (10 µmol/l, 6 h) increased Rac1 expression by 62±29% (n=5 separate experiments, P<0.05) (Fig. 1A). Pretreatment with simvastatin (1 µmol/l) for 15 h significantly decreased basal Rac1 protein expression and inhibited the effect of angiotensin II to 42±4.3% and 40±6.8% of control levels, respectively (n=5 separate experiments, P<0.05). Similarly, 0.1, 1 and 10 µmol/l atorvastatin concentration-dependently downregulated Rac1 expression in H9C2 cardiomyoblasts to 48±8%, 28±8% and 15±7%, respectively (Fig. 1B) (n=4 separate experiments, P<0.05). Simvastatin at concentrations of 0.1, 1, 5 and 10 µmol/l reduced angiotensin II (10 µmol/l, 6 h) stimulated Rac1 expression to 25±8%, 22±6%, 15±6% and 12±6%, respectively (Fig. 1C) (n=3 separate experiments, P<0.05).
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Northern analysis showed upregulation of Rac1 mRNA expression after stimulation with angiotensin (10 µmol/l, 6 h) to 153±16% (n=4 separate experiments, P<0.05). Treatment with simvastatin (1 µmol/l, 15 h) almost completely inhibited basal as well as stimulated Rac1 mRNA expression to levels close to the detection limit (5±4%) (Fig. 1D). Downregulation of Rac1 mRNA was time-dependent (Fig. 1E), complete inhibition of Rac1 expression was observed after 6 h (simvastatin, 1 µmol/l) and did not increase during the time period studied (up to 48 h).
Rac function depends on its membrane-associated GTP-binding activity [9]. Therefore, Rac1 expression was studied in membrane and cytosolic preparations. Angiotensin II (10 µmol/l, 6 h) increased Rac1 membrane expression by 50±13% and Rac1 cytosolic expression by 48±15% (Fig. 2A). Pretreatment with simvastatin (1 µmol/l) for 15 h decreased basal and angiotensin-stimulated Rac1 membrane expression to 10±8% of control. Basal Rac1 in the cytosol was downregulated by 65±27%, demonstrating an inhibition of Rac1 total protein expression as well as membrane translocation (n=4 separate experiments, P<0.05).
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To test the effects of angiotensin and statin on Rac1 activity, [35S]GTP
S-binding assays were performed. Angiotensin II significantly upregulated Rac1 GTP-binding by 62±17% (Fig. 2B). Simvastatin effectively inhibited both basal as well as stimulated Rac1 activity.
3.2. Statins decrease protein content and ANF mRNA expression
To determine the effects of angiotensin II and statins on cellular hypertrophy, H9C2 cardiomyoblasts were treated with angiotensin II (10 µmol/l, 6 h) and atorvastatin (0.1–1 µM, 15 h). Angiotensin II increased the cellular protein content by 26±9% (Fig. 3), which was completely inhibited by 0.1 µM atorvastatin.
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ANF has been described as a marker of the hypertrophic phenotype. In rat neonatal cardiomyocytes, treatment with angiotensin II (10 µmol/l, 6 h) increased ANF mRNA expression by 69±34% (Fig. 4A). Pretreatment with simvastatin completely inhibited ANF expression in unstimulated cardiomyocytes and, to the same extent, in angiotensin II-treated cells (8±5% of control, n=4 separate experiments, P<0.05).
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Similarly, treatment of H9C2 cardiomyoblasts for 12 h with 0.1, 1 and 10 µM atorvastatin for 15 h concentration-dependently decreased ANF mRNA expression by 20±4.2, 90±8.2, and 91±12.7%, respectively (n=4 separate experiments, P<0.05) (Fig. 4B). Simvastatin downregulated ANF mRNA by 25±9, 85±19, and 88±13%, respectively (n=4 separate experiments, P<0.05), suggesting a class effect of HMG-CoA reductase inhibitors. Downregulation of ANF in cardiomyocytes by statins was time-dependent (maximum after 12 h) and persisted for up to 96 h (data not shown).
Downregulation of ANF expression by simvastatin, 10 µM, 12 h, was completely reversed in the presence of L-mevalonate (200 µmol/l) (Fig. 4C), indicating that the statin effect is mediated by inhibition of mevalonate synthesis. To test whether downregulation of ANF was mediated by inhibition of isoprenoid synthesis, cells were treated with the specific geranylgeranyl-transferase inhibitor GGTI-286 (50 µmol/l) and the farnesyl-transferase inhibitor FTI-276 (10 nmol/l) for 15 h. Inhibition of geranylgeranylation but not inhibition of farnesylation resulted in downregulation of ANF mRNA expression by 80±17% (n=3 separate experiments, P<0.05), suggesting that geranylgeranylated proteins of the Rho family are involved in the regulation of ANF expression.
3.3. Inhibition of Rac1 GTPase downregulates ANF expression
To test whether downregulation of ANF expression by statins was mediated by inhibition of Rac1 geranylgeranylation, Rac1 was inhibited directly by overexpression of the dominant negative mutant RacN17 using electroporation (Fig. 5A), and by treatment with the glucosyltransferase of Rac, Clostridium sordellii lethal toxin (LT, 200 ng/ml, 12 h) (Fig. 5B) [11,22]. Inhibition of Rac1 by overexpression of RacN17 as well as LT significantly decreased the expression of ANF mRNA at baseline and in the presence of angiotensin II (n=4, P<0.05). In contrast, overexpression of the constitutively active mutant RacL61 did not increase ANF expression in H9C2 cells, suggesting that Rac1 activation is necessary but not sufficient for baseline and angiotensin II-stimulated ANF expression.
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3.4. Role of RhoA GTPase in the regulation of ANF expression
The regulation of RhoA expression was studied in membrane and cytosolic protein preparations. Similarly to the effects on Rac1, angiotensin II (10 µmol/l, 6 h) increased RhoA membrane expression by 64±12% (Fig. 6A). Pretreatment with simvastatin (1 µmol/l) for 15 h decreased basal and angiotensin-stimulated RhoA membrane expression to 15±13% of control. Statin downregulated RhoA expression in the cytosol to 30±11% (n=3 separate experiments, P<0.05). To test whether inhibition of RhoA membrane expression contributes to the downregulation of ANF mRNA by statins, RhoA was specifically inhibited by ADP-ribosylation with Clostridium botulinum C3 transferase (C3, 50 µg/ml) [28]. Treatment with C3 only partially inhibited ANF expression by 19±11% (Fig. 6B), (n=3 separate experiments, P<0.05). Inhibition of RhoA by overexpression of the dominant-negative RhoN19 reduced ANF mRNA by 23±8% (Fig. 6C). These experiments suggest that RhoA contributes to the regulation of ANF expression, but to a lesser extent compared to Rac1 GTPase.
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3.5. Statins downregulate MLC-2 and JNK expression in cardiomyocytes
Similarly to the effects on ANF expression, angiotensin II (10 µmol/l, 6 h) upregulated the mRNA expression of MLC-2 in neonatal rat cardiomyocytes (n=3, P<0.05). Simvastatin (and atorvastatin, data not shown) significantly decreased baseline as well as stimulated expression of MLC-2 to 25±9% of control (Fig. 7A).
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The c-Jun NH2 terminal kinase (JNK) has been reported to be regulated by Rac1 in cardiac myocytes [29]. Western analysis showed that atorvastatin (1 µmol/l, 15 h) downregulated basal as well as angiotensin II (10 µmol/l, 6 h)-induced phosphorylated JNK expression (Fig. 7B).
3.6. Statins decrease Rac1 activity and ANF expression in spontaneously hypertensive rats
To determine whether statin treatment modulates Rac1 activity in the heart in vivo, normocholesterinemic spontaneously hypertensive rats as a model of chronic pressure overload were treated with oral atorvastatin for 30 days. Immunoprecipitation of [35S]GTPS-labeled Rac1 in heart left ventricular tissue revealed downregulation of Rac1 and RhoA GTP-binding activity by 61±16% and 72±24%, respectively (n=4, P<0.05) in the statin-treated animals (Fig. 8A and B). In agreement with the cell culture data, Northern analysis showed that cardiac ANF mRNA expression was downregulated by 80±24% (n=4, P<0.05) (Fig. 9A). Similarly, real time PCR demonstrated inhibition of MLC-2 expression in the statin group (Fig. 9B). These data show that Rac1 and RhoA activity in the heart can be inhibited by oral statin treatment.
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4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
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Our findings suggest the Rho family of small GTPases as novel targets of HMG CoA reductase inhibitors in cardiomyocytes in culture as well as in vivo. Treatment with angiotensin II increases Rac1 and RhoA expression and membrane translocation, supporting the role of Rho GTPases as downstream targets of the renin–angiotensin system in the heart [30,31]. Statins inhibit Rho protein activity in cardiomyocytes by two complementary mechanisms: in addition to inhibiting basal and angiotensin II-stimulated mRNA and protein expression, statins reduce membrane-translocation and GTP-binding activity. It has previously been shown that statin treatment inhibits the isoprenylation of RhoA and Rac1 in cultured cells [17–19]. In endothelial and vascular smooth muscle cells, statins inhibit Rho function and expression in the cell membrane but upregulate Rho proteins in the cytosol by a negative feed-back mechanism [19,25]. In contrast, cytosolic and total Rho protein expression is downregulated by statins in cardiomyocytes, suggesting cell type specific regulators of Rac1 and RhoA gene transcription or mRNA stability, presumably in the cytoskeleton.
Statins time- and concentration-dependently downregulated basal and angiotensin II-induced expression of ANF and MLC-2 in cardiomyocytes. Atorvastatin and simvastatin had similar effects, revealing a class effect of statins. The effect was specific, because downregulation of ANF expression was reversed in the presence of mevalonate, and identifies an interaction of the cholesterol synthesis pathway with the signaling leading to cardiac hypertrophy. Specifically, inhibition of geranylgeranyl-transferase prevented the molecular characteristics of the hypertrophic phenotype, suggesting a role of the Rho family, which depends on geranylgeranylation for its membrane-associated activity [9,11]. Inhibition of farnesyl-transferase did not alter ANF expression, suggesting a less important role of Ras isoprenylation in this context.
To test whether the regulation of small G proteins by statins and angiotensin II influences markers of the hypertrophic phenotype, Rac1 and RhoA activity was inhibited independent of isoprenylation. Glycosylation of Rac1 by Clostridium sordellii lethal toxin as well as overexpression of the dominant-negative mutant RacN17 effectively reduced basal as well as stimulated ANF expression. In contrast, inhibition of RhoA by specific ADP ribosylation or by overexpression of the dominant negative RhoN19 only partially reduced ANF expression, demonstrating an important role of Rac1 for ANF regulation in cardiomyocytes. These experiments show that inhibition of small G protein function by statins is the mechanism of downregulation of cardiac ANF expression. In agreement with these findings, statins inhibited one of the downstream targets of Rac1 in cardiac myocytes, the c-Jun NH2 terminal kinase [29].
These data show that Rac1 and RhoA mediate angiotensin II-induced ANF expression and can be effectively inhibited by statins. To overcome the limitations of a cell culture study, spontaneously hypertensive rats were treated with atorvastatin. The primary target of statins in vivo is the HMG CoA reductase of the hepatocytes. Therefore, the most important finding of the animal experiments is the inhibition of Rac1 and RhoA activity in cardiac tissue after oral treatment with atorvastatin, identifying statins as a pharmacological intervention to inhibit Rho activity in the heart. The potential significance of this finding is underlined by the downregulation of cardiac ANF and MLC-2 mRNA in the statin group compared to animals treated with vehicle. These expression data provide the basis for future studies to determine the effects of statin treatment on cardiac physiology in vivo.
It is interesting to speculate that inhibition of cardiac Rho proteins may be beneficial by reducing the hypertrophic phenotype. Downregulation of Rac1 GTPase has been shown to protect from reoxygenation injury [32], and as part of the NAD(P)H-oxidase complex, Rac1 is necessary for the release of reactive oxygen species in several cell types, including neutrophils [33] and vascular cells [17,19]. Furthermore, inhibition of RhoA has been shown to increase endothelial nitric oxide and to inhibit endothelin expression in endothelial cells [18,20]. Therefore, targeting the Rho family in the heart may improve several aspects of hypertrophy and failure. In concert with our findings, two recent animal studies reported improvement of cardiac hypertrophy after statin treatment [34,35], and a recent study found greater reduction of left ventricular mass in hypertensive patients with hyperlipidemia treated with pravastatin and anti-hypertensive medication compared to patients treated with diet and anti-hypertensive medication, and compared to normocholesterinemic patients treated with anti-hypertensive medication alone [36]. The authors conclude that statin therapy may reduce left ventricular mass independent of cholesterol lowering.
In summary, the presented data provide evidence and rationale for using statin drugs to target small G proteins in the heart, irrespective of cholesterol levels. Therefore our findings warrant further studies testing the effects of statin drugs on cardiac hypertrophy and heart failure.
Time for primary review 25 days.
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Acknowledgments |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
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We thank P. Karczewski, K. Aktories and A. Hall for kindly providing H9C2 cells, LT and RacN17, and I. Paez-Mallez for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (U.L.) and the Universität des Saarlandes (U.L.). The study was not funded by the pharmaceutical industry.
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Notes |
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1 Ulrich Laufs and Heiko Kilter contributed equally to this study.
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