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Atorvastatin desensitizes ß-adrenergic signaling in cardiac myocytes via reduced isoprenylation of G-protein -subunits

Ulrike Mühlhäuser^*, Oliver Zolk, Thomas Rau^*, Felix Münzel, Thomas Wieland and Thomas Eschenhagen^*,1

^* Institute of Experimental and Clinical Pharmacology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany;
Institute of Clinical Pharmacology, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany; and
Institute of Pharmacology and Toxicology Mannheim, University of Heidelberg, Mannheim, Germany

¹Correspondence: Institute of Experimental and Clinical Pharmacology, University Medical Center, Hamburg-Eppendorf, Martinistr., Hamburg 52 20246, Germany. E-mail: t.eschenhagen{at}uke.uni-hamburg.de

SPECIFIC AIMS

Inhibitors of HMG-CoA reductase (statins) exert cholesterol-independent (pleiotropic) effects on blood vessels and inflammatory cells that have been attributed to reduced isoprenylation of monomeric GTPases. It is not known whether, besides their well-characterized effects on artherosclerosis and vascular function, statins affect cardiac myocyte function directly. This study evaluated the effects of statins on cardiac myocyte contractile function and the molecular mechanism of this effect. We asked whether statins also reduce isoprenylation of subunits of the large, heterotrimeric G-proteins and thereby affect ß-adrenergic signaling and regulation of force in cardiac myocytes.

PRINCIPAL FINDINGS

1. Atorvastatin reduces G₃ isoprenylation in cardiac myocytes
Historically, G subunits of heterotrimeric G-proteins have been the first proteins shown to depend on isoprenylation for their signal transducing activity. Isoprenylation of G3, the main G subtype in neonatal cardiac myocytes, results in a small upward shift in gel mobility. Cultured neonatal rat cardiac myocytes (NRCM) were treated with atorvastatin (1 µmol/l, 48 h), fractionated in cytosolic and particulate fractions, and subjected to SDS-PAGE and Western blot. Under these conditions a G3 subunit doublet was reproducibly detectable at 10 kDa, whereas extracts from control cells showed a single, slower migrating band only (Fig. 1 A). The second, faster migrating band disappeared in atorvastatin-treated NRCM after supplementation with geranylgeranyl pyrophosphate (GGPP; 10 µmol/l) and was similarly seen in NRCM treated with GGTI-298, a compound specifically blocking geranylgeranylation but not farnesylation (10 µmol/l). This suggests that the faster migrating band represented non-isoprenylated G3. This interpretation was substantiated by experiments in HEK 293 cells demonstrating that a non-isoprenylatable G3 mutant run at a single band at 10 kDa, whereas the wild-type G3 migrated slightly more slowly. The mutated G3 was found predominantly in the cytosolic wild-type, mainly in the particulate fraction. Transfection with mutated G3, but not wild-type G3, induced a partial redistribution of endogenous Gß from the particulate to the cytosolic fraction. This supports earlier findings showing that membrane translocation of Gß depends on G. Thus, the results show that atorvastatin reduces isoprenylation and membrane anchorage of G3 in cardiac myocytes.

View larger version (33K): [in this window] [in a new window]   Figure 1. Atorvastatin changes migrating properties of G3, Gß, and Gs subunits. NRCM were treated with atorvastatin (Ator; 1–10 µmol/l) for 48 h and homogenates were subfractionated into a cytosolic and particulate fraction by differential centrifugation and analyzed by Western blot. Equal amounts of protein were loaded. A) Representative Western blot for G3. The cytosol and particulate fraction in atorvastatin-treated cells show an additional band with an apparent lower molecular weight. Results are representative of 2 independent experiments. Graphs on the right show the densitometric profile of the bands in lanes 3 and 4, corrected for electrophoretic shifts of each lane. B) Representative Western blots for Gß. C) Representative Western blots for Gs of the cytosolic and particulate fraction of NRCM treated with 1 µmol/l Ator. D, E) Corresponding quantitative data are shown as mean±SE. Number in columns = number of independent experiments *P < 0.05 vs. control (Ctr).

2. Atorvastatin causes dropout of Gß into the cytosol and a reduction in total Gs, but not Gi, in cardiac myocytes
In parallel with the accumulation of non-isoprenylated G3 subunits, the cytosolic fraction of atorvastatin (1 µmol/l, 24 h) -treated NRCM contained more Gß (+123%) than the cytosol of control cells (Fig. 1B, D ). This suggests that, secondary to reduced G subunit isoprenylation, Gß subunits in atorvastatin-treated NRCM are less effectively translocated to the membrane and partly retained in the cytosol. Moreover, atorvastatin reduced the Gs content in both the particulate fraction and in total cellular content (–35%; Fig. 1C, E ). In contrast, localization and/or amount of Gi2 remained unchanged. Treatment with the geranyltransferase inhibitor GGTI-298 reduced the content of Gs in the particulate fraction similar to atorvastatin, whereas supplementation with GGPP (10 µmol/l) prevented the reduction of Gs.

3. The effects of atorvastatin on Gs are concentration- and time-dependent, and sensitive to GGPP, but not to FPP or squalene
To test for the specificity and mechanism of the statin effect, cells were treated with atorvastatin at different concentrations (0.1–10 µmol/l, 48 h), for different times (12–48 h, 0.1 µmol/l), and in the presence of different intermediates of the cholesterol-synthetizing pathway and assayed for total Gs. Significant reductions in Gs were seen at 0.1 µmol/l (–17%) after 48 h, maximal reductions at 10 µmol/l (–46%). The effect of atorvastatin at 1 µmol/l was abolished by supplementation with mevalonate (200 µmol/l), GGPP (10 µmol/l), but not FPP (10 µmol/l) or the cholesterol precursor squalene (10 µmol/l). These data show a cholesterol-independent and geranylgeranyl-dependent effect of statins.

4. Atorvastatin reduces the cAMP-increasing and positive inotropic effects of isoprenaline
The ß-adrenergic-Gs-adenylyl cyclase pathway provides the most important regulation of cardiac contractile function and so was chosen to evaluate whether the atorvastatin-mediated reduction in Gs has functional consequences. Indeed, the concentration response curve of isoprenaline (1–1000 nmol/l) for cAMP accumulation was shifted to the right (EC₅₀ 10.6 vs. 5.6 nmol/l) and the maximal effect was reduced by 10%. In contrast, the maximal effect of the receptor-independent adenylyl cyclase stimulator forskolin was increased by 22%, demonstrating that atorvastatin did not diminish cAMP generation in cardiac myocytes per se. Contractile consequences were studied in the model of 3-dimensional engineered heart tissue from neonatal rat cardiac cells (EHT; Fig. 2 D). Treatment of EHT with atorvastatin for 5 days did not affect basal contractile force, but reduced the positive inotropic effect of isoprenaline in a concentration-dependent manner by 30–50% (Fig. 2A ). The effect of atorvastatin (1 µmol/l) was completely reversed by mevalonate (Fig. 2C ).

View larger version (48K): [in this window] [in a new window]   Figure 2. Atorvastatin attenuates the positive inotropic effect of isoprenaline in EHTs. EHTs were treated with atorvastatin (Ator; 0.1–10 µmol/l) for 5 days. The contractile response to isoprenaline was evaluated in organ baths. A) EHT on a stretch device during culture. B) Isoprenaline-induced change in force of contraction ( FOC). C) FOC in % of maximal effect. D) Isoprenaline-induced FOC of EHTs treated for 5 days with Ator (2 µmol/l) in the presence or absence of mevalonate (Meva; 200 µmol/l). *P < 0.05 vs. control (Ctr).

5. Atorvastatin up to 1 µmol/l has a small antihypertrophic, but no cytotoxic effect on cardiac myocytes
Statins have been reported to interfere with cardiac growth in various animal models and, at high concentrations, can exert cytotoxic effects. In EHTs treated with 1 or 10 µmol/l, atorvastatin induced a modest reduction of myocyte bundle formation and cell organization corresponding to a slight reduction in protein synthesis in EHTs (–15%) observed at 10 µmol/l atorvastatin. In cultured NRCM, atorvastatin at 1 µmol/l regularly caused thinning of cardiac myocytes in a mevalonate-sensitive manner but did not increase the release of LDH. These data argue against a cytotoxic effect of atorvastatin in concentrations up to 1 µmol/l.

CONCLUSIONS AND SIGNIFICANCE

The present study shows that statins reduce ß-adrenergic responsiveness of cardiac myocytes. The effect is cholesterol-independent and mediated via reduced geranylgeranylation of G with the consequence of partial redistribution of Gß to a cytosolic compartment and reduction of total Gs (Fig. 3 ). The data add another facet to the pleiotropic effects of statins, which have been mainly attributed to reduced signaling through small monomeric GTPases such as Rho and rac in endothelial, smooth muscle, and inflammatory cells. The degree of functional and biochemical changes in cardiac myocytes and EHTs seen after treatment with atorvastatin is moderate, but well within the range of pleiotropic effects of statins seen previously. Atorvastatin at clinically relevant doses (40 mg) has recently been shown to reduce Rho isoprenylation in peripheral mononuclear blood cells in healthy volunteers, suggesting that the effects on heterotrimeric G-protein signaling in the present study may also occur in patients; however, this has to be tested directly. Given the eminent importance of the ß-adrenergic regulation of heart function, most prominently documented by the success of beta blockers, it appears likely that even a moderate damping of catecholamine responses by treatment with statins has a consequence, most likely a beneficial one.

View larger version (13K): [in this window] [in a new window]   Figure 3. Mechanisms of the cholesterol-independent, pleiotropic effects of HMG-CoA reductase inhibitors (statins). Diagram of the cholesterol biosynthesis pathway showing biological effects of isoprenoids as recently reviewed by Liao and Laufs (2005). Signaling molecules that have been shown to depend on isoprenylation and to be affected by treatment with statins in endothelial, smooth muscle, blood, and cardiac muscle cells are the monomeric GTPase ras, rho, rac, and cdc42. Consequently, multiple signaling pathways are modulated. Incorporated in the scheme are results of the present study indicating that statins also affect signaling through heterotrimeric G-proteins by reducing isoprenylation of G subunits. In cardiac myocytes, this effect causes a reduced response to ß-adrenergic stimulation of cAMP and force of contraction.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5067fje;

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