Statins as antioxidant therapy for preventing cardiac myocyte hypertrophy

Cardiac hypertrophy is a major cause of morbidity and mortality worldwide. The hypertrophic process is mediated, in part, by small G proteins of the Rho family. We hypothesized that statins, inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, inhibit cardiac hypertrophy by blocking Rho isoprenylation. We treated neonatal rat cardiac myocytes with angiotensin II (AngII) with and without simvastatin (Sim) and found that Sim decreased AngII-induced protein content, [³H] leucine uptake, and atrial natriuretic factor (ANF) promoter activity. These effects were associated with decreases in cell size, membrane Rho activity, superoxide anion (O₂·̄) production, and intracellular oxidation, and were reversed with L-mevalonate or geranylgeranylpyrophosphate, but not with farnesylpyrophosphate or cholesterol. Treatments with the Rho inhibitor C3 exotoxin and with cell-permeable superoxide dismutase also decreased AngII-induced O₂·̄ production and myocyte hypertrophy. Overexpression of the dominant-negative Rho mutant N17Rac1 completely inhibited AngII-induced intracellular oxidation and ANF promoter activity, while N19RhoA partially inhibited it, and N17Cdc42 had no effect. Indeed, Sim inhibited cardiac hypertrophy and decreased myocardial Rac1 activity and O₂·̄ production in rats treated with AngII infusion or subjected to transaortic constriction. These findings suggest that statins prevent the development of cardiac hypertrophy through an antioxidant mechanism involving inhibition of Rac1.

Cardiac hypertrophy represents an initial physiological adaptive response to increases in blood pressure or afterload (1). However, despite normalization of systemic blood pressure by medication, cardiac hypertrophy frequently decompensates into congestive heart failure (2). Indeed, cardiac hypertrophy is an independent risk factor in cardiovascular disease, and increases cardiovascular mortality by more than twofold (3). The hypertrophic process is characterized by the induction of immediate-early genes such as c-fos, c-jun, and erg-1; the reexpression of embryonic genes such as atrial natriuretic factor (ANF), β-myosin heavy chain, and skeletal α-actin (4); and the increased synthesis of contractile proteins such as myosin light chain-2 (MLC-2v) and cardiac α-actin (5, 6).

Stimulation of the angiotensin II (AngII) type I receptor or exposure to pressure overload induces a cardiac hypertrophic response mediated in part by the activation of the heterotrimeric G proteins (e.g., G_q) (7, 8) and small G proteins (e.g., Ras and Rho) (9–11). In particular, the Rho proteins, RhoA, Rac1, and Cdc42, are thought to play critical roles in the hypertrophic process by regulating cell morphology and contractile elements (12). For example, previous studies have shown that RhoA is required for Gα_q and α₁-receptor signaling in cardiac myocytes, and that dominant-negative mutants of RhoA attenuate cardiac myocyte hypertrophy in response to stimulation with phenylephrine (11, 13). Surprisingly, cardiac-specific overexpression of RhoA in mice does not lead to cardiac hypertrophy, but produces sinus and atrioventricular nodal dysfunction and development of ventricular failure (14). These results suggest that other pathways may contribute to the hypertrophic response. Indeed, Rac1, and not RhoA, is required for signal transduction pathways leading to cardiac myocyte hypertrophy (15).

Current treatments for cardiac hypertrophy are limited to vasodilators or afterload reducers, with few if any therapies directed at the myocardial process. The 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) reductase inhibitors, or statins, are widely prescribed cholesterol-lowering agents that decrease the incidence of myocardial infarction and ischemic stroke (16, 17). In addition to inhibiting cholesterol synthesis, the statins also inhibit the synthesis of important isoprenoid intermediates that are important lipid attachments required for the subcellular localization and function of a variety of proteins, including the Rho proteins (18, 19). The purpose of this study, therefore, was to determine whether statins can attenuate cardiac hypertrophy by inhibiting Rho proteins, and if so, to determine the potential underlying mechanism involved.

Cell culture. Relatively pure (>95%) neonatal ventricular myocytes were confirmed by their morphological features using phase-contact microscopy and immunofluorescent staining with a monoclonal anti-cardiac MHC antibody (data not shown). There were no observable adverse effects by AngII, Sim, L-mev, LDL, GGPP, FPP, GGTI-286, FTI-276, C3 TF, PEG-SOD, or PEG-CAT on cellular viability for any treatment conditions.

Statins inhibit AngII-induced cardiac myocyte hypertrophy. Cardiac myocyte hypertrophy is characterized by increased protein content (e.g., [³H]leucine uptake) and induction of fetal gene expression (e.g., ANF promoter activity). Treatment with AngII induced neonatal rat cardiac myocyte hypertrophy, as measured by changes in [³H]leucine uptake, total protein content, and ANF promoter activity (50% ± 6%, 20% ± 3%, and 180 ± 20%–fold increases, respectively; P < 0.01 for all conditions) (Figure 1a). Cotreatment with Sim (5 μM) inhibited the AngII-induced increases in [³H]leucine incorporation, total protein content, and ANF promoter activity by 71% ± 4%, 85% ± 8%, and 94% ± 5%, respectively. These inhibitory effects of Sim were completely reversed by cotreatment with L-mev or GGPP, but not with FPP or LDL (Figure 1). Treatment with Sim alone, however, did not affect basal [³H]leucine incorporation, total protein content, or ANF promoter activity. Similar effects were observed with other statins (e.g., lovastatin, atorvastatin), indicating a class effect of these agents on myocardial HMG CoA reductase.

Figure 1

Inhibition of cardiac myocyte hypertrophy by statins. (a) Effects of AngII or Sim (5 μM) with and without L-mev, GGPP, or FPP on total protein content, [³H] leucine incorporation, and ANF promoter activity. Values are expressed as mean ± SEM. *P < 0.01 compared with AngII alone. (b) Effects of Sim (5 μM) with and without LDL, GGTI, FTI, C3 TF, PEG, PEG-SOD, and PEG-CAT on AngII-induced [³H] leucine incorporation (Control). Values are expressed as mean ± SEM. *P < 0.01 compared with control.

Using selective inhibitors of geranylgeranylation and farnesylation at their optimal inhibitory concentrations, we found that GGTI-286 (30 μM), but not FTI-276 (20 nM), completely blocked AngII-induced [³H]leucine incorporation (Figure 1b). To determine whether inhibition of Rho proteins could be mediating the effects of Sim on cardiac myocyte hypertrophy, Rho proteins were directly inhibited by C3 TF (50 μg/ml) (29). Treatment with C3 TF inhibited the AngII-induced increase in [³H]leucine incorporation by 59% ± 5%. In contrast, direct activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 (30) partially reversed the inhibitory effects of Sim (data not shown). Since the Rho protein Rac1 is involved in the generation of reactive oxygen species (31, 32) and mediates signaling pathways leading to cardiac hypertrophy (15), we investigated whether superoxide anion (O₂·̄) or hydrogen peroxide (H₂O₂) could be playing a role in AngII-induced cardiac hypertrophy. Cotreatment with cell-permeable PEG-SOD, but not PEG-CAT or PEG alone, inhibited AngII-induced [³H]leucine incorporation (Figure 1b). These findings suggest that the hypertrophic process is more dependent upon O₂·̄ than on H₂O₂.

To determine whether Sim can affect cardiac myocyte cell size and sarcomeric organization, we immunostained cardiac myocytes for two sarcomere-associated proteins, desmin and α-actinin. Treatment with AngII induced significant increases in cell size and sarcomere organization (Figure 2a); cotreatment with Sim blocked these AngII-induced cellular changes. The hypertrophic process is also associated with the induction of fetal and structural cardiac genes such as ANF and MLC-2v (4, 33). Indeed, steady-state ANF and MLC-2v mRNA expression were each increased by about twofold following AngII stimulation (n = 3, P < 0.01) (Figure 2, b and c). Cotreatment with Sim inhibited AngII-induced ANF, and to a lesser extent, MLC-2v mRNA expression in cardiac myocytes. High basal expression of ANF and MLC-2v is attributed to the use of neonatal cardiac myocytes instead of adult cardiac cells. Nevertheless, these findings correlate with the inhibition of cardiac myocyte hypertrophy by Sim.

Figure 2

Inhibition of cardiac sarcomere organization, myocyte size, and fetal gene expression by statins. (a) Effects of AngII with and without Sim (5 μM) on cardiac myocyte size and sarcomere organization. Double immunofluorescent microscopy was performed using specific antibodies to desmin (upper panel, red color) and α-actinin (lower panel, green color). Experiments were performed three times with similar results. Effect of AngII with and without Sim (5 μM) on steady-state (b) ANF and (c) MLC-2v mRNA expression after 24 hours of treatment. Corresponding ethidium bromide–stained 28S ribosomal RNA was used to standardize loading. The results shown are representative of three separate experiments.

Inhibition of cardiac hypertrophy by statins is mediated by Rac1 and RhoA. Treatment with AngII increased membrane-associated RhoA and Rac1 GTP-binding activity by 147% and 87%, respectively (P < 0.05 for both) (Figure 3a). Cotreatment with Sim completely blocked AngII-induced increases in membrane-associated RhoA and Rac1 GTP-binding activity. These inhibitory effects of Sim were reversed by cotreatment with GGPP, but not FPP, indicating that Sim inhibited the membrane localization and activities of both RhoA and Rac1.

Figure 3

Inhibition of RhoA and Rac1 by statins. (a) Effect of AngII with and without Sim, GGPP, or FPP on membrane-associated Rac1 and RhoA GTP-binding activity in rat cardiac myocytes. Values are expressed as mean ± SEM. *P < 0.05 compared with unstimulated cells (Control). (b) Effects of transfection with dominant-negative Rho mutants (N17Rac1, N19RhoA, or N17Cdc42) with and without Sim (5 μM) or GGPP on AngII-induced ANF promoter activity. Values are expressed as mean ± SEM. *P < 0.01 compared with transfection with vector alone (Control). †P < 0.05 compared with AngII and dominant-negative Rho alone. Tx: DN-Rho = N17Rac1, N19RhoA, or N17Cdc42.

To assess which Rho protein mediates AngII-induced cardiac hypertrophy, we cotransfected rat cardiac myocytes with dominant-negative mutants of Rac1, RhoA, or Cdc42 and an ANF promoter–luciferase reporter construct. Transfection with dominant-negative Rac1 (N17Rac1), and to a lesser extent, RhoA (N19RhoA), inhibited AngII-induced ANF promoter activity (Figure 3b). Transfection with Cdc42 (N17Cdc42) had no inhibitory effect. Cotreatment with Sim further decreased ANF promoter activity in cells transfected with N19RhoA and N17Cdc42, but not N17Rac1. Indeed, N17Rac1 decreased ANF promoter activity to below basal levels (P < 0.05). Although cotreatment with GGPP reversed the inhibitory effects of Sim, GGPP could not reverse the inhibitory effect of N17Rac1 on ANF promoter activity. These results suggest that Rac1 is the predominant Rho protein that mediates AngII-induced cardiac hypertrophy.

Statins inhibit Rac1-induced O₂·̄ production. An important function of Rac in neutrophils (34), fibroblasts (31), and vascular smooth muscle cells (32) is facilitating the assembly of NADPH oxidase, which is a major source of O₂·̄ production in these cells. To determine whether the inhibitory effects of Sim on cardiac hypertrophy involve inhibition of O₂·̄ production, we monitored O₂·̄ released into the supernatant by cardiac myocytes using a ferricytochrome c reduction assay (25). AngII doubled O₂·̄ production (Figure 4a). This increase was blocked by cotreatment with Sim, C3 TF, and PEG-SOD (50 U/ml), but not with PEG-CAT (500 U/ml). Treatment with PEG or Sim alone had little or no effect on O₂·̄ production. Cotreatment with GGPP completely reversed the inhibitory effect of Sim on O₂·̄ production, but not the effects of C3 TF or PEG-SOD (data not shown).

Figure 4

Inhibition of superoxide anion production and intracellular oxidation by statins. (a) Effects of Sim (5 μM), GGPP, C3 TF, PEG-SOD, or PEG-CAT on AngII-induced O₂·̄ production in rat cardiac myocytes, as measured by ferricytochrome c reduction and aconitase assays. Values are expressed as mean ± SEM. *P < 0.01 compared with unstimulated cells (Control). (b) Effects of Sim (5 μM) with and without L-mev, GGPP, or FPP on AngII-induced intracellular oxidation (DCF fluorescence). Results shown were chosen from five random fields and are representative of three separate experiments.

To confirm that the effects of AngII and Sim on O₂·̄ production correspond to changes in cellular oxidative stress, we assessed total intracellular oxidation in rat cardiac myocytes by DCF fluorescence. Since H₂O₂ is the downstream dismutated product of O₂·̄, the level of DCF fluorescence, which correlates with increased levels of peroxide-derived reactive intermediates, may also serve as an useful marker for O₂·̄ production. Indeed, AngII-induced DCF fluorescence was completely blocked by the addition of PEG-CAT, which converts H₂O₂ to H₂O, but not by PEG-SOD, which enhances rather than inhibits the conversion of O₂·̄ to H₂O₂ (data not shown). Stimulation with AngII produced a 2.7-fold increase in intracellular oxidative stress, as measured by DCF fluorescence (n = 3, P < 0.05) (Figure 4b). This increase was completely blocked by cotreatment with Sim. The inhibitory effect of Sim on intracellular oxidation was reversed by L-mev and GGPP, but not by FPP, suggesting that Rho proteins were mediating the increase in O₂·̄-derived intracellular oxidation in response to AngII.

Rac1 is required for AngII-induced intracellular oxidation. To determine which Rho proteins mediate the AngII-induced increase in intracellular oxidation, we transfected rat cardiac myocytes with c-myc–tagged dominant-negative (N17 or N19) or constitutively active (L61 or L63) mutants of Rho, and performed dual fluorescence microscopy for c-myc and intracellular oxidation. Treatment with AngII increased intracellular oxidation, as determined by DCF fluorescence in nontransfected cells or cells transfected with dominant-negative RhoA or Cdc42 (Figure 5a). Transfection with dominant-negative Rac1, however, completely inhibited AngII-induced intracellular oxidation. Conversely, only cells transfected with constitutively active Rac1 showed increased intracellular oxidation; nontransfected cells or cells transfected with constitutively active RhoA or Cdc42 showed no such increase (Figure 5b). These results indicate that Rac1 mediates AngII-induced intracellular oxidation, and suggest that Sim inhibits intracellular oxidation by inhibiting Rac1.

Figure 5

Inhibition of Rac1-induced intracellular oxidation by statins. Intracellular oxidation in rat cardiac myocytes that were transfected with dominant-negative Rho mutants (N17 or N19) and stimulated with AngII (a), or transfected with constitutively active Rho mutants (L61 or L63) (b). Double fluorescent microscopy was performed to determine the expression of c-myc–tagged Rho mutants (left panel, red color) and corresponding levels of DCF fluorescence (right panel, green color). Experiments were performed three times with similar results. The arrows indicate transfected cardiac myocytes in corresponding panels.

Statins inhibit cardiac hypertrophy in vivo. To determine whether our in vitro findings have physiological relevance, we evaluated the effects of statins using two widely used models of cardiac hypertrophy (35, 36). AngII infusion caused a substantial increase in systolic blood pressure that was not affected by cotreatment with Sim (Table 1). Treatment with Sim alone had no effect on systemic blood pressure, BW, or total serum cholesterol levels. AngII infusion (200 ng/kg/day for 14 days) caused a significant increase in LV/BW ratio and LV mass. However, in the short duration of our experimental conditions, AngII did not produce significant deposition of collagen or fibrosis. The increases in LV/BW ratio and LV mass by AngII were inhibited in a concentration-dependent manner by cotreatment with Sim. Similar findings were also observed with atorvastatin, suggesting a class effect on myocardial HMG CoA reductase inhibition.

Table 1

Effects of statin on AngII-induced cardiac hypertrophy

In hearts from rats treated with AngII infusion, there were increases in membrane-associated RhoA and Rac1 GTP-binding activities, both of which were inhibited by cotreatment with Sim (Figure 6a). Indeed, AngII increased O₂·̄ production in intact, nonhomogenized rat heart tissues by 40% (from 42 ± 4 to 59 ± 5 nmol/mg, n = 6, P < 0.05), an effect that was completely blocked by cotreatment with Sim (34 ± 8 nmol/mg, n = 6, P > 0.05 compared to control) (Figure 6b). These results correlated inversely with a 41% decrease in aconitase activity in rat heart tissues after AngII infusion (from 440 ± 27 to 260 ± 41 nmol/min/mg, n = 6, P < 0.005), which was completely restored by cotreatment with Sim (460 ± 31 nmol/min/mg, n = 6, P > 0.05 compared to control). These findings suggest that inhibition of Rho proteins contributed to the statin’s inhibitory effect on AngII-induced O₂·̄ production in vivo.

Figure 6

Inhibition of Rac1 and superoxide anion production in rat hearts. Effects of AngII (200 ng/kg/min) infusion with and without Sim (20 mg/kg/day for 14 days) on (a) membrane-associated Rac1 and RhoA GTP-binding activities and (b) aconitase activity and O₂·̄ released from intact rat hearts. Values are expressed as mean ± SEM. *P < 0.05 compared with vehicle-treated animals (Control). †P < 0.05 compared with animals treated with AngII alone.

To determine whether the antihypertrophic effect of statins is a central mechanism that could be generalized to other etiologies of cardiac hypertrophy, we investigated the effects of Sim in a pressure-overload model of cardiac hypertrophy. TAC (aortic banding) did not affect systolic blood pressure or BW. However, TAC produced significant increases in the ratio of heart weight (HW) to BW compared with sham-operated mice (Figure 7a). These effects of TAC correlated with a 59% decrease in aconitase activity in mouse heart tissues (from 490 ± 22 to 201 ± 18 nmol/min/mg, n = 3, P < 0.01). Indeed, TAC resulted in anatomical and functional changes that were consistent with cardiac hypertrophy, such as increases in posterior wall thickness and LV end-systolic diameter, and a 25% decrease in fractional shortening or ejection fraction (Table 2). Cotreatment with Sim attenuated HW/BW ratio by approximately 50%, completely restored aconitase activity in mouse heart tissues, and improved cardiac diastolic and to a lesser extent, systolic function. Furthermore, treatment with Sim significantly decreased cardiac myocyte size, LV wall thickness, and gross cardiac dimensions (Figure 7b).

Figure 7

Inhibition of pressure overload–induced O₂·̄ production and cardiac hypertrophy by statins. (a) Effects of TAC with and without Sim (2 mg/kg/day for 4 weeks) on HW/BW ratio in mice and aconitase activity in mouse hearts. Values are expressed as mean ± SEM. *P < 0.01 compared with vehicle-treated sham-operated animals (Sham). †P < 0.05 compared with TAC treatment without Sim. (b) Cardiac hypertrophy was assessed by hematoxylin and eosin staining of heart tissues (top row; bar = 10 μm) and gross postmortem cross-sectional examination of the heart at the level just below the mitral valve (bottom row; bar = 1 mm).

Table 2

Echocardiographic analysis of mice with TAC

Statins prevent the development of cardiac hypertrophy in a cholesterol-independent manner. The mechanism is due in part to the inhibition of isoprenoid synthesis, Rho geranylgeranylation, and Rac1-induced O₂·̄ production in cardiac myocytes. Indeed, we found that the nonspecific antioxidant, N-acetylcysteine, was just as effective as statins in inhibiting AngII-induced cardiac hypertrophy (data not shown). However, further studies need to be performed to determine the precise mechanism by which N-acetylcysteine inhibits cardiac hypertrophy, and whether it involves its antioxidant effects. Nevertheless, the ability of statins to decrease O₂·̄ production via inhibition of Rac1 suggests that they may have clinical benefits beyond lipid lowering in attenuating the hypertrophic process.

Evidence is emerging that statins and antioxidants could have inhibitory effects on cardiac hypertrophy (37–39), although the mechanism for these effects remains largely unknown. For example, a recent study showed that statins could inhibit cardiac hypertrophy and fibrosis in a rabbit β-myosin heavy chain-Q⁴⁰³ mutant model of cardiac hypertrophy without affecting Rac-GTP activity (38). These results suggest that the antihypertrophic effects of statins may not be due to the inhibition of Rho proteins alone, but may also involve the inhibition of other downstream signaling pathways. In contrast to other studies, we did not observe any increase in cardiac fibrosis in our AngII infusion model, probably due to the lower concentration of AngII and the shorter treatment period used in our study. Indeed, cardiac hypertrophy and fibrosis are observed with higher concentrations of AngII (40). Our findings are in agreement with a previous study showing that statins attenuate vascular smooth muscle cell hypertrophy by inhibiting Rac-induced O₂·̄ production (32).

The antihypertrophic effect of statins could also be due, in part, to decreases in AngII type 1 receptor expression (32) or myocardial angiotensin-converting enzyme activity (37). However, the ability of statins to inhibit cardiac myocyte hypertrophy in response to other agonists such as endothelin-1 and phenylephrine, and to pressure overload, suggests that the predominant mechanism of action includes mechanisms that are distal to the AngII type 1 receptor. Statin therapy also increases vascular nitric oxide production (23), which can potentially decrease blood pressure and attenuate the hypertrophic process. However, there were no significant changes in systemic arterial systolic blood pressure in animals treated with statins in our study. Finally, nitric oxide may also contribute to the direct antihypertrophic effects of statins through favorable effects on cardiac performance and metabolism (41). Further studies with statins in eNOS^–/– mice should help clarify these issues.

Statins inhibit the synthesis of important isoprenoid intermediates such as GGPP and FPP, which are important lipid attachments for the posttranslational modification of small GTP-binding proteins such as Rho (18, 19). Rho proteins play a critical role in mediating the development of cardiac hypertrophy. For example, RhoA controls the formation of actin stress fiber and focal adhesion complexes through activation of Rho kinases and myosin light chain phosphorylation (42). Rac1 and Cdc42 regulate actin cytoskeletal processes called lamellipodia (43) and filopodia (44), which may be involved in the morphological changes associated with cardiac hypertrophy. In addition, Rho proteins could also regulate the hypertrophic process by activating downstream signaling molecules such as mitogen-activated protein (MAP) kinases (11, 13). Our finding that overexpression of dominant-negative Rac1 completely inhibited intracellular oxidation and cardiac myocyte hypertrophy, whereas RhoA caused a lesser inhibition and Cdc42 none at all, is in agreement with a previous study showing that Rac1 is required for signal transduction pathways leading to cardiac myocyte hypertrophy (15).

Since the hypertrophic response is a mechanism of physiological adaptation to increases in afterload, it is not entirely clear whether inhibition of the hypertrophic process at the expense of decreasing cardiac performance would actually be beneficial. For example, in experimental models of LV pressure overload, inhibition of the hypertrophic response leads to the rapid development of dilated cardiomyopathy and heart failure (45). Similar detrimental effects often occur following myocardial infarction, if the hypertrophic process or myocardial remodeling is inhibited (46). However, despite these concerns, our results indicate that statins not only decreased cardiac hypertrophy, but also improved cardiac performance by mostly reducing LV end-diastolic dimensions, even in the presence of persistent elevations in systemic blood pressure and afterload. The precise mechanism(s) underlying these properties of statins is unknown, but it may involve enhancing nitric oxide–mediated improvement in myocardial energy metabolism (41).

In summary, statins are effective agents for preventing the development of cardiac hypertrophy. Of clinical significance is that hypertension, not hypercholesterolemia, is the main risk factor for cardiac hypertrophy (2). These results, therefore, suggest a novel pharmacological approach to treating cardiac hypertrophy in a patient population where statin therapy may not otherwise be indicated. Supporting data from large clinical trials, however, are needed before such recommendations can be made.

We thank Stefan Frantz and Gail K. Adler for technical assistance, and Alan Hall and Wouter Moolenaar for Rho constructs. This work was supported by grants from NIH (HL-52233, HL-62602, and HL-48743, to J.K. Liao), the American Heart Association Bugher Foundation Award (to J.K. Liao), the Banyu-Merck Fellowship in Lipid Metabolism and Atherosclerosis (to M. Takemoto and H. Nakagami), and the Feodor Lynen Fellowship from the Alexander von Humboldt Stiftung (to M. Grimm).

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