Statins as antioxidant therapy for preventing cardiac myocyte hypertrophy

Abstract

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.

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.

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.

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.

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.

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.

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.

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).