Simvastatin inhibits noradrenaline-induced hypertrophy of cultured neonatal rat cardiomyocytes

Abstract

1. Oxidative mechanisms have been implicated in neonatal cardiomyocyte hypertrophy. We and others have shown that a HMG-CoA reductase inhibitor preserves endogenous antioxidant enzyme activity and inhibits cardiac hypertrophy in vivo. We therefore have examined whether noradrenaline (NA) induces the generation of reactive oxygen species (ROS) during its induction of neonatal cardiomyocyte hypertrophy and whether simvastatin, a HMG-CoA reductase inhibitor, attenuates ROS production and thus NA-induced hypertrophy of cardiomyocytes. 2. NA increased the intracellular ROS levels in a concentration-dependent manner. This increase of ROS was significantly inhibited by simvastatin and catalase. Prazosin partially suppressed NA-induced increase of ROS and beating, while preincubation with both prazosin and propranolol completely abolished NA-evoked increase of ROS and beating. Simvastatin did not affect NA-induced increase of beating. 3. The NA-induced increase of protein content was partially suppressed by prazosin and completely abolished by preincubation with both prazosin and propranolol. Simvastatin inhibited the increase of NA-induced increase of RNA content and [(3)H]-leucine incorporation in a concentration-dependent manner. Mevalonic acid (MVA) reversed the inhibition of NA-induced RNA and protein increase by simvastatin. Catalase also inhibited the NA-induced increase of RNA and protein. 4. We conclude that the inhibitory effects of simvastatin on myocyte hypertrophy were associated with its antioxidant effects and inhibition of MVA-metabolism pathway in neonatal rat cardiomyocytes. NA-induced increases of intracellular ROS and cardiomyocyte hypertrophy requires both alpha and beta adrenoceptors activation in neonatal rat cardiomyocytes. The increases of ROS induced by NA is required for hypertrophy.

Figure 1

Inhibitory effects of simvastatin on PI fluorescence increase induced by NA. On culture day 4, cultured cardiomyocytes were treated with NA (20 μmol l⁻¹), NA+SIM (1 – 1000 nmol l⁻¹), NA+SIM+MVA (100 μmol l⁻¹), NA+catalase (200 u ml⁻¹) or diluent without NA (control). After 3 days of treatment, cardiomyocytes were incubated with PI and fluorescence intensity was measured by laser confocal microscopy. (A) Representative cardiomyocytes observed by laser confocal microscopy. (B) Histograms representing the mean±s.e.mean obtained from four separate experiments in which eight visual fields were quantified. ^##P<0.01 vs control. ^$$P<0.01 vs NA. ⁺⁺P<0.01 vs NA+SIM.

Figure 2

(A) Inhibitory effects of simvastatin on NA- induced increase in [³H]-leucine incorporation. On culture day 4, cultured cardiomyocytes were treated with NA (20 μmol l⁻¹), NA+SIM (1 – 1000 nmol l⁻¹), NA+SIM+MVA (100 μmol l⁻¹), NA+catalase (200 u ml⁻¹) or diluent without NA (control). After 3 days of treatment, incorporated [³H]-leucine was counted. Each point is mean±s.e.mean obtained from four separate experiments. ^##P<0.01 vs control. ^$P<0.05, ^$$P<0.01 vs NA. ⁺⁺P<0.01 vs NA+SIM. (B) Inhibitory effects of simvastatin on increase in protein content induced by NA. On culture day 4, cultured cardiomyocytes were treated with NA (20 μmol l⁻¹), NA+SIM (1 – 1000 nmol l⁻¹), NA+SIM+MVA (100 μmol l⁻¹), NA+catalase (200 u ml⁻¹) or diluent without NA (control). After 3 days of treatment, protein content was measured. Each point is mean±s.e.mean obtained from four separate experiments. ^#P<0.05, ^##P<0.01 vs control. ^$P<0.05, ^$$P<0.01 vs NA. ⁺⁺P<0.01 vs NA+SIM.

Figure 3

Concentration-dependent increase in DCF-DA fluorescence by NA. On culture day 4, cultured cardiomyocytes were treated with NA (0.2 – 20 μmol l⁻¹), NA (20 μmol l⁻¹)+catalase (200 u ml⁻¹), NA+PRA (20 μmol l⁻¹), NA+PRO (20 μmol l⁻¹), NA+PRA+PRO or diluent without NA (control) and simultaneously with DCF-DA (5 μmol l⁻¹). After 1 h of incubation, fluorescence intensity was measured by laser confocal microscopy. (A) Representative living cardiomyocytes observed by laser confocal microscopy. (B) Histograms representing the mean±s.e.mean obtained from four separate experiments in which eight visual fields were quantified. ^#P<0.05, ^##P<0.01 vs control. ^$$P<0.01 vs NA (20 μmol l⁻¹).

Figure 4

Inhibitory effects of simvastatin on DCF-DA fluorescence increase induced by NA. On culture day 4, cultured cardiac myocytes were treated with NA (20 μmol l⁻¹), NA+SIM (10 – 1000 nmol l⁻¹) or diluent without NA (control) and simultaneously with DCF-DA (5 μmol l⁻¹). After 1 h of incubation, fluorescence intensity was measured by laser confocal microscopy. Each point is mean±s.e.mean obtained from four separate experiments in which eight visual fields were quantified. ^#P<0.05, ^##P<0.01 vs control. ^$$P<0.01 vs NA.