Blunted Akt/FOXO signalling and activation of genes controlling atrophy and fuel use in statin myopathy

Abstract

Statins are used clinically for cholesterol reduction, but statin therapy is associated with myopathic changes through a poorly defined mechanism. We used an in vivo model of statin myopathy to determine whether statins up-regulate genes associated with proteasomal- and lysosomal-mediated proteolysis and whether PDK gene expression is simultaneously up-regulated leading to the impairment of muscle carbohydrate oxidation. Animals were dosed daily with 80 mg kg(-1) day(-1) simvastatin for 4 (n = 6) and 12 days (n = 5), 88 mg kg(-1) day(-1) simvastatin for 12 days (n = 4), or vehicle (0.5% w/v hydroxypropyl-methylcellulose and 0.1% w/v polysorbate 80; Control, n = 6) for 12 days by oral gavage. We found, in biceps femoris muscle, decreased Akt(Ser473), FOXO1(Ser253) and FOXO3a(Ser253) phosphorylation in the cytosol (P < 0.05, P < 0.05, P < 0.001, respectively) and decreased phosphorylation of FOXO1 in the nucleus after 12 days simvastatin when compared to Control (P < 0.05). This was paralleled by a marked increase in the transcription of downstream targets of FOXO, i.e. MAFbx (P < 0.001), MuRF-1 (P < 0.001), cathepsin-L (P < 0.05), PDK2 (P < 0.05) and PDK4 (P < 0.05). These changes were accompanied by increased PPARalpha (P < 0.05), TNFalpha (P < 0.01), IL6 (P < 0.01), Mt1A (P < 0.01) mRNA and increased muscle glycogen (P < 0.05) compared to Control. RhoA activity decreased after 4 days simvastatin (P < 0.05); however, activity was no different from Control after 12 days. Simvastatin down-regulated PI3k/Akt signalling, independently of RhoA, and up-regulated FOXO transcription factors and downstream gene targets known to be implicated in proteasomal- and lysosomal-mediated muscle proteolysis, carbohydrate oxidation, oxidative stress and inflammation in an in vivo model of statin-induced myopathy. These changes occurred in the main before evidence of extensive myopathy or a decline in the muscle protein to DNA ratio.

Figure 1. Protein expression of Akt, FOXO1 and FOXO3a in biceps femoris muscle of Control animals and animals dosed daily with 80 mg kg⁻¹ day⁻¹ simvastatin for 4 and 12 days and 88 mg kg⁻¹ day⁻¹ simvastatin for 12 days

A, total Akt (tAkt) and phosphorylated Akt (pAkt) in the cytosolic protein fraction; B, total FOXO1 (tFOXO1) and phosphorylated FOXO1 (pFOXO1) in the cytosolic protein fraction; C, total FOXO1 (tFOXO1) and phosphorylated FOXO1 (pFOXO1) in the nuclear protein fraction; D, total FOXO3a (tFOXO3a) and phosphorylated FOXO3a (pFOXO3a) in the cytosolic protein fraction; E, total FOXO3a (tFOXO3a) and phosphorylated FOXO3a (pFOXO3a) in the nuclear protein fraction. Phosphorylated to total protein ratios are described in Results. Values expressed as mean optical density ±s.e.m.*P < 0.05, **P < 0.01, ***P < 0.001 when compared to Control. Images show a representative Western blot of the protein of interest and the corresponding actin or lamin blot.

Figure 1. Protein expression of Akt, FOXO1 and FOXO3a in biceps femoris muscle of Control animals and animals dosed daily with 80 mg kg⁻¹ day⁻¹ simvastatin for 4 and 12 days and 88 mg kg⁻¹ day⁻¹ simvastatin for 12 days

A, total Akt (tAkt) and phosphorylated Akt (pAkt) in the cytosolic protein fraction; B, total FOXO1 (tFOXO1) and phosphorylated FOXO1 (pFOXO1) in the cytosolic protein fraction; C, total FOXO1 (tFOXO1) and phosphorylated FOXO1 (pFOXO1) in the nuclear protein fraction; D, total FOXO3a (tFOXO3a) and phosphorylated FOXO3a (pFOXO3a) in the cytosolic protein fraction; E, total FOXO3a (tFOXO3a) and phosphorylated FOXO3a (pFOXO3a) in the nuclear protein fraction. Phosphorylated to total protein ratios are described in Results. Values expressed as mean optical density ±s.e.m.*P < 0.05, **P < 0.01, ***P < 0.001 when compared to Control. Images show a representative Western blot of the protein of interest and the corresponding actin or lamin blot.

Figure 2. mRNA expression (fold changes relative to Control) determined in biceps femoris muscle of Control animals and animals dosed daily with 80 mg kg⁻¹ day⁻¹ simvastatin for 4 and 12 days and 88 mg kg⁻¹ day⁻¹ simvastatin for 12 days

A, MAFbx, MuRF-1 and cathepsin-L expression; B, PDK2, PDK4 and PPARα expression; C, TNFα, IL6 and Mt1A expression. Values expressed as mean fold changes relative to Control ±s.e.m.*P < 0.05, **P < 0.01, ***P < 0.001 when compared to Control (which is set at 1).