Skeletal muscle metabolic adaptations to endurance exercise training are attainable in mice with simvastatin treatment

Abstract

We tested the hypothesis that a 6-week regimen of simvastatin would attenuate skeletal muscle adaptation to low-intensity exercise. Male C57BL/6J wildtype mice were subjected to 6-weeks of voluntary wheel running or normal cage activities with or without simvastatin treatment (20 mg/kg/d, n = 7-8 per group). Adaptations in in vivo fatigue resistance were determined by a treadmill running test, and by ankle plantarflexor contractile assessment. The tibialis anterior, gastrocnemius, and plantaris muscles were evaluated for exercised-induced mitochondrial adaptations (i.e., biogenesis, function, autophagy). There was no difference in weekly wheel running distance between control and simvastatin-treated mice (P = 0.51). Trained mice had greater treadmill running distance (296%, P<0.001), and ankle plantarflexor contractile fatigue resistance (9%, P<0.05) compared to sedentary mice, independent of simvastatin treatment. At the cellular level, trained mice had greater mitochondrial biogenesis (e.g., ~2-fold greater PGC1α expression, P<0.05) and mitochondrial content (e.g., 25% greater citrate synthase activity, P<0.05), independent of simvastatin treatment. Mitochondrial autophagy-related protein contents were greater in trained mice (e.g., 40% greater Bnip3, P<0.05), independent of simvastatin treatment. However, Drp1, a marker of mitochondrial fission, was less in simvastatin treated mice, independent of exercise training, and there was a significant interaction between training and statin treatment (P<0.022) for LC3-II protein content, a marker of autophagy flux. These data indicate that whole body and skeletal muscle adaptations to endurance exercise training are attainable with simvastatin treatment, but simvastatin may have side effects on muscle mitochondrial maintenance via autophagy, which could have long-term implications on muscle health.

Fig 1. The effects of exercise training and simvastatin treatment on treadmill running capacity and contractile fatigue resistance (n = 7–8 per exercise/treatment group).

A: Distance run during a progressive velocity treadmill running test to exhaustion. Run > Sed indicates main effect of training, P < 0.001. B: Plantarflexor torque loss following a fatiguing bout of 120 contractions. Run > Sed indicates main effect of training, P < 0.040. Torque loss is expressed as a percent of the peak torque generated during the protocol.

Fig 2. The effects of exercise training and simvastatin treatment on plantarflexor muscle torque and mass (n = 7–8 per exercise/treatment group).

A: Peak isometric torque (left bars) and peak isometric torque normalized by plantarflexor muscle mass (right bars). Con > Statin indicates a strong trend for main effect of treatment, P = 0.051. B: Plantarflexor muscle masses normalized by grams of BW. Con > Statin indicates main effect of treatment, P = 0.039. The plantarflexor muscle mass represents the combined masses of the gastrocnemius, soleus, and plantaris muscles.

Fig 3. The effects of exercise training and simvastatin treatment on mitochondrial and vascular gene expressions (n = 7–8 per exercise/treatment group).

A: Fold change relative to control sedentary mice in expression of genes involved in mitochondrial biogenesis. Run > Sed indicates main effect of training, all P < 0.002. B: Fold change relative to control sedentary mice in expression of genes involved in vascular signaling. Run > Sed indicates main effect of training, all P ≤ 0.046.

Fig 4. The effects of exercise training and simvastatin treatment on markers of autophagy.

A: Immunoblot images of Ulk1, p-Ulk1:Ulk1, Beclin1, p62, Bnip3, LC3-I, and LC3-II in plantaris muscle. β-actin was used as a loading control. B—C: Quantitative and statistical analysis of data in A (sed n = 8; sed+statin n = 7; run n = 5; run+statin n = 8). Run > Sed indicates main effect of training, all P ≤ 0.049. * indicates significantly different from control run, statin sedentary and statin run, all P ≤ 0.046.

Fig 5. The impact of simvastatin treatment on mitochondrial fusion and fission.

A: Immunoblot images of mitofusin 2 (Mfn2), and Drp1. β-actin was probed as a loading control. B: Quantitative and statistical analysis of data in A (sed n = 8; sed+statin n = 7; run n = 5; run+statin n = 8). Con > Statin indicates a main effect of treatment, P = 0.040.

Fig 6. Exercise training-induced effects on mitochondrial biogenesis and maintenance, and potential contraindicative effects of statin use.

Normal mitochondrial maintenance (black arrows) is theoretically characterized by the balance between the addition of healthy mitochondria (mitochondria with white fill) via mitochondrial biogenesis and removal of damaged mitochondria (mitochondrial with red fill) via autophagy. Herein, short-term statin use was associated with a decrease in mitochondrial fission protein Drp1 and altered autophagy-related protein LC3 content, which may contribute to disrupted mitochondrial maintenance. Long-term disruption in the mitochondrial maintenance (red dashed arrow) may lead to accumulation of damaged mitochondria. More directed research is necessary to understand the potential long-term impact of statin use on mitochondrial fission, autophagy, and function.