FoxO1 Regulates Skeletal Muscle Fiber Type Specification And Transition

Skeletal muscle is comprised of heterogeneous specialized muscle fibers that differ in their size, metabolic and contractile properties. On the basis of specific myosin heavy chain (MyHC) isoform expression, adult skeletal muscle fibers are generally categorized as MyHC I (slow oxidative), MyHC IIa (fast oxidative), MyHC IIx/d (fast oxidative-glycolytic) and MyHC IIb (fast glycolytic). Fiber composition in adult skeletal muscle is regulated in response to changes in endurance exercise, environment, or pathological conditions. The Forkhead box O1 (FoxO1) transcription factor governs muscle growth, metabolism and cell differentiation and has been shown to be involved in regulating muscle fiber type specification. However, to date the mechanism behind FoxO1-mediated fiber type diversity is still unclear. Here we constructed stably FoxO1 over-expressed C2C12 myoblast mediated by liposome and adopted endurance exercise program, immunofluorescence, quantitative real-time RT-PCR, Western blotting and phosphatase activity assay methods to explore the potential role of FoxO1 in control of specification of muscle fiber type. The main results were as follows:1. Endurance swimming training switch the fiber from fast to slow-twitch. The real-time PCR showed that mouse slow-oxidative soleus consists of about 60% MyHC I slow fibers and 40% MyHC IIa fast oxidative fibers, while fast-glycolytic gastroenemius is mainly composed of MyHC IIb and MyHC IIx fast fibers (>90%, together). After an chronic training based on 4 weeks with daily swimming training, we found an increase in the percentage of MyHC I and a concomitant decrease in MyHC IIa in soleus while an up-regulation of MyHC IIa and IIx but a down-regulation of MyHC IIb in gastroenemius of trained mice compared to untrained mice (P<0.05).2. Inactivation of FoxO1 after long-term swimming training. The real-time PCR showed that FoxO1 expression was significantly related to muscle fiber type distribution and expressed preferentially in muscle enriched in fast fibers (P<0.05). Accordingly, the comparison between gastrocnemius and soleus in control mice showed that FoxO1 protein expression was three to four times greater in the fast than in the slow muscles (P<0.05). A significant reduction of FoxO1 mRNA was detected in both slow-oxidative and fast-glycolytic muscles of trained mice when compared to untrained mice. Surprisingly, Western blotting did not detect any change in the amount of total FoxO1 protein in both soleus and gastroenemius muscles of the trained and the untrained mice. However, an increase in FoxO1 phosphorylation at Ser256 was observed in both slow and fast muscles after long-term endurance exercise.3. FoxO1 inhibits C2C12 myoblast differentiation. First we stably transfected C2C12 mouse cell line with plasmids expressing FoxO1-WT and FoxO1-A3 (the constitutively active FoxO1). Then we detected the time-course expression of early (MyoD), intermediate (myogenin) and late (MyHC) myogenic transcription factors during the differentiation of skeletal muscle C2C12 myoblasts. Overexpression of FoxO1 mutant had a little reduction in MyoD expression but a approximately 40% decrease in myogenin protein at 3 days differentiation.4. FoxO1 induces slow to fast-twitch fiber transition. In control infected C2C12 myotubes, MyHC IIx mRNA was the most highly expressed adult isoform, followed by MyHC IIb, MyHCⅠand MyHC IIa. C2C12 myotubes induced by FoxO1 was attributable to combine effects of the up-regulation of MyHC2x and the down-regulation of MyHC I, MyHC IIa and MyHC IIb. Western blotting analysis indicated FoxO1 enhanced fast MyHC protein but suppressed MyHC slow protein, which was consistent with the MyHCs transcript expression. Notably, the relative expression of the total sarcomeric MyHC protein was also decreased in FoxO1-expressing cells.5. FoxO1 decreases oxidative capacity in C2C12 myotube. We treated cells with resveratrol (RSV, 100μM), which inhibited the endogenous FoxO1 activity. The results showed treatment of cells with RSV increased the oxidative fibers related gene TnI slow and myoglobin whereas was not enough to change the ratio of fast glycolytic to slow oxidative type muscle fibers. Overexpression of FoxO1 largely blocked the RSV-induced increase in TnI slow and myoglobin expression. Importantly, in FoxO1-infected C2C12 cells addition of RSV reversed FoxO1-induced muscle fiber switch.6. FoxO1 inhibits calcineurin phosphatase activity. In this study, relative to empty vector–infected control cells, cells expressing FoxO1-A3 showed a significant decrease in endogenous calcineurin phosphatase activity. FoxO1 activation significantly decreased the mRNA abundance of modulatory calcineurin interacting protein exon 4 isoform (MCIP1.4), a direct target of the calcineurin/NFAT pathway. We tested the effect of RSV treatment on calcineurin activity and found no change. However, quantitative real-time PCR showed that inhibition of FoxO1 activity by RSV markedly increased the mRNA abundance of MCIP1.4. Furthermore, RSV antagonized FoxO1-induced down-regulation of MCIP1.4 expression.