Inhibition of FoxO transcriptional activity prevents muscle fiber atrophy during cachexia and induces hypertrophy

Abstract

Cachexia is characterized by inexorable muscle wasting that significantly affects patient prognosis and increases mortality. Therefore, understanding the molecular basis of this muscle wasting is of significant importance. Recent work showed that components of the forkhead box O (FoxO) pathway are increased in skeletal muscle during cachexia. In the current study, we tested the physiological significance of FoxO activation in the progression of muscle atrophy associated with cachexia. FoxO-DNA binding dependent transcription was blocked in the muscles of mice through injection of a dominant negative (DN) FoxO expression plasmid prior to inoculation with Lewis lung carcinoma cells or the induction of sepsis. Expression of DN FoxO inhibited the increased mRNA levels of atrogin-1, MuRF1, cathepsin L, and/or Bnip3 and inhibited muscle fiber atrophy during cancer cachexia and sepsis. Interestingly, during control conditions, expression of DN FoxO decreased myostatin expression, increased MyoD expression and satellite cell proliferation, and induced fiber hypertrophy, which required de novo protein synthesis. Collectively, these data show that FoxO-DNA binding-dependent transcription is necessary for normal muscle fiber atrophy during cancer cachexia and sepsis, and further suggest that basal levels of FoxO play an important role during normal conditions to depress satellite cell activation and limit muscle growth.

Figure 1.

Cachexia increases the mRNA expression of FoxO family members. Relative mRNA levels of FoxO1, FoxO3a, and FoxO4 in soleus (A, C) and TA muscle (B, D) at 14 d after LLC cell injection (A, B) and after 4 d of sepsis (C, D). Bars represent means ± se for 6 muscles/group. *P < 0.05 vs. control.

Figure 2.

Cachexia increases FoxO transcriptional activity. FoxO-dependent luciferase reporter activity from the soleus (A, C) and TA muscles (B, D) of control and LLC tumor-bearing mice (A, B) and control and septic mice (C, D). Muscles were injected with an empty vector or DN FoxO plasmid and harvested ∼14 d after LLC cell injection or after 4 d of sepsis. Bars represent means ± se for 6 muscles/group. *P < 0.05 vs. control empty vector.

Figure 3.

FoxO transcriptional activation is required for the cachexia-induced increase in the mRNA levels of selected atrophy-related genes. Relative mRNA levels of MuRF1, atrogin-1, cathepsin L, and Bnip3 from soleus (A, C) and TA muscles (B, D) injected with an empty vector or DN FoxO plasmid and harvested after ∼14 d from control and LLC tumor-bearing mice (A, B) or after 4 d from control and septic mice (C, D). Bars represent means ± se for 6 muscles/group. *P < 0.05 vs. control (Con) empty vector; ^†P < 0.05 vs. tumor-bearing (TB) or septic (Sep) empty vector.

Figure 4.

FoxO transcriptional activation is necessary for cachexia-induced muscle fiber atrophy. A, B, E, F) Representative cross sections taken from the TA (A, E) and soleus muscles (B, F) of control and ∼14 d LLC tumor-bearing mice (A, B), and control and 7-d septic mice (E, F), each transfected with either DsRed or DN FoxO-DsRed. Sections were incubated with wheat germ agglutinin to allow for visualization of muscle fibers and the CSA of transfected fibers (fibers expressing either DsRed or DN FoxO-DsRed) were measured and compared. Arrows indicate representative DN FoxO-DsRed expression, which we have previously shown localizes to the nucleus. C, D) Mean TA (C) and soleus (D) fiber CSA from control and LLC tumor bearing mice transfected with either DsRed or DN FoxO-DsRed in panels A and B, respectively. G, H) Mean TA (G) and soleus (H) fiber CSA from control and septic mice transfected with either DsRed or DN FoxO-DsRed in panels E and F, respectively. Bars represent means ± se for 6 muscles/group. *P < 0.05 vs. control DsRed; ^†P < 0.05 vs. tumor-bearing or septic DsRed.

Figure 5.

Inhibition of FoxO activity induces myofiber hypertrophy via increased de novo protein synthesis. A, B) Soleus and TA muscles were transfected with an empty vector or a DN FoxO expression plasmid; 4 d later, mice were injected with cycloheximide or vehicle for 6 d. Myofiber hypertrophy in soleus (A) and TA (B) muscle fibers expressing DN FoxO in vehicle-injected mice was prevented in mice injected with the protein synthesis inhibitor cycloheximide. C) Representative images and mean myotube diameter of C2C12 myotubes transfected (as myoblasts) with either DsRed or DN FoxO-DsRed and treated with vehicle or rapamycin (Rapa) for 72 h. *P < 0.05 vs. control empty vector or vehicle DsRed.

Figure 6.

Inhibition of FoxO activity activates muscle satellite cells. Soleus and TA muscles were transfected with an empty vector or DN FoxO; 7 d later, the mice were injected with bromodeoxyuridine (BrdU) for 4 d. A–D) Representative cross sections of the soleus (A, B) and TA (C, D), showing BrdU incorporation visualized by immunohistochemistry. Total number of BrdU-positive nuclei under the basal lamina (A, C) and under dystrophin (B, D) were counted in each muscle. Arrows indicate representative BrdU-positive nuclei under the basal lamina or dystrophin. E, F) Mean numbers of BrdU-positive nuclei per ×20 field under laminin and dystrophin in the soleus (E) and TA (F). G, H) Relative mRNA level of Pax7 (G) and MyoD (H) in TA and soleus muscles transfected with an empty vector or DN FoxO. Bars represent means ± se for 6 muscles/group. *P < 0.05 vs. respective empty vector group.

Figure 7.

Inhibition of FoxO activity decreases activity of the myostatin/Smad pathway. A) Relative mRNA level of myostatin in TA muscle injected and electroporated with an empty vector or DN FoxO. B, C) Smad-dependent reporter activity in C2C12 cells transfected with an empty vector or DN FoxO and differentiated for 3 d and then harvested (B) or treated with vehicle or recombinant myostatin for 12 h and then harvested (C). Data in C are normalized to their respective vehicle-treated group, and are therefore expressed as with or without (±) myostatin. D) Representative images and mean myotube diameter of C2C12 myotubes transfected (as myoblasts) with either DsRed or DN FoxO-DsRed and treated with vehicle or myostatin for 96 h. *P < 0.05 vs. control empty vector or vehicle.

Figure 8.

Proposed mechanisms for Foxo-DNA binding-dependent transcription in the maintenance of muscle fiber size. A) During normal conditions, maintenance of muscle fiber size is controlled through balancing protein synthesis and protein degradation. Growth factor (G.F.) stimulation and myostatin-mediated repression contribute to basal levels of Akt signaling and protein synthesis, mediated through mTOR. Foxo signaling, while predominantly inactive under normal conditions, regulates the transcription of myostatin, as well as genes involved in protein degradation, which at basal levels, helps to balance protein synthesis and maintain muscle fiber size. In addition, satellite cells are maintained in a quiescent state during normal conditions, which is mediated, in part, through myostatin. B) In the current study, we demonstrate that muscle hypertrophy accompanied by satellite cell proliferation and fusion into muscle fibers is induced following repression of basal levels of Foxo-DNA binding-dependent transcription. This hypertrophy required active mTOR signaling and de novo protein synthesis. Because the hypertrophy induced via repression of Foxo also required a decrease in myostatin, which is known to increase satellite cell proliferation, mTOR signaling, and protein synthesis, as well as cause hypertrophy, our data support a model in which the basal activity of Foxo maintains muscle size through regulating myostatin transcription.