Files in this Data Supplement:
Fig. S1. Synergist ablation model. (A) A graphic representation of the dorsal aspect of a rodent lower hindlimb showing the superficial gastrocnemius, which is removed during the synergist ablation (SA) surgery to reveal the underlying soleus and plantaris muscles adapted with permission from Gordon et al. (Gordon et al., 2001). The soleus is subsequently excised leaving behind only the plantaris muscle to assume the function of the gastrocnemius and soleus muscles. (B) SA for one (SA-1), two (SA-2) or four (SA-4) weeks produces a significant increase in plantaris muscle mass with >80% increase in muscle mass occurring within the first two weeks (n=4-5/group). Red dotted lines indicate 100% and 200% of sham control normalized plantaris muscle weight. Asterisk indicates significant difference (P<0.05) from SA-1. Hash indicates significant difference (P<0.05) from sham control.
Fig. S2. Hypertrophic response to synergist ablation in satellite cell-depleted muscle. (A) Satellite cell abundance was determined by Pax7 IHC co-localized with DAPI staining in SA-2 plantaris muscle from vehicle- and tamoxifen-treated animals. Scale bar: 100 µm. Arrowheads indicate satellite cells as assessed by Pax7+/DAPI+ staining. (B-D) Single fiber mechanical analyses showed no differences in ktr in maximally activating solution (maximal rate of cross-bridge cycling; B), pCa50 (Ca2+ concentration required for half-maximal activation; C) and n (Hill coefficient, indicative of the cooperativity of the contractile apparatus; D). (E) Fiber cross-sectional area analysis showed a significant increase in the number of fibers with cross-sectional area of 600-1400 µm2 in SA-2 groups (vehicle, white bar; tamoxifen, black bar; n=8/group) relative to respective sham control (vehicle, light gray bar; tamoxifen, dark gray bar; n=6/group). (F) Protein concentration (protein/muscle weight, mg/mg) was maintained following SA-2 with no difference between groups. (G) Total RNA increased in response to SA-2 but was not different between vehicle and tamoxifen groups. (H,I) Akt (H)and mTOR (I) signaling as indicated by phosphorylation of Akt (T308) and p70S6K1 (T389), respectively, were elevated in both SA-2 groups relative to sham control (n=6/group).Values are presented as mean ± s.e.m. with significant difference (P<0.05) between sham and SA-2 groups indicated by a hash.
Fig. S3. Myonuclear accretion in satellite cell-depleted muscle in response to synergist ablation. (A) Representative image from dystrophin immunohistochemistry (IHC; red) and DAPI staining (blue) showing the location of nuclei relative to the sarcolemma. Nuclei residing within the fiber, as delineated by the dystrophin stained sarcolemma, were scored as myonuclei (white arrowhead). Interstitial nuclei are indicated by yellow arrowheads. (B) Representative image of single muscle fibers isolated by NaOH digestion of SA-2 plantaris muscles following paraformaldehyde fixation at resting length and DAPI staining. Myonuclear number and fiber length and diameter were used to calculate the myonuclear domain size for vehicle and tamoxifen SA-2 groups. Scale bar: 100 µm. (C) Myonuclear length was measured in isolated single fibers from vehicle and tamoxifen SA-2 groups. Myonuclear length was significantly increased by ∼17% in tamoxifen SA-2 compared with vehicle SA-2 group. Values are presented as mean ± s.e.m. with significant difference (P<0.05) between sham and SA-2 groups indicated by a hash.
Fig. S4. High correlation between satellite cell abundance and myonuclei per fiber. Satellite cell abundance (x-axis) plotted against myonuclei/fiber (y-axis) revealed a significant correlation (r=0.834, P<0.05) between vehicle SA-2 (solid diamond) and tamoxifen SA-2 (solid square).