Binding of myostatin to activin type II receptors. (a) Analysis of purified myostatin protein. Myostatin protein preparation following the heparin column (heparin eluate) or following reverse-phase HPLC (fractions 32–34 or 35–37 containing the C-terminal region or propeptide, respectively) was electrophoresed under reducing (+βME) or nonreducing (−βME) conditions and either silver stained or subjected to Western analysis. (b) Binding of myostatin to lentil lectin. The heparin eluate was bound to lentil lectin Sepharose and eluted with methyl mannose. Samples were electrophoresed under reducing conditions, blotted, and probed with antibodies directed against the C-terminal region. Similar analysis by using antibodies directed against the pro region showed that the pro region was also retained on the column and eluted with methyl mannose (data not shown). (c) Crosslinking experiments. COS-7 cells transfected with expression constructs for the indicated receptors were incubated with ¹²⁵I-myostatin followed by the crosslinking agent disuccinimidyl suberate. Crosslinked complexes were analyzed by SDS/PAGE. Asterisk denotes predicted size for myostatin bound to an activin type II receptor. In the rightmost lane, excess unlabeled myostatin was included in the binding reaction. (d) Binding of myostatin to ActRIIB. All points represent the average of triplicate samples. (e) Scatchard analysis of the data shown in d. (f) Inhibition of myostatin binding to ActRIIB by follistatin (diamonds) and the propeptide (circles). Each experiment was carried out in triplicate, and each curve represents the average of three independent experiments.

Analysis of transgenic mice. (a) Specific expression of transgenes in skeletal muscle. Northern analysis of RNA samples prepared from female mice was carried out by using simian virus 40 sequences as a probe. On longer exposures of these blots, no expression of the transgenes was observed in liver, kidney, spleen, or heart in any of these lines. (b) Muscle analysis. Sections were stained with hematoxylin and eosin. The gastrocnemius and plantaris muscles are outlined (Left). Center shows higher magnifications of representative areas from the gastrocnemius muscle. Right shows distribution of fiber diameters. Each graph represents the composite of 450 fiber measurements from 3 animals (150 per animal), except for the F3 graph, which represents 175 measurements from the F3 founder animal. Standard deviations of fiber sizes were 9, 11, 11, and 13 μm for control, B32, C27, and F3 animals, respectively. Black and gray arrows show the mean fiber diameters for control and transgenic animals, respectively. Note that in each case, the mean fiber diameter was increased in the transgenic animals (P < 0.001).

Increased muscling in mice overexpressing a dominant-negative form of ActRIIB or full length follistatin. A control male nontransgenic mouse, a male transgenic mouse from the C11 line (dominant-negative ActRIIB), and the F3 male founder mouse (follistatin) are shown. Pictures of live mice are shown in the top row, and pictures of animals that had been killed and skinned are shown in the bottom three rows.