Figure 1.

Obestatin Activates Human Myoblast Proliferation in Regenerating Muscle

(A) Experimental scheme for the in vivo assay. Human myoblasts were isolated from the semitendinosus muscle of a 25-year-old male and implanted into the tibialis anterior (TA) muscles of Rag2^−/−IL2Rb^−/− mice immediately after cryodamage. Obestatin (300 nmol/kg in DMEM) or vehicle (DMEM) was administrated via injection into the target muscles every 24 hr up to 5 consecutive days. At 24 hr, 5 days, and 21 days after engraftment, mice were sacrificed and the TAs were dissected. (B) Proliferation of human cells after injection into tibialis anterior (TA) muscles of Rag2^−/−IL2Rb^−/− mice. (B, upper panel) Quantification of Ki67⁺ cells double-labeled with human-specific anti-Lamin A/C antibody (human cells) is shown at 24 hr and 5 days post-injury after intramuscular injection of obestatin (300 nmol/kg body weight/24 hr during 5 days) or control (DMEM) following human myoblast transplantation (n = 5 per group). Graph depicts percentage of human cell number that expressed Ki67. (B, lower panels) Representative sample sections for Ki67 and Lamin A/C expression at 5 days following cell transplantation. (C, upper panel) Detection of pH3 cells double-labeled with the human-specific anti-Lamin A/C antibody is shown at 24 hr and 5 days following cell transplantation and intramuscular injection of obestatin (300 nmol/kg body weight/24 hr during 5 days; n = 5 per group) or control (DMEM; n = 5 per group). Graph depicts percentage of human cell number that expressed pH3. (C, lower panels) Representative detection of pH3 and Lamin A/C at 24 hr following human myoblast transplantation. (D, upper panel) quantification of TUNEL⁺ cells double-labeled with human-specific anti-Lamin A/C antibody (human cells) is shown at 24 hr and 5 days post-injury after intramuscular injection of obestatin (300 nmol/kg body weight/24 hr during 5 days) or control (DMEM) following human myoblast transplantation (n = 5 per group). Graph depicts average percent of TUNEL⁺ human cells. (D, lower panels) Representative detection of Lamin A/C and TUNEL labeling at 5 days following human myoblast transplantation. (E) Representative detection of cleaved caspase-3 and Lamin A/C at 24 hr and 5 days following cell transplantation and intramuscular injection of obestatin (300 nmol/kg body weight/24 hr during 5 days; n = 5 per group) or control (DMEM; n = 5 per group). (B–D) Data are expressed as mean ± SEM. *p < 0.05 versus control values.