Introduction and Objective: GDF8 and activin A signal through the activin typeII receptor ActRIIA and ActRIIB to limit muscle growth. However, the specific role of each ActRII at regulating muscle mass remains to be elucidated. Here we pharmacologically interrogate the function of ActRIIA and ActRIIB using human myoblast differentiation model which is highly predictive of muscle growth in humans.Methods: Binding affinity of antibodies was determined via Biacore 8K and biochemical activity was measured using HTRF. Human primary skeletal muscle cells were cultured on Matrigel in growth medium (ATCC, PCS-500-030). Then myogenic differentiation was initiated by changing to the differentiation medium (ATCC, PCS-950-050). Myoblasts differentiated for 4-6 days with GDF8 or activin A alone or in the presence of anti-ActRIIA, anti-ActRIIB and dual anti-ActRIIA/IIB Bimagrumab antibodies. Following differentiation, cells were stained for MyHC and with Hoechst. Images were acquired using Opera Phenix Plus high-content system and analyzed with AI.Results: The anti-ActRIIA antibody LA01 specifically inhibited ActRIIA with a KD of 67 pM and an IC50 of 0.2 nM. The anti-ActRIIB antibody 17G05 specifically inhibited ActRIIB with a KD of 509 pM and an IC50 of 0.26 nM. Each antibody had no detectable binding to the other activin type II receptor. GDF8 or activin A treatment induced markedly atrophy of human myotube cultures. This atrophy was completely prevented by LA01, which resulted in a significant increase in myotube number, width and differentiation index. The recovery magnitude by the anti-ActRIIA antibody was comparable to that treated with bimagrumab. In contrast, 17G05 failed to relieve GDF8-induced inhibition of differentiation and showed only a moderate effect on the inhibition induced by activin A.Conclusion: These results indicate that ActRIIA plays a more prominent role than ActRIIB in the differentiation of human myoblasts. This provides a strong rationale for more presicse targeting ActRII receptors to preserve muscle in obesity treatment.
Y. Du: None.
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