Adipocyte-Derived Exosomal Circ_0000002 Affects the Myoblast Growth and Muscle Regeneration

Skeletal muscle development is strongly influenced by crosstalk between adipose tissue and muscle, yet the underlying molecular mechanisms in Ovis aries remain insufficiently defined. This study investigated the regulatory effects of adipocyte-derived exosomes on sheep primary myoblasts. Co-culture with adipocytes significantly enhanced myoblast proliferation, as indicated by increased cyclin-dependent kinase 4 (CDK4), proliferating cell nuclear antigen (PCNA), and Cyclin D1 expression, while simultaneously suppressing differentiation via reduced myogenin (MYOG), myogenic differentiation 1 (MYOD), and myosin heavy chain (MYHC) levels. Exosomes isolated from mature adipocytes (30-150 nm), expressing TSG101, CD63, and CD9, were effectively internalized by myoblasts and reproduced these effects. RNA sequencing identified circ_0000002 as one of the most abundant circular RNAs (circRNAs) in adipocyte-derived exosomes. Functional assays demonstrated that circ_0000002 promoted myoblast proliferation and inhibited differentiation. Mechanistically, circ_0000002 acted as a competing endogenous RNA (ceRNA) by sponging miR-27a, thereby relieving miR-27a-mediated repression of myostatin (MSTN). Dual-luciferase reporter assays confirmed direct interactions between circ_0000002 and miR-27a and between miR-27a and the MSTN 3' untranslated region (3´UTR). Co-transfection experiments further validated that the ceRNA-like mechanism of circ_0000002/miR-27a/MSTN regulates myoblast differentiation. In a cardiotoxin (CTX)-induced tibialis anterior injury mouse model, intramuscular administration of adipocyte-derived exosomes impaired muscle regeneration and increased MSTN expression, supporting the in vivo relevance of this pathway. Collectively, our findings reveal that exosomal circ_0000002 regulates sheep myoblast differentiation via miR-27a/MSTN ceRNA pathway. This work provides the first evidence that an adipocyte-derived exosomal circRNA mediates fat-muscle communication and highlights a potential target for improving muscle growth in sheep.