Single-cell analysis identifies stage-specific impairments in skeletal muscle myogenesis in growth-restricted fetal sheep

Introduction Placental insufficiency causes persistent deficits in myonuclear accretion and skeletal muscle growth. It remains unclear whether impairments in fetal muscle development are driven by deficits in myoblast proliferation, differentiation, and/or fusion. Methods We used flow cytometry to quantify cell cycle activity and myogenic regulatory factor expression in myoblasts isolated from sheep with placental insufficiency-induced fetal growth restriction (FGR) and normally growing controls. Results MyoD and Myogenin (MyoG) expression was analyzed in >12,000 CD56 + myoblasts per animal to quantify the distribution of cells across stages of myogenesis: cell cycle (G0/G1, S/G2), uncommitted to myogenic differentiation (MyoD-/MyoG-), committed to myogenic differentiation (MyoD+/MyoG-), actively differentiating (MyoD+/MyoG+), and terminally differentiated (MyoD-/MyoG+). FGR fetuses had a lower percentage of proliferating (EdU + ) myoblasts and a higher percentage of myoblasts in G0/G1 compared to controls. Despite reduced proliferation, the percentage of uncommitted myoblasts (MyoD-/MyoG-) was similar between groups and was not associated with skeletal muscle mass. FGR fetuses had lower percentages of myoblasts committed to myogenic differentiation (MyoD+/ MyoG-) and actively differentiating (MyoD+/MyoG+) compared to controls. However, the proportion of terminally differentiated myoblasts (MyoD-/MyoG +) was higher in FGR fetuses. Downregulation of Myomaker and Myomerger genes in FGR skeletal muscle suggests impaired myoblast fusion, which may contribute to the accumulation of terminally differentiated myoblasts in FGR. Discussion Collectively, our findings indicate that placental insufficiency disrupts myoblast progression through multiple stages of myogenesis. Impairments in proliferation and fusion may underly deficits in myonuclear accretion and skeletal muscle growth in FGR fetuses.