Myoblasts from intrauterine growth‐restricted sheep fetuses exhibit intrinsic deficiencies in proliferation that contribute to smaller semitendinosus myofibres

Key points To investigate loss of skeletal muscle mass in intrauterine growth‐restricted (IUGR) fetuses near term, which may result from myoblast dysfunction, we examined semitendinosus myofibre and myoblast morphology in placental insufficiency‐induced IUGR sheep fetuses; we also isolated and cultured IUGR fetal myoblasts to determine whether reduced rates of proliferation were due to intrinsic cellular defects or extrinsic factors associated with serum. Using tests for myogenin and pax7 to identify differentiated and undifferentiated fetal myoblasts, respectively, we found that myofibre area and percentage of myogenin‐positive nuclei were less in IUGR fetal semitendinosus muscles than in controls, but myofibre density and percentage of pax7‐positive nuclei were not different. The percentage of pax7‐positive cells that expressed proliferating cellular nuclear antigen was less in IUGR semitendinosus muscles than in controls, while in myoblasts isolated from fetal sheep and replicated and differentiated in culture, IUGR fetal myoblasts proliferated at slower rates than control myoblasts, under identical culture conditions, but the ability to differentiate was similar between treatments. Media supplemented with IUGR serum decreased replication rates in both IUGR and control myoblasts compared to media supplemented with control fetal serum. These findings show that myoblasts proliferate at slower rates in IUGR fetuses due to a combination of intrinsic cellular characteristics and extrinsic serum factors; the intrinsic defects may explain reduced skeletal muscle mass observed in IUGR newborn children and adults.Abstract Intrauterine growth restriction (IUGR) reduces skeletal muscle mass in fetuses and offspring. Our objective was to determine whether myoblast dysfunction due to intrinsic cellular deficiencies or serum factors reduces myofibre hypertrophy in IUGR fetal sheep. At 134 days, IUGR fetuses weighed 67% less ( P  < 0.05) than controls and had smaller ( P  < 0.05) carcasses and semitendinosus myofibre areas. IUGR semitendinosus muscles had similar percentages of pax7‐positive nuclei and pax7 mRNA but lower ( P  < 0.05) percentages of myogenin‐positive nuclei (7 ± 2% and 13 ± 2%), less myoD and myogenin mRNA, and fewer ( P  < 0.05) proliferating myoblasts (PNCA‐positive–pax7‐positive) than controls (44 ± 2% vs . 52 ± 1%). Primary myoblasts were isolated from hindlimb muscles, and after 3 days in growth media (20% fetal bovine serum, FBS), myoblasts from IUGR fetuses had 34% fewer ( P  < 0.05) myoD‐positive cells than controls and replicated 20% less ( P  < 0.05) during a 2 h BrdU pulse. IUGR myoblasts also replicated less ( P  < 0.05) than controls during a BrdU pulse after 3 days in media containing 10% control or IUGR fetal sheep serum (FSS). Both myoblast types replicated less ( P  < 0.05) with IUGR FSS‐supplemented media compared to control FSS‐supplemented media. In differentiation‐promoting media (2% FBS), IUGR and control myoblasts had similar percentages of myogenin‐positive nuclei after 5 days and formed similar‐sized myotubes after 7 days. We conclude that intrinsic cellular deficiencies in IUGR myoblasts and factors in IUGR serum diminish myoblast proliferation and myofibre size in IUGR fetuses, but intrinsic myoblast deficiencies do not affect differentiation. Furthermore, the persistent reduction in IUGR myoblast replication shows adaptive deficiencies that explain poor muscle growth in IUGR newborn offspring.