Skeletal myoblast proliferation and differentiation are reduced in growth restricted fetal sheep
Authors: Barooni Neeka , Chang Eileen , Brown Laura , McCurdy Carrie 
Published: May 2024

Fetal growth restriction (FGR) increases the risk of type 2 diabetes due in part to deficits in fetal skeletal muscle growth that are not fully compensated after birth. During prenatal development, muscle progenitor cells proliferate, differentiate, and fuse with existing fibers to support myofiber hypertrophy. The total number of myofibers is set in utero, and reductions in myofiber number, size, and myonuclear accretion suggest that myogenesis is impaired in FGR. Previous studies identify similar rates of proliferation and differentiation in FGR and control (CON) myoblasts exposed to enriched growth media in vitro, despite deficits in myoblast proliferation/differentiation in whole muscle of FGR fetuses. Whether muscle progenitor cells of FGR fetuses exhibit impaired myogenic capacity in vivo remains unknown. Using a novel flow cytometry approach to quantify stages of myogenesis in vivo, we aimed to identify the cellular origins of dysregulated skeletal muscle development in FGR due to placental insuffciency. Pregnant sheep were exposed to elevated temperatures (n=15) from early to mid-gestation to induce placental insuffciency and subsequent FGR, defined as birth weight 2 SD below the mean for gestational age. At 90% gestation, fetuses were weighed, and muscle progenitor cells were isolated from biceps femoris of FGR (n=8) and CON (n=5) fetuses for flow cytometry. Muscle cells were identified by anti-CD56, anti-Pax7, and live/dead dye. DAPI was used to quantify resting (G0/1) versus replicating (S/G2) myoblasts. Myogenic regulatory factors, anti-MyoD and anti-myogenin (MyoG), distinguished non-committed (MyoD-/MyoG-) and activated (MyoD+/MyoG-) myoblasts from those beginning (MyoD+/MyoG+) and completing (MyoD-/MyoG+) differentiation. Statistical significance was determined by Student’s t-tests with α=0.05. Percentages are relative to total myoblasts (live, singlets expressing Pax7/CD56). Total myoblasts per live singlets were not different between FGR and CON muscle. FGR fetuses had more muscle progenitors in G0/1 (97% vs 95%, p<0.0001) and fewer replicating myoblasts in S/G2 compared to CON (3% vs 5%, p<0.0001). Myofactor analysis found decreased activated myoblasts (MyoD+/MyoG-) in FGR versus CON (2.5% vs 5%, p<0.005), though the fraction of non-committed progenitors (MyoD-/MyoG-) was similar between groups (14% vs 15%). Despite reduced myogenic induction in FGR, there were no differences between FGR and CON myoblasts expressing MyoG in early (MyoD+, 38% vs 47%) or terminal differentiation (MyoD-, 47% vs 34%). Correlation analyses of all fetuses (n=15) revealed positive relationships between fetal weight and MyoD+/MyoG- (r=0.57) as well as MyoD+/MyoG+ myoblasts (r=0.56), while MyoD-/MyoG+ cells correlated negatively with fetal weight (r=-0.60). FGR fetuses have decreased myoblast proliferation in vivo. Myogenic lineage commitment is reduced in FGR progenitor cells, and positive correlations between fetal weight and myogenic induction suggest that deficits in muscle size and body mass may result from impaired initiation of myogenesis in utero. The elevated percentage of terminally differentiated myocytes in low birthweight fetuses may be a consequence of impaired fusion, which likely contributes to reductions in myonuclei within FGR myofibers. Since the capacity for growth is conserved in FGR skeletal muscle, indicated by retention of myoblast number and proliferation/differentiation capacity in vitro, therapies that stimulate myogenic induction and/or myoblast fusion during fetal development may be necessary/suffcient to restore muscle growth in FGR. NIH R01HD079404; Evonuk Graduate Fellowship; UO VPRI funds. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.