Primary myoblasts from intrauterine growth-restricted fetal sheep exhibit intrinsic dysfunction of proliferation and differentiation that coincides with enrichment of inflammatory cytokine signaling pathways

Abstract

Intrauterine growth restriction (IUGR) is linked to lifelong reductions in muscle mass due to intrinsic functional deficits in myoblasts, but the mechanisms underlying these deficits are not known. Our objective was to determine if the deficits were associated with changes in inflammatory and adrenergic regulation of IUGR myoblasts, as was previously observed in IUGR muscle. Primary myoblasts were isolated from IUGR fetal sheep produced by hyperthermia-induced placental insufficiency (PI-IUGR; n = 9) and their controls (n = 9) and from IUGR fetal sheep produced by maternofetal inflammation (MI-IUGR; n = 6) and their controls (n = 7). Proliferation rates were less (P < 0.05) for PI-IUGR myoblasts than their controls and were not affected by incubation with IL-6, TNF-α, norepinephrine, or insulin. IκB kinase inhibition reduced (P < 0.05) proliferation of control myoblasts modestly in basal media but substantially in TNF-α-added media and reduced (P < 0.05) PI-IUGR myoblast proliferation substantially in basal and TNF-α-added media. Proliferation was greater (P < 0.05) for MI-IUGR myoblasts than their controls and was not affected by incubation with TNF-α. Insulin increased (P < 0.05) proliferation in both MI-IUGR and control myoblasts. After 72-h differentiation, fewer (P < 0.05) PI-IUGR myoblasts were myogenin+ than controls in basal and IL-6 added media but not TNF-α-added media. Fewer (P < 0.05) PI-IUGR myoblasts were desmin+ than controls in basal media only. Incubation with norepinephrine did not affect myogenin+ or desmin+ percentages, but insulin increased (P < 0.05) both markers in control and PI-IUGR myoblasts. After 96-h differentiation, fewer (P < 0.05) MI-IUGR myoblasts were myogenin+ and desmin+ than controls regardless of media, although TNF-α reduced (P < 0.05) desmin+ myoblasts for both groups. Differentiated PI-IUGR myoblasts had greater (P < 0.05) TNFR1, ULK2, and TNF-α-stimulated TLR4 gene expression, and PI-IUGR semitendinosus muscle had greater (P < 0.05) TNFR1 and IL6 gene expression, greater (P < 0.05) c-Fos protein, and less (P < 0.05) IκBα protein. Differentiated MI-IUGR myoblasts had greater (P < 0.05) TNFR1 and IL6R gene expression, tended to have greater (P = 0.07) ULK2 gene expression, and had greater (P < 0.05) β-catenin protein and TNF-α-stimulated phosphorylation of NFκB. We conclude that these enriched components of TNF-α/TNFR1/NFκB and other inflammatory pathways in IUGR myoblasts contribute to their dysfunction and help explain impaired muscle growth in the IUGR fetus.

Daily Injection of the β2 Adrenergic Agonist Clenbuterol Improved Muscle Glucose Metabolism, Glucose-Stimulated Insulin Secretion, and Hyperlipidemia in Juvenile Lambs Following Heat-Stress-Induced Intrauterine Growth Restriction

“…Our IUGR-born lambs exhibited substantially greater circulating TNFα and IL-6 concentrations that were completely ameliorated by daily clenbuterol administration. They also exhibited evidence of enhanced muscle sensitivity to inflammatory factors, albeit less robustly than observed in IUGR fetuses [ 27 , 36 , 48 ]. β2 agonists suppress leukocytic inflammasomes and reduce cytokine secretion into the bloodstream [ 49 , 50 , 51 ].…”

Dousing the flame: reviewing the mechanisms of inflammatory programming during stress-induced intrauterine growth restriction and the potential for ω-3 polyunsaturated fatty acid intervention

“…Myoblast dysfunction is reflected in expression of key myogenic transcription factors, which hint at mechanisms for IUGR myoblast deficits. Specifically, differentiation factors myoD and myogenin were reduced in IUGR fetal myoblasts near term, whether assessed in vivo or ex vivo ( Yates et al, 2014 ; Chang et al, 2019b ; Rozance et al, 2021 ; Posont et al, 2022 ). Reduced myoblast proliferation, differentiation, and fusion slowed myonuclear accumulation in all muscle fiber types, which in IUGR fetal lambs restricted cross-sectional fiber area by as much as 60% ( Yates et al, 2016 ; Chang et al, 2019b ).…”

“…The fetal hypoxemia-hypercatecholaminemia-hypoinsulinemia cascade appears instrumental in muscle growth restriction, as experimental induction of each factor individually produced some degree of IUGR in sheep ( Philipps et al, 1991 ; Bassett and Hanson, 1998 ; Rozance et al, 2018 ). Hypoxemia-induced inflammation also limited muscle mass by increasing protein catabolism, reducing protein accretion, and altering muscle stem cell function ( Li W. et al, 2009 ; Wang et al, 2014 ; Madaro et al, 2018 ; Posont et al, 2022 ). Less severe reductions in body length, head circumference, and cannon bone length observed in IUGR fetal lambs reflected the modest nature of structural growth restriction ( Rozance et al, 2018 ; Cadaret et al, 2019c ; Sandoval et al, 2020 ).…”

“…Myoblasts undergo rate-limiting proliferation and differentiation steps before fusing with existing muscle fibers to increase the myonuclear content/protein capacity of the fiber ( Allen et al, 1979 ; Yates et al, 2012b ). Histological and ex vivo assessments indicated that differentiation capacity was impaired in myoblasts from IUGR fetal lambs ( Yates et al, 2014 ; Yates et al, 2016 ; Soto et al, 2017 ; Posont et al, 2022 ). Myoblast proliferation was also typically reduced by IUGR intrauterine conditions, although proliferation was increased in one model induced by maternofetal inflammation ( Soto et al, 2017 ; Cadaret et al, 2019a ; Posont et al, 2022 ).…”

“…Histological and ex vivo assessments indicated that differentiation capacity was impaired in myoblasts from IUGR fetal lambs ( Yates et al, 2014 ; Yates et al, 2016 ; Soto et al, 2017 ; Posont et al, 2022 ). Myoblast proliferation was also typically reduced by IUGR intrauterine conditions, although proliferation was increased in one model induced by maternofetal inflammation ( Soto et al, 2017 ; Cadaret et al, 2019a ; Posont et al, 2022 ). Myoblast dysfunction is reflected in expression of key myogenic transcription factors, which hint at mechanisms for IUGR myoblast deficits.…”