Antagomir-22-3p treatment on skeletal muscle growth in intrauterine growth restricted lambs

Susan Kay Duckett^1*Maslyn Greene¹Aliute N S Udoka¹Rhonda Reigers Powell¹Terri F Bruce¹James Levi Klotz²

• ¹Clemson University, Clemson, United States
• ²Forage-Animal Production Research Unit, Agricultural Research Service, United States Department of Agriculture, Washington D.C., District of Columbia, United States

microRNAs (miRNA) are involved in regulating gene expression and muscle development. Previous research identified miR-22-3p as differentially regulated during muscle hypertrophy and in vitro experiments found that antagomir-22-3p enhanced satellite cell proliferation. The objective of this study was to test in vivo vascular injection of antagomir-22-3p in lateral saphenous vein on miRNA and HDAC family mRNA expression in intrauterine growth restricted (IUGR) lambs. Pregnant ewes (n = 18) with twins were either fed at 100% of NRC (CON) or nutrient restricted (60% NRC; NR) from gestational d 86 to parturition. On d 12 of age, NR lambs (n = 8) were randomly selected and given a systemic injection of antagomir-22-3p (440 ug/lamb) in the lateral saphenous vein of the right leg (NR-ANT22) for three consecutive days. CON lambs (n=8) were randomly selected and given a sham injection of PBS in the lateral saphenous vein of the right leg (CON-SHAM). Blood samples were collected from each lamb weekly to monitor circulating miR-22-3p expression. Muscle samples were obtained at 24 d post-injection for miR- 22-3p and mRNA expression of potential miRNA targets. Cell-free circulating miR-22-3p expression was downregulated (P < 0.05) for NR-ANT22 compared to CON-SHAM on d 14 after injection. On d 21 after injection, miR-22-3p expression in plasma tended (P = 0.08) to be down-regulated in NR-ANT22 compared to CON-SHAM. Lamb body weight and muscle weights at harvest were similar between NR-ANT and CON- SHAM. In the semitendinosus (ST) muscle, miR-22-3p expression was down-regulated (P < 0.05) in NR-ANT22_R ST (treated) compared to NR-ANT22_L ST (non-treated) and CON-SHAM ST. In heart and semimembranosus (SM) muscles, expression of miR-22-3p was down-regulated (P < 0.05) in NR-ANT22 compared to CON-SHAM. The number of type I, IIa, and IIax muscle fibers were greater (P < 0.05) for NR-ANT22 than CON-SHAM; whereas the number of type IIx fibers was greater (P < 0.05) for CON-SHAM. NR-ANT22 treatment appears to shift muscle fibers towards more oxidative metabolism. The systemic injection of antagomir-22-3p down-regulated miR-22-3p expression, which altered expression of HDAC/SIRT genes involved in muscle fiber type conversion.