All-Trans Retinoic Acid Impacts Myogenic Gene Expression in Bovine Satellite Cells.

All-trans retinoic acid (ATRA) has been associated with the enhancement of myogenesis, the alteration of muscle fiber type, and can subsequently alter lipid metabolism through various transcriptional reactions in skeletal muscle. A completely randomized design cell culture study used orthogonal contrasts for unequally spaced treatments to examine dose effects of ATRA on expression of genes related to skeletal muscle fiber type and hypertrophy. Bovine satellite cells were isolated from the semimembranosus of two 14-month-old cross breed steers. The isolated muscle satellite cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) solution with 10% Fetal Bovine Serum, 1× Antibiotic-Antimycotic at 37ºC under a humidified atmosphere of 95% O₂ and 5% CO₂. Upon reaching 80 to 90% confluence, the growth medium was replaced with differentiation medium composed of DMEM and 2% horse serum to induce myogenic differentiation. Along with differentiation media, various doses (0, 0.01, 0.1, 1, 10^-6M) of ATRA were randomly administered to wells (n=6 wells/dose). After 96 h of incubation, cells were harvested for mRNA analysis. Real-time quantitative PCR (Rtq – PCR) was used to measure the quantity of protein kinase B (Akt), AMP-activated protein kinase α (AMPKα), glucose transporter 4 (GLUT4), myogenin, myosin heavy chain (MHC) I, MHC IIA, MHC IIX, IGF-I, PPARγ, and transforming growth factor-beta effector protein (SMAD3) mRNA relative to ribosomal protein subunit 9 (RPS9). No differences for control vs. ATRA were detected for mRNA expression of Akt (P = 0.17), AMPKα (P = 0.46), or GLUT4 (P = 0.30). A tendency (P = 0.09) for a linear increase of MHC I mRNA expression was detected with increasing doses of ATRA. Expression of PPARγ, known to induce oxidative muscle fiber type formation, linearly increased (P < 0.01) with ATRA. The mRNA expression of IGF-I was linearly increased (P < 0.01) by ATRA. The mRNA abundance of SMAD3 (P < 0.01) and myogenin (P < 0.01) both linearly increased with ATRA. A novel finding in this study is that PPARγ expression was increased by ATRA in bovine muscle cells. It is well known that ATRA inhibits expression of PPARγ and other adipogenic genes in adipocytes. In muscle cells, ATRA appears to push muscle fibers toward the MHC isoform that prefers oxidative metabolism, primarily via increased expression of genes associated with MHC I isoform and myogenic differentiation.