Recombinant Growth Hormone and Tri-iodothyronine Influence Myofiber Differentiation in Cultured Muscle Satellite Cells † 475

Abstract

The process of muscle differentiation and maturation in vivo involves the sequential expression of myosin heavy chain (MyHC) isoforms from fetal to adult phenotype. Growth and thyroid hormones are critical modulators of this developmental process in concert with variations in innervation and activation patterns. A model of in vitro muscle differentiation was tested for effect of human recombinant growth hormone (rHGH) or tri-iodothyronine (T3) on the muscle maturation process in the absence of neural influences. MM14 cells, a murine muscle satellite cell line (S.D. Hauschka, Seattle WA) were subcloned and myoblasts were expanded in parallel cultures on gelatin coated plates utilizing proliferation media (F10C + 15% horse serum) including bFGF. Subconfluent cultures (1 × 106 cells/plate)were “switched” to differentiation media and separate plates were administered rHGH or T3 at levels 1X, 10X or 100X of in vivo serum levels. Control plates received saline vehicle at the time of switch and parallel cultures were harvested every 12 hours to 144 hours after switch. Total RNA was purified and cDNA yields were quantified after reverse transcription. Equal cDNA aliquots were amplified via PCR for expression of MyHC: slow, 2A, 2B, 2X, and neonatal isoforms, muscle creatine kinase (MCK) and a constitutive marker (β-cytoplasmic actin). High resolution 6% SDS-PAGE and Western blotting defined MyHC protein composition. Untreated myoblasts rapidly formed bipolar morphological structures, underwent extensive intercellular fusion and expressed predominantly MyHC 2A and slow at the mRNA and protein level 72 hrs. post switch. In addition, low levels of neonatal and 2B myosins were present by 144 hrs post switch at both the RNA and protein levels but no 2X isoform was detected. rHGH administration at medium and high levels produced a 10 fold induction of MyHC:neo and MCK and a 2 fold induction of MyHC:2B compared to control myocytes. A reciprocal effect occurred in the presence of the highest dose of T3 whereby MyHC:neo, MyHC:slow and MCK fell to 0.1X the control values. There was no effect of T3 on MyHC:2B expression compared to controls. These effects were consistent in 3 separate myoblast expansions controlled for cell number at time of switch and β-actin levels during differentiation. These data support the hypothesis that rHGH and T3 influence muscle specific gene expression in vitro by a direct mechanism. Furthermore, rHGH enhanced expression of MyHC and MCK levels consistent with an energetic profile of a “fast” muscle phenotype.