MEF2 activation in differentiated primary human skeletal muscle cultures requires coordinated involvement of parallel pathways

The myocyte enhancer factor (MEF) 2 transcription factor is important for development of differentiated skeletal muscle. We investigated the regulation of MEF2 DNA binding in differentiated primary human skeletal muscle cells and isolated rat skeletal muscle after exposure to various stimuli. MEF2 DNA binding activity in nonstimulated ( basal) muscle cultures was almost undetectable. Exposure of cells for 20 min to 120 nM insulin, 0.1 and 1.0 mM hydrogen peroxide, osmotic stress ( 400 mM mannitol), or 1.0 mM 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside ( AICAR) led to a profound increase in MEF2 DNA binding. To study signaling pathways mediating MEF2 activity, we preincubated human skeletal muscle cell cultures or isolated rat epitrochlearis muscles with inhibitors of p38 mitogen-activated protein kinase (MAPK) (10 muM SB-203580), MEK1 (50 muM PD-98059), PKC (1 and 10 muM GF109203X), phosphatidylinositol (PI) 3-kinase (10 muM LY-294002), or AMP-activated protein kinase (AMPK; 20 muM compound C). All stimuli resulted primarily in activation of MEF2D DNA binding. Exposure of cells to osmotic or oxidative stress increased MEF2 DNA binding via pathways that were completely blocked by MAPK inhibitors and partially blocked by inhibitors of PKC, PI 3-kinase, and AMPK. In epitrochlearis muscle, MAPK inhibitors blocked contraction but not AICAR-mediated MEF2 DNA binding. Thus activation of MEF2 in skeletal muscle is regulated via parallel intracellular signaling pathways in response to insulin, cellular stress, or activation of AMPK.