Regulation of adult muscle phenotype by Ets-2

Abstract

A unique characteristic of skeletal muscle is its heterogeneous nature and ability to acquire new functional characteristics to keep up with changing demands. These plastic properties are mainly regulated by activity. Distinct patterns of electrical stimuli, exerted by motorneurons, are sufficient to activate separate transcriptional programs that affect muscle strength, speed and endurance. Elucidating the molecular pathways linking electrical stimulation to gene expression is necessary to explain the effects of training and neuromuscular disorders. This work presents erythroblastosis virus E26 oncogene homolog 2 (Ets-2) as a possible mediator in the signaling network coupling electrical signals to transcriptional responses. The Ets-2 protein has been shown to be more abundant in fast muscle, where it selectively translocates and binds an enhancer element necessary for transcription of the troponin I fast (TnIf) gene. The purpose of this work was to determine the wild type expression pattern of the two skeletal troponin I (TnI) genes, and investigate the possible effects of Ets-2 overexpression and knockdown on TnI isoform distribution. An expression vector encoding Ets-2 was transfected into the “slow” soleus muscle in rats by in vivo electroporation. Myosin heavy chain (MyHC) fiber type distribution and TnI isoform expression among the transfected fibers were analyzed, and compared to sham transfected and normal controls. In a separate series of experiments, an expression vector encoding siRNA against Ets-2 was transfected into the “fast” extensor digitorum longus (EDL). Fiber type and TnI distribution among the transfected fibers were analyzed. To investigate a possible effect on TnI expression, the fluorescent intensity of the siRNA-transfected fibers were measured and compared against normal scramble transfected and normal control fibers. Mapping of TnI expression showed the slow TnI isoform (TnIs) is confined to slow-twitch fibers and TnIf to fast-twitch fibers, however fast-twitch fibers in soleus were shown to coexpress both isoforms. Overexpression of Ets-2 had no effect on TnIf expression or MyHC composition. Knockdown of Ets-2 resulted in a 16% reduction of MyHC 2b fibers accompanied by 12% increase in 2a fibers. Measurements of TnIf fluorescence showed a minor reduction in TnIf staining intensity, however a larger sample material is needed to make any certain conclusions.