A Selective Role for MRF4 in Innervated Adult Skeletal Muscle: Na_V 1.4 Na⁺ Channel Expression Is Reduced in MRF4-Null Mice

Abstract

The factors that regulate transcription and spatial expression of the adult skeletal muscle Na+ channel, Na(V) 1.4, are poorly understood. Here we tested the role of the transcription factor MRF4, one of four basic helix-loop-helix (bHLH) factors expressed in skeletal muscle, in regulation of the Na(V) 1.4 Na+ channel. Overexpression of MRF4 in C2C12 muscle cells dramatically elevated Na(V) 1.4 reporter gene expression, indicating that MRF4 is more efficacious than the other bHLH factors expressed at high levels endogenously in these cells. In vivo, MRF4 protein was found both in extrajunctional and subsynaptic muscle nuclei. To test the importance of MRF4 in Na(V) 1.4 gene regulation in vivo, we examined Na+ channel expression in MRF4-null mice using several techniques, including Western blotting, immunocytochemistry, and electrophysiological recording. By all methods, we found that expression of the Na(V) 1.4 Na+ channel was substantially reduced in MRF4-null mice, both in the surface membrane and at neuromuscular junctions. In contrast, expression of the acetylcholine receptor, and in particular its alpha subunit, was unchanged, indicating that MRF4 regulation of Na+ channel expression was selective. Expression of the bHLH factors myf-5, MyoD, and myogenin was increased in MRF4-null mice, but these factors were not able to fully maintain Na(V) 1.4 Na+ channel expression either in the extrajunctional membrane or at the synapse. Thus, MRF4 appears to play a novel and selective role in adult muscle.

Basic helix–loop–helix factors recruit nuclear factor I to enhance expression of the NaV 1.4 Na+ channel gene

“…Although NFI is known to regulate other genes expressed in skeletal muscle, notably the GLUT4 glucose transporter [18], the mechanism presented in this paper is novel. Myogenin and especially MRF4 recruit NFI to enhance Na + channel expression, consistent with our previous observation that expression of the Na V 1.4 Na + channel is decreased in MRF4-null mice suggesting this mechanism is important not only in cell culture but also in vivo [15].…”

“…Previous work from our lab indicates that bHLH factors bound at the promoter E box influence the behavior of transcription factors bound at other sites in the 5′ flanking region of the Na V 1.4 gene, but these other factors were not identified [15,16,17]. In this work, we now demonstrate that the repressor E box binds ZEB/AREB6 and a factor we designate REB, and that mutation of this site interferes with binding of these proteins.…”

“…Utilizing the C2C12 muscle cell line as a convenient model system, we demonstrated that Na + channel expression is initiated by myogenin and maintained at the highest levels by MRF4 [15,17]. Although the bHLH factors counteract the activity of an upstream repressor located between −135 and −57 [17], the precise mechanism involved is poorly understood.…”

“…The channel protein is expressed at highest levels at neuromuscular junctions (NMJs), but also at lower levels throughout the surface membrane and in the T-tubular membranes [5,6]. Although protein-protein interactions likely "fine-tune" channel spatial distribution [7,8,9,10,11], we and others have shown that transcriptional mechanisms are very important in sculpting the development of the surface membrane and synapse [12,13,14,15].…”

JAZF1 promotes proliferation of C2C12 cells, but retards their myogenic differentiation through transcriptional repression of MEF2C and MRF4—Implications for the role of Jazf1 variants in oncogenesis and type 2 diabetes

“…Nevertheless, several investigations have provided insight into the functional roles of MRF4. MRF4 is preferentially expressed in subsynaptic nuclei and its deletion results in the reduced expression of a sodium channel (NaV1.4) and the loss of synaptic vesicles from neuromuscular junctions [39][40][41]. Moreover, knockdown of MRF4 by morpholinos in zebrafish leads to myofibril misalignment and disorganized growth of motor axons [42].…”