Differential regulation of MyoD and myogenin mRNA levels by nerve induced muscle activity

Abstract

The levels or mRNAs coding for the myogenic factors MyoD and myogemn were measured dtlrin8 synapse formation in dry©loping mu~l~ and in adult muscle, after denervation and reinnervatlon ~.nd after muscle paralysis Induced by blocking of n~uromu~cular transmission by neurotosins known to alter the density and localization of s~naptie proteins such as the acetyleholine receptor (AChR), Th~ mRNA levels of both factors d~l~nd on usalle of the neuromuscular synapses, but they change to different extents, Myogenin mRNA levels decrease drastically with inncrva lion and increase strongly following blocking of transmfs~ion whereas the level of MyoD mRNA showed only a small decrease in response to innetvatlon, denervation or muscl© paralysis by neurotoxins, Neither mRNA showed a synapse.related cellular distribution. The results suggest that nerve.induced ©l~trical muscle activity determines the cellular ratio of MyoD and myogenln mRNAs in adult muscle, are also directly involved in the regulation of proteins which are localized at the neuromuscular synapse such as the AChR. We have measured the changes in the levels of MyoD. and myogenic-specific mRNA levels during establishment, maintenance and disruption of neuromuscular transmission, during which there are dramatic changes in the level and spatial distribution of synapse-specific mRNAs and proteins. MATERIALS AND METHODS 2,1. Isolation and surgery of rat muscleTriceps muscles from adult Wistar rats (150-300 g) or rats of various postnatal ages, killed with ether, CO2 or decapitated were removed and immediately frozen in liquid nitrogen, Denervation and reinnervation experiments were performed as described previously [12], Pharmacological muscle inactivation was induced either by injection of botulinum toxin (BoTX) type A into the triceps muscle or by chronic blockade of nerve signal conduction in the sciatic nerve usmg tetrodotoxin (TTX) following procedures described by Brenner et el. [13] [16] to Biodyne membranes (Pall) and crosslinked with the UV Stratalinker from Stratagene. Hybridization was carried out following the procedure recommended by Pall. The membranes were exposed to X.ray film (Kodak X-OMAT) for 1-3 days at -70°C using intensifying screens. 2,3. Hybridization probesHybridization probes were labelled with [a-~aP]dCTP using the random primed labelling kit from Boehringer (Mannheim) F.vt~h~atlon oy uuloratttoxramsEqual Ioad/nj or total RNA wa~ ascertained by czhhltum bromide staining ot the agaro~e lids, The ribosomal 18S and 2~t,~ [~,NA band~ served as internal standards, Loadtnll increasinll amounts (2-I$ all) or total RNA showed a linear increase in ~peeiftg hybridlxation signals, In ~ome ca~es the .~ame blots were rehybrltllzed usin8 AChR ~ubuni~ or acdn mRNA ~pe¢irie probes (Witxemann et al.. in preparation) to conrlrm the specificity of the observed ehan~es in mRNA content (not shown), Autoradiogram5 were ~anned with the dual wavelenlltl~ ~¢anner from Shimadzu (CS.910). For d~ndtomelrie evaluation care was taken to scan autoradioilrams who~e ...

MyoD protein accumulates in satellite cells and is neurally regulated in regenerating myotubes and skeletal muscle fibers

“…Levels of MyoD immunoreactivity increased after denervation, a result that mirrors the effect of denervation on MyoD mRNA (Eftimie et al, 1991;Witzemann and Sakmann, 1991;Saitoh et al, 1993). Intriguingly, the myonuclei of denervated muscle were either moderately labelled or unlabelled.…”

Expression of Myosin Isoforms in Denervated, Cross‐Reinnervated, and Electrically Stimulated Rabbit Muscles

“…Manipulated muscles, which can be transformed into the opposite type by chronic electrical stimulation for example, raise the question of the nature of the primary cellular signal responsible for initiating and maintaining the transformation process. Among the possible candidates are the members of the MYOD family of basic helix-loop-helix proteins (MyoD, myogenin, myf5 and MRF4) which are sensitive to electrical activity (Saitoh et al, 1993 ;Witzemann and Sakmann, 1991) and which are known to promote the transcription of muscle-specific genes (for reviews, see Olson and Klein, 1994;Sassoon, 1993). Among these, the hypothesis that activity-dependent autoregulation of myogenic factors (particularly myogenin) expression was a prerequisite to activity-dependent regulation of acetylcholine receptor subunit gene expression has been intensely investigated (Berberich et al, 1993 ;Bessereau et al, 1993;Eftimie et al, 1991;Merlie et al, 1994;Neville et al, 1992).…”

Neuronal control of myogenic regulatory factor accumulation in fetal muscle

“…The marked increases in MRF mRNAs that occur after adult denervation (Eftimie et al, 1991;Duclert et al, 1991;Witzemann and Sakmann, 1991;Buonanno et al, 1992;Voytik et al, 1993), are followed almost immediately by increases in myoD, myogenin and MRF4 proteins (Weis, 1994;Kostrominova et al, 2000;Weis et al, 2000;Hyatt et al, 2003;Ishido et al, 2004). This increase in protein is accompanied by changes in transcript accumulation for proteins encoded for by several MRF target genes (e.g., the nicotinic acetylcholine receptor subunits [cf.…”

“…myoD and myogenin mRNAs are preferentially expressed in adult rodent fast and slow twitch muscle fibers, respectively; however, there does not appear to be an absolute correlation between the accumulation of myoD and myogenin mRNAs and specific fiber types (Hughes et al, 1993(Hughes et al, , 1997Voytik et al, 1993;Kraus and Pette, 1997). Denervation of adult, rodent hindlimb muscles results in large increases in the mRNAs for all of the MRFs by ϳ2 days after denervation (Duclert et al, 1991;Eftimie et al, 1991;Witzemann and Sakmann, 1991;Buonanno et al, 1992;Voytik et al, 1993;Hyatt et al, 2003), followed almost immediately by increases in myoD, myogenin, and MRF4 proteins (Weis, 1994;Weis et al, 2000;Kostrominova et al, 2000;Hyatt et al, 2003;Ishido et al, 2004). While the changes in individual MRF mRNAs vary with the muscles studied and with the species of rodent evaluated, myogenin mRNA reportedly exhibits the greatest increase (levels of this transcript are 40-to 200-fold higher than in control muscle at 1-week after denervation).…”