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Comparative Study
. 2007 Oct 15;584(Pt 2):637-50.
doi: 10.1113/jphysiol.2007.141457. Epub 2007 Aug 30.

De-phosphorylation of MyoD is linking nerve-evoked activity to fast myosin heavy chain expression in rodent adult skeletal muscle

Merete Ekmark  1 , Zaheer Ahmad Rana, Greg Stewart, D Grahame Hardie, Kristian Gundersen
Affiliations
Comparative Study

De-phosphorylation of MyoD is linking nerve-evoked activity to fast myosin heavy chain expression in rodent adult skeletal muscle

Merete Ekmark et al. J Physiol. .

Abstract

Elucidating the molecular pathways linking electrical activity to gene expression is necessary for understanding the effects of exercise on muscle. Fast muscles express higher levels of MyoD and lower levels of myogenin than slow muscles, and we have previously linked myogenin to expression of oxidative enzymes. We here report that in slow muscles, compared with fast, 6 times as much of the MyoD is in an inactive form phosphorylated at T115. In fast muscles, 10 h of slow electrical stimulation had no effect on the total MyoD protein level, but the fraction of phosphorylated MyoD was increased 4-fold. Longer stimulation also decreased the total level of MyoD mRNA and protein, while the level of myogenin protein was increased. Fast patterned stimulation did not have any of these effects. Overexpression of wild type MyoD had variable effects in active slow muscles, but increased expression of fast myosin heavy chain in denervated muscles. In normally active soleus muscles, MyoD mutated at T115 (but not at S200) increased the number of fibres containing fast myosin from 50% to 85% in mice and from 13% to 62% in rats. These data establish de-phosphorylated active MyoD as a link between the pattern of electrical activity and fast fibre type in adult muscles.

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Figures

Figure 1
Figure 1
Anti-pMyoDT115 antibody is specific for MyoD phosphorylated at T115 A, spots of phosphopeptide, but not dephosphopeptide, were clearly detected down to < 10 ng. B, Western blot of nuclear extracts from Hek-293 cells after transfection with pCMS-EGFP expressing EGFP only (sham), pCMS-EGFP-MyoDwt expressing wild type MyoD, pSP3-MyoD expressing MyoD with a S200A mutation, or pCMS-EGFP-MyoDA115 expressing MyoD with a T115A mutation. The blots were stained with anti-MyoD or anti-pMyoDT115. Loading was adjusted for differences in transfection efficiency by adjusting the load to yield equal signals with anti-MyoD.
Figure 2
Figure 2
EGFP expression after electroporation Cross-sections of rat muscles electroporated with expression vectors containing EGFP. Fibres expressing EGFP are encircled in green for clarity. Both muscles were sectioned 14 days after electroporation. Scalebar, 50 μm.
Figure 3
Figure 3
MyoD and myogenin mRNA and protein levels are different in the slow soleus muscle and the fast EDL muscle A, The level of mRNA for MyoD and myogenin quantified by RT-PCR relative to expression of the housekeeping gene β2-microglobulin.B, the level of MyoD and myogenin proteins in nuclear extracts quantified on Western blots stained with anti-MyoD or anti-myogenin antibodies. C, examples of representative Western blots. Data are given as mean ±s.e.m. of the ratio between normal soleus muscles and normal EDL muscles on a logarithmic scale, n= 6–7. *P≤ 0.05. Numerical values are given in Table S1.
Figure 4
Figure 4
MyoD and myogenin mRNA and protein levels change in response to electrical activity A, the level of mRNA for MyoD and myogenin quantified by RT-PCR relative to expression of the housekeeping gene β2-microglobulin (Myog). Some of the RT-PCR data were confirmed on Northern blots probed against MyoD and myogenin (upper panel). B and C, the level of MyoD and myogenin proteins in nuclear extracts quantified on Western blots stained with anti-MyoD or anti-myogenin antibodies. Examples of representative Western blots are shown above graphs. Data are given as the ratio between the levels in EDL muscles stimulated with a slow (S-stim) pattern, and contralateral normal EDLs (A and B), or as the ratio between fast-stimulated (F-stim) soleus muscles and contralateral normal solei (C). Data are given as mean ±s.e.m. of the stimulated versus normal muscles on a logarithmic scale, n= 6–10. *P≤ 0.05; **P≤ 0.01. Numerical values are given in Table S1.
Figure 5
Figure 5
Levels of T115-phosphorylated MyoD differ between EDL and soleus, and are influenced by activity The level of MyoD T115-phosphorylated protein from nuclear extracts quantified on Western blots stained with the anti-pMyoDT115 antibody in normal solei relative to EDL (A), in normal EDL stimulated with a slow pattern (S-stim) 10 h and 14 days relative to normal EDL (B), and in soleus muscles stimulated with a fast pattern (F-stim) for 14 days relative to normal solei (C). Data are given as mean ±s.e.m. on a logarithmic scale, n= 6–11, and as representative Western blots (upper panels). *P≤ 0.05. Numerical values are given in Table S1.
Figure 6
Figure 6
MyHC immuno-histochemistry on neighbouring cross sections from rat soleus Transfected fibres are encircled in green and hybrid fibres are marked with red asterisks. Notice the increased amount of hybrid fibres in MyoD-positive denervated fibres and T115A-mutated MyoD fibres. All fibres were negative for IIb MyHC (not shown). Scalebar, 100 μm.
Figure 7
Figure 7
Active MyoD shifts fibre type distribution in the fast direction Serial cross sections from mice and rats were stained with antibodies against different MyHC isoforms and typed according to Table 1 (see Methods). Data are given for non-transfected fibres (Normal), non-transfected denervated fibres (Denerv); and 14 days after transfection with plasmids overexpressing EGFP only (Sham), or EGFP combined with wild type MyoD in innervated (MyoD) or denervated fibres (MyoD Denerv); T115A-mutated MyoD (MyoD-A115); or S200A-mutated MyoD (MyoD-A200). The mutated MyoDs were investigated in innervated fibres. Numerical values and statistical analysis are given in Table S2.
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References

    1. Agell N, Bachs O, Rocamora N, Villalonga P. Modulation of the Ras/Raf/MEK/ERK pathway by Ca2+, and calmodulin. Cell Signal. 2002;14:649–654. - PubMed
    1. Allen DL, Sartorius CA, Sycuro LK, Leinwand LA. Different pathways regulate expression of the skeletal myosin heavy chain genes. J Biol Chem. 2001;276:43524–43533. - PubMed
    1. Andrews NC, Faller DV. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 1991;19:2499. - PMC - PubMed
    1. Asakura A, Fujisawa SA, Komiya T, Nabeshima Y, Nabeshima YI. MyoD and myogenin act on the chicken myosin light-chain 1 gene as distinct transcriptional factors. Mol Cell Biol. 1993;13:7153–7162. - PMC - PubMed
    1. Ausoni S, Gorza L, Schiaffino S, Gundersen K, Lomo T. Expression of myosin heavy chain isoforms in stimulated fast and slow rat muscles. J Neurosci. 1990;10:153–160. - PMC - PubMed

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