Quantification of MyoD, Myogenin, MRF4 and Id-1 by Reverse-Transcriptase Polymerase Chain Reaction in Rat Muscles — Effects of Hypothyroidism and Chronic Low-Frequency Stimulation

Authors

  • Bernhard Kraus,

    1. Fakultät für Biologie, Universität Konstanz, Germany
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  • Dirk Pette

    Corresponding author
    1. Fakultät für Biologie, Universität Konstanz, Germany
      D. Pette, Fakultät für Biologie, Universität Konstanz, Box M641, D-78457 Konstanz, Germany
      Fax: +49 7531 88 39 40.
      E-mail: dirk.pette@uni-konstanz.de
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D. Pette, Fakultät für Biologie, Universität Konstanz, Box M641, D-78457 Konstanz, Germany
Fax: +49 7531 88 39 40.
E-mail: dirk.pette@uni-konstanz.de

Abstract

A highly sensitive method of reverse-transcriptase polymerase chain reaction (RT-PCR) was established to quantify transcript levels of the myogenic regulatory factors MyoD, myogenin and MRF4 (muscle regulatory factor 4) and for Id-1 (inhibitor of differentiation), a putative negative regulator of myogenesis. The method was sensitive enough to detect mRNA amounts as low as 20 molecules. Measurements in 10 different skeletal muscles of the rat revealed that the amounts of the four factors differ by almost three orders of magnitude. Id-1 is expressed at lowest levels (≈4×105 molecules/μg RNA) and MRF4 at highest levels (≈9×107 molecules/μg RNA). In general, myogenin and MyoD mRNAs were inversely distributed in slow and fast muscles. A correlation seemed to exist between the levels of MyoD and myosin heavy chain (MHC) IIb, the fastest MHC isoform. However, as revealed by changes in the expression levels of these two regulatory factors under conditions of hypothyroidism and chronic low-frequency stimulation (CLFS), MyoD and myogenin did not seem to be strictly correlated with fast and slow myosins, respectively. Hypothyroidism led to pronounced depressions of MyoD, but only to small increases in myogenin mRNA in fast muscles. These changes were only slightly increased by CLFS. However, as previously shown, CLFS in combination with hypothyroidism induces in rat muscle pronounced fast to slow transitions in myosin expression [Kirschbaum, B. J., Kucher, H.-B., Termin, A., Kelly, A. M. & Pette, D. (1990) J. Biol. Chem. 265, 13974–13980]. These findings suggest that MyoD and myogenin may not be causally related to the development and maintenance of fiber-type diversities.

Abbreviations
bHLH

basic helix-loop-helix

CLFS

chrome low-frequency stimulation

DEIA

DNA enzyme immunoassay

EDL

extensor digitorum longus

Id-1

inhibitor of differentiation

MHC

myosin heavy chain

MRF4

muscle regulatory factor 4

RT-PCR

reverse-transcriptase polymerase chain reaction

TA

tibialis anterior

Enzymes
 

Reverse transcriptase from avian myeloblastosis virus (EC 2.7.7.49)

 

Taq polymerase (EC 2.7.7.7)

Ancillary