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

A highly sensitive method of reverse-transcriptase polymerase chain re action (RT-PCR) was established to quantify transcript levels of the m yogenic regulatory factors MyoD, myogenin and MRF4 (muscle regulatory factor 4) and for Id-1 (inhibitor of differentiation), a putative nega tive regulator of myogenesis. The method was sensitive enough to detec t mRNA amounts as low as 20 molecules. Measurements in 10 different sk eletal muscles of the rat revealed that the amounts of the four factor s differ by almost three orders of magnitude. Id-1 is expressed at low est levels (approximate to 4X10(5) molecules/mu g RNA) and MRF4 at hig hest levels (approximate to 9X10(7) molecules/mu g RNA). In general, m yogenin and MyoD mRNAs were inversely distributed in slow and fast mus cles. A correlation seemed to exist between the levels of MyoD and myo sin heavy chain (MHC) IIb, the fastest MWC isoform. However, as reveal ed by changes in the expression levels of these two regulatory factors under conditions of hypothyroidism and chronic low-frequency stimulat ion (CLFS), MyoD and myogenin did not seem to bs strictly correlated w ith fast and slow myosins, respectively. Hypothyroidism led to pronoun ced depressions of MyoD, but only to small increases in myogenin mRNA in fast muscles. These changes were only slightly increased by CLFS. H owever, as previously shown, CLFS in combination with hypothyroidism i nduces in mt muscle pronounced fast to slow transitions in myosin expr ession [Kirschbaum, B. J., Kucher. H.-B., Termin. A., Kelly, A. M. & P ette, D. (1990) J. Biol. Chem. 265, 13974-13980]. These findings sugge st that MyoD and myogenin may not be causally related to the developme nt and maintenance of fiber-type diversities.