Employing published methods for isolation of cardiac myocyte nuclei from adult rat ventricular myocardium with the use of mechanical disruption without digestive enzymes, we obtained transcriptionally active cardiac myocyte nuclei with sufficient yield and purity. The relative content of Na/K-ATPase subunit mRNAs (alpha 1, alpha 2, and beta 1) in ventricular myocardium of euthyroid rats closely matched the relative rates of transcription of the respective subunit genes determined by nuclear run-on assay. Treatment of hypothyroid rats with T(3)to elicit hyperthyroidism was associated with 2.9-, 7.5-, and seven-fold increases in the contents of alpha 1-, alpha 2, beta 1-mRNAs, respectively. In contrast, rates of transcription of the subunit genes were not changed significantly by T(3), while transcription of the 18 S ribosomal gene was stimulated identical with three-fold by the treatment. A quantitative reverse transcription-polymerase chain reaction assay for measurement of primary RNA transcripts of the beta 1 gene was developed employing a rat genomic DNA fragment that contains the first exon and part of the first intron of the beta 1 gene. The relative abundance of beta 1 primary transcripts did not change in RNA isolated from hypothyroid, euthyroid, and hyperthyroid rats. It is concluded that: (1) The relative contents of Na/K-ATPase subunit mRNAs in euthyroid adult myocardium is primarily controlled at the transcriptional level, and (2) T(3)-induced increases in the contents of Na/K-ATPase subunit mRNAs in the heart is not associated with increased rates of transcription of the subunit genes, and the effect is mediated at the post-transcriptional level.
Copyright 2000 Academic Press.