Thyroid hormone regulates the transcription of several important cardiac genes. Although the thyroid gland produces predominantly thyroxine (T(4)), it is triiodothyronine (T(3)) that is transported across the sarcolemma and binds to nuclear thyroid hormone receptor proteins; yet various studies suggest that serum T(3) levels do not accurately reflect cellular T(3) action. To address this question, we studied the dose-response relationship of T(3) administered by constant infusion in hypothyroid animals with the simultaneous in vivo transcription rate of the cardiac-specific alpha-myosin heavy chain (MHC) gene, measured by quantitating alpha-MHC heteronuclear (hn)RNA content. Constant infusion of 4 mug T(3) x kg body wt(-1) x day(-1) for 3 days normalized serum T(3) and restored transcription to euthyroid levels; in contrast, daily injections of the same dose increased alpha-MHC transcription by only 55% of that obtained by infusion. Although infusion of T(3) at 1.25 microg T(3) x kg body wt(-1) x day(-1) was not sufficient to restore serum T(3) to normal, it was capable of restoring transcription to normal at 3 days, but when administered for 12 days, transcription of alpha-MHC was found to be 50% of euthyroid levels, demonstrating a decreased sensitivity to T(3) over time. Treatment with trichostatin A (TSA) to inhibit histone deacetylation increased levels of total nuclear acetylated histone H4 by almost 50% but was without effect on the real-time PCR measures of alpha-MHC hnRNA. TSA administered together with T(3) (10 mug T(3)/kg body wt) significantly increased transcription of alpha-MHC after 30 h, thus demonstrating a potential role for histones as cofactors in the T(3) regulation of cardiac alpha-MHC transcription.