Differential regulation of the myosin heavy chain genes alpha and beta in rat atria and ventricles: role of antisense RNA

Abstract

Background: The myosin heavy chain (MHC) genes are regulated by triiodothyronine (T3) in a reciprocal and chamber-specific manner. To further our understanding of the potential mechanisms involved, we determined the T3 responsiveness of the MHC genes, alpha and beta, and the beta-MHC antisense (AS) gene in the rat ventricles and atria.

Methods: Hypothyroid rats were administered a single physiologic (1 microg) or pharmacologic (20 microg) dose of T3, and sequential measurements of beta-MHC hn- and AS RNA and alpha-MHC heterogeneous nuclear RNA from rat ventricular and atrial myocardium were performed with reverse transcription PCR.

Results: We have demonstrated that T3 treatment increases the myocyte content of an AS beta-MHC RNA in atria and ventricles that includes sequences complementary to both the first 5' and last 3' introns of the beta-MHC sense transcript. In the hypothyroid rat ventricle, beta-MHC sense RNA expression is maximal, while in the euthyroid rat ventricle, beta-MHC AS RNA is maximal. beta-MHC AS expression increased by 52 +/- 9.8% at the peak, 24 hours after injection of a physiologic dose of T3 (1 microg/animal), while beta-MHC sense RNA decreased by 41 +/- 2.2% at 36 hours, the nadir. In hypothyroid atria, beta-MHC AS RNA was induced by threefold within 6 hours of administration of 1 microg T3, demonstrating that in the atria, beta-MHC AS expression is regulated by T3, while alpha-MHC expression is not.

Conclusions: In the hypothyroid rat heart ventricle, beta-MHC AS RNA expression increases in response to T3 similar to that of alpha-MHC. Simultaneous measures of beta-MHC sense RNA are decreased, suggesting a possible mechanism for AS to regulate sense expression. In atria, while alpha-MHC is not influenced by thyroid state, beta-MHC sense and AS RNA were simultaneously and inversely altered in response to T3. This confirms a close positive relationship between T3 and beta-MHC AS RNA in both the atria and ventricles, while demonstrating for the first time that alpha- and beta-MHC expression is not coupled in the atria.

FIG. 1.

LV expression of β-myosin heavy chain heterogeneous nuclear RNA (β-MHC hnRNA) (sense) after a single injection of 1 or 20 μg triiodothyronine (T₃) to hypothyroid animals using primer set 1. Values are expressed as percent of the maximal expression in hypothyroid hearts. LV, left ventricular.

FIG. 2.

Representative gels for α- and β-myosin heavy chain (MHC) sense and antisense (AS) RNA from ventricular and atrial cardiac tissue in euthyroid and hypothyroid rat hearts. The 24- and 48-hour bands represent the amount of β-MHC sense and AS RNA in hypothyroid animals after injection of 1 μg triiodothyronine (T₃). Animals sacrificed at 48 hours received a second injection of 1 μg triiodothyronine (T₃) 24 hours after the first injection as described in “Materials and Methods” section.

FIG. 3.

β-Myosin heavy chain (MHC) sense and antisense (AS) expression after administration of a single 1 μg dose of triiodothyronine (T₃) to hypothyroid rats. Expression of β-MHC sense RNA is given as the percent of hypothyroid levels (where expression is maximal), and expression of β-MHC AS RNA is given as the percent of euthyroid levels (where expression is maximal). Reverse transcription (RT) PCR was accomplished using primer set 1 for both sense and AS RNA. Expression was measured by RT PCR as described at the various time points after a single injection of 1 μg T₃.

FIG. 4.

Expression of β-myosin heavy chain antisense (β-MHC AS) RNA at 6, 12, 24, and 36 hours after a single injection of 1 or 20 μg triiodothyronine (T₃) to hypothyroid animals using primer set 2. Expression is given as percent euthyroid AS levels.

FIG. 5.

β-Myosin heavy chain (MHC) sense and antisense (AS) expression after administration of a single 20 μg dose of triiodothyronine (T₃) to hypothyroid rats. Expression of β-MHC sense RNA is given as the percent of hypothyroid levels, and expression of β-MHC AS RNA is given as the percent of euthyroid levels. Reverse transcription PCR was accomplished as described using primer sets that target the 5′ end of each molecule (primer sets 1 and 2 for sense and AS RNA, respectively). Expression was measured at the various time points after a single injection of 20 μg T₃ to hypothyroid rats.

FIG. 6.

Expression of (A) α-myosin heavy chain heterogeneous nuclear RNA (α-MHC hnRNA), (B) β-MHC sense (hnRNA), and (C) β-MHC antisense (AS) RNA in the atria and ventricles of hypothyroid (Tx) and euthyroid (Eu) rats. *p < 0.01 versus Eu.