RESUMO
Thyroid hormone binds to specific nuclear receptors, regulating the expression of target genes, with major effects on cardiac function. Triiodothyronine (T3) increases the expression of key proteins related to calcium homeostasis, such as the sarcoplasmic reticulum calcium ATPase pump, but the detailed mechanism of gene regulation by T3 in cardiac voltage-gated calcium (Cav1.2) channels remains incompletely explored. Furthermore, the effects of T3 on Cav1.2 auxiliary subunits have not been investigated. We conducted quantitative reverse transcriptase polymerase chain reaction, Western blot, and immunofluorescence experiments in H9c2 cells derived from rat ventricular tissue, examining the effects of T3 on the expression of α1c, the principal subunit of Cav1.2 channels, and Cavß4, an auxiliary Cav1.2 subunit that regulates gene expression. The translocation of phosphorylated cyclic adenosine monophosphate response element-binding protein (pCREB) by T3 was also examined. We found that T3 has opposite effects on these channel proteins, upregulating α1c and downregulating Cavß4, and that it increases the nuclear translocation of pCREB while decreasing the translocation of Cavß4. Finally, we found that overexpression of Cavß4 represses the mRNA expression of α1c, suggesting that T3 upregulates the expression of the α1c subunit in response to a decrease in Cavß4 subunit expression.
Assuntos
Canais de Cálcio Tipo L , Miócitos Cardíacos , Animais , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/genética , Ratos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Tri-Iodotironina/farmacologia , Tri-Iodotironina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Hormônios Tireóideos/metabolismo , Linhagem Celular , Regulação para Cima/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genéticaRESUMO
The H9c2 myoblast cell line, isolated from the left ventricular tissue of rat, is currently used in vitro as a mimetic for skeletal and cardiac muscle due to its biochemical, morphological, and electrical/hormonal signaling properties. During culture, H9c2 cells acquire a myotube phenotype, where a critical component is the inclusion of retinoic acid (RA). The results from some authors on H9c2 suggested that thousands of genes respond to RA stimuli, while others report hundreds of genes responding to RA over different cell types. In this article, using a more appropriate experimental design, we first confirm the H9c2 cardiac phenotype with and without RA and report transcriptomic and physiological changes regarding calcium handling, bioenergetics, and other biological concepts. Interestingly, of the 2360 genes showing a transcriptional change, 622 genes were statistically associated with the RA response. Of these genes, only 305 were RA-specific, and the rest also showed a culture-time component. Thus, the major expression changes (from 74 to 87%) were indeed due to culture conditions over time. Unexpectedly, only a few components of the retinol pathway in KEGG responded to RA. Our results show the role of RA in the H9c2 cultures impacting the interpretation using H9c2 as an in vitro model.