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
Exercise plays an important role in cardiac health and enhances the transport of glucose in cardiac muscle by increasing the glucose transporter-4 (GLUT4) content at the cell membrane. The GLUT4 gene is a target of myocyte enhancer transcription factor 2A (MEF2A). Several transcription factors are regulated by microRNAs (miRs), small non-coding RNAs that control gene expression at the posttranscriptional level. In this study we tested the hypothesis that exercise regulates the expression of miR-223 and that MEF2A is a direct target of miR-223. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot experiments showed that GLUT4 gene expression and protein abundance increased by 30 and 23%, respectively, in the microsomal fraction immediately after exercise, and had returned to control levels after 18 h. In contrast, the increase in GLUT4 in the membrane fraction was delayed. Exercise also increased the protein abundance of transcription factors involved in GLUT4 expression. Immediately after exercise, the protein abundance of MEF2A, nuclear respiratory factor 1 (NRF1), and forkhead box O1 (FOXO1) increased by 18, 30, and 40%, respectively. qRT-PCR experiments showed that miR-223-3p and miR-223-5p expression decreased immediately after exercise by 60 and 30%, respectively, and luciferase assays indicated that MEF2A is a target of the 5p strand of miR-223. Overexpression of miR-223-5p in H9c2 cells decreased the protein abundance of MEF2A. Our results suggest that the exercise-induced increase in GLUT4 content in cardiac muscle is partly due to the posttranscriptional increase in MEF2A protein abundance caused by the decrease in miR-223-5p expression. The exercise-induced decrease in miR-223-3p expression likely contributes to the increases in NRF1 and FOXO1 abundance and GLUT4 content.