Thyroid Hormone Upregulates Cav1.2 Channels in Cardiac Cells via the Downregulation of the Channels’ β4 Subunit

Figures

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  Figure 1. Relative mRNA expression and α1c protein abundance in H9c2 cells after incubation with T3. (A,B) Means ± SEMs of relative α1c subunit mRNA expression and protein abundance in H9c2 cells from three separate experiments. Insets show representative blots under control conditions (lanes a, c, e, and g) and 2, 6, 24, and 48 h after T3 treatment (lanes b, d, f, and h, respectively). Tubulin bands were used to normalize α1c subunit density values. In panels (A,B) each symbol represents a separate experiment. Original blots are presented in Figure S1. * p < 0.05. ** p < 0.01.

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  Figure 2. Relative Cavβ4 protein abundance in H9c2 cells after incubation with T3. (A) Means ± SEMs of relative Cavβ4 protein abundance in H9c2 cells (n = 3–7). Insets show representative blots under control conditions (a, c, e, and g) and 2, 6, 24, and 48 h after T3 incubation (lanes b, d, f, and h, respectively) from four separate experiments. GAPDH bands were used to normalize Cavβ4 subunit density values. Original blots are presented in Figure S1. (B) Means ± SEMs of relative Cavβ4 subunit mRNA expression in H9c2 cells after incubation with T3. (C) Confocal microscopic images of H9c2 cells under control conditions and after T3 treatment for 2 h. Representative images from three independent experiments show the co-localization of the Cavβ4 subunit (green) with Hoechst 33342-labeled nuclei (blue). Calibration bar, 32 μm. (D) Means ± SEMs of the nuclei/cytosolic ratio from three separate experiments. In panels (A,B,D), each symbol represents a separate experiment. * p < 0.05, ** p < 0.01.

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  Figure 3. Relative α1c mRNA expression and protein abundance in H9c2 cells after Cavβ4 subunit overexpression. (A) Means ± SEMs of relative Cavβ4 protein abundance under control conditions and after Cavβ4 overexpression (n = 3). The inset shows representative blots of the Cavβ4 subunit and GAPDH bands; the latter was used to normalize Cavβ4 subunit density values. Original blots are presented in Figure S1. (B) Means ± SEMs of relative α1c subunit mRNA expression from control and Cavβ4 overexpression experiments (n = 14). (C) Means ± SEMs of α1c protein abundance from control and Cavβ4 overexpression experiments (n = 10). The inset shows representative blots of α1c subunit and tubulin bands under control conditions (lanes a and c) and after Cavβ4 overexpression (lanes b and d) from two separate experiments. Original blots are presented in Figure S1. In panels (A,B,C), each symbol represents a separate experiment. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 4. Relative abundance of pCREB in H9c2 cells after incubation with T3. Means ± SEMs of the relative abundance of pCREB under control conditions and after T3 treatment (n = 5). Each symbol represents a separate experiment. Representative blots of pCREB and tubulin under control conditions (lanes a, c, e, g, and i) and after T3 treatment for the indicated periods (lanes b, d, f, h, and j). Tubulin density values were used for normalization. Original blots are presented in Figure S1.

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  Figure 5. T3-induced translocation of pCREB to the nuclei of H9c2 cells. Confocal microscopic images of H9c2 cells under control conditions and after T3 treatment for 2 h. Representative images from three independent experiments show the co-localization of pCREB (red) with Hoechst 33342-labeled nuclei (blue). Calibration bar, 30 μm. Means ± SEMs of the nuclei/cytosolic ratio after T3 treatment for 2 and 6 h (n = 3). Each symbol represents a separate experiment. * p < 0.05.

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  Figure 6. Role of Cavβ4 in pCREB regulation by T3. Schematic showing signaling mechanisms hypothetically involved in thyroid hormone actions on the CACNA1C gene via pCREB and the release of Cavβ4 inhibition of α1c subunit expression in ventricular cardiac cells.