Id2 Represses Aldosterone-Stimulated Cardiac T-Type Calcium Channels Expression

Figures

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  Figure 1. Aldosterone reduce the Id2 expression in neonatal rat ventricular cardiomyocytes. Neonatal rat cardiomyocytes were stimulated with (A) Aldo (aldosterone) or (B) 1 µmol/L Aldo + Spirono (Spironolactone) for 24 h. Bar graphs show the Id2′s mRNA level in aldosterone (Aldo)-treated and nontreated cardiomyocytes. Id2 mRNAs were measured by real-time qPCR analysis (n = 6 for (A) and n = 3 for (B)). (C) Representative pictures of western blot experiments showing the Id2 protein (upper) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein (lower) expression levels in neonatal rat ventricular cardiomyocytes treated with 1 µmol/L aldosterone (Aldo) for 24 h. (D) The bar graph indicates the mean +/− s.e.m. of Id2 protein expression in aldosterone (Aldo)-treated (1 µmol/L) and non-treated cardiomyocytes from five independent experiments. The data were evaluated by one-way analysis of variance (ANOVA) followed by Tukey–Kramer’s post hoc test or by unpaired two-tailed Student’s t-test. Bar graphs are mean and + s.e.m, * p < 0.05, ** p < 0.01.

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  Figure 2. Effects of Id2 overexpression and siRNA knockdown on cardiomyocytes spontaneous action potential rate. (A) Id2 overexpression was achieved after 24 h transduction with adenoviral vector. Pictures of western blot experiments showing the Id2 protein (upper) and GAPDH protein (lower) expression levels in neonatal rat ventricular cardiomyocytes. (B) Id2 expression was knock down with siRNA. siRNAs were transfected for 48 h and proteins were extracted. Representative pictures of western blot experiments showing the Id2 protein (upper) and GAPDH protein (lower) expression levels in neonatal rat ventricular cardiomyocytes in control, Id2 targeting siRNA, and Luciferase (Luc) targeting siRNA. Bar graph are the mean of n = 3 independent experiments. (C) Bar graph shows the number of spontaneous action potential per min in control, aldosterone treated (1 µmol/L), Id2 overexpressing, Id2 overexpressing with aldosterone (1 µmol/L), Id2 siRNA, and Luc1 siRNA cardiomyocytes. The data were evaluated by one-way analysis of variance (ANOVA) followed by Tukey–Kramer’s post hoc test. Bar graphs are mean and + s.e.m, * p < 0.05, ** p < 0.01

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  Figure 3. Manipulation of Id2′s expression altered of voltage-gated calcium channel expression in neonatal rat ventricular cardiomyocytes and prevented aldosterone-stimulated increased. (A) 2, CaV3.1, and CaV3.2 were measured by RT-qPCR in neonatal rat ventricular cardiomyocytes treated and non-treated with 1 µmol/L Aldo for 24 h upon Id2 overexpression. Bar graphs shows the mean expression of CaV1.2 mRNA (left), CaV3.1 mRNA (middle), and CaV3.2 (right) (n = 5). (B) The mRNA expression of CaV1.2, CaV3.1, and CaV3.2 were measured by real-time qPCR in neonatal rat ventricular cardiomyocytes treated with Id2 siRNA-2, siRNA-5 or Luciferase1 siRNA for 48 h. Bar graphs shows the mean expression of CaV1.2 mRNA (left), CaV3.1 mRNA (middle), and CaV3.2 (right) (n = 3). The data were evaluated by one-way analysis of variance (ANOVA) followed by Tukey–Kramer’s post hoc test. Data are the mean ± s.e.m, * p < 0.05, ** p < 0.01. *** p < 0.001.

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  Figure 4. Manipulation of Id2′s expression altered of voltage-gated Ca2+ currents in neonatal rat ventricular cardiomyocytes. Voltage-gated Ca2+ currents were measured by whole-cell patch-clamp experiments under the voltage-clamp mode. Graphs show the mean current-voltage relationship. currents measured in ventricular neonatal rat cardiomyocytes. (A) Graphs show the mean current-voltage relationship (IV curve) of voltage-gated Ca2+ currents measured in ventricular neonatal rat cardiomyocytes. Ca2+ currents were measured from a holding potential of −90 mV and depolarized by 10 mV voltage steps to +80 mV, as described in Methods. (A) IV curves recorded from cells that were treated (Aldo, ●☐) or non-treated (Control, ■) with 1 µmol/L aldosterone for 24 h and in the same cells overexpressing Id2, treated (Id2-Ad + Aldo, ▲) or non-treated (Id2-Ad, ▼) with aldosterone (Aldo) at 1 µmol/L. Right traces are representative recordings traces for cells non-treated (Control), treated (Aldo) with aldosterone at 1 µmol/L (Aldo) (n=10 to 11 cells). (B) IV curves of Ca2+ currents recorded in non-treated cardiomyocytes (■) and cardiomyocytes transfected with Id2 siRNA (siRNA Id2-5, ▲) or control siRNA…

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  Figure 5. Id2 regulates the activity of CaV3.1 promoter. The luciferase reporter gene was placed under the control of 0.8kb of the CaV3.1 promoter. The reporter gene was transected into neonatal rat cardiomyocytes to measure the luciferase activity upon Id2 overexpression (left) or Id2 siRNA knockdown (right). Bar graphs represent the mean of the relative activity of luciferase from the CaV3.1 luciferase reporter plasmid construct. The data were evaluated by one-way analysis of variance (ANOVA) followed by Tukey–Kramer’s post hoc test. Values are mean + s.e.m., *** p < 0.001, ** p < 0.01 (n = 4–6).

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  Figure 6. Id2 expressing transgenic mice prevents the aldosterone-stimulated expression of CaV3.1 voltage-gated calcium channels in vivo. WT or cardiomyocyte-specific Id2 expressing transgenic B6D2F1/Slc mice were stimulated with Aldosterone (60 mg/Kg/day) or Saline solution for 1 week by implanting subcutaneously osmotic pumps. (A) Representative pictures of western blot experiments showing the Id2 protein (upper) and GAPDH protein (lower) expression levels from the heart of WT mice treated with saline solution or aldosterone. The bar graph is the mean of Id2 expression (n = 4). (B) Pictures of western blotting experiments showing the Id2 protein (upper) and GAPDH protein (lower) expression levels in the heart of B6D2F1/Slc WT and two cardiomyocyte-specific Id2 expressing transgenic (Tg) mice. (C) Pictures of western blotting experiments showing CaV3.1 (upper) and tubulin (lower) proteins expression levels in WT or Id2 transgenic mice treated with saline solution or aldosterone. Graph is are the mean expression of CaV3.1 and tubulin expression (n = 4). The data were evaluated by one-way analysis of variance (ANOVA) followed by Tukey–Kramer’s post hoc test or by unpaired two-tailed…