Hydrogen Peroxide-Induced Re-Expression of Repressor Element 1-Silencing Transcription Factor Contributes to Cardiac Vagal Dysfunction in Type 2 Diabetes Mellitus

Figures

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  Figure 1. Effect of H2O2 on protein expression of REST and N-type Ca2+ channels (Cav2.2-α) in differentiated NG108-15 cells. (A) Raw and quantitative data representing the REST protein levels in undifferentiated NG108-15 cells (Control), 5-day differentiated NG108-15 cells, and 5-day differentiated NG108-15 cells treated with H2O2 for 24 h, measured by Western blot analysis. (B) Raw and quantitative data representing Cav2.2-α protein levels in undifferentiated NG108-15 cells (Control), 5-day differentiated NG108-15 cells, and 5-day differentiated NG108-15 cells treated with H2O2 for 24 h. Black dots represent each individual data point. N = 6 measurements/group. Data are means ± SEM. One-way ANOVA with post-hoc Bonferroni test was used to assess statistical significance. * p < 0.05 vs. Control; # p < 0.05 vs. 5-day differentiation.

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  Figure 2. Effect of adenoviral catalase (Ad.CAT) gene transfection on REST protein expression in AVG neurons from T2DM. Raw images (top panel) and quantitative data (bottom panel) representing the REST protein levels in AVG neurons from all groups of rats, analyzed by reverse-phase protein microarray. Black dots represent each individual data point. N = 12 measurements from 6 rats/group. Data are means ± SEM. One-way ANOVA with post-hoc Bonferroni test was used to assess statistical significance. * p < 0.05 vs. sham; # p < 0.05 vs. T2DM.

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  Figure 3. Effect of REST shRNA transfection on N-type Ca2+ channel (Cav2.2-α) protein expression in AVG neurons from T2DM rats. (A) Raw and quantitative data representing the REST protein levels in AVG neurons from all groups of rats, analyzed by reverse-phase protein microarray. (B) Raw and quantitative data representing Cav2.2-α protein levels in the AVG neurons from all groups of rats. Black dots represent each individual data point. N = 12 measurements from 6 rats/group. Data are means ± SEM. One-way ANOVA with post-hoc Bonferroni test was used to assess statistical significance. * p < 0.05 vs. sham; # p < 0.05 vs. T2DM.

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  Figure 4. Effect of REST shRNA transfection on N-type Ca2+ currents in AVG neurons from T2DM rats. (A) Raw data for Ca2+ currents from sham, T2DM, and T2DM + lenti.REST shRNA rats. (B) Quantitative data for total Ca2+ currents and other types of Ca2+ currents (cells treated with ω-conotoxin GVIA, a specific N-type Ca2+ channel blocker) recorded under the test pulse at 0 mV in AVG neurons from all groups of rats. (C) Quantitative data for N-type Ca2+ currents analyzed from the original recording in AVG neurons from all groups of rats. Subtracting Ca2+ currents under treatment of ω-conotoxin GVIA from total Ca2+ currents in the original recording was used to obtain N-type Ca2+ currents. Black dots represent each individual data point. N = 6 neurons from 4 rats/group; data are means ± SEM. One-way ANOVA with post-hoc Bonferroni test was used to assess statistical significance. * p < 0.05 vs. sham; # p < 0.05 vs. T2DM.

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  Figure 5. Effect of REST shRNA transfection on neuronal excitability in AVG neurons from T2DM rats. Raw (A) and quantitative data (B) for action potentials during a 1 s current clamp with a current injection of 100 pA in AVG neurons from all groups of rats. Black dots represent each individual data point. N = 6 neurons from 4 rats/group; data are means ± SEM. One-way ANOVA with post-hoc Bonferroni test was used to assess statistical significance. * p < 0.05 vs. sham; # p < 0.05 vs. T2DM.

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  Figure 6. Effect of REST shRNA on vagal control of the ventricular function in T2DM. Left vagal efferent nerve stimulation (VNS)-induced changes of the left ventricular systolic pressure (LVSP, panel A) and the maximum rate of increase of left ventricular pressure (LV dP/dtmax, panel B) represented the vagal control of the ventricular function. N = 6 rats/group; data are means ± SEM. Two-way repeated measures ANOVA with post-hoc Bonferroni was used to test statistical significance. * p < 0.05 vs. sham; # p < 0.05 vs. T2DM.