Hydrogen Peroxide Scavenging Restores N-Type Calcium Channels in Cardiac Vagal Postganglionic Neurons and Mitigates Myocardial Infarction-Evoked Ventricular Arrhythmias in Type 2 Diabetes Mellitus

Figures

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  Figure 1. Overexpression of catalase in CVP neurons reduced intracellular H2O2 levels in T2DM rats. (A) Representative images of cytosolic H2O2 levels in isolated CVP neurons from sham, T2DM, and T2DM+Ad.CAT rats, measured by detecting fluorescence intensity of pHyPer-cyto (green color, a H2O2 sensor). (B) Quantitative data showing fluorescence intensity of pHyPer-cyto in CVP neurons in all groups. N = 40 neurons from 6 rats per group; data are means ± SEM. Statistical significance was determined by one-way ANOVA with post-hoc Bonferroni test. *P < 0.05 vs. sham; †P < 0.05 vs. T2DM.

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  Figure 2. Effect of Ad.CAT gene transfection on protein expression of catalase and N-type Ca2+ channels (Cav2.2-α) in CVP neurons in T2DM. (A) Raw images and (B) quantitative data showing protein expression of catalase in CVP neurons from all groups of rats, measured by revers–phase protein microarray. (C) Representative images and (D) quantitative data showing protein expression of Cav2.2-α in the CVP neurons from all groups of rats. N = 24 measurements from 6 rats per group. (E) Catalase activity measured in all groups. N = 6 rats per group. Ad.CAT gene transfection significantly increased T2DM-reduced catalase activity, and protein expression of catalase and Cav2.2-α in CVP neurons. Data are means ± SEM. Statistical significance was determined by one-way ANOVA with post-hoc Bonferroni test. *P < 0.05 vs. sham; †P < 0.05 vs. T2DM.

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  Figure 3. Reduction of the H2O2 levels through transfection of Ad.CAT gene increased T2DM-reduced N-type Ca2+ currents in CVP neurons in T2DM rats. BaCl2 replaced CaCl2 in the extracellular solution for Ca2+ current recording. (A) Original whole-cell patch-clamp recording of Ca2+ currents from sham, T2DM, and T2DM+Ad.CAT rats. (B) Current-voltage (I–V) curve of N-type Ca2+ currents in CVP neurons from all groups of rats. (C) Quantitative data of total Ca2+ currents, other types of Ca2+ currents, and N-type Ca2+ currents elicited by 500-ms test pulse at 0 mV from holding potential of −80 mV in CVP neurons from all groups. ω-conotoxin GVIA, a specific N-type Ca2+ channel blocker, was used to block the N-type Ca2+ channel. N-type Ca2+ currents were obtained by subtracting Ca2+ currents under treatment of ω-conotoxin GVIA from total Ca2+ currents. N = 8 neurons from 6 rats per group; data are means ± SEM. Statistical significance was determined by two-way repeated measures ANOVA with post-hoc Bonferroni test for data presented in (B). Statistical significance was determined by one-way ANOVA with post-hoc Bonferroni test for data presented in (C). *P < 0.05 vs. sham; †P < 0.05 vs. T2DM.

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  Figure 4. In vivo transfection of Ad.CAT gene restored T2DM-reduced cell excitability of CVP neurons in T2DM rats. (A) Original recording of action potentials (APs) in CVP neurons from sham, T2DM, and T2DM+Ad.CAT rats. (B,C) Quantitative data for the frequency of APs in CVP neurons from all groups. The frequency of APs was measured in a 1-s current clamp with a current injection of 100 pA. N = 8 neurons from 6 rats per group; data are means ± SEM. Statistical significance was determined by one-way ANOVA with post-hoc Bonferroni test for data presented in (B). Statistical significance was determined by two-way repeated measures ANOVA with post-hoc Bonferroni test for data presented in (C). *P < 0.05 vs. sham; †P < 0.05 vs. T2DM.

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  Figure 5. In vivo transfection of Ad.CAT gene restored T2DM-decreased intracellular Ca2+ levels in CVP neurons from T2DM rats. (A) Raw images of fluo-3/AM (green color, a calcium indicator) in isolated CVP neurons from sham, T2DM, and T2DM+Ad.CAT rats, in which the fluorescent image was captured by a confocal microscope before (F0) and during KCl (30 mM) stimulation at 30 s (FMax). (B) Mean data for the fluorescence intensity of fluo-3/AM in isolated CVP neurons from all groups of rats. N = 60 neurons from 6 rats per group; data are means ± SEM. Statistical significance was determined by one-way ANOVA with post-hoc Bonferroni test. *P < 0.05 vs. sham; †P < 0.05 vs. T2DM.

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  Figure 6. Effect of Ad.CAT gene transfection into CVP neurons on vagal control of ventricular function in T2DM rats, which was determined by changes of the left ventricular systolic pressure (LVSP) and the maximum rate of increase of left ventricular pressure (LV dP/dtmax) in response to different frequencies of left vagal efferent nerve stimulation (VNS) in anesthetized rats. (A) Representative recordings demonstrating changes of the LVSP in response to 7.5 V, 20 Hz of VNS in sham, T2DM, and T2DM+Ad.CAT rats. (B,C) Quantitative data for changes of the LVSP (B) and LV dP/dtmax (C) in response to different frequencies (1–100 Hz) of VNS in all groups of rats. In vivo Ad.CAT gene transfection into CVP neurons significantly improved T2DM-blunted vagal control of ventricular function. N = 6 rats per group; data are means ± SEM. Statistical significance was determined by two-way repeated measures ANOVA with post-hoc Bonferroni test. *P < 0.05 vs. sham; †P < 0.05 vs. T2DM.

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  Figure 7. In vivo transfection of Ad.CAT gene into CVP neurons improved T2DM-attenuated cardiac vagal activation, measured by the power spectral analysis of heart rate variability (HRV) in conscious rats. (A) Representative tracings of HRV analyzed from 24-h ECG recording in sham, T2DM, and T2DM+Ad.CAT rats. Spectral power was quantified for LF from 0.2 to 0.75 Hz and HF from 0.75 to 2.5 Hz. (B,C) Quantitative data of HF (B) and LF (C) from all groups of conscious rats. N = 6 rats per group; data are means ± SEM. Statistical significance was determined by one-way ANOVA with post-hoc Bonferroni test. *P < 0.05 vs. sham; †P < 0.05 vs. T2DM.

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  Figure 8. Reduction of intracellular H2O2 levels in CVP neurons attenuated the heterogeneity of ventricular electrical activities in conscious T2DM rats. (A) Representative tracings for QT and Tpe intervals in sham, T2DM, and T2DM+Ad.CAT rats. (B–F) Quantitative data for QT interval (B), QTc interval (C), QT dispersion (D), QTc dispersion (E), and Tpe (F) in all experimental groups. N = 6 rats per group; data are means ± SEM. Statistical significance was determined by one-way ANOVA with post-hoc Bonferroni test. *P < 0.05 vs. sham; †P < 0.05 vs. T2DM.

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  Figure 9. Effect of Ad.CAT gene transfection into CVP neurons on susceptibility to ventricular tachyarrhythmia in anesthetized T2DM rats. (A) Raw data for PES-evoked VT/VF in anesthetized sham, T2DM, and T2DM+Ad.CAT rats. (B,C) Mean data for incidence (B) and inducibility quotient (C) of PES-evoked VT/VF in all groups of rats. Ad.CAT gene transfection into CVP neurons markedly decreased the inducibility quotient of PES-evoked VT/VF in T2DM rats. N = 8 rats per group; data are means ± SEM. Statistical significance was determined by Fisher exact test for the incidence of VT/VF and one-way ANOVA with post-hoc Bonferroni test for inducibility quotient of VT/VF. *P < 0.05 vs. sham; †P < 0.05 vs. T2DM.

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  Figure 10. In vivo transfection of Ad.CAT gene into CVP neurons suppressed myocardial infarction (MI)-evoked ventricular arrhythmias in conscious T2DM rats. (A) Raw ECG recordings of VT/VF in conscious sham, T2DM, and T2DM+Ad.CAT rats. (B,C) Mean data for incidence (B) and cumulative duration of VT/VF (C) in all groups of conscious rats. N = 6 rats per group; data are means ± SEM. Statistical significances in the incidence of VT/VF and cumulative duration of VT/VF were determined by Fisher exact test and one-way ANOVA with post-hoc Bonferroni test, respectively. *P < 0.05 vs. sham; †P < 0.05 vs. T2DM.

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  Figure 11. Contribution of endogenous H2O2 elevation in CVP neurons to ventricular arrhythmogenesis and related therapeutic strategy in T2DM. In the T2DM state, the oxidative stress, as evidenced by endogenous H2O2 elevation and lowered catalase occurred in CVP neurons. Consequently, endogenous H2O2 elevation reduced the neuronal excitability through reduction of the N-type Ca2+ channel expression and activation. H2O2 overproduction in CVP neurons further attenuated the cardiac vagal activation and enhanced the susceptibility to ventricular arrhythmogenesis, including the inducibility of ventricular arrhythmias in anesthetized rats and MI-evoked ventricular arrhythmias in conscious rats. Reduction of cytosolic H2O2 levels in the AVG through in vivo Ad.CAT gene transfection markedly increased T2DM-reduced expression and activation of N-type Ca2+ channels, intracellular Ca2+ levels, and cell excitability of CVP neurons. Ad.CAT gene transfection subsequently improved impaired cardiac vagal function and suppressed ventricular arrhythmogenesis in T2DM. Therefore, targeting H2O2-N-type Ca2+ channel signaling pathway could be a potential therapeutic strategy to improve the withdrawal of cardiac…