Dynamic alterations in cardiac autonomic regulation during recurrent generalized seizures

Figures

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  Figure 1. Seizure-associated HRV alterations and cardiac autonomic regulation (A) Pre-PTZ: ECG signal before PTZ administration. No arrhythmias were observed, and the ECG remained stable. (B) PTZ phase: Representative ECG waveforms showing short-term heart rate irregularities and occasional atrioventricular (AV) block during early seizure-related muscle twitches. Zoomed-in view demonstrates the presence of regular P, QRS, and T waves in sinus beats, while non-sinus beats were excluded based on the absence of P waves or irregular R–R intervals. (C) Grade V seizure: Representative ECG showing prolonged rhythm disturbances, including recurrent AV block and transient cardiac arrest during behaviorally defined Grade V motor seizures. (D) Fatal seizure: ECG recorded during a terminal seizure, showing erratic and disorganized cardiac rhythm leading to complete asystole. EMG contamination during convulsions was excluded from HRV analysis. (E) Representative ECG waveforms from the same mouse before and after PTZ injection, showing preserved P, QRS, and T waves despite seizure activity. (F) Quantification of ECG conduction parameters before and after PTZ administration. P-wave duration and QRS…

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  Figure 2. Effects of systemic muscarinic blockade on seizure-associated HRV alterations and cardiac autonomic regulation (A) Representative ECG traces from mice pretreated with atropine before PTZ administration. The left panel shows ECG activity during minor seizure-related twitches, and the right panel shows ECG during a Grade V seizure. Zoomed-in views (lower traces) highlight regular P–QRS–T wave morphology and the absence of severe arrhythmias, demonstrating that atropine pretreatment was associated with reduced seizure-associated rhythm abnormalities. (B) Representative averaged ECG waveforms from the same mouse before and after atropine + PTZ treatment, showing well-preserved P, QRS, and T wave morphology under both conditions. (C) Quantitative analysis of ECG conduction parameters. No significant differences were found in P-wave duration, PQ interval, QRS duration, or QTc time between pre-PTZ and atropine + PTZ groups (p > 0.05 for all; n = 12), indicating that atropine did not alter intrinsic myocardial conduction. P-wave duration and QRS duration were analyzed using the Wilcoxon signed-rank test, while PQ interval and QTc time were analyzed using paired t-tests. (D) R–R interval…

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  Figure 3. Cardiac autonomic regulation following recurrent seizures (A) Representative ECG waveforms in control (left) and seizure (right) mice show preserved P, QRS, and T wave morphology in both groups. (B) Quantitative analysis of ECG conduction parameters shows no significant differences in P-wave duration, PQ interval, QRS duration, or QTc interval between groups (p > 0.05), indicating that no significant differences were detected in the measured ECG conduction parameters. (C) R–R interval plots from 12-h ECG recordings reveal markedly reduced HRV in seizure mice compared to controls, indicating persistent alterations in cardiac autonomic regulation. (D–G) Quantification of heart rate and HRV parameters. (D) Heart rate was significantly elevated in seizure mice, consistent with altered autonomic regulation, possibly involving reduced parasympathetic contribution. (E–G) Time-domain HRV analysis showed significantly lower SDNN (E), RMSSD (F), and PNN6 (G) in seizure mice, reflecting persistent alterations in cardiac autonomic regulation. Data are presented as mean ± SEM or median [IQR] for nonparametric data. Statistical comparisons were performed using unpaired t-tests or the…

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  Figure 4. Functional changes in cardiac vagal neurons (CVNs) following repeated seizures (A) Schematic illustration of retrograde labeling of CVNs by applying DiI to the sinoatrial node. Representative low-magnification (5×) and high-magnification (40×) images of DiI-labeled neurons in the nucleus ambiguus (NA) are shown together with brightfield, fluorescence, and merged views. Scale bars, 250 μm (5× image) and 25 μm (40×, brightfield, fluorescence, and merged images). (B and C) Passive membrane properties were unchanged between groups. Input resistance (Rm, B) and membrane capacitance (Cm, C) showed no significant differences (unpaired t test, p > 0.05). (D) Representative whole-cell patch-clamp recordings of CVNs spike firing in response to square-pulse current injections (300 pA and 600 pA) in control and seizure mice. Seizure mice exhibited reduced firing frequency compared to controls. (E) Quantification of spike number as a function of injected current (100–600 pA). Seizure mice showed significantly fewer spikes across increasing current injections compared to control mice, indicating impaired excitability. ∗∗∗p < 0.001, two-way ANOVA with Tukey’s multiple comparisons test. (F)…

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  Figure 5. Optical mapping analysis of cardiac electrophysiology in control and seizure mice (A) Representative optical action potential traces recorded from control and seizure mice during electrical stimulation (10 Hz, 2 s). No significant differences were observed between groups. (B) Representative activation maps showing conduction velocity (CV) distribution across the heart in control and seizure mice. (C) Quantification of conduction velocity (CV) as a function of pacing interval. No significant differences were detected between control and seizure mice. (D) Representative activation maps of action potential duration at 50% repolarization (APD50) in control and seizure mice. (E) Quantification of APD50 at different pacing intervals. No significant differences were observed between control and seizure groups. (F) Representative activation maps of action potential duration at 80% repolarization (APD80) in control and seizure mice. (G) Quantification of APD80 at different pacing intervals. No significant differences were found between groups. Statistical comparisons were performed using two-way ANOVA with Tukey’s multiple comparisons test. Analyses were based on 8 control mice and 8…

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  Figure 6. Effects of systemic muscarinic blockade on seizure duration and survival in PTZ-induced seizures (A) Comparison of seizure duration between mice that survived and those that died. Seizure duration was significantly longer in mice that did not survive. Data points represent individual seizures observed in the mice, with some mice experiencing multiple Grade V seizures. ∗∗∗p < 0.001, Mann-Whitney rank-sum test. (B) Seizure duration progressively increased across successive Grade V seizures. The second seizure lasted significantly longer than the first, and the third was significantly longer than the second. Each data point represents an individual Grade V seizure from a single mouse. Statistical comparisons were performed using the Kruskal–Wallis one-way ANOVA on ranks followed by Dunn’s post hoc test. ∗p < 0.05. (C) Atropine pretreatment did not significantly alter seizure duration across repeated Grade V seizures. Statistical comparisons were performed using two-way ANOVA with Tukey’s multiple comparisons test. Both PTZ-only and PTZ + atropine groups exhibited a similar progressive increase in seizure duration over successive episodes. (D) Survival rates in PTZ-only and PTZ +…