Targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced sustained ventricular tachycardia in conscious rats

Abstract

The Cardiac Arrhythmia Suppression Trial demonstrated that antiarrhythmic drugs not only fail to prevent sudden cardiac death, but actually increase overall mortality. These findings have been confirmed in additional trials. The "proarrhythmic" effects of most currently available antiarrhythmic drugs makes it essential that we investigate novel strategies for the prevention of sudden cardiac death. Targeted ablation of cardiac sympathetic neurons may become a therapeutic option by reducing sympathetic activity. Thus cholera toxin B subunit (CTB) conjugated to saporin (a ribosomal inactivating protein that binds to and inactivates ribosomes; CTB-SAP) was injected into both stellate ganglia to test the hypothesis that targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced, sustained ventricular tachycardia in conscious rats. Rats were randomly divided into three groups: 1) control (no injection); 2) bilateral stellate ganglia injection of CTB; and 3) bilateral stellate ganglia injection of CTB-SAP. CTB-SAP rats had a reduced susceptibility to ischemia-induced, sustained ventricular tachycardia. Associated with the reduced susceptibility to ventricular arrhythmias were a reduced number of stained neurons in the stellate ganglia and spinal cord (segments T(1)-T(4)), as well as a reduced left ventricular norepinephrine content and sympathetic innervation density. Thus CTB-SAP retrogradely transported from the stellate ganglia is effective at ablating cardiac sympathetic neurons and reducing the susceptibility to ventricular arrhythmias.

Fig. 1.

One-second analog recordings of arterial pressure and the electrocardiogram (ECG) before coronary artery occlusion (preoccclusion), at 4 min of occlusion, and at the onset of sustained ventricular tachycardia and reduction in arterial pressure (prearrhythmia) from two rats. Each rat received bilateral stellate injections of cholera toxin B (CTB; top) or CTB-saporin (SAP; bottom). The time from coronary artery occlusion to the onset of sustained ventricular tachycardia and reduction in arterial pressure was defined as the ventricular arrhythmia threshold. Coronary artery occlusion was documented by ST segment elevation. Note the higher ST segment elevation in the CTB rat compared with the CTB-SAP rat at 4 min of occlusion. CTB-SAP rats had a significantly longer ventricular arrhythmia threshold compared with control and CTB rats. In this example, the sustained ventricular tachycardia and reduction in arterial pressure occurred at 4 min and 12 s in the CTB rat compared with 10 min in the CTB-SAP rat.

Fig. 2.

Ventricular arrhythmia threshold (VAT) in the control, CTB, and CTB-SAP groups. There was no difference in the VAT between the control and CTB group. However, the VAT was significantly longer in the CTB-SAP group. Values are means ± SE. *P < 0.05, CTB-SAP vs. control or CTB.

Fig. 3.

ST-segment elevation at 4 min of occlusion in the control, CTB, and CTB-SAP groups. There were no differences in the ST-segment elevation between the control and CTB groups. However, the ST-segment elevation was significantly lower in the CTB-SAP group. Values are means ± SE. *P < 0.05, CTB-SAP vs. control or CTB.

Fig. 4.

Neuronal number (A), soma area (B), and cresyl violet-stained neurons (C) of the stellate ganglia from three rats that had no injection (control), CTB, or CTB-SAP injected into both stellate ganglia. A: counts of stellate neurons in the CTB-SAP group showed a significant reduction in the number of neurons compared with the control or CTB groups. These results are consistent with C, showing many stellate neurons in the control and CTB groups and few neurons in the CTB-SAP group. B: however, mean soma area was not different between the three groups. The counts reported in this figure are similar to counts reported in two publications (6, 21). Values are means ± SE. *P < 0.05, CTB-SAP vs. control or CTB. Scale bar (C, bottom right) = 25 μm.

Fig. 5.

Horizontal sections of spinal cord segments T₁-T₄ from two rats that had CTB (A) or CTB-SAP (B) injected into both stellate ganglia. Many CTB-immunoreactive sympathetic preganglionic neurons are identified in the CTB rat; however, none is observed in the CTB-SAP rat. CTB-immunoreactive sympathetic preganglionic neurons are seen in the intermediolateral cell column, lateral funiculus, intercalated nucleus, and central automomic nucleus. Scale bar (bottom right) = 25 μm.

Fig. 6.

Cresyl violet-stained neurons of the dorsal root ganglia from rats that had CTB (A) or CTB-SAP (B) injected into both stellate ganglia. It is clear that CTB-SAP did not alter neurons within the dorsal root ganglia. Scale bar (bottom right) = 25 μm.

Fig. 7.

Tyrosine hydroxylase-immunoreactive fiber density (A), fiber number (B), and stained sympathetic fibers (C) from rats that had no injection (control), CTB, or CTB-SAP injected into both stellate ganglia. The CTB-SAP group showed a significant reduction in sympathetic nerve fiber density and number compared with the control and CTB groups. These results are consistent with C, showing many sympathetic fibers in the control and CTB groups and few sympathetic fibers in the CTB-SAP group. Values are means ± SE. *P < 0.05, CTB-SAP vs. control or CTB.

Fig. 8.

Left ventricular norepinephrine content from rats that had no injection (control), CTB, or CTB-SAP injected into both stellate ganglia. The CTB-SAP group showed a significant reduction in norepinephrine content compared with control and CTB groups. Values are means ± SE. *P < 0.05, CTB-SAP vs. control or CTB.