Endogenous endothelin stimulates cardiac sympathetic afferents during ischaemia

Abstract

Myocardial ischaemia activates cardiac sympathetic afferents leading to chest pain and reflex cardiovascular responses. Previous studies have shown that a brief period of myocardial ischaemia increases endothelin in cardiac venous plasma draining ischaemic myocardium and that exogenous endothelin excites cutaneous group III and IV sensory nerve fibres. The present study tested the hypothesis that endogenous endothelin stimulates cardiac afferents during ischaemia through direct activation of endothelin A receptors (ET(A)Rs). Nerve activity of single unit cardiac sympathetic afferents was recorded from the left sympathetic chain or rami communicates (T(2)-T(5)) in anaesthetized cats. Single fields of 38 afferents (CV = 0.25-3.86 m s(-1)) were identified in the left or right ventricle with a stimulating electrode. Five minutes of myocardial ischaemia stimulated all 38 cardiac afferents (8 Adelta, 30 C-fibres) and the responses of these 38 afferents to chemical stimuli were further studied in the following protocols. In the first protocol, injection of endothelin 1 (ET-1, 1, 2 and 4 microg) into the left atrium (LA) stimulated seven ischaemically sensitive cardiac afferents in a dose-dependent manner. Second, BQ-123, a selective ET(A)R antagonist, abolished the responses of nine afferents to 2 microg of ET-1 injected into the left atrium and attenuated the ischaemia-related increase in activity of eight other afferents by 51%. In contrast, blockade of ET(B) receptors caused inconsistent responses to exogenous ET-1 as well as to ischaemia. Furthermore, in the absence of ET(A)R blockade, cardiac afferents responded consistently to repeated administration of ET-1 (n = 7) and to recurrent myocardial ischaemia (n = 7). Finally, using an immunocytochemical staining approach, we observed that ET(A) receptors were expressed in cardiac sensory neurons in thoracic dorsal root ganglia. Taken together, these data indicate that endogenous endothelin contributes to activation of cardiac afferents during myocardial ischaemia through direct stimulation of ET(A) receptors likely to be located in the cardiac sensory nervous system.

Figure 1. Location of the receptive fields of ischaemically sensitive cardiac afferents on epicardial surface of left ventricle

Receptive fields of cardiac afferents included in the study: ▴, ET-1 (n= 7); ⋆, repeated ET-1 (n= 7); ⋆, ET-1 + BQ-123 (n= 9); □, repeated ischaemia (n= 7); •, ischaemia + BQ-123 (n= 8).

Figure 2. Impulse activity of seven cardiac sympathetic afferents to graded doses of ET-1 (1–4 μg) injected into left atrium

Columns and error bars represent means ±s.e.m.*P < 0.05 compared with control.

Figure 4. Neurohistogram showing response of a cardiac sympathetic C-fibre (CV = 0.32 m s⁻¹) innervating the posterior wall of the left ventricle to ET-1 (2 μg, LA) before and after treatment with BQ-123 (0.1 mg kg⁻¹, i.v.)

A, administration of ET-1 increased baseline activity of this afferent from 0.68 to 1.81 imp s⁻¹. B, BQ-123 abolished the increase (0.74 to 0.73 imp s⁻¹) in discharge activity of this afferent during repeated injection off ET-1. Panels 1 and 2 are representative tracings of the discharge activity of the afferent at times indicated by the arrows above histograms.

Figure 3

Responses of peak activity of cardiac sympathetic afferents to repeated LA injection of ET-1 (2 μg) with vehicle (PBS, 1.5 ml, i.v., n= 7, A) or treatment with BQ-123 (0.1 mg kg⁻¹, i.v., n= 9, B). C, neurohistograms of summated 2 s discharge activity from all nine cardiac afferents in response to ET-1 stimulation before (a) and after (b) treatment with BQ-123. Columns and error bars represent means ±s.e.m.*P < 0.05 compared with control. †P < 0.05 post-BQ-123 vs. pre-BQ-123.

Figure 5. Neurohistogram showing response of a cardiac sympathetic Aδ-fibre (CV = 3.1 m s⁻¹) innervating the posterior wall of the left ventricle to 5 min of myocardial ischaemia before and after treatment with BQ-123 (0.1 mg kg⁻¹, i.v.)

A, 5 min of myocardial ischaemia increased baseline activity of this afferent from 0.39 to 2.42 imp s⁻¹. B, BQ-123 attenuated the increase (0.29 to 1.62 imp s⁻¹) in discharge activity of this afferent during repeated ischaemia, particularly the second peak of the biphasic response. Panels 1–4 are representative tracings of the discharge activity of the afferent at times indicated by the arrows above histograms.

Figure 6. Responses of cardiac afferents to ischaemia before and after blockade of ET_A with receptors with BQ-123

A, bar graph summarizing changes in activity of seven cardiac sympathetic afferents before (open bar) and during (filled bar) 5 min of myocardial ischaemia. B, responses of another eight cardiac sympathetic afferents to myocardial ischaemia before and after treatment with BQ-123. C, neurohistograms of summated 2 s discharge activity from all eight afferents during ischaemia before (a) and after (b) treatment with BQ-123. *P < 0.05 compared with control. †P < 0.05 post-BQ-123 vs. pre-BQ-123.

Figure 7. Confocal microscopic images showing coexistence of ET_A receptors and DiI in dorsal root ganglion (DRG) neurons at spinal T₂ level in a cat

A and B demonstrate DRG neurons stained with ET_A receptors and DiI, respectively. C is a merged image from A and B. Arrows indicate two neurons containing ET_A receptors, DiI, or both of them, respectively. Scale bars in A–C represent 100 μm.