Myocardial infarction reduces cardiac nociceptive neurotransmission through the vagal ganglia

Figure 1. Assessment of expression profiles of left versus right nodose ganglia neurons in normal and LAD-infarcted animals. (A) Creation of myocardial infarct in the LAD distribution. Left panel: representative coronary angiography. Middle panel: Coronary artery angiography after microsphere injection showing occlusion at the mid-LAD. Right panel: Representative gross image of an infarcted heart showing scar in the region vascularized by the LAD. (B) Nodose ganglia from normal and infarcted animals were removed and analyzed for immunohistochemical changes. Scale bars are 5 mm (top), 50 μm (bottom). (C) Percentages of nodose ganglia neurons from normal and (D) infarcted (MI) animals expressing P2RX3, CGRP, PIEZO2, and NOS1 were quantified. No difference in the expression profiles of neurons between the left and right nodose ganglia was found in either normal or infarcted animals for P2RX3 (normal left vs. right nodose: P = 0.82; MI left vs. right nodose: P = 0.67), CGRP (normal left vs. right nodose: P = 0.82; MI left vs. right nodose: P = 0.89), PIEZO2 (a mechanosensitive ion channel; normal left vs. right nodose: P = 0.97; MI left vs. right nodose: P = 0.67), or NOS1 (normal left vs.…

Figure 2. Immunohistochemical assessment of neurons in the porcine nodose ganglia. Representative images of nodose ganglia from normal (left) and LAD-infarcted (right) pig nodose ganglion stained and quantified for (A and B) P2RX3, (C and D) CGRP, (E and F) PIEZO2, and (G and H) NOS1. Expression of P2RX3 and CGRP were increased after chronic LAD infarction (P = 0.005 for normal vs. infarcted animals for P2RX3 and CGRP). PIEZO2 expression remained unchanged (P = 0.32). Expression of NOS1 in the nodose ganglia was reduced after LAD infarction (P = 0.002 for normal vs. infarcted animals). n = 10 pigs per group for P2RX3 and CGRP quantification, n = 5 pigs per group for PIEZO2, and n = 8 pigs per group for NOS1; data are shown as mean ± SEM; unpaired, 2-tailed Student’s t test was used for comparisons. Scale bars are 50 μm.

Figure 3. Functional analysis of nodose neuronal activity following MI. (A) Creation of right coronary artery myocardial infarct. Left panel: representative coronary angiography (RCA) while placing the percutaneous transluminal angioplasty catheter (PTA) in the RCA. The distal radiopaque marker indicates the distal end of the angioplasty catheters where microspheres are injected. Right panel: Coronary artery angiography after RCA infarct creation. Contrast dye was injected into the RCA to confirm the location of the occlusion. (B) Representative magnetic resonance imaging (MRI) images of the RCA-infarcted heart. Scar tissues were confirmed by assessing the thickness and the color of the myocardial regions in the MRI image. (C) Customized 16-channel linear microelectrode array probes were used to record nodose neurons in vivo. (D) The neural probe was inserted in the nodose ganglion. (E) Representative sorted neuronal action potentials for 3 neurons from 1 animal. Action potentials from individual neurons are illustrated in the right panel. Left ventricular pressure (LVP) and electrocardiogram (ECG) were recorded simultaneously with neural recordings. (F) Responses of 97 cardiac nodose…

Figure 4. Cardiac phase–related neural activity. (A) Representative neural activity from 3 nodose neurons. The first neuron (top, purple) fires during isovolumetric relaxation, the one in the middle fires during systole, and the third (bottom, pink) fires stochastically. The characteristic extracellular action potential for each neuron is also shown on the right. (B) Cardiac phases were divided into the following 4 phases: diastole (D), isovolumetric contraction (IC), systole (S), and isovolumetric relaxation (IR). Each dot represents a neural spike, and dots with the same color represent the spiking activity from the same neuron. The position of each dot shows the LVP at each spike time. (C) Normalized firing rate of nodose neurons in each cardiac phase. There is no significant difference between normal and RCA-infarcted animals (n = 12 for normal and n = 44 for MI animals). Median with IQR is shown, and Mann-Whitney U statistical test was used.

Figure 5. Nodose neural responses to specific cardiac interventions. (A) Percentages of neurons with significant changes in absolute firing rates in response to ventricular pacing (VP), aortic occlusion (AO), epicardial mechanical stimulation (EMS), inferior vena cava (IVC) occlusion, and nociceptive stimuli (NS) are shown. (B) Percentage of chemosensitive neurons that responded to each specific chemical stimulus is shown (capsaicin, CAPS; bradykinin, BRADY; veratridine, VERAT) in normal and MI animals. (C) Percentage of neurons that were excited or inhibited in response to each cardiac stressor is shown. The predominant response to nociceptive stimuli in normal animals was excitatory (an increase in firing rate: +) while the predominant response to nociceptive chemicals in RCA-infarcted animals was inhibitory (a decrease in firing rate: -). (D) The percentage of neurons that were excited or inhibited for each specific chemical in normal and RCA-infarcted animals is shown. Both capsaicin and bradykinin caused statistically significant decreases in firing rates. Fisher’s exact test with Dunn’s correction for multiple comparisons was used to compare the percentage of neurons in normal and MI…

Figure 6. Temporal profile of nociceptive neural responses in infarcted animals demonstrates inhibition of firing during nociceptive chemical application of capsaicin and bradykinin, which persists after removal of the nociceptive chemical. (A) Representative inhibitory response of 2 nodose neurons to bradykinin (BRADY) and capsaicin (CAPS) in a chronic RCA infarct. (B) Quantified temporal inhibitory responses of neurons (n = 80) to nociceptive stimuli in the RCA-infarcted animals show persistence of inhibition of neural firing rates even after removal of the stimulus/chemical. Data are shown as mean ± SEM. Dunn’s multiple-comparison tests were used to compare the normalized firing rate versus baseline (BL). +P ≤ 0.001 compared with BL.

Figure 7. Nonselective augmentation of satellite glial cell activation following MI in the nodose ganglia. (A) Representative images of nodose ganglia from normal (N; left) and LAD-infarcted (MI; right) pigs stained for GFAP (red) and CGRP (purple). (B) Summary of the percentage of neurons surrounded by GFAP+ satellite glial cells as a subset of all neurons, CGRP+ neurons, and CGRP– neurons is shown. There was a statistically significant increase in GFAP expression after MI (P < 0.001 for infarcted vs. normal animals). This was, however, true for both CGRP-positive (P = 0.03 for infarcted vs. normal animals) and CGRP-negative neurons (P < 0.001 for infarcted vs. normal animals). n = 5 pigs per group in all neurons. n = 5 normal pigs and n = 4 for MI animals for coexpression. Data are shown as mean ± SEM; unpaired, 2-tailed Student’s t test was used for comparisons. Scale bars are 50 μm.

Figure 8. Upregulation of inhibitory GABAergic neurotransmission specifically in CGRP-expressing nodose ganglia neurons involved in nociceptive neurotransmission. (A) Representative images of nodose ganglia from normal (N; left) and LAD-infarcted (MI; right) animals stained for GAD2 (blue) and CGRP (purple). (B) Top, summary of the percentage of GAD2+ neurons as a subset of all neurons, CGRP+ neurons, and CGRP– neurons is shown. Although overall expression of GAD2 was unchanged between normal and LAD-infarcted animals (P = 0.80), the percentage of neurons that coexpress both GAD2 and CGRP was increased after MI (P = 0.04 for LAD-infarcted vs. normal animals). Coexpression of GAD2 was decreased in neurons that do not express CGRP (P = 0.006 for normal vs. LAD-infarcted animals). n = 5 pigs per group. Bottom, summary of the percentage of GABA+ neurons as a subset of all neurons, CGRP+ neurons, and CGRP– neurons is shown. Similar to GAD2, GABA showed no overall difference (P = 0.57) while its expression in CGRP+ neurons was increased (P = 0.02) in MI animals. However, expression of GABA in CGRP– neurons was similar between normal and MI animals (P = 0.89). (C) Representative images of nodose…