Spinal nociceptive denervation impedes subsequent chronic autonomic remodeling after myocardial infarction in male swine

Figures

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  Fig. 1. Experimental protocol. A Schematic representation of the experimental groups and their respective interventions. B Percutaneous creation of myocardial infarction (MI) via injection of microspheres in the distal left anterior descending (LAD) coronary artery after percutaneous angioplasty balloon inflation. Following injection, flow is significantly decreased in the mid and distal LAD (red arrows) and C ST-segment elevation is observed on the surface ECG. D At the time of terminal studies, a 56-electrode sock was placed around the ventricles to record local unipolar electrograms, from which E repolarization time (RT) and activation time (AT) were measured and activation recovery interval (ARI) calculated in all infarcted animals. F For epidural injections, an epidural catheter (white arrow) was advanced to the C7-T1 dorsal epidural space and position confirmed by contrast injection under fluoroscopic guidance. In terminal procedures involving the group of animals that underwent acute epidural RTX injections after chronic MI, catheter position was secondarily confirmed at the end of experiments by cut down to the epidural space and removal of epidural fat. Asterisk placed at…

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  Fig. 2. Immunohistochemical confirmation of successful ablation of cervicothoracic spinal nociceptive neurons and fibers. A Representative images of calcitonin gene-related peptide (CGRP) immunostaining of the dorsal root ganglia, and B spinal cord dorsal horn from a chronic vehicle and an epidural cRTX (chronic resiniferatoxin-treated) animal. C A significant decrease in CGRP expressing neurons in the dorsal root ganglia (DRG) and D dorsal horn of epidural cRTX (n = 7) vs vehicle animals (n = 5) is observed, confirming successful chemoablation of spinal nociceptive cardiac afferents in animals that received RTX. E Expression of purinergic receptor P2X (P2RX3) and F Piezo-type mechanosensitive ion channel component 2 (PIEZO2) in the cervicothoracic DRG was unchanged between cRTX (n = 5) vs vehicle treated, infarcted animals (n = 5). Similarly, expression levels of G P2RX3 and H PIEZO2 in the cervicothoracic dorsal horn were not different between vehicle-treated (n = 5) versus cRTX animals (n = 5). I Comparison of CGRP expressing neurons in the thoracolumbar (T12-L1) DRGs, or J dorsal horn of vehicle-treated (n = 5) versus cRTX animals (n = 5), demonstrated no difference, suggesting that…

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  Fig. 3. Effects of epidural RTX prior to MI on cardiac hemodynamics and vagal tone four to six weeks after MI. A–D Baseline hemodynamic parameters were not different between vehicle (n = 13) and epidural cRTX (n = 11) animals. E Example of phenylephrine-evoked baroreflex responses in a vehicle and epidural cRTX (resiniferatoxin-treated, infarcted) animal. F Baroreflex sensitivity (BRS) was significantly improved in epidural cRTX (n = 11) compared to vehicle animals (n = 13). G Schematic image of ventral interventricular ganglionated plexus (VIVGP) neural recordings and identification of VIVGP neurons. A magnified image of the neural recording electrode in a VIVGP is shown. The left atrial appendage is being retracted to fully expose the VIVGP. A picture of the neural probe and a schematic of the tip with the most distal 4 recording electrodes are shown. There was no difference in basal firing frequency of all (H) or non-VNS-responsive (I) neurons. J However, the basal firing frequency of VNS-responsive neurons (e.g., parasympathetic postganglionic neurons) was significantly higher in epidural cRTX (n = 6) compared to vehicle animals (n = 5). Differences in heart rate (HR), left…

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  Fig. 4. Effects of spinal afferent ablation with epidural RTX prior to MI on reflex-evoked cardiac function four-to-six weeks after MI. A Representative 2D-map of electrophysiological changes evoked by epicardial application of bradykinin in a vehicle (left) and an epidural cRTX (RTX-treated, infarcted) animal (right). B–D Quantified hemodynamic and electrophysiological responses to epicardial application of bradykinin in vehicle-treated (n = 10) and cRTX (n = 11) infarcted animals are shown. E Representation of hemodynamic changes evoked by epicardial application of capsaicin. F–H Quantified hemodynamic and electrophysiological changes in repolarization time (RT) and dispersion of RT upon epicardial application of capsaicin in vehicle (n = 10) and cRTX (n = 11) animals. Differences in heart rate (HR), left ventricular systolic pressure (LVSP), dP/dtmax, dP/dtmin, RT and dispersion responses upon bradykinin and capsaicin administration between vehicle and epidural cRTX animals were assessed using the two-sided unpaired Student’s t-test. CS: Capsaicin. Epidural vehicle-treated animals that subsequently were infarcted are denoted as Vehicle+MI and epidural RTX-treated animals that were…

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  Fig. 5. Proteomic analyses demonstrate a decrease in inflammatory and stress response pathways in infarcted animals with epidural RTX treatment prior to MI compared to vehicle-treated, infarcted animals four-to-six weeks post-infarction. A Schematic representation of plasma protein isolation to proteomic data analyses. B Volcano plot showing the differentially expressed proteins in vehicle and cRTX (resiniferatoxin-treated, infarcted) animals. C Proteomic pathway analyses conducted on expression data against the Gene Ontology database show a significant decrease in oxidative stress and inflammatory pathways in cRTX infarcted animals compared to vehicle-treated, infarcted animals. Pathways associated with extracellular signal-regulated kinase 1 (ERK1) and extracellular signal-regulated kinase 2 (ERK2) cascades were upregulated in cRTX animals, which have been described to be fundamental for cardiomyocyte homeostasis and stress responses. Differentially expressed proteins were identified using a two-sided Student’s t-test. Pathway analysis was performed against the Gene Ontology database using Rapid Integration of Term Annotation and Network. Animals that received vehicle (epidural saline)…

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  Fig. 6. Immunohistochemical changes associated with ablation of cervicothoracic spinal nociceptive nerves with epidural RTX. A Representative images of immunostaining of spinal horns in cRTX and vehicle animals for glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (IBA1; markers of glial activation and microglia, respectively (scale bars represent 500 µm). Quantified data show decreased IBA1 (B) and GFAP (C) immunoreactivity in RTX-treated (cRTX; n = 5) vs vehicle-treated (n = 5) infarcted animals. D Representative images of immunostaining of the spinal horn for CD3 (marker of T lymphocytes). E Representative image of immunohistochemical staining of GFAP in T1 (thoraic level 1) dorsal root ganglia (scale bars represent 20 µm). F Quantified data show a significant reduction in the amount of CD3 positive T lymphocytes in cRTX (n = 5) vs vehicle-treated (n = 5) infarcted animals, suggesting reduced inflammation. G Quantified data showed a significant decrease in glial activation in the high thoracic dorsal root ganglia of cRTX (n = 5) versus vehicle (n = 5) animals. These effects of epidural RTX administration on reducing inflammation and glial activation…

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  Fig. 7. Effects of spinal nociceptive afferent ablation prior to MI on ventricular APD, DoR, refractoriness, and ventricular arrhythmia inducibility post-infarction. A Administration of cervicothoracic epidural RTX prior to MI did not alter global baseline activation recovery intervals (ARI), or B regional ARIs four-to-six weeks post-MI. C Examples of border zone electrograms from a vehicle (top) and RTX treated (cRTX+MI, bottom) animal showing ARIs from these regions. D In cRTX animals, global dispersion of repolarization (DoR) was not significantly different from vehicle-treated infarcted animals, however, E regional analysies demonstrated that ARI dispersion was greatest in border zone regions in both vehicle and RTX treated animals, but that this border zone dispersion was significantly less in infarcted animals treated with RTX. F Left atrial ERP (LA ERP) was not significantly different between vehicle-treated and cRTX animals. G Example of ventricular ERP (VERP) measurements showing the last-captured extra-stimulus (S2) at interval of 270 ms. H Ventricular ERP (VERP) was significantly prolonged in RTX treated animals, and I RT-dispersion of the S2 paced beat was significantly…

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  Fig. 8. Temporal hemodynamic responses and electrophysiological effects of acute cervicothoracic spinal nociceptive afferent ablation with epidural RTX in the setting of chronic MI. The acute effects of epidural RTX (aRTX) were assessed to determine the kinetics and tolerability of RTX in infarcted animals (n = 15). Within 1-h of administration, RTX increased A heart rate (HR), B left ventricular systolic pressure (LVSP), C left ventricular inotropy (dP/dtmax), and D left ventricular lusitropy (dP/dtmin). Left ventricular (LV) lusitropy remained significantly elevated, whereas LV inotropy and LVSP were decreased 4 h after epidural RTX administration. E Percentage change in HR, LVSP, LV inotropy and lusitropy over time are shown. *p < 0.05 vs baseline. F Beat-to-beat changes in RR interval vs LVSP were plotted to assess baroreflex sensitivity (BRS) before, 1-h, and 4-h post-RTX injection. G BRS was significantly improved by 4-h post-RTX (n = 10). H Acute epidural RTX shortened activation recovery intervals (ARI) at 1- and 4-h after administration. I When corrected for heart rate (ARIc), this effect was no longer significant. J Epidural RTX did not alter atrial ERP (AERP) in chronic MI…

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  Fig. 9. Spinal nociceptive afferent signaling contributes to post-myocardial infarction neuroinflammation, oxidative stress, glial activation, and sympatho-vagal imbalance, resulting in increased dispersion of repolarization and ventricular arrhythmias (VT/VF), a process that is mitigated by interruption of this signaling with epidural resiniferatoxin (RTX). GFAP glial fibrillary acid protein, TRPV1 transient receptor potential vanilloid 1, CGRP calcitonin gene-related peptide.