Neural Mechanisms of Heart–Lung Crosstalk in Chronic Heart Failure

Abstract

Background:

The cardiac sympathetic afferent reflex (CSAR) and pulmonary spinal afferent reflex (PSAR) amplify sympathetic activity and may contribute to chronic heart failure (CHF). We hypothesized that cardiac injury triggers a cardiac nerve-mediated cytokine uptake cascade that propagates neural inflammation in thoracic dorsal root ganglia (DRGs) to drive reciprocal cardiopulmonary afferent sensitization through suppression of voltage-gated potassium (Kv) channels after myocardial infarction (MI).

Method:

MI was induced in rats by coronary ligation. Molecular profiling, immunofluorescence, tissue clearing, and functional assays were used to assess neuroinflammation and reflex responses.

Results:

Post-MI, thoracic DRGs showed macrophage infiltration, glial activation, cytokine upregulation, and reduced Kv channel expression. Bulk RNA-seq identified enrichment of macrophage activation–related genes, and in vitro studies confirmed that pro-inflammatory cytokines and activated macrophages suppressed Kv channels and increased DRG neuron excitability. Epicardial injection of biotinylated TNF-α demonstrated cardiac afferent–mediated cytokine transport to DRGs, inducing macrophage infiltration via a cytokine receptor-dependent mechanism. Anti-inflammatory interventions including oral minocycline, systemic macrophage depletion, and local epidural delivery of thermo-responsive hydrogel-forming dexamethasone prodrug (ProGel-Dex) significantly reduced DRG neuroinflammation, restored Kv channel levels, and attenuated exaggerated CSAR and PSAR responses. Minocycline and ProGel-Dex also improved cardiac chamber dilation in the post-MI rats.

Conclusions:

These findings identify a neural inflammatory cascade involving cytokine uptake, glial activation, and macrophage activation as a contributor to cardiopulmonary afferent sensitization after MI. Targeting DRG inflammation, particularly with sustained dexamethasone delivery using ProGel-Dex, offers a prospect of precision medicine to dampen pathological sympathetic activation and improve cardiac outcomes in CHF.

Introduction

Chronic heart failure (CHF) is a progressive disease that affects approximately six million Americans, with over 660,000 new diagnoses each year.¹ Exaggerated sympatho-excitation, a hallmark of CHF, is a critical factor in the development and progression of the CHF state.^2,3 Sympathetic dysfunction takes the form of persistent and adverse activation of sympathetic nerve outflow to the heart, kidneys and other organs, causing an array of chronic effects that aggravate the disease progression^4,5. Previous studies from our laboratory have shown that the enhanced cardiac sympathetic afferent reflex (CSAR), a sympathoexcitatory reflex originating in the heart, is a critical contributor to the elevated sympathetic tone in CHF.^6–9 More recently, we published new evidence showing that chronic and selective ablation of cardiac spinal afferents with the potent neurotoxin resiniferatoxin (RTX) markedly attenuates the deleterious cardiac remodeling and cardiac dysfunction in CHF.⁷ However, the mechanisms underlying the enhanced CSAR in the CHF state are not fully understood.

Inflammation plays an important role in the development and progression of CHF, contributing to cardiac remodeling and peripheral vascular disturbances.¹⁰ Increased levels of inflammatory cytokines, especially TNFα, IL-6, and IL-1β, have been found in CHF, with particularly high concentrations in patients with the most severe heart failure.¹¹ It is also well recognized that inflammation, such as microglia activation in the central nervous system (CNS), is involved in the pathogenesis of sympatho-excitation in CHF¹². Microglia, resident macrophages in the brain, are thought to modulate the pathologic and regenerative states of the brain by producing a variety of molecules including neurotrophic and neurotoxic factors.¹³ Studies have demonstrated that microglial activation in the hypothalamic paraventricular nucleus (PVN) following MI mediated the increase of sympathetic nerve activity in animals with reduced left ventricular function.^12,14 On the other hand, whether inflammation occurred in the peripheral nervous system, such as in sensory ganglia, in CHF remains unclear. It is well established that macrophages can infiltrate the stellate ganglia and drive an enhanced Ca²⁺ phenotype in dysautonomia¹⁵ as well as injured nerve tissue such as the dorsal root ganglia (DRG), which activates the development of neuropathic pain following peripheral nerve injury.¹⁶ Whether similar macrophage activation occurs in thoracic T1-T4 DRGs (the most predominant sensory ganglia innervating both heart and lung) post-MI has not been examined. In this study, our first goal was to determine whether macrophages are activated in the thoracic T1-T4 DRGs in a time-dependent manner in the post-MI state. Our second goal was to define how activated macrophages influence thoracic visceral sensory afferents. We employed an in vitro co-culture system to probe macrophage-neuron interactions. We also tested a novel cardiac afferent–mediated cytokine uptake hypothesis by injecting biotinylated cytokines into the epicardium and tracking their transport to thoracic DRGs.

Because the lungs and heart anatomically share common sympathetic afferent (i.e. T1-T4 DRGs) input, macrophage infiltration and activation in these DRGs could also sensitize pulmonary afferents post-MI. Previous work from our laboratory has shown that activation of pulmonary spinal afferents by application of bradykinin and capsaicin to the surface of the lung resulted in a reflex increase in mean arterial pressure (MAP), heart rate, and renal sympathetic nerve activity (RSNA) in the anesthetized, vagotomized rat, demonstrating an excitatory pulmonary spinal afferent reflex (PSAR) in normal animals.¹⁷ We therefore hypothesized that the PSAR is reciprocally sensitized in CHF and that this sensitization is induced by macrophage activation in T1-T4 DRGs common to both heart and lung.

Methods

Data availability

The datasets generated and/or analyzed during the current study are available in the Figshare repository (10.6084/m9.figshare.32085987). The bioinformatic datasets generated during the current study are available in the NCBI GEO archive (GSE315346). Additional data are available from the corresponding author upon reasonable request.

Expanded Methods and Statistics Analysis

Detailed descriptions of all experimental methods and statistical analyses are provided in the online Expanded Methods section. For detailed information on all materials and reagents, please refer to the Major Resources Table in the Supplemental Materials.

All animal experimentation was approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center (UNMC) and performed in accordance with the National Institutes of Health’s Guide for Use and Care of Laboratory Animals and in accordance with the ARRIVE guidelines.¹⁸ Adult male and female Sprague-Dawley rats weighing 180–200g were purchased from Charles Rivers. The experimental flow chart for all rat experiments is shown in Supplemental Figure S1. Male rats were primarily used for these mechanistic studies to reduce biological variability and establish the underlying neuroimmune mechanisms in a single sex. A selected experiment was subsequently performed in both male and female rats to evaluate the generalizability of the findings of increased proinflammatory biomarkers in thoracic DRGs post-MI. A total of 419 male rats were used, including 194 sham/normal rats and 225 MI rats. In addition, 16 female rats were included in the study experiments, consisting of 8 sham and 8 MI animals.

For cytokine uptake experiments, ~10-week-old male TNFα receptor knockout (p55/p75 KO; Stock No.003243, Jackson Laboratory) and their wild-type controls (B6129SF2/J, Jackson Laboratory) were used. For tissue-clearing experiments, male heterozygous Cx3cr1CreER::TdTomato (mT/mG) mice were generated by crossing homozygous Cx3cr1CreER males (Stock No. 020940, Jackson Laboratory) with homozygous TdTomato (mT/mG) females (Stock No. 007676, Jackson Laboratory). A total of 34 male mice were used in this study.

Results

1. Myocardial infarction causes time-dependent macrophage activation in thoracic T1-T4 DRGs

We examined macrophage infiltration and activation in DRGs at different time points after MI. T1–T4 DRG sections were stained for ionized calcium-binding adaptor molecule-1 (Iba-1), a macrophage marker in the DRG.³⁶ Compared with sham-operated rats, the number of Iba-1–immunoreactive cells were significantly increased at 1 week post-MI and remained elevated through 8 weeks, indicating sustained macrophage infiltration into thoracic DRGs after MI (Figure 1A and 1B).

Figure 1. Macrophage activation in the post-MI rats.

(A) Triple immunofluorescence for Iba1, NF200, and IB4 in T1–T4 DRGs of sham and MI rats at 1, 4, and 8 weeks post-MI. White arrows indicate Iba1-positive cells; white bar=50 μm. (B) Quantification of Iba1-positive cells(n=5 sham, 5 MI-1w, 7 MI-4w, 5 MI-8w); one-way ANOVA with Holm–Šídák correction for 3 comparisons versus sham. (C) SEM images showing increased macrophages in T1–T4 DRGs of 8-week MI rats versus sham (n=3/group). Red asterisks and white arrows indicate DRG neurons and macrophages, respectively; B, blood vessels; G, glial cells. (D) Western blot analysis and quantification of IRF8 protein in T1–T4 DRGs of sham and MI rats at 1, 4, and 8 weeks. n=6/group at week 1, n=10/group at week 4, and n=15/group at week 8; two-way ANOVA with Bonferroni–Šídák correction for 3 comparisons (MI versus sham at each time point).

Staining for NF200 (A-fiber neuronal marker) and IB4 (C-fiber neuronal marker) showed macrophage accumulation around both A- and C-fiber DRG neurons. Tissue clearing and 3D imaging of thoracic DRGs from CX3CR1CreER-ROSAmT/mG tdTomato-EGFP reporter mice further demonstrated abundant CX3CR1-positive macrophages at 4 weeks post-MI, whereas sham-operated mice showed only scattered CX3CR1-positive cells (Supplemental Video).

SEM provided additional high-resolution evidence of macrophages surrounding DRG neurons, with increased macrophages in T1–T4 DRGs of male rats at 8 weeks post-MI compared with sham rats (Figure 1C and Supplemental Figure S2). To quantify macrophage activation, we assessed Interferon Regulatory Factor 8 (IRF8), a transcription factor critical for driving macrophages toward a reactive phenotype.³⁷ IRF8 protein expression was significantly increased in male rats at 4 and 8 weeks post-MI (Figure 1D) and in female rats at 4 weeks post-MI (Supplemental Figure S3).

2. Myocardial infarction upregulates pro-inflammatory gene expression in thoracic T1–T4 DRGs

To determine whether MI-induced macrophage activation was associated with increased inflammatory gene expression, we performed RNA-seq on T1–T4 DRGs from 8-week post-MI rats (Figure 2 and Table S15). Differential Gene Expression (DEG) analysis identified 41 DEGs (29 upregulated and 12 downregulated; FDR≤0.15, log₂FC≥0.5). Irf7 expression was significantly increased after MI (Log₂FC=0.67; P = 1.0E-5, MI n=5; sham n=4) (Figure 2A). Gene Ontology (GO) analysis highlighted gene groups associated with terms related to contractile proteins and immune responses (Figure 2B). KEGG pathway analysis again revealed enrichment in pathways centered on immune responses, contractile components, and phagosome (Figure 2C). At the gene level, there was increased expression of contractile genes (Myl9, Actg2, Acta2, Myh11, Cnn1) and immune/inflammatory response genes (Cst3, Slfn4, Irf7, C4b, Pla2g2a, Ifit1), and genes associated with negative regulation of neuronal growth and inhibitory neurotransmission (Cbln4, Rtn4lp1). In contrast, sham DRG exhibited relatively higher levels of ECM/adhesion markers (Thbs4, Pcdhga3, Fndc7) and positive regulation of neuronal growth (Vgf, Elk1). (Figure 2D). Real-time PCR confirmed significantly increased IL-6, but not TNFα or IL-1β, mRNA in T1–T4 DRGs at 4 weeks post-MI (Supplemental Figure S4).

Figure 2. T1-T4 DRG transcriptional changes 8 weeks post-MI.

(A) Volcano plot of differentially expressed genes (DEGs) in T1-T4 DRGs from rats 8 weeks post-MI versus sham (MI n=5; sham n=4). Thresholds: FDR≤0.15 and |log₂FC|≥0.5. Upregulated (red) and downregulated (blue) genes are shown with Irf7 highlighted. (B) B, Gene Ontology enrichment analysis. C, KEGG pathway enrichment analysis. D, Heatmap of representative DEGs grouped by functional category; values are gene-wise Z-scores. Columns represent individual DRGs (MI, left; sham, right).

3. Macrophage activation decreases Kv channel protein levels in 50B11 DRG neurons in vitro

Because voltage-gated K+ (Kv) channels regulate DRG neuronal excitability and nociceptive signaling,³⁸ we examined whether activated macrophages suppress Kv channel expression. 50B11 DRG neurons were indirectly co-cultured for 72 h with LPS-pretreated BV2 microglia or RAW264.7 macrophages. 50B11 DRG neurons were indirectly co-cultured for 72 h with LPS-pretreated BV2 microglia or RAW264.7 macrophages (Figure 3A).

Figure 3. Effects of macrophages and cytokines on Kv channel expression in 50B11 neurons and T1–T4 DRGs after MI.

A-G, Kv channel protein expression in 50B11 DRG neurons after co-culture with BV2 or RAW264.7 cells. A Co-culture schematic. B, C, Representative gels; D–G, quantification of Kv isoforms with or without etanercept (10 ng/ml). BV2: n=4 control, 6 LPS, 6 LPS+etanercept; one-way ANOVA with Holm–Šídák correction for 3 comparisons. RAW264.7: n=8/group; Welch’s t-test for Kv1.4, Kv3.4, and Kv4.3 and Mann–Whitney U test for Kv4.2. H–L, Representative gels (H) and quantification (I–L) after 3-day exposure to pro- or anti-inflammatory cytokines; Welch’s t-test or Mann–Whitney U test as specified in Table S9. M–Q, Kv channel protein expression in T1–T4 DRGs of sham and MI rats at 1, 4, and 8 weeks. Kv1.4: n=6, 5, and 6/group; Kv3.4: n=6, 4, and 4/group; Kv4.2: n=6, 5, and 4/group; Kv4.3: sham n=4, 5, and 4 and MI n=4, 4, and 4 at weeks 1, 4, and 8, respectively; two-way ANOVA with Bonferroni–Šídák correction for 3 comparisons.

LPS-pretreated BV2 and RAW264.7 cells significantly reduced Kv channel protein expression in 50B11 neurons (BV2: Kv1.4 P = 0.010, Kv3.4 P = 5.2E-12, Kv4.2 P = 1.1E-11, Kv4.3 P = 1.1E-7; RAW264.7: Kv1.4 P = 0.89, Kv3.4 P = 0.014, Kv4.2 P = 6.2E-4, Kv4.3 P = 0.036, Figure 3B–G). Notably, the TNFα inhibitor Etanercept (10 ng/ml) failed to significantly block these effects. These findings show that co-culture with LPS-pretreated BV2 or RAW264.7 cells was associated with reduced Kv channel protein expression in 50B11 neurons.

4. Pro-inflammatory cytokines reduce Kv channel expression in 50B11 cells in vitro

Because etanercept did not block the effects of activated microglia, we examined whether other inflammatory cytokines could contribute to Kv channel downregulation. 50B11 cells were exposed for 3 days to TNFα, IL-1β, IL-6, IFNγ, IL-4, or IL-10, and protein levels of Kv1.4, Kv3.4, Kv4.2, and Kv4.3 were assessed. Kv1.4 expression increased after TNFα treatment (P = 0.027, n=8, Figure 3H–L) but decreased after IFNγ treatment (P = 0.020, n=8). Kv3.4 expression decreased after TNFα, IL-1β, IL-6, IFNγ, and IL-4 treatment (TNFα P = 0.0041, IL-1β P = 0.0011, IL-6 P = 0.048, IFNγ: P = 7.0E-4, and IL-4 P = 0.029; n=8). Kv4.2 decreased after IL-6 and IFNγ treatment (IL-6 P = 0.030 and IFNγ P = 0.044, n=8; Figure 3H–L), while Kv4.3 decreased after IL-6 treatment (P = 0.0048; n=8; Figure 3H–L). Overall, several pro-inflammatory cytokines reduced expression of multiple Kv channel isoforms, whereas IL-4 and IL-10 produced variable effects.

5. Protein expression of Kv channel isoforms is decreased in T1-T4 DRGs of post-MI rats

We next assessed Kv channel expression in T1–T4 DRGs at 1, 4, and 8 weeks post-MI (Figure 3M–Q). Kv1.4, Kv3.4, Kv4.2, and Kv4.3 protein levels were significantly reduced after MI (Kv1.4 P_MI = 9.4E-4, Kv3.4 P_MI = 0.0040, Kv4.2 P_MI = 0.027, Kv4.3 P_MI = 0.015, n=6). Post-hoc testing showed no statistical differences in Kv channel expression one week post-MI, whereas Kv1.4 was decreased 4 and 8 weeks post-MI (P = 0.015 and 4.5E-4, respectively), Kv3.4 was decreased 8 weeks post-MI (P = 0.013), and Kv4.3 was decreased 4 weeks post-MI (P = 0.026). Immunofluorescence confirmed a significant reduction in Kv-positive DRG neurons at 4 weeks post-MI (Table S1; Supplemental Figure S5A–D).

6. Chronic oral minocycline attenuates macrophage activation, restores Kv channel function, and normalizes exaggerated CSAR and PSAR in post-MI rats

To determine the functional contribution of macrophage activation to DRG neuronal dysfunction, we co-cultured acutely dissociated T1–T4 DRG neurons with LPS-pretreated BV2 or RAW264.7 cells for 24 hours (non-LPS-treated cells as control). Two weeks prior to dissociation, DiI was injected into the subepicardium of the left ventricle to retrogradely label cardiac-specific afferent DRG neurons.³⁹ Whole-cell patch-clamp recordings were performed in IB4-positive cardiac C-fiber neurons. LPS-pretreated BV2 or RAW264.7 cells altered the Kv current (Ito) I–V relationship in cardiac DRG neurons, with significant group × voltage interactions for BV2 [F(12, 299) = 34.24, P = 3.30×10⁻⁴⁹] and RAW264.7 [F(12, 336) = 87.33, P = 1.60×10⁻⁹⁵] (Figure 4G, H, Table S9).

Figure 4. Effects of minocycline on IRF8, Kv channels, Kv currents, and cardiopulmonary spinal afferent reflexes after MI.

A, B, IRF8 protein expression; n=4/group; two-way ANOVA with Tukey correction for 6 comparisons. C, D, Kv channel protein expression. Kv1.4: n=4 sham, 6 MI, 6 MI+Mino; Kv3.4: n=4, 6, 6; Kv4.2: n=6, 6, 4; Kv4.3: n=4, 6, 6; one-way ANOVA with Holm–Šídák correction for 2 comparisons for each isoform. E, F, Kv currents; n=13 sham, 22 MI+Veh, and 22 MI+Mino neurons from 6–8 rats/group. G, H, Effects of LPS-pretreated BV2 or RAW264.7 cells on Kv currents in cardiac DRG neurons; n=14 BV2, 13 LPS-BV2, 14 RAW264.7, and 16 LPS-RAW264.7 neurons from 6 rats/group. For F, H, filled circles indicate Holm–Šídák-adjusted P<0.05 and open circles adjusted P≥0.05 at the indicated membrane potential. Exact P values and statistical details are provided in Tables S9 and S10. Error bars=SD. I–P, MAP, heart rate, and RSNA responses to epicardial or pulmonary BK in sham, vehicle-treated MI, and minocycline-treated MI rats. n=6 sham, 6 MI, and 7 MI+Mino for both CSAR and PSAR; one-way ANOVA with Holm–Šídák correction for 2 comparisons, except heart rate, analyzed by Kruskal–Wallis test with Dunn correction for 2 comparisons.

We next tested whether macrophage inhibition could rescue these effects in vivo. Minocycline (20 mg/kg/day in drinking water), a macrophage deactivator,^20,40 reduced IRF8 expression in T1–T4 DRGs by ~76% in 4-week post-MI rats, restoring levels to those of sham rats (Figure 4A, B). Minocycline also Minocycline also increased Kv channel protein expression and Ito current density in post-MI DRGs, with a significant group × voltage interaction [F(12, 504) = 46.18, P = 2.39×10⁻⁷³]; (Figure 4C, D), and significant differences from −40 to +40 mV (Figure 4C–F; Table S10).

Functionally, minocycline significantly attenuated the exaggerated CSAR and PSAR responses induced by bradykinin (BK, 10 μg/ml) in vagotomized 4-week post-MI rats, bringing them to sham levels (Figure 4I–P, Tables S2 and S3). There was no significant difference in infarct size (34.50±2.67% vs. 36.13±2.82%, Veh vs. Minocycline, P = 0.68 by Welch’s t-test) between MI rats treated with vehicle or minocycline. In a separate cohort, 6-week oral minocycline treatment initiated 3 days before MI reduced systolic and diastolic LV volumes but did not significantly alter ejection fraction or fractional shortening (Supplemental Figure S6).

7. Liposomal clodronate–mediated macrophage depletion attenuates exaggerated CSAR and PSAR in post-MI rats

To independently confirm the role of macrophages, we depleted peripheral macrophages using tail vein injection of clodronate liposomes. CD68-positive macrophages in the heart, lung, and spleen were completely depleted in post-MI rats following treatment (Supplemental Figure S7). In thoracic DRGs, Iba-1–positive cells were significantly reduced in 4-week post-MI rats treated with clodronate (Figure 5A, B).

Figure 5. Effects of clodronate liposomes on macrophage infiltration and cardiopulmonary spinal afferent reflexes after MI.

A, B, Iba1-positive macrophages in thoracic DRGs of sham, vehicle-treated MI, and clodronate liposome (CL)-treated MI rats; white bar=50 μm. n=5/group; one-way ANOVA with Holm–Šídák correction for 2 comparisons. C–F, CSAR and G–J, PSAR responses. CSAR: n=6 sham, 6 MI, 7 MI+CL; PSAR: n=6 sham, 7 MI, 8 MI+CL. One-way ANOVA with Holm–Šídák correction for 2 comparisons, except RSNA in both protocols, analyzed by Kruskal–Wallis test with Dunn correction for 2 comparisons.

Clodronate treatment significantly reduced the exaggerated CSAR and PSAR responses to BK (10 μg/ml) in vagotomized post-MI rats (Figure 5C, D; Tables S4 and S5). There was no significant difference in infarct size (36.83±3.28% vs. 37.33±2.70%, Veh vs. Liposomal Clodronate, P = 0.91 by Welch’s t-test) between MI rats treated with vehicle or liposomal clodronate.

8. Local epidural delivery of ProGel-Dex restores Kv channel function, reduces neuronal hyperexcitability, and delays cardiac remodeling in post-MI rats.

We next tested local modulation of macrophage activation by epidural delivery of ProGel-Dex (a thermoresponsive dexamethasone prodrug that forms hydrogel upon injection in vivo^22,23) to T1–T4 DRGs. ProGel-Dex normalized IRF8 protein expression (Figure 6A, B) and restored downregulated Kv channel expression (Figure 6C, D) in thoracic DRGs of post-MI rats.

Figure 6. Effects of epidural ProGel-Dex on Kv channels, neuronal excitability, spinal afferent reflexes, and cardiac function after MI.

Effects of ProGel-Dex on IRF8 (A, B), Kv channel expression (C, D), Kv currents (E–I), neuronal excitability (J–M), CSAR and PSAR (N–Q), and cardiac function (R–W) in sham and 4-week post-MI rats. A, B, IRF8: n=4 sham, 6 MI, 6 MI+ProGel-Dex; one-way ANOVA with Holm–Šídák correction for 2 comparisons. C, D, Kv1.4: n=4, 6, 6; Kv3.4: n=4, 6, 6; Kv4.2: n=5, 5, 5; Kv4.3: n=4, 6, 6 for sham, MI, and MI+ProGel-Dex, respectively. One-way ANOVA with Holm–Šídák correction for 2 comparisons, except Kv4.3, analyzed by Brown–Forsythe and Welch ANOVA with Dunnett T3 correction. E–I, Kv currents: n=17 sham, 19 MI+Veh, 21 MI+ProGel-Dex neurons for total Ito (F); n=6, 12, and 7, respectively, for the neuronal subpopulation in G–I. Filled circles indicate Holm–Šídák-adjusted P<0.05; open circles, adjusted P≥0.05. Exact P values and statistical details are in Tables S11–S14. Error bars=SD. J–M, action potentials: n=6 sham, 8 MI, 8 MI+ProGel-Dex. Amplitude, Welch ANOVA with Dunnett T3 correction; threshold and resting membrane potential, one-way ANOVA with Holm–Šídák correction. N–Q, CSAR and PSAR: n=6 sham, 6 MI, 7 MI+ProGel-Dex for each protocol; one-way ANOVA with Holm–Šídák correction for 2 comparisons, except PSAR heart rate, analyzed by Brown–Forsythe and Welch ANOVA with Dunnett T3 correction. R–W, echocardiography: n=10 MI and 7 MI+ProGel-Dex; Welch’s t-test for ejection fraction and LV end-diastolic diameter and Mann–Whitney U test for fractional shortening, LV end-diastolic volume, LV end-systolic volume, and LV end-systolic diameter.

Patch-clamp recordings showed that MI reduced Ito and IA current densities, whereas IK did not differ significantly at the group level but showed a significant group × voltage interaction (Figure 6E–I). MI also increased action potential number, lowered threshold potential, and depolarized the resting membrane potential (Figure 6J–M). Compared with MI+Veh, ProGel-Dex significantly altered the Ito I–V relationship in the complete neuronal population [F(12, 456) = 52.99, P = 1.31×10⁻⁷⁸] and neuronal subpopulation [F(12, 204) = 27.90, P = 9.64×10⁻³⁷]. Significant group × voltage interactions were also observed for IA [F(12, 204) = 13.34, P = 4.02×10⁻²⁰] and IK [F(12, 204) = 8.00, P = 3.25×10⁻¹²], although no individual voltage-specific comparison remained significant after Holm–Šídák adjustment (Figure 6F–I; Tables S11–S14).

Functionally, ProGel-Dex prevented the exaggerated CSAR and PSAR responses to BK in 4-week post-MI rats (Figure 6N–Q; Tables S6 and S7). Echocardiography showed reduced LV chamber dilation but no significant improvement in basal systolic function (Figure 6R–W). There was no significant difference in infarct size (36.50±1.95% vs. 35.57±1.74%, Veh vs. ProGel-Dex, P = 0.73 by Welch’s t-test) between MI rats treated with vehicle or ProGel-Dex. These results show that ProGel-Dex treatment was associated with reduced IRF8 expression, increased Kv channel expression and current density, reduced neuronal excitability, and attenuated CSAR and PSAR responses after MI.

9. Cytokine uptake by cardiac sensory afferents triggers macrophage recruitment to thoracic DRGs

To investigate why macrophages are activated in DRGs after MI, we hypothesized that cytokines produced by the injured heart are taken up by cardiac sensory nerve terminals and retrogradely transported to DRG somata, where they are released to recruit macrophages. To test this, we injected biotin-labeled TNFα (1 μL, 1.25 ng/μL, three injection sites in the left ventricular subepicardium) in rats. Saline-injected rats and L4/L5 DRGs served as controls. Biotin-TNFα was detected in T1–T4 DRG neuronal somata at day 1 post-injection, but not in controls (Figure 7A, B). Uptake occurred predominantly in NF200-positive A-fiber neurons and, to a lesser extent, CGRP-positive peptidergic C-fiber neurons, but not IB4-positive non-peptidergic C-fiber neurons. Similar results were obtained with biotinylated IL-6. Biotin-TNFα was also detected in stellate ganglia neurons, indicating sympathetic uptake (Supplemental Figure S8).

Figure 7. Cytokine uptake and satellite glial cell activation in thoracic DRGs after myocardial injury.

A, B, Immunofluorescence showing uptake and retrograde transport of biotinylated TNFα injected into the LV subepicardium by NF200- and CGRP-positive, but not IB4-positive, spinal afferents (n=5/group). C, Biotinylated TNFα in satellite glial cells (SGCs) 3 days after injection (n=5/group). D, Time course of SGC activation in T1–T4 DRGs after MI; day 3, n=13 sham and 16 MI; day 7, n=4/group; week 4, n=7/group; week 8, n=4/group; two-way ANOVA with Bonferroni–Šídák correction for 4 comparisons. E, Increased MCP-1 expression in SGCs at day 3 post-MI (n=5/group). F, MCP-1 protein measured by ELISA; n=13 sham, 5 MI-3d, 4 MI-1w, 10 MI-4w; one-way ANOVA with Holm–Šídák correction for 3 comparisons versus sham. IB4, non-peptidergic C-fiber marker; NF200, A-fiber marker; CGRP, peptidergic C-fiber marker; GFAP, SGC marker.

At day 3, biotin-TNFα was released into adjacent GFAP-positive satellite glial cells (SGCs, Figure 7C). GFAP expression in DRGs was significantly elevated from day 3 through 8 weeks post-MI (P_MI = 1.3E-11, Figure 7D), preceding the peak of IRF8 expression. MCP-1 levels, assessed by ELISA, were transiently but significantly increased at day 3 post-MI (P = 0.0498, Figure 7E, F) and localized primarily in GFAP-positive cells, implicating SGCs as an early cytokine source. Biotin-TNFα injection was also associated with increased Iba-1–positive macrophages in DRGs by day 7 (Supplemental Figure S9).

To determine whether cytokine uptake depends on TNFα receptors, we injected biotin-TNFα into wild-type (WT) and p55/p75 double knockout (KO) mice. In WT mice, biotin-TNFα was detected in A-fiber DRG neurons at day 1, and macrophage numbers increased by day 7 (Figure 8A, B). In contrast, biotin-TNFα was not detected in DRG neurons, and increased macrophage recruitment was not observed in p55/p75 KO mice. No biotin-TNFα labeling or increased macrophage number was observed in saline-injected WT or KO mice or in L4–L6 DRGs from either genotype.

Figure 8. TNFα receptor-dependent cytokine transport and macrophage recruitment to thoracic DRGs.

Immunofluorescence showing retrograde transport of biotinylated TNFα to T1–T4 DRGs at day 1 (A) and subsequent Iba1-positive macrophage recruitment at day 7 (B) after LV subepicardial injection in wild-type and p55/p75 knockout mice. n=5/group. IB4, C-fiber neuron marker; NF200, A-fiber neuron marker. White bar=50 μm.

Discussion

This study provides evidence that macrophages are activated in a time-dependent manner in cardiopulmonary sensory ganglia after MI, shaping the inflammatory milieu within the DRG microenvironment. Our in vitro co-culture experiments demonstrated that activated macrophages sensitize cardiopulmonary DRG neurons, most likely through downregulation of Kv channels. In vivo, we found that MI produced a time-dependent downregulation of Kv channels in T1–T4 DRG neurons. Importantly, minocycline, systemic macrophage depletion with clodronate liposomes, and local epidural delivery of ProGel-Dex attenuated DRG inflammation and exaggerated CSAR and PSAR responses. Minocycline and ProGel-Dex also reduced adverse ventricular remodeling without improving resting systolic function. These findings identify macrophage activation in thoracic DRGs as a potential mechanism linking myocardial injury to persistent cardiopulmonary afferent sensitization and provide evidence for a neural mechanism of heart–lung communication after MI.

Cardiac sympathetic afferents have traditionally been viewed primarily as pathways conveying cardiac nociceptive information to the central nervous system. However, increasing evidence^6–8,41,42 indicates that these afferents also participate in cardiovascular autonomic regulation and become sensitized in chronic heart failure. Activation of cardiac sympathetic afferents increases sympathetic outflow and impairs baroreflex function^7–11,39,40, whereas cardiac sympathetic afferent denervation attenuates adverse cardiac remodeling. Our previous studies⁷ further demonstrated that ablation of TRPV1-positive cardiac afferents improves cardiac and renal dysfunction in heart failure. The present findings extend these observations by identifying a peripheral neuroimmune mechanism that may contribute to persistent cardiac afferent sensitization after MI.

A major finding is that MI produces a time-dependent inflammatory response in T1–T4 DRGs. Macrophage numbers increased as early as 1 week after MI, whereas the protein expression of IRF8, a proinflammatory marker³⁷, was elevated at 4 and 8 weeks, suggesting that macrophage accumulation precedes the development of a more pronounced pro-inflammatory phenotype. Although the phenotype of the early infiltrating cells was not directly determined, this temporal pattern raises the possibility that recruited macrophages initially have a less inflammatory phenotype and subsequently acquire pro-inflammatory characteristics during post-MI remodeling. This interpretation remains speculative and will require direct characterization of macrophage populations at different stages after MI. Nevertheless, the combined Iba1 staining, IRF8 expression, tissue-clearing, electron microscopy, and RNA-sequencing data provide convergent evidence for macrophage involvement in thoracic DRG inflammation after MI.

The functional significance of this neuroinflammatory response is supported by our electrophysiological findings. Activated macrophages reduced Kv channel expression and suppressed Ito currents in cardiopulmonary DRG neurons, resulting in increased neuronal excitability. Individual inflammatory cytokines, including TNF-α, IL-1β, IL-6, and IFN-γ, each contributed to Kv channel downregulation, suggesting that macrophage-mediated neuronal sensitization is unlikely to depend on a single inflammatory mediator. These observations are consistent with evidence that reduced potassium conductances increase neuronal excitability in cardiovascular dysautonomia⁴³ and pain⁴⁴. Our findings therefore provide a mechanistic link between macrophage activation in DRGs and enhanced cardiopulmonary afferent activity after MI.

An important question is how myocardial injury initiates inflammation within anatomically distant DRGs. Conventional models of neuroinflammation emphasize neuronal signaling within the spinal cord, including ATP-dependent activation of microglia.^45,46. Such mechanisms do not readily explain the macrophage accumulation observed within DRGs, where neuronal synaptic transmission is fundamentally different from that in the dorsal horn. Previous studies have demonstrated that peripheral sensory nerve terminals can internalize molecules such as nerve growth factor^47–50 and transport them retrogradely to neuronal somata. Cytokines have also been shown to undergo axonal transport in models of neuropathic pain⁵¹. Building on that foundation, our results demonstrate that sensory nerve terminals within peripherally innervated target organs can indeed mediate cytokine uptake and retrograde transport to DRG somata in vivo. Moreover, while Myers et al.⁵¹ focused on neuropathic pain mechanisms by targeting the sciatic nerve, our work revealed a broader phenomenon: autonomic nerve endings, such as cardiac sympathetic efferents, can also take up cytokines from the heart and transport them back to the stellate ganglia soma. Thus, our study expands the concept beyond sensory nerves to show that both sensory and autonomic nerve terminals can internalize cytokines from a damaged organ in a non-neuropathic pain context.

Our data further supports a pathway in which cytokine uptake by peripheral nerve terminals initiates a local neuroimmune response within the DRG. Biotinylated TNF-α and IL-6 administered to the heart were detected in T1–T4 DRG neurons and stellate ganglia and were associated with GFAP-positive satellite glial cells and increased MCP-1 production. Importantly, TNF-α uptake and subsequent macrophage infiltration were prevented in TNFR-deficient mice. These findings support a model in which cytokines released from injured myocardium are internalized by cardiac sensory nerve terminals, transported to neuronal somata, and communicated to surrounding satellite glial cells, which then promote macrophage recruitment through chemokine signaling. This mechanism expands the concept of cytokine-mediated neural signaling beyond neuropathic pain and suggests that organ-derived inflammatory mediators can directly influence peripheral autonomic and sensory ganglia.

The therapeutic experiments provide additional evidence that DRG inflammation contributes to post-MI afferent sensitization. Minocycline treatment prevented the increase in IRF8 and attenuated the exaggerated CSAR. Because minocycline crosses the blood–brain barrier and can affect central microglia^20,40, however, its effects cannot be attributed exclusively to DRG macrophages. Likewise, systemic clodronate liposomes substantially depleted macrophages in peripheral organs but did not completely eliminate macrophages in the DRGs. Nevertheless, the reduction in CSAR despite incomplete DRG macrophage depletion supports an important contribution of macrophage-driven inflammation to afferent sensitization. These pharmacological approaches therefore provide complementary, although not completely selective, evidence for a causal role of neuroinflammation.

ProGel-Dex provided a more localized strategy for suppressing thoracic ganglionic inflammation. Unlike systemic minocycline or clodronate, epidural administration was intended to deliver the anti-inflammatory effect directly to the T1–T4 region. ProGel-Dex is a thermoresponsive macromolecular dexamethasone prodrug that undergoes a liquid-to-gel transition at physiological temperature and is designed to provide prolonged local drug exposure. Previous studies have demonstrated sustained therapeutic effects of ProGel-Dex in inflammatory disease models^22,52. In the present study, a single epidural administration after MI suppressed macrophage activation, restored Kv channel function and neuronal excitability, and attenuated exaggerated CSAR and PSAR responses. ProGel-Dex also reduced ventricular chamber dilation and systolic and diastolic volumes without significantly altering resting ejection fraction. These findings are consistent with our previous work showing that reducing pathological cardiac afferent activity can attenuate adverse cardiac remodeling.⁷ The reduction in left ventricular end-systolic volume (LVESV) may be especially meaningful because LVESV integrates ventricular emptying and structural remodeling and has been associated with prognosis after MI². Thus, modulation of pathological cardiopulmonary afferent signaling may influence ventricular remodeling even when global systolic function remains unchanged.

An additional novel finding is that pulmonary spinal afferents become sensitized after MI. Activation of pulmonary spinal afferents with bradykinin produced exaggerated pressor and sympatho-excitatory responses four weeks after MI, and these responses were prevented by minocycline, clodronate, or ProGel-Dex. Although pulmonary sensory regulation has been dominated by studies of vagal afferents⁵³, our findings indicate that pulmonary spinal afferents also contribute to sympatho-excitatory cardiovascular responses and can be sensitized following myocardial injury. Because the T1–T4 DRGs contain neurons associated with both cardiac and pulmonary innervation, inflammation within these ganglia provides a plausible substrate for cross-sensitization between cardiac and pulmonary afferent pathways. Together, these data support a novel paradigm in which myocardial injury initiates a neuro-inflammatory cascade involving macrophage activation in autonomic ganglia shared by the heart and lungs, leading to cross-organ afferent sensitization. Given that DRGs contain heterogeneous neurons that innervate multiple organs, cross-sensitization of afferent pathways provides a plausible mechanism by which pathology in one organ can influence sensory function in another. To our knowledge, this represents a new form of organ-to-organ interaction mediated by the peripheral nervous system, which may extend beyond the heart–lung axis.

Several limitations should be considered. First, the temporal relationship between macrophage accumulation and macrophage activation remains incompletely defined. Iba1-positive cells increased at 1 week, whereas IRF8 increased at later time points. Because we did not directly characterize macrophage phenotypes, the possibility that early infiltrating macrophages differ functionally from later pro-inflammatory macrophages remains unresolved. Second, the temporal profile of cytokine expressions in the DRGs was not comprehensively assessed. Although increased IL-6 expression was detected at 4 weeks, earlier cytokine changes were not systematically examined, limiting our ability to establish the precise relationship between cytokine signaling, macrophage activation, and Kv channel dysregulation. The cytokine experiments also cannot exclude contributions from other inflammatory mediators or signaling pathways.

Third, the CSAR and PSAR experiments were performed after bilateral vagotomy to isolate spinal afferent-mediated reflexes. Although this approach is established for studying these pathways, it removes vagal input and therefore does not reproduce the intact autonomic state. Recent work⁵⁴ suggests that vagal afferents may also contribute to cardiac remodeling after MI, emphasizing the need to investigate interactions between vagal and spinal afferent pathways in future studies. Fourth, most experiments were conducted in male animals, limiting our ability to determine whether the neuroinflammatory response and its functional consequences differ between sexes.

Finally, the in vivo release kinetics of ProGel-Dex after epidural administration were not directly measured. Our interpretation of prolonged drug availability is based on prior studies demonstrating sustained ProGel-Dex retention and dexamethasone exposure in other tissues. Thus, the persistent suppression of IRF8 observed for four weeks provides evidence of prolonged biological activity but does not establish the precise release profile within the epidural space or DRGs. Epidural delivery may also expose adjacent spinal segments. Although our preliminary analysis did not detect increased IRF8 or GFAP in T9–L1 DRGs at four weeks, future pharmacokinetic and biodistribution studies will be needed to define the spatial and temporal characteristics of ProGel-Dex delivery. In addition, patch-clamp experiments were performed at room temperature, and therefore the electrophysiological properties observed ex vivo may differ from those at physiological temperature.

In conclusion, our findings identify a neuroinflammatory cascade within T1–T4 DRGs after MI characterized by glial activation, macrophage accumulation, inflammatory signaling, and increased cardiopulmonary afferent excitability. We propose that cytokine uptake by peripheral nerve terminals and retrograde transport to sensory ganglia contributes to satellite glial activation and macrophage recruitment, thereby promoting persistent afferent sensitization (Supplemental Figure S10). Local suppression of this inflammatory response with ProGel-Dex attenuated both cardiac and pulmonary spinal afferent reflexes and reduced adverse ventricular remodeling. These findings identify DRG inflammation as a potential therapeutic target for pathological cardiopulmonary neural signaling after MI and suggest a previously unrecognized neural mechanism linking cardiac injury with pulmonary sensory dysfunction.

Supplementary Material

A List of Supplemental Materials

Supplemental Materials

Expanded Materials & Methods

Online Figures S1-S10

Major Resources Table

Data Set

References 18–35

Uncropped Western Blots

Online Video 1

Online Video 2

Novelty and Significance.

What Is Known?

• The heart and lung share thoracic spinal sensory pathways that coordinate cardiopulmonary function and sympathetic activity.

• Myocardial infarction (MI) produces persistent sensitization of cardiac sympathetic afferent reflexes and contributes to sympathetic overactivation in chronic heart failure.

• Whether injury in one organ can induce neuroinflammation within shared sensory ganglia and thereby alter afferent signaling from another organ remains unknown.

What New Information Does This Article Contribute?

• We identify a previously unrecognized heart–lung neural crosstalk mechanism whereby myocardial injury induces neuroinflammation in shared thoracic dorsal root ganglia (DRGs) and remotely sensitizes pulmonary spinal afferent signaling.

• We demonstrate that inflammatory cytokines from the injured heart are transported by cardiac afferents to thoracic DRGs, promoting macrophage and glial activation and suppressing neuronal voltage-gated potassium (Kv) channels to increase sensory neuron excitability.

• Neuroinflammatory remodeling of shared DRGs produces reciprocal sensitization of cardiac and pulmonary spinal afferent reflexes, while targeted suppression of DRG inflammation attenuates these pathological reflexes and improves post-MI cardiac remodeling.

Novelty and Significance.

This study identifies a previously unrecognized heart–lung neural crosstalk mechanism through which injury to one organ remotely alters sensory signaling from another. We demonstrate that myocardial infarction initiates inflammatory signaling from the injured heart that is transported through cardiac afferents to shared thoracic dorsal root ganglia (DRGs), where it promotes macrophage and glial activation and suppresses neuronal voltage-gated potassium channels. This neuroinflammatory remodeling of shared sensory ganglia increases neuronal excitability and sensitizes both cardiac and pulmonary spinal afferent reflexes, revealing a mechanism by which cardiac injury can propagate pathological neural consequences to the lung. Thus, the DRG functions as a critical neural interface linking inflammation in the heart to altered pulmonary sensory signaling and sympathetic overactivation. Importantly, pharmacological suppression of DRG inflammation, particularly sustained local dexamethasone delivery with ProGel-Dex, attenuates pathological cardiopulmonary reflexes and improves post-MI cardiac remodeling. These findings establish neuroinflammation of shared sensory ganglia as a mechanistic basis for heart–lung crosstalk and identify the DRG as a potential therapeutic target for interrupting pathological cardiopulmonary neural signaling in heart failure.

Non-standard Abbreviations and Acronyms

BBB

blood-brain barrier

BK

bradykinin

CHF

chronic heart failure

CNS

central nervous system

CPC

combined PACT-CUBIC tissue-clearing protocol

CSAR

cardiac sympathetic afferent reflex

DEG

differentially expressed gene

Dex

dexamethasone

FDR

false discovery rate

FBS

fetal bovine serum

GFAP

glial fibrillary acidic protein

GO

Gene Ontology

HPMA

N-(2-hydroxypropyl)methacrylamide

IB4

isolectin B4

Iba1

ionized calcium-binding adaptor molecule 1

IRF8

interferon regulatory factor 8

IS

infarct size

Ito

transient outward potassium current

IA

4-aminopyridine–sensitive potassium current

IK

tetraethylammonium-sensitive potassium current

LPS

lipopolysaccharide

LVESV

left ventricular end-systolic volume

MAP

mean arterial pressure

MCP-1

monocyte chemoattractant protein-1

NGF

nerve growth factor

PSAR

pulmonary spinal afferent reflex

PFA

paraformaldehyde

RSNA

renal sympathetic nerve activity

RTX

resiniferatoxin

SGC

satellite glial cell

SEM

scanning electron microscopy

TMM

trimmed mean of M-values

TNFR

tumor necrosis factor receptor