Cardiac macrophages prevent sudden death during heart stress

Heart failure is a growing global burden. While research has focused largely on left ventricular (LV) dysfunction, right heart failure is increasingly recognized as a serious unmet medical need and is associated with adverse outcomes, including increased risk of serious arrhythmias and sudden cardiac death. The molecular mechanisms underlying right ventricular (RV) dysfunction remain poorly understood. Immune cells, particularly macrophages, have diverse roles in cardiac homeostasis and disease, contributing to LV remodeling and adaptive responses after injury, yet their roles during RV stress are less clear. This study investigates whether cardiac immune cells, especially macrophages and their effector amphiregulin (AREG), are essential for survival under acute RV stress by maintaining cardiac electrical conduction via gap junctional communication.

Prior work has shown that immune cells regulate cardiac homeostasis and pathology, with macrophages implicated in both adverse LV remodeling and in promoting adaptive responses and healing after myocardial infarction. Other immune cells (lymphocytes, mast cells) also influence LV remodeling. RV dysfunction is associated with increased risk of arrhythmia and sudden death, but immune cell roles in RV stress have been understudied. Gap junctions, particularly those composed of connexin 43 (Cx43), are central to myocardial electrical coupling; phosphorylation state and localization of Cx43 modulate conduction and arrhythmia susceptibility. EGFR ligands, including AREG, can activate signaling pathways (e.g., MEK/ERK) that influence cellular communication. These observations motivated testing whether macrophage-derived AREG regulates gap junction integrity and conduction during RV stress.

Animal models: Male C57BL/6J mice and Areg knockout (Areg−/−) mice were used. Acute RV pressure overload was induced by pulmonary artery banding (PAB) to raise RV pressure from 5–10 mmHg to 30–35 mmHg. Immune cell contributions were probed using depletion or genetic models: macrophages depleted with clodronate liposomes; granulocytes with Ly6G antibody; CD4+ and CD8+ T cell knockouts; B cell–deficient (Rag2KO); additional monocyte/macrophage lineage manipulation using Cx3cr1CreERT2;R26 reporter mice. Bone marrow transplantation (BMT) created chimeras by reconstituting lethally irradiated recipients with WT or Areg−/− donor marrow to assess hematopoietic macrophage-derived AREG effects. ECG telemetry: Long-term ECGs in freely moving mice assessed baseline arrhythmias and those induced by PAB or β-adrenergic stimulation. β-adrenergic stress: isoproterenol (5 mg/kg, intraperitoneal) was administered to test susceptibility to arrhythmias and sudden death. Ex vivo electrophysiology: Langendorff-perfused hearts underwent optical mapping and programmed stimulation/burst pacing to assess inducibility of ventricular tachycardia/ventricular fibrillation (VT/VF) and conduction properties. Gap junction assays: Neonatal mouse cardiomyocyte cultures were used for scrape loading/dye transfer to quantify gap junctional intercellular communication (GJIC) with or without cardiac resident macrophages and with recombinant AREG. Pharmacologic inhibitors tested EGFR/MEK/ERK signaling involvement (AG1478, U0126, FR180204). A Cx43 hemichannel-selective inhibitor was used to test the contribution of hemichannels to arrhythmia susceptibility in Areg−/− mice under isoproterenol stress. Molecular and imaging analyses: Western blotting assessed Cx43 phosphorylation status; immunohistochemistry localized Cx43 in myocardium (intercalated discs vs lateralization). Recombinant AREG was administered intraperitoneally to test rescue of Cx43 localization. HeLa cells transfected with Cx43-GFP evaluated AREG-induced gap junction plaque formation and phosphorylation, with or without EGFR pathway inhibitors. Conduction velocity was measured in cultured cardiomyocytes with motion imaging; flow cytometry characterized cardiac immune cell populations; single-cell RNA-seq datasets were mined to identify cell types expressing Areg and other EGFR ligands. Statistics: Group comparisons used t-tests or ANOVA with multiple-comparison corrections as appropriate; survival analyzed by Kaplan–Meier and log-rank tests; VT/VF induction frequencies compared with t-tests.

- Macrophages are essential for survival after acute RV pressure overload: macrophage depletion with clodronate caused high early mortality after PAB; 57% died within 4 hours. After PAB, 61% of clodronate-treated mice (n=7) developed advanced AV block leading to ventricular arrest. - Areg−/− mice displayed baseline conduction disturbances (sinus arrest/SA block, intermittent complete block, PVCs) on telemetry that were absent in WT. Under RV pressure overload (PAB), >85% of Areg−/− mice died within 24 hours, with AV block observed and no detectable histological or functional RV changes at that time point. - Hematopoietic deficiency of AREG confers susceptibility: in BMT experiments, recipients of Areg−/− bone marrow exhibited high mortality after PAB (57% within hours; 80% within 7 days) and AV block, whereas WT bone marrow recipients were protected. - β-adrenergic stimulation unmasks lethal vulnerability: isoproterenol (5 mg/kg) caused sudden death in ~57% of Areg−/− mice within 2 days and induced severe arrhythmias (advanced AV block, ventricular fibrillation). WT mice had only infrequent isolated PVCs and no deaths. - Pro-arrhythmic substrate in Areg−/− hearts: VT/VF was inducible in 6/6 Areg−/− hearts versus 1/6 WT hearts during ex vivo programmed stimulation. - Cx43 disorganization underlies conduction defects: in WT hearts, Cx43 localized to intercalated discs; in Areg−/− hearts, Cx43 was mislocalized (lateralization). Recombinant AREG administration rapidly restored Cx43 localization toward intercalated discs. - Inhibition of Cx43 hemichannels reduced isoproterenol-induced mortality in Areg−/− mice, implicating aberrant hemichannel activity when gap junctions are disorganized. - AREG enhances gap junctional coupling: co-culture of cardiomyocytes with cardiac macrophages or addition of AREG increased dye transfer in scrape-loading assays and increased conduction velocity in vitro. EGFR pathway inhibitors (AG1478, U0126, FR180204) significantly attenuated AREG-mediated dye transfer. - AREG promotes Cx43 phosphorylation and gap junction plaque formation: in Cx43-GFP–transfected HeLa cells, AREG increased gap junction plaque size and induced Cx43 phosphorylation; these effects were blocked by EGFR/MEK/ERK inhibitors. Full Cx43 phosphorylation required sites targeted by PKA, CK1, and MAPK pathways. - Among EGFR ligands expressed by cardiac macrophages, Areg and Hbegf were detected, but only AREG increased Cx43 phosphorylation and cardiomyocyte conduction velocity, indicating ligand-specific effects.

The study demonstrates that cardiac macrophages are critical for maintaining myocardial impulse conduction during acute cardiac stress, particularly RV pressure overload. Macrophage-derived amphiregulin (AREG) preserves and enhances intercellular electrical coupling by promoting Cx43 phosphorylation and stabilizing its localization at intercalated discs, facilitating functional gap junction formation. Loss of AREG leads to Cx43 disorganization (lateralization), impaired conduction, and susceptibility to advanced AV block and ventricular arrhythmias, culminating in sudden death during RV pressure overload or β-adrenergic stimulation. Pharmacologic inhibition of EGFR/MEK/ERK signaling blunted AREG’s enhancement of GJIC, identifying this pathway as a principal mediator of Cx43 phosphorylation and gap junction assembly in this context. The heightened VT/VF inducibility in Areg−/− hearts and the rescue of Cx43 organization by recombinant AREG support a causal link between macrophage-derived AREG, gap junction integrity, and arrhythmia prevention. These findings extend the role of resident cardiac macrophages beyond structural remodeling to dynamic, ongoing regulation of electrophysiological coupling, suggesting that targeting AREG signaling could offer therapeutic strategies to prevent lethal arrhythmias and sudden death during acute cardiac stresses.

Cardiac resident macrophages protect against lethal arrhythmias during acute cardiac stress by secreting amphiregulin, which enhances Cx43 phosphorylation and gap junction stability via EGFR/MEK/ERK signaling, thereby maintaining myocardial electrical conduction. AREG deficiency—globally or in hematopoietic/macrophage lineages—disrupts Cx43 organization and predisposes to AV block and ventricular arrhythmias under RV pressure overload and β-adrenergic stress. Recombinant AREG can acutely restore Cx43 localization, and hemichannel inhibition mitigates stress-induced mortality in Areg−/− mice, underscoring therapeutic potential. Future studies should define optimal modalities and timing for AREG-based interventions, delineate additional signaling nodes that modulate Cx43 phosphorylation, and evaluate translational relevance in large-animal models and human cardiac disease.

The work is primarily in mouse models and neonatal cardiomyocyte cultures, which may limit direct clinical translatability. While EGFR/MEK/ERK signaling was identified as a major pathway for AREG-induced Cx43 phosphorylation, Cx43 harbors multiple phosphorylation sites regulated by several kinases, indicating mechanistic complexity beyond a single pathway. The study focuses on Cx43-mediated coupling; other AREG targets or macrophage-derived factors may also contribute to conduction and arrhythmia phenotypes. Some datasets (e.g., single-cell RNA-seq reanalysis) are indirect and inferential regarding cellular sources of AREG in stress contexts.