Background: Acute myocardial infarction (AMI) is effectively treated by timely reperfusion; however, up to 10% of patients develop life-threatening ventricular arrhythmias (VA) within the
first 48 hours post-reperfusion, contributing significantly to early mortality. During this critical time window, neutrophils (nGs) rapidly infiltrate the injured myocardium. Although nGs are central regulators of inflammation, their contribution to arrhythmogenesis in the reperfused heart remains poorly defined. Simultaneously, AMI disrupts the cardiac autonomic nervous system (ANS) leading to regionally heterogeneous denervation and compensatory hyperinnervation, leading to electrical instability.
Aim:Visualization and quantification of sympathetic nerve alterations and their spatial proximity to infiltrating neutrophils in the acute phase of reperfused myocardial infarction. Methods:Reperfused acute myocardial infarction (repAMI) was induced in mice by transient occlusion of the left anterior descending coronary artery followed by 24 hours reperfusion. For whole-heart sympathetic nerve visualization, samples were cleared and immunolabeled using a lightsheet-compatible protocol and stained for tyrosine hydroxylase (TH) and Ly6G to detect sympathetic fibers and neutrophils, respectively. In parallel, cryosections were subjected to immunohistochemistry for TH and Ly6G to validate regional innervation changes and immune cell localization at higher spatial resolution. Statistical significance was assessed using one-way ANOVA. A p-value <0.05 was considered statistically significant.
Results: Light-sheet 3D imaging revealed a pronounced regional remodeling of sympathetic nerve fibers 24 hours after repAMI: Nerve fiber density was reduced by 87.7% in the infarct area, while the border zone exhibited a 140% increase, compared to controls (Figure 1). This pattern of infarct denervation and border-zone hyperinnervation was confirmed by immunohistochemistry. Quantification of TH⁺ nerve fibers showed significantly higher spots in the border region (183.0 ± 29.0 spots/ROI, P= <0.0001) and remote myocardium (244.0 ± 5.0 spots/ROI, P= <0.0001) relative to the infarct (37.0 ± 8.3 spots/ROI). Neutrophil infiltration followed a distinct regional pattern, with high accumulation in the infarct (123.0 ± 17.6 spots/ROI, P= <0.0001) and border zone (111.7 ± 27.9 spots/ROI, P= <0.0001), and lower levels in the remote myocardium (15.0 ± 5.1). Spatial proximity analysis demonstrated that significantly more Ly6G⁺neutrophils were located within 50 µm of TH⁺ nerve fibers in infarct and border regions (81.7 ± 18.5, 72.0 ± 17.2, 75.0 ± 11.8 spots/ROI, P=0.0001, P=0.0001 respectively), a pattern not observed in remote, control hearts and not present at distances >50 µm.
Conclusion: These findings indicate early and region-specific sympathetic remodeling coinciding with close neutrophil–nerve fiber spatial association, suggesting a potential inflammatory–neurogenic interaction in the acute post-reperfusion phase.
