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Sympathetic Denervation of Peri-Infarct Myocardium Requires the p75 Neurotrophin Receptor

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Sympathetic Denervation of Peri-Infarct Myocardium Requires the p75 Neurotrophin Receptor

Christina U Lorentz et al. Exp Neurol.

Abstract

Development of cardiac sympathetic heterogeneity after myocardial infarction contributes to ventricular arrhythmias and sudden cardiac death. Regions of sympathetic hyperinnervation and denervation appear in the viable myocardium beyond the infarcted area. While elevated nerve growth factor (NGF) is implicated in sympathetic hyperinnervation, the mechanisms underlying denervation are unknown. Recent studies show that selective activation of the p75 neurotrophin receptor (p75(NTR)) in sympathetic neurons causes axon degeneration. We used mice that lack p75(NTR) to test the hypothesis that activation of p75(NTR) causes peri-infarct sympathetic denervation after cardiac ischemia-reperfusion. Wild type hearts exhibited sympathetic denervation adjacent to the infarct 24h and 3 days after ischemia-reperfusion, but no peri-infarct sympathetic denervation occurred in p75(NTR)-/- mice. Sympathetic hyperinnervation was found in the distal peri-infarct myocardium in both genotypes 3 days after MI, and hyperinnervation was increased in the p75(NTR)-/- mice. By 7 days after ischemia-reperfusion, cardiac sympathetic innervation density returned back to sham-operated levels in both genotypes, indicating that axonal pruning did not require p75(NTR). Prior studies revealed that proNGF is elevated in the damaged left ventricle after ischemia-reperfusion, as is mRNA encoding brain-derived neurotrophic factor (BDNF). ProNGF and BDNF preferentially bind p75(NTR) rather than TrkA on sympathetic neurons. Immunohistochemistry using Bdnf-HA mice confirmed the presence of BDNF or proBDNF in the infarct after ischemia-reperfusion. Thus, at least two p75(NTR) ligands are elevated in the left ventricle after ischemia-reperfusion where they may stimulate p75(NTR)-dependent denervation of peri-infarct myocardium. In contrast, NGF-induced sympathetic hyperinnervation in the distal peri-infarct ventricle is attenuated by p75(NTR).

Keywords: 123I-MIBG; Axon degeneration; BDNF; Brain-derived neurotrophic factor; HA; Ischemia–reperfusion; LAD; MI; NE; NGF; Pruning; Regeneration; SCG; TH; TrkA; brain-derived neurotrophic factor; hemagglutinin; iodine-123 meta-iodobenzylguanidine; left anterior descending coronary artery; myocardial infarction; nerve growth factor; norepinephrine; p75 neurotrophin receptor; p75(NTR); superior cervical ganglia; tropomyosin-related receptor kinase A; tyrosine hydroxylase.

Figures

Figure 1
Figure 1
Method for quantification of sympathetic innervation. A) Cartoon cross section of the left and right ventricles showing the 11 fields of view analyzed (A–K). Pictures A and B were taken from the right ventricle, C–J were taken from the subepicardium of the left ventricle, and K was taken from the septum. B) Cartoon cross section of the left and right ventricles showing the variable location of the infarct after ischemia-reperfusion. The proximal peri-infarct region (P1) was defined as the field of view adjacent to the infarct and the distal peri-infarct region (P2) was defined as the field of view adjacent to P1 (530 μm from the infarct). C) Photomicrograph depicting the infarct 7 days after ischemia-reperfusion and the adjacent viable myocardium. D) Cartoon cross section of the regions quantified. One representative picture within the infarct was averaged over the four sections analyzed. E) Quantification of sympathetic fiber density in sham-operated animals from WT (black bars) and p75NTR−/− mice (white bars) from three fields of view (D, E, and H) that correspond to P2, P1, and infarct regions after MI respectively, mean ± SEM, n=8.
Figure 2
Figure 2
Sympathetic denervation occurs in the infarct after ischemia-reperfusion. Quantification of sympathetic fiber density in the infarct 24 hours, 3 days, and 7 days after ischemia- reperfusion from WT (black squares) and p75NTR−/− mice (open squares) compared to sham operated animals, mean ± SEM, ***p< 0.001, n=4 (24 hrs) or n=6–8 (sham, 3 day, 7 day).
Figure 3
Figure 3
Sympathetic innervation density 24 hours after ischemia-reperfusion in WT and p75NTR−/− peri-infarct left ventricle. A) Quantification of sympathetic fiber density in the proximal peri-infarct region (P1) and distal peri-infarct region (P2) 24 hours after ischemia-reperfusion from WT (black squares) and B) p75NTR−/− mice (open squares), mean ± SEM, *p< 0.05, n=4–8. C) Representative picture from WT sham operated left ventricle D) and from p75NTR−/− left ventricle. E) Representative picture from the proximal peri-infarct region (peri-infarct 1, P1) from WT mice and F) from p75NTR−/− mice. G) Representative picture from the distal peri-infarct region (peri-infarct 2, P2) from WT mice and H) p75NTR−/− mice. Scale bars= 100μm
Figure 4
Figure 4
Sympathetic innervation density 3 days after ischemia-reperfusion in WT and p75NTR−/− peri-infarct left ventricle. A) Quantification of sympathetic fiber density in the proximal peri-infarct region (P1) and distal peri-infarct region (P2) 3 days after ischemia-reperfusion from WT (black squares) and B) p75NTR−/− mice (open squares), mean ± SEM, *p< 0.05, **p< 0.01, ***p< 0.001, n=6–8. C) Representative picture from WT sham operated left ventricle D) and from p75NTR−/− left ventricle. E) Representative picture from the proximal peri-infarct region (peri-infarct 1, P1) 3 days after ischemia-reperfusion from WT mice and F) from p75NTR−/− mice. G) Representative picture from the distal peri-infarct region (peri-infarct 2, P2) 3 days after ischemia-reperfusion from WT mice and H) p75NTR−/− mice. Scale bars= 100μm
Figure 5
Figure 5. NGF does not stimulate TH activity
A) Sympathetic neurons were treated with NGF (100ng/ml) for 2 or 20hrs and TH activity was assessed by quantifying the rate of L-DOPA production. This graph shows a representative experiment done in quadruplicate. Since TH protein levels did not differ significantly between replicates or treatment groups, the data are expressed as the amount of L-DOPA produced over time. B) Dissociated sympathetic neurons were treated with NGF (100ng/ml) for 2, 5, and 15 minutes, and blotted for phosphorylated (pSer31) and total (TH) tyrosine hydroxylase. Phospho-ERK1/2 (pERK1/2) was a positive control for NGF stimulation. C) Quantification of phospho-Ser31 TH relative to total TH. Data are calculated as percent of control and averaged across 3 independent experiments (mean ± SEM).
Figure 6
Figure 6
Sympathetic innervation density 7 days after ischemia-reperfusion in WT and p75NTR−/− peri-infarct left ventricle. A) Quantification of sympathetic fiber density in the proximal peri-infarct region (P1) and distal peri-infarct region (P2) 7 days after ischemia-reperfusion from WT (black squares) and B) p75NTR−/− mice (open squares), mean ± SEM, n=6–8. C) Representative picture from WT peri-infarct region 7 days after ischemia-reperfusion shows heterogeneous sympathetic innervation with distinct regions of denervated myocardium next hyperinnervated regions, scale bar= 100μm.
Figure 7
Figure 7
BDNF protein is elevated in the left ventricle 24 hours after ischemia-reperfusion. Representative pictures of immunofluorescence detection of BDNF using an antibody to HA in the left ventricle from Bdnf-HA mice 24 hrs after sham surgery (A) or ischemia-reperfusion (B), scale bar A, B= 100μm. C, D) Co-immunofluorescence detection of BDNF-HA (green), and in red the neutrophil marker Ly-6B.2 (C) or macrophage marker Mac2 (D). Scale bar C, D= 25μm. Arrows highlight cells labeled for a single marker and arrowheads highlight neutrophils positive for both HA and Ly-6B.2.

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