Sympathetic hyperinnervation and inflammatory cell NGF synthesis following myocardial infarction in rats

Abstract

Sympathetic hyperinnervation occurs in human ventricular tissue after myocardial infarction and may contribute to arrhythmias. Aberrant sympathetic sprouting is associated with elevated nerve growth factor (NGF) in many contexts, including ventricular hyperinnervation. However, it is unclear whether cardiomyocytes or other cell types are responsible for increased NGF synthesis. In this study, left coronary arteries were ligated and ventricular tissue examined in rats 1–28 days post-infarction. Infarct and peri-infarct tissue was essentially devoid of sensory and parasympathetic nerves at all time points. However, areas of increased sympathetic nerve density were observed in the peri-infarct zone between post-ligation days 4–14. Hyperinnervation occurred in regions containing accumulations of macrophages and myofibroblasts. To assess whether these inflammatory cells synthesize NGF, sections were processed for NGF in situ hybridization and immunohistochemistry. Both macrophage1 antigen-positive macrophages and α-smooth muscle actin-immunoreactive myofibroblasts expressed NGF in areas where they were closely proximate to sympathetic nerves. To investigate whether NGF produced by peri-infarct cells induces sympathetic outgrowth, we co-cultured adult sympathetic ganglia with peri-infarct explants. Neurite outgrowth from sympathetic ganglia was significantly greater at post-ligation days 7–14 as compared to control tissue. Addition of an NGF function-blocking antibody prevented the increased neurite outgrowth induced by peri-infarct tissue. These findings provide evidence that inflammatory cell NGF synthesis plays a causal role in sympathetic hyperinnervation following myocardial infarction.

Introduction

Arrhythmias are frequent complications of myocardial infarction, occurring most often in the first 30 days following the event (Solomon et al., 2005). While increased sympathetic drive and altered ventricular conduction are clearly contributory (Schomig, 1990, Du et al., 1999, Lal et al., 2005), recent evidence suggests that sympathetic axon remodeling in ventricular myocardium may be a major factor. Postmortem analyses of infarcted human myocardium show abnormal increases in numbers of sympathetic axons adjacent to the site of injury (Cao et al., 2000a). Ischemic injury appears to be the cause of the remodeling, as coronary artery ligation induces hyperinnervation in dogs (Lai et al., 2000, Zhou et al., 2004) and rats (Ahonen et al., 1975, Paessens and Borchard, 1980, Holmgren et al., 1981, Vracko et al., 1990, Kaye et al., 2000, Li et al., 2004). Through enhanced norepinephrine-mediated myocardial depolarization, abnormally increased sympathetic nerve density may be proarrhythmogenic and represent a significant factor in post-infarct sudden cardiac death.

The cellular and molecular mechanisms leading to post-infarct sympathetic hyperinnervation remain incompletely understood. However, the neurotrophin nerve growth factor (NGF) is strongly implicated. NGF is a potent growth and survival factor for sympathetic neurons (Korsching and Thoenen, 1985, Glebova and Ginty, 2004). Recent evidence indicates that NGF expression is up-regulated in the region of the infarction (Hiltunen et al., 2001, Zhou et al., 2004), and infusion of NGF into the stellate ganglion results in cardiac hyperinnervation in canine hearts (Cao et al., 2000b). However, the cell types involved in post-infarct cardiac NGF production and the neural selectivity of the response are not well defined. Similarly, while exogenous NGF can cause sympathetic sprouting there is no definitive evidence that endogenous NGF is required for cardiac sympathetic sprouting in the post-infarct heart.

The objective of the present study was to assess whether inflammatory cells, which are present in the infarcted myocardium (Vracko and Thorning, 1991, Desmouliére et al., 1996) and may synthesize NGF in other contexts (Matsuda et al., 1998, Hasan et al., 2000, Aloe, 2004, Kawamoto and Matsuda, 2004), serve as a local source of NGF which in turn initiates sympathetic sprouting. Accordingly, we assessed whether inflammatory cells express NGF and whether expression is temporo-spatially consistent with the observed pattern of hyperinnervation. Additionally, the requirement for NGF release by this tissue in inducing sympathetic sprouting was assessed by antibody neutralization explant culture studies. The present study provides novel evidence that inflammatory myofibroblasts and macrophages play a central role in peri-infarct NGF synthesis, and that inflammatory cell-derived NGF is required for and sufficient to induce selective sprouting of cardiac sympathetic axons.