Decreased adrenoceptor stimulation in heart failure rats reduces NGF expression by cardiac parasympathetic neurons

Abstract

Postganglionic cardiac parasympathetic and sympathetic nerves are physically proximate in atrial cardiac tissue allowing reciprocal inhibition of neurotransmitter release, depending on demands from central cardiovascular centers or reflex pathways. Parasympathetic cardiac ganglion (CG) neurons synthesize and release the sympathetic neurotrophin nerve growth factor (NGF), which may serve to maintain these close connections. In this study we investigated whether NGF synthesis by CG neurons is altered in heart failure, and whether norepinephrine from sympathetic neurons promotes NGF synthesis. NGF and proNGF immunoreactivity in CG neurons in heart failure rats following chronic coronary artery ligation was investigated. NGF immunoreactivity was decreased significantly in heart failure rats compared to sham-operated animals, whereas proNGF expression was unchanged. Changes in neurochemistry of CG neurons included attenuated expression of the cholinergic marker vesicular acetylcholine transporter, and increased expression of the neuropeptide vasoactive intestinal polypeptide. To further investigate norepinephrine's role in promoting NGF synthesis, we cultured CG neurons treated with adrenergic receptor (AR) agonists. An 82% increase in NGF mRNA levels was detected after 1h of isoproterenol (β-AR agonist) treatment, which increased an additional 22% at 24h. Antagonist treatment blocked isoproterenol-induced increases in NGF transcripts. In contrast, the α-AR agonist phenylephrine did not alter NGF mRNA expression. These results are consistent with β-AR mediated maintenance of NGF synthesis in CG neurons. In heart failure, a decrease in NGF synthesis by CG neurons may potentially contribute to reduced connections with adjacent sympathetic nerves.

Keywords: Adrenergic receptor; Autonomic; Cardiac ganglion; Heart failure; Nerve growth factor; Parasympathetic.

Figure 1

ProNGF (A, C) and NGFβ (B, D) immunoreactive neurons in cardiac ganglia from rats after coronary artery ligation (CAL) or sham surgery. ProNGF immunoreactivity (-ir) was prominent within neuronal soma (white arrowheads; A, C), nerve bundles (nb; B) and vascular smooth muscle (v; C). Percentage of proNGF-ir neurons was stable between groups (E). NGFβ-ir was prominent in cardiac ganglia from sham rats (B, white arrowheads); however after CAL, NGFβ-ir was present in fewer neurons with most not displaying NGFβ-ir (grey arrowheads; 34% reduction, p=0.001, D, E). Data are mean ±SEM, ***p<0.001. Scale bar in D= 40μm for all panels.

Figure 2

VAChT (A, B), VIP (C, D) and VMAT (E, F) immunoreactive neurons in cardiac ganglia (CG) from rats after coronary artery ligation (CAL) or sham surgery. Cytoplasmic VAChT immunoreactivity (-ir) was strong in 60% of CG neurons (white arrowheads, A). Following CAL, VAChT-ir was less prominent (grey arrowheads= negative neurons, B; 22% reduction, E). VIP-ir was present in a small subpopulation of CG neurons; after CAL, the percentage of VIP-ir neurons increased by 47% (D, E; p=0.04). VMAT-ir was observed in nerve fibers (E, arrows) and a small subpopulation of CG neuronal soma (32%, E, G) in sham rats; this percentage did not significantly alter after CAL (F, G). Data are mean ±SEM, *<0.05, ***p<0.001. Scale bar in D= 50μm for all panels.

Figure 3

VAChT immunoreactivity (-ir) in cultured parasympathetic cardiac ganglion (CG; A, B) and pterygopalatine (PPG; C, D) neurons. CG neurons in vitro stained strongly in neuronal soma (arrowhead) and neurites for the pan-neuronal marker peripherin (A) and VAChT (B), VAChT-ir being prominent in 60% of neurons, very similar to that in vivo (E). Cultured PPG neurons stained strongly in neuronal soma (arrowhead) and processes for both the pan-neuronal marker PGP9.5 (PGP; C) and VAChT (D) with a similar percentage (59%) to that in vivo (E) and to that in CG neurons. Data are mean ±SEM. Scale bar in D= 40μm for all panels.

Figure 4

NGF mRNA quantitation in cardiac ganglion neurons after adrenergic agonist treatment. For isoproterenol (IsoP; A), a significant increase (p<0.01) over control levels was seen after 1 and 24 hr of treatment that was blocked by propranolol (Prop). In contrast, neither 1 nor 24 hr treatment with phenylephrine (PhE) altered NGF transcript expression over controls (B). Data are mean ±SEM, *<0.05, ***p<0.001.