Heterogeneous ventricular sympathetic innervation, altered beta-adrenergic receptor expression, and rhythm instability in mice lacking the p75 neurotrophin receptor

Abstract

Sympathetic nerves stimulate cardiac function through the release of norepinephrine and the activation of cardiac beta(1)-adrenergic receptors. The sympathetic innervation of the heart is sculpted during development by chemoattractive factors including nerve growth factor (NGF) and the chemorepulsive factor semaphorin 3a. NGF acts through the TrkA receptor and the p75 neurotrophin receptor (p75(NTR)) in sympathetic neurons. NGF stimulates sympathetic axon extension into the heart through TrkA, but p75(NTR) modulates multiple coreceptors that can either stimulate or inhibit axon outgrowth. In mice lacking p75(NTR), the sympathetic innervation density in target tissues ranges from denervation to hyperinnervation. Recent studies have revealed significant changes in the sympathetic innervation density of p75NTR-deficient (p75(NTR-/-)) atria between early postnatal development and adulthood. We examined the innervation of adult p75(NTR-/-) ventricles and discovered that the subendocardium of the p75(NTR-/-) left ventricle was essentially devoid of sympathetic nerve fibers, whereas the innervation density of the subepicardium was normal. This phenotype is similar to that seen in mice overexpressing semaphorin 3a, and we found that sympathetic axons lacking p75(NTR) are more sensitive to semaphorin 3a in vitro than control neurons. The lack of subendocardial innervation was associated with decreased dP/dt, altered cardiac beta(1)-adrenergic receptor expression and sensitivity, and a significant increase in spontaneous ventricular arrhythmias. The lack of p75(NTR) also resulted in increased tyrosine hydroxylase content in cardiac sympathetic neurons and elevated norepinephrine in the right ventricle, where innervation density was normal.

Fig. 1.

Fiber distribution is altered in the p75 neurotrophin receptor-deficient (p75^NTR−/−) left ventricle (LV). Sympathetic fiber distribution was identified by tyrosine hydroxylase (TH) immunoreactivity. A: representative pictures taken at ×5 magnification showing the entire width of the LV free wall revealing the altered pattern of sympathetic innervation in p75^NTR−/− mice (right) compared with wild-type (WT) mice (left). This low magnification was not used for quantitative analysis. The edges of the tissue have been outlined for clarity. Scale bar = 0.25 mm. B: sympathetic fiber distribution in the right ventricle (RV) of WT mice (left) and p75^NTR−/− mice (right). Scale bar = 0.1 mm. C: example of the threshold discrimination used to determine innervation density in which the TH-positive nerves above the threshold are highlighted. Innervation density was then expressed as the percent area that was above the determined threshold (TH⁺ fibers). Scale bar = 0.05 mm. D: quantification of sympathetic fiber density in the subendocardium (en and Endo) and subepicardium (ep and Epi) of WT and p75^NTR−/− mice. ns, Not significant. Values are means ± SE; n = 4. ***P < 0.001. E: quantification of sympathetic fiber density of the RV in WT and p75^NTR−/− mice. Values are means ± SE; n = 4.

Fig. 2.

Innervation density is normal in the p75^NTR−/− subepicardium. A and B: representative pictures at ×20 magnification of TH⁺ fibers in the WT (A) and p75^NTR−/− (B) subepicardium that were used for quantitative analysis. Scale bar = 0.1 mm. C: diagram showing the regions (areas A–E) analyzed to further characterize the innervation density of WT and p75^NTR−/− ventricles. D–F: quantification of sympathetic fiber density in the WT and p75^NTR−/− RV (D), interventricular septum (E), and LV (F). Values are means ± SE; n = 7.

Fig. 3.

p75^NTR−/− sympathetic axons are more sensitive to semaphorin 3a (Sema3a) growth cone collapse/inhibition. A: Representative stellate ganglia explants at time 0 (left) and 6 h later (right). Scale bar = 0.1 mm. B: quantification of axon growth rate from WT and p75^NTR−/− sympathetic ganglia in the presence of 0, 120, or 240 ng/ml Sema3a. Values are means ± SE; n = 3. *P < 0.05; **P < 0.01; ***P < 0.001. C: neuropilin (NP)-1 (left) and plexin A4 (right) mRNA normalized to GAPDH in WT and p75^NTR−/− stellate ganglia. Values are means ± SE; n = 5.

Fig. 4.

Mice lacking p75^NTR have increased spontaneous ventricular arrhythmias. A: representative ECG showing spontaneous premature ventricular complexes (PVCs) in a p75^NTR−/− mouse. B: p75^NTR−/− mice experienced more spontaneous PVCs than WT mice. Values are means ± SE; n = 8–9. *P < 0.05. C: number of PVCs in WT and p75^NTR−/− mice during 2 h of the awake phase and sleep phase. Values are means ± SE; n = 8–9. ***P < 0.01.

Fig. 5.

dP/dt_max and dP/dt_min are low in p75^NTR−/− mice. A: baseline dP/dt_max in WT and p75^NTR−/− mice. B: dP/dt_min in WT and p75^NTR−/− mice. Values are means ± SE; n = 6–7. *P < 0.05.

Fig. 6.

p75^NTR−/− mice have an enhanced response to β₁-adrenergic receptor (β₁AR) stimulation in the denervated subendocardium. A and B: peak radial thickening velocity of the subepicardium at early diastole in WT and p75^NTR−/− mice as determined by tissue Doppler analysis before (A) and after (B) an intraperitoneal injection of dobutamine (32 μg/kg). C and D: peak radial thickening velocity of the subendocardium in WT and p75^NTR−/− mice determined at early diastole before (C) or after (D) an intraperitoneal injection of dobutamine (32 μg/kg). Values are means ± SE; n = 4. *P < 0.05.

Fig. 7.

β₁AR expression is low, and its distribution is altered in the p75^NTR−/− LV. A: β₁AR protein in the subendocardium and subepicardium of WT (left; open bars) and p75^NTR−/− mice (right; shaded bars) normalized to total protein. Values are means ± SE; n = 4. *P < 0.05. B: representative Western blot (top) and quantification (bottom) of β₁ARs and actin in WT and p75^NTR−/− LVs. Values are means ± SE; n = 6. *P < 0.05; **P < 0.01.

Fig. 8.

Norephinephrine (NE) content, NE uptake, and TH levels in the LV. A: NE content in the p75^NTR−/− LV was not significantly different than that in the WT LV. Values are means ± SE; n = 5–6. B: NE is elevated in the RV of p75^NTR−/− mice. Values are means ± SE; n = 5–6. *P < 0.05. C: NE uptake in p75^−/− mouse ventricles is identical to uptake in WT ventricles. Values are means ± SE; n = 6. D: TH protein, normalized to the pan-neuronal marker protein gene product 9.5 (PGP9.5), is elevated in the LV of p75^NTR−/− mice compared with WT mice. Values are means ± SE; n = 5–6. **P < 0.01.