Christina U. Lorentz

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

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.

Regulation of cardiac innervation and function via the p75 neurotrophin receptor

Homeostatic regulation of cardiac function is dependent on the balance of inputs from the sympathetic and parasympathetic nervous systems. We investigated whether the p75 neurotrophin receptor plays a developmental role in cardiac innervation by analyzing sympathetic and parasympathetic fibers in the atria of p75 knockout and wildtype mice at several stages of postnatal development, and examining the effect on control of heart rate. We found that parasympathetic innervation of the atria in p75-/- mice was similar to wildtype at all time points, but that the density of sympathetic innervation was dynamically regulated. Compared to wildtype mice, the p75-/- mice had less innervation at postnatal day 4, an increase at day 28, and decreased innervation in adult mice. These changes reflect defects in initial fiber in-growth and the timing of the normal developmental decrease in sympathetic innervation density in the atria. Thus, p75 regulates both the growth and stability of cardiac sympathetic fibers. The distribution of sympathetic fibers was also altered, so that many regions lacked innervation. Basal heart rate was depressed in adult p75-/- mice, and these mice exhibited a diminished heart rate response to restraint stress. This resulted from the lack of sympathetic innervation rather than increased parasympathetic transmission or a direct effect of p75 in cardiac cells. Norepinephrine was elevated in p75-/- atria, but stimulating norepinephrine release with tyramine produced less tachycardia in p75-/- mice than wild type mice. This suggests that altered density and distribution of sympathetic fibers in p75-/- atria impairs the control of heart rate.

Sympathetic denervation of peri-infarct myocardium requires the p75 neurotrophin receptor.

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).

Altered norepinephrine content and ventricular function in p75NTR-/- mice after myocardial infarction.

Cardiac sympathetic neurons stimulate heart rate and the force of contraction through release of norepinephrine. Nerve growth factor modulates sympathetic transmission through activation of TrkA and p75NTR. Nerve growth factor plays an important role in post-infarct sympathetic remodeling. We used mice lacking p75NTR to examine the effect of altered nerve growth factor signaling on sympathetic neuropeptide expression, cardiac norepinephrine, and ventricular function after myocardial infarction. Infarct size was similar in wildtype and p75NTR-/- mice after ischemia-reperfusion surgery. Likewise, mRNAs encoding vasoactive intestinal peptide, galanin, and pituitary adenylate cyclase activating peptides were identical in wildtype and p75NTR-/- cardiac sympathetic neurons, as was expression of the TrkA neurotrophin receptor. Norepinephrine content was elevated in the base of the p75NTR-/- ventricle compared to wildtype, but levels were identical below the site of occlusion. Left ventricular pressure, dP/dt(MAX), and dP/dt(MIN) were measured under isoflurane anesthesia 3 and 7 days after surgery. Ventricular pressure decreased significantly 3 days after infarction, and deficits in dP/dt(MAX) were revealed by stimulating beta receptors with dobutamine and release of endogenous norepinephrine with tyramine. dP/dt(MIN) was not altered by genotype or surgical group. Few differences were observed between genotypes 3 days after surgery, in contrast to low pressure and dP/dt(MAX) previously reported in control p75NTR-/- animals. Seven days after surgery ventricular pressure and dP/dt(MAX) were significantly lower in p75NTR-/- hearts compared to WT hearts. Thus, the lack of p75NTR did not enhance cardiac function after myocardial infarction.

BMP7‐Induced Dendritic Growth in Sympathetic Neurons Requires p75NTR Signaling

Dendritic morphology is a critical determinant of neuronal connectivity, and in postganglionic sympathetic neurons, tonic activity correlates directly with the size of the dendritic arbor. Thus, identifying signaling mechanisms that regulate dendritic arborization of sympathetic neurons is important to understanding how functional neural circuitry is established and maintained in the sympathetic nervous system. Bone morphogenetic proteins (BMPs) promote dendritic growth in sympathetic neurons; however, downstream signaling events that link BMP receptor activation to dendritic growth are poorly characterized. We previously reported that BMP7 upregulates p75NTR mRNA in cultured sympathetic neurons. This receptor is implicated in controlling dendritic growth in central neurons but whether p75NTR regulates dendritic growth in peripheral neurons is not known. Here, we demonstrate that BMP7 increases p75NTR protein in cultured sympathetic neurons, and this effect is blocked by pharmacologic inhibition of signaling via BMP type I receptor. BMP7 does not trigger dendritic growth in sympathetic neurons dissociated from superior cervical ganglia (SCG) of p75NTR nullizygous mice, and overexpression of p75NTR in p75NTR−/− neurons is sufficient to cause dendritic growth even in the absence of BMP7. Morphometric analyses of SCG from wild‐type versus p75NTR nullizygous mice at 3, 6, and 12 to 16 weeks of age indicated that genetic deletion of p75NTR does not prevent dendritic growth but does stunt dendritic maturation in sympathetic neurons. These data support the hypotheses that p75NTR is involved in downstream signaling events that mediate BMP7‐induced dendritic growth in sympathetic neurons, and suggest that p75NTR signaling positively modulates dendritic complexity in sympathetic neurons in vivo.

Priming with BTCP, a dopamine reuptake blocker, reinstates cocaine-seeking and enhances cocaine cue-induced reinstatement

N-[1-(2-benzo[b]thiophenyl)cyclohexyl]piperidine (BTCP), a potent dopamine reuptake inhibitor, substitutes for the reinforcing effects of cocaine and meets other criteria for possible agonist pharmacotherapeutic potential. The purpose of this study was to determine (1) whether BTCP modifies reinstatement of cocaine-seeking elicited by cocaine-related environmental stimuli and (2) whether this compound produces priming effects. Male Wistar rats were trained to associate discriminative stimuli (S(D)) with cocaine availability (0.25 mg/infusion) versus non-reward and then were subjected to repeated extinction sessions during which the reinforcer and S(D) were withheld. Subsequent presentation of the cocaine S(D) produced recovery of cocaine-seeking. BTCP (2.5-30 mg/kg; i.p.) did not attenuate the conditioned reinstatement induced by the cocaine S(D) but, rather, potentiated this effect at 10 mg/kg. To test whether BTCP, by itself, exerts priming effects, different groups of rats were trained to self-administer cocaine (0.25 mg/infusion) for 2 weeks. After a 2-week extinction period, BTCP (5, 10 and 20 mg/kg, i.p.) reinstated cocaine-seeking, showing that BTCP not only increases cocaine-seeking induced by cocaine-related stimuli but also produces priming effects following abstinence. The results suggest that, in cocaine abstinent rats, BTCP produces cocaine-like effects.

Factor XI contributes to myocardial ischemia-reperfusion injury in mice

Key Points Inhibiting contact activation of factor XI during reperfusion of acute myocardial ischemia reduces infarct size in mice. Factor XII/XI contact axis inhibition may improve the outcome of coronary artery recanalization in acute myocardial infarction.