Post-infarct cardiac sympathetic hyperactivity regulates galanin expression.

The neuropeptide galanin is elevated in the cardiac sympathetic innervation after myocardial infarction (MI). Galanin inhibits vagal transmission and may support the regeneration of sympathetic nerves, thereby contributing to the development of arrhythmia and sudden cardiac death after MI. The reason for increased galanin production in sympathetic neurons after myocardial infarction is not known. Cardiac sympathetic neurons are activated chronically after cardiac ischemia-reperfusion, and activation of sympathetic neurons in culture stimulates galanin expression. Therefore, we tested the hypothesis that increased sympathetic nerve activity stimulates galanin expression in cardiac sympathetic neurons after myocardial infarction. To test this hypothesis we used TGR(ASrAOGEN) transgenic rats, which lack brain angiotensinogen and do not exhibit post-infarct sympathetic hyperactivity. Hearts and stellate ganglia were collected 1 week after ischemia-reperfusion. Galanin mRNA was quantified by real-time PCR and peptide content was assayed by enzyme-linked immunosorbent assay. Galanin mRNA increased approximately 3-fold after MI in cardiac sympathetic neurons of both genotypes compared to unoperated and sham controls. Left ventricular galanin content, however, increased after MI only in Sprague-Dawley rats and not in AOGEN rats. These data suggest that post-infarct cardiac sympathetic hyperactivity stimulates galanin peptide production but is not required for increased galanin mRNA expression.

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.

Postinfarct sympathetic hyperactivity differentially stimulates expression of tyrosine hydroxylase and norepinephrine transporter.

The balance between norepinephrine (NE) synthesis, release, and reuptake is disrupted after acute myocardial infarction, resulting in elevated extracellular NE. Stimulation of sympathetic neurons in vitro increases NE synthesis and the synthetic enzyme tyrosine hydroxylase (TH) to a greater extent than it increases NE reuptake and the NE transporter (NET), which removes NE from the extracellular space. We used TGR(ASrAOGEN) transgenic rats, which lack postinfarct sympathetic hyperactivity, to test the hypothesis that increased cardiac sympathetic nerve activity accounts for the imbalance in TH and NET expression in these neurons after myocardial infarction. TH and NET mRNA levels were identical in the stellate ganglia of unoperated TGR(ASrAOGEN) rats compared with Sprague Dawley (SD) controls, but the threefold increase in TH and twofold increase in NET mRNA seen in the stellate ganglia of SD rats 1 wk after ischemia-reperfusion was absent in TGR(ASrAOGEN) rats. Similarly, the increase in TH and NET protein observed in the base of the SD ventricle was absent in the base of the TGR (ASrAOGEN) ventricle. Neuronal TH content was depleted in the left ventricle of both genotypes, whereas NET was unchanged. Basal heart rate and cardiac function were similar in both genotypes, but TGR(ASrAOGEN) hearts were more sensitive to the beta-agonist dobutamine. Tyramine-induced release of endogenous NE generated similar changes in ventricular pressure and contractility in both genotypes, but postinfarct relaxation was enhanced in TGR(ASrAOGEN) hearts. These data support the hypothesis that postinfarct sympathetic hyperactivity is the major stimulus increasing TH and NET expression in cardiac neurons.

The lack of cardiotrophin-1 alters expression of interleukin-6 and leukemia inhibitory factor mRNA but does not impair cardiac injury response.

Cardiotrophin-1 (CT-1) was identified as a growth factor for cardiac myocytes and CT-1 protects myocytes from cell death. Adult CT-1(-/-) mice exhibit neural deficits including the loss of preganglionic sympathetic neurons, but their autonomic and cardiac parameters have not been examined. We used these mice to determine if the absence of CT-1 or loss of preganglionic sympathetic input altered heart rate, left ventricular pressure, cardiac contractility (dP/dt), or cell death following ischemia-reperfusion. Basal heart rate was increased in CT-1(-/-) mice, and this difference was abolished by ganglionic block. Left ventricular pressure and dP/dt were unchanged. Dobutamine stimulated similar increases in heart rate and dP/dt in both genotypes, but ventricular pressure was significantly lower in CT-1 nulls. Cardiac expression of interleukin-6 (IL-6) mRNA was increased significantly in CT-1 null mice, while leukemia inhibitory factor (LIF) mRNA was unchanged. Infarct size normalized to area at risk was no different in CT-1(-/-) mice (33.8+/-1.0% vs. 37.7+/-3.2% WT) 24h after ischemia-reperfusion. Induction of IL-6 mRNA after infarct was significantly abrogated in CT-1 null mice compared to wild-type mice, but LIF mRNA-induction remained significant in CT-1 null mice and might contribute to cardiac protection in the absence of CT-1.

LEX032, a novel recombinant serpin, protects the brain after transient focal ischemia.

This investigation examined the effectiveness of a serine protease inhibitor (LEX032) when used as a cerebral protective agent after ischemia. Focal cerebral ischemia in the rat was produced by intravascular occlusion of the middle cerebral artery for a period of 30 min. Just prior to thread withdrawal (i.e., reperfusion), rats received an iv bolus administration of either vehicle or LEX032 (50 mg/kg), an optimal dose chosen based on previous studies. Somatosensory evoked potentials (SSEP's) were monitored prior to, during, and for a period of 60 min after removal of occlusion. The animals were allowed to recover for 24 h after the ischemic insult. Cortical activity in the occluded region, as assessed by SSEPs, returned much sooner in the LEX032-treated animals (10 +/- 6 min) than in the untreated animals (40 +/- 25 min). On a scale ranging from 0 to 3, with three indicating the most severely injured, the LEX032 animals had a significantly better neurologic score (1.0 +/- 0.9) than the untreated animals (2.3 +/- 0.5) 24 h after ischemia. The improved neurobehavior was related to a 55% reduction in brain injury as assessed by TTC staining. LEX032-treated animals had significantly (P < 0.01) smaller infarcts (115 +/- 40 mm3) compared to vehicle-treated animals (263 +/- 13 mm3). In a separate group of animals (n = 6/group), leukocyte infiltration, as evaluated by tissue myeloperoxidase activity (MPO U/g tissue wt), was also significantly (P < 0.05) lower in the LEX032-treated animals (1.4 +/- 0.3) compared to vehicle-treated animals (3.6 +/- 0.7). This data demonstrates that LEX032 reduces brain injury and suggests that serine protease inhibitors may reduce ischemia/reperfusion injury by decreasing leukocyte activation and migration.