Diana C. Parrish

Cardiac ischemia-reperfusion regulates sympathetic neuropeptide expression through gp130-dependent and independent mechanisms

Cardiac function is regulated by a balance of sympathetic and parasympathetic transmission. Neuropeptide Y (NPY) and galanin (GAL) released from cardiac sympathetic neurons inhibits parasympathetic transmission in the heart. Sympathetic peptides may contribute to autonomic imbalance, which is characterized by increased sympathetic and decreased parasympathetic transmission and contributes to life threatening cardiovascular pathologies. Several gp130 cytokines are increased in the heart after myocardial infarction (MI), and these cytokines stimulate neuropeptide expression in sympathetic neurons. We used mice whose sympathetic neurons lack the gp130 receptor (gp130(DBH-Cre/lox) mice) to ask if cytokine activation of gp130 regulated neuropeptide expression in cardiac sympathetic nerves after MI. Myocardial infarction decreased NPY mRNA through a gp130 independent mechanism and increased VIP and PACAP mRNA via gp130, while GAL mRNA was unchanged. Immunohistochemistry revealed a gp130-dependent increase in PACAP38 in cells of the stellate ganglion after MI, and PACAP was detected in pre-ganglionic fibers of all genotypes and surgical groups. VIP was identified in a few sympathetic nerve fibers in all genotypes and surgical groups. GAL and PACAP38 were not detected in sham hearts, but peptide immunoreactivity was high in the infarct three days after MI. Surprisingly, peptides were abundant in cells that co-labeled with macrophage markers F4/80 and MAC2, but were not detected in sympathetic axons. PACAP protects cardiac myocytes from apoptosis, and GAL stimulates axon regeneration in addition to inhibiting parasympathetic transmission. Thus, these peptides may play an important role in cardiac and neuronal remodeling after ischemia-reperfusion.

Infarction‐induced cytokines cause local depletion of tyrosine hydroxylase in cardiac sympathetic nerves

Myocardial infarction causes a heterogeneity of noradrenergic transmission that contributes to the development of ventricular arrhythmias and sudden cardiac death. Ischaemia‐induced alterations in sympathetic transmission include regional variations in cardiac noradrenaline (NA) and in tyrosine hydroxylase, the rate‐limiting enzyme in NA synthesis. Inflammatory cytokines that act through gp130 are elevated in the heart after myocardial infarction. These cytokines decrease expression of tyrosine hydroxylase in sympathetic neurons, and indirect evidence suggests that they contribute to the local depletion of tyrosine hydroxylase in the damaged left ventricle. However, gp130 cytokines are also important for the survival of cardiac myocytes following damage to the heart. To examine the effect of cytokines on tyrosine hydroxylase and NA content in cardiac nerves we used gp130DBH‐Cre/lox mice, which have a deletion of the gp130 receptor in neurons expressing dopamine β‐hydroxylase. The absence of neuronal gp130 prevented the loss of tyrosine hydroxylase in cardiac sympathetic nerves innervating the left ventricle 1 week after ischaemia–reperfusion compared with wild‐type C57BL/6J mice. Surprisingly, restoration of tyrosine hydroxylase in the damaged ventricle did not return neuronal NA content to normal levels. Noradrenaline uptake into cardiac nerves was significantly lower in gp130 knockout mice, contributing to the lack of neuronal NA stores. There were no significant differences in left ventricular peak systolic pressure, dP/dtmax or dP/dtmin between the two genotypes after myocardial infarction, but ganglionic blockade revealed differences in autonomic tone between the genotypes. Stimulation of the heart with dobutamine or release of endogenous NA with tyramine generated similar responses in both genotypes. Thus, the removal of gp130 from sympathetic neurons prevents the post‐infarct depletion of tyrosine hydroxylase in the left ventricle, but does not alter NA content or cardiac function.

Cytokines inhibit norepinephrine transporter expression by decreasing Hand2

Functional noradrenergic transmission requires the coordinate expression of enzymes involved in norepinephrine (NE) synthesis, as well as the norepinephrine transporter (NET) which removes NE from the synapse. Inflammatory cytokines acting through gp130 can suppress the noradrenergic phenotype in sympathetic neurons. This occurs in a subset of sympathetic neurons during development and also occurs in adult neurons after injury. For example, cytokines suppress noradrenergic function in sympathetic neurons after axotomy and during heart failure. The molecular basis for suppression of noradrenergic genes is not well understood, but previous studies implicated a reduction of Phox2a in cytokine suppression of dopamine beta hydroxylase. We used sympathetic neurons and neuroblastoma cells to investigate the role of Phox2a in cytokine suppression of NET transcription. Chromatin immunoprecipitation experiments revealed that Phox2a did not bind the NET promoter, and overexpression of Phox2a did not prevent cytokine suppression of NET transcription. Hand2 and Gata3 are transcription factors that induce noradrenergic genes during development and are present in mature sympathetic neurons. Both Hand2 and Gata3 were decreased by cytokines in sympathetic neurons and neuroblastoma cells. Overexpression of either Hand2 or Gata3 was sufficient to rescue NET transcription following suppression by cytokines. We examined expression of these genes following axotomy to determine if their expression was altered following nerve injury. NET and Hand2 mRNAs decreased significantly in sympathetic neurons 48 h after axotomy, but Gata3 mRNA was unchanged. These data suggest that cytokines can inhibit NET expression through downregulation of Hand2 or Gata3 in cultured sympathetic neurons, but axotomy in adult animals selectively suppresses Hand2 expression.

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).

Absence of gp130 in dopamine β-hydroxylase-expressing neurons leads to autonomic imbalance and increased reperfusion arrhythmias

Inflammatory cytokines that act through glycoprotein (gp)130 are elevated in the heart after myocardial infarction and in heart failure. These cytokines are potent regulators of neurotransmitter and neuropeptide production in sympathetic neurons but are also important for the survival of cardiac myocytes after damage to the heart. To examine the effect of gp130 cytokines on cardiac nerves, we used gp130(DBH-Cre/lox) mice, which have a selective deletion of the gp130 cytokine receptor in neurons expressing dopamine beta-hydroxylase (DBH). Basal sympathetic parameters, including norepinephrine (NE) content, tyrosine hydroxylase expression, NE transporter expression, and sympathetic innervation density, appeared normal in gp130(DBH-Cre/lox) compared with wild-type mice. Likewise, basal cardiovascular parameters measured under isoflurane anesthesia were similar in both genotypes, including mean arterial pressure, left ventricular peak systolic pressure, dP/dt(max), and dP/dt(min). However, pharmacological interventions revealed an autonomic imbalance in gp130(DBH-Cre/lox) mice that was correlated with an increased incidence of premature ventricular complexes after reperfusion. Stimulation of NE release with tyramine and infusion of the beta-agonist dobutamine revealed blunted adrenergic transmission that correlated with decreased beta-receptor expression in gp130(DBH-Cre/lox) hearts. Due to the developmental expression of the DBH-Cre transgene in parasympathetic ganglia, gp130 was eliminated. Cholinergic transmission was impaired in gp130(DBH-Cre/lox) hearts due to decreased parasympathetic drive, but tyrosine hydroxylase immunohistochemistry in the brain stem revealed that catecholaminergic nuclei appeared grossly normal. Thus, the apparently normal basal parameters in gp130(DBH-Cre/lox) mice mask an autonomic imbalance that includes alterations in sympathetic and parasympathetic transmission.

Transient denervation of viable myocardium after myocardial infarction does not alter arrhythmia susceptibility

Cardiac sympathetic nerves stimulate heart rate and force of contraction. Myocardial infarction (MI) leads to the loss of sympathetic nerves within the heart, and clinical studies have indicated that sympathetic denervation is a risk factor for arrhythmias and cardiac arrest. Two distinct types of denervation have been identified in the mouse heart after MI caused by ischemia-reperfusion: transient denervation of peri-infarct myocardium and sustained denervation of the infarct. Sustained denervation is linked to increased arrhythmia risk, but it is not known whether acute nerve loss in peri-infarct myocardium also contributes to arrhythmia risk. Peri-infarct sympathetic denervation requires the p75 neurotrophin receptor (p75NTR), but removal of p75NTR alters the pattern of sympathetic innervation in the heart and increases spontaneous arrhythmias. Therefore, we targeted the p75NTR coreceptor sortilin and the p75NTR-induced protease tumor necrosis factor-α-converting enzyme/A disintegrin and metalloproteinase domain 17 (TACE/ADAM17) to selectively block peri-infarct denervation. Sympathetic nerve density was quantified using immunohistochemistry for tyrosine hydroxylase. Genetic deletion of sortilin had no effect on the timing or extent of axon degeneration, but inhibition of TACE/ADAM17 with the protease inhibitor marimastat prevented the loss of axons from viable myocardium. We then asked whether retention of nerves in peri-infarct myocardium had an impact on cardiac electrophysiology 3 days after MI using ex vivo optical mapping of transmembrane potential and intracellular Ca2+. Preventing acute denervation of viable myocardium after MI did not significantly alter cardiac electrophysiology or Ca2+ handling, suggesting that transient denervation at this early time point has minimal impact on arrhythmia risk. NEW & NOTEWORTHY Sympathetic denervation after myocardial infarction is a risk factor for arrhythmias. We asked whether transient loss of nerves in viable myocardium contributed to arrhythmia risk. We found that targeting protease activity could prevent acute peri-infarct denervation but that it did not significantly alter cardiac electrophysiology or Ca2+ handling 3 days after myocardial infarction.

Infarction-induced cytokines cause local depletion of tyrosine hydroxylase in cardiac sympathetic nerves: Cytokine suppression of tyrosine hydroxylase

Myocardial infarction causes a heterogeneity of noradrenergic transmission that contributes to the development of ventricular arrhythmias and sudden cardiac death. Ischaemia‐induced alterations in sympathetic transmission include regional variations in cardiac noradrenaline (NA) and in tyrosine hydroxylase, the rate‐limiting enzyme in NA synthesis. Inflammatory cytokines that act through gp130 are elevated in the heart after myocardial infarction. These cytokines decrease expression of tyrosine hydroxylase in sympathetic neurons, and indirect evidence suggests that they contribute to the local depletion of tyrosine hydroxylase in the damaged left ventricle. However, gp130 cytokines are also important for the survival of cardiac myocytes following damage to the heart. To examine the effect of cytokines on tyrosine hydroxylase and NA content in cardiac nerves we used gp130DBH‐Cre/lox mice, which have a deletion of the gp130 receptor in neurons expressing dopamine β‐hydroxylase. The absence of neuronal gp130 prevented the loss of tyrosine hydroxylase in cardiac sympathetic nerves innervating the left ventricle 1 week after ischaemia–reperfusion compared with wild‐type C57BL/6J mice. Surprisingly, restoration of tyrosine hydroxylase in the damaged ventricle did not return neuronal NA content to normal levels. Noradrenaline uptake into cardiac nerves was significantly lower in gp130 knockout mice, contributing to the lack of neuronal NA stores. There were no significant differences in left ventricular peak systolic pressure, dP/dtmax or dP/dtmin between the two genotypes after myocardial infarction, but ganglionic blockade revealed differences in autonomic tone between the genotypes. Stimulation of the heart with dobutamine or release of endogenous NA with tyramine generated similar responses in both genotypes. Thus, the removal of gp130 from sympathetic neurons prevents the post‐infarct depletion of tyrosine hydroxylase in the left ventricle, but does not alter NA content or cardiac function.

Infarction-induced cytokines cause local depletion of tyrosine hydroxylase in cardiac sympathetic nerves: Experimental Physiology-Research Paper

Myocardial infarction causes a heterogeneity of noradrenergic transmission that contributes to the development of ventricular arrhythmias and sudden cardiac death. Ischaemia‐induced alterations in sympathetic transmission include regional variations in cardiac noradrenaline (NA) and in tyrosine hydroxylase, the rate‐limiting enzyme in NA synthesis. Inflammatory cytokines that act through gp130 are elevated in the heart after myocardial infarction. These cytokines decrease expression of tyrosine hydroxylase in sympathetic neurons, and indirect evidence suggests that they contribute to the local depletion of tyrosine hydroxylase in the damaged left ventricle. However, gp130 cytokines are also important for the survival of cardiac myocytes following damage to the heart. To examine the effect of cytokines on tyrosine hydroxylase and NA content in cardiac nerves we used gp130DBH‐Cre/lox mice, which have a deletion of the gp130 receptor in neurons expressing dopamine β‐hydroxylase. The absence of neuronal gp130 prevented the loss of tyrosine hydroxylase in cardiac sympathetic nerves innervating the left ventricle 1 week after ischaemia–reperfusion compared with wild‐type C57BL/6J mice. Surprisingly, restoration of tyrosine hydroxylase in the damaged ventricle did not return neuronal NA content to normal levels. Noradrenaline uptake into cardiac nerves was significantly lower in gp130 knockout mice, contributing to the lack of neuronal NA stores. There were no significant differences in left ventricular peak systolic pressure, dP/dtmax or dP/dtmin between the two genotypes after myocardial infarction, but ganglionic blockade revealed differences in autonomic tone between the genotypes. Stimulation of the heart with dobutamine or release of endogenous NA with tyramine generated similar responses in both genotypes. Thus, the removal of gp130 from sympathetic neurons prevents the post‐infarct depletion of tyrosine hydroxylase in the left ventricle, but does not alter NA content or cardiac function.