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.

Loss of chondroitin sulfate proteoglycan sulfation allows delayed sympathetic reinnervation after cardiac ischemia-reperfusion.

Sympathetic nerve loss in the heart predicts the risk of ventricular arrhythmias after myocardial infarction (MI) in patients. Sympathetic denervation after cardiac ischemia-reperfusion is sustained by matrix components chondroitin sulfate proteoglycans (CSPGs) in the cardiac scar. We showed that 4,6-sulfation of CSPGs was critical for preventing nerve growth into the scar. Promoting early reinnervation with therapeutics reduces arrhythmias during the first 2 weeks after MI, but the longer-term consequences of restoring innervation are unknown. Therefore, we asked if the beneficial effects of early reinnervation were sustained. We compared cardiac function and arrhythmia susceptibility 40 days after MI in mice treated on Days 3-10 with vehicle or with intracellular sigma peptide to restore innervation. Surprisingly, both groups had normal innervation density in the cardiac scar 40 days after MI, indicating delayed reinnervation of the infarct in vehicle-treated mice. That coincided with similar cardiac function and arrhythmia susceptibility in the two groups. We investigated the mechanism allowing delayed reinnervation of the cardiac scar. We found that CSPG 4,6-sulfation, which is elevated early after ischemia-reperfusion, was reduced to control levels allowing reinnervation of the infarct. Thus, remodeling of extracellular matrix weeks after injury leads to remodeling of sympathetic neurons in the heart.

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.

Chondroitin sulfate proteoglycan 4,6 sulfation regulates sympathetic nerve regeneration after myocardial infarction.

Sympathetic denervation of the heart following ischemia/reperfusion induced myocardial infarction (MI) is sustained by chondroitin sulfate proteoglycans (CSPGs) in the cardiac scar. Denervation predicts risk of sudden cardiac death in humans. Blocking CSPG signaling restores sympathetic axon outgrowth into the cardiac scar, decreasing arrhythmia susceptibility. Axon growth inhibition by CSPGs can depend on the sulfation status of the glycosaminoglycan (CS-GAG) side chains. Tandem sulfation of CS-GAGs at the 4th (4S) and 6th (6S) positions of n-acetyl-galactosamine inhibits outgrowth in several types of central neurons, but we don't know if sulfation is similarly critical during peripheral nerve regeneration. We asked if CSPG sulfation prevented sympathetic axon outgrowth after MI. Reducing 4S with the 4-sulfatase enzyme Arylsulfatase-B (ARSB) enhanced outgrowth of dissociated rat sympathetic neurons over CSPGs. Likewise, reducing 4S with ARSB restored axon outgrowth from mouse sympathetic ganglia co-cultured with cardiac scar tissue. We quantified enzymes responsible for adding and removing sulfation, and found that CHST15 (4S dependent 6-sulfotransferase) was upregulated, and ARSB was downregulated after MI. This suggests a mechanism for production and maintenance of sulfated CSPGs in the cardiac scar. We decreased 4S,6S CS-GAGs in vivo by transient siRNA knockdown of Chst15 after MI, and found that reducing 4S,6S restored tyrosine hydroxylase (TH) positive sympathetic nerve fibers in the cardiac scar. Reinnervation reduced isoproterenol induced arrhythmias. Our results suggest that modulating CSPG-sulfation after MI may be a therapeutic target to promote sympathetic nerve regeneration in the cardiac scar and reduce post-MI cardiac arrhythmias.

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 gp130(DBH-Cre/lox) mice, which have a deletion of the gp130 receptor in neurons expressing dopamine beta-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/dt(max) or dP/dt(min) 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.

Absence of gp130 in dopamine beta-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.

Measurement science in the circulatory system.

The dynamics of the cellular and molecular constituents of the circulatory system are regulated by the biophysical properties of the heart, vasculature and blood cells and proteins. In this review, we discuss measurement techniques that have been developed to characterize the physical and mechanical parameters of the circulatory system across length scales ranging from the tissue scale (centimeter) to the molecular scale (nanometer) and time scales of years to milliseconds. We compare the utility of measurement techniques as a function of spatial resolution and penetration depth from both a diagnostic and research perspective. Together, this review provides an overview of the utility of measurement science techniques to study the spatial systems of the circulatory system in health and disease.

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).

ProNGF, a cytokine induced after myocardial infarction in humans, targets pericytes to promote microvascular damage and activation.

Treatment of acute cardiac ischemia focuses on reestablishment of blood flow in coronary arteries. However, impaired microvascular perfusion damages peri-infarct tissue, despite arterial patency. Identification of cytokines that induce microvascular dysfunction would provide new targets to limit microvascular damage. Pro-nerve growth factor (NGF), the precursor of NGF, is a well characterized cytokine in the brain induced by injury. ProNGF activates p75 neurotrophin receptor (p75(NTR)) and sortilin receptors to mediate proapoptotic responses. We describe induction of proNGF by cardiomyocytes, and p75(NTR) in human arterioles after fatal myocardial infarction, but not with unrelated pathologies. After mouse cardiac ischemia-reperfusion (I-R) injury, rapid up-regulation of proNGF by cardiomyocytes and p75(NTR) by microvascular pericytes is observed. To identify proNGF actions, we generated a mouse expressing a mutant Ngf allele with impaired processing of proNGF to mature NGF. The proNGF-expressing mouse exhibits cardiac microvascular endothelial activation, a decrease in pericyte process length, and increased vascular permeability, leading to lethal cardiomyopathy in adulthood. Deletion of p75(NTR) in proNGF-expressing mice rescues the phenotype, confirming the importance of p75(NTR)-expressing pericytes in the development of microvascular injury. Furthermore, deficiency in p75(NTR) limits infarct size after I-R. These studies identify novel, nonneuronal actions for proNGF and suggest that proNGF represents a new target to limit microvascular dysfunction.

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.

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.