Ryan T. Gardner

Targeting protein tyrosine phosphatase σ after myocardial infarction restores cardiac sympathetic innervation and prevents arrhythmias

Millions of people suffer a myocardial infarction (MI) every year, and those who survive have increased risk of arrhythmias and sudden cardiac death. Recent clinical studies have identified sympathetic denervation as a predictor of increased arrhythmia susceptibility. Chondroitin sulfate proteoglycans present in the cardiac scar after MI prevent sympathetic reinnervation by binding the neuronal protein tyrosine phosphatase receptor σ (PTPσ). Here we show that the absence of PTPσ, or pharmacologic modulation of PTPσ by the novel intracellular sigma peptide (ISP) beginning 3 days after injury, restores sympathetic innervation to the scar and markedly reduces arrhythmia susceptibility. Using optical mapping we observe increased dispersion of action potential duration, supersensitivity to β-adrenergic receptor stimulation and Ca2+ mishandling following MI. Sympathetic reinnervation prevents these changes and renders hearts remarkably resistant to induced arrhythmias.

Infarct-derived chondroitin sulfate proteoglycans prevent sympathetic reinnervation after cardiac ischemia-reperfusion injury

Sympathetic nerves can regenerate after injury to reinnervate target tissues. Sympathetic regeneration is well documented after chronic cardiac ischemia, so we were surprised that the cardiac infarct remained denervated following ischemia-reperfusion (I-R). We used mice to ask if the lack of sympathetic regeneration into the scar was due to blockade by inhibitory extracellular matrix within the infarct. We found that chondroitin sulfate proteoglycans (CSPGs) were present in the infarct after I-R, but not after chronic ischemia, and that CSPGs caused inhibition of sympathetic axon outgrowth in vitro. Ventricle explants after I-R and chronic ischemia stimulated sympathetic axon outgrowth that was blocked by nerve growth factor antibodies. However, growth in I-R cocultures was asymmetrical, with axons growing toward the heart tissue consistently shorter than axons growing in other directions. Growth toward I-R explants was rescued by adding chondroitinase ABC to the cocultures, suggesting that I-R infarct-derived CSPGs prevented axon extension. Sympathetic ganglia lacking protein tyrosine phosphatase sigma (PTPRS) were not inhibited by CSPGs or I-R explants in vitro, suggesting PTPRS is the major CSPG receptor in sympathetic neurons. To test directly if infarct-derived CSPGs prevented cardiac reinnervation, we performed I-R in ptprs−/− and ptprs+/− mice. Cardiac infarcts in ptprs−/− mice were hyperinnervated, while infarcts in ptprs+/− littermates were denervated, confirming that CSPGs prevent sympathetic reinnervation of the cardiac scar after I-R. This is the first example of CSPGs preventing sympathetic reinnervation of an autonomic target following injury, and may have important consequences for cardiac function and arrhythmia susceptibility after myocardial infarction.

Myocardial infarction causes transient cholinergic transdifferentiation of cardiac sympathetic nerves via gp130

Sympathetic and parasympathetic control of the heart is a classic example of norepinephrine (NE) and acetylcholine (ACh) triggering opposing actions. Sympathetic NE increases heart rate and contractility through activation of β receptors, whereas parasympathetic ACh slows the heart through muscarinic receptors. Sympathetic neurons can undergo a developmental transition from production of NE to ACh and we provide evidence that mouse cardiac sympathetic nerves transiently produce ACh after myocardial infarction (MI). ACh levels increased in viable heart tissue 10–14 d after MI, returning to control levels at 21 d, whereas NE levels were stable. At the same time, the genes required for ACh synthesis increased in stellate ganglia, which contain most of the sympathetic neurons projecting to the heart. Immunohistochemistry 14 d after MI revealed choline acetyltransferase (ChAT) in stellate sympathetic neurons and vesicular ACh transporter immunoreactivity in tyrosine hydroxylase-positive cardiac sympathetic fibers. Finally, selective deletion of the ChAT gene from adult sympathetic neurons prevented the infarction-induced increase in cardiac ACh. Deletion of the gp130 cytokine receptor from sympathetic neurons prevented the induction of cholinergic genes after MI, suggesting that inflammatory cytokines induce the transient acquisition of a cholinergic phenotype in cardiac sympathetic neurons. Ex vivo experiments examining the effect of NE and ACh on rabbit cardiac action potential duration revealed that ACh blunted both the NE-stimulated decrease in cardiac action potential duration and increase in myocyte calcium transients. This raises the possibility that sympathetic co-release of ACh and NE may impair adaptation to high heart rates and increase arrhythmia susceptibility. SIGNIFICANCE STATEMENT Sympathetic neurons normally make norepinephrine (NE), which increases heart rate and the contractility of cardiac myocytes. We found that, after myocardial infarction, the sympathetic neurons innervating the heart begin to make acetylcholine (ACh), which slows heart rate and decreases contractility. Several lines of evidence confirmed that the source of ACh was sympathetic nerves rather than parasympathetic nerves that are the normal source of ACh in the heart. Global application of NE with or without ACh to ex vivo hearts showed that ACh partially reversed the NE-stimulated decrease in cardiac action potential duration and increase in myocyte calcium transients. That suggests that sympathetic co-release of ACh and NE may impair adaptation to high heart rates and increase arrhythmia susceptibility.

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.

Molecular Mechanisms of Sympathetic Remodeling and Arrhythmias

A variety of pathological conditions can increase risk for development of ventricular arrhythmias in humans including diabetes mellitus, obesity, myocardial infarction (MI), and heart failure. Many of these diseases involve global disruption of the autonomic nervous system, including increased sympathetic drive and parasympathetic withdrawal, but another common factor among these disorders is sympathetic dysfunction within the heart. Treatments that target cardiac sympathetic transmission, including β-blockers and ganglionectomy, prolong life and decrease arrhythmias.1–5 Sympathetic control of the heart under normal conditions occurs primarily via norepinephrine acting on β adrenergic receptors (β-AR) to stimulate increases in heart rate (chronotropy), conduction velocity (dromotropy), and contractility (inotropy). These positive effects of sympathetic stimulation allow myocytes to meet increased cardiac demands during stress or exercise, serving to maintain homeostasis. The nervous system adapts to changing conditions, however, and sympathetic neurons undergo structural and functional alterations in response to injury and disease. There are at least 4 types of sympathetic remodeling that occur during conditions of increased arrhythmia susceptibility: hyperinnervation (increased nerve density), denervation (decreased nerve density), altered neurotransmitter or neuropeptide production and increased neuronal excitability. Rubart and Zipes6 proposed a model to explain how these diverse changes in sympathetic transmission might contribute to arrhythmia generation, suggesting that inappropriate heterogeneity of norepinephrine release within the heart leads to differential electric remodeling of cardiac myocytes and predisposes the heart to electric instability. Many studies now support the hypothesis that heterogeneity of noradrenergic transmission increases the risk of arrhythmia and have identified some of the mechanisms that underlie neuronal remodeling. This review will examine the mechanisms of sympathetic remodeling and will connect neural changes to increased arrhythmia susceptibility. We will focus on ventricular arrhythmias because atrial arrhythmias were reviewed recently.7 Regional hyperinnervation was the first type of neural remodeling linked to arrhythmia generation …

Systemic Inhibition of CREB is Well-tolerated in vivo

cAMP-response element binding protein (CREB) is a nuclear transcription factor activated by multiple extracellular signals including growth factors and hormones. These extracellular cues activate CREB through phosphorylation at Ser133 by various protein serine/threonine kinases. Once phosphorylated, it promotes its association with transcription coactivators CREB-binding protein (CBP) and its paralog p300 to activate CREB-dependent gene transcription. Tumor tissues of different origins have been shown to present overexpression and/or overactivation of CREB, indicating CREB as a potential cancer drug target. We previously identified 666-15 as a potent inhibitor of CREB with efficacious anti-cancer activity both in vitro and in vivo. Herein, we investigated the specificity of 666-15 and evaluated its potential in vivo toxicity. We found that 666-15 was fairly selective in inhibiting CREB. 666-15 was also found to be readily bioavailable to achieve pharmacologically relevant concentrations for CREB inhibition. Furthermore, the mice treated with 666-15 showed no evidence of changes in body weight, complete blood count, blood chemistry profile, cardiac contractility and tissue histologies from liver, kidney and heart. For the first time, these results demonstrate that pharmacological inhibition of CREB is well-tolerated in vivo and indicate that such inhibitors should be promising cancer therapeutics.