Nitroxyl (HNO) for Treatment of Acute Heart Failure

The loss of contractile function is a hallmark of heart failure. Although increasing intracellular Ca²⁺ is a possible strategy for improving contraction, current inotropic agents that achieve this by raising intracellular cAMP levels, such as β-agonists and phosphodiesterase inhibitors, are generally deleterious when administered as long-term therapy due to arrhythmia and myocardial damage. Nitroxyl donors have been shown to improve cardiac function in normal and failing dogs, and in isolated cardiomyocytes they increase fractional shortening and Ca²⁺ transients, independently from cAMP/PKA or cGMP/PKG signaling. Instead, nitroxyl targets cysteines in the EC-coupling machinery and myofilament proteins, reversibly modifying them to enhance Ca²⁺ handling and myofilament Ca²⁺ sensitivity. Phase I–IIa trials with CXL-1020, a novel pure HNO donor, reported declines in left and right heart filling pressures and systemic vascular resistance, and increased cardiac output and stroke volume index. These findings support the concept of nitroxyl donors as attractive agents for the treatment of acute decompensated heart failure.     

p53-Induced inflammation exacerbates cardiac dysfunction during pressure overload

The rates of death and disability caused by severe heart failure are still unacceptably high. There is evidence that the sterile inflammatory response has a critical role in the progression of cardiac remodeling in the failing heart. The p53 signaling pathway has been implicated in heart failure, but the pathological link between p53 and inflammation in the failing heart is largely unknown. Here we demonstrate a critical role of p53-induced inflammation in heart failure. Expression of p53 was increased in cardiac endothelial cells and bone marrow cells in response to pressure overload, leading to up-regulation of intercellular adhesion molecule-1 (ICAM1) expression by endothelial cells and integrin expression by bone marrow cells. Deletion of p53 from endothelial cells or bone marrow cells significantly reduced ICAM1 or integrin expression, respectively, as well as decreasing cardiac inflammation and ameliorating systolic dysfunction during pressure overload. Conversely, overexpression of p53 in bone marrow cells led to an increase of integrin expression and cardiac inflammation that reduced systolic function. Norepinephrine markedly increased p53 expression in endothelial cells and macrophages. Reducing β₂-adrenergic receptor expression in endothelial cells or bone marrow cells attenuated cardiac inflammation and improved systolic dysfunction during pressure overload. These results suggest that activation of the sympathetic nervous system promotes cardiac inflammation by up-regulating ICAM1 and integrin expression via p53 signaling to exacerbate cardiac dysfunction. Inhibition of p53-induced inflammation may be a novel therapeutic strategy for heart failure.     

Abnormalities of calcium metabolism and myocardial contractility depression in the failing heart

Heart failure (HF) is characterized by molecular and cellular defects which jointly contribute to decreased cardiac pump function. During the development of the initial cardiac damage which leads to HF, adaptive responses activate physiological countermeasures to overcome depressed cardiac function and to maintain blood supply to vital organs in demand of nutrients. However, during the chronic course of most HF syndromes, these compensatory mechanisms are sustained beyond months and contribute to progressive maladaptive remodeling of the heart which is associated with a worse outcome. Of pathophysiological significance are mechanisms which directly control cardiac contractile function including ion- and receptor-mediated intracellular signaling pathways. Importantly, signaling cascades of stress adaptation such as intracellular calcium (Ca²⁺) and 3′-5′-cyclic adenosine monophosphate (cAMP) become dysregulated in HF directly contributing to adverse cardiac remodeling and depression of systolic and diastolic function. Here, we provide an update about Ca²⁺ and cAMP dependent signaling changes in HF, how these changes affect cardiac function, and novel therapeutic strategies which directly address the signaling defects.     

Human cholangiocarcinoma development is associated with dysregulation of opioidergic modulation of cholangiocyte growth

Background/AimsIncidence of cholangiocarcinoma is increasing worldwide, yet remaining highly aggressive and with poor prognosis. The mechanisms that drive cholangiocyte transition towards malignant phenotype are obscure. Cholangiocyte benign proliferation is subjected to a self-limiting mechanism based on the autocrine release of endogenous opioid peptides. Despite the presence of both, ligands interact with δ opioid receptor (OR), but not with μOR, with the consequent inhibition of cell growth. We aimed to verify whether cholangiocarcinoma growth is associated with failure of opioidergic regulation of growth control.MethodsWe evaluated the effects of OR selective agonists on cholangiocarcinoma cell proliferation, migration and apoptosis. Intracellular signals were also characterised.ResultsActivation of μOR, but not δOR, increases cholangiocarcinoma cell growth. Such an effect is mediated by ERK1/2, PI3K and Ca²⁺–CamKIIα cascades, but not by cAMP/PKA and PKCα. μOR activation also enhances cholangiocarcinoma cell migration and reduces death by apoptosis. The anti-apoptotic effect of μOR was PI3K dependent.ConclusionsOur data indicate that cholangiocarcinoma growth is associated with altered opioidergic regulation of cholangiocyte biology, thus opening new scenarios for future surveillance or early diagnostic strategies for cholangiocarcinoma.     

Cellular localization and adaptive changes of the cardiac delta opioid receptor system in an experimental model of heart failure in rats

The role of the cardiac opioid system in congestive heart failure (CHF) is not fully understood. Therefore, this project investigated the cellular localization of delta opioid receptors (DOR) in left ventricle (LV) myocardium and adaptive changes in DOR and its endogenous ligand, the precursor peptide proenkephalin (PENK), during CHF. Following IRB approval, DOR localization was determined by radioligand binding using [H³]Naltrindole and by double immunofluorescence confocal analysis in the LV of male Wistar rats. Additionally, 28 days following an infrarenal aortocaval fistula (ACF) the extent of CHF and adaptions in left ventricular DOR and PENK expression were examined by hemodynamic measurements, RT-PCR, and Western blot. DOR specific membrane binding sites were identified in LV myocardium. DOR were colocalized with L-type Ca²⁺-channels (Ca_v1.2) as well as with intracellular ryanodine receptors (RyR) of the sarcoplasmatic reticulum. Following ACF severe congestive heart failure developed in all rats and was accompanied by up-regulation of DOR and PENK on mRNA as well as receptor proteins representing consecutive adaptations. These findings might suggest that the cardiac delta opioid system possesses the ability to play a regulatory role in the cardiomyocyte calcium homeostasis, especially in response to heart failure.     

Ethanol alters opioid regulation of Ca(2+) influx through L-type Ca(2+) channels in PC12 cells

Background: Studies at the behavioral and synaptic level show that effects of ethanol on the central nervous system can involve the opioid signaling system. These interactions may alter the function of a common downstream target. In this study, we examined Ca²⁺ channel function as a potential downstream target of interactions between ethanol and μ or κ opioid receptor signaling. Methods: The studies were carried out in a model system, undifferentiated PC12 cells transfected with μ or κ opioid receptors. The PC12 cells express L‐type Ca²⁺ channels, which were activated by K⁺ depolarization. Ca²⁺ imaging was used to measure relative Ca²⁺ flux during K⁺ depolarization and the modulation of Ca²⁺ flux by opioids and ethanol. Results: Ethanol, μ receptor activation, and κ receptor activation all reduced the amplitude of the Ca²⁺ signal produced by K⁺ depolarization. Pretreatment with ethanol or combined treatment with ethanol and μ or κ receptor agonists caused a reduction in the amplitude of the Ca²⁺ signal that was comparable to or smaller than that observed for the individual drugs alone, indicating an interaction by the drugs at a downstream target (or targets) that limited the modulation of Ca²⁺ flux through L‐type Ca²⁺ channels. Conclusions: These studies provide evidence for a cellular mechanism that could play an important role in ethanol regulation of synaptic transmission and behavior through interactions with the opioid signaling.     

Myocardial Phosphodiesterases and Regulation of Cardiac Contractility in Health and Cardiac Disease

Phosphodiesterase (PDE) inhibitors are potent cardiotonic agents used for parenteral inotropic support in heart failure. Contractile effects of these agents are mediated through cAMP-protein kinase A-induced stimulation of I _Ca2+ which ultimately results in increased Ca²⁺-induced sarcoplasmic reticulum Ca²⁺ release. A number of additional effects such as increases in sarcoplasmic reticulum Ca²⁺ stores, stimulation of reverse mode Na⁺–Ca²⁺ exchange, direct or cAMP-mediated effects on sarcoplasmic reticulum ryanodine receptor, stimulation of the voltage-sensitive sarcoplasmic reticulum Ca²⁺ release mechanism, as well as A₁ adenosine receptor blockade could contribute to positive inotropic responses to PDE inhibitors. Moreover, some PDE inhibitors exhibit Ca²⁺ sensitizer properties as they could increase the affinity of troponin C Ca²⁺-binding sites as well as reduce Ca²⁺ threshold for thin myofilament sliding and facilitate cross-bridge cycling. Inotropic responses to PDE inhibitors are significantly reduced in cardiac disease, an effect largely attributed to downregulation of cAMP-mediated signalling due to sustained sympathetic activation. Four PDE isoenzymes (PDE1, PDE2, PDE3 and PDE4) are present in myocardial tissue of various mammalian species, of which PDE3 and PDE4 are particularly involved in regulation of cardiac myocyte contraction. PDE cAMP-hydrolysing activity is preserved in compensated cardiac hypertrophy but significantly reduced in animal models of heart failure. However, clinical studies have not revealed any changes in distribution profile as well as kinetic and regulatory properties of myocardial PDEs in failing human hearts. A reduction of PDE inhibitors-induced contractile responses in heart failure has therefore been ascribed to reduced cAMP synthesis due to uncoupling of adenylyl cyclase from β-adrenoreceptor. In cardiac myocytes, PDEs are targeted to distinct subcellular compartments by scaffolding proteins such as myomegalin, mAKAP and β-arrestins. Over subcellular microdomains, cAMP hydrolysis by PDE3 and PDE4 allows to control the activity of local pools of protein kinase A and therefore the extent of protein kinase A-mediated phosphorylation of cellular proteins.     

Preservation of myocardial beta-adrenergic receptor signaling delays the development of heart failure after myocardial infarction

When the heart fails, there is often a constellation of biochemical alterations of the beta-adrenergic receptor (betaAR) signaling system, leading to the loss of cardiac inotropic reserve. betaAR down-regulation and functional uncoupling are mediated through enhanced activity of the betaAR kinase (betaARK1), the expression of which is increased in ischemic and failing myocardium. These changes are widely viewed as representing an adaptive mechanism, which protects the heart against chronic activation. In this study, we demonstrate, using in vivo intracoronary adenoviral-mediated gene delivery of a peptide inhibitor of betaARK1 (betaARKct), that the desensitization and down-regulation of betaARs seen in the failing heart may actually be maladaptive. In a rabbit model of heart failure induced by myocardial infarction, which recapitulates the biochemical betaAR abnormalities seen in human heart failure, delivery of the betaARKct transgene at the time of myocardial infarction prevents the rise in betaARK1 activity and expression and thereby maintains betaAR density and signaling at normal levels. Rather than leading to deleterious effects, cardiac function is improved, and the development of heart failure is delayed. These results appear to challenge the notion that dampening of betaAR signaling in the failing heart is protective, and they may lead to novel therapeutic strategies to treat heart disease via inhibition of betaARK1 and preservation of myocardial betaAR function.     

The ATPase activity of cardiac myosin from failing and hypertropied hearts.

We have measured the EDTA and Ca²⁺ activated ATPase activity of myosin from hearts of normal dogs, dogs in gross heart failure and dogs with cardiac hypertrophy without failure. Heart failure was caused by systolic and/or diastolic hemodynamic loads. Under one or more assay conditions, depressed ATPase activity was consistently found for myosin from failing hearts. In contrast to myosin from hearts with diastolic loads, myosin from failing hearts with pulmonic stenosis in the presence of Ca²⁺ and 0.1 m KCl showed normal mean ATPase activity with several preparations having elevated activity. Elevated ATPase activities were obtained for myosin from the non-failing, but hypertrophied hearts of dogs with aortic stenosis. The data suggest that the interaction of ionic strength and pH with the ATPase active site of suggest that the interaction of ionic strength and pH with the ATPase active site of myosin from failing hearts is altered, thus modifying the ATPase activity in the presence of Ca²⁺ or EDTA. The altered interaction may be modified by the presence of hypertrophy, resulting in normal or increased activity in the presence of Ca²⁺ and decreased activity in the presence of EDTA.     

Modulation of sarcoplasmic reticulum Ca<Superscript>2+</Superscript> cycling in systolic and diastolic heart failure associated with aging

Hypertension, atherosclerosis, and resultant chronic heart failure (HF) reach epidemic proportions among older persons, and the clinical manifestations and the prognoses of these worsen with increasing age. Thus, age per se is the major risk factor for cardiovascular disease. Changes in cardiac cell phenotype that occur with normal aging, as well as in HF associated with aging, include deficits in ß-adrenergic receptor (ß-AR) signaling, increased generation of reactive oxygen species (ROS), and altered excitation–contraction (EC) coupling that involves prolongation of the action potential (AP), intracellular Ca²⁺ (Ca _i ²⁺ ) transient and contraction, and blunted force- and relaxation-frequency responses. Evidence suggests that altered sarcoplasmic reticulum (SR) Ca²⁺ uptake, storage, and release play central role in these changes, which also involve sarcolemmal L-type Ca²⁺ channel (LCC), Na⁺–Ca²⁺ exchanger (NCX), and K⁺ channels. We review the age-associated changes in the expression and function of Ca²⁺ transporting proteins, and functional consequences of these changes at the cardiac myocyte and organ levels. We also review sexual dimorphism and self-renewal of the heart in the context of cardiac aging and HF.     

Phospholamban: A Promising Therapeutic Target in Heart Failure?

Dilated cardiomyopathy and end-stage heart failure result in characteristic functional, biochemical and molecular alterations. Multiple defects in cardiac excitation-contraction coupling have been suggested to underlie disturbed myocardial function and progressive remodeling. Ca²⁺ uptake and release by the sarcoplasmic reticulum (SR) have been shown to be altered in various animal models and human conditions. This review will focus on SR Ca²⁺ ATPase and its regulatory protein, phospholamban, as potential therapeutic targets. We summarize structural and genetic approaches, which have helped to elucidate the physiological role of phospholamban as a principal regulator of cardiac contractility and -adrenergic stimulation in the heart. These findings are extended to the clinical arena, indicating a phospholamban/SR Ca²⁺ ATPase mismatch in human heart failure. Evidence is then provided, using genetically engineered mouse models, that SR dysfunction may play a key role in the onset and progression of heart failure. Phospholamban deficiency may prevent such left ventricular dysfunction and its progression to heart failure in some of the animal models with dilated cardiomyopathy. Based on these findings, we discuss the question of whether and how interfering with the phospholamban/SR Ca²⁺ ATPase interaction may be a promising therapeutic approach for heart failure.     

Physiologic and emerging pathophysiologic role of cardiac calcium channels

The link between cardiac contractile dysfunction in patients with end-stage heart failure and aberrant myocardial intracellular calcium handling is now well established. The precise intracellular protein(s) responsible for this breakdown in calcium handling is at present unclear. However, a number of distinct sarcolemmal (L-type, N-type, T-type, P-type, Q-type) and sarcoplasmic reticular (calcium release, ryanodine) calcium channels that have been defined on a biophysical, biochemical, and molecular basis lend valuable insights into possible factors that may contribute to the abnormal calcium handling in the hearts of these patients. What is now clear is that cardiac muscle contraction is a rigorously regulated event that follows the organized cycling of calcium from the sarcoplasmic reticulum (SR) into the cytosol and back into the SR, and that this cycle follows the graded entry of trigger calcium that enters the cell through the voltage-sensitive calcium channel. Furthermore, the voltage-dependent properties of potential-dependent calcium channels provide the underpinning for the vascular selectivity of the clinically available calcium channel drugs. Moreover, it has also been reported that the efficacy of these agents is augmented in pathologic (ischemic) tissue owing to the state dependence of these channels. Recently, the basis for the critical role of the SR in calcium signaling has started to emerge. The SR calcium handling proteins (SR calcium release channel/ryanodine receptor, SR Ca²⁺ ATPase, phospholamban, calsequestrin) play a critical role in maintaining intracellular free ionized calcium concentrations ([Ca²⁺ _i), which therefore regulate systolic and diastolic function on a beat-to-beat basis within the cardiac cell. This rigorous control of [Ca²⁺]_i is in part the result of the highly developed junctional regions of the cardiac SR. Elucidation of the calcium handling process in these regions and the potential damage resulting from cardiovascular disease has been greatly aided by the invaluable molecular tool, ryandine. From an expanding volume of information provided by animal models of ischemia, hypertrophy, and heart failure, it now appears that changes in cardiac voltage-sensitive calcium channels are likely to be the result of a secondary process that may not be directly linked to the onset of these cardiovascular diseases. Conversely, the regulation of ryanodine receptors has been suggested to be a mechanism initiating the decline in myocardial contractility leading to heart failure. These reports have been supported by studies demonstrating SR calcium release channel/ryanodine receptor changes in pressure overload hypertrophy and myocardial ischemia. Further support for the role of SR calcium release channels in cardiovascular disease is found in reports that couple leaking SR channels with ischemia and cardiac failure. These results suggest that changes in SR calcium handling proteins may be critically linked to cardiovascular disease. A more central question stemming from these results is exactly how these altered SR calcium handling proteins are involved with the onset and progression of cardiovascular disease. Application of transgenic technologies and animal models of chronic heart failure that parallel the human condition will provide the means necessary for unequivocally determining if the apparent adaptive changes in calcium handling are associated with the onset and progression of this syndrome.     

Effects of cyclic adenosine monophosphate (cAMP) on sarcoplasmic reticulum Ca2+-loading in failing rabbit and human cardiac trabeculae

The response of cardiac SR Ca²⁺-loading to cAMP in failing rabbit and human myocardium was examined. Right ventricular (RV) trabeculae were isolated and mounted for isometric tension measurement. They were treated with saponin to permeabilise the sarcolemma but retain SR function, and bathed in a mock intracellular solution including adenosine triphosphate (ATP) and buffered calcium. Caffeine (10 mM) was used to release calcium from the SR. The amplitude of the caffeine-induced contracture was used as a quantitative gauge of the calcium content of the SR. Trabeculae were isolated from rabbits with coronary ligation-induced heart failure (LIG, n=11), sham operated controls (SH, n=10), isoprenaline-infused rabbits (ISO, 7 days mini-osmotic pump 100 μg/kg·h; n=7) and saline-infused controls (SAL, n=7). Failing human RV trabeculae were obtained at the time of cardiac transplantation. Failing rabbit trabeculae demonstrated increased baseline caffeine-induced contractures compared with controls, the response to cAMP was similar in the two groups (LIG 9.3±2.8 vs SH 10.6±3.2% F_max; P=0.55). There was no difference in the baseline SR Ca²⁺-loading in ISO trabeculae compared with SAL controls but there was a marked difference in the response to cAMP (11.1±5.4 vs 4.2±2.1% F_max, P=0.02). SR Ca²⁺-loading in failing human RV trabeculae was related to the severity of LV dysfunction (r=0.59, P=0.04) and demonstrated a marked cAMP-induced enhancement of caffeine-contracture (20.2±4.7% increase of F_max) which was greater in patients with low compared with high ejection fraction. While β-receptors are known to be down regulated in heart failure these results suggest that the scope for cAMP-mediated enhancement of SR Ca²⁺-loading is maintained. Received: 12 March 1998, Accepted: 3 June 1998     

Cardiac adrenomedullin: its role in cardiac hypertrophy and heart failure

Co-localization of adrenomedullin (AM) and its receptor components such as calcitonin receptor like receptor (CRLR), receptor activity modifying protein (RAMP)2 and RAMP3 in peripheral tissues, including the heart, kidney, and vasculature, suggests an important role for the peptide as a regulator of cardiovascular function. Indeed, we previously reported that AM gene expression and / or immunoreactivity are increased in the ventricles of cardiac hypertrophy and heart failure. Recently, we also found that not only levels of AM peptide and AM gene expression, but also mRNA levels of CRLR, RAMP2 and RAMP3 are increased in cardiac hypertrophy and failing heart. Cardiac myocytes and fibroblast produce and secrete two molecular forms of AM and express CRLR, RAMP2 and RAMP3, and AM is known to have inhibitory effect of collagen synthesis and antiproliferative effect in cardiac fibroblasts. Stimulation by IL-1beta significantly increased gene expression of AM and its receptor components in cardiac fibroblasts. Preincubated IL-1beta elevated the intracellular cAMP response to exogenous administered AM. AM antisense oligodeoxynucleotide treatment significantly lowered AM levels in cultured medium. IL-1beta significantly increased (3)H-proline incorporation and AM antisense oligodeoxynucleotide treatment further increased (3)H-proline incorporation. Collectively, these results support a protective role for increased AM in the cardiac hypertrophy and heart failure. Then, we tested the effects of acute administration of AM in experimental and human heart failure, because AM has hemodynamic effects including vasodilation, increases in cardiac contractility, cardiac output, diuresis, and natriuresis. We observed profound and sustained cardiovascular, hormonal and renal effects. These effects may incorporate many of the therapeutic goals of heart failure management.     

Na+ Channel Activators as Positive Inotropic Agents for the Treatment of Chronic Heart Failure

The Na⁺ channel agonists DPI 201-106, BDF 9148 and BDF 9198 are a new group of positive inotropic agents which increase cardiac contractility in a cAMP independent manner. The most likely mechanism by which positive inotropy is mediated is an enhancement of Na⁺/Ca²⁺ exchange activity in response to a Na⁺ channel agonist induced increase in the cardiac myocyte intracellular Na⁺ concentration. While the positive inotropic effect of drugs which exert their effects in a cAMP dependent manner is blunted in failing compared to nonfailing myocardium, the efficacy and potency of Na⁺ channel agonists is not only maintained, but enhanced in failing myocardium. This finding makes these substances interesting for the treatment of patients with heart failure. The positive inotropic effects of the Na⁺ channel agonists, however, are accompanied by a potential increase in the incidence of cardiac arrhythmias. These side effects might limit the clinical use of Na⁺ channel agonists and demand future development of Na⁺ channel modulators without significant arrhythmogenic effects.     

Reorganized PKA-AKAP associations in the failing human heart

Here we reveal that the characterization of large-scale re-arrangements of signaling scaffolds induced by heart failure can serve as a novel concept to identify more specific therapeutic targets. In the mammalian heart, the cAMP pathway, with the cAMP-dependent protein kinase (PKA) in a central role, acts directly downstream of adrenergic receptors to mediate cardiac contractility and rhythm. Heart failure, characterized by severe alterations in adrenergic stimulation is, amongst other interventions, often treated with β-blockers. Contrasting results, however, have shown both beneficial and detrimental effects of decreased cAMP levels in failing hearts. We hypothesize that the origin of this behavior lies in the complex spatiotemporal organization of the regulatory subunit of PKA (PKA-R), which associates tightly with various A-kinase anchoring proteins (AKAPs) to specifically localize PKA's activity. Using chemical proteomics directly applied to human patient and control heart tissue we demonstrate that the association profile of PKA-R with several AKAPs is severely altered in the failing heart, for instance effecting the interaction between PKA and the novel AKAP SPHKAP was 6-fold upregulated upon failing heart conditions. Also a significant increase in captured cGMP-dependent protein kinase (PKG) and phosphodiesterase 2 (PDE2) was observed. The observed altered profiles can already explain many aspects of the aberrant cAMP-response in the failing human heart, validating that this dataset may provide a resource for several novel, more specific, treatment options. This article is part of a Special Issue entitled "Local Signaling in Myocytes".