Curr Heart Fail Rep (2014) 11:227–235 DOI 10.1007/s11897-014-0210-z

DECOMPENSATED HEART FAILURE (JE HO, SECTION EDITOR)

Nitroxyl (HNO) for Treatment of Acute Heart Failure Alessia Arcaro & Giuseppe Lembo & Carlo G. Tocchetti

Published online: 1 July 2014 # Springer Science+Business Media New York 2014

Abstract 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 Ca 2+ 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.

Keywords Nitroxyl . Heart failure . Cardiac function . Cardiomyocytes . Inotropic drugs . Reactive oxygen species . Reactive nitrogen species . Experimental animal studies . Clinical studies A. Arcaro : G. Lembo IRCCS Neuromed, Pozzilli, IS, Italy G. Lembo La Sapienza University, Rome, Italy C. G. Tocchetti (*) Clinica Montevergine, Via Mario Malzoni, 83013 Mercogliano, AV, Italy e-mail: [email protected]

Introduction Loss of contractile function is a hallmark of many forms of heart failure (HF) [1]. Nitroxyl (HNO, the one-electronreduced product of nitric oxide (NO•) [2, 3] has emerged as a molecule with a variety of pharmacological properties pertinent to the management of heart failure, and its donors may prove useful for inotropic and dilator support in HF. HNO exhibits different chemical and physiological properties from NO [2] and from reactive oxygen and other nitrogen species. Studies have indicated that HNO donors induce positive inotropy/lusitropy in vivo in animals with normal [4] and failing hearts [5], and improve cardiac function in patients with acutely decompensated HF [6••]. HNO action is highly redox-sensitive in that it rapidly interacts with protein thiols, mostly on cysteine residues, resulting in the formation of reversible disulfides that induce biological effects by changing the conformation of the target protein [7••, 8••, 9••]. Herein we review HNO chemical and pharmaceutical properties, and how HNO donors can mitigate E-C coupling and myofilament abnormalities that occur in the failing heart. Lastly, we describe results of the first clinical study of a pure HNO donor in patients with decompensated HF.

Inotropic alterations in Heart Failure In the cardiomyocyte, contraction is stimulated by the influx of a small quantity of Ca2+ via voltage-gated (L-type) channels, followed by Ca2+ released from the sarcoplasmic reticulum (SR) via ryanodine receptor channels (RyR2) [10]. This event, known as Ca2+-induced Ca2+ release, makes Ca2+ available to the myofilaments and their regulatory proteins, resulting in myofilament contraction. At the end of systole, Ca2+ is pumped back into the SR via the ATP-dependent SR Ca2+-ATPase (SERCA2a) or excluded from the cell by the

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Na+-Ca2+ exchanger [11]. The function of SERCA2a is substantially regulated by phospholamban (PLN), which inhibits the ATPase when de-phosphorylated but enhances its function when phosphorylated by several kinases, including protein kinase A (PKA) [12•]. β-adrenergic stimulation via cyclic adenosine monophosphate (cAMP) and PKA-signaling increases phosphorylation of the L-type Ca2+ channel, RyR2, PLN, and multiple myofilament proteins, enhancing contraction magnitude and kinetics [11, 13, 14•]. In the failing heart, Ca2+ uptake into the SR and sarcoreticular Ca2+ concentrations are impaired, defects that are attributed to reduced SERCA2a expression, phospholamban phosphorylation, and depleted SR Ca2+ stores due to abnormal RyR2 leakage of Ca2+ [15–17]. The failing heart also exhibits oxidative stress [18, 19•, 20], altering EC-coupling and myofilament function [7••]. Myofilament responsiveness to Ca2+ can also be impaired by isoform switching of key myofilament proteins [1], with a decline in the expression of α-myosin heavy chain [21].

Targeting Depressed Contractility While pharmacological efforts to enhance contractility have generally relied on β-adrenergic agonists or phosphodiesterase type III inhibitors to raise intracellular cAMP levels, this tactic has resulted in deleterious side effects when used as chronic therapy [1, 22, 23, 24•, 25••]. Another approach is to enhance myofilament Ca2+ sensitivity [26, 27••]; both levosimendan and, more recently, omecamtiv mecarbil represent drugs aimed at achieving this. Levosimendan stimulates troponin C binding to Ca2+, while also inhibiting PDE3A and stimulating ATP-sensitive K+ channels in smooth muscle, thereby acting as a vasodilator as well. Despite early optimism, the clinical effects of levosimendan have been mixed, with arrhythmia and cardiostimulation concerns leading to limited approval in Europe [24•, 28]. Omecamtiv mecarbil, a myosin ATPase activator that appears to affect contractility only by prolonging the duration of systole, was recently studied in a phase IIb trial, and showed some improvement in heart function but did not alter the course of acute hospitalization for decompensated heart failure [29, 30•]. Istaroxime represents yet another approach, which is based on blockade of the Na/K ATPase and putative enhancement of SR Ca2+ uptake [31]. Phase IIa studies reported some improvement in hemodynamics, although results of a recent phase III trial were negative. Lastly, gene therapy based on enhancing SERCA2a expression is presently being studied in phase IIb trials, and early data appear promising [32•, 33•]. HNO represents an alternative approach. It was first discovered to induce both venous and arterial dilation and positive inotropy in intact failing hearts, and since then, mechanistic studies have revealed multiple pathways that combine the strategies of these other approaches.

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HNO Donors and Chemistry While there are many HNO donors currently available [34, 35•], not all of them are amenable for in vivo or even in vitro studies. The HNO-releasing agents most commonly used for animal studies are the prototypic HNO donor Angeli’s salt (AS, Na2N2O3) [36], Piloty’s acid (PhSO2NHOH) and its derivatives, isopropylamine-NO• (IPA/NO) [37], and acyloxy nitroso compounds such as 1-nitrosocyclohexyl acetate (NCA, also known as the “blue compound”) [8••, 38]. Of these, AS is most widely used, despite its very short half-life as well as the fact that it is a co-generator of NO2-. The latter requires the use of proper controls to exclude this potential confounding effect [36]. Newer pure HNO donors include CXL-1020, a congener of Piloty’s acid that decomposes to HNO and an inactive organic byproduct [6••]. HNO is chemically different from NO• and other nitrosoredox species in that it is not a free radical and therefore cannot be detected by electron paramagnetic/electron spin resonance [7••]. While the chemistry and mechanisms of action of HNO are becoming better understood, a major debate continues as to whether the molecule is generated endogenously. With the lack of a definitive method to determine native HNO generation in biological systems, the question remains unanswered. Unlike NO•, HNO reacts with higher-oxidation-state metals [Fe(III), Cu(II), and Mn(III)], and therefore it can affect key metalloproteins either by activating them, as in the case of soluble guanylyl cyclase (sGC) [39], or by inhibiting their function, as shown for peroxidases and monooxygenases like cytochrome P450 [40]. Among the more distinguishing biochemical traits of HNO versus NO is its rapid reversible reactivity with thiols. Thiolbased “redox switches” [41] can sense the local redox environment through the unique chemical properties of their thiol side chain. These sensors oscillate between a reduced and oxidized state, responding to ROS or RNS with a continuum of posttranslational modifications [42]. HNO can modify these cysteine residues in two distinct ways. The first is via direct targeting of reactive thiols (thiolates) in cysteine residues to form N-hydroxysulfenamide (RSNHOH) [43•]. Second, if an additional thiol resides in the near vicinity, a disulfide bond can form [44]. Under mild oxidizing conditions, the appearance of inter- and/or intra-disulfide bonds often leads to a change in protein function due to a conformational change. These modifications can be reversed by cellular reducing factors belonging to the thioredoxin and glutathione systems [45•]. However, the PKA of a given cysteine and the location of the molecular target likely compartmentalize HNO reactivity, conferring specificity of action and capacity to modify thiols despite the presence of high cellular GSH. Indeed, HNO readily reacts with thiols of very low PKA and targets that are preferentially located in highly hydrophobic cellular regions [7••].

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HNO Enhances Ca2+ Cycling The proteins involved in EC-coupling are modulated not only by their phosphorylation status but also by their redox conditions [46, 47•]. Interestingly, such structures harbor critical thiol groups in cysteine residues (Fig. 1) that form redox reactive switches under the control of endogenous signaling molecules such as NO•, RNS, or ROS [48]. These regulator species are constitutively produced during muscle contraction and exposure to βadrenergic stimulation [49] to modulate muscle contraction and relaxation [50]. The chemical modifications include reversible post-transcriptional S-nitrosylation to irreversible carbonylation or oxidation, depending upon concentration, length of exposure, and specifics of the chemical reaction with sulfhydryl groups of the signaling molecule [51–53]. For example, S-nitrosylation of the RyR2 channel increases its open probability [50], while oxidation of thiols in the RyR2, L-type Ca2+ channels (LTCC), and myofilament proteins results in a loss of function [53–56]. HNO donors enhance the function of isolated rodent (mouse and rat) myocytes, increasing shortening magnitude and relaxation rates. These inotropic effects are sustained so long as HNO is present. The augmented whole Ca2+ transient that accompanies these effects results from HNO’s ability to enhance Ca2+ cycling into and out of the SR. This is due to HNO stimulation of RyR2 release enhancing SR Ca2+ fractional release [57], and faster SR Ca2+ re-uptake due to HNO’s modulation of SERCA2a/PLN interaction [9••]. Interestingly, HNO does not alter L-type channel function [58], but it increases the open probability of the Ca2+ release channels in RyR2 and the Ca2+ spark frequency (Fig. 1). Neither NO• donors [57] nor nitrite [58] reproduce these effects. A similar HNO stimulatory action has been described for skeletal Ca2+ release channels (RyR1) [59]. In that study, HNO’s effects on both RyR2 and RyR1 were suppressed by addition of dithiothreitol, a sulfhydryl reducing agent, indicating that HNO targets hyperactive cysteine groups in these channels.

Fig. 1 Mechanism of action of HNO donors

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Enhanced Ca2+ is the result of two mechanisms, which are not mutually exclusive. Lancel et al. demonstrated that HNO increases the maximal activation of SERCA2a via Sglutathiolation at cysteine 674 in rat ventricular myocytes [60]. This effect was blocked by the reducing agent dithiothreitol (DTT). In contrast, our group found that HNO inotropy/ lusitropy requires the presence of PLN [9••]. Substituting the three cysteines present in the PLN transmembrane domain with alanine residues abolishes the stimulatory action of HNO, consistent with HNO targeting these residues to alter the structural interaction of PLN with SERCA2a, and thus the PLN inhibitory brake [61]. HNO enhances SR Ca(2+) uptake in WT but not in PLN(-/-) SR-vesicles. Using spectroscopic studies in insect cell microsomes expressing SERCA2a ± PLN, the authors showed that HNO increases Ca(2+)-dependent SERCA2a conformational flexibility, but only when PLN was present. In intact cardiomyocytes, HNO achieves this effect by stabilizing PLN in an oligomeric disulfide bond-dependent configuration, decreasing the amount of free inhibitory monomeric PLN available [9••]. These effects are similar in outcome to PKA phosphorylation [62], but by a completely different mechanism.

HNO Enhances Myofilament Ca2+ Sensitivity HNO also acts at the myofilament level to enhance force generation. When directly applied to isolated intact cardiac muscle (right ventricular trabeculae), HNO enhances myofilament responsiveness to Ca2+, augmenting myocardial contractility more than the whole-cell Ca2+ transient [63], suggesting that HNO also functions as a Ca2+ sensitizing agent. Again, the alteration of intracellular redox conditions with dithiothreitol prevented HNO-induced augmentation in muscle force development, consistent with the theory that HNO targets thiols [63]. Gao and colleagues recently found that HNO exerts direct effects on myofilament proteins, increasing both maximum force (Fmax) and Ca2+ sensitivity in intact and skinned cardiac muscles by promoting reversible disulfide bond formation between crucial cysteine residues [8••] (Fig. 1). Using mass spectrometry (MS) coupled with a modified biotin switch assay, the following HNO-targeted residues were identified: Cys257 in the actin subdomain 4, Cys190 in tropomyosin (TM), Cys81 in myosin light chain 1 (MLC1), and Cys37 in the myosin heavy chain’s (MHC) head region. The authors reported that treatment with HNO resulted in the formation of an actin–TM heterodimer, which correlated with increased Ca2+ sensitivity, and dimeric forms of MHC and MLC1 associated with increased force generation. All of these effects were readily reversed by DTT. The action of HNO on myocyte mechanical properties appears to be independent of cyclic guanosine monophosphate (cGMP) and cAMP [57, 64], focused rather on its redoxdependent controlling mechanisms (Fig. 1). The fact that HNO action appears to be preserved in failing [6••] or in β-

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adrenergic desensitized [65] myocytes reveals that HNO can target thiol residues that are not readily available to generalized oxidation that might affect failing cardiac cells [66•].

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distinct from NO•. In particular, HNO is able to activate KV and KATP channels in resistance arteries [77•].

HNO in Failing Hearts In Vivo HNO in Normal Hearts In Vivo The inotropic effects detailed in various in vitro studies were, in fact, first reported in intact hearts in conscious mammals [4, 5]. While NO• donors have modest or even negative effects on contractility, particularly at higher doses, [67], HNO donors promptly increased contractility and accelerated relaxation. In vivo HNO also displayed potent vascular effects, reducing ventricular diastolic volume (preload) in normal hearts and arterial resistance (afterload) and preload in failing hearts. Dissection of HNO primary cardiac versus vascular loading effects required use of pressure-volume analysis. When AS/HNO was administered intravenously to conscious dogs, increases in maximal ventricular stiffness (elastance) independent from loading changes were revealed, as was improved ventricular relaxation [4]. These effects persisted after autonomic blockade and/or restoration of ventricular preload volume. Unlike β-agonists or PDE3A inhibitors, the effects of AS/HNO were not diminished by β-adrenergic receptor blockade, and in contrast to NO donors, the effects of AS/HNO were additive to those of β-adrenergic stimulants. However, HNO-stimulated cardiac function was fully prevented by coadministration of N-acetyl-L-cysteine, confirming its redox dependence. This combination of properties was not mimicked by NO•, nitrate, or peroxynitrite [68, 69]. Paolocci and colleagues [4] first suggested a link between AS/HNO contractile effects in vivo and the release of calcitonin gene-related peptide (CGRP) from nonadrenergicnoncholinergic fibers. Subsequent studies [70], however, showed that CGRP-inotropy was mediated by norepinephrine released from sympathetic efferent fibers and fully prevented by beta blockade. Canine myocytes did not express CGRP receptors, and thus CGRP release could not account for HNOinduced inotropy/lusitropy. Vascular studies have also found that CGRP is not fully responsible for HNO-induced vasodilation [71], and so the significance of this initial observation, while true, is unclear. Interestingly, unlike its myocardial effects, HNO vasodilation has also been attributed to sGC activation [71, 72]. Already in the early 1990s, Fukuto et al. [73] reported that AS/HNO was able to relax both rabbit aorta and bovine intrapulmonary artery by means of a sGCdependent mechanism. Indeed, HNO has important vasoprotective properties. Like NO•, HNO is able to inhibit platelet aggregation [74] and proliferation of vascular smooth muscle cells [38, 75, 76]. In addition, HNO can be putatively generated within the vasculature, and it was recently suggested that it also serves as an endothelium-derived relaxing factor (EDRF). Significantly, HNO targets signaling pathways

Abnormalities of EC-coupling processes in which both cardiac contractility and muscle relaxation velocity are reduced are centrally involved in the onset of HF, and these alterations may originate, at least in part, from redox imbalance of the myocardium [78•]. In HF, ROS and RNS are generated by multiple sources, leading to a reduced bioavailability of NO•. In addition to decreased antioxidant defenses, the reduction in NO• can result in a further increase in ROS due to the uncoupling of NOS. Other consequent events associated with the onset and progression of HF include cardiomyocyte maladaptive hypertrophy, extracellular matrix remodeling, abnormal tissue energetics, loss in viable myocardium, vascular and capillary alterations, and inflammation [18, 19•, 66•]. Further, ROS/RNS may dampen contractile reserve and relaxation stimulated by β-agonists, either by altering the βagonists per se [70] or by negatively interacting with their associated signaling pathways. For instance, endogenous NO• and nitrates are known to attenuate the response of β-adrenergic stimulation in both experimental and human HF [67]. This effect represents a major drawback when a combination of β-agonists and unloading agents is the desired treatment modality in patients with HF. As such, inotropic/lusitropic agents that do not lose their efficacy in the HF setting or display a facile reaction toward ROS/RNS should be ideal for the treatment of patients with acutely decompensated heart failure [7••]. Importantly, in the setting of experimental HF induced by tachypacing in a dog model, HNO donors such as AS improved both contractility and relaxation to a similar extent as in control preparations [5]. Furthermore, when HNO was administered concomitantly with β-agonist mimetics such as dobutamine, they were additive in supporting myocardial contraction, as opposed to NO/nitrite that blunted dobutamine-induced enhancement in function. Interestingly, preliminary studies performed in neonatal rat cardiomyocytes by Irvine and colleagues [79] also revealed that AS/HNO may counter angiotensin II-induced hypertrophy in a cGMPdependent fashion, hinting at the possibility that HNO may counter chamber remodeling and dysfunction in hearts subjected to chronic volume or pressure overload [7••]. Current evidence shows that HNO cardiac action is fully preserved in CHF preparations in the face of altered tissue and vascular redox conditions, and furthermore, that its action in vitro is resistant to both the development of tolerance and scavenging by superoxide (O2-•) [77•, 80•]. These findings are fully consistent with the HNO chemistry and with previous in vivo studies in which it was demonstrated that repeated infusions of AS/HNO did not lead to any loss in efficacy [4].

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Table 1 Limitations of major current and investigational inotropes. From Goldhaber JI, Hamilton MA, “Role of Inotropic Agents in the Treatment

of Heart Failure,” Circulation, 2010;121:1655–1660, adapted with permission from Wolters Kluwer Health

Drug

Mechanism

Indication

Effect on mortality

Digoxin

Na-K pump inhibitor, raises SR Ca2+

Recurrent HF admissions

Dobutamine

Beta agonist, widespread cAMP/PKA-dependent phosphorylation; increases extracellular Ca2+ entry via LTCC; raises SR Ca2+, synchronizes Ca2+ sparks, stimulates remodeling Beta agonist, widespread cAMP/PKA-dependent phosphorylation; raises SR Ca2+, stimulates remodeling Increases cAMP levels through PDE inhibition Myofilament Ca2+ sensitization, PDE inhibitor

Shock, palliative use in end-stage HF

Neutral. Increased mortality if long-term treatment discontinued Increased

Dopamine

Milrinone Levosimendan

Shock

Increased

Shock, palliative use in end-stage HF Shock

Increased Increased

These HNO effects are not exclusive to AS, but have been confirmed by the use of other HNO donors such as IPA/NO [37] (chemically unrelated to AS), as well as NCA [8••, 38] and the novel compound CXL-1020 [6••]. CXL-1020 (developed by Cardioxyl Pharmaceuticals, founded in 2005) is a pure HNO donor that has been proven particularly efficacious not only in normal and failing cardiac myocytes in vitro, but also in vivo in two canine models of cardiac failure (ischemic and tachypacinginduced HF). In particular, in anesthetized dogs with coronary microembolization-induced HF, CXL-1020 reduced left ventricular end-diastolic pressure and myocardial oxygen consumption while increasing ejection fraction and maximal ventricular power index. In conscious dogs with tachypacing-induced HF, CXL1020 increased contractility assessed by end-systolic elastance and provided veno-arterial dilation, with negligible alterations in heart rate.

Therapeutic Potential of HNO Donors in the Treatment of HF Despite huge efforts and decades of active experimental and clinical research [81, 82], congestive heart failure (CHF) still represents a “therapeutic challenge.” Inotropic agents that use intracellular cAMP levels to leverage myocardial performance (i.e., β-agonists and phosphodiesterase inhibitors) are deleterious in the long term. [1, 22, 23, 24•, 25••]. Accordingly, their use in patients with CHF is currently limited to palliative care and bridge to transplantation or mechanical assist device implantation (Table 1 [23]). There is a paucity of safe and effective therapies that enhance left ventricular (LV) function while also aiding in decongestion. A plethora of animal studies have demonstrated the ability of antioxidant supplements to prevent LV remodeling and to improve function in several models of CHF. When translated to human CHF settings, however, these interventions invariably fail, in some cases even resulting in increased mortality, unless the antioxidant effect is combined with other pharmacological activities, as is the case of the β-blocker carvedilol [83].

On the other hand, the studies reviewed in this manuscript show that HNO improves myocardial function by direct positive and cAMP-independent lusitropic and inotropic effects and by combined venous and arterial dilation. HNO targets selective cysteine residues (negatively charged, or thiolates) resulting in covalent bonding and/or formation of a reversible disulfide. In myocytes, HNO enhances sarcoplasmic reticular calcium uptake and release via cysteine modifications on SERCA2a, phospholamban, and the ryanodine receptor, and also improves myofilament calcium sensitivity. HNO does not alter L-type calcium channel current (LTCC) or total SR calcium load. Importantly, the effects of HNO on the heart are a) independent of cAMP or cGMP, b) equipotent in normal and failing myocardia, c) not arrhythmogenic [6••], d) minimally affected by β-adrenergic receptor blockade, and, e) unlike NO donors, are additive to agents stimulating the cAMP/PKA pathway (e.g., beta-receptor agonists). In essence, the properties of HNO render its donors an ideal class of inotropic and unloading agents for treating patients with acutely decompensated CHF (Table 2). The first clinical trial Table 2 Main effects of HNO donors, future directions, and potential limitations Main effects of HNO donors • Positive inotropy/lusitropy • Balanced vasodilation • LV unloading effects • No alterations in Ca2+ homeostasis • No arrhythmogenesis • Antiaggregant effects • Anti-inflammatory and antiproliferative actions • Preconditioning action [84, 85•] Future directions and potential limitations • HNO effects in a more chronic setting (cardiac remodeling) • HNO effects on renal function • HNO effects on myocardial energetics • At certain doses, HNO may display some neurotoxicity [86, 87]

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employing CXL-1020 as a pure HNO donor fully supports the experimental evidence obtained thus far [6••]. Importantly, by isolating the response to HNO, our most recent studies [6••] excluded potential confounding effects of nitrites and overcame AS instability that had made sustained (4–6 hours) infusions impossible. In our recently completed clinical study (ClinicalTrials.gov NCT010960430), CXL-1020 was infused intravenously, using a placebo-controlled six-hour forced titration design, at doses of 1–20 μg/kg per minute in patients hospitalized for hemodynamic assessment of HF before transplantation or for treatment of decompensated HF requiring hemodynamic monitoring [6••]. Inclusion criteria were a mean cardiac index of ≤2.5 L/min and a mean pulmonary capillary wedge pressure or pulmonary artery diastolic pressure of >20 mm Hg at baseline. Exclusion criteria were a heart rate (HR) of 95 mm Hg at baseline before randomization. Also excluded were patients with atrial fibrillation without adequate rate control or with evidence of clinically significant non-sustained ventricular tachycardia (10 beats or at a rate of >120 beats per minute) in the preceding 12 hours. CXL-1020 produced a decline in diastolic filling pressures, modest fall in SVR, and rise in cardiac output as a result of increased stroke volume without change in heart rate, all consistent with what was previously reported in animal preparations. The clinical study identified a threshold dose of 10–20 μg/kg/min of CXL-1020 for hemodynamic effects. CXL-1020 was well-tolerated, with few apparent side effects [6••] or adverse trends in routine laboratory parameters. The results of studies of CXL-1020, AS, and other novel HNO donors confirm that the hemodynamic effects of HNO are a class phenomenon independent of the donor. These effects distinguish HNO donors from other classes of inotropes or inodilators, and provide a strong rationale for continuing studies to develop donors with optimized pharmacological and clinical efficacy for the treatment of congestive heart failure. Unfortunately, long-lasting CXL-1020 infusions (12–24 hours) at 20 μg/kg/min produced an inflammatory irritation at the intravenous infusion site. As a result, Cardioxyl Pharmaceuticals has ceased further development of CXL-1020 as a human therapeutic and has now advanced its improved second-generation HNO donor, CXL-1427, into the clinic, having recently initiated the first phase I trial (www. cardioxyl.com). This is a promising new compound that causes no venous irritation, while keeping the full spectrum of HNO experimental hemodynamic effects [6••].

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preparations in vitro as well as in failing canine hearts in vivo. The recent accomplishment of the first clinical study with the new HNO donor CXL-1020 tested proof-of-concept for patients with decompensated HF. CXL-1020 enhanced cardiac performance while unloading the left ventricle in patients with decompensated HF. This first cell-to-human evaluation of a pure HNO donor suggests the potential efficacy and utility of this pharmacological approach to improving the function of a failing heart [6••]. Further studies with recently developed long-lasting HNO donors will be needed to assess the effects of HNO on LV remodeling in a more chronic setting as well as its potential effects in critical areas such as renal function and myocardial energetics. Acknowledgments We are grateful to Drs. David Kass and Nazareno Paolocci (Johns Hopkins Medical Institutions, Baltimore, MD, USA) for critically revising the manuscript. Compliance with Ethics Guidelines Conflict of Interest Alessia Arcaro declares that she has no conflict of interest. Giuseppe Lembo declares that he has no conflict of interest. Carlo G. Tocchetti, along with Drs. David A. Kass and Nazareno Paolocci, is a co-inventor of Canadian patent no. 2,613,477, “Thiol Sensitive Positive Inotropes,” issued December 3, 2013. The same patent is currently pending in the United States. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

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6.••

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