NCX 2057, a novel NO-releasing derivative of ferulic acid,suppresses inflammatory and nociceptive responses in in vitro and in vivo models

Background and purpose:

We previously reported that NCX 2057, a compound comprising a nitric oxide (NO)-releasing moiety and the natural antioxidant, ferulic acid (FA), inhibits pro-inflammatory mediators through NO-mediated gene regulation. Here, we have assessed the activities of NCX 2057 in models of inflammatory and neuropathic pain, and characterized its effects on cyclooxygenase (COX)-1 and COX-2.

Experimental approach:

Anti-nociceptive and anti-inflammatory activities of NCX 2057 were measured in vitro and in vivo in models of inflammatory (carrageenan) and neuropathic (chronic constriction injury; CCI) pain. Effects of NCX 2057 were measured on COX-1 and COX-2 activities in RAW 264.7 macrophages.

Key results:

NCX 2057 dose-dependently inhibited single motor unit responses to noxious mechanical stimulation (ID₅₀= 100 µmol·kg⁻¹) and wind-up responses in rats with paw inflammation induced by carrageenan. Moreover, NCX 2057 inhibited allodynic responses following CCI of the sciatic nerve [ipsilateral Paw Withdrawal Threshold (g): vehicle: 41.4 ± 3.3; NCX 2057: 76.3 ± 4.8 FA: 37.9 ± 15.5 at 175 µmol·kg⁻¹]. NCX 2057 reversed carrageenan-induced hyperalgesic responses in mice and inhibited prostaglandin E₂ formation in paw exudates. Finally, NCX 2057 competitively inhibited COX-1 and COX-2 activities in whole RAW macophages (IC₅₀= 14.7 ± 7.4 and 21.6 ± 7.5 µM, respectively). None of these properties were exhibited by equivalent treatments with FA or standard NO donor compounds.

Conclusions and implications:

These studies indicate that NCX 2057 is effective in chronic inflammatory and neuropathic pain models, probably because of its particular combination of anti-COX, antioxidant and NO-releasing properties.     

Functional expression of the Na+/Ca2+ exchanger in the embryonic mouse heart

The Na(+)/Ca(2+) exchanger (NCX) is one of the earliest functional genes and is currently assumed to compensate at least in part for the rudimentary sarcoplasmic reticulum in the developing mouse heart. However, to date little is known about the functional expression of NCX during development. This prompted us to investigate the NCX current (I(NCX)) in very early (embryonic day E8.5-E9.5 post coitum), early (E10.5-E11.5), middle (E13.5) and late (E16.5) stage mouse embryonic cardiomyocytes. For standard I(NCX) measurements, [Ca(2+)](i) was buffered to 150 nmol/l and voltage ramps were applied from +60 mV to -120 mV. At very early stages of development, we observed a prominent role of the I(NCX) Ca(2+) inward mode in elevating the cytosolic Ca(2+) concentration ([Ca(2+)](i)). Accordingly, a high I(NCX) density was observed (+60 mV: 4.6+/-0.7 pA/pF, n=14). Likewise, we found a strong Ca(2+) outward mode of I(NCX) (-120 mV: -3.9+/-0.7 pA/pF, n=14). At later stages, however, I(NCX) Ca(2+) inward mode was reduced by 54+/-6% (n=15, p<0.0001) in ventricular and 68+/-10% (n=9, p<0.0006) in atrial cells. For the outward mode, a reduction by 43+/-10% (n=15, p<0.01) in ventricular and 62+/-11% (n=9, p<0.004) in atrial cardiomyocytes was observed. By contrast, NCX isoform expression and the reversal potential did not significantly change during development. Thus, NCX displays a prominent Ca(2+) inward and outward mode during early embryonic heart development pointing to its important contribution to maintain [Ca(2+)](i) homeostasis. The functional and protein expression of NCX declines during further development.     

Targeted GLUT-4 deficiency in the heart induces cardiomyocyte hypertrophy and impaired contractility linked with Ca and proton flux dysregulation

There is clinical evidence to suggest that impaired myocardial glucose uptake contributes to the pathogenesis of hypertrophic, insulin-resistant cardiomyopathy. The goal of this study was to determine whether cardiac deficiency of the insulin-sensitive glucose transporter, GLUT4, has deleterious effect on cardiomyocyte excitation-contraction coupling. Cre-Lox mouse models of cardiac GLUT4 knockdown (KD, 85% reduction) and knockout (KO, > 95% reduction), which exhibit similar systemic hyperinsulinemic and hyperglycemic states, were investigated. The Ca²⁺ current (I_Ca) and Na⁺-Ca²⁺ exchanger (NCX) fluxes, Na⁺-H⁺ exchanger (NHE) activity, and contractile performance of GLUT4-deficient myocytes was examined using whole-cell patch-clamp, epifluorescence, and imaging techniques. GLUT4-KO exhibited significant cardiac enlargement characterized by cardiomyocyte hypertrophy (40% increase in cell area) and fibrosis. GLUT4-KO myocyte contractility was significantly diminished, with reduced mean maximum shortening (5.0 ± 0.4% vs. 6.2 ± 0.6%, 5 Hz). Maximal rates of shortening and relaxation were also reduced (20-25%), and latency was delayed. In GLUT4-KO myocytes, the I_Ca density was decreased (− 2.80 ± 0.29 vs. − 5.30 ± 0.70 pA/pF), and mean I_NCX was significantly increased in both outward (by 60%) and inward (by 100%) directions. GLUT4-KO expression levels of SERCA2 and RyR2 were reduced by approximately 50%. NHE-mediated H⁺ flux in response to NH₄Cl acid loading was markedly elevated GLUT4-KO myocytes, associated with doubled expression of NHE1. These findings demonstrate that, independent of systemic endocrinological disturbance, cardiac GLUT4 deficiency per se provides a lesion sufficient to induce profound alterations in cardiomyocyte Ca²⁺ and pH homeostasis. Our investigation identifies the cardiac GLUT4 as a potential primary molecular therapeutic target in ameliorating the functional deficits associated with insulin-resistant cardiomyopathy.     

In vitro metabolism of a nitroderivative of acetylsalicylic acid (NCX4016) by rat liver: LC and LC–MS studies

The metabolism of a nitroderivative of acetylsalicylic acid, benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX4016), the lead compound of a new class of NO-releasing non steroidal-antiinflammatory drugs has been studied in vitro in rat liver subcellular fractions (S 9000×g, microsomes, cytosol). Samples were extracted with CH₃CN (2 vol.) containing 1% H₃PO₄ (2 M), vortexed for 3 min and then centrifuged for 5 min at 5000 rpm. Supernatants were diluted with 0.02 M phosphoric acid and analysed by reverse-phase LC. Linearity of calibration for NCX4016 and metabolites was observed over the range 0.25–50 μg/ml with coefficients of determination greater than 0.9996. Extraction efficiency from spiked liver samples ranged from 85 to 95% for all the analytes. In the S 9000×g fraction, NCX4016 undergoes rapid metabolization, with the formation of salicylic acid (SA) and [3-(nitrooxymethyl)phenol] (HBN). HBN is then rapidly metabolised to 3-hydroxybenzylalcohol (HBA), and mainly to a new metabolic species, whose formation takes place specifically in the liver cell cytosol. LC–MS analysis (electrospray ionisation) of the cytosol extract in negative and positive-ion modes furnished deprotonated [M−H]⁻ and protonated [M+H]⁺ molecular ions at m/z 412 and 414, respectively, accompanied by the typical clusters with sodium. MS/MS analysis in negative-ion mode, by selection and collision of the ion at m/z 412, gave a fragmentation pattern characterized by the ions at m/z 272 and 254, which allowed to assign the structure of 1-(glutathion-S-yl)methylene-3-hydroxy-benzene, a conjugated product between GSH and the benzyl carbon atom of HBN. In rat liver cytosol HBN is completely metabolised to this thioether adduct within 30 min incubation; the process is enzymatically mediated by GSH transferase and strictly dependent on GSH availability. The relevance of this new metabolic pathway in NCX4016 detoxification by rat liver is discussed.     

Effects of estrogen via estrogen receptors on parvalbumin levels in cardiac myocytes of ovariectomized rats

The study investigated the effects of estrogen on parvalbumin (PV) levels in cardiac myocytes of ovariectomized rats, which is a model system for postmenopausal woman. Parvalbumin acts as a relaxing factor in cardiac myocytes. Adult female Wistar rats, 12 weeks old, were randomly divided into 5 groups of 10: sham-operated (SHAM), ovariectomized (OVX), and OVX receiving estrogen replacement of 10 μg/kg (Es10), 20 μg/kg (Es20) and 40 μg/kg (Es40). After 10 weeks, serum estrogen levels were measured and the α and β estrogen receptors in cardiac myocytes were investigated by immunohistochemistry. PV levels were examined by immunohistochemistry and Western blot analysis. Cardiac myocytes of all animals showed strong staining intensities for α immunoreactive (Es α-ir), but weak staining for β immunoreactive (Es β-ir) estrogen receptors. The Es α-ir was reduced in the cardiac myocytes of the OVX groups, but increased in the Es10, Es20 and Es40 groups. We therefore suggest that estrogen effects are mediated via Es α receptors rather than Es β receptors in female rat hearts. Estrogen and PV immunoreactive (PV-ir) levels and the intensity of the PV band observed in the OVX group were less than those of the SHAM group. In the Es10, Es20 and Es40 groups, the increased intensity of the PV-ir and PV bands correlated with the increased estrogen levels. The low PV levels in cardiac myocytes induced by low estrogen were restored by estrogen replacement therapy. Therefore a reduction of PV may lead to diastolic dysfunction in menopause.     

2019 HRS expert consensus statement on evaluation,risk stratification,and management of arrhythmogenic cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM) is an arrhythmogenic disorder of the myocardium not secondary to ischemic, hypertensive, or valvular heart disease. ACM incorporates a broad spectrum of genetic, systemic, infectious, and inflammatory disorders. This designation includes, but is not limited to, arrhythmogenic right/left ventricular cardiomyopathy, cardiac amyloidosis, sarcoidosis, Chagas disease, and left ventricular noncompaction. The ACM phenotype overlaps with other cardiomyopathies, particularly dilated cardiomyopathy with arrhythmia presentation that may be associated with ventricular dilatation and/or impaired systolic function. This expert consensus statement provides the clinician with guidance on evaluation and management of ACM and includes clinically relevant information on genetics and disease mechanisms. PICO questions were utilized to evaluate contemporary evidence and provide clinical guidance related to exercise in arrhythmogenic right ventricular cardiomyopathy. Recommendations were developed and approved by an expert writing group, after a systematic literature search with evidence tables, and discussion of their own clinical experience, to present the current knowledge in the field. Each recommendation is presented using the Class of Recommendation and Level of Evidence system formulated by the American College of Cardiology and the American Heart Association and is accompanied by references and explanatory text to provide essential context. The ongoing recognition of the genetic basis of ACM provides the opportunity to examine the diverse triggers and potential common pathway for the development of disease and arrhythmia.     

Reactive oxygen species directly modify sodium–calcium exchanger activity in a splice variant-dependent manner

The sodium–calcium exchanger isoform 1 (NCX1) operating in calcium-efflux mode plays an important role in maintaining calcium homeostasis in the heart. Paradoxically, activity of NCX1 in calcium-influx mode contributes to the pathological intracellular calcium overload during cardiac ischemia–reperfusion injury. Reactive oxygen species (ROS) also contribute to myocardial dysfunction in ischemia–reperfusion and are reported to alter NCX1 activity. However, the molecular mechanism(s) by which ROS modifies NCX1 activity have not been elucidated. Therefore, the effects of the ROS, H₂O₂, on recombinant NCX1 splice variants were studied using the patch-clamp technique. H₂O₂ irreversibly increased calcium-influx mode activity in the cardiac NCX1.1 splice variant, without affecting calcium-efflux mode activity. In direct contrast, H₂O₂ inhibited the calcium-influx mode of the vascular NCX1.3 splice variant indicating that these disparate effects of H₂O₂ may be dependent on the exon complement of the alternative splicing region. Using NCX1 splice variants with various exon compositions, the mutually exclusive exons A and B were found to bestow the differential effects of H₂O₂ on NCX1 function. As NCX1 inhibition is a potential therapeutic strategy for ischemia–reperfusion injury, the effects of the NCX1 inhibitor KB-R7943 were examined. KB-R7943 was ~ 7-fold less potent at inhibiting NCX1 activity after H₂O₂ modification. In summary, this study provides insights into the molecular regulation of NCX1 by ROS and indicates that ROS may elicit differential effects in various tissues depending on the exon composition of the splice variant expressed. These results also highlight that the potency of NCX1 inhibitors may be impaired under conditions of oxidative stress.     

Microarray analysis of rat hippocampus exposed to excitotoxicity: reversal Na(+)/Ca(2+) exchanger NCX3 is overexpressed in glial cells

Multiple factors are involved in the glutamate-induced excitotoxicity phenomenon, such as overload of ionotropic and metabotropic receptors, excess Ca(2+) influx, nitric oxide synthase activation, oxidative damage due to increase in free radicals, and release of endogenous polyamine, among others. In order to attempt a more integrated approach to address this issue, we established, by microarray analysis, the hippocampus gene expression profiles under glutamate-induced excitotoxicity conditions. Increased gene expression is mainly related to excitotoxicity (CaMKII, glypican 2, GFAP, NCX3, IL-2, and Gmeb2) or with cell damage response (dynactin and Ecel1). Several genes that augmented their expression are related to glutamatergic system modulation, in particular with NMDA receptor modulation and calcium homeostasis (IL-2, CaMKII, acrosin, Gmeb2, hAChE, Slc83a, and SP1 factor). Conversely, among genes that diminished their expression, we found the Syngap 1, which is downregulated by CaMKII, and the MHC II, which is downregulated by glutamate. Changes observed in gene expression induced by monosodium glutamate (MSG) neonatal treatment in the hippocampus are consistent with the activation of the mechanisms that modulate NMDA receptor function as well as with the implementation of plastic response to cell damage and intracellular calcium homeostasis. Regarding this aspect, we report here that NCX3/Slc8a3, a Na(+)/Ca(2+) membrane exchanger, is highly expressed in astrocytes, both in vitro and in vivo, in response to glutamate-induced excitotoxicity. Hence, the results of this analysis present a broad view of the expression profile elicited by MSG neonatal treatment, and lead us to suggest the possible molecular pathways of action and reaction involved under this experimental model of excitotoxicity.     

SEA0400 fails to alter the magnitude of intracellular Ca2+ transients and contractions in Langendorff-perfused guinea pig heart

SEA0400 is a recently developed inhibitor of the Na(+)/Ca(2+) exchanger (NCX) shown to suppress both forward and reverse mode operation of NCX. Present experiments were designed to study the effect of partial blockade of NCX on Ca handling and contractility in Langendorff-perfused guinea pig hearts loaded with the fluorescent Ca-sensitive dye fura-2. Left ventricular pressure and intracellular calcium concentration ([Ca(2+)](i)) were synchronously recorded before and after cumulative superfusion with 0.3 and 1 muM SEA0400. SEA0400 caused no significant change in the systolic and diastolic values of left ventricular pressure and [Ca(2+)](i). Accordingly, pulse pressure and amplitude of the [Ca(2+)](i) transient also remained unchanged in the presence of SEA0400. SEA0400 had no influence either on the time required to reach peak values of pressure and [Ca(2+)](i) or on half relaxation time. On the other hand, both 0.3 and 1 muM SEA0400 significantly increased the decay time constant of [Ca(2+)](i) transients, obtained by fitting its descending limb between 30% and 90% of relaxation, from 127 +/- 7 to 165 +/- 7 and 177 +/- 14 ms, respectively (P < 0.05, n = 6). In contrast to the guinea pig hearts, rat hearts responded to SEA0400 treatment with increased [Ca(2+)](i) transients and contractility. These interspecies differences observed in the effect of SEA0400 can be explained by the known differences in calcium handling between the two species.