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(Circulation. 2004;109:406-411.)
© 2004 American Heart Association, Inc.

Basic Science Reports

N-Acetylcysteine Prevents the Deleterious Effect of Tumor Necrosis Factor- on Calcium Transients and Contraction in Adult Rat Cardiomyocytes

Michel Cailleret, MSci^*; Aïssata Amadou, MSci^*; Nathalie Andrieu-Abadie, PhD; Artur Nawrocki, PhD; Christophe Adamy, PhD; Bouziane Ait-Mamar, MD; François Rocaries, PhD; Martin Best-Belpomme, MD, PhD; Thierry Levade, MD, PhD; Catherine Pavoine, PhD; Françoise Pecker, PhD

From INSERM Unité 581, Hôpital Henri Mondor, Créteil (M.C., A.A., A.N., C.A., B.A.-M., M.B.-B., C.P., F.P.), INSERM Unité 466, CHU Rangueil, Toulouse (N.A.-A., T.L.), and Ecole Superieure d’Ingenieurs en Electrotechnique et Electronique, Noisy-Le-Grand (F.R.), France. Dr Nawrocki is now at the Medical Academy of Bialystok, Department of Physiology, Bialystok, Poland.

Correspondence to Françoise Pecker, INSERM Unité 99, Hôpital Henri Mondor, 94010 Créteil, France. E-mail francoise.pecker{at}im3.inserm.fr

Received December 16, 2002; de novo received August 12, 2003; accepted September 19, 2003.

	Abstract

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Background— The negative effect of tumor necrosis factor- (TNF-) on heart contraction, which is mediated by sphingosine, is a major component in heart failure. Because the cellular level of glutathione may limit sphingosine production via the inhibition of the Mg-dependent neutral sphingomyelinase (N-SMase), we hypothesized that cardiac glutathione status might determine the negative contractile response to TNF-.

Methods and Results— We examined the effects of TNF- in isolated cardiomyocytes obtained from control rats or rats that were given the glutathione precursor N-acetylcysteine (NAC, 100 mg IP per animal). In cardiomyocytes obtained from control rats, 25 ng/mL TNF- increased reactive oxygen species generation and N-SMase activity (500% and 34% over basal, respectively) and decreased the amplitude of [Ca²⁺]_i in response to electrical stimulation (22% below basal). NAC treatment increased cardiac glutathione content by 42%. In cardiomyocytes obtained from NAC-treated rats, 25 ng/mL TNF- had no effect on reactive oxygen species production or N-SMase activity but increased the amplitude of [Ca²⁺]_i transients and contraction in response to electrical stimulation by 40% to 50% over basal after 20 minutes. This was associated with a hastened relaxation (20% reduction in t_1/2 compared with basal) and an increased phosphorylation of both Ser¹⁶- and Thr¹⁷-phospholamban residues (260% and 115% of maximal isoproterenol effect, respectively).

Conclusions— It is concluded that cardiac glutathione status, by controlling N-SMase activation, determines the severity of the adverse effects of TNF- on heart contraction. Glutathione supplementation may therefore provide therapeutic benefits for vulnerable hearts.

Key Words: tumor necrosis factor- • phospholamban • N-acetylcysteine • glutathione • sphingomyelinase

	Introduction

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High expression in the myocardium and high levels in serum of the proinflammatory cytokine tumor necrosis factor- (TNF-) are causally linked to the progression of heart failure.^1–4 TNF- exerts a dual effect on heart contraction^5,6 that we could reproduce in isolated, electrically stimulated adult rat cardiomyocytes. At low concentrations, TNF- increases the amplitude of [Ca²⁺]_i transients and contraction in response to electrical stimulation, whereas at high concentrations, it impairs electrically stimulated [Ca²⁺]_i transients and contraction.⁷ The negative inotropic effect of TNF- relies on arachidonic acid, released on activation of the cytosolic phospholipase A₂, and sphingosine.^7,8 We proposed that sphingosine was produced by downstream ceramide production after activation by arachidonic acid of the Mg-dependent neutral sphingomyelinase (N-SMase).⁷ In such a hypothesis, under conditions in which N-SMase activation would be impaired, the negative effect of high TNF- concentrations should be abrogated. Furthermore, the positive effect on [Ca²⁺]_i transients and contraction, which is detectable at low TNF- concentrations and is not mediated by sphingosine, might be unmasked.

Heart failure is associated with oxidative stress and depletion in the tripeptide glutathione, which ensures the maintenance of cell redox status.⁹ Independently of the reduced/oxidized glutathione ratio (GSH/GSSG), cellular glutathione depletion stimulates N-SMase activity.^10–12 The aim of the present study was therefore to look for a possible link between cardiac glutathione content, N-SMase activity, and the contractile response to TNF-. We examined reactive oxygen species (ROS) generation, N-SMase activity, electrically stimulated [Ca²⁺]_i transients, and contraction in response to TNF- in isolated cardiomyocytes obtained from rats that were given the glutathione precursor N-acetylcysteine (NAC).

	Methods

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Cardiomyocyte Isolation
The care and the use of the animals were in accordance with institutional guidelines. Adult, male Wistar rats (180 to 250 g, Janvier, LeGenest St Isle, France) were used. Calcium-tolerant myocytes were isolated by cardiac retrograde aortic perfusion as described by Delcayre et al.¹³ Freshly isolated cardiomyocytes were plated on laminin (3 µg/mL; Sigma) and cultured for up to 2 days according to Sambrano et al.¹⁴ When indicated, rats were treated with NAC (Sigma) administered twice intraperitoneally (100 mg per animal), 48 and 24 hours before anesthesia. As shown in Figure 3, exogenously added GSH 10 mmol/L did not affect the response of cardiomyocytes to electrical stimulation either in the basal state or in the presence of TNF-. Thus, to avoid possible leakage of cellular GSH, 10 mmol/L GSH was added to the heart perfusion, cardiomyocyte attachment, fura 2-AM loading, and [Ca²⁺]_i imaging media when cardiomyocytes were obtained from NAC-treated rats.

View larger version (19K): [in this window] [in a new window]   Figure 3. Exogenously added GSM reproduced effects of in vivo NAC treatment. After preincubation for 2 to 3 hours and loading with fura 2-AM in a medium supplemented with either 10 mmol/L GSH, 50 µmol/L NAC, 50 µmol/L GSM, 10 µmol/L butylated hydroxyanisol (BHA), or 1 µg/mL vitamin (Vit) E, cardiomyocytes isolated from control rats were electrically stimulated in a medium supplemented with molecule tested at given concentration and with or without 25 ng/mL TNF-. Amplitude of [Ca2+]i transients after 30 minutes is normalized to control value determined at time zero. Values are mean±SEM of effects observed on 5 cells obtained from 3 different isolations. *P<0.05 vs corresponding basal value; P<0.05 vs corresponding control value.

Measurement of [Ca²⁺]_i Transients and Cell Fractional Shortening
Plating of cardiomyocytes on laminin, loading with fura 2-AM (Molecular Probes), field electrical stimulation (square waves, 0.5 Hz), [Ca²⁺]_i imaging, and fractional shortening were performed as previously described.^7,15 Results are shown as mean±SEM for 5 to 10 cells obtained from 3 different isolations, or as typical.

Measurement of Intracellular ROS
Cardiomyocytes attached on laminin (10⁵ cells per 2-cm-diameter dish) were loaded with 5 µmol/L 2N,7N-dichlorodihydrofluorescein diacetate (H₂DCF-DA) (Molecular Probes) in the conditions described for fura 2-AM loading.^7,15 DCF fluorescence was recorded at an excitation wavelength of 480 nm and a 510-nm filter.¹⁶ Data are expressed as percent of the maximal effect produced by 3.5 mmol/L H₂O₂ and are mean±SEM for 5 to 10 cells obtained from 3 different isolations.

N-SMase Activity
Cardiomyocytes attached on laminin (6.5x10⁵ cells per 9-cm-diameter dish) were bathed at 37°C in a saline buffer (mmol/L: glucose 10, NaCl 130, KCl 5, HEPES 50, pH 7.4, MgCl₂ 1, CaCl₂ 2), with or without 25 ng/mL TNF-. After 30 minutes, cells were harvested by scraping and frozen in liquid nitrogen. Aliquots were assayed for N-SMase activity as previously described.¹⁷ Values are mean±SEM for 3 to 5 isolations.

Analysis of Site-Specific Phospholamban Phosphorylation in Isolated Hearts
Hearts were perfused in the Langendorff mode with a modified Krebs-Henseleit perfusion medium containing (mmol/L) NaCl 135, KCl 4.75, KH₂PO₄ 1.19, Na₂HPO₄ 16, NaHCO₃ 25, sucrose 134, HEPES 10, and glucose 10, gassed with 95% O₂/5% CO₂ (pH 7.4, 37°C). When stated, 1 µmol/L isoproterenol (Sigma) for 2 minutes or TNF- (Peprotech Inc) at varying concentrations and for 10 minutes was added to the perfusion medium. Left ventricles were homogenized at 4°C with an Ultra-Turrax T25 (Janke-Kunkel) in buffer containing (mmol/L) Tris-HCl 10, pH 7.4, Na₄P₂O₇ 10, sucrose 300, dithiothreitol 1, vanadate 0.4, phenylmethanesulfonyl fluoride 0.3, and NaF 50. Homogenates were centrifuged at 8000g for 20 minutes.

For Western blot analysis, supernatant proteins were solubilized in Laemmli loading buffer, boiled for 5 minutes, and resolved with 18% SDS-polyacrylamide gels using a 6% stacking gel. Proteins were transferred to polyvinylidene difluoride membranes (0.22 µm, Millipore) by electroblotting. Membranes were next incubated with the primary antibodies P-Ser¹⁶– or P-Thr¹⁷–phospholamban (PLB) (1:1250 dilution; Cyclacel Ltd), then with peroxidase-conjugated donkey anti-rabbit IgG (1:5000 dilution, Jackson Immunoresearch). The peroxidase activity was visualized with an enhanced chemiluminescent detection kit, ECL PLUS (Amersham Biosciences). Loading and protein transfers were verified with a mouse actin antibody (AC 15, Sigma).

Dot blots were performed for quantification of PLB phosphorylation with 0.22-µm nitrocellulose membrane (Schleicher and Schuell). For standardization, samples (0.03 to 2.5 µg proteins) of pooled ventricular tissues obtained from hearts perfused with 1 µmol/L isoproterenol for 2 minutes were applied in parallel. Immunodetection of phosphorylated PLB was performed as described above for the Western blots. P-Ser¹⁶– or P-Thr¹⁷–PLB was quantified by scanning densitometry (NIH image 1.6; David Chow and Jai Evans) and calculated from the standard curve established with isoproterenol samples, the 2.5-µg sample of isoproterenol being taken as 100%. Values are mean±SEM for 3 to 5 hearts.

Measurement of Total Glutathione Content in Heart
Total glutathione (GSH+GSSG) was measured according to a modification of the method of Tietze.¹⁸ Values are mean±SEM for 3 hearts.

Statistical Analysis
Results were analyzed by the Student 2-tailed t test or repeated-measures ANOVA and post hoc multiple comparison testing between control and treatment groups (Dunnett test), as appropriate. Differences were considered statistically significant at a value of P<0.05.

	Results

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NAC Treatment Increases GSH Content in the Heart
NAC treatment (100 mg IP per animal, administered twice, 48 and 24 hours before anesthesia) increased cardiac GSH+GSSG by 42±6% (P<0.05), suggesting that NAC augmented the buffering capacity against oxidative stress of the heart (Table 1).

View this table: [in this window] [in a new window]   TABLE 1. Effects of NAC Treatment on Total Glutathione Content in Heart, ROS Generation, and N-SMase Activity in Isolated Cardiomyocytes

NAC Treatment Blunts ROS Generation and N-SMase Activation Elicited by TNF-
In cardiomyocytes obtained from control rats, ROS generation, in the basal state and after 30 minutes, reached 10.8±4.1% of the maximal increase produced by H₂O₂ (P<0.05, Table 1). In those cardiomyocytes isolated from control rats, 25 ng/mL TNF- increased ROS generation by 6-fold, which after 30 minutes reached 66.2±6.2% of H₂O₂ maximal response (P<0.05; Table 1). In contrast, no ROS generation was detected in cardiomyocytes obtained from NAC-treated rats, either in the basal state or in response to TNF- (Table 1). Those data proved that NAC treatment improved cardiomyocyte defense against oxidative stress and were relevant to the high glutathione level measured in NAC-treated rat hearts. In addition, N-SMase activation triggered by 25 ng/mL TNF- in cardiomyocytes obtained from control rats (1.25±0.09 versus 0.93±0.11 nmol · h^-1 · mg protein^-1 for basal, P<0.05) was abrogated in cardiomyocytes obtained from NAC-treated rats (0.97±0.12 versus 0.82±0.12 nmol · h^-1 · mg protein^-1 for basal). Those data agreed with previous observations in other cell types reporting that glutathione impaired TNF-–induced N-SMase activation.^11,19 Taken together, those findings support the proposal that NAC treatment can antagonize cell responses to TNF-.

NAC Treatment Abrogates the Negative Inotropic Effect of TNF- and Unmasks a Positive Effect
We have previously reported that TNF- exerted a dual, positive and negative, effect on [Ca²⁺]_i transients.⁷ At 10 ng/mL, TNF- increased the amplitude of [Ca²⁺]_i transients in cardiomyocytes obtained from control hearts (24±8% over control after 20 minutes, P<0.05, Figure 1A). In contrast, at higher concentrations (25 ng/mL), TNF- decreased the amplitude of [Ca²⁺]_i transients (22±6% below control levels, P<0.05). The negative effect of 25 ng/mL TNF- was significant after 10 minutes and developed for 30 minutes (Figure 1B). As also shown in Figure 1, not only the negative effect of high TNF- concentrations on [Ca²⁺]_i transients was abrogated in electrically stimulated cardiomyocytes⁷ obtained from NAC-treated rats, but a positive effect was also unmasked. After 20 minutes of perfusion, 25 to 50 ng/mL TNF- increased the amplitude of [Ca²⁺]_i transients by 40% to 50% over basal (P<0.05) (Figure 1A). The positive effect of TNF- was detected from 10 minutes and developed for at least 30 minutes (Figure 1B). The typical traces of [Ca²⁺]_i transients and fractional shortening in Figure 2 show the correlation between the increase in the amplitude of [Ca²⁺]_i transients and that of contraction in cardiomyocytes obtained from hearts of NAC-treated rats electrically stimulated and exposed to TNF-. After 20 minutes of exposure to 25 ng/mL TNF-, the amplitudes of [Ca²⁺]_i transients and fractional shortening were increased by 40% over the control value at time zero. Maximal 2- to 3-fold increases over basal were observed after 30 minutes (Figure 2). The positive inotropic effect of TNF- was associated with a lusitropic potency (Table 2). After 30 minutes of exposure to 25 ng/mL TNF-, the decay half-times (t_1/2) of both [Ca²⁺]_i transients (480±37 versus 609±35 ms, P<0.05) and contraction (486±12 ms versus 614±32 ms, P<0.05) were reduced to 80% of the basal values. Times to peak were reduced similarly (P<0.05; Table 2). It should be noted that oxidative stress induced by H₂O₂ (200 µmol/L) abrogated the positive effect of TNF- on the amplitude of [Ca²⁺]_i transients and contraction in cardiomyocytes obtained from NAC-treated rats (not shown).

View larger version (20K): [in this window] [in a new window]   Figure 1. NAC treatment abrogates negative effect and unmasks a positive effect of high TNF- concentrations on [Ca2+]i transients of electrically stimulated cardiomyocytes. Cardiomyocytes obtained from either control or NAC-treated rats were loaded with fura 2-AM and electrically stimulated at 0.5 Hz. A, Cardiomyocytes were exposed for 20 minutes to indicated concentration of TNF-. B, Time course of effect of 25 ng/mL TNF-. Amplitude of [Ca2+]i transients is normalized to control value determined at time zero. Values are mean±SEM of effects observed on 5 cells obtained from 3 different isolations. *P<0.05 vs basal; P<0.05 vs control rat.

View larger version (22K): [in this window] [in a new window]   Figure 2. Time course of positive effect of 25 ng/mL TNF- on [Ca2+]i transients and fractional shortening of electrically stimulated cardiomyocytes isolated from NAC-treated rats. Typical traces of simultaneously recorded [Ca2+]i transients (top) and fractional shortening (bottom) of cardiomyocytes isolated from NAC-treated rats, loaded with fura 2-AM, electrically stimulated at 0.5 Hz, and exposed to 25 ng/mL TNF-. Tracings are representative of 6 cells obtained from 3 different isolations.

View this table: [in this window] [in a new window]   TABLE 2. Effects of TNF- on the Kinetics of Cytosolic [Ca2+]i Transient and Contraction of Electrically Stimulated Cardiomyocytes Isolated From Control or NAC-Treated Rats

In Isolated Cardiomyocytes, Exogenously Added S-Methylglutathione Reproduced the Effects of In Vivo NAC Treatment
We asked whether the effects of NAC treatment relied on the increase in total cellular glutathione content rather than on cell redox status. Therefore, we examined the effects of 2- to 3-hour preincubations with either antioxidants or the glutathione donor S-methylglutathione (GSM) on the response to TNF- of isolated cardiomyocytes obtained from control rats. Butylated hydroxyanisol (10 µmol/L) and vitamin E (1 µg/mL) blunted the negative effect of 25 ng/mL TNF- on the amplitude of electrically stimulated [Ca²⁺]_i transients (P<0.05; Figure 3). Nevertheless, neither antioxidant unmasked a positive effect of TNF- at high concentrations. Conversely, glutathione repletion, achieved with 50 µmol/L GSM, did reproduce the effects of in vivo NAC treatment, unmasking a positive effect of 25 ng/mL TNF- (+50±0.17%, P<0.05; Figure 3). GSM had no effect on the amplitude of basal [Ca²⁺]_i transients. NAC had an intricate effect on the amplitude of [Ca²⁺]_i transients. Like both other antioxidants, NAC blunted the negative effect of TNF-. However, per se, it increased the amplitude of basal [Ca²⁺]_i (Figure 3). Thus, although a positive effect on [Ca²⁺]_i transients was observed with TNF- after preincubation with NAC, this effect could not be considered a specific TNF- effect. These data show that only the in vitro preincubation with the glutathione donor GSM could reproduce the in vivo effect of NAC.

NAC Treatment Promotes PLB Phosphorylation by TNF-
Relaxation depends on the activity of the sarcoplasmic reticulum Ca²⁺-ATPase, the activity of which is essentially controlled by its inhibitor dephosphorylated PLB. Phosphorylation of PLB reverses this inhibition, thereby accelerating Ca²⁺ uptake into the sarcoplasmic reticulum and facilitating cardiac relaxation.²⁰ In response to ß-adrenergic stimulation in vivo, PLB is phosphorylated at 2 adjacent amino residues, Ser¹⁶ and Thr¹⁷.^21,22 We looked for PLB phosphorylation as a possible component of the positive effect of TNF-. Because phosphorylation of the Thr¹⁷-PLB residue depends on the beating state of the cardiomyocyte,²² the experiments were performed on isolated, spontaneously beating, perfused hearts obtained from rats treated or not with NAC. The heart was perfused with medium with or without either 1 µmol/L isoproterenol for 2 minutes or with 10 or 25 ng/mL TNF- for 10 minutes. Figure 4 shows typical Western blots of site-specific Ser¹⁶- and Thr¹⁷-PLB phosphorylation induced by TNF- and quantification of the effects assessed by densitometric evaluations of dot blots. In control hearts, TNF- had no significant effect on the phosphorylation of either Ser¹⁶- or Thr¹⁷-PLB residues. NAC treatment also did not significantly affect basal phosphorylation of Ser¹⁶ and Thr¹⁷ residues. In contrast, in hearts obtained from NAC-treated rats, 10 to 25 ng/mL TNF- produced a dramatic increase in the phosphorylation of both Ser¹⁶- and Thr¹⁷-PLB residues (+260±70%, P<0.05, and +115±10% of isoproterenol, P<0.05, respectively, Figure 4).

View larger version (28K): [in this window] [in a new window]   Figure 4. TNF- triggers Ser16-PLB and Thr17-PLB phosphorylation in NAC-treated rat hearts. Hearts obtained from control rats were perfused with either 1 µmol/L isoproterenol for 2 minutes or indicated concentration of TNF- for 10 minutes. Hearts obtained from NAC-treated rats were perfused for 10 minutes with perfusion medium added with 10 mmol/L GSH and indicated concentration of TNF-. A, Autoradiograms are representative of Western blots probed with specific P-Ser16– or P-Thr17–PLB antibodies (left and right, respectively), each lane representing a separate isolated rat heart. B, Densitometric evaluations of dot blots probed with specific P-Ser16– or P-Thr17–PLB antibodies (left and right, respectively) are expressed as percentage of 1 µmol/L isoproterenol- induced Ser16- or Thr17-PLB phosphorylation, respectively, and are mean±SEM of 3 to 5 different hearts. *P<0.05 vs basal value; P<0.05 vs corresponding control rat.

	Discussion

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The new findings of this study are twofold. First, in vivo administration of NAC, which increases heart glutathione content, protects isolated cardiomyocytes against ROS generation and the negative effect of [Ca²⁺]_i transients and contraction produced by high TNF- concentrations. Second, such an anti–TNF- action of NAC is related to N-SMase inhibition.

Because it is a key player in the maintenance of cell antioxidant status, glutathione and its precursor NAC are generally matched with antioxidants. Clearly, a distinction should be made between the total cellular glutathione content and antioxidant status. Indeed, the decreases in both glutathione content and antioxidant status, related to an increase in oxidative stress, are crucial elements in the pathogenesis of heart failure. However, glutathione has other, nonrelated antioxidant effects. Thus, reduced as well as oxidized glutathione inhibits N-SMase activity.²³ Accordingly, only glutathione replenishment can compensate for glutathione depletion. Indeed, when added exogenously to isolated cardiomyocytes, only the glutathione donor GSM could reproduce the effects of in vivo NAC treatment. NAC added in vitro, which does not increase glutathione content in normoxic isolated cardiomyocytes,²⁴ solely shifts the negative contractile effect of TNF- to a null effect, as do the antioxidants butylated hydroxyanisol and vitamin E. Taken together, these data support the proposal that the effects of NAC in vivo treatment described here (1) are caused by the conversion of NAC into glutathione and are not imputable to a NAC byproduct and (2) are linked to an increase in glutathione content rather than a scavenging of antioxidants.

A previous study on rat neonatal cardiomyocytes showed that TNF- inhibited isoproterenol-induced PLB phosphorylation by stimulating the ceramide-activated phosphatase, phosphatase 2A.²⁵ We show here that the beneficial effect of NAC treatment is related to an increase in PLB phosphorylation in response to TNF-. Because NAC treatment triggers N-SMase inhibition, an increase in PLB phosphorylation might result from phosphatase 2A inhibition caused by a drop in ceramide. This highlights the implication of ceramide, in addition to sphingosine, and further strengthens the role of N-SMase in the negative contractile effect of TNF-.

In summary, the present study shows that administration of NAC suppresses the deleterious effect of TNF- on heart contraction. Importantly, NAC also inhibits the nuclear factor-B–dependent cardiac hypertrophic response to TNF-.²⁶ Taken together, those findings point out the unique property of NAC in antagonizing 2 major components of heart failure. However, the limitation of this study is that it addresses the protective effect of NAC against the acute negative inotropic effects of TNF-. The long-term beneficial effects of NAC treatment in animal models of developing heart failure remain to be investigated. Nevertheless, the absence of functional data does not detract from the fact that NAC abrogates TNF- effects deleterious to cardiomyocytes, a finding that raises the possibility of a new therapeutic option for human heart failure, consistent with the administration of NAC to minimize the impact of reperfusion injury in the treatment of acute myocardial infarction.²⁷ In conclusion, NAC might be regarded as a more general therapeutic tool for the management of vulnerable, aging, or failing hearts that are particularly exposed to TNF-.

	Acknowledgments

 
This work was supported by the Institut National de la Santé et de la Recherche Médicale, the French Ministère de la Recherche et de la Technologie, the North Atlantic Treaty Organization, and Université Paris XII. We thank N. Defer and S. Lotersztajn for helpful discussions and J. Hanoune and G. Guellaën for their continuing support.

	Footnotes

 
*These authors contributed equally to this work.

	References

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