Objective: Norepinephrine (NE)-induced apoptosis in cardiomyocytes is an important cause of heart failure. Previous studies revealed that reactive oxygen species (ROS) are involved in apoptosis. Tumor necrosis factor-α (TNF), a well-known mediator that stimulates apoptosis, is not only produced by macrophages but also by cardiomyocytes. Until now, the role of TNF and its relationship to ROS in NE-induced apoptosis of cardiomyocytes have never been investigated. Methods: Neonatal rat cardiomyocytes were treated with various concentrations of NE. Apoptosis of cardiomyocytes was determined using the TUNEL assay. The level of secreted TNF was measured by ELISA and TNF mRNA expression was determined by semiquantitative reverse transcriptional polymerase chain reaction. Caspase activity was measured by a fluorogenic protease assay kit. Anti-TNF antibodies, caspase inhibitors and antioxidants (N-acetyl-l-cysteine or vitamin C) were added to determine if they could inhibit the apoptotic effect of NE. Results: NE induced apoptosis of cardiomyocytes in a dose- and time-dependent manner. NE up-regulated TNF mRNA expression and increased TNF secretion and caspase-2,-3,-6, and -9 activities. A neutralizing anti-TNF antibody and caspase-2 and -3 inhibitors significantly attenuated NE-induced apoptosis. Antioxidants completely abrogated NE-induced TNF secretion, caspase activation, and apoptotic death. Conclusion: NE induced apoptosis in neonatal rat cardiomyocytes through a ROS–TNF–caspase signaling pathway.
Apoptosis is a form of programmed cell death that contributes to the pathogenesis of a number of human diseases . Heart failure is the final common pathway of diverse etiologies that result in impaired systolic and diastolic function with high morbidity and mortality . Accumulating evidence shows that apoptosis of cardiomyocytes plays an important role in causing heart failure [3–5]. However, the mechanism by which cardiomyocytes fall into apoptosis is still unclear.
Increased sympathetic nerve activity in the myocardium is a central feature of patients with heart failure [6–10]. Norepinephrine (NE), the primary transmitter of the sympathetic nervous system, is able to induce apoptosis of cardiomyocytes in many studies [10–13]. NE exerts its effect by binding to G-protein-coupled adrenergic receptors and then activating the cAMP–protein kinase A pathway [10–13]. However, the gene and terminal protein that are activated by this pathway remain unidentified.
Tumor necrosis factor-α (TNF), a pleiotropic cytokine, is a well-known mediator to induce apoptosis . It is not only produced by macrophages but also by cardiomyocytes . Because apoptosis of cardiomyocytes and high levels of NE and TNF coexist in the failing heart [3–10,15–20], we hypothesize that TNF is the downstream protein and plays a pivotal role in NE-induced apoptosis of cardiomyocytes. Previous report demonstrated that reactive oxygen species (ROS) could induce cardiomyocyte apoptosis . Furthermore, β-adrenergic receptor-stimulated apoptosis is mediated by ROS-dependent pathway . The purpose of the present study was to investigate the role of TNF and its relationship to ROS in NE-induced apoptosis of cardiomyocytes. Here we found that treatment with high concentrations of NE stimulates TNF gene expression and then induces apoptosis in cultured rat cardiomyocytes. Antioxidants completely abrogate NE-mediated TNF secretion and apoptotic death.
Vitamin C (Vit-C, ascorbic acid), N-acetyl-l-cysteine (NAC), NE, pancreatin, 4′,6-diamidino-2-phenylindole-2 HCl (DAPI), monoclonal anti-desmin antibody and cytosine β-d-arabino-furanoside were purchased from Sigma. Anti-rat TNF neutralizing antibody (500-P72) and anti-rat-IL-6 neutralizing antibody (500-P73) were obtained from PeproTech EC LTD, UK. Anti-rat-IL-6 non-neutralizing antibody was obtained from Santa Cruz. Anti-rat TNF capture antibody (26971E), OptEIA™ rat TNF set, and TMB substrate reagent set were obtained from Pharmingen (San Diego, CA, USA). 1st-STRAND™ cDNA synthesis kit was purchased from Clontech (USA). Caspase-2 inhibitor (Z-VDVAD-FMK), caspase-3 inhibitor (Z-DEVD-FMK), caspase-8 inhibitor (Z-IETD-FMK), and caspase-9 inhibitor (Z-LEHD-FMK) were purchased from KAMIYA Biochemical.
2.2. Cardiomyocyte culture
Cardiomyocyte cultures were prepared from 1-day-old neonatal Sprague–Dawley rat hearts as described elsewhere . Isolated cardiomyocytes were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 IU/ml penicillin, and 0.1 μg/ml streptomycin. To inhibit non-cardiomyocyte cell proliferation, cytosine arabinose (10 μM; Sigma-Aldrich) was added throughout the culture period. The medium was changed every 2 days. After 4 days in culture, the medium was replaced with serum-free medium. Cardiomyocyte cultures thus obtained were more than 95% pure, as revealed by observation of their contractile characteristics under a phase-contrast microscope and by indirect immunofluorescence staining with monoclonal antibodies directed against desmin (1:200; Sigma) (Fig. 1) as described previously . This project was approved by the Institutional Animal Care and Use Committees of the Taichung Veterans General Hospital. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
2.3. Apoptosis determination
Cardiomyocytes were treated with NE for the indicated time period. Both floating and adherent cells were collected for apoptosis determination. These collected cells were washed with cold phosphate-buffered saline, and fixed with 2% paraformaldehyde at room temperature for 30 min. In situ cell death was detected in cultured cardiomyocytes by using Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) assay kit (Roche Diagnostics, Meylan, France) according to the manufacturer's instructions. Cells were then washed and double stained with 1 μg/ml DAPI at room temperature for another 20 min. The labeled cardiomyocytes were examined by fluorescence microscopy to quantify apoptotic cells. The percentage of TUNEL-positive cardiomyocytes (with green fluorescent nuclei) was measured at 200 × magnification in five randomly chosen fields of each of nine independent experiments. The proportion of TUNEL-positive cardiomyocytes was expressed as a percentage of the total cells counted.
2.4. TNF measurement
Primary cultured rat cardiomyocytes were treated with or without NE for the indicated time points. After incubation, the medium was collected and TNF levels were quantified using an ELISA assay kit specific for rat TNF with a lower limit of detectability of 15 pg/ml (Pharmingen). The assay was performed according to the instructions provided by the manufacturer. Results are expressed as picograms of TNF per milliliter of medium.
2.5. RNA analysis
Total RNA was extracted from primary cultured neonatal rat cardiomyocytes using the guanidium isothiocyanate–chloroform method . For semiquantitative reverse transcriptional polymerase chain reaction (RT-PCR) analysis of TNF expression, total RNA (2 μg) was annealed with random primers at 70 °C for 10 min. The cDNA was synthesized using a 1st-STRAND™ cDNA Synthesis Kit (Clontech) in 20 μl of solution containing 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 10 mM dithiothreitol, 500 μM dNTP, and 200 units of reverse transcriptase at 42 °C for 50 min. For PCR amplification of TNF-specific cDNA, the reaction mixture (30 μl) was prepared on ice and contained 20 mM Tris–HCl (pH 8.4), 50 mM KCl, and 2 mM MgCl2, 1 unit of Taq polymerase (Advanced Biotechnologies, UK), 200 μM of each dNTP, 100 pmol of both forward and reverse primers, and 2.5 μg of cDNA product. The reaction conditions were denaturing cDNA for 7 min at 95 °C and submitted to multiple cycles of amplification (1 cycle: 95 °C, 50 s; 63 °C, 70 s; 72 °C, 60 s) followed by a final extension of 7 min at 72 °C in a Bio-Rad icycler (Bio-Rad). For each combination of primers, the kinetics of PCR amplification was studied. The number of cycles corresponding to the plateau was determined. PCR was performed at an exponential range. PCR primers derived from rat sequences were as follows: TNF forward primer 5′-TACTGAACTTCGGGGTGATTGGTCC-3′; reverse primer 5′-CAGCCTTGTCCCTTGAAGAGAACC-3′. GAPDH forward primer 5′-TATGACAACTCCCTCAAGAT-3′; reverse primer 5′-AGATCCACAACGGATACATT-3′. We analyzed the expression of the GAPDH gene as an internal control. PCR products were separated by electrophoresis on 2% agarose gels and stained with ethidium bromide. Gels were analyzed using the EverGene Image System. Bands with the expected size of 295 base pairs (bp) for TNF, 317 bp for GAPDH were detected.
2.6. Caspase-activity assay
Caspase-1, 2,-3,-6,-8, and -9 activities were measured by caspase fluorogenic protease assay kit (R&D System Minneapolis, USA). Briefly, cardiomyocytes were lysed to collect their intracellular contents. The cell lysate then was tested for caspase activity by addition of a caspase-specific peptide substrate that is conjugated with a fluorescent reporter molecule 7-amino-4-trifluoromethyl coumarin. The cleavage of the peptide by the caspase releases the fluorochrome that when excited by light at the 400 nm wavelength emits fluorescence at 505 nm , the fluorescence signal detected with a fluorometer, or a fluorescent microplate reader (Fluoroskan Ascent; Labsystems, Finland). Each caspase activity was determined by comparing the results of the treatment with the level of the control.
2.7. Statistical analysis
All data are expressed as mean±S.D. of at least nine independent experiments. For statistical analysis, we performed an unpaired two-tailed Student's t-test. The mean values of two groups were considered significantly different if *p<0.05, **p<0.01, or ***p<0.001.
3.1. NE-induced apoptosis in a dose- and time-dependent manner
To quantify the effect of NE on cardiomyocyte death, neonatal rat cardiomyocytes were exposed to various concentrations of NE, and apoptosis was estimated after 48 h of culture by TUNEL assay (Fig. 2A). As shown in Fig. 2B, treatment of cardiomyocytes with 1 μM NE failed to induce apoptosis, whereas increasing NE concentration from 10, 50, 100, to 200 μM caused a dose-dependent apoptosis in serum-free conditions, approximately 20±7%, 39±12%, 58±14%, and 92±9% apoptotic cells were detected in these samples, respectively. In control cultures only 9±3% apoptotic cells was observed. To examine the kinetics of apoptotic death induced by NE treatment, we performed a time-course analysis. The results in Fig. 2C indicate that NE (50 and 100 μM) caused time-dependent cell death in cardiomyocytes. Approximately 16±6%, 38±11%, and 65±14% apoptotic cells were observed after 12, 24, and 48 h of incubation, respectively, with 100 μM NE. These results demonstrate that NE triggers apoptosis in rat neonatal cardiomyocytes only at high concentrations (10–200 μM).
3.2. Effect of NE on TNF secretion
Previous studies report that elevation of NE concentrations in the plasma of experimental rats with liver dysfunction have been shown to stimulate the production of TNF from Kupffer cells both in vivo and in vitro . To determine whether the production of TNF was regulated by NE in cardiomyocytes, primary cultured cells were exposed to a series concentrations (10, 50, 100, and 200 μM) of NE for 48 h, then culture media were collected and levels of secreted TNF were measured by ELISA assay. Fig. 3A shows that NE potently induced TNF secretion from cardiomyocytes. This NE-mediated increase in the levels of TNF secretion was statistically significant at 50 μM NE and was maximal at 200 μM, giving a half-maximal action approximately at 70 μM. The time dependence of TNF secretion in the medium was also studied. As shown in Fig. 3B, elevated amounts of TNF were first observed after 24 h of incubation with 100 μM of NE, with a maximal secretion occurring by 48 h (112±15 pg/ml). To further verify whether NE regulates the gene expression of TNF, a semiquantitative RT-PCR analysis was performed. The results showed that treatment of cardiomyocytes with 100 μM NE markedly increased the levels of steady-state TNF mRNA (Fig. 4). NE-induced expression of TNF transcripts was first observed after 24-h exposure. However, TNF mRNA was not detectable in control cultures. These observations indicate that treatment of cardiomyocytes with a high concentration of NE could induce TNF mRNA production.
3.3. Attenuation of NE-induced apoptosis by neutralizing anti-TNF antibody
To elucidate whether NE-induced TNF production plays a critical role in NE-triggered apoptotic cell death in cardiomyocytes, antibody neutralization experiments were performed. We used two kinds of anti-TNF antibodies. One was the neutralizing anti-TNF antibody which can interfere the interaction between TNF and its receptor and subsequently inhibit the activation of TNF receptor. The other is the non-neutralizing anti-TNF antibody which binds with TNF but does not inhibit the TNF activity. After 48 h of treatment, both floating and adherent cells were collected and the apoptosis was estimated by TUNEL assay. Fig. 5 shows that the apoptotic effect of NE on cardiomyocytes was significantly attenuated by exogenously administered neutralizing anti-TNF antibody. A concentration of 2 μg/ml neutralizing antibody inhibited NE-induced apoptosis by 60%. However, co-incubation with non-neutralizing anti-TNF antibody or anti-IL-6 antibody did not affect NE-induced apoptotic cell death. These observations suggest that NE mediates apoptosis in cardiomyocytes, at least partially, through a TNF-dependent process.
3.4. NE leads to the activation of caspase-2, -3, -6, and -9
Previous study found that caspases were activated by NE in cardiomyocytes. To provide the activation state of caspases in NE-treated cardiomyocytes, six synthetic oligopeptide substrates were used: Ac-WEHD-AFC for caspase-1, -4, and -5; Ac-VDVAD-AFC for caspase-2; Ac-DEVD-AFC for caspase-3, -7, and -10; Ac-VEID-AFC for caspase-6; Ac-IETD-AFC for caspase-8; Ac-LEHD-AFC for caspase-9. As shown in Fig. 6A, caspase-2 and -3 (perhaps also caspase-7 and -10) were clearly activated at 24 h, earlier than other caspases, and maximal activities were observed at 36 h. Caspase-6 and -9 were significantly activated at 36 h NE treatment. However, caspase-1 and -8 activity did not significantly change during NE treatment. To define the involvement of specific caspase in NE-induced cardiomyocyte apoptosis, caspase inhibitors were used. Results showed that treatment with Z-VDVAD-FMK (caspase-2 inhibitor) and Z-DEVD-FMK (caspase-3 inhibitor) significantly prevent NE-triggered apoptosis, but Z-IETD-FMK (caspase-8 inhibitor) or Z-LEHD-FMK (caspase-9 inhibitor) did not (Fig. 6B).
3.5. Prevention of NE-triggered apoptotic death, TNF secretion, and caspase activation by antioxidant
Accumulating evidence indicated that reactive oxygen species (ROS) can cause cardiomyocyte apoptosis [21,28,29]. To clarify whether the NE-induced cell death, TNF production and caspases activation are mediated via a ROS-dependent pathway. Cardiomyocytes were treated with NE in the presence or absence of antioxidant (NAC or Vit-C). Antioxidant agents had no significant effects in the control cultures, but completely abrogated NE-induced TNF secretion (Fig. 7A), caspase activation (Fig. 7B), and apoptotic death (Fig. 7C).
The sympathetic nervous system has been viewed as the physiologically critical mechanism for cardiovascular response to increase circulatory needs during acute stress, augmenting cardiac rate and contractility, and changing peripheral vascular tone . However, excessive and prolonged activity of the system plays a pivotal role in the natural progression of heart failure . This includes early activation of cardiac adrenergic drive that is followed by an increasing magnitude of generalized sympathetic activation, with worsening heart failure. The hypersympathetic activity can negatively impact the heart in several ways, including down-regulating β1-receptors, exerting direct toxic effects on the myocardium, and contributing to myocardial remodeling and life-threatening arrhythmias .
NE, the primary transmitter of the sympathetic nervous system, is able to induce apoptosis of cardiomyocytes in many studies [9–13]. It signals via adrenergic receptors that are coupled to G-proteins. Pharmacological studies of cardiomyocytes in vitro demonstrate that stimulation of β1-adrenergic receptor induces apoptosis that is cAMP-dependent and involves the voltage-dependent calcium influx channel [9–13]. However, the gene and terminal protein that are activated by this pathway remain unidentified. The interesting finding of the present study is that upregulation of TNF production is involved in NE-induced apoptosis in rat neonatal cardiomyocytes. Similarly, recent studies have also demonstrated a connection between NE and TNF observed in vivo and in vitro in the hepatic system . The causal relationship between TNF and NE has been demonstrated by TNF inhibition that prevented NE-induced apoptosis in vitro and attenuated liver dysfunction in vivo [27,30]. These results suggest that there may be biologically important cross-talk between the sympathetic system and cytokines in the heart. Functionally, this cross-talk may lead to self-sustaining or self-amplifying autocrine/paracrine feedback circuits in the failing heart. The result is that TNF and/or NE production becomes sustained or increases to the extent where the toxic effects of these molecules are sufficient to contribute to disease progression in the failing heart. Whether this mechanism provides a satisfactory explanation for the sustained expression of TNF and/or increased activation of the sympathetic system that has been observed in the failing human heart will require further study.
Previous experimental studies and clinical work suggest that TNF plays a pathological role in heart failure [14–20]. The expression of this cytokine is limited in the normal heart but can be significantly induced in failing hearts in response to certain humoral factors or mechanical stress . These findings suggest that the myocardium represents an important source for TNF production in heart failure. In the present study, we indicated that NE stimulates TNF mRNA expression and protein secretion in rat neonatal cardiomyocytes in a dose-dependent manner. The amount of TNF produced by treatment with NE was ranged from 30 to 150 pg/ml, which is similar to that observed in patients with heart failure [18,19]. In the immune system, the biosynthesis of TNF is regulated primarily at the translational level [31,32]. Nevertheless, this study shows that NE stimulates not only the release of TNF but also the synthesis of TNF mRNA in cardiomyocytes. These observations suggest that the biosynthesis of TNF by NE in rat neonatal cardiomyocytes is regulated at least partial at the transcriptional level. In addition, our findings suggest that neutralizing anti-TNF antibody can significantly abrogate the apoptotic effect of NE on neonatal rat cardiomyocytes. It implies that anti-TNF treatment may prevent cardiomyocyte apoptosis in patients with heart failure. Recent studies have shown that treatment with etanercept, a TNF antagonist, led to a significant dose-dependent improvement in left ventricular structure and function and a trend toward improvement in patient functional status . However, some other clinical trials of anti-TNF therapy have demonstrated no clinical benefit . The reason of the different results is not clear, but a more specific TNF antagonist with fewer side effects and a further study are demanded.
In previous in vitro studies, NE at a concentration of 10 μM induced apoptosis in adult cardiomyocytes by 20–30% [10–13]. The present study shows that NE at the concentration of 10, 50, 100, and 200 μM causes 20±7%, 39±12%, 58±14%, and 92±9% apoptosis in neonatal cardiomyocytes, respectively. The apoptotic effect of 10 μM NE is similar between adult and neonatal cardiomyocytes. To observe significant protective effects of anti-TNF antibodies, caspase inhibitors and antioxidants, the present study chose approximately 50% apoptotic dose of 100 μM NE to undergo experiment, although it is unusual in clinical condition.
Classically TNF induces apoptosis through binding to TNF receptor and then activating caspase-8. Recent studies demonstrate that RAIDD/CRADD, a novel adaptor molecule of TNF receptor, interacts with prodomain of caspase-2 and then activates caspase-2 . Accumulating evidences indicate that TNF operates apoptotic death with caspase-2 acting as the upstream initiator in the activation of caspases [36,37]. However, events subsequent to caspase-2 activation remain largely unknown. Paroni et al.  reports that caspase-2 is a regulative caspase, which is able to induce mitochondrial dysfunction and subsequent activate apoptosome through direct or indirect Bid processing. Another report demonstrates that caspase-2 acts upstream of mitochondria to promote cytochrome c release and subsequent activation of caspase-9 and -3 . In this study, NE induces the activation of caspase-2, -3, -6, and -9 but not caspase-1 and -8. It implicates that TNF operates its apoptotic effect through RAIDD/CRADD–caspase-2 pathway and not classical FADD–caspase-8 pathway. Furthermore, our observations show that treatment with inhibitors of caspase-2 or -3 abolish the NE-induced apoptosis, but the inhibitors of caspase-8 or -9 do not. It is possible that the activation of caspase-9 could be the downstream of the caspase-2-dependent Bid processing or the result of a caspase amplification loop during the apoptotic response. Thus, inhibitor of caspase-9 does not have any effects against NE-induced-apoptosis. However, additional studies are needed to characterize the mechanism of caspase-2 activation as well as subsequent events leading to the engagement of the downstream apoptotic pathway.
Growing evidence demonstrate that ROS may mediate cardiomyocyte apoptosis [28–30]. Administration of antioxidants had been shown to reduce cardiomyocyte apoptosis in rat after a large myocardial infarction . It had been reported that increased hydroxyl free radical generation was observed in the rat heart after NE administration and cardiac sympathetic nerve stimulation [41,42]. Recently, Remondino et al.  demonstrated that β-adrenergic receptor-stimulated apoptosis is mediated by ROS-dependent pathway in rat cardiomyocytes. In addition, Qin et al.  provided evidence that the induction of cardiomyocyte apoptosis was associated with an increase in tissue oxidative stress in NE-treated ferrets, this apoptotic effect was effectively prevented by co-administration of antioxidant vitamins. In this study, we found that treatment with antioxidant agents (such as Vit-C and NAC) completely blocked TNF production, caspases activation and apoptosis induced by NE. Our observations implicate that ROS could act as the upstream initiators of NE-induced apoptosis in cardiomyocytes. These results suggest that ROS are involved in the regulation of TNF protein production and apoptosis upon NE treatment. In consistence with our observations, a recent study shows that ROS may contribute in an important manner to initiating cardiomyocyte apoptosis through TNF expression . Many studies demonstrate that ROS regulated the expression of TNF via activation of redox-sensitive transcription factor, NFκB . At present, the exact mechanisms responsible for the effect of ROS on the NE-mediated TNF production and caspases activation in cardiomyocytes have not been clearly defined, and require further investigation.
In conclusion, the present study provides genetic and biochemical evidence that high concentration of NE induces cardiomyocyte apoptosis in vitro via a ROS–TNF–caspases signaling pathway. Antioxidants and anti-TNF treatments can drastically block this apoptosis, suggesting antioxidants and anti-TNF strategies may be useful in the management of heart failure induced by NE.
This study was supported by grants from Taichung Veterans General Hospital and Cardiac Children's Foundation, Taiwan (CCF 01-11).