Abstract: Recombinant human neuregulin-1 (rhNRG-1) improves cardiac function in animal models of doxorubicin (DOX)-induced cardiomyopathy, but the underlying mechanism remains largely unknown. Here, we confirm a role for rhNRG-1 in attenuating DOX-induced autophagy and define the signaling pathways through365医学网 转载请注明
which it mediates some of its effects. Neonatal rat ventricular myocytes were subjected to different treatments both to induce autophagy and to determine the effects of rhNRG-1 on the process. The rhNRG-1 inhibited DOX-induced autophagy, reduced reactive oxygen species production and increased protein expression of Bcl-2,365医学网 转载请注明
effects that were recapitulated when the cells were treated with the antioxidant N-acetylcysteine. These effects were blocked by the phosphatidylinositol 3-kinase inhibitor LY294002, pointing to the involvement of the Akt pathway in mediating the process. Inhibition of Bcl-2 expression with small interfering RNA silencing also365医学网 转载请注明
inhibited rhNRG-1’s ability to attenuate DOX-induced autophagy. The rhNRG-1 is a potent inhibitor of DOX-induced autophagy and multiple signaling pathways, including Akt and activation of reactive oxygen species, play important roles in the anti-autophagy effect. The rhNRG-1 is a novel drug that may be effectively therapeutically in protecting further damage in DOX-induced damaged myocardium.
Key Words: neuregulin, doxorubicin, autophagy, cardiotoxicity, Akt, Bcl-2
Doxorubicin (DOX) is an effective antineoplastic drug and is frequently used in the treatment of hematologic and solid tumors including leukemia, breast cancer, and sarcomas. However, its clinical benefit is limited by its cardiotoxicity.1,2 DOX-induced cardiomyopathy is characterized by irreversible left ventricular dysfunction and congestive heart failure with a poor prognosis.3,4 To date, treatments that to protect the heart from DOX-induced damage are limited.3 Therefore,the approaches to treating DOX-induced cardiotoxicity remains a critical issue in both cardiology and oncology.
Recent studies suggest that increased autophagy accompanies overt presentation of cardiac damage and may contribute to DOX-induced cardiotoxicity.5–8 Autophagy is a cellular process of “self-digestion” through the lysosomal degradation pathway and is highly conserved across multiple phyla. Elucidated first in lower organisms such as yeast,9 it is a multiple-step highly regulated mechanism for degrading proteins and organelles that have become damaged or need to be recycled for other reasons, such as utilization during calorie deprivation.9–11 Autophagy also has been implicated in various pathological365医学网 转载请注明
processes and some data suggest that autophagy may serve as a cell death mechanism under certain circumstances. This autophagy-dependent cell death has been defined as “autophagic cell death” or “type II programmed cell death.”12
Neuregulin (NRG)-1, a member of the NRG family, is expressed in many cell types and organs, including the heart. NRG-1/erbB signaling is essential for embryonic cardiac development. Postnatal conditional erbB2 deficiency in cardiomyocytes results in severe cardiomyopathy and enhanced myocyte susceptibility to DOX-induced death.13,14 Recombinant human neuregulin-1 (rhNRG-1, part of NRG-1) is a 61-amino acid peptide containing an epidermal growth factor-like domain, which is the domain necessary for erbB2/4 activation. Previously, we reported that rhNRG-1 could improve cardiac function in patients suffering from congestive heart failure, with significant increases in left ventricular ejection fraction. Treatment also decreased end systolic and diastolic volume,15 demonstrating a beneficial effect on pathological remodeling. It has been reported that rhNRG-1 can activate erb2/4 heterodimerization, thus improving cardiac function and survival in animal models of DOX-induced cardiomyopathy.16 However, the underlying molecular mechanism remains to be defined and the role of365医学网 转载请注明
autophagy in the process is unclear.
Based on these considerations, we tested the hypothesis that rhNRG-1 can attenuate DOX-induced autophagy using neonatal rat ventricular myocytes (NRVMs) as a model system. Here, we investigate the roles of rhNRG-1/erbB signaling and autophagy in DOX cardiotoxicity using the NRVM model of365医学网 转载请注明
DOX-induced cell autophagy.5–8 We demonstrate that rhNRG-1 attenuated DOX-induced autophagy and this is accompanied by Akt activation and reactive oxygen species (ROS) reduction, resulting in the upregulation of Bcl-2 protein levels. These signaling processes partially contribute to the ability of365医学网 转载请注明
rhNRG-1 to inhibit DOX-induced autophagy.
RhNRG-1, was a kind gift from Prof Zhou, Zensun Sci & Tech Ltd (Shanghai, China), N-acetylcysteine (NAC), DOX were obtained from Sigma-Aldrich (St Louis, MO). The 5(6)-365医学网 转载请注明
carboxy-20, 70-dichlorofluorescein diacetate (DCFH-DA) was from Molecular Probes Inc (Eugene, OR). LY294002 and primary antibodies against light chain 3 (LC3) of microtubuleassociated protein 1, Beclin 1, Bcl-2, phospho-Akt, Akt, and b-actin were obtained from Cell Signaling Technology (Danvers, MA). Horseradish peroxidase–conjugated secondary antibodies were purchased from Beyotime (Beijing, China).
We cultured NRVMs from 2-day-old Sprague–Dawley rats as previously described.17 The animal protocol was approved by the Fu Wai Hospital Animal Care and Use Committee and is in accordance with “Guide for the Care and Use of Laboratory Animals” published by the US National Institute of Health (National Institute of Health Publication No. 85-23, revised 1996). In brief, hearts were washed, the atria removed, and ventricles minced after dissection in N-2-hydroxyethylpiperazine-N0-2-ethane sulfonic acid (HEPES)-buffered saline solution containing 130 mM of NaCl, 3 mM of KCl, 1 mM of NaH2PO4,4 mM of glucose, and 20 mM of HEPES (pH adjusted to 7.35 with NaOH). We dispersed the tissues in a series of incubations at 378C in HEPES-buffered saline solution containing 1.2 mg/mL365医学网 转载请注明
of pancreatin and 0.14 mg/mL of collagenase (Worthington).After centrifugation, cells were resuspended in Dulbecco modified eagle medium (DMEM)/F-12 medium (GIBCO) containing 5％ (vol/vol) heat-inactivated horse serum, 0.1 mM of ascorbate,insulin-transferring sodium selenite media supplement, 100 U/mL of penicillin, 100 mg/mL of streptomycin, and 0.1 mM of bromodeoxyuridine. We preplated the dissociated cells at 378C for 1 hour, diluted them to 1 · 106 cells per milliliter and plated them in culture dishes coated with 10 mg/mL of laminin.
Detection of Intracelluar ROS Levels
The intracellular ROS levels were detected using a DCFH-DA probe. Briefly, cells were incubated with DCFH-DA (5 mM) in serum-free DMEM at 378C in a 5％ of CO2 incubator for 20 minutes, washed twice with phosphate buffered saline, and fluorescent images were acquired from a laser confocal microscope (Zeiss LSM 510 META,Berlin, Germany). Measurement of dihydrodichlorofluorescein (DCF) fluorescence intensity was performed using ImageJ v.1.42q. For each photograph, the cellular and the background average fluorescence signals were collected by365医学网 转载请注明
manually tracing the shape of cells. Results are displayed in a ratio metric fashion normalized for control conditions.
Transient Transfection With Green365医学网 转载请注明
To detect autophagy, cells were plated onto coverslips coated with 0.01％ (wt/vol) poly-L-lysine, grown for 16 hours, and then transiently transfected with the green fluorescent protein (GFP)-LC3 plasmid using FuGENETM HD (Roche) according to the manufacturer’s protocol. The cells were then incubated for 24 hours before treatment with the indicated drugs. The percentage of cells with more than 5 GFP-LC3 dots, which were considered to be undergoing active autophagy, was then quantified. We imaged it use a laser scanning confocal microscope (Zeiss LSM5 10 META).
Gene Silencing with Small Interfering RNA
SignalSilence Control small interfering RNA (siRNA) and SignalSilence Bcl-2 siRNA were purchased from Cell Signaling Technology. The siRNA transfection was performed using FuGENETM HD (Roche) according to the manufacturer’s protocol.
Immunoblotting and Immunoprecipitation
After the designated treatment was performed, cells from each group were lysed using radio-immunoprecipitation assay buffer containing 20 mm of Tris–HCl (pH 7.4), 150 mm of NaCl, 1％ of Nonidet P-40, 0.5％ of sodium deoxycholate, 0.1％ of sodium dodecyl sulfate (SDS), 0.004％ of sodium azide, 1％ of phenylmethanesulfonyl fluoride, 1％ of sodium orthovanadate, and 1％ of protease365医学网 转载请注明
inhibitor cocktail at 48C. The lysate was cleared by 10- minute centrifugation at 48C and 12,000g after which the supernates were collected. Protein concentration was determined using a bicinchoninic acid assay. Proteins (100 mg) were subjected to 12％ SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes. The membranes were blocked for 1 hour in 1％ of skim365医学网 转载请注明
milk and incubated overnight at 48C with the primary antibodies. The membranes were then probed using horseradish peroxidase–conjugated goat antirabbit immunoglobulin G.Antigen–antibody complexes were detected by means of enhanced chemiluminescence (American Biosciences365医学网 转载请注明
Crop, Blauvelt, NJ). The protein expression levels were determined by analyzing the signals captured on the nitrocellulose membranes using a Chemi-doc image analyzer (Bio-Rad).
For immunoprecipitation, protein concentration was determined using a bicinchoninic acid assay. Protein (250 mg) was taken and antibodies (2.5 mg/mL) were incubated for 2 hours at 48C with protein G-conjugated agarose beads (30 mL) and then washed 5 times with lysis buffer. To immunoprecipitate endogenous Beclin 1 and endogenous Bcl-2, anti-Beclin 1 immunoprecipitates were subjected to SDS-PAGE and Bcl-2 was detected by immunoblot analysis.
The results are expressed as mean 6 standard error of mean. The statistical significance was calculated by 1-way analysis of variance followed by the Tukey posthoc test for multiple comparisons. Two groups were evaluated by the Student t test. P , 0.05 was considered statistically significant.
The rhNRG-1 Inhibits365医学网 转载请注明
To confirm previous data and the usefulness of the NRVM system, we determined if upregulation of welldefined markers of autophagy and the levels of ROS production occurred as a result of DOX treatment. Cultures were plated, treated with DOX for 18 hours, and the proteins isolated and subjected to PAGE and Western analyses. As expected, autophagy was unregulated, with LC3-II and Beclin365医学网 转载请注明
1 significantly increased compared with protein levels in untreated cells (Fig. 1A). And the production of intracellular ROS was activated too (Fig. 1B). We also found that no differences were observed in the expression of LC3-II, Beclin 1, Bcl-2, and the levels of ROS production between control cells treated with rhNRG-1, LY294002, and NAC and untreated cells (Figs. 1A, B).
We then determined whether rhNRG-1 could attenuate DOX-induced autophagy. The pretreatment of rhNRG-1 (10,100, or 1000 ng/mL) attenuated the DOX-induced decrease in NRVMs viabilities in a concentration-dependent way, and 1000 ng/mL of rhNRG-1 shows a marked protection (data not365医学网 转载请注明
shown). Thus, we chose 1 mg/mL of rhNRG-1 for future experiments. The NRVMs were treated with rhNRG-1 for 18 hours and biochemical markers of cell autophagy were determined (Fig. 2). Western blot analyses showed that rhNRG-1 pretreatment significantly attenuated LC3-II and Beclin 1 protein expression (Fig. 2A). The formation of puncta with the GFP-LC3 fusion protein is a well-characterized365医学网 转载请注明
method for visualizing autophagosomes.18 To further confirm that rhNRG-1 could attenuate cell autophagy, we transfected the GFP-LC3 plasmid into NRVMs and subjected the cultures to combined treatment with rhNRG-1 and DOX for 18 hours. Microscopic examination showed the characteristic punctate fluorescent pattern of GFP-LC3, indicating autophagosome formation and the occurrence of autophagy (Fig. 2B). LC3-II and Beclin 1 protein levels were therefore consistent with the cell fluorescence data.
Reduction of Intracellular ROS Is Associated365医学网 转载请注明
With the Anti-autophagy Effects of rhNRG-1
Free radical generation and cardiomyocyte oxidative stress occur soon after exposure to DOX and significantly contribute to the drug’s cardiotoxic effects.19,20 We wished to determine if rhNRG-1 treatment affected ROS production,thus implicating that mechanism in the attenuation of autophagy that was observed as a result of rhNRG-1 treatment.Intracellular ROS was determined via fluorescent detection of DCF.17 The rhNRG-1 pretreatment significantly inhibited DOX-induced ROS production and was almost as effective as the antioxidant NAC (Fig. 3A). To determine whether ROS production plays a role in DOX-induced autophagy, we transfected NRVMs with a GFP-LC3 plasmid to observe fluorescent puncta directly and complemented those assays with analyses of LC3-II and Beclin 1. These data show that pretreatment with NAC for 1 hour significantly decreases the LC3-II and Beclin 1 levels and abrogated the formation of GFP-LC3 puncta induced by DOX (Figs. 3B, C). These results therefore suggest that NAC pretreatment can rescue cells from DOX-induced autophagy and that rhNRG-1–induced reduction of autophagy in DOX-damaged NRVMs occurs at least partially via inhibition of intracellular ROS.
The rhNRG-1–induced Reduction of ROS and365医学网 转载请注明
Cell Autophagy Through Akt Signaling
The phosphatidylinositol 3-kinase (PI3K)–Akt pathway is the central regulator of the NRG-1–erbB signaling network. NRG-1 promotes survival and growth of cardiomyocytes by activating the PI3K–Akt pathway.21 To determine the role of Akt activation in rhNRG-1–induced reduction of cell autophagy, we used LY294002, a common PI3K inhibitor, to pretreat NRVMs for 1 hour before rhNRG-1 treatment. The results showed that pretreatment with the PI3K inhibitor LY294002365医学网 转载请注明
inhibited rhNRG-1–increased serine phosphorylation of Akt (Fig. 4A). These findings indicate th, at rhNRG-1 induces Akt phosphorylation via the PI3K/Akt pathway. The combination of rhNRG-1 and DOX increased the levels of phosphorylated rhNRG-1. However, no differences were observed in Akt365医学网 转载请注明
phosphorylation between cells treated with NAC and nontreated cells in the presence of DOX or rhNRG-1 (Fig. 4E). Taken together, these results indicate that rhNRG-1– induced reduction of autophagy in DOX-damaged NRVMs is at least partially dependent on Akt activation and the365医学网 转载请注明
reduction of intracellular ROS occurs downstream of this event.
Bcl-2 Mediates the Anti-autophagy Effect365医学网 转载请注明
Beclin 1 is a mammalian autophagy gene24 that was originally identified as a Bcl-2–interacting protein.25 Bcl-2 inhibits beclin 1–dependent autophagy in yeast and mammalian cells, and cardiac Bcl-2 transgenic expression also inhibits autophagy in murine heart cells.18 To evaluate whether the interaction between Bcl-2 and Beclin 1 might play a role in the anti-autophagy effect of rhNRG-1, we assayed Bcl-2 and Beclin 1 expression levels by Western blot. Pretreatment of the cells with rhNRG-1–increased Bcl-2 and decreased Beclin 1 (Fig. 5A), consistent with the hypothesis that Bcl-2 is a potential downstream effector of the rhNRG-1 actions. Protein interactions in the cells were confirmed365医学网 转载请注明
using coimmunoprecipitation assays. DOX-treated NRVMs show minimal levels of Bcl-2–Beclin 1 in the coimmunoprecipitates, consistent with previous data.7 However, comparing with DOX alone, pretreatment with rhNRG-1 results in significantly higher levels of Bcl-2 coimmunoprecipitating with Beclin 1 (Fig.5B, lane 2 vs lane 6). To further test the role of Bcl-2 in rhNRG-1–induced autophagy inhibition, we determined if diminished Bcl-2 levels could attenuate or abolish the ability of rhNRG-1365医学网 转载请注明
to inhibit autophagy. We used Bcl-2–RNA interference (RNAi) to decrease Bcl-2 expression in the NRVMs (Fig. 6A). Cells were infected with Bcl-2–RNAi or scrambled control (CON)-RNAi365医学网 转载请注明
followed by treatment with rhNRG-1 and DOX. The Bcl-2 siRNA treatment markedly attenuated the inhibitory effect of rhNRG-1 on autophagy (Figs. 6B, C), indicating that Bcl-2 indeed partly mediates the anti-autophagic effects of rhNRG-1.
Akt and intracellular ROS have been reported to regulate Beclin 1 and Bcl-2 expressions.22,26 We examined whether Akt and ROS played a role in regulating the formation of the Beclin 1–Bcl-2 complex after pretreatment of NRVMs with rhNRG-1. The results showed that the ROS scavenger, NAC, inhibits the DOX-induced low level of Bcl-2 coimmunoprecipitating with Beclin 1(Fig. 5B, lane 2vs lane 8). Moreover, during pretreatment with rhNRG-1,inhibiting Akt activity via LY294002, also attenuated formation of the Bcl-2–Beclin 1 complex as assayed by coimmunoprecipitation (Fig. 5B, lane 6 vs. lane 7). Taken together, these findings suggest that Bcl-2 is involved in the rhNRG-1–365医学网 转载请注明
induced reduction of autophagy.
DOX significantly reduces NRG-1 protein expression in the heart.27 In the heterozygous NRG-1 gene-ablated mice,DOX-induced severe cardiac injury, higher mortality, and worse left ventricular dysfunction compared with wild-type mice.28 Based on work in isolated cardiomyocytes, current365医学网 转载请注明
data showed that a lot of processes are regulated by NRG-1 signaling, including cell growth,29 myofilament structure and organization,30,31 survival,29 and myocyte matrix coupling.32 To our knowledge, the present study shows for the first time that rhNRG-1 attenuates DOX-induced autophagy via Akt/ROS/Bcl-2 signaling in cardiomyocytes.
Until now, 31 members of spliced variants of NRG-1 have been identified.33 Their isoforms differ in their tissuespecific expression patterns and their biological activities,thereby contributing to the great diversity of the functions of NRG-1 and different groups have used different ligands. In this365医学网 转载请注明
study, rhNRG-1 was focused on due to the fact that rhNRG-1 was administered intravenously to clinically relevant chronic rat models of DOX-induced cardiomyopathy, and cardiac function and survival were improved. However, DOX did not alter hemodynamic or cardiac contractility in normal animals. In the present study, we also found that no differences were observed in the expression of LC3, Beclin 1, Bcl-2, and the levels of ROS production between cells treated with rhNRG-1 and nontreated cells. The effects of rhNRG-1–treated cells were almost the same as NRVMs that were treated with LY294002 or NAC alone (Figs. 1A, B). These results showed that, in the present study, rhNRG-1 counteracted the effects of DOX without additive actions and the antagonistic action only occurs in the365医学网 转载请注明
presence of DOX. Making rhNRG-1 a promising therapeutic agent for DOX-induced cardiomyopathy.
The PI3K/Akt pathway can play a central role in the survival of cardiomyocytes mediated by NRG-1, as treatment with a PI3K inhibitor or overexpression of a dominant-negative Akt isoform both negate NRG-1’s effects.23,34,35 We therefore examined whether the PI3K substrate Akt was activated by365医学网 转载请注明
rhNRG-1 in cardiomyocytes. The rhNRG-1 caused the activation of Akt, which was inhibited by the PI3K inhibitor LY294002 (Fig. 3A). The PI3K-I/PKB pathway is involved in the negative modulation of autophagy. Active PI3K transfers to the inner surface of the cell membrane, phosphorylating365医学网 转载请注明
phosphatidylinositol, and leading to the activation of protein kinase B (Akt/PKB) and other enzymes. This is followed by activation of mammalian targets of rapamycin,36,37 which negatively regulates multiple autophagy-related proteins to prevent autophagy activation. In the present study, we found that pretreatment with 1 mg/mL of rhNRG-1 attenuated DOX-induced autophagy in NRVMs and that attenuation was dependent on Akt activity. However, further studies are needed to identify the necessity and sufficiency of the specific pathways downstream of Akt that are fully responsible for protection from DOX-induced injury.
ROS generation can be one of the critical events for initiating myocardial damage.38 Treatment with antioxidant agents attenuate DOX-induced cardiotoxicity.39,40 Recent data suggest that many stimuli, including nutrient starvation, hypoxia, and chemotherapy drugs, can induce cell autophagy via activation of ROS signaling.41 To examine the antioxidant role of rhNRG-1, we performed a test for free radicals based on the oxidation of DCHF-DA inside the cytoplasm. The rhNRG-1 attenuated DOX-induced DCF fluorescence to a degree similar to the antioxidant, NAC. Although the DCF test has some limitations,42 our data suggest that ROS is involved in DOX-induced autophagy and subsequent autophagic cell death in NRVMs. Regulation of ROS is complex: however, we confirmed that the antioxidant effect is at least partially dependent on the PI3K/Akt pathway, a result that is365医学网 转载请注明
consistent with previous reports.43,44
Akt/ROS signaling upregulated Bcl-2 expression, which may play a role in the anti-autophagy effects of rhNRG. We showed that the ability of rhNRG-1 to inhibit autophagy was mediated at least partly by its downstream effector Bcl-2.2 This was further supported by the observations that inhibition of365医学网 转载请注明
autophagy by rhNRG-1 was attenuated by using Bcl-2 siRNA. Beclin 1 is an important participant in mammalian autophagy.In Beclin 12/2 animals, autophagy was significantly attenuated compared with wild-type controls.45 Beclin 1 interacted with class III PI3K and the Beclin 1/PI3K-III complex is involved in autophagosome formation. Bcl-2 could inhibit the formation of the Beclin 1/Vsp34 PI3K complex and Beclin 1–associated class III PI3K activity, thus inhibiting Beclin 1–dependent autophagy. Potentially, interaction between Bcl-2 and Beclin 1 might act as a rheostat, maintaining autophagy activity at optimal levels for cell survival rather than cell death.18
The limitation of the study is that in vitro studies may lead to results that do not totally correspond to the circumstances occurring around a living organism. The peak plasma levels of rhNRG-1 achieved in the phase 2 clinical trials15,46 is 9.35 ng/mL (data not published). The concentrations of rhNRG- 1 and DOX that were selected for the present in vitro study was much higher when compare with the plasma levels of rhNRG-1 and DOX46 that occurred in patients. So to determine the effective dose, further studies in vivo are required in the future.
Many studies proposing to define a role for NRG-1 have been carried out. Overall, the data support the concept that NRG-1 promotes the growth and survival of isolated cardiac myocytes. But because of the complex nature of the molecule, the area is somewhat confusing because different groups have used in different ligands.47 In this study, we focused on rhNRG-1 because it is the only one whose safety and efficacy have been assessed in chronic heart failure patients.15 Our data showed that rhNRG-1 attenuated DOXinduced autophagy by activating Akt-mediated reduction of ROS, resulting in upregulation of Bcl-2. We propose the hypothesis that rhNRG-1 is a novel drug for the protection365医学网 转载请注明
of DOX-induced damaged myocardium and should be a focus of future experiments.
We thank Prof J. Robbins, Division of Molecular Cardiovascular Biology, Children’s Hospital Research Foundation, Cincinnati, for editing the manuscript.
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