Atrogin-1/MAFbx Enhances Simulated Ischemia/Reperfusion-induced Apoptosis in Cardiomyocytes through Degradation of MAPK Phosphatase-1 and Sustained JNK Activation*

Atrogin-1/MAFbx is a major atrophy-related E3 ubiquitin ligase that is expressed specifically in striated muscle. Although the contribution of atrogin-1 to cardiac and muscle hypertrophy/atrophy has been examined extensively, it remains unclear whether atrogin-1 plays an essential role in the simulated ischemia/reperfusion-induced apoptosis of primary cardiomyocytes. Here we showed that atrogin-1 markedly enhanced ischemia/reperfusion-induced apoptosis in cardiomyocytes via activation of JNK signaling. Overexpression of atrogin-1 increased phosphorylation of JNK and c-Jun and decreased phosphorylation of Foxo3a. In addition, atrogin-1 decreased Bcl-2, increased Bax, and enhanced the activation of caspases. Furthermore, JNK inhibitor SP600125 markedly blocked the effect of atrogin-1 on cell apoptosis and the expression of apoptotic-related proteins and caspases. Importantly, atrogin-1 induced sustained activation of JNK through a mechanism that involved degradation of MAPK phosphatase-1 (MKP-1) protein. Atrogin-1 interacted with and triggered MKP-1 for ubiquitin-mediated degradation. In contrast, proteasome inhibitors markedly blocked the degradation of MKP-1. Taken together, these results demonstrate that atrogin-1 promotes degradation of MKP-1 through the ubiquitin-proteasome pathway, thereby leading to persistent activation of JNK signaling and further cardiomyocyte apoptosis following ischemia/reperfusion injury.

Apoptosis in cardiac myocytes appears to be an early event and is well recognized to be responsible for myocardial infarction following ischemia/reperfusion (I/R) 2 injury. However, the intracellular signaling pathways that are involved in stimulus recognition and progression to apoptosis in cardiomyocytes following I/R injury remain to be elucidated. It is reported that activation of the mitogen-activated protein kinase (MAPK) signaling cascades plays an essential role in this progress (1)(2)(3). It is believed that activation of p38 is initiated by ischemia and sustained during reperfusion, whereas sustained activation of JNK occurs only during reperfusion and is involved in apoptosis (1,2). Although I/R is found to be the activator of apoptosis via JNK activation, the mechanism by which I/R prolongs the JNK activation is not fully understood.
MAPKs become activated by dual phosphorylation on Ser/ Thr and Tyr residues of TEY sites within the activation loop, whereas dephosphorylation of these residues by MAPK phosphatases (MKPs) terminates such activation (4 -8). Thus, MKPs play an important role in negatively regulating MAPK signaling. MKPs constitute a family of 11 dual-specificity phosphatases that exhibit differential specificity toward MAPK substrates. Among these phosphatases, MKP-1(also known CL100/DUSP1) is identified as the first member of this family (9 -12) and preferentially dephosphorylates and inactivates both JNK and p38 (13)(14)(15). Recent studies have shown that MKP-1-deficient macrophage cells exhibit prolonged JNK activation as well as enhanced production of tumor necrosis factor-␣ and interleukin-6 compared with wild-type cells (16 -18). Several observations demonstrate that MKP-1 is overexpressed in a number of cell types, and its overexpression has been shown to protect cells from apoptosis induced by I/R injury, cisplatin, ethanol, and H 2 O 2 through inactivation of JNK (14,15,19,20). Taken together, these data demonstrate that MKP-1 plays a role in the regulation of stress-responsive JNK-mediated apoptosis.
MKP-1 has been reported to be a labile protein whose stabilization can be enhanced by proteasome inhibitors (21). Choi et al. reported that activation of PKC␦ triggers MKP-1 degradation via the ubiquitin-proteasome pathway (22). Furthermore, MKP-1 DEF motif is necessary for active ERK2 binding to initiate site-specific phosphorylation and serves as an essential recognition domain for the Skp1/Cul1/Skp2 (SCF Skp2 ) ubiq-uitin ligase complex, leading to MKP-1 polyubiquitination and subsequent proteasomal degradation (23). More recently, Venugopal et al. (20) demonstrate that ethanol-induced oxidative stress causes the sustained activation of JNK and apoptosis in hepatocytes via the enhancement of MKP-1 degradation. Although MKP-1 is degraded by the ubiquitin-proteasome system, there is no evidence indicating that atrogin-1 modulates this process in cardiomyocytes.
Atrogin-1/MAFbx is a F-box protein containing an E3 ligase and expressed specifically in striated (cardiac and skeletal) muscle (24,25). Remarkably, atrogin-1 is up-regulated markedly in skeletal muscle in response to a variety of stimuli that leads to the loss of muscle mass (24 -28). Recent study has demonstrated that the induction of atrogin-1 in failing hearts and the up-regulation in response to either tumor necrosis factor-␣ or H 2 O 2 are associated with activation of MAPKs (28,29). Furthermore, our previous data have shown that atrogin-1 overexpression inhibits cardiac hypertrophy through calcineurin/ nuclear factor of activated T cells and phosphatidylinositol 3-kinase/Akt/Foxo signaling pathways (30,31), indicating atrogin-1 plays a key role in regulating protein degradation in cardiac and skeletal muscle hypertrophy/atrophy. Nevertheless, the direct role of atrogin-1 and the downstream targets in cardiomyocyte apoptosis after I/R injury remain unclear.
The present study was designed to examine whether the expression of atrogin-1 affects apoptosis of cardiomyocytes in response to I/R. Our findings define the mechanisms by which atrogin-1 promotes MKP-1 degradation through the ubiquitinproteasome system, thereby enhancing activation of JNK1/2 signaling and cardiomyocyte apoptosis. Overall, these results for the first time show that atrogin-1 is an important mediator in controlling apoptosis of cardiomyocytes following I/R injury.
Simulated I/R Protocol-After 24 h of adenovirus infection, the cells were subjected to simulated ischemia for 1 h by replacing the cell medium with an "ischemia buffer" that contained

FIGURE 1. Atrogin-1 promotes I/R-induced apoptosis in cardiomyocytes.
A, cardiomyocytes were infected with adenovirus vector or atrogin-1 and exposed to 1-h ischemia followed reperfusion for 0 -24 h. Apoptotic cells were observed using a TUNEL assay. At least 120 cells per dish were counted, and each treatment was performed in triplicate. Results are expressed as means Ϯ S.E. for three independent experiments. *, p Ͻ 0.01 versus Ad-control; **, p Ͻ 0.001 versus Ad-control. B, cardiomyocytes were cultured and infected with adenovirus siRNA-control or siRNA-atrogin-1 for 24 h, and then treated with normoxia or I/R for 24 h. Apoptotic cells were observed using TUNEL assay (middle panel, TUNEL-positive cardiomyocytes stained in red fluorescent color). The "GFP" indicates the efficiency of adenovirus infection. A representative field is shown for each condition. C, quantitative analysis of TUNEL-positive cells was from three independent experiments, and at least 120 cells per dish were counted. Results are expressed as means Ϯ S.E. for three independent experiments. *, p Ͻ 0.001 versus siRNA-control.
Cell Viability and TUNEL Assays-Cell viability was determined by Trypan blue exclusion assay (33). Apoptosis was analyzed by TUNEL staining as reported previously (33). Cells were counterstained with 4Ј,6-diamidino-2-phenylindole and finally examined by fluorescent microscopy. The number of TUNELpositive cells was analyzed using National Institutes of Health Image software.
Western Blot Analysis-Protein samples were prepared from cultured neonatal cardiomyocytes, H9c2 cells using extraction buffer as described previously (30). The membranes were incubated with primary antibodies as indicated overnight and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. The blots were developed using a chemiluminescent system, and the bands were scanned, and densitometry analysis was performed with Gel-pro 4.5 Analyzer (Media Cybernetics).
Immunoprecipitations and GST Pulldown Assays-Immunoprecipitations were performed as described (30). Briefly, H9c2 cells were cotransfected with Myc-atrogin-1. Endogenous MKP-1 proteins were immunoprecipitated for 2 h at 4°C with anti-MKP-1 antibody. The beads were washed and analyzed by immunoblotting with anti-Myc or anti-MKP-1 antibodies. GST fusion protein was prepared, and GST pulldown assays were performed as described previously (30). Briefly, H9c2 cells were lysed for 30 min in lysis buffer containing 50 mM Tris, pH 7.6, 150 mM NaCl, 5 mM MgCl 2 , 0.5% Triton X-100, 5 mM dithiothreitol, 10 mM NaF, and protease inhibitors. Lysates were precleared with GST beads for 1 h and incubated with GST or GST-atrogin-1 fusion proteins for 1 h at 4°C. Protein-bound GST beads were washed with lysis buffer and analyzed by Western blot with anti-MKP-1 antibody.
Statistical Analysis-Data are presented as means Ϯ S.E. Differences between groups were evaluated for statistical significance using Student's t test. p Ͻ 0.05 was regarded as significant.

Atrogin-1 Enhances I/R-induced Apoptosis in Neonatal
Cardiomyocytes-To investigate the role of atrogin-1 in I/Rinduced apoptosis, neonatal cardiomyocytes were infected with Ad-atrogin-1 or Ad-control for 24 h and then were exposed to 1 h of simulated ischemia followed by 24 h reperfusion. As shown in Fig. 1A, TUNEL-positive cells were not significantly different between two groups within 2 h of reperfusion. With an extended reperfusion period of 4 h, TUNEL-positive cells in atrogin-1 infection markedly increased as compared with the control, indicating that atrogin-1 overexpression potentiated I/R-induced apoptosis in cardiomyocytes. To further determine the involvement of endogenous atrogin-1 in apoptosis, cardiomyocytes were infected with siRNA-atrogin-1 or siRNAcontrol followed by I/R for 24 h. The percentage of TUNELpositive cells in siRNA-atrogin-1 infection markedly reduced as compared with siRNA-control (Fig. 1, B and C), suggesting that knockdown of atrogin-1 inhibited I/R-induced apoptosis in cardiomyocytes. Taken together, these findings demonstrate that atrogin-1 plays a proapoptotic role in cardiomyocytes following I/R injury.
Effect of Atrogin-1 on the Level of Akt, JNK1/2, ERK1/2 and p38 Phosphorylation-To elucidate the signaling pathways involved in atrogin-1-induced cell apoptosis, we first assessed the kinetics of Akt, JNK1/2, ERK1/2, and p38 activities in cardiomyocytes during I/R stimulation. As shown in Fig. 2 (A, C and D), the level of Akt, ERK1/2, and p38 phosphorylation in Ad-atrogin-1 infection was equivalent to that of the control following reperfusion. In contrast, there was a significant increase in the level of JNK1/2 phosphorylation in Ad-atrogin-1 infection and reached a maximum at 24 h of reperfusion as compared with the control (Fig. 2B). These results demonstrate that atrogin-1 selectively up-regulates the activation of JNK1/2 in cardiomyocytes after I/R injury.
Atrogin-1 Enhances I/R-induced Apoptosis via JNK1/2 Signaling Pathway-To determine the relationship between atrogin-1-induced apoptosis and activation of MAPKs and Akt, cardiomyocytes were treated with p38 inhibitor SB203580, JNK inhibitor SP600125, MEK1/2 inhibitor U0126, and phosphatidylinositol 3-kinase inhibitor wortmannin. The results in Fig.  3A showed that these inhibitors could completely block the phosphorylation of p38, JNK1/2, ERK1/2, and Akt. Interestingly, JNK inhibition with SP600125 only significantly increased the percentage of viable cells and decreased the number of TUNEL-positive cells in atrogin-1 infection as compared with the control (Fig. 3, B and C), suggesting that the JNK pathway is the major factor of atrogin-1-induced apoptosis in cardiomyocytes after I/R injury.
Atrogin-1 Enhances the Level of c-Jun Phosphorylation-JNK1/2 targets the transcription factor, c-Jun. Overexpression of atrogin-1 clearly increased c-Jun and JNK1/2 phosphorylation compared with the control. In contrast, knockdown of atrogin-1 by siRNA was efficient and decreased the levels of c-Jun and JNK1/2 phosphorylation during I/R stimulation (Fig.  4, A and B).
Atrogin-1 Down-regulates Foxo3a Phosphorylation-Our recent data demonstrate that atrogin-1 decreases Foxo3a phos-  Fig. 1A. The protein levels of atrogin-1, total and phospho-JNK1/2, and total and phospho-c-Jun were examined by Western blotting using indicated antibodies. B, cardiomyocytes were infected and treated as in Fig. 1B. The protein levels of atrogin-1, total and phospho-JNK1/2, and total and phospho-c-Jun were examined as in A. C, cardiomyocytes were infected and treated as in Fig. 1A. The protein levels of total and phospho-Foxo3a were examined by Western blotting using indicated antibodies. D, cardiomyocytes were infected and treated as in Fig. 1B. The protein levels of total and phospho-Foxo3a were examined as in C. A representative blot is shown for each condition.
phorylation and positively regulates Foxo3a activity with lysine 63-linked ubiquitin chains. Thus, we next examined whether atrogin-1 also affects Foxo3a phosphorylation in cardiomyocytes after I/R. Consistent with our previous data (31), atrogin-1 overexpression resulted in markedly decrease of Foxo3a phosphorylation, whereas depletion of atrogin-1 by siRNA had an opposite effect (Fig. 4, C and D). These results indicate that atrogin-1 also enhances Foxo3a activation. Atrogin-1 Modulates the Expression of Bcl-2, Bax, and Cleaved Caspases through JNK Signaling-To investigate whether atrogin-1 could regulate the expression of apoptosisrelated proteins and caspases, cardiomyocytes were infected with Ad-atrogin-1 or siRNA-atrogin-1 and stimulated with I/R. As shown in Fig. 5A and B, overexpression of atrogin-1 markedly decreased the level of Bcl-2 protein, and increased the level of Bax protein, cleaved caspase-9 and caspase-3. In contrast, knockdown of atrogin-1 had an opposite effect. These results indicate that atrogin-1 is involved in the regulation of Bcl-2, Bax, and caspases implicated in cardiomyocyte apoptosis.
To explore the mechanisms for atrogin-1 regulating the expression of Bcl-2 and Bax in cardiomyocytes following I/R, we first examined the relationship between atrogin-1 and Bcl-2 or Bax. Coimmunoprecipitation assays demonstrated that atrogin-1 did not directly interact with Bcl-2 or Bax in vivo (data not shown), indicating that Bcl-2 and Bax are not the targets of atrogin-1 in this assay. We therefore tested the involvement of JNK signaling using JNK-specific inhibitor SP600125. As shown in Fig. 5 (A and B), JNK inhibition with SP600125 eventually attenuated JNK phosphorylation and markedly blocked the effect of atrogin-1 on the expression of Bcl-2, Bax, cleaved caspase-9, and caspase-3 compared with the control. These results demonstrate that JNK activation by atrogin-1 functions as an upstream regulator of Bcl-2 and Bax, thereby leading to caspases activation and cell apoptosis.
Atrogin-1 Down-regulates the Level of MKP-1 Protein-Because MKP-1 plays a negative regulator of JNK activation, we then determine whether sustained activation of JNK1/2 induced by atrogin-1 is associated with down-regulation of MKP-1 in cardiomyocytes following I/R injury. As shown in Fig.  6A, the level of MKP-1 was significantly increased after 1 h of reperfusion in both atrogin-1 and control groups. However, MKP-1 protein in atrogin-1 infection substantially decreased after 2 h of reperfusion as compared with the control. In contrast, knockdown of endogenous atrogin-1 markedly resulted in the increase of MKP-1 protein level as compared with siRNAcontrol at the end of reperfusion (Fig. 6B), indicating that atrogin-1 participates in the down-regulation of MKP-1 in cardiomyocytes after I/R injury.

Atrogin-1 Interacts with and Decreases Endogenous MKP-1 in H9c2
Cells-We next assessed whether atrogin-1 interacts with endogenous MKP-1 in rat cardiac myoblast H9c2 cells by coimmunoprecipitation assays. As shown in Fig. 7A, MKP-1 was detected in the Myc immune complex precipitated with a specific rabbit Myc antibody, whereas no MKP-1 was found in the immune complex precipitated with a nonspecific rabbit IgG. Furthermore, endogenous MKP-1 in H9c2 cells was pulled down by GST-atrogin-1 fusion protein purified from bacteria but not GST alone (Fig. 7B). Thus, these results suggest that atrogin-1 directly associates with MKP-1 in vivo and in vitro.

Atrogin-1 Induces Cardiomyocyte Apoptosis via MKP-1
in the down-regulation of endogenous MKP-1 protein in H9c2 cells. Indeed, overexpression of atrogin-1 caused a markedly decrease of endogenous MKP-1 protein and increase of JNK1/2 phosphorylation in a dose-dependent manner (Fig. 7C). In contrast, knockdown of endogenous atrogin-1 by siRNA resulted in an increase of endogenous MKP-1 protein and down-regulation of JNK1/2 phosphorylation (Fig. 7D). However, MKP-2 and MKP-3 were not affected markedly by atrogin-1 expression (data not shown), excluding the involvement of MKP-2 and MKP-3 in atrogin-1-induced JNK activation. Taken together, these findings demonstrate that atrogin-1 specifically downregulates MKP-1 protein level. Atrogin-1 Promotes the Ubiquitination and Degradation of Endogenous MKP-1-It has been recently shown that the MKP-1 protein is targeted for proteasomal degradation in breast cancer cell line MCF-7 and HT22 cells (15,22). To determine that the down-regulation of MKP-1 protein by atrogin-1 is mediated through ubiquitin-proteasome degradation, we compared the half-life of endogenous MKP-1 protein in cells transfected with empty vector or atrogin-1. As shown in Fig.  8A, ectopic expression of atrogin-1 resulted in a rapid decrease in the MKP-1 protein as compared with vector control, and this effect was abolished completely by the proteasome inhibitor MG132 (Fig. 8B), indicating that atrogin-1 targets MKP-1 protein for proteasome degradation. Because MKP-1 has been identified as a ubiquitination target in MCF-7 and HT22 cells (15,22), we examined whether atrogin-1 as an E3 ligase affects ubiquitination of MKP-1 in H9c2 cells. Indeed, ubiquitination of MKP-1 was confirmed and enhanced markedly by the transfection of atrogin-1 in H9c2 cells (Fig. 8C). Moreover, ubiquitination of MKP-1 was enhanced further by MG132 (Fig. 8D). Taken together, these results demonstrate that atrogin-1 promotes the ubiquitination and proteasome degradation of MKP-1 protein.

DISCUSSION
Atrogin-1 (also known as muscle atrophy F-box) is first identified as a crucial participant and is induced early during the atrophy process (25,26). Mice lacking atrogin-1 are resistant to muscle atrophy following denervation (26), indicating that atrogin-1 targets key muscle protein(s) for destruction, although the identity of these component(s) is still unclear. Atrogin-1 is a cardiac-and skeletal muscle-specific F-box protein that binds to Skp1, Cul1, and Roc1, the common components of SCF ubiquitin ligase complexes (26,30). We previously found that atrogin-1 inhibits pathologic and physiological hypertrophy in vivo and in vitro (30,31). Furthermore, atrogin-1 mediates part of the effects of statin in muscle atrophy (16). These observations suggest that atrogin-1 may be a critical mediator of the heart and muscle atrophy. However, the role of atrogin-1 in cardiomyocyte apoptosis after I/R injury remains unknown. In the present study, we demonstrate that atrogin-1 enhances I/R-induced apoptosis in cardiomyocytes. This augmentation is associated with the reduced expression of MKP-1 and the sustained activation of JNK signaling. This study also provides evidence that atrogin-1 down-regulates the level of MKP-1 protein through ubiquitination-mediated proteasomal degradation.
Previous studies have shown that JNK is activated in response to various stress and mitogenic stimuli and plays an important role in apoptosis in the heart and cultured cardiomyocytes (1,2). JNK was shown to be activated by I/R injury in transgenic hearts (34). Furthermore, JNK inhibition has been recently reported to actually protect cardiac myocytes from I/R-induced apoptosis (35)(36)(37). Therefore, activation of JNK plays a critical role in controlling cell apoptosis and survival. In the present study, we found that atrogin-1-induced apoptosis of cardiomyocytes was associated with the increase of JNK phosphorylation. Moreover, treatment with SP600125, a specific inhibitor of JNK, markedly decreased apoptotic cells in atrogin-1 infection (Fig. 3). These results suggest that atrogin-1induced apoptosis in cardiomyocytes selectively relies upon the activation of JNK1/2 signaling.
It may be significant that JNK is markedly enhanced by atrogin-1 overexpression following I/R injury, because JNK and its downstream targets are known to play a key role in mitochondrial-driven apoptosis of cardiomyocytes. First, stimulation of JNK activity induces apoptosis through a mechanism that involves c-Jun activation (38). Indeed, we found that atrogin-1 expression significantly increased the level of c-Jun phosphorylation (Fig. 4). Second, there has been much evidence reporting that the JNK pathway controls the activity and expression of several members of the Bcl-2 family proteins involving the intrinsic mitochondria apoptotic pathway (41,(43)(44)(45). Activation of JNK signaling can trigger Bcl-2 down-regulation through proteasome degradation (39) and increase Bax expression through p53 in several cell types (40,41), thereby leading to cytochrome c release from mitochondria and caspase-3 activation (42)(43)(44). Moreover, Bax was shown to bind to Bcl-2 and Bcl-X L , resulting in cell death through the release of cytochrome c and Apaf-1 from mitochondria and thus the activation of caspases, thereby enhancing cell apoptosis (45,46). Consistent with these data, our results showed that atrogin-1 overexpression markedly decreased the level of Bcl-2, increased the expression of Bax and the activation of caspases. Importantly, treatment with JNK inhibitor SP600125 markedly abolished the effects of atrogin-1 on the alteration of Bcl-2, Bax, cleaved caspases (Fig. 5, A and B), indicating that the atrogin-1-mediated JNK activation is a major upstream regulator of Bcl-2 and Bax expression. Collectively, these results demonstrate that the apoptosis-modulating properties of atrogin-1 are mediated by the activation of JNK and its downstream effectors, including c-Jun, Bcl-2, and Bax in cardiomyocytes following I/R injury.
Although atrogin-1 is suggested to markedly contribute to I/R-induced apoptosis via JNK activation in cardiomyocytes mentioned above, there may be other factors that play a role in atrogin-1-induced apoptosis beyond this mechanism. FOXO3a is a member of the forkhead transcription factor family and plays an important role in cell cycle arrest and apoptosis of various cell lines (47). Foxo proteins have been shown to be involved in the transactivation of Bim and FasL, which induce cell death through mitochondria-and death receptor-dependent mechanisms. Our previous data have shown that Foxo3a is a target of atrogin-1 in Akt-induced cardiac hypertrophy, and atrogin-1 positively regulates Foxo3a activation through lysine-63-linked ubiquitination (30), suggesting Foxo3a may participate atrogin-1-induced apoptosis in cardiomyocytes. Indeed, our data showed that atrogin-1 down-regulated Foxo3a phosphorylation in cardiomyocytes following I/R injury. These results indicate that Foxo3a, in part, involves in atrogin-1-induced apoptosis in cardiomyocytes.
There are 11 members of the MKP family that have unique and overlapping substrate specificity toward MAPKs. MKP-1 is identified as the first member of this family and is inducible in response to stress (9 -12). It has been shown that overexpression of MKP-1 can inhibit apoptosis in several cell lines, and this effect was mainly associated with its ability to inactivate JNK or p38 (13,14,48). Importantly, MKP-1 has been shown to be targeted for ubiquitin-mediated proteasomal degradation, whereas the degradation of MKP-1 protein is attenuated by proteasome inhibitors (21). Recent studies suggested that degradation of MKP-1 was triggered by ERK and PKC␦ signaling pathways through activation of the ubiquitin-proteasome pathway, which contributes to the sustained activation of ERK1/2 and JNK1/2, thereby providing a positive-feedback mechanism (22,23). Venugopal et al. (20) demonstrated that ethanol causes the sustained activation of JNK and apoptosis in hepatocytes via the enhancement of MKP-1 degradation. More importantly, F-box protein Skp2, a vital E3 ligase, promotes MKP-1 polyubiquitination and subsequent destruction via the 26 S proteasome (23). These reports suggest to us that MKP-1 degradation might play a role in atrogin-1-induced sus- tained JNK activation and subsequent apoptosis in cardiomyocytes. In the present study, we showed that overexpression of atrogin-1 down-regulated MKP-1 protein level and resulted in persistent activation of JNK1/2 (Figs. 6A and 7C). In contrast, knockdown of atrogin-1 increased expression of MKP-1 and decreased phosphorylation of JNK1/2 (Figs. 6B and 7D). More importantly, atrogin-1 interacted with MKP-1 and promoted its ubiquitination and degradation by proteasome (Fig. 8, C and D), whereas atrogin-1 did not affect MKP-2 and MKP3 protein level, suggesting that atrogin-1-mediated down-regulation is specific for MKP-1 protein. Taken together, we conclude that atrogin-1 promotes the ubiquitination and proteasome degradation of MKP-1, which leads to sustained JNK1/2 activation.
In conclusion, our results suggest a newly described mechanism by which E3 ligase atrogin-1 may induce apoptosis in neonatal cardiomyocytes following I/R injury. Atrogin-1 interacts with MKP-1 and promotes its proteasomal degradation, thereby leading to sustained activation of JNK. The proposed pathway is presented in Fig. 9. Future studies are required to determine the exact mechanisms by which atrogin-1 promotes degradation of MKP-1 and the role of atrogin-1 in I/R-induced apoptosis in animal model in vivo. Strategies for reducing atrogin-1 should be examined for their potential to expand therapeutic options for protecting the heart against I/R injury.