Effects of MEK5/ERK5 Association on Small Ubiquitin-Related Modification of ERK5: Implications for Diabetic Ventricular Dysfunction After Myocardial Infarction
Diabetes mellitus (DM) contributes to the exacerbation of left ventricle (LV) dysfunction after myocardial infarction (MI). Activation of ERK5, an atypical mitogen activated protein kinase with transcriptional activity, inhibits apoptosis and LV dysfunction after doxorubicin treatment. SUMOylation has been proposed as a negative regulator of various transcription factors. In the current study, we investigated the role of ERK5-SUMOylation in ERK5 transcriptional activity as well as on DM-mediated exacerbation of LV dysfunction and apoptosis after MI. ERK5 wild-type transcriptional activity was inhibited by Ubc9 (SUMO E2 conjugase) or PIAS1 (E3 ligase), but not in the ERK5-SUMOylation-site defective mutant (K6R/K22R). H2O2 and high glucose, 2 well-known mediators of diabetes, induced ERK5-SUMOylation, and the K6R/K22R mutant, dominant negative form of Ubc9, and siRNA-PIAS1 reversed H2O2-mediated reduction of ERK5 transcriptional activity in cardiomyocytes, indicating the presence of SUMOylation-dependent ERK5 transcriptional repression. Constitutively active form of MEK5α (CA-MEK5α) inhibited ERK5-SUMOylation independent of kinase activity, but dependent on MEK5-ERK5 association. To investigate the pathological role of ERK5-SUMOylation in DM mice after MI, we used cardiac specific CA-MEK5α transgenic mice (CA-MEK5α-Tg). MI was induced in streptozotocin (STZ)-injected (DM+MI group) or vehicle-injected mice (MI group) by ligating the left coronary artery. The ERK5-SUMOylation was increased in the DM+MI, but not in the MI group. ERK5-SUMOylation, the exacerbation of LV dysfunction, and the number of TUNEL-positive cells in DM+MI was significantly inhibited in CA-MEK5α-Tg mice. Of note, we could not detect any difference of cardiac function after MI in non-diabetic CA-MEK5α-Tg and non-transgenic littermate control mice. These results demonstrated that ERK5 transcriptional activity is subject to downregulation by diabetes-dependent SUMOylation, which resulted in a proapoptotic condition contributing to poor post-MI LV function.
Diabetes is an independent risk factor for both mortality and morbidity after myocardial infarction (MI).1,2 A number of clinical studies have shown that the post-MI left ventricular (LV) function is significantly worse in diabetic compared with nondiabetic patients.3,4 However, what is lacking is a plausible relationship between diabetes and any of the known regulators of myocyte apoptosis known to play a significant role in the post-MI cardiac dysfunction. Previously, we have demonstrated that downregulation of phosphodiesterase 3A (PDE3A) is associated with apoptosis and induction of inducible cAMP early repressor (ICER), a proapoptotic transcriptional repressor, providing a mechanistic framework for how angiotensin II (Ang II) regulates myocyte apoptosis.5,6 Sustained elevation of ICER favors apoptosis through inhibition of cAMP response element binding protein (CREB)-mediated transcription and downregulation of Bcl-2. Interactions between PDE3A and ICER constitute an autoregulatory positive feedback loop (PDE3A-ICER feedback loop) likely to determine the fate of injured myocytes. ERK5, an atypical mitogen activated protein kinase with transcriptional activity, negatively regulates the executor PDE3A-ICER feedback loop and subsequent apoptosis.7
Our recent data indicated that ERK5 transcriptional activity itself is subjected to downregulation by reactive oxygen species (ROS) and advanced glycogen endproducts (AGE)-dependent small ubiquitin-related modification (SUMO), and inhibits KLF2 and eNOS expression in endothelial cells.8 ERK5-SUMOylation at the NH2-terminal region (K6 and K22) significantly inhibits COOH-terminal ERK5 transcriptional activity.8 Posttranslational modification by SUMO commonly regulates the assembly and disassembly of protein complexes, protein localization, stability, and function.9 SUMOylation is known to be highly substoichiometric because it often generates intermediates that result in new protein interactions or conformational states that persist even after SUMO removal.9
In the current study, we showed that ERK5 is 1 of the major targets of SUMOylation in diabetic hearts. We found that SUMOylation-dependent ERK5 transcriptional repression induced by ROS and high glucose. Constitutively active form of MEK5α (CA-MEK5α) inhibited ERK5-SUMOylation independent of its kinase activity but dependent on MEK5-ERK5 association. Diabetic hearts demonstrating exacerbation of LV dysfunction after myocardial infarction (MI) were accompanied by enhanced ERK5-SUMOylation. The inhibition of ERK5-SUMOylation by CA-MEK5α significantly improved the cardiac function after MI in diabetic mice but not in nondiabetic mice. These data suggested the importance of ERK5-SUMOylation on ROS-mediated ERK5 transcriptional repression, which contributes to poor cardiac function after MI in diabetes.
Materials and Methods
Details on reagents, antibodies, plasmid and adenovirus vectors construction, cell culture, mammalian 1-hybrid analysis and transfection of cells, immunoprecipitation (SUMOylation assay) and Western blot analysis, transfection of the PIAS1 siRNA, analysis of apoptosis, animal models, streptozotocin (STZ) injections, coronary ligation surgery, echocardiographic analysis and in vivo hemodynamic measurements, and statistical analysis are also provided in the online Data Supplement (available online at http://circres. ahajournals.org).
Streptozotocin (STZ)-Induced Hyperglycemia Exacerbates LV Dysfunction and Failure After an Experimental MI
Diabetes adversely affects LV dysfunction after MI and leads to a higher incidence of heart failure, but the exact mechanism of diabetes-mediated exacerbation of LV dysfunction remains largely unknown. First, we established a diabetic mouse model in our laboratory and demonstrated the development of heart failure after MI as previously described.10 Four groups of mice were studied. Diabetes mellitus (DM) was induced in male FVB mice (5 to 8 weeks old and 25 to 35 g body weight, The Jackson Laboratory, Bar Harbor, ME) by intraperitoneal injection of STZ (200 mg/kg body weight).10,11 Tail vein blood glucose samples were measured 6 days after injection to ensure induction of hyperglycemia. As a control, vehicle (0.1 mol/L citrate buffer, pH 4.5) was injected in another group of mice. At 7 days after injection, MI was induced in STZ-injected (DM+MI group) or vehicle-injected mice (MI group) by ligating the left coronary artery.10,12 We have carefully characterized the fasting glucose level after different doses of intraperitoneal injection of STZ in FVB mice and found that one injection of 200 mg/kg body weight STZ resulted in fasting blood glucose (FBG) of around 200 mg/dL at 1 week after injection. Because mice with extremely high glucose level (> 400 mg/dL) did not tolerate the coronary ligation surgery well, we chose a STZ dose that resulted in this moderately high FBG as the diabetic mice model for our study. A sham operation without ligating the coronary artery was also performed in additional groups of STZ-injected (DM group) and saline-injected mice (control group). All 4 groups of mice (control, DM, MI, and DM+MI) were followed up for another 1 week (1-week post-MI study). STZ treatment significantly increased random blood sugar (BS) levels 2 weeks after injection (Figure 1A, left panel). Of note, we did not observe any significant body weight difference between the MI and DM+MI groups at 1 week after injection and at the time of surgery (Figure 1A, right panel), and at 1 week after surgery (supplemental Table I). Survival at 1 week after MI was significantly lower in the DM+MI versus MI group (Figure 1B).
We could not detect any difference in LV weight/tibial length (TL) between the MI and DM+MI groups. But lung weight/TL was increased in the MI group, which was significantly exacerbated in the DM+MI group (Figure 1C), suggesting a more severe congestive heart failure in the DM+MI group than MI group. Echocardiography 1 week after ligation showed LV dilatation and dysfunction in the MI group but with both measures exaggerated in the DM+MI group (Figure 1D and 1E; supplemental Table I).
ERK5-SUMOylation in DM+MI Model
Recently, we have reported the importance of ERK5-SUMOylation on regulating its transcriptional activity.8 H2O2 and advanced glycation end products (AGE), 2 well-known mediators of diabetes, negatively regulated ERK5 transcriptional activity via ERK5-SUMOylation in endothelial cells. Because ERK5 demonstrates a cardio-protective effect,7 we investigated the effect of STZ-mediated hyperglycemia and MI on the ERK5-SUMOylation. As shown in Figure 2, we found a slight increase of ERK5-SUMOylation in both the DM (with sham operation) and MI (without STZ treatment) groups. In contrast, the ERK5-SUMOylation was significantly exaggerated in the DM+MI group. These data suggested the possible role of ERK5-SUMOylation on the exaggerated LV dysfunction and development of heart failure after MI in DM mice. In contrast, a reduction of ERK5 phosphorylation was observed in both MI and DM+MI groups compared with non-MI control and DM groups. No significant difference in ERK5 phosphorylation was observed in MI versus DM+MI groups, suggesting that ERK5 phosphorylation and its subsequent kinase activation may not be involved in the exaggerated LV dysfunction after MI in DM mice.
We investigated the possible cardio-toxic effect of STZ, as opposed to hyperglycemia, by administering insulin (60 U/kg subcutaneous b.i.d. NPH human insulin [Humulin N; Eli Lilly]) 1 to 2 days before the coronary ligation surgery to normalize the FBG and determined infarct size, cardiac function, and the biochemical markers such as ERK5-SUMOylation after MI as previously described.13 We demonstrated that preoperative insulin treatment of STZ-injected mice decreased the FBG to 157.6±49.5 mg/dL. Such preoperative insulin-treatment did not result in significant differences in basal cardiac function from nondiabetic MI control (data not shown). In addition, we could not detect any increase in ERK5-SUMOylation after insulin-treatment, which was observed in the hyperglycemic DM+MI mice (Figure 2B). Therefore, we concluded that the exacerbation of cardiac damage and ERK5-SUMOylation in DM+MI model was attributable to hyperglycemia and not to STZ toxicity.
One of the major concerns of this study is whether the observed differences between the groups can be accounted for by metabolic perturbations (particularly malnutrition and weight loss in the diabetic animals), or by the direct effects of STZ on the myocardium. We carefully examined this issue and found that both MI alone and DM+MI animals had equally low body weights, but DM+MI mice showed greater cardiac damage (Figure 1A; supplemental Table I).
H2O2 Inhibited ERK5 Transcriptional Activity and Induced Apoptosis via ERK5-SUMOylation
Because we found ERK5-SUMOylation in the DM+MI group, and ERK5-SUMOylation could decrease ERK5 transcriptional activity and possibly its cardio-protective effect, we investigated whether H2O2 and high glucose can induce endogenous ERK5-SUMOylation. As shown in Figure 2C and 2D, H2O2 significantly increased ERK5-SUMOylation in a time- and dose-dependent manner. High glucose, but not mannitol (20 mmol/L) as a control, also increased ERK5-SUMOylation (Figure 2E). Previously, we reported that H2O2 and advanced glycation end products (AGE) inhibited ERK5 transcriptional activity via ERK5-SUMOylation in endothelial cells.8 Because we found that H2O2 and high glucose could induce ERK5-SUMOylation in cardiomyocytes (Figure 2C through 2E), we investigated whether ERK5-SUMOylation was important in the H2O2-mediated reduction of ERK5 transcriptional activity in cardiomyocytes. If SUMOylation of ERK5 underlies the H2O2-mediated reduction of its transcriptional activity, inhibition of SUMOylation by coexpression of the dominant negative form of Ubc9 (DN-Ubc9) should interfere with this reduction in transcriptional activity. As shown in Figure 3A, H2O2 (30 μmol/L) decreased ERK5 transcriptional activity by approximately 56%, but this reduction in the transcriptional activity was less in DN-Ubc9 transfected cardiomyocytes in a DN-Ubc9 dose-dependent manner.
Next, we compared the H2O2 (30 μmol/L)-induced reduction of transcriptional activity in the wild-type and SUMOylation sites-mutant (K6R/K22R) ERK5. As shown in Figure 3B, H2O2-induced reduction of ERK5 transcriptional activity was significantly less in the ERK5-K6R/K22R mutant. To further confirm the involvement of ERK5-SUMOylation in transcriptional regulation, we tested whether deletion of PIAS1 using PIAS1 siRNA could inhibit the H2O2-mediated reduction of ERK5 transcriptional activity. PIAS1 siRNA inhibited PIAS1 expression and H2O2-mediated ERK5-SUMOylation in cardiomyocytes (Figure 3C). Furthermore, PIAS1 siRNA significantly prevented H2O2-mediated reduction of ERK5 transcriptional activity (Figure 3D). Taken together, these data strongly suggested the critical role of ERK5-SUMOylation in H2O2-mediated reduction of ERK5 transcriptional activity.
We have reported previously the importance of ERK5 in the regulation of cardiomyocyte apoptosis.7 Therefore, we investigated whether ERK5-SUMOylation was involved in the H2O2-mediated apoptosis. As shown in Figure 4, in this model the deletion of PIAS1 significantly inhibited H2O2-mediated apoptosis. To further support the critical role of ERK5-SUMOylation in regulating cardiomyocyte apoptosis, we generated 2 adenovirus vectors expressing the ERK5 wild-type and ERK5 K6R/K22R mutant. Sixteen hours after transduction with adenovirus vector containing Xpress tagged-ERK5 wild-type (Ad-ERK5-WT) or ERK5 K6R/K22R mutant (Ad-ERK5 K6R/K22R), cardiomyocytes were treated with H2O2 (100 μmol/L) for 24 hours, and apoptosis was measured by TUNEL staining. We found that expression of ERK5-SUMOylation site-mutant significantly inhibited H2O2-induced apoptosis (Figure 4C). No difference in the amount of ERK5 wild-type and K6R/K22R mutant expression was confirmed in samples by immunoblotting with anti-Xpress antibody (Figure 4C). Of note, we needed to increase the concentration of H2O2 from 30 μmol/L to 100 μmol/L in this set of experiments because we could not detect significant increase of TUNEL positive cells in Ad-ERK5 WT tranduced cells with 30 μmol/L H2O2 treatment, probably because of the protective effect of ERK5 wild-type overexpression.
Interaction of MEK5α With ERK5 Inhibits ERK5-SUMOylation but not MEK5α-Mediated ERK5 Kinase Activation
It has been reported that ERK5 activation inhibits MEF2-SUMOylation via MEF2-Ser179 phosphorylation and increases MEF2 transcriptional activity.14 Therefore, we investigated whether MEK5 activation induced by CA-MEK5α can similarly inhibit Ubc9 and SUMO3-mediated ERK5-SUMOylation. As shown in Figure 5A, CA-MEK5α significantly inhibited Ubc9 and SUMO3-mediated ERK5-SUMOylation (lanes 1 through 3). These data suggested that CA-MEK5α could potentially transcriptionally activate ERK5 via inhibiting SUMOylation and provides a novel mechanism of MEK5α activation of ERK5 via inhibition of ERK5-SUMOylation.
To determine the effect of MEK5α-mediated ERK5 phosphorylation on ERK5-SUMOylation, we mutated the TEY phosphorylation motif of ERK5, which is critical for ERK5 kinase activation,15 and investigated whether these mutations can affect the CA-MEK5α–mediated inhibition of ERK5-SUMOylation. As shown in Figure 5A (lanes 4 through 6), although it is well known that the ERK5 kinase cannot be activated without the TEY motif phosphorylation,15 mutation of this motif had no effect on CA-MEK5α–mediated reduction of ERK5-SUMOylation. This suggested that both ERK5 kinase activation and TEY motif phosphorylation were not involved in the CA-MEK5α–induced inhibition of ERK5-SUMOylation. Of note, despite the presence of CA-MEK5α–induced inhibition of ERK5-SUMOylation, no significant difference in the SUMO conjugated 75- and 110-kDa proteins and other SUMO conjugated proteins between 30 to 280 kDa with or without CA-MEK5α transfection was noted. These data suggested that CA-MEK5α may have a unique regulatory role in ERK5-SUMOylation distinct from the general SUMOylation process induced by Ubc9/SUMO3.
Residues 78 to 139 of ERK5 constitutes the MEK5α-binding domain.16 To investigate the contribution of MEK5-ERK5 association on CA-MEK5α–induced inhibition of ERK5-SUMOylation, we generated 2 deletion mutants of the MEK5α-ERK5 association site, Δ72 to 139 and Δ90 to 130, with respective deletions of the numbered residues. Coimmunoprecipitation studies confirmed the lack of association between these Δ-mutants and MEK5α (Figure 5B). Cotransfection of HA-SUMO3 and Ubc9 induced ERK5-SUMOylation, which was inhibited by CA-MEK5α induction (Figure 5C, lanes 1 through 3). As shown in Figure 5C (lanes 4 through 7), both MEK5 binding site Δ-mutants were SUMOylated, but CA-MEK5α no longer substantially inhibited ERK5-SUMOylation compared with WT. These data indicate the critical role of ERK5-MEK5 association, but not ERK5 kinase activation and TEY motif phosphorylation, on CA-MEK5α–mediated inhibition of ERK5- SUMOylation.
ERK5-SUMOylation, PDE3A-ICER Feedback Loop, and Apoptosis in DM+MI Mice Was Significantly Inhibited in CA-MEK5α-Tg Mice
As shown in Figure 2, we found that ERK5-SUMOylation was significantly increased in DM+MI mice heart. However, the involvement of ERK5-SUMOylation on diabetes-mediated exacerbation of LV dysfunction after MI remains unclear. Because CA-MEK5α–mediated MEK5-ERK5 association inhibited ERK5-SUMOylation in vitro we investigated whether DM-mediated ERK5-SUMOylation might be inhibited in cardiac specific CA-MEK5α-Tg mice. As shown in Figure 6A (top panel), ERK5-SUMOylation was significantly increased at 1 week after MI in diabetic nontransgenic littermate control (NLC) mice. In contrast, we did not find any significant MI-induced increase in ERK5-SUMOylation in diabetic CA-MEK5α-Tg mice. Previously, we have reported on the critical role of ERK5 activation in regulating the PDE3A/ICER feedback loop in a heart failure model.7 Because we found that ERK5-SUMOylation inhibited ERK5 transcriptional activity, we determined whether the induction of PDE3A/ICER feedback loop in DM+MI mice can also be inhibited in the CA-MEK5α-Tg mice. As shown in Figure 6C, we found a reduction of PDE3A and ICER induction at 1 week after MI in diabetic NLC mice but significantly inhibited in CA-MEK5α-Tg mice. ERK5 phosphorylation was decreased in diabetic NLC mice after MI as we have shown in Figure 2A, but was recovered in CA-MEK5α-Tg mice (Figure 6A, 2nd panel from the top). No significant differences in Akt and activation between NLC and CA-MEK5α-Tg mice were observed in this model (Figure 6B, top and 2nd from the top). In contrast to ERK5, we found that ERK1/2 phosphorylation was increased in both diabetic NLC and CA-MEK5α-Tg mice after MI, which suggested the different regulatory mechanism of ERK1/2 compared with ERK5 after MI in DM mice (Figure 6B, 3rd from the top and bottom). Thus ERK5 activation as well as subsequent inhibition of ERK5-SUMOylation regulates the PDE3A/ICER feedback loop in DM+MI model.
Apoptosis is believed to be important for the development of heart failure.17 In NLC mice hearts 1 week after MI, the number of in situ TUNEL-positive cells increased to 0.32±0.12%, with a greater increase to 0.91±0.39% in the remote areas from the DM+MI model (Figure 7B). We found significantly decreased TUNEL-positive cells in diabetic CA-MEK5α-Tg mice compared with diabetic NLC mice at the same time point after MI (Figure 7B). These data support the antagonistic role of ERK5 activation in DM-mediated exacerbation of cardiac apoptosis after MI.
Cardiac Dysfunction One Week After MI in DM, but not in Non-DM Mice, Was Rescued in CA-MEK5α-Tg Mice
Because we found an inhibitory role for CA-MEK5α on ERK5-SUMOylation and subsequent PDE3A and the ICER feedback loop and apoptosis, we next investigated whether the inhibition of ERK5-SUMOylation by CA-MEK5α can prevent the exacerbation of LV dysfunction and heart failure after MI in DM mice in vivo. No survival difference was noted between NLC and CA-MEK5α-Tg mice at the 1-week time point after MI in nondiabetic mice (Figure 8A). In contrast, in STZ-treated DM mice, the survival rate for CA-MEK5α was significantly higher than for NLC mice (Figure 8B). Random BS levels were elevated in both NLC and CA-MEK5α-Tg mice 1 week after STZ injection and at the time of surgery, but we did not find any difference in the body weight among these groups (Figure 8C).
One week after coronary ligation, both LV weight/TL and lung weight/TL were increased in diabetic NLC mice. In CA-MEK5α-Tg mice lung weight/TL was significantly decreased, consistent with the idea that MEK5α activation reduces cardiac dysfunction and heart failure after MI in diabetic mice (Figure 8D; supplemental Table I). In NLC mice, echocardiographic analysis showed that both LVEDD and LVESD after MI in DM mice were greater than in sham, and FS% also significantly decreased in DM+MI group (Figure 8E; supplemental Table I). Increased in LVEDD and LVESD and the reduction of FS% after MI in diabetic NLC mice were significantly prevented in CA-MEK5α-Tg mice (Figure 8E). Of note, there was no significant difference in cardiac function (FS%) between NLC and CA-MEK5α-Tg mice at the same time point after MI in nondiabetic mice (Figure 8F), confirming the unique role of MEK5α activation on DM-mediated exacerbation of LV dysfunction after MI. In agreement with echocardiographic data, we observed a significant decrease in developed pressure (DP) and dP/dt max in diabetic NLC mice after MI. In contrast, diabetic CA-MEK5α-Tg mice showed significantly higher DP and dP/dt max (Figure 8G; supplemental Table II). Thus physiological data also confirmed the critical role of MEK5 activation in ameliorating the exacerbation of cardiac dysfunction after MI by DM.
In this study we found that ERK5-SUMOylation was significantly increased after MI in diabetic mice. Diabetic CA-MEK5α-Tg mice demonstrated reduced ERK5-SUMOylation and improved LV function and lung weight/TL after MI in comparison to the NLC mice. These data strongly suggested that the activation of MEK5, which inhibited diabetes-mediated ERK5-SUMOylation after MI, was cardio-protective against STZ-induced exacerbation of LV dysfunction after MI. Because both H2O2 and high glucose increased ERK5-SUMOylation, it is most likely that ROS production may be involved in this process, but the exact mechanism remains unclear. DN-Ubc9 and PIAS1 siRNA impaired ERK5-SUMOylation suggesting the involvement of Ubc9 and PIAS1 in ERK5-SUMOylation. Recently, the involvement of PIAS1 phosphorylation on regulating downstream events has been reported. PIAS1 can act by selectively inhibiting the recruitment of NF-κB/STAT1 to the endogenous gene products. IKKα is associated with PIAS1 and mediates the Ser 90 phosphorylation of PIAS1, which in turn is required for PIAS1 to block the promoter binding of p65. In this report, the importance of both Ser 90 phosphorylation and SUMO ligase activity of PIAS1 to repress transcriptional activity was proposed. However, the mutant of S90A PIAS1 did not decrease its ability to induce protein SUMOylation compared with wild type, suggesting additional regulatory mechanism of PIAS1 E3 ligase activity in addition to SUMOylation.18 The possible link between phosphorylation and SUMOylation is intriguing provided that ROS or high glucose–mediated kinase activation could be subject to PIAS1 phosphorylation coupled to ERK5-SUMOylation. Future research should focus on identification of the molecular mechanism of ROS and high glucose–mediated ERK5-SUMOylation because this process appears to be involved in the exacerbation of diabetes-mediated LV dysfunction after MI.
An expanded Discussion section is available in the Online Data Supplement.
Sources of Funding
This work is supported by grants from the Mochida Memorial Foundation for Medical and Pharmaceutical Research to Dr Tetsuro Shishido, the America Heart Association to Dr Woo (Postdoctoral fellowship 0625957T), and from the National Institutes of Health to Dr Abe (GM-071485, HL-077789, and HL-088637), Dr Yan (HL-077789 and HL-088400), and Dr Yang (GM-071485 and HL-088637). Drs Abe and Yan are recipients of Established Investigator Awards of the American Heart Association (0740013N and 0740021N).
↵*T.S., C.-H.W., and B.D. contributed equally to this study.
Original received November 18, 2007; revision received April 27, 2008; accepted April 30, 2008.
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