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Originally published In Press as doi:10.1074/jbc.M703048200 on May 24, 2007

J. Biol. Chem., Vol. 282, Issue 30, 21945-21952, July 27, 2007
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Diminished GATA4 Protein Levels Contribute to Hyperglycemia-induced Cardiomyocyte Injury*

Satoru Kobayashi{ddagger}12, Kai Mao{ddagger}1, Hanqiao Zheng{ddagger}, Xuejun Wang{ddagger}, Cam Patterson§, Timothy D. O'Connell{ddagger}, and Qiangrong Liang{ddagger}3

From the {ddagger}Cardiovascular Research Institute, Sanford Research, University of South Dakota Sanford School of Medicine, Sioux Falls, South Dakota 57105 and §Carolina Cardiovascular Biology Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

Received for publication, April 11, 2007 , and in revised form, May 24, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyperglycemia is an independent risk factor for diabetic heart failure. However, the mechanisms that mediate hyperglycemia-induced cardiac damage remain poorly understood. The transcription factor GATA4 is essential for cardiac homeostasis, and its protein levels are dramatically reduced in the heart in response to diverse pathologic stresses. In this study, we investigated if hyperglycemia affects GATA4 expression in cardiomyocytes and if enhancing GATA4 signaling could attenuate hyperglycemia-induced cardiomyocyte injury. In cultured rat cardiomyocytes, high glucose (HG, 25 or 40 mM) markedly reduced GATA4 protein levels as compared with normal glucose (NG, 5.5 mM). Equal amount of mannitol did not affect GATA4 protein expression (NG, 100 ± 12%; mannitol, 97 ± 8%, versus HG, 43 ± 16%, p < 0.05). The GATA4 mRNA content, either steady-state or polysome-associated, remained unchanged. HG-induced GATA4 reduction was reversed by MG262, a specific proteasome inhibitor. HG did not activate the ubiquitin proteasome system (UPS) in cardiomyocytes as indicated by a UPS reporter, nor did it increase the peptidase activities or protein expression of the proteasomal subunits. However, the mRNA levels of ubiquitin-protein isopeptide ligase (E3) carboxyl terminus of Hsp70-interacting protein (CHIP) were markedly increased in HG-treated cardiomyocytes. CHIP overexpression promoted GATA4 protein degradation, whereas small interfering RNA-mediated CHIP knockdown prevented HG-induced GATA4 depletion. Moreover, overexpression of GATA4 blocked HG-induced cardiomyocyte death. Also, GATA4 protein levels were diminished in the hearts of streptozotocin and db/db diabetic mice (44 ± 7% and 67 ± 13% of control, p < 0.05), which correlated with increased CHIP mRNA abundance. In summary, increased GATA4 protein degradation may be an important mechanism that contributes to hyperglycemic cardiotoxicity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Diabetes is a major risk factor for the development of various cardiovascular diseases, including atherosclerosis, hypertension, and diabetic cardiomyopathy, which collectively constitute the leading causes of mortality from diabetes. Diabetic cardiomyopathy, independent of vascular pathology, is now recognized as an important causative factor for the heightened risk of heart failure and mortality in diabetic patients (1, 2). The characteristic metabolic abnormalities of diabetes include hyperlipidemia, hyperinsulinemia (type 2 diabetes), and hyperglycemia. The development of diabetic cardiomyopathy correlates with the duration and severity of hyperglycemia. Hyperglycemia induces cardiac damage through a number of biochemical mechanisms, including the formation of advanced glycation end products (3), altered calcium homeostasis (4), enhanced renin-angiotensin system (5), and protein kinase C activation (6). Ultimately, it is thought that hyperglycemia increases oxidative stress inducing cardiomyocyte death leading to heart failure in animals (7, 8) and humans (9) with diabetes. In fact, high concentrations of glucose induce the generation of reactive oxygen species (ROS)4 and cell death in cultured cardiomyocytes (7, 10–13). The importance of oxidative stress in diabetic cardiomyopathy is underscored by the ability of various antioxidants to prevent diabetic cardiac damage in animal studies (10, 14, 15). However, antioxidants are not efficacious in human diabetic cardiomyopathy (16, 17), suggesting that mechanisms other than ROS generation might be involved.

GATA4 is a cardiac-enriched zinc finger-containing transcription factor that belongs to the GATA superfamily, which is composed of six members. GATA4 regulates the expression of various cardiac genes ranging from contractile proteins to peptide hormones and transcription factors (18). Accordingly, GATA4 is essential for several developmental processes (18) and a number of adaptive responses in the heart, including myocyte survival and cardiac hypertrophy (19, 20). As a survival factor, GATA4 contributes to cytoprotection in cardiomyocytes induced by endothelin-1 (21) and ischemic preconditioning (22, 23). GATA-4 is markedly reduced in endotoxin or infarction-induced failing hearts, and treatments that improve cardiac function also restore GATA4 levels (24, 25). Moreover, the anticancer drug doxorubicin depletes GATA4 protein levels and induces apoptosis in cardiomyocytes, which is prevented by overexpression of GATA4 (26). In addition, genetic inactivation of GATA4 in the heart induces apoptosis and impairs cardiac function (20). Collectively, these results indicate that GATA4 is an essential myocardial survival factor that can protect against cardiac injury elicited by a wide variety of pathological conditions. However, it remains unclear if GATA4 plays any role in hyperglycemia-induced cardiomyocyte injury and diabetic cardiomyopathy.

In this study, we examined the effect of hyperglycemia on the survival factor GATA4 in cultured cardiomyocytes to identify novel mechanisms of diabetic cardiomyopathy. Our results demonstrate that high glucose induces GATA4 depletion in cardiomyocytes by increased degradation of GATA4 protein through the ubiquitin proteasome system (UPS). This is likely mediated by high glucose-induced expression of the E3-ubiquitin ligase CHIP. We also found that overexpression of GATA4 prevents hyperglycemia-induced GATA4 depletion and myocyte death. In addition, in mouse models of both type 1 and type 2 diabetes, GATA4 levels are decreased, and CHIP levels are increased. In summary, our findings identify a potentially novel mechanism to explain hyperglycemic cardiotoxicity and diabetic cardiomyopathy.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Neonatal Rat Ventricular Cardiomyocyte (NRVC) Culture and High Glucose Treatment—NRVCs were isolated from 0- to 2-day-old Harlan Sprague-Dawley rat neonates using a kit from Worthington as described previously (27). For hyperglycemia studies, the NRVCs were cultured in Dulbecco's modified essential medium (DMEM containing 1% of penicillin/streptomycin; Invitrogen) with 5.5, 25, or 40 mmol/liter of glucose. To control for changes in medium osmolarity caused by increased glucose concentration, NRVCs were cultured in medium containing equivalent amounts of mannitol.

Western Blot Analysis—Protein extracts were prepared, and the Western blot analysis was performed as described previously (27). Primary antibodies were obtained from the following vendors: GATA4, Bcl2, NKX2.5, Bax, and GFP from Santa Cruz Biotechnology; {alpha}-actinin from Sigma; 20 S proteasome subunits {alpha}2-7, beta1, and beta2 and 19 S proteasome subunits RPN2 and RPT5 from Biomol; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Research Diagnostics Inc. (Concord, MA).

Semi-quantitative RT-PCR—RT-PCR was carried out using the TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA) as described (27). Two µl of the cDNA was used for PCR amplification. The gene-specific primers for GATA4, Bcl2, Bax, and GAPDH were described previously (27). The primer sequences for other genes are as follows: atrogin1 forward, 5'-ACTGGACTTCTCGACTGCCAT, and reverse, 5'-CTCCATCCGATACACCCACAT; MuRF1 forward, 5'-AACACAACCTCTGCCGGAA, and reverse, 5'-TGCAAGGTGTTTGGGGCT; CHIP forward, 5'-AGGGCAAGGAGGAAAAGGA, and reverse, 5'-TGGCAATGGCCTCATCATAA; 18 S ribosomal RNA forward, 5'-GTCCCCAACTTCTTAGAG, and reverse, 5'-CACCTACGGAAACCTTGTTAC.

Analysis of Polysome-associated mRNA—The polysome-associated GATA4 mRNA was analyzed according to published procedures (28). NRVCs were cultured in DMEM containing 5.5 or 40 mmol/liter glucose for 3 days. Cells were washed with ice-cold phosphate-buffered saline and lysed in polysome lysis buffer containing 100 mmol/liter KCl, 10 mmol/liter MgCl2, 1 mmol/liter dithiothreitol, 0.5% Nonidet P-40, 100 µg/ml cycloheximide, 10 units/ml of RNase inhibitor, and 10 mmol/liter Tris-HCl, pH 7.4. The amounts of RNA containing cytoplasmic lysates equivalent to 20 A at 260 nm were loaded onto a linear sucrose gradient in polysome lysis buffer. Gradients were centrifuged at 256,000 x g for 100 min at 4 °C. From the top of gradients, 35x 300-µl fractions were sequentially removed and their absorbances at 260 nm were measured and plotted. The polysome-bound RNAs were extracted from each sample pooled from three continuous fractions and used for RT-PCR.

Gene Silencing with siRNA—GATA4 siRNA was designed based on sequence specific for both mouse and rat GATA4 cDNA (5'-GGAGGGGATTCAAACCAGA-3') and purchased from Ambion (Austin, TX). Three different pre-designed siRNAs targeting rat CHIP mRNA (28–30) and human GAPDH siRNA were also obtained from Ambion. siRNA transfection with a final concentration of 50 nmol/liter was performed using Lipofectamine RNAiMAX according to the manufacturer's instructions (Invitrogen). Briefly, 0.7 x 106 NRVCs were transfected in 3 ml of serum-free and antibiotics-free DMEM containing 500 µl of Opti-MEM (Invitrogen), 6 µl of Lipofectamine RNAiMAX, and 50 nmol/liter of each siRNA. The media were replaced 12 h later with fresh serum-free media containing 5.5 or 40 mmol/liter glucose. The cells were harvested 60 h later for RNA extraction or Western blotting.

Measurement of Myocyte Death—Myocyte death was measured by propidium iodide (PI) staining (nonspecific indicator of cell death), TUNEL staining (apoptosis), and DNA laddering (apoptosis). Myocytes were cultured for 72 h with different concentrations of glucose. PI (Roche Applied Science) was added directly to the culture medium, and myocytes were photographed under both phase contrast and fluorescent conditions. For TUNEL staining, myocytes were labeled by terminal deoxynucleotidyltransferase-mediated nick-end labeling (TUNEL) using the In Situ Cell Death Detection kit (Roche Applied Science). Myocytes were counter-stained with 4,6-diamidino-2-phenylindole (DAPI) and Alexa Fluor 488-conjugated phalloidin. For both PI and TUNEL staining at least 200 myocytes were examined from each sample, and each condition was measured in triplicate. For DNA laddering, we used a semi-quantitative PCR-based DNA laddering kit (Maxim Biotech, San Francisco, CA) as described (29).

Adenoviral Constructs—Adenoviruses expressing beta-galactosidase, GATA4, CHIP, atrogin1 (muscle atrophy F-box), or MuRF1 (muscle ring finger protein-1) were described previously (19, 30–32).

Measurement of UPS Activity and 20 S Peptidase Activity—UPS proteolytic function in NRVCs was assayed by an adenovirus encoding a GFPu reporter (33). The chymotrypsin-, caspase-, and trypsin-like activities of 20 S proteasome were determined by using the synthetic fluorogenic peptide substrates as described previously (33).


Figure 1
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  		FIGURE 1.
HG (25 and 40 mmol/liter) treatment for 72 h diminished GATA4 protein levels in NRVCs (A and B), but NG (5. 5 mmol/liter) plus 34.5 mmol/liter mannitol (Man) did not have any effect (B). HG did not affect the steady-state levels of GATA4 mRNA as determined by RT-PCR (B, lower panel). C, quantification of GATA4 protein levels in NRVCs. n = 4; *, p < 0.05 versus NG. D, absorption profile at 260 nm of the polysomes was identical between NG- and HG-treated NRVC. E, HG did not affect the amount of GATA4 mRNA bound to each of the polysomal fractions as shown by RT-PCR.

 
Diabetic Mouse Models—Type 1 diabetes was induced in mice by injecting 2-month-old FVB mice with a single dose of streptozotocin (STZ) (intraperitoneally, 150 mg/kg body weight in 10 mmol/liter sodium citrate, pH 4.5), a well established agent that destroys pancreatic beta cells (34). Fasting blood glucose content of 15 mmol/liter or greater was considered diabetic, whereas vehicle-treated mice were used as controls. The heart tissues from type 2 diabetic db/db mice and their control db/+ mice were kindly provided by Dr. Paul Epstein, University of Louisville. The db/db mice are a well characterized insulin-resistant diabetic model with early onset cardiomyopathy (35). All protocols involving animal use were reviewed and approved by the Internal Animal Care and Use Committee at the University of South Dakota.

Statistical Analysis—Data are expressed as mean ± S.E. In all experiments, values were compared by two-tailed Student's t test or one-way analysis of variance, and p < 0.01 or 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
High Glucose Diminishes the Cardiac Transcription/Survival Factor GATA4—Hyperglycemia is an independent risk factor for diabetic heart failure (2). In cultured cardiomyocytes, high glucose induces ROS generation and apoptosis (7, 10–13). In animal models of diabetes, antioxidants attenuate heart failure, suggesting a mechanistic link between hyperglycemia-induced ROS generation, myocyte death, and heart failure (10, 14, 15). However, antioxidants show limited efficacy in preventing diabetic heart failure in humans, indicating other mechanisms might contribute (16, 17).


Figure 2
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  		FIGURE 2.
The proteasome inhibitor MG262 completely reversed HG-induced GATA4 reduction in cardiomyocytes. NRVCs were exposed to NG or HG for 72 h in the absence or presence of 25 nmol/liter of MG262. The protein levels were determined by Western blotting analysis.

 


Figure 3
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  		FIGURE 3.
HG did not enhance UPS proteolytic activity in NRVCs. A, HG slightly increased the accumulation of GFPu protein, a UPS reporter introduced into NRVCs by adenoviral infection. B, HG and mannitol (Man) (5.5 mM glucose + 34.5 mM mannitol) did not affect trypsin-like or chymotrypsin-like activity of the 20 S proteasome, but they inhibited the caspase-like activity. n = 6; *, p < 0.05 versus NG. C, HG did not affect the protein expression of 20 S proteasomal subunits ({alpha}2-7, beta1, and beta2) or 19 S proteasomal subunits (RPN2 and RPT5).

 
Diverse pathological insults deplete GATA4 protein levels in the heart, including endotoxin (24), doxorubicin (36), and myocardial infarction (25). To identify alternative mechanisms to explain diabetic heart injury, we examined the effect of high glucose (HG) on expression of the cardiac-specific transcription/survival factor GATA4 in cultured NRVC. HG (25 or 40 mmol/liter) markedly reduced GATA4 protein levels in NRVCs as compared with normal glucose (5.5 mmol/liter, NG) (Fig. 1A). Increased culture medium osmolarity did not explain the reduction in GATA4 induced by HG in NRVCs, because normalizing osmolarity with mannitol (5.5 mmol/liter glucose + 34.5 mmol/liter mannitol) did not alter GATA4 protein levels (Fig. 1, B and C;NG100 ± 12%, mannitol 97 ± 8% versus HG 43 ± 16%, n = 4, p < 0.05). Conversely, HG did not alter GATA4 mRNA levels in NRVCs as determined by RT-PCR (lower panel, Fig. 1B), suggesting a post-transcriptional regulation of GATA4 protein levels by HG. Moreover, HG did not significantly reduce the protein levels of NKX2.5 (Fig. 1B), another cardiac specific transcription factor, suggesting that the HG effect on GATA4 is relatively specific.


Figure 4
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  		FIGURE 4.
The E3-ubiquitin ligase CHIP mediated HG-induced GATA4 depletion. A, HG-induced mRNA expression of CHIP but not atrogin1 or MuRF1 in cultured NRVCs as shown by RT-PCR. B, adenovirus-mediated gene transfer of CHIP, but not atrogin1 or MuRF1, depleted GATA4 protein levels. C, CHIP mRNA was reduced by three specific but different siRNA oligonucleotides with number 28 being the most effective. Note: CHIP siRNA did not affect GATA4 mRNA as determined by RT-PCR. D, CHIP gene silencing by siRNA number 28 prevented HG-induced GATA4 protein depletion. Note: siRNA against human GAPDH did not reduce GAPDH protein in NRVCs.

 
To examine post-transcriptional regulation of GATA4 by high glucose, we measured the association of GATA4 mRNA with high molecular weight polysome complexes to assess the rate of GATA4 translation in NRVCs cultured with NG or HG. Cytoplasmic cell lysates were passed through a sucrose gradient to separate polysome-bound mRNA, which was then analyzed by RT-PCR. HG had no effect on the absorption profile of polysomes at 260 nm in NRVCs (Fig. 1D). More importantly, HG had no effect on the relative abundance of GATA4 mRNA bound to each of the polysomal fractions (Fig. 1E), indicating that the levels of polysome-associated GATA4 mRNA were not reduced in HG-treated NRVCs. In summary, these findings demonstrate that HG depleted GATA4 in NRVCs by mechanisms other than reduced GATA4 transcription or translation.

HG-induced GATA4 Reduction Is Mediated by UPS—UPS-mediated protein degradation is a multistep process involving ubiquitination of the target protein followed by proteasome-mediated degradation. Protein ubiquitination is regulated by a cascade of enzymatic reactions involving the ubiquitin-activating enzyme, a ubiquitin-conjugating enzyme, and an E3. Multiple ubiquitin moieties are conjugated to the substrate, which permits recognition and degradation of the substrate by the 26 S proteasome (37). The 26 S proteasome is composed of two 19 S regulatory complexes and a catalytic core 20 S proteasome. The 19 S complex recognizes and removes the ubiquitin moieties from labeled proteins and transfers them to the 20 S proteasome for degradation. The specific peptidase activities residing in the 20 S proteasome are responsible for protein degradation.

To determine whether HG-induced GATA4 depletion is regulated by the UPS, NRVCs were cultured in HG with MG262 (25 nmol/liter), a specific proteasome inhibitor. HG-induced GATA4 depletion was completely reversed by MG262 (Fig. 2). In fact, MG262 increased GATA4 levels in both NG- and HG-treated NRVCs, indicating that UPS-mediated GATA4 degradation is an important regulator of basal GATA4 levels as well (Fig. 2). These results suggest that HG-induced GATA4 depletion was likely mediated by the UPS.

GATA4 depletion, we measured the effect of HG on UPS activity by using a previously described GFPu UPS reporter (33). The GFPu protein carries a consensus degradation signal known as degron CL1 that renders the protein sensitive to UPS-mediated degradation. The protein level and the fluorescence intensity of GFPu are inversely related to the UPS proteolytic activity (33). Unexpectedly, HG did not increase UPS-mediated degradation of GFPu in NRVCs, shown by the slightly increased levels of the GFPu protein (Fig. 3A). In addition, we measured trypsin-like, caspase-like, and chymotrypsin-like activities associated with the 20 S proteasome. However, HG inhibited caspase-like activity and had no effect on trypsin- and chymotrypsin-like activity in NRVCs (Fig. 3B). The 20 S and 19 S proteasome each contains multiple subunits with distinct structural and functional roles (37). Once again however, HG had no effect on protein levels of the 20 S ({alpha}2-7, beta1, and beta2) or 19 S (RPN2 and RPT5) subunits in NRVCs (Fig. 3C). Mannitol mimicked the effect of HG and slightly inhibited the UPS activity (Fig. 3A, slightly increased GFPu levels), significantly reduced caspase-like activity (Fig. 3B), and had no effect on 20 S or 19 S protein levels (Fig. 3C). In summary, HG induced-GATA4 depletion was not associated with a general activation of the UPS, and UPS proteolytic activity does not appear to be rate-limiting in GATA4 protein degradation. Alternatively, increased ubiquitination per se might enhance protein degradation simply by directing more modified GATA4 protein to the 26 S proteasome even if the proteasome activity is moderately reduced.

Increased Expression of the E3 Ubiquitin Ligase CHIP Mediates HG-induced GATA4 Depletion—Ubiquitin E3 ligases determine the relative specificity of substrates that undergo proteasome-mediated degradation (37). Therefore, HG might activate a specific E3 ligase that facilitates GATA4 ubiquitination and degradation even if proteasome activity is moderately inhibited. We screened several E3 ligases that are expressed in cardiomyocytes, including atrogin1, MuRF1, and CHIP. HG dramatically induced mRNA expression of CHIP but not atrogin1 or MuRF1 in cultured NRVCs as determined by RT-PCR (Fig. 4A). Importantly, adenovirus-mediated gene transfer of CHIP, but not atrogin1 or MuRF1, depleted GATA4 protein levels in NRVCs (Fig. 4B), whereas CHIP gene silencing by three different siRNAs dramatically reduced CHIP mRNA levels (Fig. 4C), which prevented high glucose-induced GATA4 depletion (Fig. 4D). These results strongly support the hypothesis that CHIP may be the E3 ligase that is responsible for hyperglycemia-induced GATA4 degradation.

Overexpression of GATA4 Prevents HG-induced GATA4 Degradation and Myocyte Death—High glucose results in ROS generation and cardiomyocyte apoptosis (7). Our results demonstrate that HG also depletes GATA4, possibly increasing susceptibility to HG-induced cell death and suggesting a novel mechanism for hyperglycemic cardiotoxicity. Therefore, to test the hypothesis that GATA4 overexpression could rescue myocytes from HG-induced GATA4 depletion and cell death, we infected NRVCs with adenovirus encoding GATA4 (AdGATA4) or beta-galactosidase (Adbetagal), cultured them in the presence of NG or HG, and measured GATA4 levels and cell death. Overexpression of GATA4 prevented GATA4 depletion and up-regulated Bcl-2 expression in NRVCs (Fig. 5A). Correspondingly, overexpression of GATA4 prevented HG-induced cell death as indicated by reduced PI-positive cells (Fig. 5, B and C), TUNEL-positive cells (Fig. 6, A and B), and DNA laddering (Fig. 6C). Increased GATA4 also reduced HG-induced ROS generation measured by CM-H2DCFDA fluorescent intensity (data not shown). In summary, GATA4 overexpression is sufficient to prevent HG-induced GATA4 depletion and attenuate hyperglycemia-induced cardiotoxicity.

Diabetes Depletes GATA4 and Increases CHIP in the Mouse Heart—To determine whether HG-induced GATA4 depletion and cardiomyocyte death translate in vivo, we measured GATA4 and CHIP expression levels in hearts from type 1 or type 2 diabetic mouse models. Type 1 diabetes was induced in 2-month-old FVB mice with STZ. In this model, cardiac GATA4 protein levels started to decrease as early as day 4 after STZ dosing and reached lowest levels at 4 weeks (Fig. 7, A–C, STZ 44 ± 7% versus control 100 ± 9.6%, n = 4, p < 0.05). However, semi-quantitative RT-PCR showed that GATA4 mRNA levels remained unchanged in the diabetic heart (lower panel, Fig. 7B). We also measured cardiac GATA4 levels in 4-month-old db/db mice, a type 2 diabetes model. Similarly, GATA4 protein levels but not mRNA levels were reduced in db/db diabetic hearts compared with db/+ nondiabetic controls (Fig. 7, D and E, db/db 67 ± 13% versus db/+ 100 ± 10%, n = 4, p < 0.05). Furthermore, we found that CHIP mRNA levels were markedly increased in STZ and db/db diabetic mouse hearts as determined by RT-PCR (Fig. 8, A and B, STZ 235 ± 9.8% versus control 100 ± 18%; db/db 173 ± 13% versus db/+ 100 ± 9.6%, n = 4, p < 0.01). In contrast, the mRNA levels of MuRF1 and atrogin1 were not changed in STZ diabetic hearts and significantly reduced in db/db diabetic hearts. In summary, diabetes, both type 1 and type 2, diminished GATA4 and increased CHIP, which correlated with our findings in NRVCs, suggesting a novel mechanism that might mediate hyperglycemic cardiotoxicity.


Figure 5
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  		FIGURE 5.
Overexpression of GATA4 in NRVCs prevented HG-induced GATA4 reduction and myocyte death. NRVCs were infected with Adbetagal or AdGATA4 and then exposed to NG (5.5 mM), HG (25 or 40 mM), or mannitol (5.5 mM glucose + 34.5 mM mannitol) for 72 h. GATA4 protein and mRNA levels were determined by Western blot analysis (left panel of A) and RT-PCR (right panel of A), respectively. Myocyte death was determined by propidium iodide (PI) staining (B). PI positive NRVCs were expressed as the percentage of total myocytes counted under phase contrast (C). Shown are representative results of several experiments in triplicate. n = 3; #, p < 0.05; *, p < 0.01 versus 5.5 mM glucose.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
More than two-thirds of diabetic patients die from heart disease. A major mechanism proposed for diabetic cardiac damage is oxidative stress that is presumably induced by hyperglycemia (2). The ability of various antioxidants to attenuate diabetic heart failure has been firmly established in animal studies (10, 14, 15). However, clinical trials have not reproduced these results (16, 17). Thus, to reduce diabetic heart failure, further studies are warranted to identify and better understand the molecular targets and signaling mechanisms that mediate diabetic cardiac damage. In this study, we showed that the protein levels of GATA4, a transcription factor essential for cardiomyocyte growth and survival, were markedly diminished in cardiomyocytes by HG and in the hearts of STZ-induced type 1 diabetic mice and db/db type 2 diabetic mice. Our results demonstrate that hyperglycemia-induced GATA4 depletion is likely caused by the increased expression of the E3-ubiquitin ligase CHIP that mediates GATA4 ubiquitination and degradation by the UPS. We also found that overexpression of GATA4 prevented hyperglycemia-induced GATA4 depletion and myocyte death, consistent with previous studies that suggest a cardioprotective role of GATA4 in response to diverse pathological insults, including endotoxin (24), doxorubicin (26, 36), and myocardial infarction (25). In summary, our findings identify a potentially novel mechanism to explain hyperglycemic cardiotoxicity and diabetic cardiomyopathy.


Figure 6
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  		FIGURE 6.
Overexpression of GATA4 blocked HG-induced apoptosis in NRVCs as shown by TUNEL labeling (A and B) and DNA laddering (C). NRVCs were treated as in Fig. 5 and stained with TUNEL reagents (green), Alexa Fluor 488-conjugated-phalloidin (red), and 4,6-diamidino-2-phenylindole (DAPI, blue). TUNEL-positive nuclei (green) were expressed as percentage of the total myocyte nuclei (blue). Shown are representative results of several experiments in triplicate. n = 3; *, p < 0.01 versus 5.5 mM glucose. Semi-quantitative PCR amplification of GAPDH gene showed equal amounts of genomic DNA used for the DNA laddering assay.

 
The conclusion that hyperglycemia-induced GATA4 depletion is because of enhanced protein degradation by the UPS is based on two observations. First, hyperglycemia-induced GATA4 depletion was not caused by a repressed mRNA transcription or protein synthesis as indicated by unchanged steady-state levels of mRNA or the amount of mRNA associated with polysome. Second, the proteasome inhibitor MG262 completely reversed HG-induced GATA4 depletion. Despite these results, we found that HG did not increase UPS proteolytic activity in cardiomyocytes, as determined by the GFPu reporter, nor did it elevate peptidase activities in the 20 S proteasome. In fact, HG inhibited the caspase-like activity of the 20 S proteasome. These results indicate that the proteasome may have substantial amounts of reserve in its proteolytic capacity, which is not rate-limiting in GATA4 protein degradation in response to HG. This may explain why hyperglycemia was able to diminish GATA4 protein levels even when the proteolytic function of the UPS was moderately reduced, as assessed by either GFPu reporter or peptidase activity. Alternatively, the GFPu surrogate reporter may not truthfully reflect the proteolytic capacity of the UPS toward all endogenous proteins, especially for signaling molecules like GATA4 that do not contain the same degradation signal as CL1.

The enhanced degradation of specific proteins could be caused by elevated UPS proteolytic activity or more likely by an increased E3-ubiquitin ligase in the case that only a selected group of proteins is degraded. E3 ligases select target proteins for ubiquitination, and the increased ubiquitination per se may result in enhanced protein degradation simply by directing more modified protein into the 26 S proteasome with or without a change in its proteolytic activity (37). Indeed, we found that the mRNA levels of CHIP were markedly increased in HG-treated cardiomyocytes and diabetic hearts. CHIP is the first identified E3 ligase that associates with molecular chaperones heat shock protein (Hsp) 70 and Hsp90 (38). CHIP mediates the ubiquitination and degradation of several proteins. including Raf1 (39) and neuronal nitric-oxide synthase (40). Given its ability to up-regulate CHIP, it is not surprising that hyperglycemia was able to promote GATA4 degradation even when it paradoxically inhibited the caspase-like activity in the 20 S proteasome. Additionally, we showed that adenovirus-mediated gene transfer of CHIP decreased GATA4 protein levels in cardiomyocytes, whereas siRNA-mediated gene knocking down of CHIP prevented HG-induced GATA4 depletion, suggesting that CHIP may be indeed the E3 ligase that is responsible for hyperglycemia-induced GATA4 degradation. If that is the case, development of specific CHIP inhibitors that can restore GATA4 levels may be an effective strategy for reducing diabetic cardiac injury. However, too much inhibition of CHIP activity may not be desirable because the hearts of CHIP knock-out mice show increased sensitivity to ischemia-reperfusion injury (41), which suggests an important role for CHIP in maintaining normal cardiac homeostasis.


Figure 7
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  		FIGURE 7.
GATA4 protein levels were reduced in diabetic mouse hearts. A, GATA4 protein levels were reduced in the heart of STZ-treated FVB mice at all time points after intraperitoneal injection. Vehicle-treated mice were used as control (Con). B, cardiac GATA4 protein (upper panel) and mRNA (lower panel) levels at 4 weeks after STZ injection. C, quantification of cardiac GATA4 protein levels 4 weeks after STZ injection. D, GATA4 protein (upper) and mRNA (lower) levels in 4-month-old db/db diabetic mouse heart. E, quantification of cardiac GATA4 protein levels in db/db mice. n = 4; *, p < 0.05 versus control.

 


Figure 8
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  		FIGURE 8.
The mRNA expression of CHIP was markedly increased in STZ (A) and db/db (B) diabetic mouse hearts as determined by semi-quantitative RT-PCR. In contrast, the mRNA levels of MuRF1 and atrogin1 were not changed in STZ diabetic hearts and significantly reduced in db/db diabetic hearts. The mRNA levels were quantified by densitometry. n = 4; **, p < 0.01; *, p < 0.05 versus control.

 
Besides hyperglycemia, a number of other pathological insults are also able to deplete GATA4 protein levels in the heart such as endotoxin (24), doxorubicin (36), and myocardial infarction (25). The mechanisms whereby GATA4 is depleted in cardiomyocytes are not necessarily the same under different pathological conditions. For example, doxorubicin-induced GATA4 depletion is mediated by both repressed gene transcription (26) and enhanced protein degradation by the increased UPS activity (42). Thus, proteasomal inhibition not only reverses GATA4 depletion but also attenuates doxorubicin-induced cardiomyocyte death.5 However, because the proteasomal function was somewhat impaired in HG-treated NRVCs as determined by the GFPu reporter or the peptidase activity, further proteasomal inhibition is probably not a viable strategy to reduce HG-induced cardiotoxicity despite the fact that the proteasome inhibitor MG262 can completely reverse GATA4 depletion. Alternative strategies that can enhance GATA4 gene expression or increase the stability of GATA4 protein by post-translational modifications may turn out to be more beneficial in reducing hyperglycemia-induced cardiotoxicity.

In summary, we demonstrate that GATA4 protein levels were markedly reduced in cardiomyocytes by high glucose. The reduction in GATA4 protein levels was associated with an up-regulation of CHIP, an E3 ligase that likely promotes GATA4 ubiquitination and degradation through the UPS. Overexpression of GATA4 prevented GATA4 depletion and attenuated hyperglycemia-induced cardiomyocyte injury. Additionally, GATA4 levels were decreased, and CHIP levels were increased in the hearts of both type 1 and type 2 diabetic mouse models. Together, these results shed new light on the mechanism of hyperglycemic cardiotoxicity and may suggest novel therapeutic strategy for managing diabetic cardiomyopathy.


   FOOTNOTES
 
* This work was supported in part by Grant P20 RR-017662 (Project 3) from the National Center for Research Resources (to Q. L.) of the National Institutes of Health and American Heart Association Scientist Development Grant 0435308N (to Q. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 Supported by an American Heart Association postdoctoral fellowship. Back

3 To whom correspondence should be addressed. Tel.: 605-328-1308; Fax: 605-328-1301; E-mail: qliang{at}usd.edu.

4 The abbreviations used are: ROS, reactive oxygen species; STZ, streptozotocin; NRVC, neonatal rat ventricular cardiomyocytes; NG, normal glucose; HG, high glucose; RT, reverse transcriptase; UPS, ubiquitin proteasome system; CHIP, carboxyl terminus of Hsp70-interacting protein; MuRF1, muscle ring finger protein-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; E3, ubiquitin-protein isopeptide ligase; TUNEL, terminal deoxynucleotidyltransferase-mediated nick-end labeling; DMEM, Dulbecco's modified Eagle's medium; siRNA, small interfering RNA; PI, propidium iodide. Back

5 S. Kobayashi and Q. Liang, unpublished observations. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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