The ubiquitin–proteasome system contributes to the inflammatory injury in ischemic diabetic myocardium: the role of glycemic control

Cardiovascular Pathology 18 (2009) 332 – 345

Original Article

The ubiquitin–proteasome system contributes to the inflammatory injury in ischemic diabetic myocardium: the role of glycemic control Raffaele Marfella a,c,⁎,1 , Clara Di Filippo b,c,1 , Michele Portoghese d , Mario Siniscalchi a , Simone Martis d , Franca Ferraraccio e , Salvatore Guastafierro a , Gianfranco Nicoletti a , Michelangela Barbieri a , Antonino Coppola f , Francesco Rossi a,c , Giuseppe Paolisso b,c , Michele D'Amico a,c a

Department of Geriatrics and Metabolic Diseases, Second University of Naples, Naples, Italy b Department of Experimental Medicine, Second University of Naples, Naples, Italy c “Centro di Eccellenza Cardiovascolare”, Second University of Naples, Naples, Italy d Cardiovascular Surgery Unit, Sassari Hospital, Italy e Department of Clinical, Public and Preventive Medicine, Second University of Naples, Naples, Italy f Intensive Coronary Unit, Hospital Santa Maria della Misericordia, Sorrento, Italy Received 10 May 2008; received in revised form 30 August 2008; accepted 30 September 2008

Abstract Background: Because the ubiquitin–proteasome pathway (UPS) is required for activation of nuclear factor kappa beta (NFkB), a transcription factor that regulates inflammatory genes, we evaluated the UPS activity, NFkB activation, and tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, in ischemic specimens of diabetic myocardium and relate them to the glycemic control (HbA1c), oxidative stress (nitrotyrosine, a modified amino acid produced by reactive O2), and cardiac outcome (echocardiographic parameters). Moreover, the role of UPS, NFkB, and TNF-alpha in the cardiac tissue injury of acute ischemia/reperfusion (I/R) was evaluated in streptozotocin (STZ)-hyperglycemic rats. Finally, this study aimed to elucidate whether an intervention on UPS with bortezomib, an inhibitor of UPS, may counteract the extensive myocardial infarction and increased inflammatory reaction into the hyperglycemic myocardium. Methods: Ventricular biopsy specimens from 16 nondiabetic and 18 type 2 diabetic patients presenting with unstable angina who underwent coronary artery bypass were collected during coronary bypass surgery. Ejection fraction (EF); myocardial performance index (MPI), which measures both systolic and diastolic function, immunostaining, and cardiac levels of nitrotyrosine; UPS activity; NFkB; and TNF-alpha were investigated in both ischemic human myocardium and heart tissue from STZ-hyperglycemic rats subject to a myocardial ischemia/reperfusion procedure. Results: We found that diabetic patients had higher MPI (Pb.041) and reduced EF (Pb.008) compared with nondiabetic patients. Diabetic specimens had higher nitrotyrosine, UPS activity, NFkB, and TNF-alpha levels compared with nondiabetic patients (Pb.001). This was mirrored by consistently high levels of UPS and inflammatory markers in STZ-infarcted hearts, associated with high myocardial damage. In contrast, lesions from normoglycemic animals as well as from hyperglycemic rats treated with bortezomib showed low levels of ubiquitin– proteasome activity, inflammation, and myocardial damage (Pb.01). Conclusions: By contributing to the increased inflammation, the UPS overactivity may enhance the risk of complication during myocardial ischemia in diabetic patients. © 2009 Elsevier Inc. All rights reserved. Keywords: Ubiquitin–proteasome system; Inflammatory injury; Ischemic diabetic myocardium; Glycemic control

This study was funded by the Ministerial Funds for Scientific Research. The authors do not have any financial associations that might pose a conflict of interest in connection with the submitted article. ⁎ Corresponding author. Piazza Miraglia 2, 80138 Naples, Italy. Tel.: +39 081 5665010; fax: +39 081 5096142. E-mail address: [email protected] (R. Marfella). 1 Shared first authorship. 1054-8807/08/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2008.09.008

1. Introduction During myocardial infarction (MI), glucose values in excess of 110–144 mg/dl are associated with a threefold increase in mortality and a higher risk of heart failure [1,2].

R. Marfella et al. / Cardiovascular Pathology 18 (2009) 332–345

Although the mechanisms underlying this association are not fully understood, evidence that use of insulin to lower glucose concentrations decreases mortality in patients with diabetes who have MI [3] suggests that hyperglycemia is not simply an epiphenomenon of a stress response, but an important and potentially modifiable risk factor for poor outcome. A growing body of evidence suggests that proinflammatory cytokines are released within the host myocardium and modulate tissue injury and adaptation after injury [4]. Indeed, the consequences of a robust inflammatory cytokine up-regulation after a myocardial ischemic event can be unfavorable, leading to acute cardiac rupture or chronic dilatation, paving the way for infarction extension and heart failure [5]. There is emerging evidence that the ubiquitin–proteasome system (UPS), the major pathway for nonlysosomal intracellular protein degradation in eukaryotic cells, induces inflammation [6]. Indeed, the UPS is required for activation of nuclear factor kappa B (NFkB), a central transcription factor that regulates inflammatory genes, by degradation of its inhibitory IkB proteins [7]. Although it has been demonstrated that diabetes may up-regulate the UPS in rat muscle [8] and in human atherosclerotic plaques [9], still no evidence exists about the potential role of the UPS in the modulation of inflammation and in the evolution of myocardial ischemia of diabetic patients. We hypothesized that, by increasing ubiquitin–proteasome activity, diabetes may enhance the inflammatory potential of ischemic myocardial tissue, worsening cardiac tissue injury. To address these issues two separate studies were conducted. One evaluated the ubiquitin–proteasome activity and proinflammatory cytokine levels such as TNF-α in myocardial biopsies obtained from patients with and without type 2 diabetes who were admitted to emergency wards for unstable angina and had to undergo coronary bypass graft surgery (CABG) and relate them to the glycemic control. Moreover, because hyperglycemia up-regulates the oxidative stress and this in turn may activate the UPS [10], the localization of nitrotyrosine, a modified amino acid produced by reactive O2 [11], in ischemic myocardium was measured quantitatively. The other was aimed to elucidate whether an intervention on UPS with bortezomib may counteract the extensive MI and increased inflammatory reaction into the myocardium occurring in case of ischemic event. We investigated the effect of bortezomib, an inhibitor of ubiquitin–proteasome activity, on the ischemic heart tissue of streptozotocin (STZ)-hyperglycemic rats.

2. Methods 2.1. Human study The study group comprised 16 nondiabetic and 18 type 2 diabetic patients presenting with unstable angina without

333

previous MI and who were to undergo CABG. The assessment of the type of unstable angina was based on Braunwald's classification [12]; they had evidence of myocardial ischemia at rest within 48 h, defined as angina or heart failure without Q waves on the electrocardiogram and without an increase in serum levels of troponin I (Class III/B). Patients were admitted into the emergency wards of the Sassari Hospital from January 2001 to January 2005. The investigation conforms with the principles outlined in the Declaration of Helsinki for use of human tissue or subjects. The criteria for enrollment included the need for coronary bypass surgery in unstable angina [13]. Patients with interfering noncardiac diseases, such as inflammatory disorders, malignancy, or infection, and three-vessel disease, were not eligible for the study. The study protocol was approved by the institutional ethics committee at Sassari Hospital. Written informed consent was obtained from all patients. Routine analyses were obtained on admission before coronary angiography and full medical therapy, including βblockers and/or calcium antagonists, low-dosage aspirin, and nitrates. Venous blood for troponin I (Behring Diagnostics, normal values b2.0 μg/l) levels was collected in EDTAcoated tubes immediately after the patients arrived at the emergency department. 2.2. Echocardiographic assessment Patients enrolled in the study underwent two-dimensional echocardiography before starting full medical therapy and surgery. The study was performed using a standardized protocol and phased-array echocardiographs with M-mode, two-dimensional, and pulsed, continuouswave, and colorflow Doppler capabilities. The ejection fraction was calculated from area measurements using the area-length method applied to the average apical area [14]. Measurements were made with a computerized review station equipped with a digitizing tablet and monitor screen overlay for calibration and measurement performance. Doppler velocities and time intervals were measured from mitral inflow and left ventricular outflow recordings. Isovolumetric relaxation time (IRT) was the time interval from cessation of left ventricular outflow to onset of mitral inflow, the ejection time (ET) was the time interval between the onset and the cessation of left ventricular outflow, and the mitral early diastolic flow deceleration time was the time interval between the peak early diastolic flow velocity and the end of the early diastolic flow. The total systolic time interval was measured from the cessation of one mitral flow to the beginning of the following mitral inflow. Isovolumetric contracting time (ICT) was calculated by subtracting ET and IRT from the total systolic time interval. The ratio of velocity time intervals (vti) of mitral early (E) and late (A) diastolic flows (Evti/Avti) was calculated. The myocardial performance index (MPI) was calculated as (IRT+ICT)/ET.

334

R. Marfella et al. / Cardiovascular Pathology 18 (2009) 332–345

2.3. Biopsy of myocardium After the induction of anesthesia and median sternotomy, the heart of each patient was examined, and 3-mm partialthickness biopsy specimens were taken from the left ventricle (LV) area. Specimens were taken from the area of presumed ischemia (ischemic area) and from an area of the ventricle free of coronary disease (nonischemic area) that could have caused ischemia. In this way, each patient served as his or her own control. All biopsies were performed before CABG, during ventilation with a fraction of inspired oxygen of 40% and peripheral oxygen saturations of N95%. 2.3.1. Analysis of specimens Half of each biopsy specimen was fixed in formalin, sectioned to a thickness of 5 μm, mounted on slides, and stained with hematoxylin and eosin. The mounted specimens were then examined for evidence of ischemia or kept for immunohistochemistry. The other half of the specimen was frozen in liquid nitrogen for the following ELISA analysis. 2.4. Proof of concept study in rats 2.4.1. Hyperglycemic rats Experiments were conducted in male Sprague-Dawley rats (4–6 months old and weighing 250 g) according to a procedure previously described [8]. Particularly, STZ was administered at 70 mg/kg ip and human regular insulin (1.5±0.5 U/day) was applied to rats in order to yield blood glucose levels of 22 mmol/l for 8 days. All experimental procedures were approved by the Animal Care Committee of Naples College. Rats were allocated to one of the four following groups: (1) normoglycemic rats (six sham-operated and six infarcted animals); (2) hyperglycemic rats (six sham-operated and six infarcted animals); and (3) hyperglycemic rats pretreated with bortezomib (six sham-operated and six infarcted animals); rats were injected through a forepaw vein with 0.05 mg/kg bortezomib (Velcade; Millennium Pharmaceuticals, Cambridge, UK). The first intravenous injection was performed 1 h before and 1 h postischemic procedures (total dosage 0.1 mg/kg). No significant side effects of the injection dosage were observed in our study. Mild diarrhea was observed in one animal. (4) Normoglycemic rats were pre-treated with bortezomib (six shamoperated and six infarcted animals). Bortezomib was dissolved in saline (vehicle). 2.4.2. In vivo I/R injury The procedure has been previously described [9]. I/R procedure was done in the STZ-treated animals after 8 days of stable hyperglycemia. Briefly, ligation of the left anterior descending coronary artery (LADCA) close to its origin was applied to induce I/R. Coronary artery occlusion was done for a 25-min period and reperfusion

occurred for 2 h. Measurement of area at risk (AR) and infarct size (IS) was assessed by staining with Evans blue dye and then triphenyltetrazolium chloride assisted by computerized planimetry using an image analysis software program. In another set of experiments at the end of 2 h of reperfusion, staining was omitted and the tissue of the area at risk was cut perpendicular to the long axis into two halves. Myocardial tissues were frozen in liquid nitrogen for the following ELISA and Western blotting analysis. 2.5. Immunohistochemistry After the surgical procedure, samples were immediately frozen in isopentane and cooled in liquid nitrogen. Serial sections were incubated with specific antibodies: antiubiquitin, anti-proteasome 20S, anti CD68 and anti-CD3 (Dako); anti–IkB-β, anti-iNOS, anti-myoglobin (Santa Cruz); anti–tumor necrosis factor-alpha (TNF-α) (R&D). Specific antibodies that selectively recognize the activated form of NFkB (p65 and p50 subunits, Santa-Cruz) were used. Nitrotyrosine was determined by anti-nitrotyrosine rabbit polyclonal antibody (DBA, Milan). iNOS quantization was scored for intensity of immunostaining. The specimens were analyzed by an expert pathologist (intraobserver variability 6%) blinded to the patient's diagnosis. 2.6. Quantitative analysis for histology We determined the area occupied by CD3- and CD68positive–rich areas. Analysis of experiments was performed with a PC-based, 24-bit color, image-analysis system. In brief, electronic images were digitized with a Leica color video camera (IM500, Leica Microsystem). A color threshold mask for immunostaining was defined to detect the red color by sampling, and the same threshold was applied to all specimens. The percentage of the total area with positive color for each section was recorded. 2.7. Biochemical assays Myocardial tissues were lysed and centrifuged for 10 min at 10,000×g at 4°C. After centrifugation, ubiquitin, IkB-β, and TNF-α levels were quantified in heart tissue using a specific ELISA kit (Santa Cruz; R&D Systems; Imgenex). Nuclear extracts from heart specimens were obtained as described by Ohlsson et al. [15]. We used a specific antibody that selectively recognizes the activated form of the NFkB subunit p65. NF-kB binding to kB sites was assessed using the Trans-AM NF-kB p65 transcription factor assay kit (Active Motif Europe, Rixensart). In this assay, an oligonucleotide containing the NF-kB consensus site is attached to a 96-well plate. The active form of NFkB contained in cell extracts specifically binds to this oligonucleotide and can be revealed by incubation with antibodies. Thereafter, whole cell extracts were prepared,

R. Marfella et al. / Cardiovascular Pathology 18 (2009) 332–345

and 10 μg of total cellular proteins was analyzed for p65 binding to kB oligonucleotide according to the manufacturer's instructions. In addition, we analyzed the expression of the activated p50 subunit by specific Trans-AM NF-kB p50 transcription factor assay kit (Active Motif, Rixensart). For the quantitative measurement of the proteasome 20S activity, a specific SDS-activation kit (Boston-Biochem) was used following company instructions: the most common assessment of 20S activity in vitro is done by measuring the hydrolysis of the fluorogenic peptidyl substrate Suc-Leu-Leu-Val-Tyr-AMC by the SDSactivated proteasome. This substrate is cleaved by the chymotryptic-like activity of the proteasome releasing free AMC (7-amino-4-methylcoumarin) which can be efficiently detected using a fluorimeter. For quantitative measurements of the proteasome, the fluorimeter may be calibrated by generating a standard curve using AMC. This calibration allows for the calculation of the exact specific activity of the 20S on each individual fluorimeter. Nitrotyrosine was assayed into heart tissue with a kit supplied by Hycult Biotech. 2.8. Polyubiquitinated proteins Polyubiquitinated proteins in rat ischemic myocardial tissue were analyzed by Western blotting. Mouse MAb (IgM) to polyubiquitinated proteins (clone FK1) (Affinity Research Products) was used. The protein (10 μg) from above the homogenates was boiled for 5 min and then subjected to SDS/PAGE (7.5%). After the transfer of proteins onto the nitrocellulose, the blots were blocked with 5% nonfat dry milk in 20 mM Tris/HCl (pH 7.5), 500 mM NaCl plus 0.1% Tween 20 (TBS-T). The membranes were subsequently incubated in agitation at 4°C overnight in 1% BSA-TBS-T buffer containing the specific antibody against polyubiquitinated proteins (clone FK1). After being washed four times with TBS-T, the blots were incubated for 1 h at room temperature with the second antibody conjugated to peroxidase, washed four times with TBS-T, developed with ECL detection reagents (Amersham, UK) for 1 min, and then exposed to X-Omat film (Eastman Kodak Co., Rochester, NY, USA). The densitometric measurements were performed using the gel image system Fluor-S equipped with the analysis software Quantity One (BioRad, Italy). 2.9. Measurement of O2− Production of O2− was measured as the superoxide dismutase-inhibitable reduction of cytochrome c, as previously described [16]. 2.10. Statistical analysis Data are presented as mean±S.D. Continuous variables were compared among the groups of patients with one-way

335

Table 1 Characteristics of study patients Nondiabetic patients Diabetic patients No. of patients Age (years) Male/female Diabetes duration (years) BMI (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Heart rate (bpm) Blood glucose (mmol/l) Insulin (μU/ml) HbA1c (%) Total cholesterol (mmol/l) HDL Cholesterol (mmol/l) Triglycerides (mmol/l) Smokers [% (n)] Serum creatinine (μmol/l) Previous disorder [% (n)] Angina pectoris Hypertension Active therapy [% (n)] β-Blocker Diuretic ACE Inhibitors Statin Nitroglycerin Sulfonylureas Metformin Insulin [% (n)] Left ventricular mass index (g/m2) Myocardial performance index Pulmonary venous flow (PVFs/PVFd ratio) Mitral deceleration (ms) Early/late diastolic ratio Ejection fraction (%) Troponin I (μg/l) Vessel disease Left main coronary artery stenosis [% (n)] Stenosis of LAD and LCA [% (n)] Macrophage-rich ischemic areas (%) T-lymphocytes-rich ischemic areas (%)

16 60±7 8/8 – 26±0.6 119±6 80±8 71±3 6±0.9 ⁎ 8.06±3.3 ⁎ 6.1±0.7 ⁎ 6.70±0.05 1.27±0.03 2.22±0.18 50 (9) 77.8±2.5

18 59±8 10/8 18±7 28±0.9 121±12 81±9 75±4 12.9±1.7 10.6±2.4 8.3±1.5 6.81±0.05 1.25±0.04 2.51±0.16 44 (8) 78.3±2.9

38 (6) 32 (5)

39 (7) 39 (7)

19 (3) 13 (2) 19 (3) 37 (6) 87 (14) – – – 121.1±8 ⁎

17 (3) 17 (3) 33 (6) 39 (7) 88 (16) 22 (4) 33 (6) 44 (8) 135±9

0.51±0.11 ⁎ 1.31±0.1 ⁎

0.62±0.12 1.55±0.2

150±11 ⁎ 0.9±0.1 ⁎ 38.9±6.5 ⁎ 1.0±0.7

168±19 1.1±0.1 32.7±6.3 1.1±0.9

62 (10)

61 (11)

37 (6)

39 (7)

7±3 ⁎

26±8

11±6 ⁎

71±14

BMI=body mass index; LAD=left anterior descending artery; LCA=left circumflex artery. Data are means±S.D. ⁎ Pb.05 vs. diabetic patients.

analysis of variance for normally distributed data, and Kruskal–Wallis test for non-normally distributed data. When differences were found among the groups, Bonferroni correction was used to make pairwise comparisons. To investigate the independent contribution of HbA1c in the modification of both ubiquitin-poteasome activity and hemodynamic parameters, a multiple regression analysis was performed. A P value ≤.05 was considered statistically significant. All calculations were performed using the computer program SPSS 12.

336

R. Marfella et al. / Cardiovascular Pathology 18 (2009) 332–345

3. Results 3.1. Preoperative characteristics of the patients and classification of ventricular specimens The characteristics and cardiac measurements of the patients before CABG and heart biopsy are shown in Table 1. The unstable angina therapy was similar in both groups (data not shown). There were no significant differences in the indication for CABG among the groups (Table 1). On admission, patients with diabetes had significantly higher plasma glucose levels and adult hemoglobin (HbA1c) values compared with nondiabetic patients (Pb.001). All ventricular biopsy specimens were examined by light microscopy for evidence of ischemia by a cardiac pathologist who was unaware of the patients' identity. There were no significant differences in vessel stenosis between the diabetic and nondiabetic patients; moreover, the ischemic areas evaluated by a cardiac pathologist were not different between the groups. Echocardiographic/Doppler measurements are presented in Table 1. Diabetic patients had lower ejection fraction (Pb.008) than nondiabetic patients. Moreover, they

had an increased MPI (Pb.02) and reduced transmitral Doppler flow (Pb.05) and pulmonary venous flow analysis (Pb.041), compared with nondiabetic patients. HbA1c levels were positively correlated with MPI (r=0.34, Pb.01) and negatively correlated with ejection fraction (r=−0.50, Pb.001). 3.2. Oxidative stress in the ischemic heart 3.2.1. Human biopsies. iNOS Significantly intense immunostaining of iNOS was present in both nonischemic and ischemic diabetic tissue compared with nondiabetic tissues (Pb.001) (Fig. 1). iNOS protein expression was present in specimens from the nonischemic areas in both groups, although at a higher level in the diabetic group (Pb.001). Nondiabetic patients with acute ischemia had expression of iNOS in the specimens from the ischemic area significantly higher compared with iNOS expression in the nonischemic area (Pb.001). Diabetic patients with acute ischemia had significantly higher iNOS expression in specimens from the ischemic area compared with the iNOS expression in

Fig. 1. (Panel A) Nitrotyrosine levels and immunostaining score for iNOS in myocardial specimens. (Boxplot displays the median, 25th, and 75th percentiles, range, and extreme values.) *Pb.001 compared with type 2 diabetic patients. †Compared with ischemic area. (Panel B) Representative sections show immunochemistry for nitrotyrosine (×630) and iNOS (×630) in myocardial specimen. These results are typical of nondiabetic and type 2 diabetic ischemic myocardial specimens.

R. Marfella et al. / Cardiovascular Pathology 18 (2009) 332–345

337

Fig. 2. Immunostaining of nitrotyrosine, proteasome 20S, and ubiquitin (×200) of consecutive serial sections of ischemic heart specimens.

the nonischemic area (Pb.001). Notably, iNOS expression in ischemic specimens was strongly dependent on glycemic control, as also reflected by the statistically significant correlation (R=0.52, Pb.001) between plasma HbA1c and iNOS expression. 3.2.2. O2− Significantly higher levels of O2− were present in ischemic diabetic tissue compared with ischemic nondiabetic tissue (2.7±0.8 vs. 7.3±1.1 pmol/l, Pb.001) (Fig. 2). In nonischemic areas, significantly higher levels of O2− were present in diabetic tissue compared with nondiabetic tissue (Pb.001). Moreover, O2- levels in ischemic specimens were strongly dependent on glycemic control, as also reflected by the statistically significant correlation between plasma HbA1c and O2− concentrations (R=0.59, Pb.001). 3.2.3. Nitrotyrosine When immunostaining for the nitrotyrosine antigen was compared, differences were found between tissues from diabetic and nondiabetic ischemic specimens. Significantly intense immunostaining as well as higher nitrotyrosine

levels were present in ischemic diabetic tissue compared with ischemic nondiabetic tissues (Pb.001) (Fig. 1). In the nonischemic areas, significantly intense immunostaining and nitrotyrosine levels were present in diabetic compared with nondiabetic patients (Pb.001). Specifically, nitrotyrosine immunoreactivity was seen in both cardiomyocytes and macrophages of the diabetic ischemic area; on the other hand, in nondiabetic patients these immunoreactivity was seen essentially in the inflammatory cells (Fig. 2). 3.2.4. Rat tissues To determine the role of hyperglycemia on ischemic myocardial injury as well as oxidative stress generation in ischemic myocardium, we evaluated heart injury and the levels of nitrotyrosine during the I/R study in both normoglycemic and STZ-hyperglycemic rats. In hyperglycemic rats, blood glucose averaged 6.2±0.2 mmol/l in basal conditions and rose to 22.9±1.7 mmol/l 15 h after STZ administration. Therapy with regular insulin intraperitoneally (1.5±0.5 U/day) was begun and adjusted to yield blood glucose levels of 22 mmol/l for 8 days. Insulin plasma levels decreased significantly in all groups of rats

338

R. Marfella et al. / Cardiovascular Pathology 18 (2009) 332–345

Fig. 3. Nitrotyrosine levels and immunostaining score for iNOS in noninfarcted and infarcted hearts of hyperglycemic, bortezomib-treated hyperglycemic, and normoglycemic rats. *Pb.05 compared with both infarcted and noninfarcted hyperglycemic group. †Pb.05 compared with bortezomib-treated hyperglycemic rats. ‡Pb.05 compared with noninfarcted hearts.

after treatment with STZ (11.8±1.1 mU/l). Heart rate during myocardial ischemia was comparable among all groups (data not shown). Occlusion of the LADCA and subsequent reperfusion in normoglycemic rats produced a marked damage in the rat left ventricle as evidenced by histology, which was reliably measured at the 2-h time point. There was not any significant difference in the AR of LV among groups. AR was 55±2% of the LV in normoglycemic rats and 49±1.2% of this portion of the LV was infarcted. The infarct size as a percentage of the AR (56%) was significantly greater in hyperglycemic rats as compared with normoglycemic rats (STZ-hyperglycemic, IS/AR 62.1±2.3%, Pb.01 vs. normoglycemic). Bortezomib, which inhibits ubiquitin–proteasome activity, provided a mild protection (∼6%) against the infarction in normoglycemic rats, while it afforded a strong protection (∼34%) in hyperglycemic rats (Pb.01 vs. normoglycemic). Occlusion/reperfusion of the LADCA produced significant differences in levels of nitrotyrosine and iNOS between heart tissues from infarcted normoglycemic rats and infarcted STZ-hyperglycemic rats (Fig. 3). These were strongly reduced by bortezomib (Fig. 3). Nitrotyrosine and iNOS levels were virtually absent in the noninfarcted normoglycemic rats (Fig. 3) and bortezomib-treated normoglycemic rats. These data indicate that diabetes/hyperglycemia produce massive nitrotyrosine levels, probably by iNOS up-regulation, which can be counteracted by blocking the UPS system with bortezomib.

3.3. Ubiquitin–proteasome activity in the ischemic heart To determine the effects of up-regulation of oxidative stress on ubiquitin–proteasome activity, we measured ubiquitin and proteasome 20S activity in both human and rat ischemic myocardium. 3.3.1. Human myocardium In specimens from the nonischemic areas, ubiquitin and proteasome 20S activity was present in both diabetic and nondiabetic patients, although at a higher level in the diabetic group (Pb.001). Fig. 4 shows that diabetic ischemic patients had detectable steady-state levels of ubiquitin and proteasome 20S activity in specimens from the ischemic region. In nondiabetic ischemic patients, ubiquitin and proteasome 20S activity was slightly detected in specimens from the ischemic area of the ventricle; these values are only 19.7% and 22.6% of the ubiquitin and proteasome 20S levels seen in diabetic ischemic areas (Pb.001) (Fig. 4). Notably, ubiquitin– proteasome activity in ischemic specimens was strongly dependent on oxidative stress, as also reflected by the statistically significant correlation between nitrotyrosine levels and both ubiquitin (R=0.49, Pb.001) and proteasome 20S (R=0.60, Pb.001) concentrations. Strong immunostaining for both ubiquitin and proteasome 20S was seen in biopsy from the nonischemic and ischemic areas of diabetic patients. In contrast, ubiquitin and proteasome 20S activity was almost undetectable in both

R. Marfella et al. / Cardiovascular Pathology 18 (2009) 332–345

339

Fig. 4. (Panel A) Levels of proteasome 20S by a specific SDS-activation kit and ubiquitin levels by ELISA kit in myocardial specimens. *Pb.001 compared with type 2 diabetic patients. (Panel B) Proteasome 20S (×630) and ubiquitin (×630) by immunohistochemistry in ischemic myocardial specimens.

nonischemic and ischemic areas of nondiabetic patients (Fig. 4). Ubiquitin and proteasome 20S immunoreactivity was seen in both cardiomyocytes and macrophages of diabetic ischemic area; on the other hand, in nondiabetic patients this immunoreactivity was seen essentially in the inflammatory cells (Fig. 2).

not shown). Moreover, the levels of mouse MAb (FK1), specific for polyubiquitinated proteins, were correlated with the degree of glycemia (R=0.39, Pb.01), showing that glycemic control may influence ubiquitination-mediated enzymatic activity. 3.4. Inflammation in the ischemic heart

3.3.2. Rat myocardium In basal conditions, cardiac ubiquitin–proteasome activity was not detectable in normoglycemic rats. Hyperglycemic rats had high ubiquitin levels and proteasome 20S activity already present into the heart after 8 days of hyperglycemia. However, we observed that at both ubiquitin levels, polyubiquitinated protein and proteasome 20S activity was (i) low in noninfarcted normoglycemic hearts; (ii) high in infarcted hyperglycemic hearts; and (iii) had intermediate levels in bortezomibtreated hyperglycemic hearts (Fig. 5). Overall application of an ischemia/reperfusion procedure to these rats caused an increase in UPS activity and ubiquitin in all the groups; however, bortezomib significantly reduced the UPS activity with a more marked effect into the heart of STZ-hyperglycemic rats (Fig. 5). The bortezomib treatment in normoglycemic rats did not evoke a significant reduction in UPS activity during the I/R study (data

3.4.1. Human myocardium Compared with nondiabetic patients, diabetic patients had a significantly greater portion of ischemic myocardium area occupied by macrophages (Table 1) (Fig. 6). To determine the effects of up-regulation of ubiquitin– proteasome activity on inflammation, we measured NFkB activation and TNF-α levels in human ischemic myocardium. In both nonischemic and ischemic specimens, NFkB activation, as reflected by the selective analysis of the activated form of both p50 and p65, and TNF-α levels were significantly higher in diabetic heart as compared with nondiabetic heart (Pb.01) (Fig. 7). Immunohistochemistry revealed higher staining of both p50 and p65 and TNF-α in ischemic specimens from diabetic patients compared with nondiabetic patients (Fig. 7). Moreover, immunohistochemistry and quantitative analyses revealed lower staining and levels for IkB-β

340

R. Marfella et al. / Cardiovascular Pathology 18 (2009) 332–345

Fig. 5. (Panel A) Ubiquitin levels and proteasome 20S activation in noninfarcted and infarcted hearts of hyperglycemic, bortezomib-treated hyperglycemic, and normoglycemic rats. *Pb.05 compared with both infarcted and noninfarcted hyperglycemic group. †Pb.05 compared with bortezomibtreated hyperglycemic rats. ‡Pb.05 compared with noninfarcted hearts. (Panel B) Western blot analysis of polyubiquitinated protein contents (MAbFK1) in noninfarcted and infarcted hearts of hyperglycemic, bortezomib-treated hyperglycemic, and normoglycemic rats. *Pb.05 compared with both infarcted and noninfarcted hyperglycemic group. †Pb.05 compared with bortezomib-treated hyperglycemic rats. ‡Pb.05 compared with noninfarcted hearts.

in ischemic diabetic hearts as compared with ischemic nondiabetic hearts (Fig. 7). Notably, inflammation in ischemic specimens was strongly dependent on proteasome 20S activity, as also reflected by the statistically significant correlation between proteasome 20S levels and TNF-α concentrations (R=0.51, Pb.001) as well as IkB-β concentrations (R=−0.32, Pb.01). Finally, there was a significant inverse correlation between IkB-β concentrations and NFkB activation (p50: R=−0.48, Pb.001; p65: R=−0.39, Pb.01). In multiple regression analysis, HbA1c levels were significant predictors of ejection fraction (adjusted r 2 =0.35, Pb.01), MPI (adjusted r 2 =0.30, Pb.05), nitrotyrosine levels (adjusted r2=0.32, Pb.01), proteasome 20S activity (adjusted r2=0.47, Pb.001), and TNF-α cardiac levels (adjusted r2=0.39, Pb.01); inclusion of age, sex, body mass index, blood pressure, cholesterol and triglyceride levels, smoking status, previous disorders, and active therapy did not add any explanatory information to our results.

3.4.2. Rat study As discussed earlier, the inflammatory markers NFkB and TNF-α followed the same trend of change observed for the oxidative markers and UPS activity seen throughout the study in STZ-hyperglycemic rats (Fig. 8). Particularly, to define the role of hyperglycemia on upregulation of the inflammation on ischemic myocardial injury, we evaluated NFkB activation and TNF-α levels during the I/R study in both normoglycemic and hyperglycemic rats. Compared with normoglycemic ischemic rats, hyperglycemic rats (all) had a significantly greater portion of the infarcted tissue occupied by macrophages (20±3% vs. 6±3%; Pb.01) and T-lymphocytes (58±12% vs. 14±8%; Pb.01). Compared to the hyperglycemic rats, bortezomib-treated hyperglycemic rats presented a significantly smaller portion of the infarcted area occupied by macrophages (Pb.01) and Tlymphocytes (Pb.01). The bortezomib-treatment in normoglycemic rats did not evoke a significant reduction of

R. Marfella et al. / Cardiovascular Pathology 18 (2009) 332–345

341

Fig. 6. Representative sections show immunochemistry for CD68 (macrophages) and CD3 T-lymphocytes) in diabetic and nondiabetic ischemic specimens (×200).

both NFkB activation and TNF-α levels during the I/R study (data not shown). We would hypothesize that hyperglycemia and ischemia have a similar and probably synergistic effect on heart inflammatory burden.

4. Discussion In the present report, we provide evidence for the functional involvement of the UPS in the NFkB-dependent inflammation up-regulation in human diabetic myocardium during acute ischemia. In particular, the present findings (I) identify differences in ubiquitin–proteasome activity in diabetic vs. nondiabetic human ischemic heart specimens; (II) recognize the oxidative stress and the inflammatory up-regulation, as evidenced by the higher levels of both iNOS and nitrotyrosine as well as higher NFkB-dependent TNF-α levels, in diabetic vs. nondiabetic human ischemic myocardium; and (III) relate the presence of ubiquitin– proteasome overactivity in both human ischemic cardiomyocytes and macrophages to the glycemic control as well as to an exaggerated inflammatory response that might promote increase in tissue injury leading to cardiac dilatation and heart failure [4]. Moreover, our study demonstrates an association between ubiquitin–proteasome activity and functional cardiac outcome in diabetic patients with myocardial ischemia: ubiquitin–proteasome overactivity amplifies inflammatory immune reaction and worsens functional cardiac outcome as evidenced by a

reduced ejection fraction and an increased MPI. The increase in the MPI, which measures both systolic and diastolic parameters of ventricular function [17], indicates a worse functional outcome during myocardial ischemia in diabetic patients. Moreover, the diminished diastolic filling time, the prolongation of mitral regurgitation, and the diminished effective ejection time in the diabetic patients suggest that diabetes may influence cardiac synchronization during MI. Studies have identified dyssynchrony between right and left ventricular contraction and relaxation as an independent predictor of heart failure and cardiac mortality in patients with heart failure [18]. The results of the present study support an association between ventricular dyssynchrony and blood glucose levels in diabetic patients with myocardial ischemia, as evidenced by a correlation between HbA1c and MPI as well as ejection fraction. As for mechanisms behind this association, hyperglycemia may be responsible for more extensive cardiac inflammation through the activation of the ubiquitin–proteasome pathway. Experimental studies about the role of the UPS in myocardial ischemia have been controversial [19]. However, in our study, macrophages were more abundant in diabetic myocardium and represented the major source of ubiquitin–proteasome activity, suggesting the presence of an active inflammatory reaction in diabetic ischemic heart. Moreover, the ubiquitin–proteasome activity was upregulated not only in inflammatory cells but also in myocytes from ischemic diabetic patients, suggesting an active role of diabetic myocardium in the inflammatory

342

R. Marfella et al. / Cardiovascular Pathology 18 (2009) 332–345

Fig. 7. (Panel A) Levels of IkB-β, activated NFkB (specific Trans-AM p50 and NFkB p65 subunits assay kit), and TNF-α in myocardial specimens.*Pb.001 compared with type 2 diabetic patients. (Panel B) Representative sections show immunohistochemistry for IkB-β (×630), activated NFkB p65 (×630) and p50 (×630), and TNF-α (×630).

progression of ischemia. In agreement with the difference in ubiquitin–proteasome staining pattern, the histological milieu of the ischemic tissues appears different with regard to cellularity, but not in the degree of coronary stenosis, suggesting that diabetic and nondiabetic ischemic tissues are only different in regard to inflammatory burden. These

data are consistent with previous findings that the inflammatory response during myocardial ischemia was greater in diabetic than in nondiabetic patients [20]. Moreover, an association between diabetes and up-regulation of inflammatory cytokines such as interleukin-6 and TNF-α has been shown in ischemic myocardium of STZ-

R. Marfella et al. / Cardiovascular Pathology 18 (2009) 332–345

343

Fig. 8. IkB-β and TNF-α levels and NFkB p50 and p65 in noninfarcted and infarcted hearts of hyperglycemic, bortezomib-treated hyperglycemic, and normoglycemic rats. *Pb.05 compared with both infarcted and noninfarcted hyperglycemic group. †Pb.05 compared with bortezomib-treated hyperglycemic rats. ‡Pb.05 compared with noninfarcted hearts.

diabetic rats [21]. Hence, the differences in ischemic myocardium behavior likely stem from the differences in the presence of stimuli (i.e., oxidative stress, as evidenced by high iNOS expression and nitrotyrosine levels) for selective expression of ubiquitin–proteasome, capable of increasing tissue injury via NFkB induction. NFkB is normally bound to IkB in the cytosol; this binding prevents its movement into the nucleus [22]. Various cellular stimuli, such as oxidative stress, induce ubiquitination of phosphorylated IkBs and subsequent degradation by the proteasome [23,24]. Degradation of IkBs results in unmasking of the nuclear localization signal of NFkB dimers, which subsequently translocates to the nucleus, where it induces the transcription of proinflammatory cytokines like TNF-α that play a central role in myocardial ischemic injury [25]. Our findings also suggest that diabetes may induce degradation of IkBs via ubiquitin– proteasome overactivity, thus enhancing NFkB activation. Previous reports evidenced the involvement of the UPS in NFkB activation, particularly under conditions of aggravated oxidative stress. Oxidative stress is the common factor underlying diabetes and cardiovascular disease, and may explain the presence of inflammation in all these conditions [26]. Although it is well recognized that

inflammation is one manifestation of oxidative stress, and the pathways that generate the mediators of inflammation, such as interleukins, are all induced by oxidative stress [27], the mechanism by which oxidative stress may be involved in the inflammatory process of type 2 diabetic myocardium is not fully clarified. In this context, our data hypothesize that oxidative stress, which increases ubiquitin–proteasome activity, may mediate inflammatory activity in diabetic ischemic myocardium. Of note, it has been shown that oxidative stress can stimulate the ubiquitin system in the macrophages by inducing the expression of components of its enzymatic machinery such as ubiquitinbinding proteins [10,28]. Notably, the intriguing and novel unfavorable mechanism of the UPS in human diabetes is supported in this study not only by the observation that ubiquitin–proteasome activity is higher in diabetic ischemic specimens with respect to nondiabetic ischemic specimens, but also by the information that it is strongly correlated with the intensity of glycemic control as reflected by HbA1c. In this context, hyperglycemia-induced oxidative stress may be the common soil that relates glycemic control to ubiquitin–proteasome overactivity in diabetic patients. Recently, it was demonstrated that in both rat and human hearts, exposure to high glucose

344

R. Marfella et al. / Cardiovascular Pathology 18 (2009) 332–345

increases iNOS gene expression, paralleled by a simultaneous increase in both NO and O2− production [16,21]. The interaction of O2− with NO is very rapid and leads to the production of a potent oxidant, peroxynitrite [29]. In our study, detection of higher levels of both iNOS and nitrotyrosine levels in diabetic ischemic hearts is strongly suggestive of increased generation of peroxynitrite. Because it has been reported that peroxynitrite may increase ubiquitin–proteasome activity [30], hyperglycemia-induced oxidative stress possibly serves as a key activator of this enzymatic machinery, leading to induction of inflammatory gene expression. To test this hypothesis, we devised an experimental model of ischemic myocardial injury in the STZ-hyperglycemic rats. The hyperglycemia during ischemia/reperfusion study produced an extension of the infarcted area and enhanced nitrotyrosine levels, ubiquitin proteasome activity, and NF-kB and TNF-α levels, as well as macrophages infiltration in the periinfarcted area of the ischemic myocardium, whereas ischemic lesions of normoglycemic rats had little oxidative stress, ubiquitin proteasome activity, and inflammation as well as macrophages infiltration. Moreover, recent evidences suggest that diabetic hyperglycemia activates the proteasome via peroxynitrite and results in the ubiquitination and degradation of guanosine 5′-triphosphate cyclohydrolase I, the limiting enzyme for tetrahydrobiopterin synthesis, and subsequent impairs endothelium-dependent vessel relaxation [31]. These findings support our hypothesis that poor glycemic control in human ischemic heart can cause an abrupt increase in the levels of oxidative stress and inflammation via ubiquitin–proteasome overactivity, resulting in expansion of ischemic injury and the potential for infarction extension and heart failure. Proinflammatory cytokines are not constitutively expressed in the normal heart [32], and are rapidly released in the central ischemic zone during infarction but are usually maximal in the border zone [33,34]. Exuberant host inflammatory response may increase ischemic injury and involve the noninfarcted zone, mediating an important remodeling process in the entire myocardium [35]. Ischemic stress represents a potent trigger for cytokine production, but hyperglycemia-induced oxidative stress is also a potent regulator. It has been suggested that cytokines not only induce reactive oxygen species (ROS) production but also are themselves induced by ROS [36]. Based on these data, we postulated that hyperglycemia-induced oxidative stress was responsible for the up-regulated inflammatory state of ischemic myocardium in diabetic patients, linking the induction of NF-kB and TNF-α to the levels of ROS. To clarify the molecular basis by which hyperglycemia-induced oxidative stress could influence the inflammatory state of ischemic myocardium, we evaluated the effects of bortezomib, an inhibitor of ubiquitin– proteasome activity, on ischemic injury as well as on NFkB activity and TNF-α in hyperglycemic rats. Here we provide evidence that the bortezomib treatment may

prevent the inflammatory burden on the progression of ischemic injury in the heart of hyperglycemic rats by reducing ubiquitin–proteasome activity. Indeed, at the same level of blood glucose levels, ischemic hearts of hyperglycemic rats treated with bortezomib had the lowest level of ubiquitin and proteasome 20S activity, inflammatory cells, NF-kB activation, and cytokine levels associated with the reduced infarcted area. Particularly, the reduced levels of ubiquitin following bortezomib treatment may be due to the reduced oxidative stimulus coming from reduced NFkB– cytokine-mediated inflammatory input. Thus, hyperglycemic rats assigned to bortezomib had lesser cytokine secretion in response to myocardial ischemia, which contributes to cardiac remodeling and eventual host outcome. In particular, the reduced ubiquitin–proteasome activity seen in ischemic hearts of the hyperglycemic rats treated with bortezomib suggests decreased IkB degradation and hence NFkB activation. In conclusion, this study demonstrates the enhanced ubiquitin–proteasome activity in ischemic lesions of diabetic patients and provides evidence that the activation of this system in inflammatory cells as well as in ischemic myocytes is associated with NFkB-dependent increase in inflammation. The increased ubiquitin–proteasome activity in response to ischemia, strictly related to hyperglycemia, may interfere with cardiac remodeling in the ischemic myocardium. Whether this plays a role in the poor prognosis of the diabetic heart awaits further elucidations. Because recent clinical data [2] have raised the possibility that hyperglycemia at the time of MI may be an important and potentially modifiable risk factor for poor outcome, these findings are potentially important from a fundamental standpoint because they identify a potential mechanism by which hyperglycemia may influence the evolution of myocardial ischemia. From a practical standpoint, these findings raise the interesting possibility that modification of the ubiquitin–proteasome activity, for example by selective proteasome inhibitors, might provide a novel form of therapy for myocardial ischemia of diabetic patients. References [1] Stone PH, Muller JE, Hartwell T, York BJ, Rutherford JD, Parker CB, Turi ZG, et al. The effects of diabetes mellitus on prognosis and serial left ventricular function after acute myocardial infarction: contribution of both coronary disease and diastolic left ventricular dysfunction to the adverse prognosis. J Am Coll Cardiol 1989;14:49–57. [2] Umpierrez GE, Isaacs SD, Bazargan N, You X, Thaler LM, Kitabchi AE. Hyperglycemia: an independent marker of in-hospital mortality in patients with undiagnosed diabetes. J Endocrinol Metab 2002;87: 978–82. [3] Malmberg K, Ryden L, Efendic S, Herlitz J, Nicol P, Waldenstrom A, et al. On behalf of the DIGAMI Study Group: a randomised trial of insulin-glucose infusion followed by subcutaneous insulin treatment in diabetic patients with acute myocardial infarction: effects on mortality it 1 year. J Am Coll Cardiol 1995;26:57–65. [4] Nian M, Lee P, Khaper N, Liu P. Inflammatory cytokines and postmyocardial infarction remodeling. Circ Res 2004;94:1543–53.

R. Marfella et al. / Cardiovascular Pathology 18 (2009) 332–345 [5] Bonvini RF, Hendiri T, Camenzind E. Inflammatory response postmyocardial infarction and reperfusion: a new therapeutic target? Eur Heart J 2005;7(suppl I):I27–I36. [6] Hershko A, Ciechanover A, Varshavsky A. Basic medical research award. The ubiquitin system. Nat Med 2000;6:1073–81. [7] Palombella VJ, Rando OJ, Goldberg AL, Maniatis T. The ubiquitin– proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B. Cell 1994;78: 773–85. [8] Lecker SH, Solomon V, Price SR, Kwon YT, Mitch WE, Goldberg AL. Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats. J Clin Invest 1999; 104:1411–20. [9] Marfella R, D'Amico M, Esposito K, Baldi A, Di Filippo C, Siniscalchi M, et al. The ubiquitin–proteasome system and inflammatory activity in diabetic atherosclerotic plaques: effects of rosiglitazone treatment. Diabetes 2006;55:622–32. [10] Kikuchi, Furukawa Y, Kubo N. Induction of ubiquitin-conjugating enzyme by aggregated low density lipoprotein in human macrophages and its implications for atherosclerosis. Arterioscler Thromb Vasc Biol 2000;20:128–34. [11] Wang P, Zweier JL. Measurement of nitric oxide and peroxynitrite generation in the postischemic heart. Evidence for peroxynitritemediated reperfusion injury. J Biol Chem 1996;271:29223–30. [12] Hamm CW, Braunwald E. A classification of unstable angina revisited. Circulation 2000;102:118–22. [13] Eagle KA, Guyton RA, Davidoff R, Ewy GA, Fonger J, Gardner TJ, et al. ACC/AHA guidelines for coronary artery bypass graft surgery: executive summary and recommendations: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (committee to revise the 1991 guidelines for coronary artery bypass graft surgery). Circulation 1999;100: 1464–80. [14] Gardin JM, Adams DB, Douglas PS, Feigenbaum H, Forst DH, Fraser AG, et al. American Society of Echocardiography: recommendations for a standardized report for adult transthoracic echocardiography. J Am Soc Echocardiogr 2002;15:275–90. [15] Ohlsson BG, Englund MCO, Karlsson ALK, Knutsen E, Erixon C, Skribeck H, et al. Oxidized low density lipoprotein inhibits lipopolysaccharide-induced binding of nuclear factor-kB to DNA and the subsequent expression of tumor necrosis factor-α and interleukin-1β in macrophages. J Clin Invest 1996;98:78–89. [16] Marfella R, Esposito K, Nappo F, Siniscalchi M, Sasso FC, Portoghese M, et al. Expression of angiogenic factors during acute coronary syndromes in human type 2 diabetes. Diabetes 2004;53:2383–91. [17] Poulsen SH, Jensen SE, Tei C, Seward JB, Egstrup K. Value of the Doppler index of myocardial performance in the early phase of acute myocardial infarction. J Am Soc Echocardiogr 2000;13:723–30. [18] Aaronson KD, Schwartz JS, Chen TM, Wong KL, Goin JE, Mancini DM. Development and prospective validation of a clinical index to predict survival in ambulatory patients referred for cardiac transplant evaluation. Circulation 1997;95:2660–7. [19] Zolk O, Schenke C, Sarikas A. The ubiquitin–proteasome system: focus on the heart. Cardiovasc Res 2006;70:410–21. [20] Marfella R, Siniscalchi M, Esposito K, Sellitto A, De Fanis U, Romano C, et al. Effects of stress hyperglycemia on acute myocardial infarction:

[21]

[22] [23]

[24]

[25]

[26]

[27]

[28] [29] [30] [31]

[32]

[33]

[34]

[35]

[36]

345

role of inflammatory immune process in functional cardiac outcome. Diabetes Care 2003;26:3129–35. Marfella R, Di Filippo C, Esposito K, Nappo F, Piegari E, Cuzzocrea S, Berrino L, Rossi F, Giugliano D, D'Amico M. Absence of inducible nitric oxide synthase reduces myocardial damage during ischemia reperfusion in streptozotocin-induced hyperglycemic mice. Diabetes 2004;53:454–62. Siebenlist U, Franzoso G, Brown K. Structure, regulation and function of NF-kappa B. Annu Rev Cell Biol 1994;10:405–55. Herrmann J, Gulati R, Napoli C, Woodrum JE, Lerman LO, Rodriguez-Porcel M, et al. Oxidative stress-related increase in ubiquitination in early coronary atherosclerosis. FASEB J 2003;17: 1730–2. Herrmann J, Ciechanover A, Lerman LO, Lerman A. The ubiquitin– proteasome system in cardiovascular diseases—a hypothesis extended. Cardiovasc Res 2004;61:11–21. Sun M, Dawood F, Wen WH, Chen M, Dixon I, Kirshenbaum LA, Liu PP. Excessive tumor necrosis factor activation after infarction contributes to susceptibility of myocardial rupture and left ventricular dysfunction. Circulation 2004;110:3221–8. Ceriello A, Motz E. Is oxidative stress the pathogenic mechanism underlying insulin resistance, diabetes, and cardiovascular disease? The common soil hypothesis revisited. Arterioscler Thromb Vasc Biol 2004;24:816–23. Roebuck KA. Oxidant stress regulation of IL-8 and ICAM-1 gene expression: differential activation and binding of the transcription factors AP-1 and NF-kappaB. Int J Mol Med 1999;4:223–30. Ishii T, Itoh K, Sato H, Bannai S. Oxidative stress-inducible proteins in macrophages. Free Radic Res 1999;31:351–5. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol 1996;271:C1424–37. Powell SR. The ubiquitin–proteasome system in cardiac physiology and pathology. Am J Physiol Heart Circ Physiol 2006;214:989–97. Xu J, Wu Y, Song P, Zhang M, Wang S, Zou MH. Proteasomedependent degradation of guanosine 5′-triphosphate cyclohydrolase I causes tetrahydrobiopterin deficiency in diabetes mellitus. Circulation 2007;116:944–53. Kapadia S, Lee J, Torre-Amione G, Birdsall HH, Ma TS, et al. Tumor necrosis factor-alpha gene and protein expression in adult feline myocardium after endotoxin administration. J Clin Invest 1995;96: 1042–52. Ono K, Matsumori A, Shioi T, Furukawa Y, Sasayama S. Cytokine gene expression after myocardial infarction in rat hearts: possible implication in left ventricular remodeling. Circulation 1998;98: 149–56. Deten A, Volz HC, Briest W, Zimmer HG. Cardiac cytokine expression is upregulated in the acute phase after myocardial infarction. Experimental studies in rats. Cardiovasc Res 2002;55:329–40. Irwin M, Mak S, Mann D, Qu R, Dawood F, Wen WH, et al. Tissue expression and immunolocalization of tumour necrosis factor-alpha in post infarction-dysfunctional myocardium. Circulation 1999;99: 1492–8. Nakamura K, Fushimi K, Kouchi H, Mihara K, Miyazaki M, Ohe T, et al. Inhibitory effects of antioxidants on neonatal rat cardiac myocyte hypertrophy induced by tumor necrosis factor-alpha and angiotensin II. Circulation 1998;98:794–9.