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Am J Physiol Heart Circ Physiol 289: H361-H367, 2005. First published February 11, 2005; doi:10.1152/ajpheart.01285.2004
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Cardioprotection with palm tocotrienol: antioxidant activity of tocotrienol is linked with its ability to stabilize proteasomes

Samarjit Das,1,4 Saul R. Powell,2 Ping Wang,2 Andras Divald,2 Kalanithi Nesaretnam,3 Arpad Tosaki,4 Gerald A. Cordis,1 Nilanjana Maulik,1 and Dipak K. Das1

1Cardiovascular Research Center, University of Connecticut School of Medicine, Farmington, Connecticut; 2Long Island Jewish Medical Center Campus of the Albert Einstein College of Medicine, New Hyde Park, New York; 3Malaysian Palm Oil Board, Kuala Lumpur, Malaysia; and 4University of Debrecen, Debrecen, Hungary

Submitted 20 December 2004 ; accepted in final form 8 February 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tocotrienols, isomers of vitamin E, have been found to possess many health benefits. The present study was designed to determine whether tocotrienol has a direct cardioprotective role. Isolated rat hearts were perfused for 15 min with Krebs-Ringer bicarbonate buffer in the absence or presence of palm tocotrienol derived from the tocotrienol-rich fraction (0.035%) of palm oil (TRF). In another group of studies, the hearts were preperfused for 15 min in the presence of a c-Src inhibitor, 4-amino-5-(4-methylphenyl)-7-(t-butyl)-pyrazolo-3,4-d-pyrimidine (PPI). The hearts were then subjected to 30 min of global ischemia followed by 2 h of reperfusion. As expected, ischemia-reperfusion caused ventricular dysfunction, electrical rhythm disturbances, and increased myocardial infarct size. PPI or TRF could reverse the ischemia-reperfusion-mediated cardiac dysfunction. Ischemia-reperfusion also upregulated c-Src expression and phosphorylation. Although TRF only minimally affected c-Src expression, it significantly inhibited the phosphorylation of c-Src. Ischemia-reperfusion reduced 20S and 26S proteasome activities, an effect prevented by TRF pretreatment. PPI exerted a cardioprotective effect that is not mediated by the proteasome but, rather, through direct inhibition of c-Src. The results of this study support a role for c-Src in postischemic cardiac injury and dysfunction and demonstrate direct cardioprotective effects of TRF. The cardioprotective properties of TRF appear to be due to inhibition of c-Src activation and proteasome stabilization.

tocotrienol-rich fraction; ischemia-reperfusion; heart; c-Src

TOCOTRIENOLS ARE THE ISOFORMS of vitamin E that are abundant in cereal grains, including soybeans, barley, oats, rice bran, and palm oil. They differ from corresponding tocopherols only in their aliphatic tail. The isoforms of tocotrienols differ in their methyl substitution in the chromanol head and a 16-carbon hydrocarbon tail; the {alpha}-form contains three methyl groups, the {beta}- and {gamma}-forms have two methyl groups, and the {delta}-form has only one methyl group (41). Recent studies have demonstrated many health benefits of tocotrienols, including their anticancer and tumor-suppressive activities and their ability to lower cholesterol (11, 31).

Dietary tocotrienol derived from plant sources, especially palm oil, has been found to be beneficial against a variety of degenerative diseases. For example, supplementation with dietary tocotrienols from a tocotrienol-rich fraction of palm oil (TRF) reduced the concentration of plasma cholesterol and apolipoprotein B, thromboxane B2, and platelet factor 4, indicating its ability to protect against endothelial dysfunction and platelet aggregation (32). Dietary tocotrienols reduce concentrations of plasma cholesterol, apolipoprotein B, thromboxane B2, and platelet factor 4 in pigs with inherited hyperlipidemias (34). Supplementation with TRF reduced plasma cholesterol levels in a human pilot study (33). Tocotrienols from TRF inhibited the proliferation of human breast cancer cell lines (25). Tocotrienols also suppressed the growth of murine B16 melanomas in vitro and in vivo (18). In neuronal cells, tocotrienols inhibited glutamate-induced pp60c-Src kinase activation of HT4 neuronal cells (39). Many of the signaling pathways involved in cell cycling and proliferation and apoptosis are regulated by the ubiquitin-proteasome system (10, 12, 27). Recent studies (4, 29) have shown that the proteasome may be inactivated during myocardial ischemia and have postulated a role for this complex in determining cardiac cell survival or death. In view of protective effects of tocotrienols in a variety of degenerative diseases, we hypothesized that tocotrienol could protect the hearts from ischemia-reperfusion injury.

We recently showed that ischemia of the isolated rat heart results in inhibition of the 26S proteasome and have hypothesized that the degree of inhibition may be related to the extent of recovery during the postischemic period (29). In these same studies, preischemic perfusion of the isolated rat heart with the proteasome inhibitor MG132 resulted in dose-dependent decrements in postischemic recovery associated with increased accumulation of ubiquitinated proteins in the myocardium, thus supporting this hypothesis. Because many in vitro studies in a variety of cell lines (for review see Refs. 12 and 27), including cardiomyocytes (3), and in at least one study in the isolated rat heart (28) show that proteasome inhibition of sufficient degree and duration can lead to apoptosis, we examined the possibility that TRF may preserve the proteasome activity in the postischemic heart.

Isolated rat hearts were perfused with TRF (0.035%) for 15 min before they were subjected to 30 min of ischemia and 2 h of reperfusion. The results indicated that TRF was able to improve postischemic ventricular dysfunction and reduce myocardial infarct size and incidence of ventricular arrhythmias. TRF also reduced the ischemia-reperfusion-induced increase in c-Src phosphorylation and stabilized 20S and 26S proteasome activities.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. TRF was supplied by the Malaysia Palm Oil Board. All chemicals were purchased from Sigma Chemical (St. Louis, MO) unless otherwise mentioned.

Animals. All animals used in this study received humane care in compliance with the principles of laboratory animal care formulated by the National Society for Medical Research and the National Institutes of Health Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, revised 1985]. The experimental procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (250–300 g body wt) were fed ad libitum regular rat chow and had free access to water until the start of the experimental procedure. The rats were randomly assigned to one of three groups: the control group comprised isolated hearts perfused with Krebs-Henseleit bicarbonate buffer (KHB) for 15 min, and hearts of the experimental groups were perfused with TRF (0.035%) or 5 µM 4-amino-5-(4-methylphenyl)-7-(t-butyl)-pyrazolo-3,4-d-pyrimidine (PPI) under identical conditions. All hearts were then subjected to 30 min of ischemia followed by 2 h of reperfusion.

Isolated working heart preparation. Rats were anesthetized with pentobarbital sodium (80 mg/kg ip; Abbott Laboratories, North Chicago, IL), and heparin sodium (500 IU/kg iv; Elkins-Sinn, Cherry Hill, NJ) was administered for anticoagulation. After sufficient depth of anesthesia was ensured, a thoracotomy was performed and hearts were perfused in the retrograde Langendorff mode at 37°C at a constant perfusion pressure of 100 cmH2O (10 kPa) for a 5-min washout period (8). The perfusion buffer consisted of a modified KHB (in mM: 118 NaCl, 4.7 KCl, 1.7 CaCl2, 25 NaHCO3, 1.2 KH2PO4, 1.2 MgSO4, and 10 glucose). The Langendorff preparation was switched to the working mode after the washout period, as previously described (4). The working mode was introduced by switching the flow to the left atrium from the aortic root with a constant preload of 17 cmH2O and an afterload of 100 cmH2O.

After 10 min, when cardiac function had attained a steady state, baseline functional parameters were recorded. The circuit was then returned to the retrograde mode, and hearts were perfused for 15 min with KHB (control) or TRF. This perfusion period was followed by 5 min of washout with KHB; then the hearts were subjected to global ischemia for 30 min followed by 2 h of reperfusion. Reperfusion was in the retrograde mode for the first 10 min to allow for postischemic stabilization and, thereafter, in the antegrade working mode to allow for assessment of functional parameters, which were recorded at 10, 30, 60, and 120 min of reperfusion.

Cardiac function assessment. Aortic pressure was measured using a pressure transducer (model P23XL, Gould Instrument Systems, Valley View, OH) connected to a sidearm of the aortic cannula; the signal was amplified using a signal conditioner (series 6600, Gould) and monitored on a real-time data acquisition and analysis system (CORDAT II, Triton Technologies, San Diego, CA) (8). Heart rate, left ventricular developed pressure (defined as the difference between maximum systolic and diastolic aortic pressures), and the first derivative of developed pressure (dP/dt) were derived or calculated from the continuously obtained pressure signal (4). Aortic flow (AF) was measured using a calibrated flowmeter (Gilmont Instrument, Barrington, IL), and coronary flow was measured by timed collection of the coronary effluent dripping from the heart.

Infarct size estimation. At the end of reperfusion, a 10% (wt/vol) solution of triphenyltetrazolium chloride in phosphate buffer was infused into the aortic cannula for 20 min at 37°C (16, 17). The hearts were excised and stored at –70°C. Sections (0.8 mm) of frozen heart were fixed in 2% paraformaldehyde, placed between two coverslips, and digitally imaged using a scan maker (model 600z, Microtek). To quantitate the areas of interest in pixels, NIH Image 5.1 (a public-domain software package) was used. The infarct size was quantified and expressed in pixels.

Tdt-mediated dUTP nick end labeling assay for assessment of apoptotic cell death. Immunohistochemical detection of apoptotic cells was carried out using Tdt-mediated dUTP nick end labeling (16). The sections were incubated again with mouse monoclonal antibody recognizing cardiac myosin heavy chain to specifically recognize apoptotic cardiomyocytes. The fluorescence staining was viewed with a confocal laser microscope. The number of apoptotic cells was counted and expressed as a percentage of the total myocyte population.

Ventricular arrhythmias. An epicardial ECG recording was made with a polygraph (Gould) during each experiment, with two silver electrodes attached directly to the heart (40). ECG data were analyzed to determine the incidence and duration of ventricular fibrillation (VF) and whether VF was nonsustained (spontaneously returning to sinus rhythm) or sustained (persisting throughout reperfusion); the heart was considered to be in VF if an irregular undulating baseline was present on the ECG.

Western blot analysis. Left ventricles from the hearts were homogenized in a buffer containing (in mM) 25 Tris·HCl, 25 NaCl, 1 orthovanadate, 10 NaF, 10 pyrophosphate, 10 okadaic acid, 0.5 EDTA, and 1 PMSF (38). One hundred micrograms of protein of each heart homogenate were incubated with 1 µg of antibody against c-Src or phosphorylated c-Src (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4°C. The immune complexes were precipitated with protein A-Sepharose, and immunoprecipitates were separated by SDS-PAGE and immobilized on a polyvinylidene difluoride membrane. The membrane was immunoblotted with PY20 to evaluate the phosphorylation of the compounds. The membrane was stripped and reblotted with specific antibodies against glucose-6-phosphate dehydrogenase, which served as the loading control. The resulting blots were digitized, normalized against the loading control, and subjected to densitometric scanning using a standard NIH Image program.

Proteasome activity. Proteasome activity was determined in cell lysate as described by Grune et al. (13). Briefly, cardiac tissue was homogenized in HEPES buffer (137 mM NaCl, 4.6 mM KCl, 1.1 mM KH2PO4, 0.6 mM MgSO4, 1 mM EDTA, 1 mM DTT, and 0.01% digitonin) without protease inhibitors at 4°C and then centrifuged at 10,000 g to obtain the soluble fraction. Cell supernatant (50 µg of protein) was incubated in 50 mmol/l Tris·HCl buffer, pH 7.8, containing 20 mM KCl, 0.5 mM MgCl2, and 1 mM DTT for 1 h with 75 µM succinyl-LLVY-methylcoumarin (Biomol Research Laboratory, Plymouth Meeting, PA). Hydrolysis was stopped by addition of ice-cold ethanol and dilution with 0.125 mol/l sodium borate, pH 9.0, and fluorescence products were monitored at 380-nm excitation and 440-nm emission. The reaction was carried out in the absence and presence of the proteasome inhibitor lactacystin (5 µM; Biomol Research Laboratory) to differentiate between nonproteasome- and proteasome-mediated peptide hydrolysis and with or without 0.0625–0.125 mmol/l ATP (with or without lactacystin) to differentiate between 20S and 26S proteasome, respectively. Results are expressed as percentage of control, because storage of tissue samples, even at –80°C, can result in interassay variation. Care was taken to avoid freeze thawing of tissue samples more than once and to match experimental samples with preischemic controls that had been stored for similar amounts of time.

Statistical analysis. The values for myocardial functional parameters, total and infarct volumes, and infarct sizes are expressed as means ± SE. ANOVA was followed by Bonferroni's correction to test for any differences between the mean values of all groups. If differences between groups were established, the values of the treated groups were compared with those of the control group by a modified t-test. Because the duration of VF was not Gaussian distributed, Wilcoxon's test (nonparametric distribution) was used, and then a 2 x n table was constructed from the overall {chi}2 test. Proteasome results were analyzed using a one-way ANOVA followed by Tukey's post hoc analysis. The results were considered significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of TRF on ventricular function. There were no differences in baseline function among the six groups. In general, there were no significant differences between TRF and control in heart rate and coronary flow (Table 1). As expected, on reperfusion, the absolute values of all functional parameters were decreased in all groups compared with the respective baseline values. TRF displayed significant recovery of postischemic myocardial function. The cardioprotective effects of TRF were evidenced by significant differences in the left ventricular dP/dt from 30 min of reperfusion onward, the difference is especially apparent at 60 and 120 of reperfusion and also in left ventricular developed pressure at 120 min of reperfusion. AF was markedly higher in the TRF group from 30 min of perfusion onward. This is also confirmed from the AF value, which is markedly lower throughout the reperfusion period (Table 1). Similar to TRF, PPI also improved postischemic ventricular recovery.


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  		Table 1. Effects of palm tocotrienol on postischemic ventricular function

 
Effects of TRF on myocardial infarct size. Infarct size (percentage of infarct vs. total area at risk) was significantly higher in the hearts subjected to 30 min of ischemia and 2 h of reperfusion than in the hearts that were not subjected to the ischemia-reperfusion protocol (almost at the baseline level, data not shown). The values were noticeably reduced in TRF and PPI groups compared with the group subjected to ischemia-reperfusion: 25.1 ± 2.45 and 25.6 ± 2.33%, respectively, vs. 33.43 ± 2.44% (Fig. 1, top). The infarct size was further reduced when a combination of TRF and PPI was used (20.5 ± 2.2% vs. 33.43 ± 2.44%; Fig. 1, top).



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  		Fig. 1. Effects of tocotrienol-rich fraction of palm oil (TRF) and 4-amino-5-(4-methylphenyl)-7-(t-butyl)-pyrazolo-3,4-d-pyrimidine (PPI) on myocardial infarct size and cardiomyocyte apoptosis. Isolated hearts from control (n = 6) and TRF-preperfused rats in the absence or presence of PPI (n = 6 each) were subjected to 30 min of global ischemia followed by 2 h of reperfusion in the working mode. Infarct size was measured by triphenyltetrazolium chloride dye method, and cardiomyocyte apoptosis was evaluated by Tdt-mediated dUTP nick end labeling in conjunction with antibody against {alpha}-myosin heavy chain. Values are means ± SE. *P < 0.05 vs. control.

 
Effects of TRF on cardiomyocyte apoptosis. Ischemia-reperfusion caused the cells to undergo apoptosis, as expected. The percentage of apoptotic cardiomyocytes was significantly reduced in the TRF and PPI groups compared with the control group: 5.7 ± 1.3 and 6.6 ± 2.2%, respectively, vs. 22.0 ± 1.7% (Fig. 1, bottom). As observed for the infarct size, the apoptotic cardiomyocytes were also further reduced when a combination of TRF and PPI was used: 4.8 ± 0.8% vs. 22.0 ± 1.7% (Fig. 1, bottom).

Effects of TRF on ventricular arrhythmias. The total incidence of VF (sustained and nonsustained) was significantly reduced with TRF and PPI from its control value of 90% to 30% and 40%, respectively (Fig. 2), indicating antiarrhythmic effects of tocotrienol and PPI.



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  		Fig. 2. Effects of TRF in the presence or absence of PPI on ventricular fibrillation (VF) and ventricular tachycardia (VT). Isolated hearts from control rats (n = 10) and TRF-preperfused rats in the absence or presences of PPI (n = 10) were subjected to 30 min of global ischemia. VF and VT were determined by contractibility of the heart and with the help of cardiac functions. *P < 0.05 vs. control.

 
Effects of TRF on c-Src expression and phosphorylation. Because previous studies indicated negative regulation of c-Src with vitamin E, we determined whether TRF could reduce the ischemia-reperfusion-induced upregulation of c-Src. The results show that ischemia-reperfusion significantly increased the induction and phosphorylation of c-Src activities (Fig. 3). TRF minimally, but significantly, affected the ischemia-reperfusion-mediated increase in c-Src induction. TRF-mediated reduction of c-Src phosphorylation was highly significant. PPI completely abolished c-Src expression (data not shown).



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  		Fig. 3. Western blot analysis of phosphorylated c-Src (p-c-Src) and c-Src protein. Results are from 2 different hearts for each group. PPI completely blocked expression of c-Src; hence, it is not shown. G6PD, glucose-6-phosphate dehydrogenase.

 
Effects of TRF on ischemic and postischemic proteasome activities. As illustrated in Fig. 4, 20S and 26S proteasome activities were significantly (P < 0.05) depressed by 45 and 46%, respectively, after ischemia, similar to our previously reported findings (29). Preischemic treatment of hearts with TRF not only prevented this decrease, but it actually appeared to activate both proteasomes to levels significantly (P < 0.05) higher than the ischemic values. After 120 min of reperfusion, 20S proteasome activity recovered to a level not different from baseline, and TRF had no effect. However, 26S proteasome activity was still significantly (P < 0.05) depressed by 58%, which was prevented in the TRF-treated hearts (Fig. 4). Treatment of hearts with PPI had no protective effects on postischemic proteasome activities (data not shown).



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  		Fig. 4. TRF prevents ischemia-associated decreases in proteasome activities. Hearts treated with TRF and subjected to ischemia and reperfusion were harvested, and 20S and 26S proteasome activities were determined. Values are means ± SE of 5–11 hearts, expressed as percentage of baseline (1,261 ± 210.6 and 612 ± 95 fluorescence units·h–1·mg protein–1 for 20S and 26S proteosome, respectively). Isch 30, 30 min of ischemia; Rep 120, 120 min of reperfusion. *P < 0.05 vs. baseline. #P < 0.05 vs. 30 min of ischemia.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
There are several salient features of the present study. 1) Palm tocotrienol was found to provide cardioprotection, as evidenced by reduction of the ischemia-reperfusion-mediated increase in ventricular dysfunction, ventricular arrhythmias, and myocardial infarct size. 2) Palm tocotrienol reduced ischemia-reperfusion-induced activation of c-Src activities. 3) Tocotrienol stabilized proteasomes by preventing the ischemia-reperfusion-mediated reduction of 26S and 20S proteasomes. The results of the study thus showed, for the first time, that beneficial effects of tocotrienol are due to its ability to reduce c-Src activation, which is linked with the stabilization of proteasomes. Tocotrienols have extremely short half-lives; after oral ingestion, they are not recognized by {alpha}-tocotrienol transport protein, which also accounts for their low bioavailability. For this reason, TRF was used in an acute experiment to determine its immediate effects on the ischemic-reperfused myocardium. The results indicate that tocotrienol readily blocks the ischemia-reperfusion-mediated increase in Src kinase activation and proteasome inactivation, thereby providing cardioprotection.

The Src kinases belong to the family of nonreceptor tyrosine kinases, which mediate a wide variety of intracellular signaling, including those mediating DNA synthesis and proliferation. Activation of Src kinase is associated with many degenerative diseases, including cardiovascular diseases, oncogenesis, and neurodegenerative diseases (23). In mammalian tumors expressing the neu protooncogene, c-Src tyrosine kinase activity is elevated (24). Markedly elevated levels of c-Src kinase activity were detected in human skin tumors (1). Myocardial ischemia-reperfusion caused an induction of c-Src protein expression (17); inhibition of c-Src with PPI reduces the extent of cellular injury.

Signaling pathways of Src kinase involve its activation through the activated cell surface receptors. For example, binding of ligand to platelet-derived growth factor receptors causes ligand to be associated with and to activate the Src family kinases (35), which trigger a cascade of events leading to entry into the S phase and subsequent DNA replication (2). The Src kinases are also involved in progression of the G2-to-M transition of the cell cycle (37). In addition, Src kinases can also transduce signals in response to cell-cell or cell-matrix adhesion (19).

The present studies confirm previous studies (4, 29) that presented evidence of inactivation of 20S and 26S proteasomes during myocardial ischemia-reperfusion. When proteasome is inhibited, cell cycle regulatory proteins and proapoptotic factors, normally inactivated by this complex, can accumulate. Several studies have shown that many activated protein kinases, such as protein kinase C (22) and several members of the Src family of kinases (14, 15, 26), undergo suicide regulation, whereby the activated form is rapidly ubiquitinated and, thereby, becomes a target for the 26S proteasome. For example, Blk, an Src family member, is recognized by the E3 ubiquitin-protein ligase E6AP, which promotes its ubiquitination and subsequent degradation by the 26S proteasome (26). Other studies (27, 36) indicate that Src itself is degraded in a ubiquitin-dependent manner and that the active form is specifically targeted for degradation, thus indicating a negative regulatory function for the proteasome. It is conceivable that decreased proteasome activity in postischemic hearts accounts for part of the observed increased Src and phosphorylated Src, which normally signals ubiquitination (36). This interpretation is strongly supported by the observation that preserving proteasome activity and, in particular, 26S proteasome activity, mitigates this increase. On the other hand, PPI has no protective effects on postischemic proteasome activities but, rather, directly inhibits c-Src, thus exerting an overall cytoprotective effect that is not mediated through the proteasome. In combination, these results support the conclusion that c-Src activation has a large role in postischemic cardiac injury and dysfunction.

The notion that preserving proteasome function in the postischemic heart can be protective may appear to be at odds with two studies (5, 30) that suggest protective effects on the ischemic myocardium of the proteasome inhibitor PS-519 (Millennium Pharmaceuticals). These studies (5, 30) used the inhibitor to limit the inflammatory response by decreasing leukocyte adhesion to endothelial cells. One of these studies (5) demonstrated positive effects in the leukocyte-supplemented crystalloid-perfused heart preparation but failed to observe any effect of the inhibitor, positive or negative, in the absence of the leukocytes. Although both of these studies (5, 30) determined peripheral leukocyte 20S proteasome activity, neither measured myocardial 20S or 26S proteasome activity, nor did they measure levels of ubiquitin-conjugated proteins, and it is not clear whether the beneficial effect was related to myocardial proteasomes. The ability of proteasome inhibitors to decrease the inflammatory response has been well documented (7) and, besides effects on leukocyte adhesion, has been attributed to decreased ubiquitin-mediated degradation of NF-{kappa}B nuclear translocation (6). Whether a proteasome inhibitor has a beneficial (anti-inflammatory) or negative (proapoptotic) effect is notoriously dose related (21) and will be somewhat dependent on degrees of proteasome activity in the different tissues (i.e., leukocyte vs. heart). When little or no proteasome inhibition is present, such as after brief ischemia, a decrease in leukocyte-mediated inflammation may be beneficial. However, in the presence of decreased proteasome activity, an inhibitor that adds to ischemia-mediated proteasome inhibition may tilt the cell toward death, but an agent, such as TRF, which protects the proteasome, may be beneficial, as shown in this study.

The mechanism by which TRF preserves proteasome activity is not completely understood from these studies. It is tempting to speculate that TRF acts as an antioxidant, preventing oxidative inactivation during ischemia. The 20S and 26S proteasomes have been shown to be vulnerable to oxidative inactivation, with 26S proteasome significantly more vulnerable (36). Indeed, Bulteau et al. (4) showed that subunits of the 20S proteasome are oxidatively modified during myocardial ischemia. However, a recent study (20) suggests that certain antioxidants isolated from cruciferous vegetables are capable of upregulating expression of several subunits of the proteasome, an effect observed 24 h after treatment. In light of the rather short treatment and posttreatment intervals used in the present experiments, it is unlikely that proteasome upregulation could account for the increased proteasome activity. Nonetheless, proteasome activity was increased to levels greater than baseline, indicating activation. This suggests mechanisms that include other than simple antioxidation, possibly redox effects, as has been suggested for the 20S proteasome (9).

In summary, ischemia-reperfusion caused ventricular dysfunction, electrical rhythm disturbances, and increased myocardial infarct size. PPI or TRF could reverse the ischemia-reperfusion-mediated cardiac dysfunction. Ischemia-reperfusion also upregulated c-Src expression and phosphorylation. Although TRF only minimally affected c-Src expression, it significantly inhibited the phosphorylation of c-Src. Ischemia-reperfusion reduced 20S and 26S proteasome activities, an effect prevented by TRF pretreatment. PPI exerted a cardioprotective effect that is not mediated by the proteasome but, rather, through direct inhibition of c-Src. The results of this study support a role for c-Src in postischemic cardiac injury and dysfunction and demonstrate direct cardioprotective effects of TRF. The cardioprotective properties of TRF appear to be due to inhibition of c-Src activation and proteasome stabilization.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grants HL-56803, HL-22559, HL-33889, HL-69910, HL-63317, and HL-68936.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. K. Das, Cardiovascular Research Center, Univ. of Connecticut, School of Medicine, Farmington, CT 06030-1110 (E-mail: ddas{at}neuron.uchc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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