Effective clearance of oxidized, damaged, and/or misfolded proteins in the cell by the ubiquitin-proteasome system (UPS) is critical for cell homeostasis, survival and function. We hypothesized that in the aging heart, generation of free radicals could impair UPS where the associated build-up of polyubiquitinated proteins could trigger programmed cell death. To test this, we used young (4 months old) and aged (24 months old) rats to analyze polyubiquitinated proteins, proteasome activity and programmed cell death in the ventricular tissue samples. Our studies reveal excessive deposition of polyubiquitinated proteins in the ventricular tissue extracts of old rats when compared to younger rats. The increased ubiquitination was accompanied by a significant decrease in 20S proteasome activity. Since the loss of proteasome-mediated clearance of ubiquitinated proteins is linked to programmed cell death, we measured TUNEL activity in aged rat heart and compared with younger animals. Aged animal hearts showed a substantial increase in programmed cell death as evidenced by TUNEL positive nuclei and DNA fragmentation. Analyses of cell death/survival pathways support our findings in terms of age-associated increase in the nuclear localization of p53, Bax/Bcl2 ratio and cleaved (active) caspase-3 and decreased expression of cellular inhibitor of apoptosis (cIAP1). Administration of grape seed extract (GSE) as a source of antioxidants significantly reduced these age-associated deleterious changes suggesting that free radicals primarily contribute to impaired UPS function and increased programmed cell death and that administration of antioxidants during aging could protect cardiac muscle cells and preserve ventricular function.
It is well established that aging is accompanied by increases in the levels of oxygen-derived free radicals and pro-oxidants [
Degradation of proteins by the ubiquitin (Ub) proteasome pathway is critical in regulating the levels of several cellular proteins where UPS eliminates damaged and/or unwanted cellular proteins, such as misfolded proteins, oxidized proteins, and proapoptotic proteins [2-6]. However, the efficiency of the UPS to degrade cellular proteins declines with age, leading to accumulation of intracellular ubiquitinated proteins destined for degradation [7-9]. In the aging heart, studies have confirmed that accumulation of misfolded proteins, damaged or oxidized proteins and proapoptotic proteins in cardiomyocytes due to compromised UPS function contributes significantly to cardiac failure [10-12]. In support of these findings, ubiquitination of cellular proteins for their subsequent elimination was found to be critical for the compensatory mechanism of the stressed heart [13,14] .
Therefore, improving UPS function in the aging heart could be one potential mechanism to prevent age-associated cardiomyocyte loss and function.
Grape seed extract is a natural extract from the seeds of Vitis vinifera. Grape seeds contain high levels of phytochemicals, which have been correlated with a decreased risk of chronic diseases. When administered, GSE is known to function as an effective free radical scavenger that reduces lipid peroxidation [
Since several studies demonstrate that protein damage by oxygen-derived free radicals is high during aging [
The following antibodies were obtained commercially: ubiquitin for Western analysis, and ubiquitin for microscopy were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Caspase-3, cleaved caspase-3 was purchased from Cell Signaling, (Beverly, MA). GAPDH was purchased from Fitzgerald (Concord, MA). Horseradish peroxidase-labeled secondary antibodies were purchased from Promega (Madison, WI). Actin antibody was purchased from Sigma (St. Louis, MO). cIAP1 was obtained from R & D Systems (Minneapolis, MN), and Alexa Fluor secondary antibodies was purchased from Invitrogen (Carlsbad, CA). ApopTag Fluorescein in situ Apoptosis Detection Kit for TUNEL assays was obtained from Chemicon, (Billerica, MA). 20S proteasome fluorometric (AMC) assay kit WAS OBTAINED from EMD Millipore (Millipore Corporation, MA). For nuclear and cytoplasmic extrac-tion from ventricular tissue samples, Pierce (Thermo Scientific, Rockford, IL) NE-PER extraction kit was used. DNA and RNA extractions were performed using Qiagen (Valencia, CA) extraction kits.
All other chemicals used were of analytical grade and were obtained from Sigma (St. Louis, MO), SRL (Sisco Research Laboratories Pvt. Ltd, India) and CDH (Central Drug House Pvt. Ltd., Mumbai, India).
Grapes, as large clusters with red berries, were bought from a local supermarket in Chennai and identified as Vitis vinifera. Grape seeds were removed from the grapes, air dried for 1 week and milled to a particle size of <0.4 mm. The grape seed powder (100 g) was macerated for 12 h at room temperature three times with 800 ml of 100 mM acetate buffer, pH 4.8, in water/acetone (30:70, v/v), each time. The three macerates were combined and concentrated until no acetone was left using a rotary evaporator under reduced pressure and a water bath temperature < 35˚C. The concentrated solution was extracted four times with 200 ml of ethyl acetate each time. The extracts were combined, evaporated to remove ethyl acetate and grape seed polyphenols was obtained as a lyophilized powder [
All experiments were conducted in accordance with guidelines approved by the Institutional Animal Ethical Committee (IAEC. No 02/024/09). Young (4-month-old) and aged (24-month-old) male Wistar albino rats were used throughout the study. Since the average life span of the Wistar albino rat is 2(1/2) to 3(1/2) years and the aged animals were defined as those that had achieved the age at which one half of the population ordinarily die (median survival time), we chose 24 months rats for the older group. Rats were divided into four groups and each group consisted of six animals: Group I, young control rats; Group II, young rats supplemented with grape seed extract; Group III, aged control rats; Group IV, aged rats supplemented with grape seed extract.
Grape seed extract (50 mg/kg body weight/day) dissolved in physiological saline was administered orally using an intragastric canula for 30 days as described previously [
Heart tissue was excised, weighed and immediately transferred to ice-cold physiological saline. After several washes, 10% homogenate was prepared in ice-cold 100 mM Tris-HCL buffer, pH 7.4. The heart homogenate was used for the biochemical analysis. Triton X-100 soluble and insoluble fractions were prepared from fresh tissue samples as before [
Nuclear isolation was performed as per the manufacturer’s (Pierce) protocol with minor modifications. Briefly, 50 mg of heart tissue was homogenized in 1 ml PBS with protease and phosphatase inhibitor cocktails. The homogenate was centrifuged at 1000× g for 8 min. To the pellet, 500 μl of cytoplasmic extraction buffer I (CERI) with inhibitors was added. After suspending the pellet, the solution was incubated on ice for 15 min, followed by the addition of 27 μl CERII. The contents were incubated on ice for 2 min, centrifuged at 16,000× g for 15 min, and the supernatant (cytoplasmic proteins) was mixed with an equal volume of 2X SDS sample buffer. The pellet was washed with PBS, suspended in 250 μl of nuclear extraction buffer with inhibitors and incubated on ice for 40 min, then vortexed for 10 sec every 10 min. After centrifugation at 16,000× g for 15 min at 4˚C, the supernatant, consisting of nuclear proteins, was mixed with an equal volume of 2X SDS sample buffer.
Amount of nuclear level of p53, cytosolic level of Bcl2, Bax, cIAP1, and Caspase-3, ubiquitin were assessed by Western blot using 4% - 12% SDS-polyacrylamide gels. The gels were blotted on to PVDF membrane. The membranes were then blocked in 10% nonfat milk in Trisbuffered saline with 0.2% Tween 20 (TBS-T) at room temperature for 1 h, and probed with the following primary antibodies diluted in TBS-T: anti-ubiquitin, Bcl-2, Bax, p53 and cIAP1 mouse monoclonal antibodies (1:300 dilution), and anti-pro-/cleaved caspase-3 rabbit polyclonal antibodies (1:2000 dilution) for 3 h at room temperature. Following incubation with secondary antimouse or rabbit IgG linked to horseradish peroxidase at a 1:5000 dilution for 45 min, the band was visualized using enhanced chemiluminescence and X-ray film. Band intensity was measured by using NIH-ImageJ. Quantification for each protein of interest was accomplished by first normalizing the protein band in each ventricle to a control endogenous protein (either GAPDH or actin).
The 20S subunits were purified as described [
The proteasome assay was performed using 20S proteasome fluorometric (AMC) assay kit (Millipore Corporation). The degradation of the fluoropeptide Suc-LeuLeu-Val-Tyr-AMC (chymotrypsin like) was measured after addition of the substrate to the tissue homogenates. 10 μl of heart tissue homogenates were incubated with 985 μl of reaction buffer (500 mM HEPES, pH 7.6 and 10 mM EDTA) for 5 minutes. 5 μl of 200× (2 mM of substrate solution diluted in reaction buffer) and 10 μl of 3% SDS were added. The mixture was incubated for 60 minutes at 37˚C, and the reaction was then stopped by addition of an equal volume of ice-cold 96% ethanol. The fluorescence determination was performed at 380 nm excitation and 460 nm emission using free AMC as a standard, and the measurements were corrected by subtracting the background fluorescence (without SucLLVY-AMC).
Fresh frozen tissue samples were placed in OCT freezing solution and left at -80˚C. Tissue sections (12 μm thick) were fixed with 2% paraformaldehyde, permeabilized in 2% SDS for 5 min at room temperature, and blocked with 10% donkey serum for 1 h at room temperature.
Primary anti-ubiquitin antibody (1:100, Dako) was added overnight at 4˚C. Sections were washed in PBS and 200 μl of DAPI was added. Slides were then incubated with Alexa Fluor secondary antibodies for 2 h. After the secondary antibody incubation, the sections were washed with PBS, mounted in Anti-Fade, and subjected to laser scanning confocal microscopy (Olympus IX81, Japan) at 40× magnification. The images were processed with Adobe Photoshop.
TUNEL (TdT-mediated dUTP Nick End Labeling) assay was performed as per manufacturer’s protocol with slight modifications. Briefly, fresh frozen tissue samples were used for TUNEL studies. Tissue sections (12 μm thick) were prepared and incubated in 1% PFA for 10 min at room temperature and washed twice with PBS each for 5 min. Then, the slides were incubated at -20˚C for 5 min in ethanol:acetic acid solution (2:1 dilution) for permeabilization and washed subsequently with PBS and then with 75 ml of equilibration buffer for 1 min. The enzymatic reaction was performed by incubating the tissue sections for 1 hour at 37˚C with 55 μl of terminal deoxynucleotidyl transferase (Reaction Buffer and TdT). After incubation, the tissues were soaked in wash buffer at room temperature for 10 min. The slides were washed three times each for 1 min and incubated in 65 ml of anti-digoxigenin fluorescein. Followed by 3 washes in PBS, the sections were stained for specific proteins or nucleus and mounted using anti-Fade and analyzed by confocal microscopy.
Total RNA extraction from the heart muscle was performed using Qiagen’s RNeasy Minikit. Total RNA was solubilized in RNase-free H2O, and quantified in duplicate by measuring the optical density (OD) at 260 nm. Purity of RNA was assured by examining the OD260/ OD280 ratio. Total RNA (100 - 150 ng) was subjected to a total volume of 50 μl RT-PCR containing 0.6 μM final concentrations of forward and reverse primers for cIAP1 and β-actin. Thermal cycling conditions were started with RT reaction for 1 h at 50˚C followed by initial activation of Taq DNA polymerase for 10 min at 94˚C. cIAP1 and β-actin cDNA amplification was started with initial thermo-cycle at 94˚C for 30 s and then at 59˚C for 45 s for primer annealing. Extension was done at 72˚C for 45 s and the final extension was at 72˚C for 10 min. PCR was performed using a programmed thermocycler (Thermocycler gradient; Eppendorf, Germany). The primer pairs used for RT-PCR amplification are as follows: cIAP1: 5’-TCCCTGTCATCTCACCATGA-3’ and 5’-TGTCTAGCATCAGGCCACAG-3’; β-actin: 5’-GCCATGTACGTAGCCATCCA-3’ and 5’-GAACCGCTCAT-TGCCGATAG-3’. After amplification, the RTPCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide. Images were captured and subjected to densitometric analysis.
The level of DNA fragmentation was measured as described previously [
Differences in the proteasome activity between the groups were compared by one-way ANOVA followed by a Tukey test for multiple comparisons. Statistical significance was defined as P < 0.05.
In the normal heart, polyubiquitination marks the damaged proteins for their subsequent effective elimination by the 26S proteasome. However, since the volume of damaged/oxidized proteins in the aged heart is substantially increased due to excessive generation of reactive oxygen species, it is possible that the ubiquitinated protein level increases in the aged heart with a corresponding decrease in the proteasome function. Furthermore, such an intracellular buildup of polyubiquitinated proteins due to the ineffective clearance by the UPS could eventually lead to programmed cell death. Therefore, to gain insight into potential consequences of age-dependent increase in the polyubiquitinated protein level in the heart, we performed immunohistochemical studies to establish whether aged rat hearts exhibit enhanced protein ubiquitination accompanied by increased programed cell death (
We performed additional biochemical experiments to confirm our immunohistochemical studies. To show that the polyubiquitinated protein level was increased in old rats that could be reverted with GSE treatment, immunoblots using anti-ubiquitin antibody was performed in Triton X-100 soluble and insoluble samples prepared from young and old rat hearts treated with ±GSE (
Administration of GSE was found to partially reverse this trend in protein ubiquitination. Finally to confirm that the old heart samples had higher levels of programmed cell death that could be controlled with GSE treatment, DNA fragmentation was analyzed. DNA fragmentation in the aged rat hearts was high when compared to young rat heart samples. However, in GSE fed rats, DNA fragmentation was substantially attenuated and the DNA migration pattern resembled similar to young rat hearts (
Accumulation of polyubiquitinated proteins in the aging rat heart could be due to excessive generation of damaged proteins with their subsequent ubiquitination and/or decreased clearance of polyubiquitinated proteins due to less efficient proteasome function. Therefore, we next measured proteasome activity in young and old rat hearts and then explored if administration of GSE could improve proteasome function. 20S proteasome activity in the heart extracts was measured using a fluorescence conjugated peptide substrate [
As shown in
Enhanced programmed cell death in older hearts indicates changes in the regulators of cell survival and programmed cell death. Therefore, we next analyzed known key components regulating programmed cell death. For this, we measured the cellular levels of the following parameters in old and young rat hearts after GSE administration: The transcription factor p53 is a tumor suppressor protein that functions by controlling cell cycle progression and/or promoting apoptosis under stress conditions. Normally, the nuclear level of the tumor suppressor protein p53 is maintained at low levels and accumulates in response to various types of stress. Nuclear localization of p53 has been shown to activate mitochondrial pathways for apoptosis by increasing Bax/Bcl2 ratio [30,31]. We observed an elevated level of p53 in the nuclear and cytoplasmic fractions of aged rat hearts and compared them with young rat heart samples (
Since p53 has been known for its role as a transcriptional factor for the expression of Bax and other proapoptotic factors [30,31], we next measured the mRNA and protein levels of Bax, a proapoptotic factor, and Bcl-2, a prosurvival factor in aged and young hearts treated with ±GSE. In untreated rats, the Bax level in the aged animal group when compared to younger animals, showed a significant (>2.5 fold) increase at the protein (
towards that of young rats, while no change by GSE was observed in young rate hearts. Similarly, the level of Bcl-2 as a pro-survival marker was assessed. Immunoblot analyses also showed a notable decrease in the Bcl-2 level in the aged rat heart (
Finally, we measured the level of cellular inhibitor of apoptosis (cIAP1), a member of E3 ubiquitin ligase that promotes cell survival through ubiquitin-mediated elimination of caspases. Measurement of cIAP1 at the mRNA expression level was significantly decreased in aged rat hearts (
activity and the associated increase in programmed cell death. This was also reflected at the protein level as evidenced from the immunoblot analysis that showed a substantial decrease in cIAP1 level in the aged heart when compared to young rat hearts (
The UPS has been shown as the main nonlysosomal degradation mechanism for the removal of defective proteins, which have often undergone free radical-derived modifications. The data in the present study demonstrate changes associated with aging and its reversal by the administration of GSE that promotes proteasome-mediated degradation of deleterious proteins in the heart. Additionally, this study shows an age-dependent decline in the peptidase activity of 20S proteasome in the heart which could be corrected by the administration of GSE (
Although the decreased proteasome function in the aging heart could be partly due to the excessive generation of oxidatively damaged proteins, other independent changes in the proteasome composition, as part of the aging process, might also contribute to the overall loss of proteasomal function. Additional studies to measure the level, composition and posttranslational modifications of proteasome machinery will help decipher the mechanism of age-associated changes in the proteasome function. In support of this idea, an earlier study indicates that the natural antioxidants have ability to enhance the expression of proteasome subunits, thus offering increased protection against various oxidants [
Our study was also designed to explore whether the build-up of damaged macromolecules during aging is associated with changes in cell survival/death mechanism and increased programmed cell death. Comparing young vs. older rats, the major findings in the aging rat heart include: 1) transcriptional activation of p53, and the associated increase in the Bax/Bcl-2 ratio, 2) age-dependent decline in the level of cellular inhibitor of apoptosis (cIAP1) with the resulting activation of caspases, and 3) reversal of all these age-associated changes and cardiomyocyte loss following treatment with GSE.
For cellular homeostasis, clearance of damaged proteins, such as oxidized proteins, mis-folded proteins, pro-apoptotic proteins, etc is critical, and the UPS serves as a major mechanism for their removal. Therefore, loss of proteasomal function as observed in the aging heart could lead to accumulation of ubiquitinated proteins meant for degradation, and this condition triggers programmed cell death in the form of either apoptotic or autophagic cell death. In this context, previous studies showed that cardiomyocyte apoptosis increases in parallel with proteasome activity depression [
It has been demonstrated that the anti-oxidant proanthocyanidins present in the GSE could function as an anti-apoptotic regulator by down-regulating pro-apoptotic genes [
p53 has been shown to regulate apoptosis by inhibiting the anti-apoptotic effects of Bcl-2 homologues while inducing the expression of pro-apoptotic genes such as PUMA, Bax, and Bak that mediate the release of cytochrome c from mitochondria and induce cell death [41-43]. Therefore, accumulation of p53 due to proteasome insufficiency and the associated changes in the pro-apoptotic gene products could be responsible for the increased cell death that was observed in the aged heart. The proteins of the Bcl-2 family have been suggested to play a pivotal role as intracellular checkpoint in the apoptotic signal transduction [
We also explored the role of cIAP family proteins that function as RING-finger E3 ligases and specifically target and ubiquitinate caspases and other associated pro-apoptotic proteins [
In summary, the build-up of deleterious proteins due to the functional loss of the UPS can cause myocardial cell loss, a major contributing factor to the development of heart failure. The present work show an age-associated increase in many cell death markers, such as TUNEL and DNA fragmentation which are accompanied by increased levels of p53 in the nucleus, Bax/Bcl-2 ratio, and active caspase-3 and decreased cIAP1 production. Importantly, administration of GSE shows antiapoptotic effects by minimizing these aging associated changes in the heart. Therefore, GSE has therapeutic benefits in reversing many of the aging related deleterious changes and improves ventricular function.
This authors wish to thank Dr. Dhan Kuppuswamy for his helpful suggestions in designing this work.