Skip Navigation

Cardiovascular Research 2007 74(3):445-455; doi:10.1016/j.cardiores.2007.02.016
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Srivastava, S.
Right arrow Articles by Prabhu, S. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Srivastava, S.
Right arrow Articles by Prabhu, S. D.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2007, European Society of Cardiology

Downregulation of CuZn-superoxide dismutase contributes to β-adrenergic receptor-mediated oxidative stress in the heart

Sanjay Srivastavaa, Bysani Chandrasekarb, Yan Gua, Jianzhu Luoa, Tariq Hamida, Bradford G. Hilla and Sumanth D. Prabhua,*

aLouisville VAMC and Institute of Molecular Cardiology, Department of Medicine, University of Louisville, Louisville, KY, United States
bMedicine/Cardiology, University of Texas Health Science Center, San Antonio, TX, United States

* Corresponding author. Medicine/Cardiology, University of Louisville, ACB, 3rd Floor, 550 South Jackson Street, Louisville, KY 40202, United States. Tel.: +1 502 852 7959; fax: +1 502 852 7147. Email address: sprabhu{at}louisville.edu

Received 21 April 2006; revised 5 January 2007; accepted 8 February 2007


   Abstract
Top
 Abstract
1. Methods
 2. Results
 3. Discussion
 Acknowledgments
 References
 
Objective: Sustained β-adrenergic receptor (β-AR) activation augments oxidative stress in the heart; whether alterations in antioxidant enzymes contribute to this effect is unknown.

Methods and results: Adult male Wistar rats were implanted with osmotic minipumps to infuse either L-isoproterenol (ISO, 25 µg/kg/h) or saline (SAL). After 7-days, ISO-treated hearts exhibited significant (p<0.005): 1) concentric hypertrophy and augmentation of systolic function, 2) reductions of end-systolic wall stress, and 3) augmentation of oxidative stress, with a ~3-fold increase in 4-hydroxy-2-nonenal-and malondialdehyde-protein adducts. ISO-treated hearts also exhibited significant (p<0.01) reductions of CuZn-superoxide dismutase (SOD) enzyme activity (30%), protein (40%), and mRNA (60%), without changes in Mn–SOD, catalase, or glutathione peroxidase. Elk-1 and YinYang1 (YY1) are transcription factors that positively and negatively regulate CuZn–SOD expression, respectively. ISO-treated hearts exhibited a 3-fold increase in YY1 and a 2-fold reduction in Elk-1 DNA binding activity, strongly favoring CuZn–SOD gene repression. In isolated cardiomyocytes, sustained (24 h) ISO stimulation significantly (p<0.01) increased reactive oxygen species (ROS), an effect blocked by CGP20712A, a β1-AR antagonist, but not by ICI118,551, a β2-AR antagonist. CuZn–SOD downregulation paralleled the increase in ROS, and were similarly blocked by β1- but not β2-AR blockade. There were no changes in CuZn–SOD mRNA stability or myocyte size with ISO treatment. However, nuclear run-on revealed a 40% reduction in CuZn–SOD mRNA expression (p<0.01), consistent with transcriptional repression. ISO also depressed total cellular antioxidant capacity, reduced glutathione (GSH) levels, and the GSH:GSSG ratio. Moreover, CuZn–SOD siRNA transfection of H9c2 cardiomyocytes to suppress CuZn–SOD protein by ~40–50% (analogous to the in vivo changes) induced cellular apoptosis.

Conclusions: Sustained β-AR stimulation transcriptionally downregulates CuZn–SOD in myocardium via the β1-AR, thereby contributing to β-AR-mediated oxidative stress.

KEYWORDS β-adrenergic receptor; Oxidative stress; Copper-zinc superoxide dismutase

Sustained adrenergic activation contributes to the progression of heart failure (HF) [1]. Chronic norepinephrine exposure induces contractile dysfunction, myocyte toxicity, and apoptosis; these effects are mediated primarily via the β1-adrenergic receptor (β1-AR) [1–3]. In contrast, β-AR blockade in HF improves survival and attenuates pathological left ventricular (LV) remodeling [1,4,5]. The mechanisms underlying these effects remain incompletely defined. Acute β-AR stimulation rapidly generates reactive oxygen species (ROS) that can modulate cellular and functional responses in the heart, suggesting a role for ROS in short-term cardiac regulation [6,7]. Over the long-term, however, β-AR-mediated oxidative stress results in detrimental effects. This is supported by the findings that chronic isoproterenol (ISO) stimulation in vivo induces ROS-dependent myocardial fibrosis [7], and that antioxidants prevent cardiomyocyte apoptosis resulting from chronic norepinephrine administration [8].

ROS are derived from mitochondrial and cellular oxidases that generate superoxide (O2Formula) [9]. As β-AR stimulation increases mitochondrial O2 consumption, there is an attendant increase in obligatory electron leakage and mitochondrial ROS production [10–12]. Conversely, ROS generation is efficiently balanced by antioxidant enzymes that scavenge ROS and limit toxicity. Of particular importance are copper–zinc (CuZn) and manganese (Mn) superoxide dismutase (SOD) that dismutate O2Formula to H2O2, and glutathione peroxidase (GPX) and catalase, which convert H2O2 to water [9]. Long-term alterations in these enzymes could chronically modulate ROS metabolism and, especially in the face of pathologically increased ROS generation, impact oxidative stress. Importantly, whether changes in antioxidant defenses play a role in chronic β-AR-mediated oxidative stress in the heart is unknown. Accordingly, we examined the effects of sustained, low-level in vivo β-AR activation on antioxidant enzymes and oxidative stress in the rat heart. Our results establish a novel cellular mechanism that contributes to β-AR-dependent cardiac oxidative stress: the β1-AR mediated downregulation of CuZn–SOD.


   1. Methods
Top
 Abstract
 1. Methods
2. Results
 3. Discussion
 Acknowledgments
 References
 
1.1 In vivo β-AR stimulation model and experimental protocol
All studies were performed in compliance with the NIH Guide for the Care and Use of Laboratory Animals (DHHS publication No. [NIH] 85–23, revised 1996). Adult male Wistar rats (250–350 g) were anesthetized with IM ketamine (44 mg/kg) and xylazine (5.4 mg/kg). Alzet osmotic minipumps (1.0 µl/h continuous infusion) containing either L-ISO 25 µg/kg/h (n=22) or saline (SAL, n=22) were implanted in the peritoneal cavity. The ISO dose was four-fold lower than in our previous study [13]. In 16 animals/group, echocardiography (Toshiba 380 Powervision, 10 MHz transducer) was performed under half-dose anesthesia 1 day prior and 7 days after minipump implantation [4,5]. LV hypertrophy was indexed by end-diastolic (ED) wall thickness and systolic function by fractional shortening (FS) and mean velocity of circumferential fiber shortening (Vcf, FS/ejection time) [14]. Following the final study, the heart was rapidly excised and rinsed in ice-cold physiological saline. The ventricles were dissected and weighed separately. A short-axis LV section was formalin-fixed for 16 h, dehydrated in ethanol, and paraffin-embedded for histological studies. The remaining LV was snap-frozen in liquid nitrogen and stored at –80 °C for biochemical/molecular studies.

In 9 animals/group, blood pressure (BP) measurements were performed under anesthesia near-simultaneously with echocardiographic measurements. Body temperature was maintained at 37 °C with heating pads. A Millar 1.4 Fr high fidelity pressure catheter was advanced via the carotid artery into the ascending aorta. After stabilization for 10–15 min, aortic BP was recorded for 1–2 min using a PowerLab 8/SP system. The catheter was removed, the artery ligated, and the tissue harvested as above. End-systolic pressure was taken to occur at the aortic pressure dicrotic notch and end-systolic meridional wall stress ({sigma}, g/cm2) was determined as follows: [1.35(ESP)(ESD)]/[4 h(1+{h/ESD})], where ESP is end-systolic pressure, ESD is end-systolic diameter, and h is end-systolic wall thickness [14].

1.2 Cardiomyocyte stimulation and myocyte ROS, antioxidant capacity, and glutathione levels
From an additional 12 rats, LV myocytes were isolated using modified Langendorff perfusion and collagenase digestion as described previously [4,15]. Myocytes (104 rod-shaped cells/cm2) were maintained in serum-free DMEM culture medium (supplemented with albumin 0.2%, L-carnitine 2 mM, creatine 5 mM, taurine 5 mM, L-glutamine 1.3 mM, insulin 0.1 µM, triiodothyronine 0.1 nM, pyruvate 2.5 mM, and penicillin/streptomycin 0.1%) at 37 °C and 5% CO2. Cell preparations contained ≥75–80% viable, calcium-tolerant, non-contracting, rod-shaped myocytes. Cells remained quiescent in media for at least 1 h prior to experimentation.

Myocytes were exposed to one of the following for 20–24 h: 1) media alone (control), 2) ISO 10–7 M, 3) ISO 10–7 M plus CGP20712A, 300 nM (selective β1–AR antagonist), 4) ISO 10–7 M plus ICI118,551, 300 nM (selective β2-AR antagonist), 5) ISO 10–7 M plus metoprolol 100 nM (predominantly β1-AR antagonism). The doses chosen have previously been shown to achieve pharmacologic receptor blockade [1,3,4]. Intracellular ROS were determined using the probe 2',7'-dichlorofluorescin diacetate (DCFH-DA) as described previously [15]. DCFH is oxidized by ROS to highly fluorescent dichlorofluorescein (DCF). DCF-fluorescence was normalized and expressed per 104 viable cells. Cell length and width were measured using video-based digital edge detection (SoftEdge Acquisition, IonOptix) as previously described [15]. Total myocyte antioxidant capacity was determined using the TAC-Peroxyl assay kit (Northwest Life Science Specialties) following the manufacturer's instructions. The antioxidant capacity was determined by a luminescence assay and compared to the effects of Trolox, a water-soluble Vitamin E analog.

Reduced (GSH) and oxidized glutathione (GSSG) levels were measured in cardiomyocytes using a commercially available kit (Glutathione Assay Kit, Cayman Chemical) as previously described [16]. Cells (2.5x105) were homogenized in phosphate buffered saline (pH 7.0, 4 °C). After centrifugation at 2000 xg for 2 min, the supernatant was collected and deproteinated by adding equal volumes of 10% metaphosphoric acid, vortexed, incubated for 5 min at 22 °C, and spun for 2 min in a microcentrifuge. The supernatant was collected and assayed for both GSH and GSSG content according to the manufacturer's instructions.

1.3 Antioxidant enzyme activity
CuZn–SOD and Mn–SOD enzyme activities were measured as described by Dieterich et al. [17]. Briefly, total SOD activity was determined in LV homogenate cytosolic fraction by monitoring the inhibition of pyrogallol auto-oxidation at 420 nm. Residual Mn–SOD activity was measured in a separate reaction by adding 1 mM NaCN to the sample to inhibit CuZn–SOD. The difference between the two was taken as CuZn–SOD activity and expressed as U/mg protein, where 1 U is the amount required to inhibit pyrogallol oxidation by 50%. For Mn–SOD activity, tissue was homogenized in 10 volumes of buffer A (in mM: mannitol 220, sucrose 70, EGTA 2, and Mops 5, pH 7.4) and centrifuged at 500 xg for 10 min at 4 °C. The supernatant was passed through cheesecloth, collected and centrifuged at 5000 xg for 20 min at 4 °C to isolate the mitochondrial pellet. The mitochondria were washed twice with 10 volumes of buffer A and resuspended in buffer A with 1.0 mM MnCl2 at a concentration of 4 mg/ml. Mitochondrial Mn–SOD activity was then determined using pyrogallol auto-oxidation as above.

GPX and catalase enzyme activity were determined as described by Li et al. [18]. GPX activity (nmol NADPH/min/mg protein) was determined in the cytosolic fraction using GSH oxidation coupled to the disappearance of reduced NADPH at 340 nm by glutathione reductase (GSHR). Catalase activity (µmol/mg protein) was measured in the cytosolic fraction by monitoring the disappearance of H2O2 at 240 nm [18].

1.4 Western immunoblotting and slot blots
Total protein extraction, SDS-PAGE Western blotting, and immunodetection using electro-chemiluminescence (ECL) were performed as previously described [4,5,19]. Western blotting for Mn–SOD was performed using mitochondrial protein, prepared as described above. Anti-Mn–SOD, CuZn–SOD, and catalase were obtained from The Binding Site Limited (London) and anti-GPX antibodies from Cortex Biochem. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldehyde dehydrogenase 2 (ALDH2) were used as loading controls for cytosolic and mitochondrial proteins, respectively. Protein bands were quantitated by image scanning densitometry (arbitrary units) and normalized to the protein loading control. The measured ratio of optical densities for each sample was then normalized to the average ratio value for all the saline treated tissue samples (or control myocyte samples) and expressed as a percent.

Tissue oxidative stress was quantified by the abundance of free (unbound) and protein adducted 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA). Myocardial levels of free HNE and MDA were measured in LV homogenate using the LPO 586 kit (Bioxytech) as per the manufacturer's instructions. Protein-HNE adducts were quantitated using slot blots. Protein from tissue homogenate (1.0 µg) was loaded in the wells of a Bio-Dot apparatus (Biorad), and microfiltered through nitrocellulose membranes under vacuum. Protein-HNE adducts were probed using polyclonal anti-KLH–HNE antibody raised in-house using previously published protocols [20]. Amido Black staining was used to normalize protein loading. Intensity of the immunoreactive bands was quantitated by ImageQuant TL software. Standard SDS-PAGE Western blotting (non-reduced) was also performed using these antibodies.

1.5 CuZn–SOD gene expression, transcription, and mRNA stability
CuZn–SOD mRNA expression was analyzed by Northern blotting [5,19,21] using CuZn–SOD cDNA [21]. 28S rRNA was used as an internal control. mRNA levels were expressed as the ratio of optical densities of the CuZn–SOD transcripts (0.7 and 0.9 kb) to 28S. In isolated cardiomyocytes, we also evaluated CuZn–SOD gene transcription using nuclear run-on assays (to determine nuclear CuZn–SOD mRNA levels), and CuZn–SOD mRNA stability by actinomycin D pulse followed by Northern blotting as previously described [22].

1.6 YinYang1 (YY1) and Elk-1 DNA binding
YY1 and Elk-1 DNA binding activities were quantified by electrophoretic mobility shift assay (EMSA). Nuclear protein extraction from frozen myocardium, EMSA, and densitometry were performed as previously described [19]. Consensus double-stranded oligonucleotides containing the YY-1 binding site (sense, 5'-GATCGAGCATCCATCTTGGCTCAC-3') and Elk1 binding site (sense, 5'-CATCGGCTTGCCTAGGAAGCGCAAGG-3') were used as probes [23]. Specificity of YY1 and Elk1 DNA binding activity was confirmed in competition studies using mutant constructs (YY1, 5'-GATCGAGCATAACGCTTGGCTCAC-3; Elk-1, 5'-CATCGGCTTGCCTACTCTTAGCAAGG-3'). In the gel supershift assay, the protein extract (10 µg) was preincubated for 40 min on ice with either rabbit-anti-Elk-1- or YY1-specific polyclonal antibodies (1 µg, Santa Cruz) or control IgG (1 µg) prior to the addition of respective 32P-labeled double stranded consensus oligonucleotide. Absence of protein extract, competition with 100-fold molar excess unlabeled respective consensus, and mutant oligonucleotide served as controls.

1.7 Immunohistochemistry
Formalin-fixed, paraffin-embedded short-axis LV sections (5 µm) were de-paraffinized, rehydrated, and serially incubated in 3% H2O2, 1:100 dilution of IgG purified primary antibody (anti-CuZn–SOD or anti-MDA [Academy Bio-Med]) for 45 min, LINK-rat (DAKO LAB2 Rat Kit) for 20 min, LABEL-Rat for 20 min, DAB buffer for 10 min, and hematoxylin for 1 min. After incubation, the slides were rinsed with PBS. Normal rabbit IgG, at equal concentration, was used as control. Secondary antibody was linked to horseradish peroxidase and 3'-3 diaminobenzidine (DAB) was used as the chromogen. Immunostaining was quantified with a MetaMorph 4.5 imaging system (Universal Imaging Corp). Staining intensity for each sample was normalized to the average intensity for all the saline treated samples and expressed as a percent.

1.8 CuZn–SOD gene silencing and apoptosis measurement in H9c2 cells
H9c2 cardiomyocytes (ATCC) were cultured in DMEM media (Invitrogen) containing 10% FBS and penicillin/streptomycin at 37 °C and 5% CO2. Cells were passaged at 70% confluence and used up to the 7th passage. For CuZn–SOD siRNA silencing, cells were transfected with 100 nM SMARTpool CuZn–SOD siRNA (Dharmacon) for 72 h. After treatment, cells were scraped and collected by centrifugation in cold PBS. Total cell lysates were prepared in RIPA buffer, and 20–50 µg protein was used to determine CuZn–SOD and poly-ADP ribose polymerase (PARP, a caspase-3 substrate and apoptotic marker) levels by SDS-PAGE Western blotting using commercially available antibodies.

1.9 Statistical analysis
Comparisons between two groups (i.e., SAL vs. ISO) were performed using an unpaired t-test. With multiple group comparisons (i.e., myocyte studies), two-way ANOVA was performed, followed by a Student–Newman–Keuls test for post-hoc comparison. A p value of <0.05 was considered significant. Group data are presented as mean±SD.


   2. Results
Top
 Abstract
 1. Methods
 2. Results
3. Discussion
 Acknowledgments
 References
 
2.1 Sustained β-AR activation in vivo induces load-independent concentric LV hypertrophy
Fig. 1 shows M-mode echocardiograms from an animal at baseline and after 7 d of ISO infusion. ISO increased heart rate (HR), wall thickness, and FS, and reduced chamber diameter. Table 1 displays echocardiographic, hemodynamic, and morphometric group data. While ISO significantly increased HR, there were no changes in arterial BP, and an actual decrease in end-systolic wall stress. Despite reduced overall afterload, the ISO group exhibited marked concentric hypertrophy, with increases in wall thickness and reductions in chamber diameter, and augmented systolic function, indicated by greater FS and Vcf. LV and RV hypertrophy were confirmed by normalized LV and RV weights. Histological examination (Figs. 2 and 3Go) revealed no appreciable inflammatory cell infiltration or myocardial necrosis. Thus, with this low dose of ISO (4.2 mg/kg over 7 days), we observed load-independent concentric hypertrophy and enhanced systolic function without inflammation or myocardial toxicity. This is dramatically different from the dilated cardiomyopathy induced by high doses of ISO (>170 mg/kg) [24].


Figure 1
View larger version (77K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 M-mode echocardiograms from one animal before and after 7-day isoproterenol (ISO) infusion (25 µg/kg/h). ISO induced concentric hypertrophy and augmented heart rate and systolic function. AW, anterior wall; PW, posterior wall; EDD, end-diastolic diameter; ESD, end-systolic diameter.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Echocardiography, hemodynamics, and gravimetry after 7-day infusion

 

Figure 2
View larger version (33K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 A, Slot-blots of protein-4-hydroxy-2-nonenal(HNE) adducts in saline(SAL)-and isoproterenol(ISO)-treated hearts. By densitometry, there was a 3.5-fold increase in protein-HNE adducts in ISO-treated hearts (n=6 per group, *p<0.001). B, Western-blots for HNE-adducted proteins in SAL-and ISO-treated hearts. Several proteins over a broad molecular weight range exhibited HNE-modification. C, Myocardial levels of free HNE and malondialdehyde (MDA) in SAL- and ISO-treated hearts. There was a 2-fold greater abundance of these aldehydes in ISO-treated hearts (n=6 per group, *p<0.01). D, Immunohistochemistry for protein-MDA adducts revealed a ~3-fold increase in staining intensity in ISO-treated hearts (*p<0.001, n=6 per group).

 

Figure 3
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 A, Western blotting and densitometry for CuZn–SOD, Mn–SOD, catalase, and GPX in saline(SAL)- and isoproterenol(ISO)-treated hearts (black and gray bars, respectively). ISO decreased CuZn–SOD protein (~40% reduction, *p<0.005) without alterations in the other antioxidant enzymes. B, Immunostaining for CuZn–SOD and quantitation (normalized to SAL) similarly revealed a ~40% reduction in CuZn–SOD staining in ISO-treated hearts (*p<0.003). n=6 per group. C, Northern blotting for CuZn–SOD mRNA. Intensity of 0.7 and 0.9 kb were combined and their average intensity is shown in the densitometric analysis. There was a 60% reduction in CuZn–SOD mRNA levels in ISO-treated hearts (*p<0.01, n=6 per group).

 
2.2 Sustained β-AR activation increases myocardial oxidative stress
As compared to SAL, ISO-treated hearts exhibited a 3.5-fold increase in HNE-modified proteins by immunoblotting (Fig. 2A), indicating increased oxidative stress and lipid peroxidation. Additionally, Western blotting revealed a broad range of HNE-modified proteins (Fig. 2B), and measurement of free aldehydes revealed a ~2-fold increase in unbound HNE and MDA in ISO-treated hearts (Fig. 2C). Immunohistochemistry (Fig. 2D) confirmed increased protein-MDA adducts with ~3-fold greater immunoreactivity in ISO-treated hearts, primarily in cardiomyocytes. Myocytes in ISO-treated hearts were also hypertrophied, and there was no discernible inflammatory cell infiltration.

2.3 Sustained β-AR activation downregulates myocardial CuZn–SOD
As demonstrated by the group data in Table 2, sustained in vivo β-AR activation selectively reduced CuZn–SOD activity (30% decrease) without altering that of Mn–SOD, GPX, or catalase. Given the selective reduction of CuZn–SOD activity, we next evaluated whether translational and/or transcriptional mechanisms contributed to this effect. CuZn–SOD protein abundance (Western blotting, Fig. 3A) decreased in a parallel fashion (~40% decline) in ISO-treated hearts. In contrast, Mn–SOD, GPX, and catalase protein levels were unchanged. CuZn–SOD immunostaining (Fig. 3B) revealed immunoreactivity primarily in myocytes under both conditions, but with reduced intensity (~40%decrease) in ISO-treated hearts consistent with the Western blotting results. Fig. 3C displays Northern blotting for CuZn–SOD gene expression. As indicated, there was a highly significant 60% overall reduction in CuZn–SOD mRNA levels in ISO-treated hearts as compared to SAL. As elegantly demonstrated by Chang et al. [23], the cis-elements of the CuZn–SOD gene promoter include a negative regulatory element (NRE) and positive regulatory element (PRE) that bind the transcription factors YY1 and Elk-1, respectively. These transcription factors are largely responsible for dynamic, coordinated changes in CuZn–SOD gene expression. Fig. 4 shows YY1 and Elk-1 EMSAs. ISO-treated hearts exhibited a marked (2.5-fold) increase in YY1 DNA binding and a 3-fold reduction in Elk-1 DNA binding, favoring suppression of CuZn–SOD gene transcription.


View this table:
[in this window]
[in a new window]

  		Table 2 Cardiac antioxidant enzyme activity after 7-day infusion

 

Figure 4
View larger version (54K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 YY1 and Elk-1 EMSAs and corresponding densitometry. ISO-treated hearts exhibited a 2.5-fold increase in YY1 DNA binding and a 3-fold reduction in Elk-1 DNA binding (*p<0.001, n=6 per group) (black arrowhead). Specificity of YY1 and Elk-1 proteins in the nuclear extracts was confirmed in supershift assays using YY1-or Elk-1-specific antibodies. Normal rabbit IgG served as a control.

 
2.4 ROS generation and CuZn–SOD downregulation occur via the β1-AR
The results of β-AR stimulation studies in isolated cardiomyocytes are shown in Fig. 5. ISO (10–7 M) exposure for 24 h increased cellular DCF fluorescence ~3-fold reflecting greater intracellular ROS (Fig. 5A). This was prevented by co-incubation with CGP, a β1-AR antagonist, but not by ICI, a β2-AR antagonist. In accordance with the in vivo studies, Northern blotting (Fig. 5B) revealed ISO-induced downregulation of CuZn–SOD gene expression in cardiomyocytes, an effect also dependent on the β1-AR as it was prevented by both CGP and nanomolar concentrations of metoprolol but not by ICI. Western blotting (Fig. 5C) confirmed that reduced CuZn–SOD protein paralleled mRNA downregulation and was mediated by the β1-AR. To further examine the mechanisms of CuZn–SOD downregulation, CuZn–SOD mRNA half-life following actinomycin D pulse was evaluated in both control- and ISO-treated myocytes (Fig. 5D); CuZn–SOD mRNA stability was similar in both groups. Moreover, nuclear run-on assays (Fig. 5E) revealed reduced nuclear CuZn–SOD mRNA levels in ISO-treated myocytes, confirming ISO-mediated transcriptional repression as suggested by the in vivo studies. Fig. 5F shows that ISO-treatment also significantly decreased cellular GSH and the GSH:GSSG ratio consistent with oxidant stress. Importantly, ISO-treated myocytes exhibited ~40% reduction in Trolox equivalents (Fig. 5G), indicating that diminished cellular antioxidant capacity contributed to the altered redox state and oxidative stress. Thus, taken together, these studies indicate that transcriptional repression of CuZn–SOD impacted both antioxidant defenses and oxidant stress, and that these effects occurred via the β1-AR. Notably, there were no changes in myocyte size with 24 h of ISO stimulation (vs. control, cell length: 113±25 vs. 110±18 µm, p=NS; cell width: 32±7 vs. 33±8 µm, p=NS), suggesting that the above effects were not dependent on cell hypertrophy.


Figure 5
View larger version (43K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 β-AR stimulation studies in rat cardiomyocytes. A, Reactive oxygen species (ROS) assessed by DCF fluorescence after 24 h of exposure to either no drug, ISO, ISO plus CGP, a β1-AR antagonist, or ISO plus ICI, a β2-AR antagonist. There was a β1-AR-dependent ~3-fold increase in myocyte ROS (n=8 per group, *p<0.01 vs. control). B, Northern blotting for CuZn–SOD from myocytes treated as in A, with an additional group co-incubated with 100 nM metoprolol, a predominantly β1-AR antagonist. There was β1-AR-mediated downregulation of CuZn–SOD gene expression. C, Western blotting for CuZn–SOD in similarly treated myocytes (n=8 per group) also revealed β1-AR-dependent attenuation of CuZn–SOD abundance (*p<0.001 vs. control). D, CuZn–SOD mRNA half-life in ISO-treated and control myocytes (n=5 per group) as evaluated by actinomycin D pulse followed by Northern blotting at the time points indicated. There was no change in CuZn–SOD mRNA stability in ISO-treated myocytes. E, CuZn–SOD gene transcription as evaluated by nuclear run-on assays revealed a significant decrease in nuclear CuZn–SOD mRNA levels following ISO-exposure (n=3 per group, *p<0.005) indicating transcriptional repression. GAPDH was used as an internal control and TOPO 2.1 as a vector control. F, Reduced and oxidized glutathione (GSH and GSSG) levels revealed significantly decreased GSH and GSH:GSSG ratio in ISO-treated myocytes indicative of oxidative stress (n=7 per group, *p<0.01 vs. control). G, Antioxidant capacity as evaluated by Trolox equivalents revealed a ~40% decrease in ISO-treated myocytes (n=5 per group, *p<0.01 vs. control).

 
2.5 Isolated CuZn–SOD suppression induces apoptosis in H9c2 cells
To determine the functional impact of isolated CuZn–SOD downregulation, we transfected CuZn–SOD siRNA into H9c2 cells (a rat cardiomyocyte cell line) (Fig. 6). Phase contrast microscopy (Fig. 6A) revealed that as compared to control (non-transfected) and cells transfected with non-specific (NS) siRNA, cells transfected with CuZn–SOD siRNA had reduced viability and cell number. Western blotting (Fig. 6B–C) revealed that a ~40–50% decrease in CuZn–SOD protein was effected by CuZn–SOD siRNA transfection, an amount similar to ISO-mediated effects in vivo. This was associated with increased cleavage of PARP, a downstream target of caspase-3 and marker of apoptosis (~60% decrease in uncleaved PARP, Fig. 6C). Together the data suggest that isolated loss of CuZn–SOD and attendant oxidative stress in cardiomyocytes can have important functional consequences, with increased apoptosis and reduced cell survival.


Figure 6
View larger version (43K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 A, Phase contrast images demonstrating reduced viability and cell number in H9c2 cells transfected with CuZn–SOD siRNA (72 h) as compared to both non-transfected cells (control) and cells transfected with non-specific (NS) siRNA. Representative Western blots (B) and densitometry (C) for CuZn–SOD and poly-ADP ribose polymerase (PARP), a caspase-3 substrate and apoptotic marker. CuZn–SOD siRNA transfection reduced CuZn–SOD protein by ~50% and this was accompanied by increased PARP cleavage (~60% decrease in uncleaved PARP), indicating apoptosis (n=4/group).

 

   3. Discussion
Top
 Abstract
 1. Methods
 2. Results
 3. Discussion
Acknowledgments
 References
 
This study demonstrates that sustained β-AR stimulation transcriptionally downregulates CuZn–SOD in myocardium, thereby reducing antioxidant capacity and contributing to β-AR-mediated oxidative stress, and that these effects occur via the β1-AR. Several lines of evidence established this conclusion. First, ISO stimulation for 7 days in vivo, at low doses that produced load-independent concentric hypertrophy without chamber dilatation, robustly increased oxidative stress in the heart. Second, ISO selectively depressed CuZn–SOD at both transcriptional and translational levels with reduced gene expression, protein abundance, and enzyme activity, but without influencing Mn–SOD, GPX, or catalase. Third, ISO impacted YY1 and Elk-1, the major transcriptional regulators of inducible CuZn–SOD gene expression, in divergent manners, by augmenting YY1 but reducing Elk-1 DNA binding. This response would favor suppression of CuZn–SOD gene expression. Fourth, ISO-induced oxidative stress, CuZn–SOD downregulation, and loss of antioxidant capacity were recapitulated in isolated cardiomyocytes and localized to the β1-AR, the primary receptor subtype responsible for the toxic effects of catecholamines. Moreover, diminished CuZn–SOD mRNA levels occurred in the nucleus, confirming transcriptional repression, and were not related to changes in CuZn–SOD mRNA stability. Fifth, selective CuZn–SOD suppression in H9c2 cardiomyocytes induced apoptosis and reduced cell viability suggesting that CuZn–SOD downregulation is of functional importance. Taken together, the data support CuZn–SOD downregulation as a novel, heretofore-unrecognized mechanism contributing to β-AR-mediated oxidative stress in the heart.

3.1 Cardiac β-AR activation and ROS
Under normal conditions, β-AR-induced ROS generation in myocardium may serve to dynamically modulate the ensuing mechanical response [6]. However, sustained β-AR-activation and attendant oxidative stress, such as occurs in HF [1,9], can have long-term deleterious effects. Zhang et al. [7] recently demonstrated that whereas β-AR-mediated ROS generation acutely stimulated mitogen-activated protein kinase signaling, prolonged stimulation induced hypertrophy and ROS-dependent myocardial fibrosis. Similarly, chronic norepinephrine administration at clinically-relevant doses induced myocardial apoptosis that was prevented by antioxidants [8]. Our study extends these findings by demonstrating that sustained low-level β-AR stimulation in vivo (0.6 mg/kg/day ISO- 5-fold lower than Zhang et al. [7] and 40-fold lower than ISO-cardiomyopathy models [24]) also induces myocardial oxidative stress, with a 2- to 3-fold increase in unbound and protein-bound HNE and MDA (Fig. 2). Importantly, this occurred without changes in BP or an increase in wall stress (Table 1) and without the myocardial toxicity or chamber dilatation characteristic of high-dose ISO models, suggesting load-independent effects that more closely approximate β-AR-induced responses during the early stages of HF.

3.2 Cardiac β-AR activation and antioxidant enzymes
β-AR stimulation is well known to increase myocardial oxygen consumption [10,12,25]. As enhanced mitochondrial O2Formula production necessarily accompanies any increase in oxygen consumption (1–2% of oxygen reduced during mitochondrial respiration normally forms O2Formula due to electron leakage from the respiratory chain [11]), increased mitochondrial ROS due to augmented oxygen consumption is a commonly invoked mechanism for β-AR-mediated oxidative stress [10,12]. Although the translation of increased ROS generation into oxidative stress also depends on the attendant antioxidant capacity, the relationship between the β-AR and antioxidant enzymes in the heart has heretofore received little attention. Using both intact animals and isolated myocytes, we demonstrate that prolonged β-AR stimulation of the heart increases oxidative stress, alters redox state, and depresses total antioxidant capacity (Figs. 2 and 5Go). Reduced antioxidant capacity was related to selective CuZn–SOD downregulation, without changes in Mn–SOD, catalase, or GPX (Figs. 3 and 5Go and Table 2). This selective loss of CuZn–SOD function was localized to the β1-AR, and fundamentally occurred at the transcriptional level, as evidenced by reduced total and nuclear CuZn–SOD mRNA levels, preserved CuZn–SOD mRNA stability, augmented YY1 DNA binding, and diminished Elk-1 DNA binding (Figs. 4 and 5Go). These findings have several important implications.

First, β-AR-mediated oxidative stress in the heart appears multifactorial, related not only to increased ROS generation but also to reduced capacity to detoxify O2Formula via CuZn–SOD. Indeed, prior work has demonstrated increased tissue superoxide in rat hearts chronically stimulated with ISO [7]. Although the absolute reduction in CuZn–SOD abundance/activity (~30–40%) is not extreme, it is important to consider that this occurs on the backdrop of β-AR-mediated augmentation of mitochondrial O2Formula production, thus magnifying its impact on tissue oxidative stress. Furthermore, as the functional impact of oxidative stress is cumulative over time (due to, for example, ongoing lipid peroxidation and the accumulation of protein–aldehyde adducts [19]), even moderate reductions in CuZn–SOD can result in significant cellular alterations over an extended period such as during chronic HF. This notion is supported by our findings that sustained, selective CuZn–SOD suppression in H9c2 cells (to levels achieved in vivo with ISO administration) increased apoptosis (Fig. 6), an important ROS-dependent effect of β-AR stimulation in myocytes [8,26]. Thus, these results indicate that even moderate CuZn–SOD reductions can have important functional effects.

Second, although sustained β-AR activation would be anticipated to increase mitochondrial O2Formula generation, there was no accompanying compensatory increase in mitochondrial Mn–SOD. This suggests that cardiac pathologies characterized by long-term β-AR activation would be quite susceptible to oxidative mitochondrial DNA damage and the activation of mitochondrial apoptotic pathways. Indeed, these phenomena are characteristic derangements in the failing heart [4,27]. Third, both the CuZn–SOD downregulation and oxidative stress induced by sustained β-AR activation are β1-AR-specific (Fig. 5). These results are consistent with prior work demonstrating that the β1-AR subtype is primarily responsible for the deleterious consequences of catecholamines [1]. β-AR-dependent myocyte apoptosis, a ROS-dependent phenomenon [26], is selectively coupled to the β1-AR, whereas β2-AR stimulation is anti-apoptotic [3]. Similarly, whereas transgenic mice with relatively low levels of cardiac-specific β1-AR overexpression develop dilated cardiomyopathy [28], similar levels of β2-AR overexpression leads to enhanced cardiac function and no increase in mortality [29].

3.3 Cardiac β-AR activation and CuZn–SOD gene expression
While CuZn–SOD is ubiquitous and constitutively expressed, CuZn–SOD mRNA expression is also dynamically regulated and can be either induced or repressed depending on the particular stimulus. An elegant analysis of the rat CuZn–SOD promoter has shown two positive regulatory elements (PRE) encompassing nucleotides –576 to –412 and –305 to –55, and one negative regulatory element (NRE) from –412 to –305 bp [23]. The –576 to –412 bp PRE contains specific binding sites for the ternary complex factor Elk-1, which enhances CuZn–SOD transcription during stress. The NRE is bound by the zinc finger transcription factor YY1, a powerful repressor of CuZn–SOD gene transcription. Elk-1 and YY1 are the primary transcriptional regulators of dynamic changes in rat CuZn–SOD expression [23]. The importance of CuZn–SOD induction (as opposed to basal expression) is underscored by studies showing that the deleterious effects of CuZn–SOD deficiency in vivo manifest during the response to injury rather than during basal, unstimulated conditions. CuZn–SOD null mice exhibit no evidence of myocardial oxidative stress or contractile dysfunction at baseline [30]. However, they are more susceptible to reperfusion injury following ischemia, with greater post-ischemic contractile dysfunction, infarct size, and ROS formation than wild-type mice [30]. Thus, dynamic changes in CuZn–SOD expression may be of particular importance during acute and/or chronic stress as part of an adaptive cytoprotective response. A key finding of our study is that sustained β-AR stimulation markedly reduced Elk-1 DNA binding while simultaneously increasing YY1 DNA binding (Fig. 4), a profile favoring CuZn–SOD gene repression [23]. Furthermore, this pattern of activation would diminish the inducible CuZn–SOD response in the heart, presumably increasing susceptibility to (patho) physiologic stressors.

To our knowledge, this is the first demonstration of a link between the β-AR and Elk-1 and YY1 in the heart. However, prior work has demonstrated that β-AR stimulation increases Elk-1 phosphorylation in neuronal cells [31]. Additionally, YY1 abundance and activity are increased in human HF, a state characterized by chronic β-AR activity, and suppresses {alpha}-myosin heavy chain gene expression (a component of the "fetal" gene program) [32]. Thus, it follows that augmented YY1 activity may be an important mechanism underlying the pathologic response to sustained β-AR stimulation, and additional evaluation of this signaling pathway is warranted.

In summary, we have shown that sustained, low-level β-AR stimulation reduces antioxidant capacity in cardiomyocytes and downregulates CuZn–SOD gene expression in the heart via the β1-AR, highlighting a novel mechanism of β-AR-mediated oxidative stress. This effect was selective for CuZn–SOD as there was no change in Mn–SOD, GPX, or catalase levels, and was related, at least in part, to transcriptional repression due to augmented YY1 and reduced Elk-1 DNA binding activity. These results suggest that reduced antioxidant capacity contributes importantly to β-AR-mediated oxidative stress, and that analogous changes in the CuZn–SOD axis may occur during the development and/or progression of pathologic states that are characterized by chronic β-AR activation.


   Acknowledgments
Top
 Abstract
 1. Methods
 2. Results
 3. Discussion
 Acknowledgments
References
 
This work was supported by VA Merit Awards, the Jewish Hospital Foundation, and NIH grants ES11860, HL078825, HL65618, and HL68020.


   Notes
 
Time for primary review 29 days


   References
Top
 Abstract
 1. Methods
 2. Results
 3. Discussion
 Acknowledgments
 References
 

  1. Bristow M.R. β-Adrenergic receptor blockade in chronic heart failure. Circulation (2000) 101:558–569.[Free Full Text]
  2. Mann D.L., Kent R.L., Parsons B., Cooper G. IV. Adrenergic effects on the biology of the adult mammalian cardiocyte. Circulation (1992) 85:790–804.[Abstract/Free Full Text]
  3. Communal C., Singh K., Sawyer D.B., Colucci W.S. Opposing effects of β1- and β2-adrenergic receptors on cardiac myocyte apoptosis: role of a pertussis toxin-sensitive G protein. Circulation (1999) 100:2210–2212.[Abstract/Free Full Text]
  4. Prabhu S.D., Wang G., Luo J., Gu Y., Ping P., Chandrasekar B. β-Adrenergic receptor blockade modulates Bcl-XS expression and reduces apoptosis in failing myocardium. J Mol Cell Cardiol (2003) 35:483–493.[CrossRef][Web of Science][Medline]
  5. Prabhu S.D., Chandrasekar B., Murray D.R., Freeman G.L. β-Adrenergic blockade in developing heart failure: effects on myocardial inflammatory cytokines, nitric oxide, and remodeling. Circulation (2000) 101:2103–2109.[Abstract/Free Full Text]
  6. Mak S., Newton G.E. Vitamin C augments the inotropic response to dobutamine in humans with normal left ventricular function. Circulation (2001) 103:826–830.[Abstract/Free Full Text]
  7. Zhang G.X., Kimura S., Nishiyama A., Shokoji T., Rahman M., Yao L., et al. Cardiac oxidative stress in acute and chronic isoproterenol-infused rats. Cardiovasc Res (2005) 65:230–238.[Abstract/Free Full Text]
  8. Qin F., Rounds N.K., Mao W., Kawai K., Liang C.S. Antioxidant vitamins prevent cardiomyocyte apoptosis produced by norepinephrine infusion in ferrets. Cardiovasc Res (2001) 51:736–748.[Abstract/Free Full Text]
  9. Sawyer D.B., Siwik D.A., Xiao L., Pimentel D.R., Singh K., Colucci W.S. Role of oxidative stress in myocardial hypertrophy and failure. J Mol Cell Cardiol (2002) 34:379–388.[CrossRef][Web of Science][Medline]
  10. Opie L.H., Thandroyen F.T., Muller C., Bricknell O.L. Adrenaline-induced "oxygen-wastage" and enzyme release from working rat heart. Effects of calcium antagonism, β-blockade, nicotinic acid and coronary artery ligation. J Mol Cell Cardiol (1979) 11:1073–1094.[CrossRef][Web of Science][Medline]
  11. Cadenas E., Davies K.J. Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med (2000) 29:222–230.[CrossRef][Web of Science][Medline]
  12. Givertz M.M., Sawyer D.B., Colucci W.S. Antioxidants and myocardial contractility: illuminating the "Dark Side" of beta-adrenergic receptor activation? Circulation (2001) 103:782–783.[Free Full Text]
  13. Murray D.R., Prabhu S.D., Chandrasekar B. Chronic β-adrenergic stimulation induces myocardial proinflammatory cytokine expression. Circulation (2000) 101:2338–2341.[Abstract/Free Full Text]
  14. Syed F., Diwan A., Hahn H.S. Murine echocardiography: a practical approach for phenotyping genetically manipulated and surgically modeled mice. J Am Soc Echocardiogr (2005) 18:982–990.[CrossRef][Web of Science][Medline]
  15. Luo J., Xuan Y.-T., Gu Y., Prabhu S.D. Prolonged oxidative stress inverts the cardiac force-frequency relation: role of altered calcium handling and myofilament calcium responsiveness. J Mol Cell Cardiol (2006) 40:64–75.[CrossRef][Web of Science][Medline]
  16. Chandrasekar B., Colston J.T., Geimer J., Cortez D., Freeman G.L. Induction of nuclear factor-{kappa}B but not {kappa}B-responsive cytokine expression during myocardial reperfusion injury after neutropenia. Free Radic Biol Med (2000) 28:1579–1588.[CrossRef][Web of Science][Medline]
  17. Dieterich S., Bieligk U., Beulich K., Hasenfuss G., Prestle J. Gene expression of antioxidative enzymes in the human heart: increased expression of catalase in the end-stage failing heart. Circulation (2000) 101:33–39.[Abstract/Free Full Text]
  18. Li T., Danelisen I., Bello-Klein A., Singal P.K. Effects of probucol on changes of antioxidant enzymes in adriamycin-induced cardiomyopathy in rats. Cardiovasc Res (2000) 46:523–530.[Abstract/Free Full Text]
  19. Srivastava S., Chandrasekar B., Bhatnagar A., Prabhu S.D. Lipid-peroxidation derived aldehydes and oxidative stress in the failing heart: role of aldose reductase. Am J Physiol Heart Circ Physiol (2002) 283:H2612–H2619.[Abstract/Free Full Text]
  20. Uchida K., Itakura K., Kawakishi S., Hiai H., Toyokuni S., Stadtman E.R. Characterization of epitopes recognized by 4-hydroxy-2-nonenal specific antibodies. Arch Biochem Biophys (1995) 324:241–248.[CrossRef][Web of Science][Medline]
  21. Chandrasekar B., Colston J.T., Freeman G.L. Induction of proinflammatory cytokine and antioxidant enzyme gene expression following brief myocardial ischaemia. Clin Exp Immunol (1997) 108:346–351.[CrossRef][Web of Science][Medline]
  22. Chandrasekar B., Marelli-Berg F.M., Tone M., Bysani S., Prabhu S.D., Murray D.R. Isoproterenol induces interleukin-18 expression via β2-AR, PI3 kinase, Akt, I{kappa}B kinase, and nuclear factor-{kappa}B signaling. Biochem Biophys Res Commun (2004) 319:304–311.[CrossRef][Web of Science][Medline]
  23. Chang M.S., Yoo H.Y., Rho H.M. Positive and negative regulatory elements in the upstream region of the rat Cu/Zn superoxide dismutase gene. Biochem J (1999) 339:335–341.[CrossRef][Web of Science][Medline]
  24. Teerlink J.R., Pfeffer J.M., Pfeffer M.A. Progressive ventricular remodeling in response to diffuse isoproterenol-induced myocardial necrosis in rats. Circ Res (1994) 75:105–113.[Abstract/Free Full Text]
  25. Prabhu S.D., Freeman G.L. Altered LV inotropic reserve and mechanoenergetics early in the development of heart failure. Am J Physiol (2000) 278:H698–H705.[Web of Science]
  26. Remondino A., Kwon S.H., Communal C., Pimentel D.R., Sawyer D.B., Singh K., et al. β-adrenergic receptor-stimulated apoptosis in cardiac myocytes is mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of the mitochondrial pathway. Circ Res (2003) 92:136–138.[Abstract/Free Full Text]
  27. Ide T., Tsutsui H., Hayashidani S., Kang D., Suematsu N., Nakamura K., et al. Mitochondrial DNA damage and dysfunction associated with oxidative stress in failing hearts after myocardial infarction. Circ Res (2001) 88:529–535.[Abstract/Free Full Text]
  28. Engelhardt S., Hein L., Wiesmann F., Lohse M.J. Progressive hypertrophy and heart failure in β1-adrenergic receptor transgenic mice. Proc Natl Acad Sci U S A (1999) 96:7059–7064.[Abstract/Free Full Text]
  29. Liggett S.B., Tepe N.M., Lorenz J.N., Canning A.M., Jantz T.D., Mitarai S., et al. Early and delayed consequences of β2-adrenergic receptor overexpression in mouse hearts: critical role for expression level. Circulation (2000) 101:1707–1714.[Abstract/Free Full Text]
  30. Yoshida T., Maulik N., Engelman R.M., Ho Y.S., Das D.K. Targeted disruption of the mouse SOD I gene makes the hearts vulnerable to ischemic reperfusion injury. Circ Res (2000) 86:264–269.[Abstract/Free Full Text]
  31. Storm S.M., Khawaja XZ. Probing for drug-induced multiplex signal transduction pathways using high resolution two-dimensional gel electrophoresis: application to beta-adrenoceptor stimulation in the rat C6 glioma cell. Brain Res Mol Brain Res (1999) 71:50–60.[CrossRef][Medline]
  32. Sucharov C.C., Mariner P., Long C., Bristow M., Leinwand L. Yin Yang 1 is increased in human heart failure and represses the activity of the human alpha-myosin heavy chain promoter. J Biol Chem (2003) 278:31233–31239.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 CirculationHome page
T. Hamid, Y. Gu, R. V. Ortines, C. Bhattacharya, G. Wang, Y.-T. Xuan, and S. D. Prabhu
Divergent Tumor Necrosis Factor Receptor-Related Remodeling Responses in Heart Failure: Role of Nuclear Factor-{kappa}B and Inflammatory Activation
Circulation, March 17, 2009; 119(10): 1386 - 1397.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Tapryal, C. Mukhopadhyay, D. Das, P. L. Fox, and C. K. Mukhopadhyay
Reactive Oxygen Species Regulate Ceruloplasmin by a Novel mRNA Decay Mechanism Involving Its 3'-Untranslated Region: IMPLICATIONS IN NEURODEGENERATIVE DISEASES
J. Biol. Chem., January 16, 2009; 284(3): 1873 - 1883.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
G. K. Soukhova-O'Hare, R. V. Ortines, Y. Gu, A. D. Nozdrachev, S. D. Prabhu, and D. Gozal
Postnatal Intermittent Hypoxia and Developmental Programming of Hypertension in Spontaneously Hypertensive Rats: The Role of Reactive Oxygen Species and L-Ca2+ Channels
Hypertension, July 1, 2008; 52(1): 156 - 162.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
I. Murtaza, H.-X. Wang, X. Feng, N. Alenina, M. Bader, B. S. Prabhakar, and P.-F. Li
Down-regulation of Catalase and Oxidative Modification of Protein Kinase CK2 Lead to the Failure of Apoptosis Repressor with Caspase Recruitment Domain to Inhibit Cardiomyocyte Hypertrophy
J. Biol. Chem., March 7, 2008; 283(10): 5996 - 6004.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. Luo, B. G. Hill, Y. Gu, J. Cai, S. Srivastava, A. Bhatnagar, and S. D. Prabhu
Mechanisms of acrolein-induced myocardial dysfunction: implications for environmental and endogenous aldehyde exposure
Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3673 - H3684.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Srivastava, S.
Right arrow Articles by Prabhu, S. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Srivastava, S.
Right arrow Articles by Prabhu, S. D.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?