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Am J Physiol Heart Circ Physiol 289: H1960-H1967, 2005. First published July 1, 2005; doi:10.1152/ajpheart.00328.2005
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Proteasome degradation of GRK2 during ischemia and ventricular tachyarrhythmias in a canine model of myocardial infarction

Xichun Yu,1,2,* Shijun Huang,1,2,* Eugene Patterson,2 Marion W. Garrett,1 Kenneth M. Kaufman,3 Jordan P. Metcalf,4 Meili Zhu,3 Samuel T. Dunn,5 and David C. Kem1,2

1Endocrinology, 2Cardiac Arrhythmia Research Institute, and 4Pulmonary and Critical Care, Department of Medicine, and 5Department of Pathology, University of Oklahoma Health Sciences Center and Veterans Affairs Medical Center, Oklahoma City; and 3Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma

Submitted 4 April 2005 ; accepted in final form 28 June 2005


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Arrhythmia-prone subepicardial border zone (EBZ) tissue demonstrates decreased G protein receptor kinase 2 (GRK2) activity and increased sensitivity to isoproterenol 6–24 h after coronary artery ligation (CAL) in the dog. With the use of a semiquantitative immunofluorescence technique, the relative fluorescence intensity (RF) of GRK2 in EBZ decreased to 24% of that in a remote site (RS) (P < 0.01, n = 30 cells from 3 dogs), whereas GRK5 RF did not change. Confocal studies of cardiac tissue from transgenic mice overexpressing GRK2 validated the use of a semilogarithmic relationship between RF and GRK2 activity. As shown with the use of quantitative real-time RT-PCR, both GRK2 and GRK5 mRNA were not decreased at 24 h in EBZ (n = 6 dogs) relative to RS control, indicating that the decrease of GRK2 in the EBZ is likely due to posttranscriptional degradation following CAL. Pretreatment of six dogs with the selective proteasome inhibitor bortezomib provided 100% (EBZ) and 50% (infarct) protection against loss of GRK2 at 24 h. There was an absence of rapid (>300 beats/min) and very rapid (>360 beats/min) ventricular triplets that are highly predictive of sudden cardiac death during ECG monitoring in the bortezomib-pretreated animals in contrast to nonpretreated infarcted animals. We have demonstrated that the dramatic decrease in GRK2 in cardiac ischemic tissue can be largely blocked by prior proteasome blockade and that this is associated with significant cardioprotection against malignant ventricular tachyarrhythmias.

{beta}-adrenergic receptor; G protein receptor kinase 2; sudden cardiac death; ventricular arrhythmias; immunofluorescence microscopy

G PROTEIN-COUPLED RECEPTOR KINASE 2 (GRK2) regulates {beta}-adrenergic receptor ({beta}-AR) sensitivity by phosphorylation of agonist-occupied receptors (6, 15, 27). Upregulation of GRK2 expression with its associated decrease in sensitivity to {beta}-agonists has been observed in several pathological processes, including congestive heart failure (31), acute global ischemia (32), and myocardial hypertrophy (7). This decreased cardiac responsiveness to {beta}-AR agonists also occurs in mice with transgenic overexpression of either GRK2 (14) or its homolog, GRK5 (30). Conversely, when GRK2 activity is suppressed in transgenic mice overexpressing a peptide inhibitor of GRK2, the heart becomes increasingly responsive to {beta}-AR agonists (14).

Sudden cardiac death (SCD), the most common cause of death in the Western world, is frequently related to recurrent ventricular arrhythmias arising from a thin layer or strand of surviving myocardium in close proximity to the infarcted tissue (9, 18, 22, 23). Two peaks of SCD are observed in dogs: one occurs within 30 min, and the second is observed in a period from 6–24 h after coronary artery ligation (CAL). In the later period, the SCD is generally characterized by episodic tachyarrhythmias subsequently triggering a sustained ventricular tachycardia and ventricular fibrillation (10, 19, 21–23). In the dog, the late arrhythmias represent primarily sustained reentrant tachycardia from the subepicardial border zone tissue (EBZ). We have reported a marked decrease in total GRK2 activity in whole slices of canine EBZ tissue obtained during the subacute period 6–24 period after infarction, resulting in a loss of the ability of the EBZ tissue to become desensitized to {beta}-adrenergic stimulation (33, 34). This heightened sensitivity predisposes this tissue to {beta}-agonist-induced ventricular tachyarrhythmias and provides a biochemical rationale for the effectiveness of {beta}-blockers in preventing SCD. Further study of the mechanisms by which GRK2 is rapidly decreased in the EBZ tissue is of paramount importance. The present study uses semiquantitative immunofluorescence microscopy of single cells to document this loss of GRK2 and thereby minimizes the potentially confounding contribution of noncardiomyocyte cell populations in the thin rim of ischemic EBZ tissue in the 24-h ligated canine model of SCD. It examines the mechanism(s) by which GRK2 is differentially decreased by ischemia and demonstrates how this process can be reversed by specific blockade of a candidate proteolytic system. The favorable outcome of this intervention clearly demonstrates the cardioprotective effects of GRK2 from malignant tachyarrhythmias that lead to SCD in this model.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ischemic dog model. Seventeen (11 control and 6 bortezomib treated) male dogs (30–45 lb) had a two-stage ligation of the left anterior descending coronary artery (LAD) below the first branch, as described by Harris and colleagues (10, 11), during pentobarbital sodium (30 mg/kg iv) anesthesia. A two-stage ligation of the coronary artery (a stenosis formed by ligating against and then removing a 20-gauge hypodermic needle, followed 15 min later by complete ligation of the artery) prevents or suppresses the initial lethal ventricular arrhythmias observed after abrupt coronary artery occlusion (10, 11). The dogs were given 0.4 mg/kg nalbuphine for postoperative analgesia and were then allowed to awaken from anesthesia. A 24-h ambulatory ECG was obtained from leads on the thoracic surface. At 24 h after CAL, dogs were reanesthetized, a thoracotomy was performed, and the heart was removed and immersed in and perfused with ice-cold Tyrode buffer via the left main coronary artery. The outer 1.0 mm of EBZ tissue overlying the infarct was shaved by scalpel dissection. A cross section of the left ventricle was stained with triphenyltetrazolium chloride (TTZ; 0.1%) to determine the degree of viable and nonviable tissue (Fig. 1). Remote site (RS) control tissue was obtained from a superior portion of the lateral left ventricle, perfused via the left circumflex coronary artery. The infarct sample was obtained from the middle of the region and was characterized by darkened, reddish myocardium. The endocardial surface was also shaved, but because of the thin nature of this tissue, it contained a significant amount of infarct tissue (Fig. 1). The tissues were rapidly (within 10 min) divided into small aliquots and stored in liquid N2. RS and EBZ-equivalent tissues were obtained from three similarly anesthetized control dogs that had not been previously operated on for LAD ligation. However, these hearts were removed within 10 min of administering the intravenous pentobarbital anesthesia to minimize stress-induced catecholamines. The animal protocols were approved by the Institutional Animal Care and Use Committee and conform to the Helsinki International Guidelines for animal experimentation.



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  		Fig. 1. A cross section of the ventricle stained with triphenyltetrazolium chloride and histological studies. The control remote site tissue (A) was taken from a higher plane 2.5–3.0 cm above the margin of the infarct. This region demonstrates normal ventricular histology. The subepicardial border zone tissue (B) was shaved from the epicardial surface of the infarct and is 1.5–2.0 mm thick. This tissue shows some patchy changes and mild leukocyte infiltration typical of this region. The infarct (C) demonstrates typical cell dissolution and leukocyte infiltration. The endocardial tissue (D) was shaved from the endocardial surface of the infarct and reveals a thin rim of subendocardial tissue (arrows) and significant infarct tissue.

 
Bortezomib treatment. Six dogs were injected through a forepaw vein with 0.0875 mg/kg bortezomib (Velcade; Millennium Pharmaceuticals, Cambridge, MA). The other 11 dogs were injected with saline (control). The first intravenous injection was given 1 h before LAD ligation and repeated at 5 h post-LAD ligation (total dosage 0.175 mg/kg). This dosage for dogs was determined after consultation with the pharmacology division (Millennium Pharmaceuticals, unpublished data). The elimination half-life of this highly selective proteasome inhibitor ranges from 5 to 15 h in different species (mouse, rat, monkey, and human) because of tight binding and was therefore estimated at 6–8 h in dogs. The maximal tolerated dosage for dogs was 0.2 mg/kg. This dosage was therefore reduced to 0.175 mg/kg for these studies to avoid side effects. In monkeys, dosages of >3 mg/kg iv are associated with progressive hypotension and thrombocytopenia with subsequent death. There are limited data available for dogs, but these studies (Millennium Pharmaceuticals, unpublished data) suggest a similar response to these dosages. No significant side effects of the injection dosage (i.e., hypotension or unusual tachycardia other than that associated with the ischemia) were observed in our study. Mild diarrhea was observed in one animal. Blood samples for measurement of proteasome activity were obtained at 1 h before LAD ligation and 0, 3, 6, and 24 h after LAD ligation.

Proteasome activity. Packed whole blood lysate proteasome activity was measured using a modification of the technique as described (17). Heparinized blood was diluted 1:1 (vol/vol) with saline and centrifuged at 500 g for 30 min at room temperature. The blood cell pellet was washed with cold PBS, centrifuged at 400 g for 5 min at 4°C, resuspended in cold PBS, and centrifuged at 6,600 g for 10 min at 4°C. The cell pellet was then frozen at –80°C for 20S proteasome activity assay. Blood cells were lysed with EDTA (5 mM; pH 8.0) for 1 h and centrifuged at 6,600 g for 10 min at 4°C. The resultant supernatant was then used in the assay. We used 0.4 mM Suc-Ala-Ala-Phe-7-amino-4-methylcoumarin (AMC; Bachem, Torrance, CA) as substrate. The assay was run with and without adding bortezomib (final concentration 10 µM). Bortezomib reduces overall proteolysis by specifically inhibiting proteasome peptidase activity. The time course for fluorescence was measured with the Victor multilabel counter (Perkin Elmer Life Sciences, Boston, MA) at time 0 and every 5 min for 90 min to estimate the amount of AMC liberated (excitation 355 nm, emission 460 nm). Slopes were calculated with Microsoft Excel. Final results were expressed as fluorescence units per 100 µg of protein per minute.

Immunoblot analysis. GRK2 immunodetection was performed on detergent-solubilized extracts after immunoprecipitation. Hearts were homogenized in ice-cold RIPA buffer (50 mM Tris, pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 5 mM EGTA, 10 mM sodium pyrophosphate, and 1 mM PMSF). GRK2 was immunoprecipitated from 1 ml of extract (250 µg) with 1.2 µg of polyclonal anti-GRK2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and 20 µl of a 50% slurry of protein A/G-agarose conjugate agitated overnight at 4°C. Immune complexes were washed three times in ice-cold RIPA, resuspended in 25 µl of loading buffer, heated at 95°C for 5 min, and then electrophoresed and transferred to nitrocellulose. The GRK2 protein was detected using the monoclonal anti-GRK2 antibody (Upstate Biotechnology, Lake Placid, NY) and enhanced chemiluminescence (ECL; Amersham). Quantification was performed by densitometric scanning and using Image QuaNT software (Molecular Dynamics, Sunnyvale, CA).

Confocal microscopy. For immunolocalization, 1.5-mm blocks of canine ventricular myocardium were frozen in OCT and sectioned at 10-µm increments. Sections were fixed with acetone at –20°C for 10 min, blocked in diluted normal goat serum for 20 min, and incubated with GRK isoform-specific antibodies as listed in Immunoblot analysis (Santa Cruz Biotechnology) in blocking buffer for 1 h, followed by incubation with Alexa 488-labeled goat anti-rabbit IgG (Molecular Probes, Eugene, OR) for 1 h. Control sections lacking primary antibody or treated with peptide preabsorbed antibody were included in each batch of slides and showed no significant staining. Slides were mounted with Slowfade mounting medium.

Immunofluorescence quantification. Tissue sections were analyzed using a Leica TCS NT confocal microscope. Raster lines were placed longitudinally in ventricular cells at x40 magnification, and relative fluorescence intensity (RF) was measured using the Leica confocal software. Background fluorescence was subtracted, and RF was generated as the mean (SD) for each raster line. These were averaged and provided an estimate of RF and cell-to-cell variability. Confocal RF studies were also performed on frozen sections of cardiac tissue obtained from the left ventricles of transgenic mice with a 20- and 3-fold overexpression of GRK2 over control (2) to develop a standard curve. These mice were obtained from Dr. Walter Koch, Jefferson University, Philadelphia, PA.

Real-time RT-PCR. GRK2 and GRK5 mRNA expressions were determined using real-time quantitative RT-PCR. Total RNA was extracted from 30 mg of frozen tissue using the RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Real-time RT-PCR was performed with the SuperScript First-Strand synthesis system (Invitrogen, Carlsbad, CA) and the SYBR Green PCR kit (Applied Biosystems, Foster City, CA) on an ABI PRISM 7700 sequence detector (Applied Biosystems). The thermal cycling conditions comprised 10 min of polymerase activation at 95°C and 40 cycles at 95°C for 15 s, 60°C for 60 s, 95°C for 15 s, 60°C for 20 s, and 95°C for 15 s with a ramp time of 20 min. Each gene assay includes triplicates of the cDNA reaction, a no-template control reaction, and a RNA control reaction. Gene expressions of GRK2 and GRK5 were determined as the amounts of their mRNAs relative to mRNA for GAPDH by using the standard curve method described in the ABI 7700 sequence detector User Bulletin 2. The primer sequences for GRK2 were forward, 5'-ACCAGGAACTCTACCGCAACTTT-3', and reverse, 5'-TTTTCTTGCGGGCCTCCATT-3'. The primer sequences for GRK5 were forward, 5'-GGATGTTGGACCCTCCCTTCATT-3', and reverse, 5'-ACGCCTTTCACGGTGGAGAA-3'. The primer sequences for GAPDH were forward, 5'-CAGTGACACCCACTCTTCCA-3', and reverse, 5'-CCGGTTGCTGTAGCCAAATT-3'.

ECG analysis. Full-disclosure ECG records were obtained at 10 mm/s for the entire 2- to 24-h postocclusion period. All measurements and calculations were made by direct visual observation and manual counting.

Statistics. Normalized immunoblot and real-time RT-PCR data were analyzed using a two-tailed nonparametric sign test. Other data are expressed as means ± SE. Normally distributed data were examined using one-way ANOVA. Bonferroni's multiple comparison test was used for posttest analysis. Significance was ascribed to P values <0.05.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Each of the dogs, after the two-stage CAL, developed distinct Q waves on the anterior ECG leads. The cardiac tissue demonstrated a 1.0- to 1.5-mm surviving layer of myocardium on the epicardial surfaces of the infarct and a much thinner (0.2–0.5 mm) layer on the endocardial surface (Fig. 1). This was confirmed by obtaining a TTZ vital stain of a strip of tissue taken through the center of the infarct. Histological examination of the control RS, ischemic EBZ, and infarct tissues (Fig. 1) demonstrated typical changes expected 24 h after a transmural infarct.

To validate the immunofluorescence technique for analysis of single cells, we performed several studies on cardiac tissue obtained from three control dogs. The sections were examined with both bright-field and fluorescence microscopy and demonstrated typical morphology, including the Z bands where the T tubules and their adrenergic signal transduction apparatus are typically concentrated (Fig. 2, A and B). Analysis of the bright-field and GRK2 immunofluorescence using a longitudinal raster line demonstrated a consistent near alignment of the greatest fluorescence intensity at or adjacent to the Z bands of the ventricular tissue (Fig. 2C). This fluorescence was not present in control tissues stained with peptide preabsorbed antibody or when the primary antibody was omitted (Fig. 2, D–F).



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  		Fig. 2. G protein receptor kinase 2 (GRK2) immunofluorescence of a normal dog heart. A: bright-field x100 image of section. B: GRK2 immunofluorescence of same section. C: when relative GRK2 fluorescence intensity (RF) was measured along the raster line, peak RF (green) nearly coincided with the nadir (Z band) of the bright-field image visual intensity (red). D: negative control (without primary antibody) bright-field image. E: negative control for GRK2 immunofluorescence. F: negative control RF and bright-field image visual intensity demonstrates no congruity of the Z-band region with background fluorescence.

 
To determine whether the GRK2 immunofluorescence techniques were applicable for semiquantitative estimates of GRK2 expression and activity, we used two different transgenic mouse models (n = 3/group) with either 3- or 20-fold GRK2 overexpression, as measured by phosphorylation of rhodopsin from dark-adapted bovine retina (5). GRK2 RF in ventricular myocytes was obtained from 10 longitudinal cells from each animal. These data were pooled for each animal group and are shown in Fig. 3A. A highly significant relationship was observed with a semilogarithmic plot of GRK2 activity and RF.



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  		Fig. 3. A: to validate use of quantitative immunofluorescence for estimation of GRK2 expression and activity, confocal microscopy studies were performed on cardiac tissues from transgenic mice exhibiting overexpressing GRK2 activity 3 and 20 times greater than that of control animals (measured by dark-adapted rhodopsin phosphorylation). When RF (mean ± SE) is plotted as a semilogarithmic function of GRK2 activity, there is a linear relationship of RF to GRK2 activity for the respective transgenic models. B: immunofluorescence staining for GRK2 (top) and GRK5 (bottom) in frozen sections taken from the remote site (RS; left) and ischemic subepicardial border zone (EBZ; right) tissue of the 24-h left anterior descending coronary artery (LAD)-ligated dog. C and D: RF values for GRK2 and GRK5 staining respectively, in EBZ and RS tissue. There was a >50% decrease in GRK2 RF in the EBZ compared with RS tissue (*P < 0.01, n = 30 cells from 3 dogs), whereas no significant change in GRK5 RF was found between EBZ and RS tissue [P > 0.05, NS (not significant)]. The difference in RF values for GRK2 and GRK5 are dependent on staining conditions rather than relative expression, because different antibodies were used for each. All specimens for the specific stain were performed at the same time for internal comparison purposes.

 
We used the same technique to examine the RF of the EBZ and RS tissues from our 24-h infarcted dogs. These data, shown in Fig. 3, B and C, demonstrate a significant decrease in GRK2 RF in the EBZ tissue compared with the RS control tissue. Importantly, the antibody-specific immunofluorescence for a related isoform, GRK5, did not differ significantly in the same tissues (Fig. 3, B and D).

To determine whether the rapid decrease in GRK2 following myocardial ischemia, as shown both by immunoblot (33, 34) and in the present study by immunofluorescence microscopy, represents a transcriptional downregulation or a posttranscriptional event, we used quantitative real-time RT-PCR to compare GRK2 and GRK5 mRNA levels in the ischemic and control tissues. Although there was a numerical increase in GRK2 mRNA, normalized to levels of GAPDH mRNA, in the EBZ and infarct tissues (Fig. 4) compared with RS tissue, this did not reach significance (P > 0.05, n = 6). GRK2 mRNA in the RS tissue was also increased over that in control nonoperated heart tissue. Similar changes in GRK5 mRNA levels, normalized to levels of GAPDH mRNA, were also observed in the respective tissues.



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  		Fig. 4. Real-time PCR analysis of GRK2 (left) and GRK5 (right) mRNAs from RS, EBZ, and infarct (INF) tissues of 6 LAD-ligated dogs. Levels of GRK2 and GRK5 mRNAs are normalized to levels of GAPDH mRNA and are expressed as averages of triplicates (±SE). Although there was a numerical increase in the ratios of GRK2 and GRK5 to GAPDH mRNA for the EBZ and INF tissue compared with RS tissue, this did not reach significance (P > 0.05, n = 6).

 
The proteasome pathway has been implicated in the posttranscriptional degradation of GRK2 (25). We used the specific proteasome inhibitor bortezomib to determine whether blockade of this pathway might alter the frequency of malignant ventricular arrhythmias and loss of GRK2 in this animal model. These animals were given bortezomib (0.175 mg/kg iv) divided into two infusions, 1 h before and 5 h after ligation of the LAD coronary artery. The effect of this infusion on whole blood cell proteasome activity (expressed as fluorescence·100 µg protein–1·min–1) is shown in Fig. 5. The proteasome activity in the dog's whole blood was maximally suppressed at 0 h at the time of LAD ligation and remained suppressed for the remainder of the 24-h study period.



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  		Fig. 5. Bortezomib suppression of whole blood proteasome activity. The first dosage (0.0875 mg/kg) was given intravenously at 1 h before LAD ligation and the second at 5 h after LAD ligation. Blood samples were obtained at the designated times. Proteasome activity, expressed as fluorescence·100 µg protein–1·min–1, was suppressed during the whole 0- to 24-h period (*P < 0.05, n = 4).

 
As previously reported, GRK2 expression after 24 h measured by immunoblotting is significantly suppressed in both EBZ and infarct tissue taken from the saline control infarct animals (33, 34). Figure 6A shows typical GRK2 expression in a LAD-ligated dog and in a bortezomib-pretreated dog 24 h after LAD ligation. There was a significant decrease of GRK2 in the EBZ and infarct tissue (a variable decrease was also observed in endocardial tissue GRK2, data not shown) compared with the RS control tissue typical of that observed previously in saline-treated ligated animals. Bortezomib treatment conferred remarkable protection against the expected loss in GRK2 expression in the EBZ tissue with levels in this region remaining similar to levels in their control RS tissue. There was also a significant protection for GRK2 in the infarct tissue up to 50% of the RS (Fig. 6B). Endocardial tissue was similarly studied and had a GRK2 expression measured by immunoblotting of 40% of the RS tissue.



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  		Fig. 6. A: representative immunoblots of GRK2 in tissue taken from a saline-treated 24-h LAD-ligated dog (left) and a bortezomib-treated ligated dog (right). B: relative densities of GRK2 for RS, EBZ, INF, and endocardial (EN) tissue (n = 6) from bortezomib-pretreated dog hearts are expressed as percentages of values in nonoperated normal (NL) dog cardiac tissue (100% NL), and mean values are shown. GRK2 level in EBZ tissue was not significantly different (P > 0.05, n = 6) from that in RS tissues from the same hearts. The relative GRK2 density in blots from INF tissue was reduced to 50 ± 14% (*P < 0.05, n = 6) of that in blots from RS tissue, indicating that some degree of preservation of GRK2 occurred from bortezomib infused before LAD ligation. The EN tissue was protected similarly to the INF tissues (40 ± 10% of RS, *P < 0.05, n = 6) but less so than the EBZ tissues. This may reflect the difficulty in obtaining endocardial tissue without contamination by the adjacent infarct.

 
Holter ECG monitoring of the bortezomib-pretreated animals demonstrated a virtually complete absence of the very rapid triplets (≥360 beats/min) whose frequency is directly correlated with the occurrence of SCD in this animal model (Fig. 7A) (21–23). This observation is in marked contrast to that observed in all survivors of the nonpretreated infarct animals (P < 0.05) and especially in those dying from SCD. This observation of a marked beneficial effect even extended to those rapid triplets with a somewhat slower rate of >300 beats/min (Fig. 7B). When analysis was extended to the most rapid triplet observed within a given hour (Fig. 8), there again was a marked diminution (P < 0.05) in this analysis. None of the 6 bortezomib-pretreated animals died, whereas 5 of 11 control infarct animals died within the 24-h observation period (P = 0.1023).



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  		Fig. 7. Rapid ventricular triplets in control and bortezomib treatment groups. A: incidence (means ± SE) of very rapid ventricular triplets (all ≥360 beats/min) per hour for control and bortezomib-treated groups. Daggers indicate times of death of 5 dogs in the control group with ventricular fibrillation (VF). BPM, beats/min. The saline control animals had a significantly greater number of triplets at the indicated time points (P < 0.05). B: all of the rapid triplets (all ≥300 beats/min) for both groups. The saline control animals had a significantly greater number of total triplets at the indicated time points and the 2 curves are significantly different (P < 0.05).

 


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  		Fig. 8. Mean intrinsic rate (beats/min) for the fastest ventricular triplet observed in each dog during each hour for the 4- to 24-h period following LAD ligation in control and bortezomib-treated animals. The difference in the mean maximal triplet rates (means ± SE) between the 2 groups was significant from 6 to 24 h (P < 0.05). The saline control group consistently had faster triplets compared with the bortezomib group, which would put them at greater risk for developing monomorphic ventricular tachycardia and subsequent ventricular fibrillation.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The EBZ tissue represents an important site for the genesis of fatal sustained ventricular tachycardia and SCD during the 5- to 24-h period following CAL in the dog (10, 19, 21–23). Despite this important issue, relatively few studies are made of this tissue because of its relatively small size and volume and the consequent difficulty in performing biochemical and enzymatic assays to correlate with electrophysiological studies in this critical thin rim of ischemic cardiomyocytes. We have reported a rapid and consistent decrease in GRK2 expression and activity in the EBZ tissue overlying the myocardial infarct in a canine model (33, 34). This decrease has been evident by 6 h after ligation of the LAD coronary artery, persisting up to 96 h. This was associated with an increase in the percentage of high-affinity {beta}-AR in the EBZ tissue and with a loss in the ability of this tissue to desensitize normally to {beta}-agonist stimulation of adenylyl cyclase activity. These observations have major clinical implications, because they provide a rationale for the increased propensity of this tissue for {beta}-AR agonist-stimulated proarrhythmic early and delayed afterdepolarizations (9, 18, 22, 23). GRK2 serves as one of several mechanisms for inhibiting maximal and/or sustained {beta}-AR signal transduction, but its overexpression in tissues does not completely block {beta}-AR function (4, 6, 15, 27). A marked deficiency of GRK2 as observed in the EBZ tissues might be expected to exaggerate the risk for an electrophysiological myocardial event. This observation would be consistent with an acquired tissue hypersensitivity to adrenergic agonists as one of several potential biochemical mechanisms placing myocardial tissue "at risk" for such events. In any event, such a loss of a {beta}-AR modulator would support the use and clinical effectiveness of {beta}-blockers when these pharmaceutical agents are used for first-line prevention of fatal cardiac arrhythmias (29). Patterson et al. (22) previously demonstrated that acute pretreatment of our animal model with the {beta}-blocker nadolol markedly suppresses the malignant tachyarrhythmias and has a beneficial effect in preventing SCD. This may seem paradoxical, because prior treatment with a {beta}-blocker for 1–3 wk already has been demonstrated to suppress cardiac GRK2 expression in animals (12, 26). However, if the {beta}-blocker therapy is sustained, then any loss of GRK2 will be more than compensated for by the continued {beta}-blockade. If there is withdrawal of a short-acting {beta}-blocker, such as can occur in humans, any lag in reexpression of suppressed GRK2 might lead to a transient increased sensitivity to endogenous {beta}-agonists. The issue of a post-{beta}-blockade withdrawal hypersensitivity and its clinical sequelae has been a controversial issue (8, 16) and is neither addressed nor resolved in this study.

Because the methods that have previously been used to study the thin rim of EBZ cells are not easily adaptable to smaller animals or to multiple assays, we have developed an immunofluorescence technique using specific antibodies in conjunction with confocal laser microscopy to examine individual cardiomyocytes. This also permits us to exclude noncardiomyocytes that potentially dilute and/or interfere with analysis of whole tissue slices. We have adapted this single-cell analytic technique to identify selective changes in the EBZ tissues taken from the same animal model used in previous studies in our laboratory. However, arrhythmias leading to SCD are the primary outcome in the present study. These are difficult to interpret in small animals because their electrophysiology is quite different from humans.

The subcellular localization of GRK2 in myocardial tissues validates our technique by virtue that focal increases in the cardiac tissue immunofluorescence signal match the expected colocalization of intracellular GRK2 with adrenergic receptors (35). The GRK2 immunofluorescence is predominantly localized to the region around the myocardial T tubules where {beta}-AR and other G protein receptors are concentrated. Because inactive GRK2 is present in the cytosol near its targeted domains and, once activated, becomes membrane bound to the G protein receptor-{beta}{gamma} membrane complex (24), our data are consistent with such a construct. Such localization with GRK5 was not observed, suggesting a more widespread distribution in the cellular membrane structures. In addition, analysis of cardiac tissue from transgenic mice with known GRK2 activity provides reassuring evidence for quantification of GRK2 expression. We were able to demonstrate a clear decrease in GRK2 expression when we applied this technique to tissues previously demonstrated to have decreased GRK2 by immunoblotting and phosphorylation assays (33, 34).

GRK2 immunofluorescence was significantly decreased in cells from the EBZ tissues compared with the RS control tissues taken from the same animal. In previous studies using GRK2 activity measurement and GRK2 immunoblot analysis, there was a decrease to <20% of the RS tissue (33, 34). With the described immunofluorescence technique, the EBZ RF values decreased to ~24% of RS. These small differences may relate to the difficulty in completely eliminating background fluorescence. We also measured GRK5 immunofluorescence as a control for the GRK2, because GRK5 is not decreased in 24-h ischemic cardiac tissue (34). There was no significant change in GRK5 RF values, compatible with our previous observation that GRK5 expression is unchanged in the EBZ and infarct tissue despite the marked drop in GRK2 expression and activity (33, 34). The observed decrease in GRK2 is therefore selective and does not represent a nonspecific degradation of GRK enzyme expression.

We have demonstrated that changes in GRK2 transcription likely do not account for the decrease in GRK2 in the EBZ tissues. In fact, there was a rise in both GRK2 and GRK5 mRNA normalized to levels of GAPDH mRNA in the EBZ tissue and in the infarct tissue 24 h after LAD ligation. This modest increase of GRK2 and GRK5 mRNA in these ischemic/infarcted tissues was not unexpected. There is precedence, because mRNAs encoding several other cardiac proteins, including cardiotrophin-1, TNF-{alpha}, renin, and angiotensinogen, also have been reported to increase in ischemic border zone and/or infarct tissue 24–48 h and longer after LAD ligation (3, 13, 20). It is possible that a biphasic response might occur shortly after production of the ischemia, but the concurrent changes of GRK2 and GRK5 mRNA, occurring despite opposite effects on their protein expression, make this unlikely. It is possible that changes in mRNA turnover might occur, but the differential response between GRK2 and GRK5 protein expression again suggests that this did not occur.

The effects of the proteasome inhibitor bortezomib on both ventricular tachyarrhythmias and on tissue expression of GRK2 were dramatic. There was a marked preservation of GRK2 in both the ischemic and infarcted tissues in five of the six pretreated animals. It is not clear why there was less preservation of the EBZ tissue in the sixth animal. Suppression of whole blood proteasome activity appeared to be consistent in this animal as well as in the others. Although there is compartmentalization of bortezomib in the body with less movement into central nervous system, eyes, and testes (1), the whole blood concentration appears to reflect those in the heart, liver, and vascular tissues. The relative preservation of GRK2 expression in the infarct tissue is likely due to the preligation (–1 h) treatment of the whole heart with bortezomib. The second infusion of bortezomib would not have equal access to this tissue through the ligated vessels leading to the infarct, whereas the ischemic tissues have access to collateral flow. This may explain why the EBZ was able to maintain almost full protection. We believe that the endocardial section had a >50% presence of infarct tissue (Fig. 1) and therefore probably reflected the infarct tissue loss of GRK2.

The effect of bortezomib in preserving GRK2 in the EBZ was accompanied by a dramatic decrease in the very rapid (>360 beats/min) and rapid (>300 beats/min) ventricular triplets observed during Holter monitoring. The GRK2 protective effect of proteasome inhibition was of greater magnitude than that observed when a similar group of dogs were pretreated with a TNF-{alpha} sequestrant etanercept (Enbrel; Amgen, Thousand Oaks, CA) (33). This marked preservation of GRK2 expression is concurrent with dramatic protection against malignant triplets that are highly associated with the onset of fatal tachyarrhythmias in this model. These data support the probability that GRK2 translocation to endosomal structures and/or a marked activation of the proteasome proteolytic pathway (25) may combine to account for the marked diminution of GRK2 protein expression in this animal model of human SCD and lead to increased sensitivity of the tissues to autonomic activity. Another highly selective proteasome inhibitor, PS-519, has been demonstrated to reduce reperfusion injury when given to a pig model (28). This agent suppressed proteasome activity to levels observed in the present study. Their data support the concept that acute, selective inhibition of the 20S proteasome may be a useful tool for examining pathophysiological sequelae of myocardial ischemia. Although the association of GRK2 preservation with antecedent proteasome inhibition is of great interest, it also is possible that bortezomib may protect or alter additional unidentified proteins that contribute to the observed antiarrhythmic effects. Such studies are difficult to construct until additional insights are gained on the multiple functions relevant to cardiac arrhythmogenesis.

In summary, our data using in vivo infarcted hearts has repetitively shown a rapid and marked decrease of GRK2 activity and protein expression in the EBZ and now in subendocardial tissues adjacent to the infarct. The increase of GRK2 mRNA in both EBZ and infarct tissue appears to make it unlikely that transcriptional changes in mRNA can account for this loss of GRK2. GRK2 endosomal translocation and proteolysis via the proteasome pathway appear to be likely candidates for loss of GRK2. When proteasome activity is inhibited before induction of ischemia, preservation of GRK2 is observed in both EBZ and infarct tissue. This suggests that protection of GRK2 expression and activity in the myocardium may provide an alternative or complementary means for decreasing the occurrence of postischemic SCD.


   GRANTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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REFERENCES
 
Support for this study was provided by the Department of Veterans Affairs Medical Center, Oklahoma City, OK, the Oklahoma Center for the Advancement of Science and Technology, the Cardiac Arrhythmia Research Institute, the American Heart Association, Oklahoma Affiliate, and grants from Dr. Doris Travis and Dr. and Mrs. William Talley.


   ACKNOWLEDGMENTS
 
Dr. Jan Pitha, Dept. of Pathology, Veterans Affairs Medical Center and University of Oklahoma Health Sciences Center, provided assistance for the histological studies of the tissue sections.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. C. Kem, Dept. of Medicine, WP1345, 920 Stanton Young Blvd., Oklahoma City, OK 73104 (e-mail: david-kem{at}ouhsc.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.

* X. Yu and S. Huang contributed equally to this work. Back


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 REFERENCES
 

  1. Adams J, Palombella VJ, Sausville EA, Johnson J, Destree A, Lazarus DD, Maas J, Pien CS, Prakash S, and Elliott PJ. Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res 59: 2615–2622, 1999.[Abstract/Free Full Text]
  2. Akhter SA, Eckhart AD, Rockman HA, Shotwell K, Lefkowitz RJ, and Koch WJ. In vivo inhibition of elevated myocardial {beta}-adrenergic receptor kinase activity in hybrid transgenic mice restores normal {beta}-adrenergic signaling and function. Circulation 100: 648–653, 1999.[Abstract/Free Full Text]
  3. Aoyama T, Takimoto Y, Pennica D, Inoue R, Shinoda E, Hattori R, Yui Y, and Sasayama S. Augmented expression of cardiotrophin-1 and its receptor component, gp130, in both left and right ventricles after myocardial infarction in the rat. J Mol Cell Cardiol 32: 1821–1830, 2000.[CrossRef][ISI][Medline]
  4. Aragay AM, Ruiz-Gomez A, Penela P, Sarnago S, Elorza A, Jimenez-Sainz MC, and Mayor F Jr. G protein-coupled receptor kinase 2 (GRK2): mechanisms of regulation and physiological functions. FEBS Lett 430: 37–40, 1998.[CrossRef][ISI][Medline]
  5. Benovic JL. Purification and characterization of beta-adrenergic receptor kinase. Methods Enzymol 200: 351–362, 1991.[ISI][Medline]
  6. Bunemann M and Hosey MM. G-protein coupled receptor kinases as modulators of G-protein signalling. J Physiol 517: 5–23, 1999.[Free Full Text]
  7. Choi DJ, Koch WJ, Hunter JJ, and Rockman HA. Mechanism of {beta}-adrenergic receptor desensitization in cardiac hypertrophy is increased {beta}-adrenergic receptor kinase. J Biol Chem 272: 17223–17229, 1997.[Abstract/Free Full Text]
  8. Croft CH, Rude RE, Gustafson N, Stone PH, Poole WK, Roberts R, Strauss HW, Raabe DS Jr, Thomas LJ, Jaffe AS, Muller J, Hoagland P, Sobel BE, Passamani ER, Braunwald E, and Willerson JT. MILIS Study Group. Abrupt withdrawal of {beta}-blockade therapy in patients with myocardial infarction: effects on infarct size, left ventricular function, and hospital course. Circulation 73: 1281–1290, 1986.[Abstract/Free Full Text]
  9. Dangman KH, Dresdner KP Jr, and Zaim S. Automatic and triggered impulse initiation in canine subepicardial ventricular muscle cells from border zones of 24-hour transmural infarcts. New mechanisms for malignant cardiac arrhythmias? Circulation 78: 1020–1030, 1988.[Abstract/Free Full Text]
  10. Harris AS. Delayed development of ventricular ectopic rhythms following experimental coronary occlusion. Circulation 1: 1318–1328, 1950.[ISI][Medline]
  11. Harris AS, Estandia A, and Tillotson RF. Ventricular ectopic rhythms and ventricular fibrillation following cardiac sympathectomy and coronary occlusion. Am J Physiol 165: 505–512, 1951.[Free Full Text]
  12. Iaccarino G, Tomhave ED, Lefkowitz RJ, and Koch WJ. Reciprocal in vivo regulation of myocardial G protein-coupled receptor kinase expression by {beta}-adrenergic receptor stimulation and blockade. Circulation 98: 1783–1789, 1998.[Abstract/Free Full Text]
  13. Irwin MW, Mak S, Mann DL, Qu R, Penninger JM, Yan A, Dawood F, Wen WH, Shou Z, and Liu P. Tissue expression and immunolocalization of tumor necrosis factor-{alpha} in postinfarction dysfunctional myocardium. Circulation 99: 1492–1498, 1999.[Abstract/Free Full Text]
  14. Koch WJ, Rockman HA, Samama P, Hamilton RA, Bond RA, Milano CA, and Lefkowitz RJ. Cardiac function in mice overexpressing the {beta}-adrenergic receptor kinase or a {beta}ARK inhibitor. Science 268: 1350–1353, 1995.[Abstract/Free Full Text]
  15. Krupnick JG and Benovic JL. The role of receptor kinases and arrestins in G protein-coupled receptor regulation. Annu Rev Pharmacol Toxicol 38: 289–319, 1998.[CrossRef][ISI][Medline]
  16. Lederballe Pedersen O, Mikkelsen E, Lanng Nielsen J, and Christensen NJ. Abrupt withdrawal of beta-blocking agents in patients with arterial hypertension. Effect on blood pressure, heart rate and plasma catecholamines and prolactin. Eur J Clin Pharmacol 15: 215–217, 1979.[CrossRef][ISI][Medline]
  17. Lightcap ES, McCormack TA, Pien CS, Chau V, Adams J, and Elliott PJ. Proteasome inhibition measurements: clinical application. Clin Chem 46: 673–683, 2000.[Abstract/Free Full Text]
  18. Mehra R, Zeiler RH, Gough WB, and El-Sherif N. Reentrant ventricular arrhythmias in the late myocardial infarction period. 9. Electrophysiologic-anatomic correlation of reentrant circuits. Circulation 67: 11–24, 1983.[Abstract/Free Full Text]
  19. Opitz CF, Mitchell GF, Pfeffer MA, and Pfeffer JM. Arrhythmias and death after coronary artery occlusion in the rat. Continuous telemetric ECG monitoring in conscious, untethered rats. Circulation 92: 253–261, 1995.[Abstract/Free Full Text]
  20. Passier RC, Smits JF, Verluyten MJ, and Daemen MJ. Expression and localization of renin and angiotensinogen in rat heart after myocardial infarction. Am J Physiol Heart Circ Physiol 271: H1040–H1048, 1996.[Abstract/Free Full Text]
  21. Patterson E, Scherlag BJ, and Lazzara R. Arrhythmias in the canine heart two to twenty-four hours after myocardial infarction. In: Electrophysiology and Pharmacology of the Heart, edited by Dangman KH and Miura DS. New York: Dekker, 1991, p. 301–330.
  22. Patterson E, Scherlag BJ, and Lazzara R. Mechanism of prevention of sudden death by nadolol: differential actions on arrhythmia triggers and substrate after myocardial infarction in the dog. J Am Coll Cardiol 8: 1365–1372, 1986.[Abstract]
  23. Patterson E, Scherlag BJ, and Lazzara R. Prevention of spontaneous sustained ventricular tachycardia in the postinfarction dog by left stellate ganglionectomy. J Cardiovasc Electrophysiol 2: 238–248, 1991.[CrossRef]
  24. Pei G, Tiberi M, Caron MG, and Lefkowitz RJ. An approach to the study of G-protein-coupled receptor kinases: an in vitro-purified membrane assay reveals differential receptor specificity and regulation by G{beta}{gamma} subunits. Proc Natl Acad Sci USA 91: 3633–3636, 1994.[Abstract/Free Full Text]
  25. Penela P, Ruiz-Gomez A, Castano JG, and Mayor F Jr. Degradation of the G protein-coupled receptor kinase 2 by the proteasome pathway. J Biol Chem 273: 35238–35244, 1998.[Abstract/Free Full Text]
  26. Ping P, Gelzer-Bell R, Roth DA, Kiel D, Insel PA, and Hammond HK. Reduced {beta}-adrenergic receptor activation decreases G-protein expression and {beta}-adrenergic receptor kinase activity in porcine heart. J Clin Invest 95: 1271–1280, 1995.[ISI][Medline]
  27. Pitcher JA, Freedman NJ, and Lefkowitz RJ. G protein-coupled receptor kinases. Annu Rev Biochem 67: 653–692, 1998.[CrossRef][ISI][Medline]
  28. Pye J, Ardeshirpour F, McCain A, Bellinger DA, Merricks E, Adams J, Elliott PJ, Pien C, Fischer TH, Baldwin AS Jr, and Nichols TC. Proteasome inhibition ablates activation of NF-{kappa}B in myocardial reperfusion and reduces reperfusion injury. Am J Physiol Heart Circ Physiol 284: H919–H926, 2003.[Abstract/Free Full Text]
  29. Reiter MJ and Reiffel JA. Importance of beta blockade in the therapy of serious ventricular arrhythmias. Am J Cardiol 82: 9I–19I, 1998.[CrossRef][ISI][Medline]
  30. Rockman HA, Choi DJ, Rahman NU, Akhter SA, Lefkowitz RJ, and Koch WJ. Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice. Proc Natl Acad Sci USA 93: 9954–9959, 1996.[Abstract/Free Full Text]
  31. Ungerer M, Bohm M, Elce JS, Erdmann E, and Lohse MJ. Altered expression of {beta}-adrenergic receptor kinase and {beta}1-adrenergic receptors in the failing human heart. Circulation 87: 454–463, 1993.[Abstract/Free Full Text]
  32. Ungerer M, Kessebohm K, Kronsbein K, Lohse MJ, and Richardt G. Activation of {beta}-adrenergic receptor kinase during myocardial ischemia. Circ Res 79: 455–460, 1996.[Abstract/Free Full Text]
  33. Yu X, Patterson E, Huang S, Garrett MW, and Kem DC. Tumor necrosis factor {alpha}, rapid ventricular tachyarrhythmias, and infarct size in canine models of myocardial infarction. J Cardiovasc Pharmacol 45: 153–159, 2005.[CrossRef][ISI][Medline]
  34. Yu X, Zhang M, Kyker K, Patterson E, Benovic JL, and Kem DC. Ischemic inactivation of G protein-coupled receptor kinase and altered desensitization of canine cardiac {beta}-adrenergic receptors. Circulation 102: 2535–2540, 2000.[Abstract/Free Full Text]
  35. Zaccolo M and Pozzan T. Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 295: 1711–1715, 2002.[Abstract/Free Full Text]



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S. Huang, E. Patterson, X. Yu, M. W. Garrett, I. De Aos, and D. C. Kem
Proteasome inhibition 1 h following ischemia protects GRK2 and prevents malignant ventricular tachyarrhythmias and SCD in a model of myocardial infarction
Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1298 - H1303.
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