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Am J Physiol Heart Circ Physiol 293: H2702-H2709, 2007. First published August 24, 2007; doi:10.1152/ajpheart.00763.2007
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Exercise training normalizes beta-adrenoceptor expression in dogs susceptible to ventricular fibrillation

Bethany J. Holycross,1 Monica Kukielka,5 Yoshinori Nishijima,3 Ruth A. Altschuld,2,3 Cynthia A. Carnes,3,4 and George E. Billman3,5

1Department of Veterinary Biosciences, 2Department of Molecular and Cellular Biochemistry, 3Dorothy M. Davis Heart and Lung Research Institute, 4College of Pharmacy, and 5Department of Physiology and Cell Biology, Ohio State University, Columbus, Ohio

Submitted 3 July 2007 ; accepted in final form 23 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous studies demonstrated an enhanced beta2-adrenoceptor (AR) responsiveness in animals susceptible to ventricular fibrillation (VF) that was eliminated by exercise training. The present study investigated the effects of endurance exercise training on beta1-AR and beta2-AR expression in dogs susceptible to VF. Myocardial ischemia was induced by a 2-min occlusion of the left circumflex artery during the last minute of exercise in dogs with healed infarctions: 20 had VF [susceptible (S)] and 13 did not [resistant (R)]. These dogs were randomly assigned to either 10-wk exercise training [treadmill running; n = 9 (S) or 8 (R)] or an equivalent sedentary period [n = 11 (S) or 5 (R)]. Left ventricular tissue beta-AR protein and mRNA were quantified by Western blot analysis and RT-PCR, respectively. Because beta2-ARs are located in caveolae, caveolin-3 was also quantified. beta1-AR gene expression decreased (~5-fold), beta2-AR gene expression was not changed, and the ratio of beta2-AR to beta1-AR gene expression was significantly increased in susceptible compared with resistant dogs. beta1-AR protein decreased (~50%) and beta2-AR protein increased (400%) in noncaveolar fractions of the cell membrane in susceptible dogs. Exercise training returned beta1-AR gene expression to levels seen in resistant animals but did not alter beta2-AR protein levels in susceptible dogs. These data suggest that beta1-AR gene expression was decreased in susceptible dogs compared with resistant dogs and, further, that exercise training improves beta1-AR gene expression, thereby restoring a more normal beta-AR balance.

beta-adrenergic receptors; myocardial infarction; sympathetic nervous system

IT IS WELL ESTABLISHED THAT cardiac beta1-adrenoceptor (AR) sensitivity decreases substantially during heart failure, whereas the beta2-AR response remains relatively constant (1, 12). As a consequence the failing heart becomes more dependent on the activation of beta2-AR for inotropic support. The activation of beta2-ARs promotes increased calcium currents without altering calcium reuptake by the sarcoplasmic reticulum (2). The resulting elevations in intracellular calcium could provoke oscillations in membrane potential that could, in turn, trigger extrasysoles. Thus, in the diseased heart, beta2-AR activation would tend to reduce cardiac electrical stability and increase the propensity for malignant arrhythmias, particularly when calcium regulation is further altered by sympathetic activation and/or myocardial ischemia.

We recently demonstrated (10, 18), in dogs with healed myocardial infarctions (MIs), that the nonselective beta-AR agonist isoproterenol provoked significantly larger heart rate and inotropic responses in those animals that were susceptible to ventricular fibrillation (VF) induced by myocardial ischemia than in those animals that were resistant to these malignant arrhythmias. The selective beta2-AR antagonist ICI-118551 reduced the isoproterenol response to a much greater extent in the susceptible animals, eliminating any differences noted between the groups (10, 18). In a similar manner, both the calcium transient amplitude and the single-cell isotonic shortening responses to isoproterenol were larger in myocytes obtained from the hearts of susceptible compared with resistant dogs, differences that were also eliminated by beta2-AR blockade but not by beta1-AR blockade (10). In the intact dog, beta2-AR blockade also prevented VF induced by acute myocardial ischemia (10). When considered together, these data demonstrate that an enhanced beta2-AR responsiveness is associated with an increased propensity for VF. One would predict that interventions that restore a more normal beta1-AR to beta2-AR balance should also protect against VF.

Regular exercise can both improve beta-AR responsiveness and decrease the incidence of malignant arrhythmias (4, 5, 7, 8, 11, 23, 25, 38, 42). We recently demonstrated (11) that a 10-wk endurance exercise program reduced beta2-AR responsiveness and prevented VF induced by acute myocardial ischemia. In contrast, beta2-AR responsiveness was further enhanced in sedentary (control) dogs that also remained prone to VF (11). These data suggest that exercise training could restore a more normal beta-AR balance after MI and could thereby prevent VF. However, it remains to be determined whether the improved beta2-AR responsiveness resulted from altered beta-AR gene expression, beta-AR protein abundance in the cell membrane, or changes in the receptor signal transduction process.

Exercise training-induced changes in the distribution of beta-ARs within the sarcolemma could also affect the response of these receptors to stimulation. beta1-ARs are widely distributed throughout the cardiac myocyte cell membrane, where agonist stimulation results in elevated intracellular cAMP and calcium flux. (33). beta2-ARs are located in caveolae, lipid-enriched invaginations of the cell membrane, both on the outer surface of the cell and throughout the t tubules (29, 37). In adult rat ventricular cardiomyocytes, it appears that beta2-ARs must be located in the caveolae to obtain close approximation to sarcoplasmic reticulum and signaling pathway proteins such as Gi and the sodium/calcium exchanger (13). Disruption of caveolae results in redistribution of beta2-ARs and a marked increase in beta2-AR agonist-induced cardiomyocyte shortening and calcium transients (13). Thus the distribution of beta2-ARs may be altered in animals susceptible to VF, and, further, exercise training may redistribute the beta2-ARs to restore a more normal beta-AR responsiveness, decreasing the risk for malignant arrhythmias.

It was therefore the purpose of this study to determine the effects of exercise training on beta2-AR and beta1-AR gene expression and protein abundance in dogs susceptible or resistant to VF. In particular, the hypothesis that endurance exercise training would restore a more normal beta2-AR to beta1-AR balance was tested. Left ventricular (LV) beta-AR protein and mRNA were quantified by Western blot and RT-PCR in tissue harvested from either sedentary (10 wk) or exercise-trained (10 wk progressively increasing treadmill running) dogs. This training program was previously shown to prevent VF in these animals (7, 11). Since beta2-ARs are located in caveolae, caveolin-3 was also quantified.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The principles governing the care and use of animals expressed by the Declaration of Helsinki and adopted by the American Physiological Society were followed at all times during this study. In addition, the Ohio State University Institutional Animal Care and Use Committee approved all the procedures used in this study.

All studies were performed on archived tissue harvested from dogs that had been randomly assigned to either exercise training (10 wk, progressively increasing treadmill running) or an equivalent sedentary time period. LV tissue from three to five dogs per treatment group was selected at random for RT-PCR and Western blot analysis. In addition, the assays were performed on LV tissue obtained from four control (i.e., no MI) dogs.

The surgical procedures, the exercise training program, and the test (exercise + ischemia) used to classify the dogs as to susceptibility to VF have been described previously (6–11, 39). A flowchart of the animals used in the present study is displayed in Fig. 1.


Figure 1
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  		Fig. 1. Flowchart illustrating the sequences of events in this study. Three to four weeks after myocardial infarction, the dogs were classified as susceptible or resistant to ventricular fibrillation (VF) with an exercise + ischemia test. The animals were then randomly assigned to either a 10-wk exercise (Ex) training program or a 10-wk sedentary period. The exercise + ischemia test was repeated at the end of the 10-wk period. n = No. of animals. defib, Defibrillate.

 
Real-Time PCR

A section of left ventricle (posterior lateral free wall, noninfarcted tissue) was snap frozen in liquid nitrogen and then stored at –80°C. Total RNA was isolated from frozen LV tissue with a ToTALLY RNA kit (Ambion, Austin TX) followed by DNase I digestion. Total RNA was quantified via spectrophotometry, and 250 ng of total RNA was used in 25-µl reverse transcription reactions utilizing TaqMan RT reagents as outlined by the manufacturer (Applied Biosystems, Foster City, CA). Real-time polymerase chain reaction (PCR) experiments were conducted in the ABI PRISM 7700 Sequence Detection System (Applied Biosystems) with the SYBR Green PCR Master Mix (24, 45). Each 25-µl reaction was performed in triplicate on a 96-well plate with 12.5 µl of SYBR Green Master Mix, 1 µl of 5 µM sense primer, 1 µl of 5 µM antisense primer, and 1.5 µl of cDNA. Thermocycler conditions were set at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 45 s, and 72°C for 45 s. Before real-time PCR analysis, each primer pair was confirmed to yield a single band at the appropriate size by conventional PCR using the above listed cycling conditions.

Primers were designed with TaqMan Probe and Primer design software and were designed to be 19–21 bp long, have a melting temperature of 59–60°C, and yield a PCR product between 52 and 63 bp long, Primers for PCR were 18S forward: 5'-GGTTGATCCTGCCAGTAGCATAT-3', 18S reverse: 5'-GTACCGGCCGTGCGTACTT-3' (GenBank accession no. AY262732); beta1 forward: 5'-TGCTACAACGACCCCAAGTG-3', beta1 reverse: 5'-AGGTACACGAAGGCCATGATG-3' (GenBank accession no. U73207); and beta2 forward: 5'-CCAGAGGCAGCTCCAGAAGA-3', beta2 reverse: 5'-CCACTTGGCTGAGGTTTTGG-3' (GenBank accession no. AY011309). Also, the primers were subjected to a BLAST search and were found to have no cross-homologies with other canine genes.

Analysis of all genes for a given dog was performed on the same 96-well plate, and dogs from the different treatment groups were randomly assigned to different 96-well plates. For a given gene, analysis parameters were consistent between plates. For each dog, a threshold cycle (Ct) for each gene was determined. A {Delta}Ct was then calculated by subtracting the Ct for 18S from the Ct obtained for the beta1-AR or beta2-AR. The average {Delta}Ct for each gene was calculated for the resistant sedentary group of dogs, and the difference between the gene expression in this group and all other treatment groups was calculated and expressed as the {Delta}{Delta}Ct. The actual fold change in expression between treatment groups is calculated as 2Formula. Values for beta2- to beta1-AR expression were calculated for each dog as {Delta}Ct beta1 – {Delta}Ct beta2 to obtain a {Delta}{Delta}Ct. Fold differences in expression were calculated as 2Formula, and the ratios beta2/beta1 were calculated as 1/fold change. Because there were no differences in the gene expression between the resistant sedentary and the resistant exercise groups, these data were combined in all subsequent analyses.

Western Blot Analysis

Frozen canine tissue was extracted by two different methods to evaluate receptor sequestered in caveolae compared with total receptor complement in the cell membrane.

SDS extraction. Approximately 100 mg of tissue was rapidly pulverized in liquid nitrogen with a mortar and pestle and placed in 500 µl of SDS buffer consisting of 1% SDS, 10 mmol/l Tris·HCl pH 7.4, 1 mmol/l orthovanadate, 0.1 mmol/l leupeptin, 0.001 mmol/l aprotinin, and 10 mg/ml PMSF as described previously (34). Lysates were boiled in a microwave oven for 15 s (1,100 W) and then centrifuged at 13,000 g for 15 min at room temperature.

Triton X-100 extraction. Approximately 100 mg of tissue was rapidly pulverized in liquid nitrogen with a mortar and pestle and then placed in 500 µl of ice-cold TE buffer containing 20 mmol/l Tris, 1 mmol/l EDTA, 0.1 mg/ml benzamidine, 10 mg/ml PMSF, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, 5 µg/ml aprotinin, and 100 µmol/l sodium orthovanadate as described previously (14). The suspension was vortexed, and Triton X-100 was added to a final concentration of 2%. Lysates were put on ice for 2 h and then centrifuged at 14,000 g for 10 min at 4°C. For each extraction, the soluble fraction was retained and stored at –80°C. Protein concentration was determined with a microBCA protein assay kit (Pierce), and 30 µg (SDS extracts) or 50 µg (Triton X-100 extracts) was heated to 95°C for 5 min in 3x SDS sample buffer (New England) and then subjected to electrophoresis (1 h, 150 V) on 10% Tris·HCl Criterion Precast Gels (Bio-Rad). Proteins were transferred for 45 min (15 V) to Immobilon-P polyvinylidene difluoride membranes with a semidry blotter (Bio-Rad). Membranes were blocked (1 h, room temperature) with 5% nonfat milk in 0.1% Tween 20 in Tris-buffered saline (TBS) and incubated overnight at 4°C in 5% BSA and 0.1% Tween 20 in TBS with rabbit polyclonal antibodies to beta1-AR or beta2-AR (Santa Cruz Biotechnology) at 1:500 dilution or mouse monoclonal anti-caveolin-3 antibodies (BD Transduction Laboratories) at 1:2,500 dilution. After three washes (5 min, room temperature) with 0.1% Tween 20 in TBS, the membranes were incubated with 1:1,000 dilution of goat anti-rabbit IgG-horseradish peroxidase (HRP) or goat anti-mouse IgM-HRP in 5% nonfat milk-0.1% Tween 20 in TBS for 1 h at room temperature. Membranes were then washed six times with 0.1% Tween 20 in TBS (15 min, room temperature). Antibody binding was detected with LumiGlo chemiluminescent subtrate (Upstate) and CL-X Posure clear blue X-ray film (Pierce). Films were scanned with ScanSuite software and quantified with ImageJ (National Institutes of Health). Densitometric comparisons were performed in samples run on the same membrane. For Western blots, three to five dog samples were analyzed per treatment group, and each analysis was performed on two or three gels. Normalization for protein loading was calculated after Ponceau staining of the membrane as described by Ping et al. (32). After normalization for loading, the data were normalized to the value of the resistant sedentary group, which was arbitrarily assigned the value of 100%. Because there were no differences in Western blot values between the resistant sedentary and the resistant exercise groups for any of the proteins examined, these data were combined in all subsequent analyses.

Echocardiography Studies

To evaluate the effects of MI on ventricular function, two-dimensional B- and M-mode echocardiograms were obtained from a subset of dogs (7 susceptible and 4 resistant) under butorphanol sedation (0.5 mg/kg im) at baseline (before surgery and infarction) and 8 wk after surgery (GE Vivid 7 echocardiograph).

Statistical Analysis

All data are reported as means ± SD. Statistical analysis was performed with a one-factor ANOVA (NCSS, Kaysville, UT). If the F-ratio exceeded a critical value (P < 0.05), the means were compared by post hoc analysis performed with the Tukey-Kramer test. The echocardiographic variables were compared with a paired t-test.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of Myocardial Infarction on Ventricular Function

In a subset of animals, echocardiograms were obtained to assess LV function and structure. In agreement with our previous studies (9, 18), MI did not induce any significant alterations in contractile function measured as LV fractional shortening in either the susceptible [n = 7; 41.3 ± 4.7% baseline vs. 40.5 ± 2.9% post-MI; P = not significant (NS)] or the resistant (n = 4; 37.5 ± 5.1% baseline vs. 37.0 ± 2.7 post-MI; P = NS) group. Furthermore, MI did not produce any significant LV dilation measured as end-diastolic diameter in either the susceptible (n = 7; 4.2 ± 0.3 cm baseline vs. 4.3 ± 0.3 cm post-MI; P = NS) or the resistant (n = 4; 4.0 ± 0.3 cm baseline vs. 4.3 ± 0.4 cm post-MI; P = NS) group. Similarly, there were no significant changes in LV end-systole diameter in either the susceptible (n = 7; 2.5 ± 0.2 cm baseline vs. 2.6 ± 0.3 cm post-MI; P = NS) or the resistant (n = 4; 2.5 ± 0.2 cm baseline vs. 2.7 ± 0.2 cm post-MI; P = NS) group.

Effect of Myocardial Infarction on beta-AR mRNA and beta-AR Protein Abundance

The effects of MI on beta-AR expression were evaluated by comparing beta-AR mRNA (real-time PCR) and protein abundance (Western blot) in LV tissue from control (no infarction) and postinfarction (sedentary resistant) dogs. The number of cycles to reach threshold ({Delta}Ct) for detection for both beta1-ARs [control (n = 4) 15.7 ± 0.8 vs. resistant (n = 4) 15.2 ± 1.6; F = 0.69, P = NS] and beta2-ARs [control (n = 4) 18.9 ± 0.6 vs. resistant (n = 4) 19.5 ± 0.6; F = 3.55, P = NS] was similar in both groups. Protein abundance (Western blot, Triton extraction) was also similar in both the infarcted and noninfarcted animals. beta1-AR [control (n = 4) 82.3 ± 37 vs. resistant (n = 4) 95.2 ± 28.6; F = 0.31, P = NS], beta2-AR [control (n = 4) 166.4 ± 78.4 vs. resistant (n = 4) 98.5 ± 28; F = 3.96, P = NS], and caveolin [control (n = 4) 133.4 ± 32.4.2 vs. resistant (n = 4) 101.1 ± 5.8; F = 0.09, P = NS] content were similar in both groups. These data suggest that beta-AR expression was not altered by MI in dogs resistant to the induction of VF. In agreement with these findings, we previously demonstrated (18) that beta-AR responsiveness was similar in susceptible and resistant dogs before MI; differences were only noted after the infarction.

Effect of Exercise Training on beta-AR mRNA

With SYBR Green real-time PCR, more cycles were required to reach a threshold {Delta}Ct for detection for beta1-ARs in the susceptible sedentary group (n = 4, {Delta}Ct = 20.5 ± 2.4) (F15 = 4.43, P < 0.05) than in either the resistant (n = 8, {Delta}Ct = 18.1 ± 1.1) or the susceptible exercise (n = 4, {Delta}Ct = 17.6 ± 1.0) group (Fig. 2). On normalization of expression to resistant dogs, the susceptible sedentary dogs had a {Delta}{Delta}Ct of –2.42 ± 2.44, equivalent to 5.3-fold lower expression of beta1-ARs (F15 = 4.39, P < 0.05), whereas the susceptible exercise dogs had a {Delta}{Delta}Ct of 0.44 ± 1.08 (a value that was not different from the resistant dogs; F15 = 4.39, P = NS). These data suggest that the expression of beta1-AR mRNA was lower in susceptible than in resistant animals and, further, that exercise training normalized beta1-AR mRNA expression such that differences were no longer detected between the susceptible and resistant groups. In contrast, beta2-AR expression was similar in resistant (n = 8, {Delta}Ct = 21.5 ± 0.8), susceptible sedentary (n = 4, {Delta}Ct = 20.9 ± 2.2), and susceptible exercise (n = 4, {Delta}Ct = 22.0 ± 0.4) animals (Fig. 3). When beta2-AR expression was compared with beta1-AR expression, it became apparent that dogs susceptible to VF have a significantly higher ratio of beta2-AR to beta1-AR (1.30 ± 1.5) compared with either resistant (0.11 ± 0.05) or susceptible exercise dogs (0.06 ± 0.04) (F15 = 7.01, P < 0.01; Fig. 4).


Figure 2
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  		Fig. 2. Expression of beta1-adrenoceptor as determined by real-time PCR using SYBR Green. After PCR, the cycle where threshold amounts of beta1-adrenergic receptor are detected (Ct) was determined and then the Ct for detection of the internal standard, 18S mRNA, was subtracted, resulting in a {Delta}Ct. Higher values for {Delta}Ct indicate a lower expression of the target gene. Susceptible sedentary group expressed significantly less beta1-adrenoceptor mRNA in cardiac tissue than resistant group of dogs (*P < 0.05). n = No. of dogs per treatment group; each dog sample performed in triplicate. Data are plotted as means ± SD.

 

Figure 3
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  		Fig. 3. Expression of beta2-adrenoceptor as determined by real-time PCR using SYBR Green. After PCR, the cycle where threshold amounts of beta2-adrenergic receptor are detected (Ct) was determined and then the Ct for detection of the internal standard, 18S mRNA, was subtracted, resulting in a {Delta}Ct. Higher values for {Delta}Ct indicate a lower expression of the target gene. There were no differences between groups. n = No. of dogs per treatment group; each dog sample performed in triplicate. Data are plotted as means ± SD.

 

Figure 4
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  		Fig. 4. beta2-to-beta1 adrenoceptor ratio in the left ventricle, as determined by real-time PCR, was significantly greater in susceptible sedentary dogs than in resistant or susceptible exercise-trained animals (*P < 0.01). n = No. of dogs per treatment group; each dog sample performed in triplicate. Data are plotted as means ± SD.

 
Effect of Exercise Training on beta-AR Protein Abundance

To determine whether mRNA expression changes were translated into differences in receptor protein, Western blot analysis was performed utilizing two different methods for protein solubilization. Triton X-100 preferentially extracts proteins located outside of lipid rafts or caveolae (34), whereas analysis of extracts obtained from boiling in SDS provides total protein content of the cell (34). beta1-ARs were less abundant in Triton X-100 extracts in sedentary dogs susceptible to VF (n = 4, 54 ± 14%) than in resistant dogs (n = 8, 100 ± 34%) or in exercise-trained susceptible dogs (n = 4, 78 ± 18%) (F15 = 3.88, P < 0.05; Fig. 5A). This same tendency was apparent in the SDS extraction, but because of sample variability significant differences (F14 = 0.62, P = NS) were not detected [susceptible sedentary (n = 3) 66 ± 76.2%; susceptible exercise (n = 4), 113 ± 54%; resistant (n = 8) 100 ± 51%] (Fig. 5B).


Figure 5
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  		Fig. 5. beta1-Adrenoceptor protein content in canine left ventricle as determined by Western blot analysis after tissue extraction by Triton X-100 (A) or SDS (B). Protein content is expressed as % of the amount in the resistant dogs. Fifty micrograms of protein was loaded per lane on Triton X-100 gels, and 30 µg of protein was loaded per lane on SDS gels. n = No. of dogs per treatment group; each sample analyzed on 2 or 3 gels. In the Triton X-100-extracted samples, beta1 protein content in susceptible (Sus) sedentary (Sed) dogs was significantly less than that in resistant (Res) dogs (*P < 0.05). Data are plotted as means ± SD.

 
beta2-AR content was significantly less in resistant dogs than in either susceptible treatment group when Triton X-100 was used to isolate the protein. These data suggest that beta2-ARs were sequestered in nonextractable caveolae in the resistant group [susceptible sedentary (n = 4) 318 ± 132%; susceptible exercise (n = 4) 268 ± 52%; resistant (n = 8) 100 ± 48.1%] (F15 = 12.87, P < 0.001; Fig. 6A). This pattern was not repeated with SDS extraction; the quantity of beta2-ARs was equivalent in all treatment groups [resistant (n = 8) 100 ± 45.3%; susceptible exercise (n = 4) 82 ± 48%; susceptible sedentary (n = 3) 88 ± 12%] (F14 = 0.27, P = NS; Fig. 6B). These protein quantities were consistent with beta2-AR mRNA expression as determined by RT-PCR reported above.


Figure 6
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  		Fig. 6. beta2-Adrenoceptor protein content in canine left ventricle as determined by Western blot analysis after tissue extraction by Triton X-100 (A) or SDS (B). Protein content is expressed as % of the amount in the resistant dogs. Fifty micrograms of protein was loaded per lane on Triton X-100 gels, and 30 µg of protein was loaded per lane on SDS gels. n = No. of dogs per treatment group; each sample analyzed on 2 or 3 gels. beta2-Adrenoceptor protein content in Triton X-100 samples was 3-fold lower in resistant dogs compared with either susceptible sedentary or susceptible exercise-trained group (*P < 0.001). There were no differences in the groups after SDS extraction. Data are plotted as means ± SD.

 
The amount of caveolin-3 extracted by Triton X-100 was significantly (F15 = 4.66, P < 0.05) less in the susceptible exercise-trained group (n = 4, 54 ± 28%) than in either the resistant (n = 8, 100 ± 27%) or susceptible sedentary group (n = 4, 108 ± 26%) (Fig. 7A), although total caveolin-3 protein was not different between groups when extracted by SDS [resistant (n = 8) 100 ± 39.6%; susceptible sedentary (n = 3) 113 ± 42%; susceptible exercise (n = 4) 100 ± 30%] (F14 = 0.14, P = NS; Fig. 7B).


Figure 7
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  		Fig. 7. Caveolin-3 protein content in canine left ventricle as determined by Western blot analysis after tissue extraction by Triton X-100 (A) or SDS (B). Protein content is expressed as % of the amount in the resistant dogs. Fifty micrograms of protein was loaded per lane on Triton X-100 gels, and 30 µg of protein was loaded per lane on SDS gels. n = No. of dogs per treatment group; each sample analyzed on 2 or 3 gels. Exercise decreased caveolin-3 protein detectable after Triton X-100 extraction in susceptible dogs (*P < 0.05). Data are plotted as means ± SD.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings of the present study are as follows: 1) beta1-AR gene expression and protein content were decreased in dogs susceptible to arrhythmias; 2) a 10-wk endurance exercise training regimen restored beta1-AR expression and protein content in dogs susceptible to VF; 3) beta2-AR gene expression and protein content were not different between dogs susceptible or resistant to arrhythmias; and 4) in contrast to the susceptible dogs, beta2-ARs from the resistant animals resisted extraction by Triton X-100. These data indicate that the beta2-ARs reside within caveolae in the resistant dogs. This partitioning into different regions of the sarcolemma could lead to alterations of beta2-AR-mediated cell signaling pathways.

In both human and animal models of heart failure, beta1-AR to beta2-AR content ratio in LV myocardium is decreased to a extent similar to that found in the present study (19, 27, 33). As a consequence, the failing heart becomes more dependent on beta2-ARs for inotropic support. However, the recruitment and activation of these "latent" beta2-ARs can also alter cardiac electrical stability, increasing the propensity for the formation of malignant arrhythmias. In fact, we previously demonstrated (10, 18) that the activation of beta2-AR provoked large increases in calcium transient amplitude and aftercontractions (cellular arrhythmias) in myocytes from dogs prone to malignant arrhythmias, while the selective beta2-AR antagonist ICI-118551 prevented VF induced by acute ischemia in animals with healed MIs.

We recently demonstrated (11) that exercise training significantly reduced both the in vivo and the in vitro contractile response to beta2-AR stimulation in animals known to be susceptible to VF. In fact, after training, the beta2-AR response of the susceptible animals was indistinguishable from that noted in dogs resistant to malignant arrhythmias. In marked contrast, the beta2-AR responsiveness increased even further in susceptible animals after the completion of a comparable sedentary period (11). The beta2-AR response was also reduced in isolated myocytes; the beta2-AR agonist zinterol elicited significantly larger increases in cell shortening in myocytes obtained from the susceptible sedentary group than in cells from either resistant or susceptible exercise-trained animals (11). As a consequence of the attenuated beta2-AR responsiveness, exercise training also completely suppressed the VF induced by acute myocardial ischemia (7, 11). However, this study did not investigate whether the improved beta2-AR responsiveness resulted from altered beta-AR gene expression or beta-AR protein abundance in the cell membrane. The present study extends these previous findings, demonstrating that exercise training elicited significant increases in beta1-AR protein and mRNA with corresponding improvements in beta2-AR to beta1-AR ratio.

In agreement with the present study, exercise conditioning has been shown to reduce cardiac sympathetic activity (38) and to improve beta-AR responsiveness in normal animals (4, 42), in aged animals (25), and in animals with cardiovascular disease (23). The improved beta1-AR responsiveness was accompanied by either reduction (4) or no change (23, 25) in beta1-AR density. For example, in normal Wistar rats, daily exercise provoked significant reductions in beta1-AR density without altering either beta2-AR or beta3-AR density (23).

The predicted molecular mass of beta1-ARs and beta2-ARs is ~51 and 47 kDa, respectively (37). On Triton X-100 extraction, immunoreactivity of beta1-ARs was detectable as a band at 65 kDa (weak), a doublet at 51 kDa, and a doublet at 45 kDa (data not shown). These molecular species correspond to those reported previously (37), and all exhibit decreased levels in susceptible sedentary vs. resistant dogs. However, the 51-kDa species was the prominent band detected after SDS extraction (65 kDa was only detected after prolonged exposure to film). Therefore the data presented here are limited to the 51-kDa species of beta1-AR for comparison purposes. With respect to the beta2-AR, Triton X-100 extraction presented immunoreactive bands at 65 and 51 kDa (not shown), each showing decreased abundance in resistant vs. susceptible animals. However, with SDS extraction only the 65-kDa immunoreactive band was apparent for the beta2-AR, and analysis of this band was performed for comparison purposes.

While beta1-ARs reside throughout the cell membrane, beta2-ARs are located in caveolae, flask-shaped invaginations in the cell membrane (13, 29, 37). It is thought that this differential cellular localization, as well as colocalization of key signaling molecules in the caveolae, is responsible for the differences in beta1-AR and beta2-AR signaling cascades noted in rat cardiomyocytes (13). Caveolae contain high concentrations of sphingolipids and cholesterol and are insoluble in Triton X-100 at 4°C, whereas cytosolic and Golgi apparatus-associated caveolins are Triton X-100 soluble (34). This could explain why caveolin-3 was detected in the Triton X-100 dog heart extracts (Fig. 7A).

Caveolae are thought to modulate excitation-contraction coupling by increasing the efficiency of the calcium-induced calcium release process rather than calcium influx (13). This recent study using adult rat ventricular myocytes suggests that Gi signaling pathway components must interact with caveolin in caveolae for effective signal transduction. Inotropic responses to beta1-AR stimulation were not affected by caveolar disruption. However, beta2-AR stimulation resulted in a three- to fivefold increase in cell shortening, calcium transients, and L-type calcium channel current (ICa,L) following caveolar disruption. Disruption of caveolae would result in redistribution of beta2-ARs to the cell membrane, thereby allowing for a closer association and interaction with beta1-ARs. As a consequence of a decreased abundance of beta1-ARs, the resulting mix of beta1-ARs and beta2-ARs on the surface of the cell membrane could lead to an increased probability of receptor heterodimerization (30, 46). Heterodimerization of beta1-ARs and beta2-ARs can produce enhanced sensitivity to isoproterenol, decreased internalization of beta2-ARs, and decreased signaling of beta2-ARs through Gi (46).

Interestingly, exercise training decreased the amount of caveolin-3 detected in Triton X-100, but not SDS, extracted samples. These data are consistent with an exercise training-induced alteration in the distribution of caveolin within caveolae. Reformation of caveolae following exercise training could restore beta2-AR signaling pathways that only occur in caveolae. These results contrast with previous studies in rat that reported no change in total caveolin-3 in senescent rats 3 mo after MI but an increase in Triton-extractable caveolin-3 after infarction (34), suggesting a redistribution of caveolin to cytosolic, Golgi, or cell membrane fractions. Furthermore, voluntary exercise training in rats provoked an increase in caveolin-3 expression (3), while cardiac hypertrophy (subsequent to 7 wk of hypertension) significantly decreased caveolin-3 extracted with SDS from the canine myocardium (31). In the present study, endurance exercise training produced a small (<10%) increase in ventricular systolic wall thickness (as measured by echocardiography) in both resistant and susceptible dogs (11), while caveolin-3 decreased in susceptible exercise-trained dogs. A similar reduction in caveolin-3 expression (28) has been reported in mice after chronic beta-AR stimulation. Therefore, species differences or differences in the model of cardiac disease may account for the observed differences in caveolin-3 expression discussed above.

Limitations of Study

LV dysfunction is known to alter beta-adrenergic responsiveness (1, 12). However, there was no evidence of ventricular dysfunction or dilation (determined by echocardiography) after MI in a subset of resistant or susceptible dogs. Furthermore, we previously reported (11) that neither baseline nor beta-AR agonist-induced contractile changes were different in the susceptible dogs before either a 10-wk sedentary or a 10-wk exercise training period; differences were only noted after the completion of the 10-wk study. Thus it is unlikely that initial differences in ventricular function can explain the results. Similarly, innate (preexisting) variability in beta-AR responsiveness could contribute to the difference noted between the susceptible and resistant dogs. However, both beta-AR mRNA and beta-AR protein abundance were similar in control (no infarct) and infarcted (resistant sedentary) dogs. These data demonstrate that beta-AR expression in the resistant dogs was similar to that in animals without infarction. Furthermore, we previously demonstrated (18) that beta-AR responsiveness was similar in both resistant and susceptible dogs before MI; the differences were only noted after infarction. When considered together, these data demonstrate that the differences in beta-AR expression occurred as a consequence of the MI rather than innate beta-AR variability. The studies were performed with whole tissue samples rather than purified myocyte preparations. As such, the differences in beta-AR expression reported in the present study could reflect exercise-induced changes in noncardiac muscle cells. However, the present results are consistent with the functional data obtained from both intact preparations and isolated myocytes (11). Thus changes in cardiac myocyte beta-AR expression were most likely responsible for the functional changes induced by exercise training.

Finally, it is important to emphasize that exercise training not only alters cardiac beta-AR balance but also enhances cardiac parasympathetic regulation (5, 7, 8, 11, 38). Because it is well established that cardiac vagal activation can prevent ischemically induced arrhythmias (40, 43), exercise training-induced changes in cardiac parasympathetic regulation could also contribute to the protection from VF noted in the present study. Exercise training did, in fact, improve cardiac parasympathetic regulation (increased heart rate variability), which was maintained even when the heart was stressed by either exercise or acute ischemia (7). Furthermore, exercise training both improved baroreflex sensitivity (BRS) and reduced cardiac mortality in patients recovering from MI (22). Thus exercise training augmented cardiac parasympathetic regulation that was associated with a reduced risk for cardiac mortality. Further investigation will be required to determine the relative contributions of alterations in cardiac parasympathetic regulation and/or beta-AR balance for the antiarrhythmic actions of exercise training.

Clinical Implications

There are at least two findings of the present study that have potentially important clinical implications: 1) an enhanced beta2-AR activation is associated with greater susceptibility to VF; and 2) exercise training can restore a more normal beta-AR balance and prevents malignant arrhythmias induced by ischemia.

Clinical studies have not extensively evaluated the contribution of beta2-ARs to cardiac mortality. A small clinical trial found that the beta2-AR agonist salbutamol increased episodes of ventricular tachycardia in patients with congestive heart failure (26). In a similar manner, there are a few case reports in which beta2-AR agonists have precipitated sudden death as a consequence of the cardiac actions of these agents in asthmatic patients (35, 36). More compelling, but indirect, evidence in support of the beta2-AR hypothesis is provided by analysis of the numerous beta-AR antagonist trials. There is overwhelming evidence that beta-AR antagonists can protect against arrhythmia formation induced by myocardial ischemia and infarction (15, 21). Indeed, this marked reduction in cardiac mortality has been verified in at least 32 trials involving ~29,000 patients (15, 21). However, if one carefully examines the clinical studies cited above, it then becomes apparent that not all beta-AR antagonists offer the same level of protection, particularly during acute MI. The majority of the studies using the beta1-AR antagonist metoprolol failed to report significant reductions in the incidence of VF during acute MI (16, 17). Furthermore, although atenolol did reduce overall mortality by 15%, the number of patients who died as the result of malignant arrhythmias was not altered (20). In contrast, propranolol therapy elicited large reductions in both overall mortality (65% decrease) and sudden cardiac death (41% decrease) in post-MI patients with persistent ST segment depression (41), a group of patients known to be at a particularly high risk for subsequent cardiac events (44). Thus a better antiarrhythmic protection can be achieved with complete (i.e., beta1- and beta2-AR), rather than selective (i.e., beta1), beta-AR blockade.

The data from the present study further demonstrate that exercise training can restore a more favorable beta-AR balance that is accompanied by a complete suppression of ischemically induced VF (11). In agreement with the present study, exercise training both improved cardiac autonomic regulation (as measured by an increase in BRS) and significantly reduced cardiac mortality during a 10-yr follow-up period (22). In fact, no patient in whom exercise training induced BRS increases of >3 ms/mmHg died during the study, while 23% (18 of 79) of the nontrained or trained patients that did not improve BRS died during the 10-year study period. Thus exercise training represents a potent nonpharmacological antiarrhythmic intervention.

In summary, although beta2-AR gene expression and total protein content were not different in dogs resistant or susceptible to VF, it appears that the membrane compartmentalization of the beta-ARs was different in the two groups; more beta2-ARs were present in the noncaveolar (i.e., Triton X-100 extractable) fraction of the cardiac membrane in dogs susceptible to VF compared with animals resistant to malignant arrhythmias. Exercise did not restore beta2-ARs to a sequestered site but did normalize the ratio of beta1-ARs to beta2-ARs, thereby restoring normal signaling pathways.


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These studies were supported by National Heart, Lung, and Blood Institute Grant HL-68609.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. E. Billman, Dept. of Physiology and Cell Biology, Ohio State Univ., 304 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210-1218 (e-mail: billman.1{at}osu.edu)

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


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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