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CARDIOVASCULAR

₂-Adrenergic Stimulation Attenuates Left Ventricular Remodeling, Decreases Apoptosis, and Improves Calcium Homeostasis in a Rodent Model of Ischemic Cardiomyopathy

Departments of Surgery (S.X., A.R.K., J.S.C., C.J.M., G.W.M., A.R.S., M.C.O.) and Medicine (S.K., I.H., A.G., J.W.), Columbia University College of Physicians and Surgeons, New York, New York; Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland (D.G., C.W.); Bernards High School, Bernardsville, New Jersey (D.H.); Faculty of Engineering, The University of Hong Kong, Hong Kong (E.X.W.); The Jack Skirball Center for Cardiovascular Research, Orangeburg, New Jersey (J.W.); and The Medical School, Nanjing University, Nanjing, People's Republic of China (J.W.)

Received December 3, 2005; accepted January 17, 2006.

	Abstract

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The benefit of the ₂-adrenergic agonist, clenbuterol, in left ventricular assist device patients with dilated cardiomyopathy has been reported, but its effect on ischemic heart failure (HF) is unknown. We investigated whether clenbuterol improves left ventricular remodeling, myocardial apoptosis and has synergy with a ₁ antagonist, metoprolol, in a model of ischemic HF. Rats were randomized to: 1) HF only; 2) HF + clenbuterol; 3) HF + metoprolol; 4) HF + clenbuterol + metoprolol; and 5) rats with sham surgery. HF was induced by left anterior descending artery (LAD) artery ligation and confirmed by decreased left ventricular fractional shortening, decreased maximum left ventricular dP/dt (dP/dt_max), and elevated left ventricular end-diastolic pressure (LVEDP) compared with sham rats (p < 0.01). After 9 weeks of oral therapy, echocardiographic, hemodynamic, and ex vivo end-diastolic pressure-volume relationship (EDPVR) measurements were obtained. Immunohistochemistry was performed for myocardial apoptosis and DNA damage markers. Levels of calcium-handling proteins were assessed by Western blot analysis. Clenbuterol-treated HF rats had increased weight gain and heart weights versus HF rats (p < 0.05). EDPVR curves revealed a leftward shift in clenbuterol rats versus metoprolol and HF rats (p < 0.05). The metoprolol-treated group had a lower LVEDP and higher dP/dt_max versus the HF group (p < 0.05). Clenbuterol and metoprolol groups had decreased myocardial apoptosis and DNA damage markers and increased DNA repair markers versus HF rats (all p < 0.01). Protein levels of the ryanodine receptor and sarcoplasmic reticulum calcium-ATPase were improved in clenbuterol-, metoprolol-, and clenbuterol+metoprolol-treated groups versus HF rats. However, as a combination therapy, there were no synergistic effects of clenbuterol+metoprolol treatment. We conclude that clenbuterol ameliorates EDPVR, apoptosis, and calcium homeostasis but does not have synergy with metoprolol in our model of ischemic HF.

Interest in clenbuterol has been recently sparked as a potential treatment for cardiac diseases, specifically in regard to improving cardiac mechanical properties. Petrou et al. (1995) found that clenbuterol led to hypertrophy of latissimus dorsi and cardiac muscle in rats. Subsequent work by this group found that clenbuterol promotes cardiac hypertrophy in rats after banding of the ascending aorta (Wong et al., 1997). Normal rat hearts treated with clenbuterol have also been shown to have elements of "physiologic" hypertrophy, with normal function, morphology, and calcium-handling mRNA levels (Wong et al., 1998). Finally, this group demonstrated improved right ventricular systolic function with clenbuterol therapy after induction of right-sided failure by pulmonary artery banding in sheep (Hon et al., 2001).

An intriguing subsequent report describes the use of clenbuterol (in combination with angiotensin-converting enzyme inhibition, ₁-selective blockade, and spironolactone) in patients with nonischemic, dilated cardiomyopathy supported with a left ventricular assist device (Yacoub et al., 2001). Ten of 15 patients treated with clenbuterol in this study had significant cardiac improvement, allowing for assist device explantation for recovery (Hon and Yacoub, 2003). This series represents a rate of myocardial recovery that is more than double that of any previously reported study. This preliminary case series has not yet been confirmed by controlled studies; in addition, the effects of clenbuterol on ischemic cardiomyopathy have not been evaluated to date in any experimental or clinical study.

The interest in the use of ₂-adrenergic agonists such as clenbuterol in heart failure also stems from the recent evidence that the toxic effects of -adrenergic stimulation is mediated primarily via ₁ receptors (Communal et al., 1999; Zhu et al., 2001, 2003), whereas ₂ receptor stimulation may be protective (Communal et al., 1999; Chesley et al., 2000; Ahmet et al., 2004). Myocardial apoptosis has been implicated as a possible mechanism in the pathogenesis of heart failure progression (Kang and Izumo, 2000) and has been correlated with the degree of left ventricular remodeling (Abbate et al., 2003). Apoptosis in postinfarction heart failure has been shown to be primarily mediated via ₁-adrenergic receptors (Prabhu et al., 2003). This raises the possibility that combination therapy with a ₁ blocker such as metoprolol and a ₂ agonist such as clenbuterol may be potentially synergistic in their effects on heart failure.

We used a well established model of ischemic chronic heart failure in rats to study: 1) the effects of clenbuterol on cardiac function and ventricular remodeling in ischemic cardiomyopathy both alone and in combination with metoprolol, and 2) the underlying effects of clenbuterol on myocardial apoptosis and calcium homeostasis. For this latter objective, we evaluated the effects of clenbuterol on markers of apoptosis, DNA damage, and DNA repair in our chronic model of heart failure. In addition, we studied the effects of clenbuterol on calcium-handling protein expression levels of the ryanodine receptor and sarcoplasmic reticulum calcium-ATPase (SERCA_2a).

	Materials and Methods

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Induction of Chronic Heart Failure. Studies were performed in compliance with the Guide for the Care and Use of Laboratory Animals and were approved by the Columbia University Institutional Animal Care and Use Committee. Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 250 to 300 g were used for all experiments.

After induction with intraperitoneal ketamine (75 mg/kg; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (5 mg/kg; Lloyd Laboratories, Shenandoah, IA), endotracheal intubation with an angiocatheter was performed. Rats were supported by a small animal ventilator (Harvard Apparatus, Holliston, MA). After performing a left thoracotomy, a sham operation (pericardiectomy only) or left anterior descending artery (LAD) ligation was performed, as previously described (Kherani et al., 2004).

A total of 69 rats were used in this study. LAD ligation surgery was performed in 60 rats, of which 37 (62%) survived 3 weeks after surgery. Another nine rats underwent a sham operation (group sham), with seven (78%) survivors. Three weeks postoperatively, echocardiography was performed to establish the baseline level of heart failure (measured by fractional shortening). The LAD ligation rats were divided into four treatment groups, matched for the degree of heart failure, and were randomly assigned to one of four therapies for 9 additional weeks: 1) rats receiving no therapy (group heart failure, n = 9); 2) rats receiving clenbuterol at 1 mg/kg/day (group clenbuterol, n = 9); 3) rats receiving metoprolol at 200 mg/kg/day (group metoprolol, n = 9); or 4) rats receiving concurrent clenbuterol at 1 mg/kg/day and metoprolol therapy at 200 mg/kg/day (group Clen+Meto, n = 10).

Of the 44 surviving rats (37 post-LAD ligation and seven sham rats), 39 (89%) survived the 12-week follow-up period. There were no differences in survival rates among the groups undergoing LAD ligation. There were also no differences in left ventricular infarct size, expressed as a percentage of the left ventricular circumference, among the groups. The LAD ligation rats all had a myocardial infarction of sufficient size to induce heart failure, spanning 20% of the left ventricular circumference (mean, 38.1 ± 12.3%; range, 20 to 63%).

Oral Pharmacotherapy. Clenbuterol (ICN Biomedicals, Aurora, OH) was sonicated and dissolved in the drinking water. Metoprolol (Sigma-Aldrich, St. Louis, MO) was dissolved in the drinking water either alone or in combination with clenbuterol. The study drug concentrations were varied to keep the dose delivered within a narrow therapeutic window based on water consumption.

Echocardiography. Under mild isoflurane anesthesia, two-dimensional echocardiography (Sonos-5500; Agilent Technologies, Palo Alto, CA) was performed 3 and 12 weeks after surgery for pre- and post-treatment measures of cardiac function and analyzed in a blinded fashion. Left ventricular anteroposterior diameter and short-axis area at the papillary muscle level were measured to obtain the left ventricular end-diastolic and end-systolic diameter and area. Fractional shortening and fractional area change were subsequently calculated.

Hemodynamic Measurements. In the terminal experiments 12 weeks after surgery, rats were anesthetized with inhaled isoflurane. A 2-French Millar catheter (Millar Instruments, Houston, TX) was inserted into the right carotid artery, and pressure measurements were collected as the catheter was advanced from the aorta into the left ventricle. Hearts were subsequently weighed and used for ex vivo determination of left ventricular end-diastolic pressure-volume relationships (EDPVR). Using Chart 4 (version 4.2.4; ADInstruments, Colorado Springs, CO), left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure, mean aortic pressure, heart rate, and maximum and minimum left ventricular dP/dt (dP/dt_max and dP/dt_min) were later obtained from hemodynamic recordings in a blinded fashion.

End-Diastolic Pressure-Volume Relationship Determinations. An angiocatheter was placed into the left ventricle through the aortic valve. A fine hemostat was placed on the atrial side of the mitral annulus to seal the left ventricle. Left ventricular pressures were measured using a 5-French Millar micromanometer introduced through the angiocatheter. While recording left ventricular pressure, saline was infused into the left ventricle in 50-µl increments using a calibrated syringe. The infused fluid was withdrawn and measured to ensure that no leakage had occurred. Using commercial software (Igor Pro, version 4.0.5.1 [EC] ; WaveMetrics, Lake Oswego, OR), values of left ventricular pressure and volume were fitted according to the equation: Pressure = Volume, where is the base constant and is an index of ventricular stiffness, as previously described (Mirsky, 1976; Kherani et al., 2004). Data were averaged to construct the mean EDPVR tracings for each group after normalizing left ventricular volumes for differences in heart weight, as reported previously (Amirhamzeh et al., 1997; Rabkin et al., 1998; Burkhoff et al., 2005). Analyses were performed in a blinded manner. Comparisons between groups were made based on normalized volume measurements at left ventricular pressures of 30 mm Hg (Burkhoff et al., 2005).

Histological Analysis. A short-axis section of the heart at the point of maximal infarction was fixed in 4% paraformaldehyde solution. Sections were embedded in paraffin, and 5-µm slices were used for trichrome staining. The infarct size was determined as a percentage of the left ventricular circumference in a blinded fashion. The remaining heart tissue was flash-frozen and stored at –80°C for Western blot analysis.

Assessments of Myocyte Apoptosis, DNA Damage, and DNA Repair Enzyme Expression. Five rat hearts from each group were randomly selected and used for analysis of apoptosis and DNA damage and repair markers. A comparable area of the infarct border zone was used for these studies. To quantify the proportion of cells with DNA fragmentation, an in situ terminal deoxynucleotidyltransferase end labeling (TUNEL) assay (Oncor, Gaithersburg, MD) was performed, as previously described (Lin et al., 2003). After deparaffinizing, sections were incubated with terminal deoxynucleotidyl transferase buffer (Boehringer Mannheim, Indianapolis, IN) containing terminal deoxynucleotidyl transferase enzyme (Boehringer Mannheim) and biotin-16-dUTP (Boehringer Mannheim). Sections were stained with diaminobenzadine and counterstained with hematoxylin. The percentage of TUNEL-positive cells was quantified as an overall percentage of counted cells.

Of all known oxidative DNA damage products, human 8-oxo-7,8-dihydrodeoxyguanine (8-oxoG) has been shown to be the most stable and important (Lin et al., 2003). The degree of DNA damage was therefore determined by staining with monoclonal 8-oxoG antibody, as previously described (Lin et al., 2003). DNA was denatured by soaking paraffin-embedded sections in HCl. Nonspecific staining sites were blocked with 10% fetal bovine serum. Slides were incubated with H₂O₂ to block endogenous peroxidase. Thereafter, slides were incubated with primary anti-8-oxoG monoclonal antibody (diluted 1:100; Trevigen, Gaithersburg, MD) and with secondary anti-mouse antibody (1:100) conjugated with streptavidin-horseradish peroxidase. Slides were stained with diaminobenzamide and counterstained with methyl green. The percentage of cells staining positive for 8-oxoG was quantified as an overall percentage of counted cells.

Human MutY homolog and 8-oxoG glycosylase are proteins that have been shown to play important roles in repairing DNA mismatch injury (Slupska et al., 1996; Arai et al., 1997). Their expression patterns were used to evaluate the DNA repair enzyme activity using both immunohistochemistry and immunoblot analysis, as we have recently described (Lin et al., 2003). Sections were blocked in 2% normal horse serum and incubated with primary human MutY homolog or 8-oxoG glycosylase antibody (both 1:100 dilution; Novus Biological, Littleton, OH). After quenching endogenous peroxidase activity with H₂O₂, the slides were incubated with secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences, Piscataway, NJ). The final reaction was achieved by incubating the sections with freshly prepared reagent containing 3-amino-9-ethylcarbazole (Sigma-Aldrich). Mounted sections were counterstained with hematoxylin. Two trained, blinded observers reviewed sections, with at least three samples scored per group. Human MutY homolog and 8-oxoG glycosylase expression was evaluated by scoring the percentage of positive staining on the section as we previously described (Wei et al., 1993, 1994). According to this semiquantitative scoring system, 0 = no staining, 1 = minimal staining (<10% positive), 2 = mild staining (10–30% positive), 3 = moderate staining (31–50% positive), and 4 = strong staining (>50% positive). The specificity of positive staining was confirmed by substitution of normal rabbit serum for the primary antiserum.

Western blot analysis for human MutY homolog and 8-oxoG glycosylase was performed as described previously (Lin et al., 2003). Protein samples were run on SDS-polyacrylamide gel electrophoresis using a 10% polyacrylamide gel. After transfer of proteins to nitrocellulose, membranes were placed in 1% powdered milk to block nonspecific binding. After reacting with the primary and secondary antibodies, the membrane was subjected to the Enhanced Chemiluminescence analysis system (Amersham Biosciences). Monoclonal antibody against actin (Ab-6; Oncogene Research Products, MA) was used to control for differences in protein loading. To ascertain specific binding of the anti-human MutY homolog or 8-oxoG glycosylase antibody, a control membrane was studied without this primary antibody.

Calcium-Handling Protein Expression Levels. Five random heart samples from each group were similarly used to analyze calcium-handling protein expression levels. Approximately 150 mg of left ventricular tissue was lysed and samples denatured at 95°C and size-fractionated using SDS-polyacrylamide gel electrophoresis using 7.5% separating and 5% stacking gels for SERCA_2a and 5% separating and 4% stacking gels for the ryanodine receptor. Proteins were transferred onto nitrocellulose. Blots were blocked in 5% nonfat milk. Blots were incubated with diluted primary antibody (anti-SERCA_2a: 1:1000, ABR Affinity BioReagents, Golden, CO; anti-ryanodine receptor: 1:2500, a gift from Dr. Andrew Marks' laboratory, Columbia University of Physicians and Surgeons; anti-tubulin: 1:1000, Sigma-Aldrich). Blots were incubated in the presence of a horseradish peroxidase-labeled secondary antibody (SERCA_2a: anti-mouse IgG, Amersham Biosciences; ryanodine receptor: anti-rabbit IgG, Amersham Biosciences) diluted 1:4000. Blots were developed using an enhanced chemiluminescence reagent (Amersham Biosciences). Optical densities of protein signals were quantified using a densitometer (Molecular Dynamics, Palo Alto, CA) in a blinded manner. SERCA_2a and ryanodine receptor protein levels were expressed relative to levels of tubulin.

Statistical Analysis. All statistical analysis was performed using SPSS 11.5 software (SPSS, Chicago, IL). Comparisons of echocardiographic and body weight data between pre- and post-treatment time points were performed with the use of repeated measures analysis of variance, with the group, infarct size, and time point as fixed factors. Comparisons between treatment groups for hemodynamic, immunohistochemistry, and protein level data were made using a two-way analysis of variance, with the group and infarct size [categorized as a large (30% of left ventricular circumference) or small infarct (<30%)] as fixed factors. For dP/dt_max and dP/dt_min, nonparametric tests (Kruskal-Wallis) were used because these variables were not normally distributed. The presence of synergy with combination pharmacological therapy was assessed with the use of multivariate analysis of variance, including interaction terms for the combination therapy cohort. Tukey's ad hoc tests were used for all group comparisons. A p value of less than 0.05 was considered significant. All data are expressed as a mean ± standard deviation.

	Results

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Echocardiographic Data. Figure 1 depicts the echocardiographic data. At 3 weeks after surgery, there was a significantly lower fractional shortening (17.4 ± 5.22%) and fractional area change (29.4 ± 6.20%) in the four LAD ligation groups compared with sham rats (fractional shortening, 50.8 ± 6.78%; fractional area change, 72.3 ± 6.89%; p < 0.001). There were no differences in the fractional shortening or fractional area change among the four LAD ligation groups before or after 9 weeks of oral pharmacotherapy.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Echocardiographic data as expressed by fractional shortening and fractional area change. Each of the LAD ligation groups had a significantly lower fractional shortening and fractional area change than the sham animals. There were no significant changes in fractional shortening or fractional area change between the baseline and endpoint parameters for any of the groups.

Body and Heart Weights. The body weight and heart weight data are shown in Table 2. The percent change in the body weight was significantly higher in the sham rats compared with the LAD ligation groups. Treatment with clenbuterol alone and in combination with metoprolol, however, led to higher increases in body weight compared with the control heart failure and metoprolol-treated groups. For heart weight, sham rats had lower weights than the LAD ligation cohorts. Clenbuterol-treated rats had significantly higher heart weights than both control heart failure and metoprolol-treated animals.

End-Diastolic Pressure-Volume Relationship Tracings. The ex vivo, passive EDPVR curves obtained are shown in Fig. 2. There was a rightward shift for heart failure, metoprolol, and Clen+Meto versus sham rats. In contrast, clenbuterol-treated rats (volume at 30 mm Hg: 0.42 ± 0.05 ml/g of heart weight) had lower left ventricular chamber volumes than either metoprolol or heart failure rats (volume at 30 mm Hg: 0.51 ± 0.08 and 0.50 ± 0.09 ml/g of heart weight, respectively; both p < 0.05) and were no different from sham rats (0.36 ± 0.03 ml/g of heart weight). This signifies that at a given left ventricular pressure, clenbuterol led to lower left ventricular volumes versus right-shifted heart failure rats with dilated and remodeled ventricles.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Ex vivo EDPVR curves, after normalization of left ventricular volumes for differences in heart weights. Clenbuterol-treated rats were shifted to the left versus both control heart failure and metoprolol rats and were no different from sham rats. This signifies that at a given left ventricular pressure, clenbuterol led to lower left ventricular volumes versus right-shifted heart failure rats with dilated and remodeled ventricles. In contrast, heart failure, metoprolol, and Clen+Meto rats had higher passive left ventricular volumes than sham rats.

View larger version (72K): [in this window] [in a new window]   Fig. 3. Representative immunohistochemistry staining patterns for the apoptosis, DNA damage, and DNA repair markers (original magnification, 1000x). A, TUNEL. B, 8-oxoG (OGG1, DNA damage). C, 8-oxoG glycosylase (OGG1, DNA repair). D, human MutY homolog (MYH, DNA repair). Representative autoradiographs of the DNA repair protein expression levels are shown (E and F) in comparison to -actin levels. 8-OxoG glycosylase and human MutY homolog levels were increased in the clenbuterol, metoprolol, and Clen+Meto groups compared with the heart failure and sham cohorts. Combination therapy with clenbuterol and metoprolol also led to higher 8-oxoG glycosylase and human MutY homolog levels than metoprolol therapy alone.

For the human MutY homolog and 8-oxoG glycosylase (markers of DNA repair), there was increased levels of both markers versus the sham group for the clenbuterol, metoprolol, and Clen+Meto animals. Clenbuterol and metoprolol treatment led to improvements both alone and in combination versus the control heart failure group. Interestingly, Clen+Meto treatment led to additive improvement in the 8-oxoG glycosylase staining pattern over either clenbuterol or metoprolol therapy alone. Of note, there was no synergy noted in the effects of Clen+Meto therapy in any of the apoptosis, DNA damage, or DNA repair marker staining patterns.

Calcium-Handling Protein Expression Levels. Figure 4 depicts the calcium-handling protein expression levels for the ryanodine receptor and SERCA_2a. Heart failure rats had decreased levels of both the ryanodine receptor and SERCA_2a versus sham rats. SERCA_2a levels were up-regulated in the clenbuterol, metoprolol, and Clen+Meto groups, although the increases reached statistical significance only for clenbuterol (p < 0.05 versus the heart failure group). Ryanodine receptor protein expression levels were normalized in the clenbuterol, metoprolol, and Clen+Meto cohorts with levels not detectably different from sham animals. The SERCA_2a protein expression level was also partially normalized by metoprolol and Clen+Meto treatment.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Calcium-handling protein expression levels of the ryanodine receptor (A) and SERCA2a (B), expressed relative to tubulin expression levels. Representative autoradiographs, with the corresponding tubulin blotting signals, are also depicted. Ryanodine receptor and SERCA2a levels were decreased in the heart failure (HF) group compared with the sham (S) group. Clenbuterol (C)-, metoprolol (M)-, and Clen+Meto (C+M)-treated rats had increased levels of the ryanodine receptor. For SERCA2a, a trend toward up-regulation was seen in all three treated groups, although the increases achieved significance only for clenbuterol.

	Discussion

Top Abstract Materials and Methods Results Discussion Conclusions References

Based on echocardiographic and histological data, we were able to achieve a significant degree of heart failure in our experimental model. The size of the left ventricular infarction attained was uniform across treatment arms. The fractional shortening and fractional area change demonstrated a significant decrease in systolic function in our chronic model of heart failure. Finally, repeat echocardiographic and direct hemodynamic data at 3 months demonstrated both systolic and diastolic dysfunction in the control heart failure group versus sham rats.

As expected, clenbuterol treatment led to significant increases in heart weight and body weight over 9 weeks of oral pharmacotherapy. Clenbuterol has been previously shown to induce "physiological" myocyte hypertrophy (Petrou et al., 1995; Wong et al., 1998) and is known to have anabolic properties (Beckett, 1992; Maltin et al., 1993; Perry, 1993; Carter and Lynch, 1994). Other ₂ agonists, such as salbutamol, cimaterol, and fenoterol, have also been shown to have anabolic characteristics (Emery et al., 1984; Martineau et al., 1992; Byrem et al., 1998). Long-acting clenbuterol has more potent anabolic effects than short-acting salbutamol unless salbutamol is continuously infused, and these anabolic effects are blocked by selective ₂ antagonists (Choo et al., 1992). The mechanism for this anabolic effect may therefore be mediated by long-acting ₂-adrenergic stimulation (Choo et al., 1992).

In contrast, metoprolol treatment led to a decreased heart rate and LVEDP and an improved dP/dt_max with oral therapy. The improvements demonstrated with metoprolol therapy are similar to previously published reports in rat postinfarction models (Prabhu et al., 2000, 2003; Ahmet et al., 2004).

The dosages used for clenbuterol and metoprolol administration in our study were based on previously published reports of use in rodents. Clenbuterol was orally administered at 1 mg/kg/day, a dose similar to previous reports of its anabolic actions in skeletal muscle (Emery et al., 1984; Choo et al., 1992; Carter and Lynch, 1994) and comparable with dosing in larger animals in a recent study of clenbuterol's effects on cardiac function after pulmonary artery banding in sheep (Hon et al., 2001). Although serum clenbuterol levels were not monitored, observed changes in heart weight and body weight with oral clenbuterol pharmacotherapy suggest effective and appropriate drug delivery. In addition, metoprolol was orally administered at 200 mg/kg/day, a dose similar to two previous reports of its use in postinfarction rodent models (Prabhu et al., 2000, 2003). Although metoprolol drug levels were also not monitored in our experiment, our finding of a decreased heart rate in the metoprolol group is strong evidence of effective therapeutic delivery.

This is the first study demonstrating decreased ventricular remodeling with clenbuterol treatment, as seen by a leftward shift in the EDPVR of the clenbuterol group. Heart failure rats exhibited substantially dilated and remodeled left ventricular chamber volumes relative to noninfarcted sham rats, with a rightward shift of the pressure-volume curve. Clenbuterol therapy, however, caused a reduction in ventricular cavity dilation, shifting EDPVR leftward, strongly suggesting that clenbuterol attenuated deleterious postinfarction left ventricular remodeling. Our findings support the report by Hon et al. (2001), who found normal diastolic relaxation and stiffness with clenbuterol therapy in a study of sheep undergoing pulmonary artery banding. Finally, our results are similar to those by Ahmet et al. (2004), who used the ₂-adrenergic agonists fenoterol and zinterol in a rodent LAD-ligation model to demonstrate that ₂-adrenergic stimulation improves diastolic function in this experimental study of chronic heart failure.

We found no evidence of systolic improvements, however, in the clenbuterol-treated cohort. There was no change in echocardiographic measurements or dP/dt_max after 9 weeks of clenbuterol therapy. This is in contrast to the findings of Hon et al. (2001), who reported improved contractile and systolic function with clenbuterol treatment of sheep after pulmonary artery banding, but similar to the findings of Wong et al. (1997), who found no significant changes in systolic function with clenbuterol-treated rats after pulmonary artery banding.

In addition, we demonstrated significantly lower levels of calcium-handling proteins with the induction of chronic ischemic heart failure and up-regulation with clenbuterol, metoprolol, and combinational clenbuterol and metoprolol treatment, as seen by the protein expression levels of the ryanodine receptor. For SERCA_2a, a trend toward up-regulation was seen in all three treated groups, although the increases in SERCA_2a achieved significance only for clenbuterol. This is the first report to evaluate the effects of clenbuterol on calcium-handling protein levels in a model of heart failure. Our study is consistent with prior experimental reports evaluating the effects of clenbuterol on SERCA_2a mRNA levels in pressure-overloaded rodents (Wong et al., 1997). Furthermore, this is consistent with a recent study reporting improved calcium-handling in isolated myocytes obtained from a small series of dilated cardiomyopathy patients treated with clenbuterol while supported with a left ventricular assist device (Terracciano et al., 2003). This up-regulation of calcium-handling proteins carries considerable clinical significance, as altered calcium homeostasis has been implicated in the pathogenesis of heart failure (Yano et al., 2003; Braz et al., 2004), and represents one possible mechanism by which clenbuterol may lead to the observed attenuation of left ventricular remodeling.

We also demonstrated that clenbuterol led to decreased myocardial apoptosis, DNA damage, and increased DNA repair, as seen with the quantitative immunohistochemistry staining of key markers of apoptosis, DNA damage, and DNA repair. In addition, Western blot analysis of 8-oxoG glycosylase and human MutY homolog protein levels confirmed these findings. Decreased myocardial apoptosis via TUNEL staining has been previously shown in both in vivo experimental models (Ahmet et al., 2004) and cultured myocytes (Communal et al., 1999; Chesley et al., 2000; Zhu et al., 2001) with therapy with other ₂-adrenergic agonists. To our knowledge, this is the first study to evaluate the effects of ₂-adrenergic stimulation on DNA damage and repair. These improvements pose another plausible mechanism by which clenbuterol therapy may attenuate left ventricular remodeling, as myocardial apoptosis has been implicated in the remodeling process (Abbate et al., 2003). Inhibition of apoptosis pathways has also been recently shown to decrease left ventricular remodeling (Chandrashekhar et al., 2004).

Interestingly, the use of clenbuterol led to similar improvements in apoptosis, DNA damage, and DNA repair as metoprolol therapy. Prior reports have demonstrated that -adrenergic receptor-mediated apoptosis is largely independent of ₂ stimulation and mostly mediated via ₁ receptor pathways (Zaugg et al., 2000). In addition, prior studies have confirmed that ₁-adrenergic antagonists (Ahmet et al., 2004) and ₂-adrenergic agonists (Communal et al., 1999; Chesley et al., 2000; Zhu et al., 2001; Ahmet et al., 2004) lead to decreased myocardial apoptosis.

The combination of the ₂-adrenergic agonist clenbuterol and the ₁ antagonist metoprolol did not lead to evidence of synergy in this study. We present evidence of additive improvements in the 8-oxoG and 8-oxoG glycosylase staining patterns in the Clen+Meto group, indicating that clenbuterol and metoprolol led to independent improvements in DNA damage and repair, but these effects were not synergistic. Chesley et al. (2000) have shown that ₂-adrenergic stimulation decreases apoptosis through a pertussis toxin-sensitive, phosphatidylinositol-3'-kinase-dependent pathway in cultured myocytes. In contrast, ₁-adrenergic-mediated apoptosis has been recently shown to occur via a calmodulin kinase II pathway (Zhu et al., 2003; Zhang et al., 2005). The independence of these pathways is further supported with the presence of an additive, but not synergistic, effect in our study. Combination therapy with clenbuterol and metoprolol, however, did not lead to the same attenuation of ventricular remodeling as with clenbuterol alone (seen with EDPVR) or to the systolic improvements observed with metoprolol therapy alone (seen with dP/dt_max), suggesting that combination therapy antagonized or counteracted each of these beneficial effects.

Limitations. The dosages used for clenbuterol and metoprolol were high-dose, raising the possibility that synergy was not seen with combination therapy because of the counteraction of ₂-adrenergic effects with the use of an imperfectly selective ₁ antagonist. As mentioned above, drug serum levels were also not measured. In addition, the mechanisms by which clenbuterol attenuated ventricular remodeling were not elucidated by this study. Improvements in calcium-handling protein levels, apoptosis, DNA damage, and DNA repair are only implicated as possible mechanisms. Future studies will better characterize the effects and pathways of chronic ₂-adrenergic stimulation in ischemic heart failure, both alone and in combination with ₁-adrenergic blockade.

	Conclusions

Top Abstract Materials and Methods Results Discussion Conclusions References

 
We demonstrated that clenbuterol attenuates ventricular remodeling and ameliorates myocardial apoptosis, DNA damage and repair, and calcium homeostasis in an experimental model of ischemic heart failure. These changes did not have synergy with metoprolol therapy. Recently initiated trials studying the effects of clenbuterol on cardiac recovery will determine whether ischemic heart failure patients will sustain benefits from this therapy.

	Acknowledgements

 
We recognize the outstanding technical assistance of Chuck Ng and Geping Zhang toward the completion of this work.

	Footnotes

 
This study was supported in part by a resident training grant from the Department of Surgery, Columbia University College of Physicians and Surgeons and was supported in part by the Ministry of Education of the People's Republic of China (IRT0430).

Results from this study were partially presented at the American Heart Association Scientific Sessions 2004; Nov 7–10, 2004; New Orleans, LA: Xydas S, Klotz S, Hay I, Chang JS, Mutrie C, Gao D, Chen L, Ng C, Wang J, and Wei C (2004) Additive effects of -1 adrenergic antagonism and -2 stimulation on myocardial apoptotic inhibition and DNA repair in a model of congestive heart failure (Abstract). Circulation 110 (Suppl): III–5.

doi:10.1124/jpet.105.099432.

ABBREVIATIONS: SERCA_2a, sarcoplasmic reticulum calcium-ATPase; LAD, left anterior descending artery; Clen+Meto, clenbuterol and metoprolol; EDPVR, end-diastolic pressure-volume relationship; LVEDP, left ventricular end-diastolic pressure; dP/dt_max, maximum left ventricular dP/dt; dP/dt_min, minimum left ventricular dP/dt; TUNEL, terminal deoxynucleotidyltransferase end labeling; 8-oxoG, 8-oxo-7,8-dihydrodeoxyguanine; SERCA_2a, sarcoplasmic reticulum calcium-ATPase.

Address correspondence to: Dr. Jie Wang, The Medical School, Nanjing University, Nanjing 210093, People's Republic of China. E-mail: jwang{at}crf.org

	References

Top Abstract Materials and Methods Results Discussion Conclusions References

 

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