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0022-3565/09/3311-178–185THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS U.S. Government work not protected by U.S. copyright JPET 331:178–185, 2009 Printed in U.S.A. Therapeutic Efficacy of a Combination of a ␤1-Adrenoreceptor(AR) Blocker and ␤2-AR Agonist in a Rat Model ofPostmyocardial Infarction Dilated Heart Failure Exceeds Thatof a ␤1-AR Blocker plus Angiotensin-Converting EnzymeInhibitor□S Ismail Ahmet, Chris Morrell, Edward G. Lakatta, and Mark I. Talan Laboratory of Cardiovascular Sciences, Intramural Research Program, National Institute on Aging, National Institutes of Health,Baltimore, Maryland Received June 3, 2009; accepted July 7, 2009 ABSTRACT
We had proposed previously a novel combination of ␤2-adre-
(nT) and sham groups. The 12-month mortality was significantly noreceptor (AR) agonist and ␤1-AR blocker that in the rat model reduced in all treatment groups (44% in ␤1-␤2⫹, 56% in ␤1- of postmyocardial infarction (MI) dilated cardiomyopathy ex- ␤2⫹ACEi, 59% in ␤1-ACEi versus 81% in nT). Bimonthly echo- ceeds the therapeutic effectiveness of either monotherapy. In cardiography revealed significant attenuation of the left ventric- the present study, we compared that treatment with a combi- ular (LV) chamber remodeling, LV functional deterioration, and nation of ␤1-AR blocker and angiotensin-converting enzyme MI expansion in all three treatment groups, but effects were inhibitor (ACEi), a current standard chronic heart failure (CHF) significantly more pronounced when treatment included a therapy. Two weeks after coronary artery ligation, rats were ␤2-AR agonist. The results indicated that a combination of divided into groups of similar average MI size, measured by ␤1-AR blocker and ␤2-AR agonist is equipotent to a combina- echocardiography, and the following 12-month treatments tion of ␤1-AR blocker and ACEi in the treatment of CHF in rats, were initiated: fenoterol (250 ␮g/kg/day), a ␤2-AR agonist, plus with the respect to mortality, and exceeds the latter with re- metoprolol (100 mg/kg/day), a ␤1-AR blocker (␤1-␤2⫹); meto- spect to cardiac remodeling and MI expansion. Thus, this novel prolol plus enalapril (20 mg/kg/day), an ACEi (␤1-ACEi); and a therapeutic regimen for CHF warrants detailed clinical investi- combination of all three drugs (␤1-␤2⫹ACEi). These treatment groups were compared with each other and with nontreated Convincing experimental data in rodent models (Pfeffer et ous effects of ␤1-adrenergic receptor (AR) stimulation on rat al., 1985, 1987) has helped to establish the beneficial clinical cardiac myocytes put forth in 1990s (Mann et al., 1992; effects of renin-angiotensin system blockade with ACE inhib- Communal et al., 1999) was swiftly translated in successful itors (ACEi) in patients with chronic heart failure (CHF) and clinical trials, reporting a 35% reduction in mortality and to include this therapy into clinical practice in 1980s (CON- significant reversal of cardiac remodeling in patients with SENSUS Trial Study Group, 1987; The SOLVD Investiga- CHF treated with ␤1-AR blockers (Hall et al., 1995; The tors, 1991; Pfeffer et al., 1992; The AIRE Study Investigators, CIBIS II Investigators, 1999; The MERIT-HF Investigators, 1993; Køber et al., 1995). Experimental evidence of deleteri- 1999). A combination of ␤1-AR blockers with ACEi (or angio-tensin receptor blockers) was subsequently recommended by This work was supported by the Intramural Research Program of the American Heart Association, and this combination is pres- National Institutes of Health National Institute on Aging.
ently considered a standard therapy for CHF (HFSA, 2006).
Article, publication date, and citation information can be found at Nevertheless, despite spectacular success in treatment dur- ing the past decade with this treatment regimen, CHF still S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
remains a major cause of morbidity and mortality, particu- ABBREVIATIONS: ACE, angiotensin-converting enzyme; ACEi, angiotensin-converting enzyme inhibitor; CHF, chronic heart failure; AR, adreno-
receptor(s); MI, myocardial infarction; SH, sham operated; LV, left ventricle; EF, ejection fraction; Echo, echocardiography; nT, nontreated; 2D,
two-dimensional; EDV, end-diastolic volume; ESV, end-systolic volume; LVM, left ventricular mass; Ees, end-systolic elastance; PRSW, preload
recruitable stroke work; Eed, end-diastolic stiffness; Ea, arterial elastance; DCM, dilated cardiomyopathy.
␤2-AR Agonist and ␤1-AR Blocker in Treatment of DCM
larly in the elderly, and a growing problem in most industri- Coronary Artery Ligation. Rats were anesthetized with isoflu-
alized countries (Mazza et al., 2005). Thus, the search for rane (2% in oxygen). The surgical procedure was performed as de- novel pharmacological approaches to treat CHF continues.
scribed previously (Hochman and Bulkey, 1982).
Evolving experimental evidence in the rat model points to Echocardiography. Echocardiography (Sonos 5500, a 12-MHz
antiapoptotic, and thus cardioprotective, properties of ␤2-AR transducer; Hewlett Packard, Andover, MA) was conducted underlight anesthesia by sodium pentobarbital (30 mg/kg i.p.) as described agonists (Communal et al., 1999; Chesley et al., 2000; Zhu et previously (Ahmet et al., 2005). In brief, parasternal long axis views al., 2001; Shizukuda and Buttrick, 2002; Ahmet et al., 2004; were obtained and recorded to ensure that the mitral and aortic Xiao et al., 2004). Furthermore, preclinical translational valves and the apex were visualized. Short axis views were recorded studies have clearly demonstrated the therapeutic effective- at the midpapillary muscle level. Endocardial area tracings, using ness of combined ␤1-AR blockers and ␤2-AR agonist therapy the leading edge method, were performed in a 2D-dimensional mode in animal models of CHF (Ahmet et al., 2005, 2008). In (short and long axis views) from digital images captured on cineloop contrast to successful translation of experimental work on to calculate end diastolic and end systolic LV areas. End-diastolic ␤1-AR blockers and ACE inhibitors in animal models to clin- volume (EDV) and end-systolic volume (ESV) were calculated by a ical practice, however, the combination of ␤1-AR blockers and modified Simpson's method. EF was then derived as EF ⫽ (EDV ⫺ ␤2-AR agonists has not been recognized in the clinical arena, ESV)/EDV ⫻ 100. Left ventricular mass (LVM) was calculated froma 2D mode. The MI size at the midpapillary muscles level was due to largely hypothetical objections: ␤2-AR stimulation estimated from 2D short axis LV images and expressed as a percent- accelerates the heart rate and, therefore, could be proar- age of the LV endocardial circumference. Infarct area was identified rhythmic (Pearce et al., 1989; Martin et al., 1998); chronic as a sharply demarcated section of the LV free wall that failed to ␤-AR stimulation leads to down-regulation and desensitiza- thicken during systole. The length of the akinetic part of the LV tion of ␤2-AR (for review, see Brodde et al., 1995) and, there- endocardial circumference was measured from freeze-frame images fore, cannot be effective. An additional barrier to the entree at end-diastole. Posterior wall thickness was measured from M- of the ␤1-AR blocker and ␤2-AR agonist-combined therapy mode. All measurements were made by a single observer who was into consideration for a clinical trial is that this combination blinded to the identity of the tracings. All measurements were off- has not been compared with the standard CHF therapeutic line averaged over three to five consecutive cardiac cycles. The re- regimen, i.e., an ACE inhibitor combined with a ␤1-AR producibility of measurements was assessed in two sets of baseline measurements in 10 randomly selected rats, and the repeated mea-sure variability did not exceed ⫾5%.
Therefore, the present study compared the effectiveness of Hemodynamic Measurements. Invasive LV pressure-volume
12-month treatment regimen of the combination of a ␤1-AR loop analyses were conducted as described previously (Ahmet et al., blocker and ␤2-AR agonist with the standard combination of 2004). Rats were anesthetized with isoflurane (2% in oxygen), intu- ␤1-AR blocker and ACE inhibitor in a well characterized rat bated, and ventilated. A bilateral thoracotomy was performed in the experimental model of post-MI dilated cardiomyopathy.
sixth intercostal space. A 1.4 French-combined pressure-conductancecatheter (Millar Instruments Inc., Houston, TX) was inserted into Materials and Methods
LV through the apex. Traditional load-dependent hemodynamic in-dices, such as EF, ⫹dP/dt, ⫺dP/dt, end-diastolic pressure, and iso- Experimental Design. Male Wistar rats (Charles River Labora-
volumic relaxation time constant (␶), were measured, and load-inde- tories, Inc., Wilmington, MA), weighing 225 to 280 g, were housed pendent indices, i.e., end-systolic elastance (Ees), preload recruitable and studied in conformance with the National Institutes of Health stroke work (PRSW), and end-diastolic stiffness (Eed) were deter- Guide for the Care and Use of Laboratory Animals (Manual 3040-2, mined or calculated. Arterial elastance (Ea) was calculated as index 1999), with approval from the Institutional Animal Care and Use of vascular tension. Arterioventricular coupling, an index of cardiac Committee. The left descending coronary artery was ligated in 224 work efficiency, was calculated as Ea/Ees.
rats. An additional 10 rats underwent a sham operation (SH) with- Histological Acquisition. Histological staining and analyses
out actual coronary ligation. Two weeks after surgery, LV dimen- were performed as described previously (Ahmet et al., 2005). In brief, sions, ejection fraction (EF), and MI size were measured by echocar- the hearts were isolated and weighed. Myocardial segments from the diography (Echo). Animals with an MI size greater than 20% but less midpapillary muscle level were imbedded in the paraffin, sectioned than 50% LV were divided into four groups of similar MI size (aver- (5 ␮m), and stained with Masson's trichrome and hematoxylin and age and variability). In three groups of rats, treatment was initiated eosin staining. MI size was expressed as an average percentage of either with a combination of metoprolol, a selective ␤1-AR blocker the LV endocardial and epicardial circumferences that were identi- plus enalapril, an ACE inhibitor (␤1-ACEi), with a combination of fied as infarct in the Masson's trichrome-staining sections.
metoprolol, and fenoterol, a selective ␤2-AR agonist (␤1-␤2⫹), or Statistical Analyses. All data are expressed as mean ⫾ S.E.M.
with a combination of metoprolol, fenoterol, and enalapril (␤1- Mortality is reported via Kaplan-Meier survival curves. Differences ␤2⫹ACEi). Fenoterol, metoprolol, and enalapril (Sigma-Aldrich, St.
among survival curves were assessed using logrank statistical anal- Louis, MO) were dissolved in the drinking water; the daily dose was yses (GraphPad Prism 4.02; GraphPad Software Inc., San Diego, adjusted to 250 ␮g/kg for fenoterol, 100 mg/kg for metoprolol, and 20 CA). For subsequent pairwise comparisons of survival curves the mg/kg for enalapril. The fourth group of animals received no treat- Bonferroni's correction for multiple comparisons was used. Reported ment (nT), and fifth group was sham-operated. Treatment was Echo indices were analyzed using the repeated measures linear started 2 weeks after coronary ligation and continued for 12 months mixed effects model. Each response variable was analyzed for main (4 weeks were counted as 1 month). Animals were inspected daily for effects of group and time as well as their interaction. If the group- signs of moribundity by a person blinded to treatment modalities.
time interactions were significantly different (statistically) among Moribund animals were euthanized, and their hearts were harvested all groups, further pairwise analyses were conducted and the out- for MI measurements. Daily records of dead or euthanized animals comes were Bonferroni-corrected for multiple comparison. Further- were used to calculate continuous mortality curves. Echocardiogra- more, post hoc Bonferroni-corrected comparisons were made for 42 phy was repeated bimonthly after the initiation of treatment. After comparisons among the group means at each time point. The model the final Echo, subsets of rats representing average Echo indices was fit using PROC MIXED in SAS 9.1 (SAS Institute, Cary, NC).
within each group were selected for invasive hemodynamic study, Power Analysis (PASS 2008, repeated measures analysis of variance; and their hearts were harvested for histological evaluation.
NCSS, Keysville, UT) indicated that for the main effect the sample Ahmet et al.
size exceeding 10 animals per group would correspond to a power more than 80%; for the interaction effect, the power for all variableswould be more than 70% for samples of 15 or larger. Group differ-ences in hemodynamic or histological data among groups were as-sessed by Student's t test or by one-way analysis of variance with Bonferroni's post hoc corrections as appropriate. Statistical signifi-cance was assumed at p ⬍ 0.05.
Early Mortality after Coronary Ligation and Treat-
Survival (%)
ment Assignment. Two hundred and twenty-four rats were
subjected to a coronary ligation. Eighty animals died withinthe first 24 h after surgery, and 14 additional rats died withinthe first 2 weeks after surgery. There was no mortality among 10 sham-operated rats. Two weeks after surgery, the 130 rats surviving coronary ligation and 10 sham-operated rats underwent echocardiography, at which time their pre-treatment (baseline) MI size, LV volumes, and EF were de- termined. Twenty-two rats, in which MI size was less than 20% or more than 50% LV, were excluded from the therapeu- tic interventions. One hundred and eight rats with an aver-age MI size of 30 ⫾ 0.67% LV were assigned to four experi- mental treatment groups (n ⫽ 27 in each group) that did not ** - p<0.01 vs nT
differ with respect to their average pretreatment Echo-de- rived LV morphometric parameters (EDV and ESV), EF, andMI size. The 10 sham-operated rats served as a control.
Fig. 1. Kaplan-Meier survival curves after induction of MI in a sham-
Table 1 lists the Echo-derived pretreatment EDV, ESV, operated group, an untreated group, and three different treatmentgroups. The number of animals at the beginning of treatment was as and EF for each experimental and sham group 2 weeks after follows: SH, 10; and nT, ␤1-ACEi, ␤1-␤2⫹, and ␤1-␤2⫹ACEi, 27 in each.
surgery. Early pretreatment LV remodeling was similar inall coronary-ligated groups and consisted of substantial in- study. The left panels show the MI expansion among differ- creases in EDV (164 –171%), ESV (397– 430%), and a 57– 62% ent experimental groups assessed bimonthly from Echo mea- decline in EF compared with SH. The thickness of posterior surements and expressed as the percentage of LV perimeter.
wall in coronary ligated rats did not vary significantly from The right panels illustrate the MI size, assessed from histo- SH at this time.
logical preparations at the termination of the study, or at the Mortality. Figure 1 illustrates the Kaplan-Meier survival
time of death. Echo-derived and histological measurements curves for the four groups of experimental animals and the were highly correlated (R2 ⫽ 0.7). In nT animals, the average sham-operated group during 1 year after initiation of treat- MI expanded from 30.1 ⫾ 1.3% LV 2 weeks after coronary ment. No SH animals died during observation. In the un- ligation to 46.2 ⫾ 1.7% LV at the end of 12 months, i.e., treated group, mortality reached 50% at 6 months and 81% at during the 12 months of observation, the MI expanded by 12 months. All treatments had improved survival, by 22 to 50%. MI expansion was attenuated in all treatment groups 37%, compared with the untreated group (p ⬍ 0.01, logrank (p ⬍ 0.001 versus nT; see Supplemental Table 1). The least test). The treatment effect became statistically significant by MI expansion, however, occurred in rats in which treatment 7th month and continued to increase with time. Among dif- included a ␤2-AR agonist, i.e., ␤1-␤2⫹ and ␤1-␤2⫹ACEi ferent treatment groups, the mortality at the end of 1 year of groups. In fact, the group ⫻ time interaction was signifi- observation was lowest in ␤1-␤2⫹ group (44%) and highest in cantly different between ␤1-ACEi group and groups with a ␤1-ACEi group (59%); the effect of each treatment on mor- ␤2-AR agonist (p ⬍ 0.001); the average MI size in ␤1-␤2⫹ tality statistically differed from the nT group (p ⬍ 0.01, post group never exceeded the pretreatment level and was signif- hoc test) but did not differ from each other.
icantly smaller than in ␤1-ACEi group at the 4th, 6th, 10th, Infarct Expansion. Figure 2 illustrates the MI expansion
and 12th month of treatment (see Supplemental Table 1).
during 12 months of treatment. The top panels present data LV Remodeling and Function. Figure 3 illustrates the
for all animals. The bottom panels illustrate the results ob- progression of LV remodeling (EDV and ESV expansion) and tained only from the rats that survived until the end of the functional decline (EF reduction) during the treatment pe- TABLE 1Pretreatment LV remodeling and MI size at 2 weeks after coronary ligation N.A., not applicable; PWth, posterior wall thickness.
*** p ⬍ 0.001 vs. SH.

␤2-AR Agonist and ␤1-AR Blocker in Treatment of DCM
Survivors at 12 mo β +*** - p<0.001 vs nT; ††† - p<0.001 vs 1 Fig. 2. Average MI size in untreated and treated rats estimated by monthly Echo (left) or on the basis of histological measurements (right) and
presented as percentage of LV. Top, all animals; bottom, animals that survived 12 months after the MI induction. ⴱⴱⴱ, p ⬍ 0.001 versus nT; †††, p ⬍
0.001 versus ␤1-ACEi (pairwise comparison, group ⫻ time interaction with Bonferroni's correction).
riod. The top panels represent average measurements from group, however, was significantly less and did not differ all animals at each time point, whereas lower panels repre- statistically from nT.
sent only animals that survived to the end of the study. Both The Echo-calculated LVM (Fig. 4A) increased in all MI the top and bottom panels reflect similar patterns. In nT groups compared with SH, and that increase in LVM was animals, the LV volume monotonically increased by 90 and reduced by all treatment modalities; however, the differences 120% for EDV and ESV, respectively, whereas EF declined with nT were statistically significant in ␤1-ACEi group only by 50%. At the end of the observation period, EDV in nT (p ⬍ 0.05; see Supplemental Table 1). Posterior wall thick- animals averaged 1170 ⫾ 75 ␮l, ESV was 1028 ⫾ 83 ␮l, and ness (Fig. 4B) became significantly reduced in all MI groups EF was 12.5 ⫾ 1.8%. For comparison (data are not shown), compared with SH. Compared with the nT group, the thin- the LV EDV in SH animals at the end of observation was only ning of posterior wall was attenuated in all treatment 506 ⫾ 13 ␮l, ESV was 242 ⫾ 10 ␮l, and EF was 52 ⫾ 1.9%.
groups, but only in the ␤1-␤2⫹ group was this attenuation The expansion of LV volumes was significantly attenuated in statistically significant (p ⬍ 0.02).
all treatment groups compared with nT (p ⬍ 0.002; see Sup- Hemodynamics. Figure 5 presents representative pres-
plemental Table 1). However, during the first 6 months of sure-volume loops recorded from SH, nT, and different treat- treatment LV expansion in the ␤1-ACEi group was similar to ment groups at the end of the study. Compared with SH, a that in the nT group. Statistical analyses with post hoc substantial rightward shift accompanied by reduction of comparisons conducted for the first 6 months revealed that stroke volume and end-systolic pressure, characteristic for both EDV and ESV in ␤1-ACEi group did not differ from nT, CHF, was observed in the nT group. All treatment groups whereas in ␤1-␤2⫹ and ␤1-␤2⫹ACEi groups, the beneficial showed significant improvement in cardiac indices, and pres- difference from nT was significant for ESV (p ⬍ 0.05) and sure-volume loop recorded from the rat from ␤ -␤ ⫹ACEi approached significance for EDV (p ⬍ 0.07). During the 12 group most closely approached the parameters of SH.
months of the study, the decline in EF was significantly Table 2 lists the results of the pressure-volume loop anal- attenuated, compared with the nT, in group with ␤2-AR yses in randomly selected subsets of rats that survived 12- agonist (p ⬍ 0.005); the preservation of EF in the ␤1-ACEi months observation, i.e., 12.5 months after induction of MI. A Ahmet et al.
End- Diastolic Volume
End- Systolic Volume
Survivors at 12 mo 500
Time (month)
* - p<0.002 vs nT; † - p<0.05 vs β1- ACEi Fig. 3. LV end-diastolic volume (left), end-systolic volume (middle), and ejection fraction (right) in untreated and treated rats estimated by bimonthly
Echo during 12 months after induction of MI. Top, all animals; bottom, only animals that survived 12 months after induction of MI. ⴱⴱ, p ⬍ 0.002
versus nT; †, p ⬍ 0.05 versus ␤1-ACEi (pairwise comparison, group ⫻ time interaction with Bonferroni's correction).
comparison of hemodynamic indices of SH and nT rats mass; thinning of posterior MI-free wall; progressive func- clearly indicates an advanced stage of CHF in nT rats: the LV tional decline; and, of course, substantial mortality. All of is greatly dilated, cardiac output and stroke volume are re- these characteristics have been described and well docu- duced by 50%, and EF is reduced 4-fold. Furthermore, a mented previously (Preffer and Braunwald, 1990; Goldman greater than 5-fold reduction in PRSW indicates a pro- and Raya, 1995; Krzemin ´ ski et al., 2008).
nounced systolic pump dysfunction; and a 3.5-fold elevation We have been committed to testing different therapeutic in Eed reflects the increased diastolic stiffness of myocar- regimens in the DCM models (Ahmet et al., 2004, 2005, dium. All three treatment groups showed a significant im- 2008). Initially, we had tested the effects of selective phar- provement of hemodynamic indices compared with the nT macological stimulation of ␤2-AR alone, or in combination group. However, the ␤1-␤2⫹ and ␤1-␤2⫹ACEi were more with ␤1-AR blocker in this in vivo rat model of postmyo- effective than ␤1-ACEi in attenuation of LV volume expan- cardial infarction DCM. The effects of 6 weeks of treatment sion and improvement of EF.
with a ␤2-AR agonist, fenoterol, alone (Ahmet et al., 2004)or in combination with a ␤1-AR blocker, metoprolol (Ahmet et al., 2005), were compared with metoprolol monotherapy.
Treatments were started 2 weeks after coronary ligation.
The rat model of permanent coronary ligation is the oldest and one of the most established models of chronic heart The progression of LV remodeling and MI expansion was failure (Selye et al., 1960). Although the ischemic-reperfusion monitored by serial echocardiography. At the endpoint of (temporary coronary occlusion) model more relevantly re- the study, cardiac function was analyzed by pressure-vol- flects the modern clinical situation of restoration of coronary ume loop measurements, and hearts were evaluated his- flow, the permanent occlusion model is better suited to study tologically. In these short-term, 6-week studies the effec- the time course of infarct expansion, remodeling, perfor- tiveness of a ␤2-AR agonist and the combination of a ␤2-AR mance decline, and morphological changes. It also produces a agonist plus a ␤1-AR blocker was similar, and both signif- more uniform pathology; therefore, it is a more suitable for icantly exceeded the effectiveness of the ␤1-AR blocker as assessment of interventional strategies for DCM. In our ex- a monotherapy in attenuation of LV dilatation and func- periment, we observed in untreated rats all aspects charac- tional decline. Both treatments that included ␤2-AR stim- teristic of post-MI development of DCM: uniform MI size; ulation also reduced myocardial apoptosis and arrested progressive MI expansion; LV dilatation; increase of LV the MI expansion. Moreover, we did not observe any

␤2-AR Agonist and ␤1-AR Blocker in Treatment of DCM
Left Ventricular Mass
Posterior Wall Thickness
Fig. 5. Representative pressure-volume loops of SH, nT, and three
treated groups recorded at the end of the study.
significantly attenuated in animals treated with the combi- nation of both drugs during the first 6 months of treatment.
MI expansion was completely prevented only in animalstreated with the drug combination; the LV functional decline was significantly attenuated during the entire year. Theepisodes of arrhythmic events were also lowest in combined treatment group. Furthermore, a reduction of cardiac ␤1-ARdensity and a reduction in chronotropic and contractile re- sponses to ␤2-AR-specific stimulation in the absence of areduction of ␤2-AR density that occurred in untreated ani- mals were precluded in rats receiving combined therapy.
This explained the additional observation of the study inwhich the beneficial effect of ␤2-AR agonist as monotherapy lasted only for the first 2 months after initiation of treat-ment, whereas the combination with a ␤1-AR blocker ex- * - p<0.05 vs SH; † - p<0.05 vs nT tended the therapeutic effectiveness throughout all 12 Fig. 4. LV mass (A) and posterior wall thickness (B) in SH, nT, and three
months of observation.
treated groups estimated by bimonthly Echo during 12 months after Thus, the effects of ␤2 AR agonist treatment in the post-MI induction of MI in all animals. ⴱ, p ⬍ 0.05 versus SH; †, p ⬍ 0.05 versus model of DCM for only 6 weeks of treatment (8 weeks post- nT (pairwise comparison, group ⫻ time interaction with Bonferroni'scorrection).
MI) showed its high therapeutic effectiveness. When we ex-tended the treatment period to 12 months, it became appar- increase in the number of arrhythmic events during ␤2-AR ent that the effect of ␤2 AR agonist monotherapy waned after 2 months. The long-term effectiveness of ␤2-AR agonist ther- These studies, however, were of a relatively short duration apy was possible only in combination with ␤1-AR blocker to (6 weeks). In the next experiment in the same model, we prevent ␤2-AR tachyphylaxis and reduce apoptosis. The use compared the effects of long-term, combined therapy with a of ␤2-AR agonist in combination with ␤1-AR blocker also ␤1-AR blocker, metoprolol, plus a ␤2-AR agonist, fenoterol, resulted in preservation of ␤1-AR density and responsiveness and either therapy alone for 12 months with survival as a of ␤2-AR to stimulation, in which reduction was observed in primary outcome (Ahmet et al., 2008). As in the short-term DCM rats (Ahmet et al., 2008). The fact that the treatment studies, therapy was started 2 weeks after permanent liga- duration in the present study lasted for 12 months is a very tion of the left descending coronary artery. Cardiac remodel- important feature of the present study.
ing, MI expansion, and LV function were assessed by serial The present study extended the series of our previous echocardiography and compared with untreated animals. A reports (Ahmet et al., 2004, 2005, 2008) demonstrating ther- mortality of 67% observed at the end of 1-year observation in apeutic effectiveness of combined therapy of a ␤1-AR blocker untreated animals was reduced to 33% in the animals and a ␤1-AR agonist in the rat model of post-MI DCM. In this treated with the combination of ␤1-AR blocker and ␤2-AR study, our goal was to specifically compare the beneficial agonist. Progressive cardiac remodeling observed in un- effects of combined ␤-AR therapy with a combination treat- treated rats or in rats treated with ␤1-AR blocker alone was ment of a ␤1-AR blocker and an ACE inhibitor, a standard Ahmet et al.
TABLE 2Hemodynamic indices in SH rats and treated and untreated rats 12.5 months after MI SH (n ⫽ 5) nT (n ⫽ 3) ␤1-ACEi (n ⫽ 6) ␤1-␤2⫹ (n ⫽ 11) ␤1-␤2⫹ACEi (n ⫽ 8) 197 ⫾ 13*a 201 ⫾ 11*a 963 ⫾ 35*a 828 ⫾ 47*a 195 ⫾ 12*a 20 ⫾ 1*a 102 ⫾ 6a 3.2 ⫾ 0.4a 2.4 ⫾ 0.3a (⫹)dP/dt (mm Hg/s) 4649 ⫾ 448*a 4943 ⫾ 337*a (⫺)dP/dt (mm Hg/s) 4046 ⫾ 307*a 49.4 ⫾ 5.1*a 48.9 ⫾ 3.3*a 36.6 ⫾ 3.0*a,b Ees (mm Hg/␮ l) Eed (103 mm Hg/␮ l) dP/dt-EDV (mm Hg/s/␮ l) 17.4 ⫾ 1.4a CO, cardiac output; EDP, end-diastolic pressure; ESP, end-systolic pressure; HR, heart rate; SV, stroke volume.
* p ⬍ 0.05 vs. SH.
a p ⬍ 0.05 vs. nT.
b p ⬍ 0.05 vs. ␤1-␤2⫹.
and widely used regimen in clinical practice. Beneficial ef- activity (Ma et al., 2001; Watanabe et al., 2004; Brower et al., fects of ␤1-AR blocker-ACE inhibitor combination in experi- 2007). The mechanisms of cardioprotection by ␤2-AR agonist mental model of post-MI CHF had been reported previously have been investigated in many studies on isolated cardio- ¨ gel et al., 1999; Fedorov et al., 2006). In this 1-year-long myocytes that discovered its marked antiapoptotic effects study, we demonstrated that DCM rats treated with com- related to activation of G signaling (Communal et al., 1999; bined ␤1-AR blocker-␤2-AR agonist therapy showed 37% in- Chesley et al., 2000; Zhu et al., 2001; Shizukuda and But- crease in survival compared with untreated rats (triple com- trick, 2002; Xiao et al., 2004). We believe that this antiapop- bination of ␤1-AR blockade-ACEi-␤2-AR agonist showed 25% totic effect of ␤2-AR stimulation was responsible for preven- improvement)—a survival benefit that did not exceed a stan- tion of cell loss and thus for attenuation of posterior wall dard therapy (an improvement of 22%) but was at least equal thinning in double and triple therapy groups of the present to it. However, with respect to cardiac remodeling and MI experiment. Because triple therapy (␤1-AR blocker ⫹ ␤2-AR expansion, the addition of ␤2-AR agonist to a therapy (␤1- agonist ⫹ ACE inhibitor) was not more effective in our ex- ␤2⫹ or ␤1-␤2⫹ACEi) clearly exceeded the effectiveness of periment than double therapy with ␤1-AR blocker ⫹ ␤2-AR ␤1-AR blocker-ACE inhibitor combination. Specifically, al- agonist, it is necessary to conclude that the primary mecha- though the ␤1-AR blocker-ACE inhibitor combination did nism responsible for the superior attenuation of LV remod- attenuate the infarct expansion observed in untreated ani- eling in the present experiment is a mechanism of double mals, combined ␤-AR therapy actually arrested the MI ex- action of ␤1-AR blocker and ␤2-AR agonist. This mechanism pansion, revealing a statistically significant improvement was elucidated in our previous study (Ahmet et al., 2008), over the ␤1-AR blocker-ACE inhibitor combination. Although i.e., preservation of ␤1-AR density and responsiveness to standard ␤1-AR blocker-ACE inhibitor combination attenu- ␤2-AR stimulation. Although coupling of ␤-AR subtypes to ated LV expansion, the addition of a ␤2-AR agonist made the specific signaling pathways can be species specific and vary treatment significantly more effective during the first 6 from mouse to rat to humans (Port and Bristow, 2001), the months of treatment. Furthermore, the combinations includ- effectiveness of therapies that are now clinically accepted for ing a ␤2-AR agonist were twice as effective as ␤1-AR blocker- humans has been demonstrated in animal experimental ACE inhibitor combination in attenuating the functional de-cline. In addition, although a standard therapy was effective models. Moreover, successful use of ␤2-AR agonist clen- in attenuating the LV mass increase, the ␤-AR combined buterol in conjunction with left ventricular assist device to treatment was effective in attenuating the thinning of pos- help to attenuate myocardial atrophy before heart transplan- terior wall, which has been interpreted to reflect myocyte cell tation (Hon and Yacoub, 2003) suggests that the translation death (Anversa et al., 1998). It is important to note that the of current finding to clinical practice seems rational.
addition of ␤2-AR agonist to the standard therapy of ␤1-AR In summary, the therapeutic utility of combined treatment blocker and ACE inhibitor significantly increased the effec- with ␤1-AR blocker and ␤2-AR agonist has been demon- tiveness of a standard therapy, with respect to LV remodel- strated in our previous studies in the rat model of post-MI ing and MI expansion; the triple therapy was not more effec- DCM. In the present preclinical study, in the same experi- tive than the ␤1-AR blocker-␤2-AR agonist combination.
mental model, we showed that inclusion of ␤2-AR agonist in The mechanisms of therapeutic effectiveness of ACE inhib- treatment regimen as double (␤1-AR blocker-␤2-AR agonist) itors in the CHF have been well characterized previously or triple (␤1-AR blocker-␤2-AR agonist-ACEi) therapy ex- (Wollert and Drexler, 1999; Anand and Florea, 2008; Werner ceeds therapeutic effectiveness of combination of ␤1-AR et al., 2008) and encompass numerous signaling pathways blocker and ACE inhibitor. Thus, the ␤1-AR blocker-␤2-AR from blocking the angiotensin II to suppression of transform- agonist combination alone or in combination with ACEi war- ing growth factor-␤ to inhibition of matrix metalloproteinase rants detailed clinical investigation as a treatment for CHF.
␤2-AR Agonist and ␤1-AR Blocker in Treatment of DCM
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