Beneficial Effects of Chronic Pharmacological Manipulation of β-Adrenoreceptor Subtype Signaling in Rodent Dilated Ischemic Cardiomyopathy
Background— Studies in isolated cardiac myocytes have demonstrated that signaling via specific β1-adrenergic receptor subtypes (β1ARs) promotes but that signaling via β2ARs protects from cell death. We hypothesized that prolonged β2AR stimulation or β1AR blockade would each protect myocytes from death and thereby ameliorate cardiac remodeling in chronic heart failure.
Methods and Results— A large myocardial infarction (MI) induced in rats by coronary artery ligation resulted in a dilated cardiomyopathy (DCM) characterized by infarct expansion and a progressive increase in left ventricular (LV) end-diastolic volume, accompanied by a reduction in ejection fraction (EF), as assessed by repeated echocardiography. Pressure-volume analysis at 8 weeks after ligation showed that diastolic stiffness (Eed) and arterial elastance (Ea) were increased, end-systolic elastance (Ees) was decreased, and arterioventricular (AV) coupling (Ea/Ees) had deteriorated. Apoptosis was present in both peri-infarct and remote myocardium. Chronic (6-week) administration of the β2AR agonists fenoterol or zinterol, starting at 2 weeks after MI, reduced the extent of LV dilation, infarct expansion, and EF decline. The β1AR blocker metoprolol did not affect the former and preserved EF to a lesser extent than did the β2AR agonists. At 8 weeks after ligation, apoptosis was reduced by all drugs but to a greater extent by β2AR agonists than by the β1AR blocker. Both β2AR agonists and the β1AR blocker improved AV coupling, the former mainly by reducing Ea and the latter mainly by increasing Ees. Only the β2AR agonists reduced the Eed and the MI size by reducing infarct expansion.
Conclusions— These results provide proof of concept for the efficacy of chronic β2AR stimulation in this DCM model.
Received October 14, 2003; de novo received January 30, 2004; revision received March 13, 2004; accepted March 26, 2004.
Dilated cardiomyopathy (DCM), characterized by compromised systolic and diastolic function, increased arterial elastance (Ea), and inefficient arterioventricular (AV) coupling, is the final common outcome of multiple cardiac disease states. Myocyte death resulting in part from apoptosis1,2 has been implicated in the evolution of DCM.3,4 Thus, protection of myocytes from cell death due to apoptosis holds promise as a therapeutic strategy with respect to delaying the initiation or progression of DCM.
Enhanced ventricular wall stress, due in part to an increased left ventricular (LV) cavity size, an increase in Ea (afterload), and excessive elaboration of catecholamines, angiotensin II, or cytokines, has been implicated in initiating or accelerating cell death. Catecholamines are elaborated excessively in chronic heart failure (CHF), and chronic exogenous administration of the mixed β-adrenergic receptor (βAR) agonist isoproterenol to rodents without heart disease results in DCM5 accompanied by cell death.6 Multiple βAR subtypes are expressed in the adult myocardium. Recent advances in β-adrenergic subtype signaling in isolated cardiac cell models indicate that stimulation of β1AR is proapoptotic,7 whereas stimulation of β2AR protects from apoptosis.8–10 Thus, there is a rationale to suggest that β2AR agonists may reduce cell death and attenuate DCM progression.
The attractive theoretical benefits of β2AR signaling with respect to direct cellular effects in protection from apoptosis, combined with its potential hemodynamic effect in reducing afterload and thus, in indirectly reducing apoptosis by reducing Ea and LV dilation, have yet to be tested in a cardiomyopathic model in the intact organism. We hypothesized that chronic stimulation of β2AR would attenuate the increase in Ea, LV dilatation, and functional decline that ensue after a large myocardial infarction (MI) as a result of permanent coronary artery ligation in rats and that these effects would be accompanied by a reduction in the extent of apoptosis. Because β1AR stimulation causes apoptosis in isolated cardiac myocytes and drugs with predominately β1AR-blocking properties have already been demonstrated to be effective in the treatment of CHF, we also compared the effects of β2AR agonists to those of a β1AR blocker.
After baseline echocardiographic assessment, the left descending coronary artery was ligated in 95 male Wistar rats, weighing 350 to 380 g (Charles River Inc, Wilmington, Mass), as described elsewhere.11 An additional 5 rats underwent sham operation without actual coronary ligation. One week after surgery, LV function and MI size in survivors were evaluated by echocardiography. Eight randomly selected, ligated animals were also euthanized at this time to assess the comparability of the echocardiographic estimate of MI size with a histological measure of MI size (see online-only Data Supplement for details). Then all remaining MI rats were divided into 4 groups of similar average MI size and variability: (1) untreated (LNT, n=13); (2) treated with a selective β2AR agonist (fenoterol, n=10); (3) treated with another selective β2AR agonist (zinterol, n=9); or (4) treated with a selective β1AR blocker (metoprolol, n=7). Echocardiographic indices of LV structure and function at this time were considered the pretreatment baseline values for each group. Beginning 2 weeks after surgery, drugs were dissolved in the drinking water. The daily dose was 250 μg/kg, 1 mg/kg, and 250 mg/kg for fenoterol, zinterol, and metoprolol, respectively. Metoprolol was started at one fourth of the full dose for the first week of treatment, increased to a half dose for the second week, and increased to a full dose thereafter.12 Echocardiography was repeated at 2 and 6 weeks after initiation of treatment (ie, 4 and 8 weeks after surgery). After the final echocardiogram, all rats underwent an invasive hemodynamic study, and hearts were harvested for histological evaluation. Fenoterol and metoprolol were purchased from Sigma; zinterol was a gift from Bristol-Myers Inc (Evansville, Ind).
Echocardiography and Hemodynamics
Rats were anesthetized with sodium pentobarbital, and standard echocardiographic measurements were made (see online-only Data Supplement [available at http://www.circulationaha.org] for details). Hemodynamic measurements by pressure-volume (PV) loop analyses were conducted as described previously.13
Myocardial sections (5 μm thick) were obtained at the midpapillary muscle level, and MI size was calculated as previously described13 (also see online-only Data Supplement [available at http://www.circulationaha.org] for details). Hematoxylin and eosin staining was used to measure myocyte diameters across the nucleus in transverse sections. Terminal dUTP nick end-labeling (TUNEL) staining (Cardio TACS, R&D Systems), followed by eosin counterstaining of the cytoplasm, was used to assess apoptosis.
All data are expressed as mean±SEM. Echocardiographically derived indices were compared by 2-way ANOVAs for repeated measures. Group differences at specific time points were assessed by a Bonferroni test. Differences in hemodynamic or histological data among sham or LNT, β2AR agonist, and β1AR blocker groups were assessed by 1-way ANOVAs, followed by a Bonferroni test. A value of P<0.05 was considered statistically significant.
Treatment Group Assignment
The 24-hour mortality rate after coronary ligation surgery was 50%. There was no later mortality. Figure 1A illustrates representative 2D, short-axis echocardiographic images of ligated rats 1 week after surgery, divided into 4 groups of equal MI size. Neither the mean nor the variance about the mean of this pretreatment echocardiographic estimate of MI size differed among groups (Table 1). Echocardiographic LV dimensions and ejection fraction (EF) also did not differ among groups (Figure 3 legend).
DCM Progression in the Absence of Treatment
The findings in Figure 2 illustrate the progression of LV remodeling during an 8-week period after coronary artery ligation in the absence of treatment. Both end-diastolic volume (EDV) and end-systolic volume (ESV) had increased significantly 1 week after surgery, and these increases were progressive until the study was terminated.
The results shown in Figure 1B illustrate representative echocardiographic images before the rats were euthanized. The average echocardiographic estimate of MI size increased from the pretreatment values by 32±5% (Table 1). The actual endocardial circumferential infarct length increased by 52±8%. Thus, infarct expansion occurred between 1 and 8 weeks after surgery.
Histological Infarct Size
Representative cardiac gross morphology (cross sections of hearts) at sacrifice is illustrated in Figure 1C. The histological MI size at 8 weeks after ligation in the absence of treatment averaged 32.2±4.0% of the LV circumference (Table 1). In the group of animals euthanized at 1 week after surgery (Figure 4, 1 week after MI), histological MI size averaged 26.1±2.1% (P<0.05 versus MI size at 8 weeks after ligation) and confirmed the echocardiographic estimate of infarct expansion between 1 and 8 weeks after surgery.
Relative Heart Weight and Myocyte Size
The heart weight–to–body weight ratio and average LV posterior wall myocyte diameter also were significantly increased (Table 1), indicating the presence of myocardial and myocyte hypertrophy.
The findings in Figure 5 illustrate representative examples of TUNEL staining of the MI border and remote areas of the myocardium. A markedly increased number of TUNEL-positive myocytes was evident in both the MI border and remote areas.
Within 1 week after coronary artery ligation, EF markedly declined (by about half) and by 8 weeks showed a modest further decline. Figure 6 illustrates representative LV PV loops recorded before euthanization at 8 weeks after coronary ligation. The average values of hemodynamic parameters are listed in Table 2. All indices of LV performance were significantly affected: EF and preload recruitable stroke work (PRSW) were reduced by 50%; dP/dt was reduced by 40%; end-systolic elastance (Ees) fell by 70%; end-diastolic elastance (Eed) increased by 300%; the isovolumic relaxation time increased by 50%; and Ea increased by 30%. As a result, the Ea-Ees ratio increased by 5-fold, indicating a marked decline in the efficiency of AV coupling.
Effect of Treatment on DCM Progression
Before the beginning of treatment (2 weeks after ligation), substantial LV dilation and functional decline had occurred, ie; post-MI DCM had already became manifest (Figure 2), and its severity did not differ among ligated groups (Table 1 and Figure 3 legend).
The results in Figure 3 illustrate the average effects of treatment on the changes in chamber dimensions from the pretreatment measurement at 1 week after ligation. The progressive increases in EDV and ESV that occurred in the absence of treatment were reduced by both β2AR agonists but not by the β1AR blocker.
In the metoprolol group, echocardiographic MI size, measured as a percentage of the LV, had increased over 6 weeks from the pretreatment value by 27±14%, ie, similar to that of the LNT group. The endocardial circumferential infarct length also increased in the metoprolol group, similar to the LNT group. In the fenoterol and zinterol groups, MI size, measured as a percentage of the LV, was reduced, but endocardial circumferential infarct length did not change during the treatment period (Table 1). Considering the enlargement of the LV cavity size during the treatment period (Figure 3), the increased relative echocardiographic MI size in both LNT and metoprolol groups likely indicates infarct expansion; on the contrary, there was no evidence of infarct expansion in the fenoterol and zinterol groups.
Histological Infarct Size
Histological MI size was highly correlated with the echocardiographic estimate of MI size (Figure 4; R2=0.88) and was significantly smaller in the fenoterol group than in the LNT or metoprolol group (Table 1).
Relative Heart Weight and Myocyte Size
The heart weight–to–body weight ratio was not affected by treatment (Table 1). LV posterior wall myocyte diameter was significantly increased in the metoprolol group compared with the sham-operated group, was significantly reduced in both fenoterol and zinterol groups in comparison with the LNT or metoprolol group (Table 1), and did not significantly differ among sham-operated, fenoterol, and zinterol groups.
The number of TUNEL-positive–stained myocytes in both fenoterol and zinterol groups was significantly less than in the LNT group, in both the MI border and remote areas (Figure 5). In the metoprolol group, TUNEL staining was also significantly less than in the LNT group in remote areas but not in the MI border area. Compared with the metoprolol group, TUNEL staining was significantly reduced in both fenoterol and zinterol groups in the MI border area but not within remote areas.
LV Function (Echocardiography)
The findings in Figure 3 illustrate the average effect of treatment on the change in EF from pretreatment baseline. In LNT and metoprolol groups, EF declined and was unchanged, respectively. In contrast, EF was improved by both β2AR agonists: with fenoterol, from 34±2% to 43±2% (P<0.05) and with zinterol, from 32±2% to 37±2% (P<0.05).
LV Function (PV Loops)
Representative PV loops for each group are illustrated in Figure 6, and average values of hemodynamic parameters in each group are listed in Table 2. In the fenoterol group compared with the LNT group, LV systolic pump function parameters PRSW, +dP/dt, and EF significantly increased; Ees also increased, but this increase did not reach statistical significance (P<0.10). For the same 2 groups, the diastolic functional parameters Eed, EDP, and isovolumic relaxation time were significantly reduced; −dP/dt was increased; the vascular afterload parameters peripheral vascular resistance (PVR) and Ea were significantly reduced; and AV coupling (Ea/Ees) was significantly improved (reduced ratio), on the basis of a reduction in Ea and the trend for Ees to increase. The profile of functional improvement in the LNT group versus the zinterol group was similar to that of fenoterol for most parameters (Table 2). In the metoprolol group compared with the LNT group, the systolic functional parameters PRSW, +dP/dt, and Ees were significantly improved, but the diastolic functional parameters Eed, EDP, isovolumic relaxation time, and −dP/dt did not differ; the vascular loading parameters PVR and Ea also did not statistically differ; and AV coupling (Ea/Ees) significantly improved, largely on the basis of an increase in Ees.
The present study compared the therapeutic effects of chronic pharmacological βAR subtype manipulation on DCM induced by a large MI in rats. This DCM model is characterized by apoptosis, infarct expansion, myocyte hypertrophy in the area remote from the MI, progressive LV dilation, and systolic and diastolic functional decline in the context of an increased Ea and inefficient AV coupling. The rationale for the study stems from previous experimental observations that signaling pathways and biological consequences of β1AR and β2AR stimulation differ and therefore, that βAR subtype stimulation potentially has different effects in the context of DCM.
Cardiac-specific overexpression of β2AR at moderate levels (30 to ≈200 times) leads to increased basal adenylyl cyclase activity and elevated contractile function without obvious cardiotoxicity, ie, hypertrophy and failure, whereas only when receptor overexpression is maintained at very high levels (350 to ≈1000 times) does it lead to these sequelae.14 In contrast to the relatively wide therapeutic window for β2AR overexpression, even low levels of β1AR overexpression (5 to ≈40 times) lead to progressive cardiac deterioration with prominent fibrosis, myocyte apoptosis, and hypertrophy.15 β2AR signaling distinctly differs from that of the β1AR.16 The β2AR is dually coupled to both Gs and Gi in many species.17 When activated, Gi signaling counteracts that of Gs, eg, on phospholamban and myofilament phospholyration,18,19 plausible reasons why most β2AR agonists induce a weaker positive inotropic effect than do β1AR agonists. Most important, β2AR stimulation protects cardiac myocytes from apoptosis, whereas β1AR stimulation is proapoptotic.8–10
The most important novel finding of our study is that fenoterol and zinterol, both selective β2AR agonists, attenuated the progression of DCM: Both reduced apoptosis and infarct expansion, attenuated LV dilatation, reduced Ea, and improved both LV systolic and diastolic functions and AV coupling. Fenoterol improved AV coupling (Ea/Ees) by reducing Ea and slightly increasing Ees. Although zinterol reduced Ea, it also slightly reduced Ees, and as a result, the improvement in AV coupling did not reach statistical significance. The reduction of the progressive LV chamber dilatation after treatment by either β2AR agonist was accompanied by both reduced myocyte apoptosis in the MI border zone, an effect likely linked to the prevention of MI expansion, and a reduction of apoptosis in the myocardium remote from the MI. These effects of both β2AR agonists were accompanied by a lack of myocyte hypertrophy within the myocardium remote from the MI.
Although the present study does not address specific pathways by which β2AR agonists protect from apoptosis, previous studies indicate that the potent antiapoptotic effect of zinterol is mediated through β2AR-Gi coupling.8 Among β2AR agonists, fenoterol specifically is the most potent in enhancing contractility in isolated cardiac myocytes, and its effect is not pertussis toxin sensitive.20 Fenoterol, however, is unique in that, unlike other β2AR selective agonists (eg, zinterol), it does not couple to Gi;20 Intriguingly, the potent antiapoptotic effect of fenoterol is also not pertussis toxin–sensitive (R.P. Xiao, MD, PhD, Senior Investigator, Laboratory of Cardiovascular Sciences, NIA, NIH, oral communication, 2004).
The effects of β2AR agonists to attenuate LV remodeling and improve LV function in the context of DCM are likely realized through activation of both peripheral vascular and cardiac β2AR signaling pathways. The vasodilating property of β2AR agonists contributes to an improvement of LV function by reduction of vascular afterload, as evidenced by reductions in Ea and PVR. The resulting reduction in LV wall stress might be an additional contributory mechanism to the reduction of apoptosis effected by β2AR agonists. This “indirect” antiapoptotic effect of β2AR agonists might be shared by carvedilol, which has proven to be beneficial in the treatment of CHF21 and exhibits an antiapoptotic effect independent of β1AR blockade.22
Metoprolol and other drugs with prominent β1AR blocker properties are widely used in the treatment of clinical CHF. Their beneficial effects on cardiac function and survival have been well documented by various large-scale clinical studies.23,24 Metoprolol, at the dose used in the present study, improved cardiac systolic function and reduced apoptosis. However, it was not as effective as β2AR agonists in this regard and did not attenuate LV chamber dilatation or improve diastolic function. Thus, after metoprolol treatment, whereas LV Ees was greater than in untreated DCM, LV Eed remained as high as in untreated DCM. Metoprolol, like fenoterol, improved AV coupling but achieved this mainly by an increase in Ees without significantly reducing Ea, as did the β2AR agonists. Although metoprolol reduced myocardial apoptosis in the LV remote from the MI, it neither reduced MI border zone apoptosis nor prevented infarct expansion or an increase in myocyte size in the myocardium remote from the MI.
In summary, the present study provides proof of concept that chronic β2AR stimulation is beneficial with regard to progressive LV dilatation and functional decline in DCM induced by coronary artery ligation. The overall results of chronic pharmacological stimulation of β2AR signaling in the present study, ie, reduced apoptosis and deleterious LV remodeling, and improved function in the context of a reduced LV afterload provide a rationale for additional studies to evaluate the effectiveness of β2AR stimulation in other CHF models. The complementary beneficial effects of β2AR stimulation and β1AR blockade on LV afterload and contractility, as well as additive effects on apoptosis demonstrated by the present results, also provide a rationale for studies to gauge whether additive myocardial protection could be achieved when these agents are used combination.
The online-only Data Supplement, which contains additional information about Methods used in this study, is available with this article at http://www.circulationaha.org.
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