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Distinct myocardial effects of beta-blocker therapy in heart failure with normal and reduced left ventricular ejection fraction

Nazha Hamdani , Walter J. Paulus , Loek van Heerebeek , Attila Borbély , Nicky M. Boontje , Marian J. Zuidwijk , Jean G.F. Bronzwaer , Warner S. Simonides , Hans W. M. Niessen , Ger J. M. Stienen , Jolanda van der Velden
DOI: http://dx.doi.org/10.1093/eurheartj/ehp189 1863-1872 First published online: 1 June 2009

Abstract

Aims Left ventricular (LV) myocardial structure and function differ in heart failure (HF) with normal (N) and reduced (R) LV ejection fraction (EF). This difference could underlie an unequal outcome of trials with β-blockers in heart failure with normal LVEF (HFNEF) and heart failure with reduced LVEF (HFREF) with mixed results observed in HFNEF and positive results in HFREF. To investigate whether β-blockers have distinct myocardial effects in HFNEF and HFREF, myocardial structure, cardiomyocyte function, and myocardial protein composition were compared in HFNEF and HFREF patients without or with β-blockers.

Methods and results Patients, free of coronary artery disease, were divided into β−HFNEF (n = 16), β+HFNEF (n = 16), β−HFREF (n = 17), and β+HFREF (n = 22) groups. Using LV endomyocardial biopsies, we assessed collagen volume fraction (CVF) and cardiomyocyte diameter (MyD) by histomorphometry, phosphorylation of myofilamentary proteins by ProQ-Diamond phosphostained 1D-gels, and expression of β-adrenergic signalling and calcium handling proteins by western immunoblotting. Cardiomyocytes were also isolated from the biopsies to measure active force (Factive), resting force (Fpassive), and calcium sensitivity (pCa50). Myocardial effects of β-blocker therapy were either shared by HFNEF and HFREF, unique to HFNEF or unique to HFREF. Higher Factive, higher pCa50, lower phosphorylation of troponin I and myosin-binding protein C, and lower β2 adrenergic receptor expression were shared. Higher Fpassive, lower CVF, lower MyD, and lower expression of stimulatory G protein were unique to HFNEF and lower expression of inhibitory G protein was unique to HFREF.

Conclusion Myocardial effects unique to either HFNEF or HFREF could contribute to the dissimilar outcome of β-blocker therapy in both HF phenotypes.

  • β-blockers
  • Heart failure
  • Myocardium
  • Diastole
  • Hypertrophy

Introduction

Over the past two decades, it became evident that more than 50% of all heart failure (HF) patients suffer of HF with normal left ventricular (LV) ejection fraction (EF).1 In heart failure with normal LVEF (HFNEF) and in heart failure with reduced LVEF (HFREF), LV structure adapts differently with concentric LV remodelling in HFNEF and eccentric LV remodelling in HFREF.24 Corresponding differences were observed at the myocardial ultrastructural level with prominent cardiomyocyte hypertrophy in HFNEF and low myofibrillar density in HFREF.4 When cardiomyocytes were isolated from LV myocardium, cardiomyocyte resting tension (Fpassive) was also higher in HFNEF than in HFREF.46 These structural and functional differences between LV myocardium in HFNEF and HFREF could underlie the unequal outcome of trials using β-blockers, angiotensin converting enzyme inhibitors (ACE-I), or angiotensin II receptor blockers (ARB) which usually yielded positive results in HFREF and mixed results in HFNEF.7,8 In line with this unequal outcome of trials, prognosis of HFREF patients improved over the last two decennia, whereas no such trend was observed in HFNEF patients.1

Large clinical trials have convincingly shown that β-blocker therapy reduces mortality and improves LV function in HFREF patients.911 In HFNEF patients, favourable effects of β-blocker therapy on mortality and LV function have not been convincingly demonstrated. After hospital discharge, HFNEF patients had improved survival when using β-blockers12 and in a community-based registry, carvedilol use was accompanied by similar 1 year mortality in HFNEF and HFREF patients but less reduction in hospitalizations in HFNEF patients.13 In the SWEDIC trial, carvedilol had no effect on mortality or hospitalizations, but ameliorated E/A ratio of HFNEF patients.14 In a similar study, however, 6 months of atenolol use had no effect on diastolic LV function of HFNEF patients with unchanged pulmonary capillary wedge pressure.15 Because of these inconsistent results, the use of β-blockers in HFNEF patients is further evaluated in clinical trials, such as the Japanese DHF study.16

To investigate whether HF therapy has indeed disparate effects on LV myocardial structure and function in HFNEF and HFREF, the present study compared LV myocardial structure, function, and protein composition in HFNEF and HFREF patients with and without β-blockers. The current widespread use of ACE-I precluded a similar comparison in patients with and without ACE-I. Endomyocardial biopsies procured in the four patient groups were used for: (i) histomorphometry of light and electron microscopic images to determine cardiomyocyte diameter (MyD), collagen volume fraction (CVF), and myofibrillar density; (ii) isolation of single cardiomyocytes to measure active force (Factive), resting force (Fpassive), and Ca2+-sensitivity (pCa50), and (iii) protein analysis to assess phosphorylation of myofilamentary proteins and the expression of proteins involved in β-adrenergic signalling and in Ca2+-handling.

Methods

The Supplementary methods section provides a detailed description of the methodology used for quantitative histomorphometry, force measurements in isolated cardiomyocytes, and protein analysis.

Patients

All patients included in the study (n = 71) had been hospitalized for worsening HF and were referred for cardiac catheterization and LV biopsy procurement because of suspicion of infiltrative or inflammatory myocardial disease. No patient had significant (>50%) coronary artery stenoses, a history of myocardial infarction, percutaneous coronary intervention, or coronary bypass surgery. Histological analysis of the biopsies showed no myocardial infiltration or inflammation. The local ethics committee approved the study protocol of Vrij Universiteit medical center (Vumc). Written informed consent was obtained from all patients, and there were no complications related to catheterization or biopsy procurement. Patients were classified as HFREF if LVEF<40% and as HFNEF if LVEF>50% and LV end-diastolic pressure (LVEDP) >16 mmHg.17 Patients with a LVEF>40% but <50% were not included. The patients had either new-onset HF or acute decompensation superimposed on chronic HF. Sixty-four percent of patients had new-onset HF and 36% had acute decompensation superimposed on chronic HF. Patients were studied after medical compensation.

Data analysis

Values are given as mean ± SEM of the observations in each patient group. Data of the β−HFNEF, β+HFNEF, β−HFREF, and β+HFREF groups were compared by two-factor ANOVA testing for β-blocker use, HFNEF/HFREF status, and their interaction (Tables 1 and 2). Subsequent comparisons (β−HFREF vs. β+HFREF and β−HFNEF vs. β+HFNEF) were performed with a Bonferroni adjusted t-test. The analyses were performed in GraphPad Prism version 4 and SPSS 12.0.

View this table:
Table 1

Clinical, haemodynamic, and echocardiographic characteristics

View this table:
Table 2

Histomorphometry, cardiomyocyte force measurements, and protein analysis

Results

Clinical and haemodynamic characteristics

Clinical, haemodynamic, and echocardiographic data are presented in Table 1. Patients on β-blocker therapy had lower heart rate and LVEDP in both HFNEF and HFREF groups. Heart failure with normal LVEF patients were older and more frequently suffered of arterial hypertension with a concomitant higher use of calcium channel and ARB. Heart failure with normal LVEF patients also had higher LV peak systolic pressure, LVEDP, LVEF, LVdP/dtmax, LVdP/dtmin, LV wall thickness, LV mass index/LV end-diastolic volume index (LVMI/LVEDVI) ratio, and smaller LVEDVI.

Histomorphometry

Histomorphometric data are summarized in Table 2 and Figure 1. Myocardial CVF was significantly lower in HFNEF than in HFREF (P < 0.0001) and only in HFNEF was β-blocker therapy associated with lower myocardial CVF (P = 0.0007). Myocyte diameter was significantly higher in HFNEF than in HFREF (P < 0.0001) and only in HFNEF was β-blocker therapy associated with reduced MyD (P < 0.0001). Myofibrillar density was also higher in HFNEF than HFREF (P < 0.0001), but was unrelated to β-blocker therapy.

Figure 1

(A) Representative examples of left ventricular myocardial histology in the four patient groups; (B) bar graphs showing effects of β-blocker therapy on collagen volume fraction (CVF), myocyte diameter (MyD), and myofibrillar density in heart failure with normal left ventricular ejection fraction (HFNEF) and heart failure with reduced LVEF patients. Only in HFNEF patients, did β-blocker therapy reduce CVF and MyD (*P < 0.001, −β vs. +β).

Force measurements in isolated cardiomyocytes

Single cardiomyocytes were attached between a force transducer and a motor (Figure 2A and B) and their sarcomere length was adjusted to 2.2 µm. Force measurements are summarized in Table 2 and Figure 2C. Factive and pCa50 were similar in HFNEF and HFREF, but Fpassive was higher in HFNEF (P = 0.0006). In both HFNEF and HFREF patients, β-blocker therapy resulted in a significant increase in Factive (P = 0.01) and pCa50 (P = 0.008). Only in HFNEF patients did β-blocker therapy raise Fpassive (P = 0.03). After protein kinase A (PKA) administration to the cardiomyocytes, Factive was comparable in all four patient groups. After PKA, Fpassive fell in all four groups but remained significantly higher in HFNEF patients with β-blockers (P = 0.001) (Figure 3A). pCa50 followed a trend similar to Fpassive by decreasing in all four groups and by remaining higher in HFNEF patients with β-blockers (P = 0.007) (Figure 3B). Hence, in vivo β-blocker therapy was associated with multiple in vitro changes in baseline and PKA-stimulated contractile function of isolated cardiomyocytes, which differed between HFNEF and HFREF groups.

Figure 2

(A) Single cardiomyocyte from a heart failure with reduced left ventricular ejection fraction (HFREF) patient glued between a force transducer and a piezoelectric motor; (B) contraction–relaxation sequence recorded in a single cardiomyocyte of a HFREF patient before (grey line) and after (black line) treatment with protein kinase A, during maximal activation (pCa 4.5) and submaximal activation (pCa 5.4); (C) bar graphs showing effects of β-blocker therapy on Factive, Fpassive, and pCa50 of single cardiomyocytes. β-blocker therapy increased Factive and pCa50 in both HFNEF and HFREF and increased Fpassive only in HFNEF (*P < 0.05, −β vs. +β).

Figure 3

(A) Protein kinase A (PKA) treatment significantly reduced Fpassive in all groups but Fpassive remained higher in the β+HFNEF patients; (B) PKA treatment significantly reduced pCa50 in all groups but pCa50 remained higher in the β+HFNEF patients (*P < 0.01, −β vs. +β; #P < 0.01, before PKA vs. after PKA).

Protein analysis

Myofilament protein phosphorylation

To explore changes in myofilamentary protein phosphorylation related to β-blocker therapy, Pro-Q Diamond-stained gels of biopsies were obtained as shown in Figure 4A. Phosphorylation status of myofilamentary proteins did not differ between HFNEF and HFREF groups. β-blocker therapy was associated with lower phosphorylation of troponin I (TnI) and of myosin-binding protein C (MyBP-C) in both HFNEF and HFREF patients (Figure 4B) and had no effect on phosphorylation status of other myofilamentary proteins (Table 2). Effect of β-blocker therapy on phosphorylation status of desmin differed with opposite changes in HFNEF and HFREF patients (P < 0.05).

Figure 4

(A) Representative Pro-Q Diamond-stained gels of a β−HFNEF, β+ HFNEF, β−HFREF, and β+HFREF myocardial sample; (B) phosphorylation status of TnI and of MyBP-C was significantly lower in β+HFNEF and β+HFREF patients compared with, β−HFNEF and β−HFREF patients, respectively. (*P < 0.05, −β vs. +β).

β-Adrenergic signalling proteins

Expression levels of proteins involved in β-adrenergic receptor signalling were determined by western immunoblotting (Table 2). Expression of β1AR, GRK2, and GRK5 was higher in HFREF than in HFNEF, but unaffected by β-blocker therapy. β-Blocker therapy was related to downregulated expression of β2AR in HFREF and HFNEF. Effects of β-blocker therapy on expression of Gs and Gi differed in HFNEF and HFREF with Gs downregulated in HFNEF and Gi downregulated in HFREF.

Calcium-handling proteins

Expression levels of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) and phospholamban (PLB) were determined by western immunoblotting (Table 2). No differences related to HFNEF–HFREF status or to β-blocker therapy were observed. The ratio of SERCA2a/PLB was higher in HFREF (1.0 ± 0.2) than in HFNEF (0.5 ± 0.08; P = 0.02) and unaffected by β-blocker therapy in both groups.

Discussion

In endomyocardial biopsies of HFNEF or HFREF patients, β-blocker therapy was associated not only with structural and functional myocardial changes shared by both HF phenotypes, but also by changes unique to each HF phenotype. The shared effects of β-blocker therapy consisted of enhanced pCa50, higher Factive at saturated [Ca2+], lower phosphorylation status of TnI and MyBP-C, and lower expression of β2AR. Effects of β-blocker therapy unique to HFNEF were reduced interstitial fibrosis, regression of cardiomyocyte hypertrophy, elevated Fpassive before and after PKA, elevated pCa50 after PKA, and reduced expression of Gs. An effect of β-blocker therapy unique to HFREF was the reduced expression of Gi. Effect of β-blocker therapy on phosphorylation status of desmin also differed between HFNEF and HFREF with opposite changes in both groups.

Myocardial effects of β-blocker therapy present in both heart failure with normal left ventricular ejection fraction and heart failure with reduced left ventricular ejection fraction

Chronic β-blocker therapy was associated with increased pCa50 of cardiomyocytes isolated from biopsies of both HFNEF and HFREF patients. Increased pCa50 implies improved cardiomyocyte contractile performance within the physiological range of Ca2+ concentrations and could be explained by the observed fall in phosphorylation status of TnI.18 Chronic β-blocker therapy increased Factive at saturated [Ca2+]. Although this Ca2+ concentration is outside the physiological range, higher Factive also reflects an increased cardiomyocyte force generating capacity. The present study observed reduced phosphorylation of MyBP-C in both β-blocker therapy groups. This reduction could relate to the lack of preload dependence of LV dP/dtmax, which was evident in both β-blocker therapy groups from the unchanged LV dP/dtmax despite lower LVEDP. Preload dependence of early LV pressure rise was recently demonstrated in transgenic mice to derive from phosphorylation of MyBP-C.19,20 Reduced phosphorylation of MyBP-C during chronic β-blocker therapy could have resulted both from lower PKA activity and from lower activity of Ca2+-calmodulin-dependent kinase (CaM-kinase). CaM-kinase can phosphorylate MyBP-C and its activity is also enhanced by β-adrenergic receptor stimulation.2123

In longitudinal studies performing serial haemodynamic investigations in HFREF patients, before and during chronic β-blocker therapy, improved myocardial contractile performance was evident from higher LVEF and lower LV filling pressures.2428 The present study confirmed lower LVEDP with β-blocker therapy in both HFNEF and HFREF groups. The enhanced pCa50 and increased Factive observed in isolated cardiomyocytes of β-blocker treated patients suggested improved cardiomyocyte contractile performance to contribute to the in vivo lowering of LVEDP induced by β-blocker therapy. The enhanced pCa50 and increased Factive could also have contributed to the lower LVEDP in the HFNEF patient group despite the increased Fpassive of the β+HFNEF cardiomyocytes.

In sequential right ventricular biopsies of patients with dilated cardiomyopathy, cardiac β-receptor density increased during metoprolol25 and carvedilol therapy.27 A subsequent study using explanted hearts corroborated these findings and also observed restoration of cardiac β-receptor density by carvedilol treatment.29 In the present study, which used LV biopsies, chronic β-blocker therapy had no effect on β1AR expression but downregulated β2AR expression. The present study, however, did not look at βAR density in membrane fractions but at βAR protein expression in homogenates. Downregulation of β2AR is beneficial for cardiac contractile performance because of less negative inotropy resulting from coupling of β2AR to Gi proteins.30 Coupling of β2AR to Gi was recently also suggested to cause the apical LV dysfunction in patients with Takotsubo cardiomyopathy.31

Previous investigations27 demonstrated that β-blocker therapy induced LV functional improvement in HFREF patients by altering expression of genes responsible for cardiomyocyte contractility. The present study also related LV functional amelioration during β-blocker therapy to improved cardiomyocyte contractility and did so in both HFREF and HFNEF patient groups. In contrast to previous studies, contractility of single cardiomyocytes was directly assessed and its improvement was evident from both higher pCa50 and Factive and linked to lower phosphorylation of TnI and MyBP-C and to reduced expression of β2AR.

Myocardial effects of β-blocker therapy unique to heart failure with normal left ventricular ejection fraction

Only in HFNEF patients, CVF was lower in the group with β-blockers. The absence of an effect of β-blockers on CVF in HFREF patients supports previous studies, which identified high myocardial fibrosis as an important predictor for poor outcome on β-blocker therapy in HFREF.32,33 The lower CVF observed in β+HFNEF patients was paralleled by a reduction of cardiomyocyte hypertrophy evident from lower MyD. In spontaneously hypertensive rats, the β-blocker bisoprolol did not reverse cardiomyocyte hypertrophy in contrast to the ACE-I perindopril.34 This study suggests that reduced renin–angiotensin system activity could possibly be involved in the observed regression of cardiomyocyte hypertrophy in the β+HFNEF patients. Both the reduced cardiomyocytes diameter (MyD) and lower CVF could have contributed to the lower LVEDP in HFNEF patients treated with β-blockers. In our previous study,5 we observed that both passive cardiomyocyte stiffness and CVF contribute to increased LVEDP. Hence, although Fpassive was increased in the β+HFNEF, the lower LVEDP in this patient group probably resulted from less cardiomyocyte hypertrophy and lower CVF.

Previous longitudinal studies showed LV mass to decrease in both HFNEF15 and HFREF patients24,26,28 as a result of β-blocker therapy. Because of smaller LVEDVI, reduction of LV mass resulted in unaltered relative wall thickness in HFREF patients. In HFNEF patients, LV volumes remained unchanged and reduction of LV mass resulted in decreased relative wall thickness. The present study also observed unchanged LVMI/LVEDVI ratio in the HFREF group and a trend for reduced LVMI/LVEDVI ratio in the HFNEF group. The reversal of LV remodelling with β-blocker therapy therefore parallels the regression of cardiomyocyte hypertrophy observed in the present study with unchanged MyD in HFREF and decreased MyD in HFNEF.

Skinned cardiomyocytes isolated from biopsies of HFNEF patients have an elevated Fpassive, which was higher than Fpassive of HFREF cardiomyocytes4 or of normal cells.5 In vitro administration of PKA corrected the high Fpassive of HFNEF cardiomyocytes, which was therefore attributed to phosphorylatable myofilamentary proteins.4 Within the HFNEF patient group, the present study observed higher Fpassive in β+HFNEF patients than in β−HFNEF patients. After in vitro administration of PKA, Fpassive was still higher in β+HFNEF patients. This finding suggests the effect of β-blocker therapy on cardiomyocyte Fpassive in HFNEF relates not only to hypophosphorylation but also to structural or other posttranslational modifications of myofilamentary proteins. The persistent elevation of pCa50 after PKA observed in β+HFNEF cardiomyocytes also supports β-blocker therapy to induce additional modifications of myofilamentary proteins.

Heart failure with reduced LVEF is usually associated with reduced β1AR density and translocation of GRK2 to the plasma membrane. These changes in β-adrenergic signalling were not observed in a recently published HFNEF rat model.35 Although the present study did not look at βAR density in membrane fractions but at βAR protein expression in homogenates, it found important differences in the expression of β1AR and GRK2 proteins between patient groups with higher myocardial expression of both proteins in HFREF than in HFNEF irrespective of the use of β-blockers. This unequal expression of β-adrenergic signalling proteins in both HF phenotypes could explain changes in other components of the β-adrenergic system by β-blocker therapy unique for each HF phenotype. Of the different components of the β-adrenergic system, Gs protein was significantly lower in HFNEF patients with β-blockers. Downregulation of β2AR-Gs signalling could be beneficial as it would favour β2AR-Gi signalling, which is known to exert antiapoptotic effects.36 Because of normal contractile LV function, potential negative inotropic effects resulting from increased β2AR-Gi signalling are less important for HFNEF patients.

Myocardial effects of β-blocker therapy unique to heart failure with reduced left ventricular ejection fraction

The reduced expression of Gi in HFREF patients treated with β-blockers could be beneficial, because Gi is upregulated in HFREF patients with a dilated hypocontractile LV and contributes to the blunted inotropic response to adrenergic stimulation observed in these patients.37,38 Desmin phosphorylation responded differently to β-blocker therapy with increased phosphorylation in HFNEF and decreased phosphorylation in HFREF. Most previous studies on myofilamentary proteins looked at desmin expression and not at desmin phosphorylation. Desmin loss has been observed in ischaemic cardiomyopathy,39 disrupted desmin in doxorubicin-induced cardiotoxicity,40 and desmin accumulation in restrictive cardiomyopathy.41 Only one study reported on desmin phosphorylation and linked desmin phosphorylation to myofibrillar disarray in cardiomyopathic hamster hearts.42 Based on this study, the reduced desmin phosphorylation in β+HFREF could be beneficial, as it would limit myofibrillar disarray in HFREF hearts.

Limitations

Patient recruitment of the present study was based on referral for diagnostic endomyocardial biopsy procurement. This resulted in a cross-sectional, non-randomized study design. Ethical restrictions only allowed for diagnostic biopsy procurement. This prevented a serial, randomized study design because follow-up biopsies would no longer serve diagnostic purposes. The cross-sectional, non-randomized study design could cause untreated patients to systematically differ from treated patients because of the presence of certain comorbidities for which β-blockers were contraindicated. Such comorbidities, which could have significantly confounded the study results, were however unlikely. Failure to use β-blockers in the β−HFREF group resulted mainly from the referring physician non-adhering to current guidelines as the majority of these patients (14/17) were started on β-blocker therapy during the same hospitalization. Furthermore, β-blockers were used in the β+HFNEF group for control of arterial hypertension (14/16) and/or previous atrial tachyarrhythmias (4/16). Prevalence of arterial hypertension was, however, similar in the β+HFNEF and β−HFNEF groups (Table 1) and, at the time of study, all patients were in regular sinus rhythm. The comparable LVEF in the β−HFREF and β+HFREF groups probably also resulted from patient recruitment based on referral for diagnostic endomyocardial biopsy procurement. This recruitment procedure introduced a bias consisting of more frequent referral for endomyocardial biopsy procurement in patients with a poor functional response to β-blocker therapy.

Conclusions

Myocardial effects associated with β-blocker therapy are either shared by HFNEF and HFREF groups, unique to HFNEF or unique to HFREF. The chronic β-blocker therapy consisted of carvedilol 12.7 ± 3.0 mg b.i.d. and bisoprolol 5.6 ± 0.8 mg b.i.d. and was on average started 12 months prior to the study. Higher pCa50, higher Factive, lower TnI phosphorylation, and lower β2AR expression are shared and beneficial for cardiomyocyte contractile performance. Lower MyD and higher Fpassive are unique to HFNEF and probably relate to the hypertrophied and stiff cardiomyocytes characteristic of HFNEF. Unchanged CVF in HFREF confirms poor clinical outcome of β-blocker therapy in HFREF patients with intense myocardial fibrosis, whereas lower Gi in HFREF contributes to improve myocardial contractile performance. Unequal outcome of β-blocker therapy in HFNEF and HFREF could relate to myocardial effects of β-blocker therapy, which are unique to either HFNEF or HFREF.

Funding

This study was supported by ICaR-VU PhD fellowship (2005 to Dr van der Velden).

Conflict of interest: none declared.

References

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