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(Circulation. 2001;104:1012.)
© 2001 American Heart Association, Inc.

Clinical Investigation and Reports

Patients With End-Stage Congestive Heart Failure Treated With ß-Adrenergic Receptor Antagonists Have Improved Ventricular Myocyte Calcium Regulatory Protein Abundance

Hajime Kubo, PhD; Kenneth B. Margulies, MD; Valentino Piacentino, III, BA; John P. Gaughan, PhD; Steven R. Houser, PhD

From the Cardiovascular Research Group, Department of Physiology and Section of Cardiology, Temple University School of Medicine, Philadelphia, Pa.

Correspondence to Steven R. Houser, PhD, Codirector, Cardiovascular Research Group, Department of Physiology, Temple University School of Medicine, 3400 N Broad St, Philadelphia, PA 19140. E-mail srhouser@ unix.temple.edu

	Abstract

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Background— Alterations in Ca²⁺-handling proteins are thought to underlie the deranged Ca²⁺ transients that contribute to deterioration of cardiac function in congestive heart failure (CHF). Clinical trials in CHF patients have shown that treatment with ß-adrenergic receptor antagonists (ßB) improves cardiac performance. The present study determined whether the abundance of Ca²⁺-handling proteins is different in failing hearts from patients treated or untreated with ß B.

Methods and Results— Ca²⁺ regulatory protein abundance was compared in LV myocardium of 10 nonfailing hearts (NF group) and 44 failing hearts (CHF group) removed at transplantation. Analysis was performed in ßB-treated (ßB-CHF) and non–ßB treated (non-ßB-CHF) patients and in 4 subgroups: ischemic cardiomyopathy (ICM, n=10), nonischemic dilated cardiomyopathy (DCM, n=10), ICM with ßB therapy (ßB-ICM, n=12), and DCM with ßB therapy (ßB-DCM, n=12). Sarcoplasmic reticulum Ca²⁺ ATPase, phospholamban, and Na⁺-Ca²⁺ exchanger protein abundance were determined by use of Western blot analysis. Ca²⁺ transients were measured with fluo-3. Sarcoplasmic reticulum Ca²⁺ ATPase was significantly less abundant whereas phospholamban and Na⁺-Ca²⁺ exchanger were not significantly altered in non-ßB-CHF versus NF. Sarcoplasmic reticulum Ca²⁺ ATPase in the ßB-ICM and ßB-DCM was greater than in non-ßB-CHF and were not different than in NF. Ca²⁺ transients in non-ßB-CHF myocytes had significantly smaller peaks and were prolonged versus NF myocytes. Ca²⁺ transients from ßB-CHF myocytes had shorter durations than in ßB-CHF myocytes.

Conclusions— ßB treatment in CHF patients can normalize the abundance of myocyte Ca²⁺ regulatory proteins and improve Ca²⁺-handling.

Key Words: heart failure • sarcoplasmic reticulumCa²⁺-transporting ATPase • sodium-calcium exchanger • proteins, calcium regulatory

	Introduction

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Progressive deterioration of cardiac pump function is a central feature of congestive heart failure (CHF). Depression of myocyte contractility is thought to underlie much of the poor pump performance of the failing human heart.^1,2 A recent study has shown that treatment of CHF patients with ß-adrenergic receptor antagonists (ß-blockers [ßB]) improves cardiac function and ventricular geometry and thereby slows (or in some cases may reverse) progression of CHF.³ Cellular and molecular bases for these ßB effects are not well understood. Our working hypothesis is that ßB slow (or reverse) progression of myocyte contractile defects in failing human heart.

Abnormal Ca²⁺ homeostasis appears to be primarily responsible for depression of myocyte contractility in CHF.^{4– 6} Peak systolic Ca²⁺ is smaller, rate of decay of the Ca²⁺ transient is slower, and diastolic Ca²⁺ can be elevated in failing versus normal ventricular myocytes, especially at faster heart rates.⁶ These observations support the hypothesis that alterations in abundance or activity of molecules that regulate systolic and diastolic Ca²⁺ are centrally involved in depressed contractility of failing heart.

Although the idea that abnormal Ca²⁺ regulation contributes to depressed cardiac performance in human CHF is well established, cellular and molecular bases of aberrant Ca²⁺ homeostasis have not been identified clearly.² The most-frequently studied Ca²⁺-regulatory molecules have been sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA), SERCA regulatory protein phospholamban (PLB), sarcolemmal Na⁺/Ca²⁺ exchanger (NCX), sarcolemmal L-type Ca²⁺ channels, and SR Ca²⁺ release channel (ryanodine receptor). Changes in the abundance or function of these molecules in human heart failure have been variable in previous studies.^7–14 In this regard, SERCA mRNA and protein abundance have been reported to be smaller in failing versus nonfailing human ventricular muscle in some studies, ^8–11 whereas others have found no significant differences.^12–14

The present research examined the hypothesis that chronic ßB therapy in CHF patients improves myocyte Ca²⁺ regulation and contractility by normalizing expression of myocyte Ca²⁺ regulatory proteins. The aim of the present research was to measure and to compare the protein abundance of SERCA, PLB, and NCX in nonfailing and failing human ventricular muscle (obtained at time of cardiac transplantation) from patients that either were or were not being treated with ßB.

	Methods

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Patients and Tissue Preparation
Protein was isolated from transmural LV myocardium from 10 nonfailing (NF group) unused donor hearts and 44 failing hearts. A total of 20 CHF patients were not (non-ßB-CHF) and 24 were receiving ßB therapy (ßB-CHF). Ten patients had ischemic dilated cardiomyopathy (ICM) and were not receiving ßB therapy (ICM group); 12 had ICM and were receiving ßB therapy (ßB-ICM group). Ten had nonischemic dilated cardiomyopathies (DCM) and were not receiving ßB therapy (DCM group); 12 had DCM and were receiving ßB therapy (ßB-DCM group).

Protein Assay
Tissue was homogenized in PBS lysis buffer containing 2% SDS (Fisher Biotech), 1% Igepal CA-630 (Sigma Chemical Co), 0.5% deoxycholate (Sigma), 5 mmol/L EDTA, and proteinase inhibitors (10 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, 5 µg/mL pepstatin A, 8 µg/mL calpain inhibitor I and II, and 200 µg/mL benzamidine). Supernatant was assayed for total protein,¹⁵ mixed with SDS sample buffer (2% SDS, 0.1 mol/L tris-Cl (pH 6.8), 20% glycerol, 100 mmol/L DTT, and 0.02% bromphenol blue) and stored at -70°C. Studies that used isolated human myocytes^16,17 and nonmyocytes used identical techniques.

Western Blot Analysis
Total protein was separated by SDS-PAGE in a buffer that contained 25 mmol/L tris base, 190 mmol/L glycine, and 0.1% SDS and transferred to nitrocellulose in a buffer that contained 25 mmol/L tris base, 190 mmol/L glycine, and 20% methanol. Nonspecific binding was blocked with TBST buffer (0.1% Tween 20, 137 mmol/L NaCl, 2.7 mmol/L KCl, and 25 mmol/L tris base, pH 7.6) that contained 5% nonfat milk. Targeted antigens were probed with monoclonal antibodies; SERCA (gift from Dr Kevin P. Campbell, University of Iowa, Iowa City), PLB (Upstate Biotechnology), NCX (Swant), cardiac -sarcomeric actin (Sigma Immuno Chemicals), and GAPDH (Biogenesis). Antibodies were labeled with horseradish peroxidase by anti-mouse Ig secondary antibody, 1:2000 dilution (Amersham Life Science). Targeted antigens were visualized with enhanced chemiluminescence assay (NEN Life Science). Cellular specificity of all antibodies was tested in LV tissue, isolated myocytes, and isolated ventricular nonmyocytes. SERCA, PLB, NCX, and cardiac actin could not be detected in nonmyocytes (data not shown). GAPDH was observed in every sample.

Films were scanned (Epson Expression 636), and band intensities were quantified with densitometric analysis by use of the NIH Image 1.62f program. Targeted bands were normalized to cardiac actin and GAPDH.

Intracellular Ca²⁺ Measurements
Fluo-3 was used to measure changes in cytosolic free Ca²⁺ (37°C) induced by 500-ms voltage-clamp steps through patch pipettes as described previously.^16–18

Statistical Analysis
Analysis of NF versus CHF samples was with an unpaired 2-tailed Student t test. Follow-up subgroup analyses (3 group, 5 group) was by ANOVA with pairwise analysis. A Bonferroni multiple comparison was used to locate significant differences among groups. A value of P<0.05 was set for statistical significance.

	Results

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Patient Characteristics
CHF duration was greatest in ßB-DCM patients (see Table 1). Heart weight/body weight ratio (HW/BW) was significantly greater in CHF (n=44) than NF (n=10; 6.7± 0.25 versus 4.9± 0.4; P<0.01). When patients were sorted by disease and treatment, HW/BW was greater in non-ßB-treated CHF patients than in those with NF hearts. HW/BW of ßB-treated CHF hearts were also greater than in NF hearts and less than in non-ß B-treated CHF hearts. However, these differences were not significant. Ejection fraction (EF) was significantly smaller in all CHF groups versus NF patients.

View this table: [in this window] [in a new window]   Table 1. Patient Characteristics

Average ßB dosages and other medications are listed in Table 2. Of the CHF patients, >90% required intravenous inotropic agents at time of transplantation, and 5 of 10 NF patients were receiving dopamine for blood pressure support.

View this table: [in this window] [in a new window]   Table 2. Medications

SERCA, PLB, and NCX in NF Versus CHF LV Samples
Ca²⁺ regulatory protein abundance was first compared in NF (n=10) versus all CHF (n=44) samples (Figure 1). Substantial patient-to-patient variability occurred in abundance of SERCA, PLB, and NCX within the CHF group. Despite this variability, SERCA (SERCA/actin) was significantly less abundant in CHF versus NF muscles (Figure 2A), whereas PLB (Figure 2B) and NCX (Figure 2C) were not significantly different.

View larger version (52K): [in this window] [in a new window]   Figure 1. Representative Western blot of protein from NF and CHF samples. NCX, SERCA, PLB pentamer and monomer, GAPDH, and cardiac-specific actin are shown. NCX bands were not seen regularly at 70 and 50 kD.

View larger version (16K): [in this window] [in a new window]   Figure 2. Mean±SEM for SERCA (A), PLB (B), NCX (C), SERCA/PLB ratio (D), and SERCA/NCX (E) protein abundance (normalized by actin) in NF (n=10) and CHF samples (n=44). * Statistical significance from NF group.

PLB interacts with SERCA and inhibits its Ca²⁺ transport rate. ¹⁹ Phosphorylation of PLB relieves this inhibition. The relative abundance of these 2 proteins and level of PLB phosphorylation influences Ca²⁺ homeostasis and cardiac myocyte contractility.^20–22 Therefore, SERCA/PLB ratio should reflect Ca²⁺ transport capacity of the SR. SERCA/PLB was significantly smaller in CHF versus NF samples (Figure 2D).

SERCA and NCX compete for cytoplasmic Ca²⁺ during the Ca²⁺ transient²³ and a high SERCA/NCX activity is associated with rapid decay of the Ca²⁺ transient. Decreases in SERCA/NCX activity should reduce the size and slow the decay rate of the Ca²⁺ transient. SERCA/NCX protein abundance of CHF samples was smaller but not significantly different from NF samples (Figure 2E).

CHF samples were grouped with respect to absence or presence of ßB treatment and compared with NF samples. Representative data are shown in Figures 3A and 3B and averages in Figure 4. SERCA was significantly less abundant in non-ßB-CHF than in NF but there was no difference between NF and ßB-CHF groups (Figure 4A). PLB and NCX were more abundant in non-ßB-CHF than in ßB-CHF or NF samples but these differences were not statistically significant (Figures 4B and 4C). SERCA/PLB and SERCA/NCX were significantly smaller in non-ßB-CHF than in NF or ß B-CHF samples, and no significant differences were seen between NF and ßB-CHF samples (Figures 4D and 4E).

View larger version (50K): [in this window] [in a new window]   Figure 3. Representative Western blot of samples (A) from NF, non-ß B-ICM, and ßB-ICM patients and (B) from NF, non-ßB-DCM and ß B-DCM patients. NCX, SERCA, PLB pentamer and monomer, GAPDH, and cardiac-specific actin are shown.

View larger version (29K): [in this window] [in a new window]   Figure 4. Mean±SEM for SERCA (A), PLB (B), NCX (C), SERCA/PLB ratio (D), and SERCA/NCX (E) protein abundance (normalized by actin) in NF (n=10), non-ßB-CHF (n=20) and ßB-CHF (n=24). *Statistical significance from NF and non-ßB-CHF group means.

CHF samples finally were subgrouped with respect to origin of disease and absence or presence of ßB treatment (Figures 3A and 3B). SERCA abundance in ICM and DCM patients who did not receive ßB was significantly less than in NF muscles (Figure 5A). Among DCM patients who received ßB drugs SERCA abundance was significantly greater than in non-ßB disease-matched counterparts. SERCA abundance in ßB-treated patients was less than but not significantly different than in NF hearts (Figure 5A). Similar results were obtained when SERCA (and all other proteins) were normalized by GAPDH (data not shown).

View larger version (30K): [in this window] [in a new window]   Figure 5. Mean±SEM for SERCA (A), PLB (B), NCX (C), SERCA/PLB (D), and SERCA/NCX (E) protein abundance (normalized by actin) in NF (n= 10), ICM (n=10), ßB-ICM (n=12), DCM (n=10), and ßB-DCM (n=12). *, , and indicate statistical significance from NF, ICM, and DCM group means.

PLB (sum of pentameric and monomeric forms) and NCX protein abundance were greater in non-ßB-treated ICM and DCM than in either NF or ßB-treated muscles (Figures 5B and 5C), but these differences failed to reach statistical significance. Identical results were obtained when PLB pentameric and monomeric forms were analyzed individually.

Ratios of SERCA/PLB and SERCA/NCX protein abundance were significantly smaller in ICM and DCM CHF patients versus NF hearts (Figures 5D and 5E). SERCA/PLB and SERCA/NCX protein abundance in ß B-treated patients was significantly greater than in disease-matched non-ßB patients and was not significantly different than in NF hearts (Figures 5D and 5E). Actin/total protein abundance was also significantly smaller in ICM versus NF (see Figure 3).

Ca²⁺ Transients in NF, Non-ßB-CHF, and ßB-Treated-CHF Myocytes
Peak systolic Ca²⁺ was smaller, Ca²⁺ transient duration was longer, and rate of decay of the transient was slower in non-ßB-CHF versus NF myocytes (Figure 6). Ca²⁺ transients in ßB-CHF myocytes had a shorter duration and faster rate of decay than non-ßB-CHF myocytes, and these were not different from NF. Peak systolic Ca²⁺ was only modestly (P>0.05) greater in ßB-CHF versus non-ßB-CHF myocytes. These experiments were performed with control of the magnitude and duration of depolarization because differences in AP duration have a significant effect on the decay of the Ca²⁺ transient in failing human ventricular myocytes through the NCX.¹⁷ Therefore, the faster rate of Ca²⁺ transient decay in ßB- versus non-ßB-CHF myocytes probably reflects enhanced SR Ca²⁺ transport.

View larger version (23K): [in this window] [in a new window]   Figure 6. A, Representative Ca2+ transients from NF, non-ßB-CHF, and ßB-CHF myocytes. B, Mean±SEM systolic and diastolic [Ca] i. C, Time to 50% decay and time constant (Tau) of decay of the Ca transient. * and ||, significant difference from NF and non-ßB-CHF group mean, respectively. and , P=0.05 vs NF and P=0.06 vs non-ßB-CHF group mean, respectively.

	Discussion

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The present data support the hypothesis that ßB treatment of CHF patients can improve myocyte Ca²⁺ handling by normalizing the abundance of Ca²⁺-regulatory proteins. Major observations are (1) that SERCA protein was significantly less abundant in CHF patients not receiving ßB than in NF patients, (2) that SERCA protein abundance was significantly greater in CHF patients treated with ßB than in CHF patients not receiving ßB, (3) that SERCA/PLB and SERCA/NCX were significantly smaller in non-ßB CHF versus NF and greater in ßB-CHF versus non-ßB-CHF patients, (4) that Ca²⁺ transients were smaller and decayed more slowly in non-ßB CHF versus NF myocytes, and (5) that Ca²⁺ transients in ßB-CHF myocytes had significantly faster rates of decay than non-ßB CHF myocytes. These results strongly support the idea that deranged Ca²⁺ homeostasis in human CHF involves alterations in abundance and activity of SR Ca²⁺ transport proteins and suggests that some of these changes can be reversed with ßB therapy. Our observations also suggest a potential mechanism (improved myocyte Ca²⁺ regulation) for the consistent yet unexplained finding that ßB improve LV function in failing human hearts.²⁴

Ca²⁺-Handling Protein Abundance and Ca²⁺ Transients in CHF
Previous studies^7–14 have shown marked variability in the abundance of SERCA, PLB, and NCX proteins among patients with CHF. Our findings of more uniform results when ßB treatment is taken into account suggest that some of this variability is related to treatment effects superimposed on disease-related alterations.

Reduction in SERCA/PLB and SERCA/NCX in CHF myocytes can explain the reduced peak and slower decay of the Ca²⁺ transient we observed in failing myocytes. Partial normalization of Ca²⁺ regulatory protein abundance in ßB-CHF myocytes can also explain the improved rate of decay of their Ca²⁺ transients. These findings support the hypothesis that the reduced abundance of SERCA protein disrupts the balance of Ca²⁺ transport between SERCA and NCX to produce defective Ca²⁺ homeostasis,^2,7,20–22 with an increased reliance on NCX^16,17,25 in failing human ventricular myocytes.

How Do ßB Work in CHF Patients?
Heightened sympathetic nerve activity in CHF helps maintain cardiac output and mean blood pressure in the face of intrinsic defects in cardiac function.^26–27 However, sustained sympathetic activity in CHF is thought to produce abnormalities in adrenergic signaling and cell physiology³ (including abnormal Ca²⁺ handling). The bases of these adrenergic cascade–related myocyte abnormalities are largely unknown. Similarly, although chronic ßB agents are effective in the treatment of heart failure,³ cellular mechanisms for their beneficial effects on cardiac function likewise are unexplained. For example, chronic treatment with carvedilol is particularly effective for improvement of LV EF among CHF patients, but this agent does not produce the alterations in ß-adrenergic receptor density observed with other ßB,²⁸ which implies modifications in myocyte phenotype at a level beyond the adrenergic receptor.

Our present study design was inadequate to prove that ßB cause beneficial changes in cardiocyte Ca²⁺ regulatory gene expression. However, our findings support a link between normalization of Ca²⁺ homeostasis and ßB treatment in CHF patients.²⁹ We cannot rule out the possibility that the pattern of Ca²⁺ regulatory gene expression in NYHA class IV CHF patients is highly variable and that only those patients with high SERCA/PLB and SERCA/NCX tolerate ßB therapies. Arguing against such a selection bias is the fact that ßB-associated increases in SERCA/PLB ratio did not correlate with EF measured before transplantation. In addition, a recent study by Gwathmey et al,³⁰ in which 1 month of treatment with the ßB carteolol increased SERCA activity and cardiac function in myopathic turkey hearts, supports a causal link between ßB therapy and expression of Ca²⁺ regulatory proteins.

We can only speculate about a mechanism for normalization of SERCA protein abundance produced by ßB treatment. This mechanism could involve elimination of ß-adrenergic receptor–mediated repression of myocyte SERCA expression. ßB also could reduce apoptosis,³¹ thereby increasing the number of functional myocytes. Still another possibility is that ßB could be working through nonmyocytes to eliminate expression of factors that depress myocyte function by decreasing SERCA expression.

Why Are ßB-Treated Hearts Still Failing?
Our results suggest that improved heart function in ßB clinical trials^3,24,29 involves normalization of aberrant Ca²⁺ regulatory protein abundance. We speculate that in early CHF, the beneficial effects of ßB on Ca²⁺-handling proteins mediate increased EF and promote beneficial remodeling. However, our data also suggest that CHF still can progress to the point at which transplantation is required in ßB-treated patients with normalized (or partially normalized) myocyte Ca²⁺ handling.

Potential explanations as to why patients with ßB treatment still required transplantation are as diverse as the mechanisms contributing to the syndrome of advanced CHF. Loss of myocytes through necrosis or apoptosis may overwhelm improvements in Ca²⁺ handling. Similarly, ongoing defects in cardiac perfusion or life-threatening arrhythmias may necessitate transplantation despite improvements in contractile function of remnant myocytes. Within the myocyte, abnormalities of electrophysiology, ultrastructure, metabolism, signaling, phosphorylation, or myofilament properties may persist despite improvements in abundance of dysregulated Ca²⁺-handling proteins. Within and beyond the heart, detrimental neurohormone and cytokine activation, vasoconstriction, and other peripheral abnormalities may drive progression of the syndrome.

Limitations
We could not control the types or concentrations of the medications that the patients were receiving. In this regard, 22 of 24 ßB-CHF patients were receiving either metoprolol¹⁶ or carvedilol.⁶ Interestingly, normalization of Ca²⁺ regulatory protein abundance was similar in all ßB-treated patients, which suggests a common mechanism. Importantly, we were unable to find a correlation between SERCA protein abundance and any other medication, including drugs that interfere with angiotensin signaling. Finally, intravenous inotropic agents, particularly milrinone (in 12 of 24 patients) did not preclude the normalized pattern of SERCA abundance in ßB-treated patients.

Summary and Conclusions
CHF patients (NYHA class IV) not treated with ßB had less SERCA protein than did those with NF hearts, and their Ca²⁺ transients were abnormal. In CHF patients treated with ßß, SERCA abundance was not different than in the NF hearts, and their Ca²⁺ transients were improved. These findings suggest that ßB-CHF therapy may cause improved cardiac performance by normalizing expression of Ca²⁺ regulatory proteins. Our observations also suggest that conventional pharmacotherapy may produce the " molecular remodeling" currently being sought through gene therapy.^32,33 Potential for long-term improvements in Ca²⁺ homeostasis in patients with NYHA class IV symptoms of heart failure also supports the use of novel adjuvants, including phosphodiesterase inhibitors³⁴ or mechanical circulatory support³⁵ to help establish ßB therapy in these clinically tenuous patients.

	Acknowledgments

 
The present study was supported by NIH grants HL-33921 and HL-61495 to Dr Houser and HL-03560 and AG-17022 to Dr Margulies. We thank the Heart Failure Unit and the transplant team at Temple University Hospital.

Received March 26, 2001; revision received June 22, 2001; accepted June 22, 2001.

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