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(Circulation. 1995;92:778-784.)
© 1995 American Heart Association, Inc.

Articles

Alterations of Sarcoplasmic Reticulum Proteins in Failing Human Dilated Cardiomyopathy

Markus Meyer, MD; Wolfgang Schillinger; Burkert Pieske, MD; Christian Holubarsch, MD; Claus Heilmann, MD; Herbert Posival, MD; Goro Kuwajima, PhD; Katsuhiko Mikoshiba, MD; Hanjörg Just, MD; Gerd Hasenfuss, MD

From the Medizinische Klinik II und III, Universität Freiburg, Germany (M.M., W.S., B.P., C. Holubarsch, C. Heilmann, H.J., G.H.); the Klinik für Thorax und Kardiovaskularchirurgie, Herzzentrum Nordrheinwestfalen, Bad Oeynhausen, Germany (H.P.); the Shionogi Institute for Medical Science, Osaka, Japan (G.K.); and the Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Japan (K.M.).

Correspondence to Gerd Hasenfuss, MD, Medizinische Klinik III, Universität Freiburg, Hugstetter Str 55, 79106 Freiburg, Germany.

	Abstract

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Background Previous studies provide considerable evidence that excitation-contraction coupling may be disturbed at the level of the sarcoplasmic reticulum (SR) in the failing human heart. Disturbed SR function may result from altered expression of calcium-handling proteins.

Methods and Results Levels of SR proteins involved in calcium release (ryanodine receptor), calcium binding (calsequestrin, calreticulin), and calcium uptake (calcium ATPase, phospholamban) were measured by Western blot analysis in nonfailing human myocardium (n=7) and in end-stage failing myocardium due to dilated cardiomyopathy (n=14). The levels of the ryanodine receptor, calsequestrin, and calreticulin were not significantly different in nonfailing and failing human myocardium. Phospholamban protein levels (pentameric form) normalized per total protein were decreased by 18% in the failing myocardium (P<.05). However, phospholamban protein levels were not significantly different in failing and nonfailing myocardium when normalization was performed per calsequestrin. Protein levels of SR calcium ATPase, normalized per total protein or per calsequestrin, were decreased by 41% (P<.001) or 33% (P<.05), respectively, in the failing myocardium. Furthermore, SR calcium ATPase was decreased relative to ryanodine receptor by 37% (P<.05) and relative to phospholamban by 28% (P<.05).

Conclusions Levels of SR proteins involved in calcium binding and release are unchanged in failing dilated cardiomyopathy. In contrast, protein levels of calcium ATPase involved in SR calcium uptake are reduced in the failing myocardium. Moreover, SR calcium ATPase is decreased relative to its inhibitory protein, phospholamban. These findings support the concept that reduced capacity of the SR to accumulate calcium may reflect a major defect in excitation-contraction coupling in human heart failure.

Key Words: sarcoplasmic reticulum • calcium • heart failure • ryanodine

	Introduction

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Recent studies performed in isolated human myocardium suggested that altered excitation-contraction coupling and, in particular, altered sarcoplasmic reticulum (SR) calcium handling may be of significant pathophysiological relevance in human heart failure.^{1 2 3 4 5} SR function may be disturbed at the level of calcium uptake by SR Ca²⁺-ATPase or its regulatory protein phospholamban at the level of calcium binding by calsequestrin and calreticulin as well as at the level of calcium release through the calcium-sensitive ryanodine receptor. Investigations on steady-state mRNA levels indicated that expression of the ryanodine receptor⁶ and calsequestrin are unchanged in failing human dilated cardiomyopathy.^{7 8} In contrast, it was observed that mRNA levels of SR Ca²⁺-ATPase and phospholamban are decreased in the failing human heart.^{9 10 11 12} However, steady-state mRNA levels cannot necessarily be assumed to be representative of protein levels, in particular because both mRNA and protein synthesis or degradation may be altered in the failing heart. No data are available on protein levels of the ryanodine receptor in human heart failure, and data on SR Ca²⁺-ATPase protein levels are controversial.^{10 13 14} Knowledge of the levels of proteins involved in SR function is important to understand the pathophysiology of the failing heart and to develop new therapeutic strategies for the treatment of heart failure. Moreover, it is important to evaluate these proteins in myocardium from the same hearts to appreciate relative changes between the different components of SR function.

Accordingly, it was the goal of the present study to quantify the ryanodine receptor by Western blot analysis in nonfailing human myocardium and in failing myocardium from hearts with dilated cardiomyopathy. In addition, protein levels of the Ca²⁺-binding proteins calsequestrin and calreticulin and protein levels of SR Ca²⁺-ATPase and phospholamban were investigated in the same hearts.

	Methods

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Patients
Levels of SR proteins were investigated in myocardium from 7 nonfailing and 14 failing human hearts. The failing myocardium was obtained from 14 explanted hearts of patients with end-stage heart failure due to dilated cardiomyopathy who were undergoing cardiac transplantation. The clinical characteristics of the patients from the examination closest to cardiac transplantation are presented in Table 1. Invasive hemodynamic data were obtained within 6 months before cardiac transplantation. Ejection fraction, measured by echocardiography, was obtained within 3 months before transplantation. Seven nonfailing hearts were obtained from brain-dead organ donors: 2 women, 5 men; mean age, 39±9 years (not different from the mean age of the failure group). The donor hearts could not be used for cardiac transplantation for technical reasons. None of these patients had a history of cardiac disease. Tissue samples of the left ventricular free wall were taken immediately after explantation, quickly frozen in liquid nitrogen, and stored at -80°C until use.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics of Patients

The study was reviewed and approved by the Ethical Committee of the University Clinics of Freiburg.

Preparation of Cardiac Tissue Homogenates
About 180 mg of myocardium devoid of fibrotic or adipose tissue, endocardium, epicardium, or great vessels was homogenized in a ninefold volume of 20 mmol/L Na-HEPES, pH 7.4, for 8x15 seconds by use of a Polytron-Homogenizer PT-K (Brinkman Instruments) at a setting of 6 with a PTA-7 unit, followed by 15 strokes of a motorized Potter-Elvehjem. The entire procedure was carried out at 4°C. The protein concentration was determined in triplicate according to Lowry et al¹⁵ with bovine serum albumin as a standard. The yield of protein per gram wet weight was calculated from the protein concentration in the homogenates. It was 140±3 and 136±3 mg/g in myocardium from nonfailing hearts and hearts with dilated cardiomyopathy, respectively (no significant differences between groups). Aliquots of the homogenates were frozen in liquid nitrogen and stored at -80°C until use.

Western Blot Analysis
Samples were solubilized in 2% sodium dodecyl sulfate (SDS), 5% 2-ß-mercaptoethanol, 10% glycerol, 0.00125% bromphenol blue, and 0.0625 mol/L Tris-Cl, pH 6.8. Lysis was performed for 10 minutes at 37°C for obtaining the pentameric form of phospholamban and the other SR proteins. Lysis was performed for 5 minutes at 95°C to dissociate the pentameric form of phospholamban into subunits. Samples were subjected to SDS-PAGE using the Laemmli buffer system¹⁶ in a Mini-Protean II Dual Slab Cell (Bio-Rad Ltd). Electrophoresis was run until complete elution of the dye front. Proteins were transferred to nitrocellulose in a Mini Trans-Blot Transfer Cell (Bio-Rad Ltd) according to the procedure of Towbin¹⁷ with the minor modification that SDS was included in the transfer buffer (25 mmol/L Tris, 192 mmol/L glycine, 0.0375% SDS, and 20% vol/vol methanol, pH 8.3). The transfer was carried out at 4°C for 2 hours at a constant voltage setting of 125 V. The transfer buffer was changed after 1 hour when an increased current generation was observed. Transfer was checked by staining of the blots in Ponceau S solution (Sigma Ltd) and staining of the remaining polyacrylamide gels in Coomassie brilliant blue G (Sigma Ltd). The blots were blocked in 5% nonfat milk diluted in Tris-buffered saline (TBS) (20 mmol/L Tris-Cl, pH 7.4, 150 mmol/L NaCl) either for 3 hours at room temperature or overnight at 4°C. The blots were washed three times for 1 minute and then three times for 5 minutes with changing volumes of TBS. Thereafter, blots were incubated in the primary antibody solution, diluted in TBS containing 0.1% Tween-20 and 1% bovine serum albumin (2.5% nonfat milk for the anti-calsequestrin antibody) for 2 hours at room temperature (Table 2; see References 18 through 22). The blots were washed three times for 1 minute and three times for 5 minutes in TBS and then incubated in the secondary antibody solution diluted in the same buffer as above for 1 hour at room temperature (Table 2). The blots were again washed three times for 1 minute and three times for 5 minutes, incubated in enhanced chemiluminescence (ECL)-detection reagents (Amersham Buchler Ltd) for 1 minute, and exposed to an X-OMAT AR x-ray film (Kodak Inc) for 30 seconds to 5 minutes. Western blot analysis of SR Ca²⁺-ATPase, phospholamban, calsequestrin, and calreticulin was performed in 7 nonfailing hearts and in 14 failing hearts. Since not enough tissue was available from 1 failing heart, Western blot analysis of the ryanodine receptor was performed in 7 nonfailing and 13 failing hearts. See Table 2 for further details.

View this table: [in this window] [in a new window]   Table 2. Conditions Used for Western Blot Analysis

Quantification of Immunoreactive Bands
The band densities were evaluated by densitometric scanning using a 2202 Ultrascan laser densitometer (LKB). Each individual value represents the mean of two independent determinations. To promote comparability of determinations from the different blots, one nonfailing reference heart was used as a standard on all blots. For each blot, normalization was performed by dividing densitometric units of each heart by the value of the reference heart from the same blot. For graphical reasons, normalized values were multiplied by a constant (densitometric units of the reference heart from blot 1). Linearity between amounts of protein and immunoreactive signals was proven for each SR protein by plotting different amounts of protein at varying exposure times against corresponding densitometric units.

Statistical Analysis
Data are expressed as mean±SEM. Comparisons between nonfailing and failing human myocardium were performed by nonpaired t test. A value of P<.05 was accepted as statistically significant.

	Results

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Ryanodine Receptor Protein Levels
Fig 1 shows Western blot analysis in failing and nonfailing human myocardium using a polyclonal antibody against the ryanodine receptor. There was no significant difference in protein levels of the ryanodine receptor between nonfailing myocardium and failing myocardium from hearts with dilated cardiomyopathy (Fig 2).

View larger version (40K): [in this window] [in a new window]   Figure 1. Western blot analysis of sarcoplasmic reticulum (SR) proteins in myocardium from nonfailing human hearts (NF) and from hearts with end-stage failing dilated cardiomyopathy (DCM). For conditions of the Western blot analysis, see Table 2. Please note that the different proteins were investigated in different blots.

View larger version (26K): [in this window] [in a new window]   Figure 2. Bar graphs showing protein levels of sarcoplasmic reticulum calcium release channel (ryanodine receptor) in nonfailing myocardium (NF) and in end-stage failing myocardium from hearts with dilated cardiomyopathy (DCM). Left, Normalization was performed per total protein recovered per gram wet weight and right, normalization per calsequestrin.

Calsequestrin and Calreticulin Protein Levels
Polyclonal antibodies were used to quantify the SR calcium binding proteins calsequestrin and calreticulin (Fig 1). There was no difference between nonfailing and failing human myocardium with respect to levels of these proteins (Fig 3).

View larger version (25K): [in this window] [in a new window]   Figure 3. Bar graphs showing protein levels of sarcoplasmic reticulum calcium binding proteins calsequestrin and calreticulin in nonfailing myocardium (NF) and in end-stage failing myocardium from hearts with dilated cardiomyopathy (DCM). Normalization was performed per total protein recovered per gram wet weight.

SR Ca²⁺-ATPase and Phospholamban Protein Levels
SR Ca²⁺-ATPase protein levels were evaluated by use of a monoclonal antibody (Fig 1). In accordance with previous measurements, we found that protein levels of SR Ca²⁺-ATPase normalized per total protein or per calsequestrin were reduced significantly, by 41% or 33%, respectively, in failing dilated cardiomyopathy (Fig 4). Phospholamban was investigated at the level of its pentameric or monomeric form, respectively, by use of a monoclonal antibody (Fig 1). Phospholamban protein levels were decreased significantly relative to total protein in dilated cardiomyopathy (Fig 5). However, when both forms of phospholamban were normalized to calsequestrin, there were no significant differences between failing and nonfailing human hearts (Fig 5). Since phospholamban inhibits SR Ca²⁺-ATPase, activity of SR Ca²⁺-ATPase may be depressed or enhanced by changes in the proportion of the levels of both proteins. Accordingly, the ratio of SR Ca²⁺-ATPase to phospholamban was calculated in nonfailing and failing human myocardium. This ratio was decreased significantly, by 28%, in failing human myocardium (Fig 6). Furthermore, the ratio of SR Ca²⁺-ATPase to ryanodine receptor was decreased significantly, by 37%, in the failing human heart.

View larger version (23K): [in this window] [in a new window]   Figure 4. Bar graphs showing protein levels of sarcoplasmic reticulum (SR) Ca2+-ATPase in nonfailing myocardium (NF) and in end-stage failing myocardium from hearts with dilated cardiomyopathy (DCM). Left, Normalization was performed per total protein recovered per gram wet weight and right, normalization per calsequestrin.

View larger version (27K): [in this window] [in a new window]   Figure 5. Bar graphs showing protein levels of phospholamban in nonfailing myocardium (NF) and in end-stage failing myocardium from hearts with dilated cardiomyopathy (DCM). Top, Both the pentameric form and the monomeric form were normalized per total protein recovered per gram wet weight. Bottom, Both forms were normalized per calsequestrin.

View larger version (22K): [in this window] [in a new window]   Figure 6. Bar graphs showing protein levels of sarcoplasmic reticulum (SR) Ca2+-ATPase in nonfailing myocardium (NF) and in end-stage failing myocardium from hearts with dilated cardiomyopathy (DCM). Left, Normalization was performed per protein levels of phospholamban (pentameric form). Right, Normalization was performed per protein levels of ryanodine receptor. SR Ca2+-ATPase normalized to the monomeric form of phospholamban tended to decrease by 22% in failing compared with nonfailing myocardium; however, this decrease was statistically not significant.

	Discussion

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The present study shows that (1) protein levels of the ryanodine receptor calsequestrin as well as calreticulin are not significantly altered in failing human myocardium due to dilated cardiomyopathy; (2) phospholamban is reduced relative to total protein but unchanged when normalization is performed to calsequestrin; (3) SR Ca²⁺-ATPase protein levels are reduced significantly relative to total protein and calsequestrin; and (4) SR Ca²⁺-ATPase protein levels are reduced significantly relative to levels of its inhibitory protein phospholamban.

The calcium sensitive SR calcium release channel (ryanodine receptor) plays a key role in excitation-contraction coupling. The ryanodine receptor is regulated by calcium, which enters the cell through voltage-gated calcium channels in the sarcolemma. Once activated by calcium influx, the channel opens and releases calcium for activation of contractile proteins.^{23 24 25} This process is called calcium-induced calcium release.²⁶ The present data indicate that protein levels of the ryanodine receptor are unchanged in heart failure due to dilated cardiomyopathy. This is consistent with recent measurements by Brillantes et al⁶ showing no significant change in ryanodine receptor mRNA levels in dilated cardiomyopathy compared with nonfailing control myocardium. Of course, the present data do not exclude the possibility that altered function of the normally expressed ryanodine receptor may be involved in disturbed excitation-contraction coupling in the failing human heart. D'Agnolo et al²⁷ recently found that the caffeine threshold of the ryanodine receptor was increased, suggesting an impaired gating mechanism of the calcium release channel in dilated cardiomyopathy. In contrast, Holmberg and Williams²⁸ reported normal properties of ryanodine receptor from failing human hearts in single-channel recordings under voltage-clamp conditions.

Calsequestrin and calreticulin are located within the lumen of the SR.^{29 30 31} Calsequestrin, a high-capacity moderate-affinity calcium binding protein, is primarily responsible for the calcium storage capacity of the SR in cardiac muscle.³¹ The present finding of unchanged calsequestrin levels in dilated cardiomyopathy is consistent with recent mRNA and protein measurements.^{7 8 14} Furthermore, several studies performed in animal models of myocardial hypertrophy and failure indicate that calsequestrin levels remain unchanged.^{32 33} Therefore, in the present study, calsequestrin protein levels were used for normalization of the other calcium regulatory proteins. Calreticulin is a major calcium binding protein of nonmuscle endoplasmic reticulum membranes.^{30 31} In addition to its apparent calcium storage role, accumulating evidence suggests that calreticulin has other regulatory functions within the cell.³⁰ This is the first time that calreticulin has been studied in human myocardium. The present analysis shows that calreticulin is present in nonfailing and failing human hearts and that levels of this protein are similar in both types of human myocardium.

Calcium transport into the SR occurs by SR Ca²⁺-ATPase, which transports two calcium ions per molecule of high-energy phosphate hydrolyzed against a high ion gradient.³¹ This pump, together with the Na⁺-Ca²⁺ exchanger and the sarcolemmal calcium ATPase, eliminates calcium from the cytosol to facilitate relaxation of the myocardium.²³ Moreover, SR Ca²⁺-ATPase is crucial for calcium accumulation within the SR and thus for the availability of calcium for systolic release through the ryanodine receptor.²³ Consistent with previous measurements, the present data confirm that SR Ca²⁺-ATPase protein levels are significantly reduced in failing human myocardium.^{10 13} Reduced expression of SR Ca²⁺-ATPase in failing human myocardium was also suggested from several studies measuring steady-state mRNA levels.^{7 9 10}

Phospholamban is the regulatory protein of SR Ca²⁺-ATPase.^{31 34 35 36} Dephosphorylated phospholamban is an inhibitor of SR Ca²⁺-ATPase activity, and phosphorylation relieves this inhibition. The inhibition has been suggested to involve direct protein-protein interaction followed by conformational changes in the SR Ca²⁺-ATPase, resulting in a decrease in the affinity of the calcium pump for calcium.³⁶ The present data show that phospholamban protein levels are significantly decreased relative to total protein in failing dilated cardiomyopathy. When phospholamban was normalized to calsequestrin, however, there was no significant difference between failing and nonfailing myocardium. The different findings depending on normalization procedure may result from the observation that calsequestrin also tended to decrease relative to total protein in the failing myocardium, which in turn may indicate a decrease in myocyte relative to nonmyocyte proteins.

Interestingly, SR Ca²⁺-ATPase protein levels were decreased to a greater proportion than protein levels of phospholamban in the failing myocardium. If we assume that the stoichiometry of phospholamban to SR Ca²⁺-ATPase determines the level of SR Ca²⁺-ATPase inhibition, this finding may indicate that in the basal low-phosphorylated state, depression of SR calcium uptake is even more pronounced than would be expected from the decrease of SR Ca²⁺-ATPase protein levels in the failing myocardium. This interpretation would be consistent with functional abnormalities observed in the failing human myocardium: (1) a decreased rate of calcium removal associated with a diminished or absent frequency potentiation of contractile force^{1 2 3 4 5 13 37} ; (2) a decreased calcium release at higher rates of stimulation as a consequence of reduced capacity of the SR to accumulate calcium for the subsequent release^{4 5 13} ; and (3) a pronounced increase in the rate of calcium removal associated with a partial normalization of the force-frequency relation after application of forskolin or isoproterenol.^{38 39} These agents, by stimulation of cAMP formation, activate protein kinase A to phosphorylate different proteins, including phospholamban.⁴⁰

Decreased protein levels of SR Ca²⁺-ATPase could result from a decreased content of SR within the myocytes from failing hearts or from a reduced density of the calcium pump within the SR membrane. The findings of unchanged protein levels of ryanodine receptor, calsequestrin, and calreticulin and decreased SR Ca²⁺-ATPase protein levels relative to calsequestrin, ryanodine receptor, and phospholamban support the latter possibility. The present data do not allow us to decide whether decreased levels of SR Ca²⁺-ATPase result from a selective downregulation of the expression of this protein or from a lack of upregulation in the presence of a hypertrophy-associated increased expression of other myocyte and nonmyocyte proteins in the failing myocardium.

Finally, it should be discussed that in a recent study, performed in a small number of hearts, Movsesian et al¹⁴ did not observe a significant difference in protein levels of SR Ca²⁺-ATPase between nonfailing human myocardium and myocardium from failing hearts with end-stage dilated cardiomyopathy. Moreover, the same group did not find a difference in SR calcium uptake between failing and nonfailing human myocardium, which also is in contrast to other studies.^{41 42} The discrepancy between the present data and the data of Movsesian et al is not clear. However, it should be mentioned that there is a wide variation in protein levels of SR Ca²⁺-ATPase even within the group of end-stage failing hearts.^{10 13} Differences in protein levels of SR Ca²⁺-ATPase within the group of failing hearts correlated closely with differences in function of the isolated failing myocardium, although hemodynamic parameters measured in the patients before cardiac transplantation did not indicate relevant differences in the degree of myocardial failure during rest.¹³ Because of the variation in protein levels of SR Ca²⁺-ATPase in the group of end-stage failing hearts and the overlap with nonfailing myocardium, statistical differences in protein levels of SR Ca²⁺-ATPase between nonfailing and failing myocardium may not be seen when the comparison is performed in a small number of hearts.

In summary, the present study shows that protein levels of the SR calcium release channel (ryanodine receptor), as well as protein levels of calsequestrin and calreticulin, are not significantly altered in end-stage failing dilated cardiomyopathy. In contrast, protein levels of SR Ca²⁺-ATPase were found to be significantly reduced relative to total protein, to calsequestrin, to the ryanodine receptor, and to phospholamban. This is consistent with the concept that reduced capacity of the SR to accumulate calcium may be of major pathophysiological relevance in failing human myocardium.

	Acknowledgments

 
This study was supported by DFG grant HA 1233/3-1. Dr Hasenfuss is an Established Investigator of the German Research Foundation (DFG, Heisenberg-Stipendium HA 1233/4-1). We are very grateful to Dr K.P. Campbell for providing the monoclonal antibody against SR Ca²⁺-ATPase.

Received November 16, 1994; revision received February 1, 1995; accepted February 8, 1995.

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