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(Circulation. 1995;92:2504-2510.)
© 1995 American Heart Association, Inc.

Articles

Effect of Ryanodine on Sarcoplasmic Reticulum Ca²⁺ Accumulation in Nonfailing and Failing Human Myocardium

Lynn R. Nimer, MD; Dolores H. Needleman, PhD; Susan L. Hamilton, PhD; Judith Krall, BS; Matthew A. Movsesian, MD

From Research Service, Salt Lake City (Utah) Veterans Affairs Medical Center (M.A.M.); the Departments of Internal Medicine (Cardiology) (L.R.N., J.K., M.A.M.) and Pharmacology (M.A.M.), University of Utah School of Medicine; and the Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Tex (D.H.N., S.L.H.).

Correspondence to Matthew Movsesian, MD, Cardiology Division, University of Utah Health Sciences Center, 50 N Medical Dr, Salt Lake City, UT 84132. E-mail matthew@cardio.med.utah.edu.

	Abstract

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Background The purpose of this study was to determine whether abnormal Ca²⁺ release through ryanodine-sensitive Ca²⁺ channels in the sarcoplasmic reticulum might contribute to the abnormal [Ca²⁺]_i homeostasis that has been described in failing human myocardium.

Methods and Results Occupancy of low-affinity ryanodine binding sites on ryanodine-sensitive Ca²⁺ channels stimulates oxalate-supported, ATP-dependent Ca²⁺ accumulation in sarcoplasmic reticulum–derived microsomes by inhibiting concurrent Ca²⁺ efflux through these channels. We examined the effects of 0.5 mmol/L ryanodine on ⁴⁵Ca²⁺ accumulation in microsomes prepared from nonfailing (n=8) and failing (n=10) human left ventricular myocardium. In the absence of ryanodine, ⁴⁵Ca²⁺ accumulation reached similar levels in microsomes from nonfailing and failing hearts. Incubation with 0.5 mmol/L ryanodine caused a 52.2±6.5% increase in peak ⁴⁵Ca²⁺ accumulation in microsomes from nonfailing hearts and a 24.3±4.1% increase in microsomes from failing hearts. The density of high-affinity ryanodine binding sites and the inhibition of [³H]ryanodine dissociation from these sites by 0.1 mmol/L ryanodine were similar in microsomes from nonfailing and failing hearts.

Conclusions These results, which demonstrate a diminished stimulation of Ca²⁺ accumulation by ryanodine in sarcoplasmic reticulum–derived microsomes from failing human myocardium that could be explained by an uncoupling of the occupancy of low-affinity ryanodine binding sites from the reduction in the open probability of these channels or by concurrent Ca²⁺ efflux through a ryanodine-insensitive mechanism, are evidence that increased efflux of Ca²⁺ from the sarcoplasmic reticulum may contribute to the abnormal [Ca²⁺]_i homeostasis described in failing human myocardium.

Key Words: sarcoplasmic reticulum • cardiomyopathy • calcium • calcium channels • heart failure

	Introduction

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Accumulation and release of Ca²⁺ by the sarcoplasmic reticulum are the principal mechanisms involved in the transient decreases and increases of cytosolic Ca²⁺ concentrations in cardiac myocytes. Ca²⁺ accumulation occurs through the activity of a Ca²⁺-transporting ATPase distributed throughout the sarcoplasmic reticulum. Ca²⁺ release occurs principally through Ca²⁺ channels in the junctional sarcoplasmic reticulum whose open probability is increased by the binding of ryanodine to high-affinity (K_d5 nmol/L) sites on these channels and decreased by the binding of ryanodine to low-affinity (K_d3 µmol/L) sites on these channels (for review, see Reference 1).

Abnormalities of [Ca²⁺]_i homeostasis have been observed in muscle strips and myocytes isolated from failing human left ventricular myocardium.^{2 3} A diminished ability to restore low cytosolic [Ca²⁺] during diastole was observed in both muscle strips and isolated myocytes, whereas a reduction in peak systolic [Ca²⁺] was observed only in isolated myocytes. These abnormalities could be explained by a decrease in the activity of the Ca²⁺-transporting ATPase of the sarcoplasmic reticulum or by increased Ca²⁺ efflux through ryanodine-sensitive Ca²⁺ channels. With respect to the former possibility, impaired Ca²⁺ transport has been observed in several animal models of heart failure (for review, see Reference 1), but the applicability of these findings to the human disease is unclear. Some investigators have reported that the steady state kinetics of Ca²⁺ transport and its modulation by phospholamban are not altered in sarcoplasmic reticulum–enriched microsomes prepared from failing human left ventricular myocardium and that protein levels of Ca²⁺-transporting ATPase and phospholamban are comparable in nonfailing and failing human myocardium, with no evidence of increased degradation of these proteins in the latter.^{4 5 6 7 8} Other investigators have found diminutions in Ca²⁺-transporting ATPase activity and protein content in failing human myocardium.^{9 10} The reason for the discrepancy in these observations is unknown.

With respect to increased Ca²⁺ efflux from the sarcoplasmic reticulum, there are several examples of abnormalities involving the function of ryanodine-sensitive Ca²⁺ channels in animal models of myocardial ischemia. The elevation of cytosolic free Ca²⁺ concentrations in ischemic rat myocardium and the reduction of Ca²⁺ accumulation in microsomes prepared from this tissue are diminished in the presence of high concentrations of ryanodine.^{11 12 13} In addition, the impairment of ATP-dependent Ca²⁺ accumulation in canine myocardial microsomes exposed to oxygen free radicals and in microsomes prepared from rat myocardium exposed to high [Ca²⁺]_o is prevented by inclusion of ryanodine at high concentrations in the reaction mixtures.^{14 15} These observations suggest that the impairment of Ca²⁺ accumulation observed in these animal models of ischemia results from concurrent Ca²⁺ efflux through ryanodine-sensitive Ca²⁺ channels whose open probability is increased under the experimental conditions and that ryanodine at high concentrations reduces the impairment by reducing the open probability of these channels. The finding that enzymatic digestion of sarcoplasmic reticulum Ca²⁺ channels by the protease calpain II results in an increase in the open probability of these channels is evidence of the plausibility of this hypothesis,¹⁶ as is the observation that the increase in Ca²⁺ efflux from the sarcoplasmic reticulum of skeletal muscle in malignant hyperthermia is attributable to a genetic structural abnormality in the ryanodine-sensitive Ca²⁺ channel of this tissue.¹⁷

There are also indications that changes in the level and function of ryanodine-sensitive Ca²⁺ channels occur in animal models of myocardial hypertrophy and failure. The increase in the amount of Ca²⁺ released by doxorubicin and caffeine from sarcoplasmic reticulum–enriched microsomes prepared from rat hearts with pressure overload–induced myocardial hypertrophy suggests that abnormal Ca²⁺ efflux through ryanodine-sensitive Ca²⁺ channels may be involved in the abnormal [Ca²⁺]_i homeostasis in this condition.¹⁸ In addition, the density of high-affinity ryanodine binding sites is reduced in myocardium from rats with pressure overload–induced myocardial hypertrophy, rabbits with doxorubicin-induced cardiomyopathy, and dogs with spontaneous or pacing-induced cardiomyopathy.^{18 19 20 21 22} In contrast, an increase in the density of ryanodine-sensitive Ca²⁺ channels has been observed in microsomes prepared from the hearts of cardiomyopathic Syrian hamsters.^{23 24 25} Somewhat surprisingly, the density of these channels was reduced in crude particulate fractions of this myocardium, as was the mRNA level for these channels.²⁵ Although no simple unifying explanation that can explain these diverse findings has been established, these results raise the possibility that changes in the function and abundance of ryanodine-sensitive Ca²⁺ channels may contribute to the pathophysiology observed in these animal models.

Whether alterations in the function and abundance of ryanodine-sensitive Ca²⁺ channels occur in human cardiomyopathy has not been determined. Single-channel Ca²⁺ current recordings in sarcoplasmic reticulum–derived membranes from failing human left ventricular myocardium were comparable to recordings made in preparations from normal canine and ovine myocardium, but comparison to preparations from nonfailing human myocardium was not performed.²⁶ Northern blot analysis of human myocardial extracts using a rabbit cDNA probe showed a significant reduction in ryanodine-sensitive Ca²⁺ channel mRNA levels in myocardium from patients with ischemic cardiomyopathy but not in myocardium from patients with idiopathic dilated cardiomyopathy, in which ryanodine-sensitive Ca²⁺ channel mRNA levels were not significantly different than in nonfailing myocardium.²⁷ To the best of our knowledge, the densities of ryanodine-sensitive Ca²⁺ channels in nonfailing and failing human left ventricular myocardium have not been compared.

To determine whether alterations in the function of ryanodine-sensitive Ca²⁺ channels might be a contributing factor in the abnormal [Ca²⁺]_i homeostasis observed in dilated cardiomyopathy in humans, we examined the effects of 0.5 mmol/L ryanodine on oxalate-supported, ATP-dependent Ca²⁺ accumulation in microsomes prepared from nonfailing and failing human left ventricular myocardium.

	Methods

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Characteristics of Patient Populations
Nonfailing left ventricular free wall myocardium was obtained from 8 unmatched organ donors, 37.6±5.9 years of age, whose left ventricular function was normal on echocardiography. Failing left ventricular free wall myocardium was obtained from the excised hearts of 10 cardiac transplant recipients, 44.5±5.4 years of age (P=NS, failing versus nonfailing), with class IV heart failure resulting from idiopathic dilated cardiomyopathy. None of the patients with heart failure had been treated with Ca²⁺ antagonists before transplantation. The left ventricular ejection fraction, determined by echocardiography, was 51.1±1.6% and 18.1±2.0% in the nonfailing and failing hearts, respectively (P=.000, failing versus nonfailing). The ß-adrenergic receptor density of crude sarcolemmal preparations, determined as described previously,²⁸ was 93.2±11.4 and 55.3±5.4 fmol/mg in nonfailing and failing myocardium, respectively (P=.014, failing versus nonfailing).

Preparation of Microsomes From Human Ventricular Myocardium
Microsomes were prepared by an adaptation of a previously published procedure.⁵ Myocardium was trimmed of epicardium and endocardium and frozen at -80°C until use. Tissue was homogenized three times in a Kinematica GmbH homogenizer for 10 seconds at setting 10 in 5 vol (vol/wt) of 0.29 mol/L sucrose, 3 mmol/L NaN₃, and 10 mmol/L MOPS (pH 6.0, 4°C) ("sucrose buffer") containing 2 mmol/L EGTA, 0.1 µmol/L pepstatin, 10 µg/mL leupeptin, 10 µg/mL antipain, 3 mmol/L benzamidine, and 0.8 mmol/L phenylmethylsulfonyl fluoride. After removal of cellular debris by two 10-minute sedimentations at 8000 rpm (5000g) in a Beckman JA-20 rotor, the homogenate was sedimented for 60 minutes at 19 000 rpm (30 000g) in a Beckman 55.2-Ti rotor. The pellet was resuspended in 0.6 mol/L KCl, 3.0 mmol/L NaN₃, and 10.0 mmol/L MOPS (pH 6.0, 30°C) and resedimented for 40 minutes at 37 000 rpm (116 000g) in a Beckman 55.2-Ti rotor before storage in sucrose buffer (without EGTA) at -80°C.

Ca²⁺-Transporting ATPase
Ca²⁺-transporting ATPase was quantified by measuring acid-stable, Ca²⁺-dependent phosphoenzyme levels at saturating Ca²⁺ and ATP concentrations following an adaptation of the method of Tada et al.²⁹ Microsomes were suspended at 0.1 mg/mL in 0.1 mol/L KCl, 1.0 mmol/L MgCl₂, 10 mmol/L NaN₃, 40 mmol/L MOPS (pH 7.0, 4°C), and either 0.1 mmol/L CaCl₂ or 1.0 mmol/L EGTA. ATP hydrolysis was initiated by the addition of 0.1 mmol/L [-³²P]ATP (Amersham Corp); the final volume of the reaction mixture was 0.5 mL. The reaction was stopped by addition of 500 µL of 10% trichloroacetic acid containing 2.0 mmol/L ATP and 0.5 mmol/L KH₂PO₄, followed immediately by addition of 100 µL of 0.63% BSA. The mixture was sedimented at 14 000 rpm (16 000g) for 5 minutes in an Eppendorf Micro Centrifuge 5415. The pellet was resuspended in 1.5 mL of 4% HClO₄ containing 30 mmol/L NaH₂PO₄ and 10 mmol/L Na₂H₂P₂O₇ and resedimented as described above. Resuspension and resedimentation were repeated three times. The final pellet was dissolved in 100 µL of 0.5 mol/L NaOH and diluted with 500 µL H₂O. ³²P content was determined by scintillation spectrometry. Ca²⁺-dependent phosphoenzyme levels were calculated by subtraction of values for ³²P incorporation measured in the presence of 1.0 mmol/L EGTA from values measured in the presence of 0.1 mmol/L Ca²⁺.

Oxalate-Supported, ATP-Dependent Ca²⁺ Accumulation
Ca²⁺ accumulation was determined by use of an adaptation of a previously published procedure.⁴ Microsomes were suspended at 0.01 mg/mL in 0.102 mol/L KCl, 5.0 mmol/L oxalic acid, 5.0 mmol/L NaN₃, 1.0 mmol/L EGTA, and 20 mmol/L MOPS (pH 7.05, 37°C). ⁴⁵CaCl₂ (Amersham Corp) and MgCl₂ were added as appropriate to yield free extravesicular Ca²⁺ and Mg²⁺ concentrations of 10.0 µmol/L and 0.4 mmol/L, respectively, using published K_d.³⁰ Reaction mixtures were incubated for 30 minutes at 37°C in the absence and presence of 0.5 mmol/L ryanodine (Calbiochem-Novabiochem Corp). Ca²⁺ accumulation was initiated by addition of 5.0 mmol/L ATP. Aliquots (50 µL) were removed at 2-minute intervals over a 12-minute time course and filtered under vacuum through 0.22-µm GS disks (Millipore Corp). The filter disks were washed six times with 1-mL aliquots of 1.0 mol/L KCl and 2.0 mmol/L EGTA. Ca²⁺ accumulation was determined by scintillation spectrometry. Ca²⁺ accumulation in each preparation was normalized to Ca²⁺-transporting ATPase content, determined as described above.

[³H]Ryanodine Binding
Microsomes (0.05 mg/mL) were incubated in the presence of 1.6 to 50.0 nmol/L [³H]ryanodine (NEN Research Products) for 16 hours at 23°C in 0.3 mol/L KCl, 0.1 mmol/L CaCl₂, 0.1 mg/mL BSA, and 20 mmol/L MOPS (pH 7.2) in the presence of 0.1 mmol/L phenylmethylsulfonyl fluoride, 0.2 mmol/L aminobenzamidine, and 1.0 µg/mL each of aprotinin, leupeptin, and pepstatin A as previously described.³¹ Samples were filtered under vacuum through Whatman GF/F glass fiber filters (VWR Scientific), which were washed five times with 5 mL ice-cold 0.3 mol/L KCl, 0.1 mmol/L CaCl₂, and 10 mmol/L MOPS (pH 7.2). Bound radioactivity was measured by scintillation spectrometry. Nonspecific binding was determined by duplicate assays in which a 100-fold excess of unlabeled ryanodine was included. Each binding curve was performed with data collected at six concentrations of [³H]ryanodine, and each assay was performed in duplicate. Values for B_max and K_d were determined by weighted nonlinear least-squares regression analysis (Gauss-Newton method) with a published computer program.³²

Dissociation of [³H]Ryanodine From High-Affinity Binding Sites
The method was adapted from a previously published procedure.³³ Microsomes (95 to 235 µg containing 0.5 pmol high-affinity ryanodine binding sites) were incubated for 15 hours at 23°C in 0.3 mol/L KCl, 0.1 mmol/L CaCl₂, 0.1 mg/mL BSA, 0.1 mmol/L phenylmethylsulfonyl fluoride, 0.2 mmol/L aminobenzamidine, and 1.0 µg/mL each of aprotinin, leupeptin, and pepstatin A, as well as 50 mmol/L MOPS (pH 7.20) ("KCl buffer") to which 20 nmol/L [³H]ryanodine and 1.0 mmol/L adenosine 5'-(ß,-methylene) were added triphosphate. The total volume of each reaction mixture was 50 µL. Dissociation of [³H]ryanodine was initiated by a 1:400 dilution of each reaction mixture into KCl buffer in the absence and presence of 0.1 mmol/L ryanodine. At the times indicated in the text, 100 µL aliquots were filtered through Whatman GF/F filters with a Millipore vacuum filtration apparatus. Filters were washed five times with 5 mL of 0.3 mol/L KCl, 0.1 mmol/L CaCl₂, and 10 mmol/L MOPS (pH 7.2, 4°C). Bound radioactivity was quantified by scintillation spectrometry.

Miscellaneous
Data are expressed as mean±SEM. Statistical comparisons were performed by use of unpaired two-tailed t tests except in the case of ryanodine dissociation, in which a repeated-measures ANOVA was performed using the GLM procedure from the SAS statistical package (SAS Institute Inc). Protein concentrations were determined as described by Bradford³⁴ with BSA as the standard. Except where indicated above, all reagents were from Sigma Chemical Co.

	Results

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Sarcoplasmic reticulum–enriched microsomes were prepared from nonfailing and failing human left ventricular myocardium by homogenization and differential sedimentation as described above. The yield of protein per wet weight of tissue was 0.43±0.05 and 0.39±0.04 mg/g in preparations from nonfailing and failing hearts, respectively. The content of functional Ca²⁺-transporting ATPase, determined by measurement of acid-stable, Ca²⁺-dependent phosphoenzyme levels at saturating Ca²⁺ and ATP concentrations, was 258±49 and 257±26 pmol/mg protein in preparations from nonfailing and failing hearts, respectively.

The effect of 0.5 mmol/L ryanodine on oxalate-supported, ATP-dependent Ca²⁺ accumulation was examined in microsomes prepared from nonfailing and failing human left ventricular myocardium. Measurements were made at a free extravesicular [Ca²⁺] of 10.0 µmol/L, at which the open probability of ryanodine-sensitive Ca²⁺ channels is high.³⁵ In the absence of ryanodine, Ca²⁺ accumulation is diminished in vesicles containing normally functioning Ca²⁺ channels because of concurrent efflux of Ca²⁺ through these channels under these conditions.³⁶ Incubation with 0.5 mmol/L ryanodine results in the occupancy of low-affinity ryanodine binding sites and a consequent decrease in the open probability of ryanodine-sensitive Ca²⁺ channels, so Ca²⁺ accumulates in vesicles containing normally functioning Ca²⁺ channels and in vesicles lacking these channels.

Fig 1 shows the results from a representative experiment. Ca²⁺ accumulation was initially linear with respect to time but invariably reached a plateau within 12 minutes, and the effects of ryanodine on maximal accumulation were more pronounced than the effects of ryanodine on the initial rate of accumulation. The effects of ryanodine on Ca²⁺ accumulation were therefore characterized with respect to both the initial rate of Ca²⁺ accumulation (Fig 2) and the peak level of Ca²⁺ accumulation (Fig 3) in each preparation. In each case, results were normalized to microsomal Ca²⁺-transporting ATPase content. In the presence of 0.5 mmol/L ryanodine, the initial rate of Ca²⁺ accumulation increased by 0.19±0.07 µmol · nmol^-1 · min^-1 in microsomes from nonfailing hearts. In microsomes from failing hearts, in contrast, the initial rate of Ca²⁺ accumulation decreased by 0.02±0.02 µmol · nmol^-1 · min^-1 in the presence of 0.5 mmol/L ryanodine (P=.005, failing versus nonfailing). Incubation with 0.5 mmol/L ryanodine thus resulted in a 19.4±4.9% increase and a 2.8±3.2% decrease in Ca²⁺ accumulation rates in microsomes from nonfailing and failing hearts, respectively (P=.002, failing versus nonfailing). Similarly, incubation with 0.5 mmol/L ryanodine increased peak levels of Ca²⁺ accumulation by 2.03±0.40 µmol/nmol in microsomes from nonfailing hearts but by only 0.94±0.18 µmol/nmol in microsomes from failing hearts (P=.170, failing versus nonfailing). Incubation with 0.5 mmol/L ryanodine thus resulted in 52.2±6.5% and 24.3±4.1% increases in peak Ca²⁺ accumulation in microsomes from nonfailing and failing hearts, respectively (P=.003, failing versus nonfailing).

View larger version (16K): [in this window] [in a new window]   Figure 1. Plot showing the effect of ryanodine on Ca2+ accumulation in sarcoplasmic reticulum–enriched microsomes. Results are from a single preparation from failing myocardium. 45Ca2+ accumulation was measured as described in "Methods" in the absence () or presence () of 0.5 mmol/L ryanodine. Each value represents the mean of two determinations.

View larger version (19K): [in this window] [in a new window]   Figure 2. Bar graph showing the effect of ryanodine on initial rates of Ca2+ accumulation in microsomes prepared from nonfailing and failing human left ventricular myocardium. Initial rates of Ca2+ accumulation were determined in the absence (striped bars) and presence (solid bars) of 0.5 mmol/L ryanodine. Values are the mean±SEM of 8 preparations from nonfailing hearts and 10 preparations from failing hearts.

View larger version (21K): [in this window] [in a new window]   Figure 3. Bar graph showing the effect of ryanodine on peak levels of Ca2+ accumulation in microsomes prepared from nonfailing and failing human left ventricular myocardium. Peak levels of Ca2+ accumulation were determined in the absence (striped bars) and presence (solid bars) of 0.5 mmol/L ryanodine. Values are the mean±SEM of 8 preparations from nonfailing hearts and 10 preparations from failing hearts.

The diminished stimulation of Ca²⁺ accumulation by 0.5 mmol/L ryanodine in microsomes prepared from failing human left ventricular myocardium might have resulted from a decrease in the density of functional ryanodine-sensitive Ca²⁺ channels in these preparations. To test this possibility, we quantified high-affinity [³H]ryanodine binding in the microsomal preparations (Fig 4). In preparations from nonfailing myocardium, the K_d for high-affinity ryanodine binding was 2.75±0.61 nmol/L, and the B_max was 3.10±0.56 pmol/mg protein. In preparations from failing myocardium, the K_d for high-affinity ryanodine binding was 3.71±0.79 nmol/L, whereas the B_max was 2.92±0.47 pmol/mg protein. These results indicate that the density of functional ryanodine-sensitive Ca²⁺ channels was comparable in microsomes from nonfailing and failing myocardium. Moreover, the ratio of functional ryanodine-sensitive Ca²⁺ channels to Ca²⁺-transporting ATPase molecules, calculated by dividing B_max by Ca²⁺-transporting ATPase content in each preparation, was 13.3±2.2 and 11.9±1.6 pmol/nmol in microsomes from nonfailing and failing hearts, respectively. The impaired stimulation of Ca²⁺ accumulation by ryanodine in microsomes from failing hearts could not therefore be attributed to a change in the level of functional ryanodine-sensitive Ca²⁺ channels relative to Ca²⁺-transporting ATPase molecules in these preparations.

View larger version (16K): [in this window] [in a new window]   Figure 4. Plot showing ryanodine binding in human myocardial microsomes. Microsomes from a single preparation from nonfailing myocardium were incubated in the presence of [3H]ryanodine as described in "Methods." Total binding (), nonspecific binding (), and specific binding () were determined as described in "Methods." Each point for total and nonspecific binding is the mean of duplicate determinations.

Alternatively, the diminished stimulation of Ca²⁺ accumulation by 0.5 mmol/L ryanodine in microsomes prepared from failing human left ventricular myocardium might have resulted from an impairment of low-affinity ryanodine binding in these preparations. Because binding to low-affinity binding sites is not saturable at concentrations of ryanodine at which the drug is soluble, we addressed this issue by examining the effect of high concentrations of ryanodine on the dissociation of ryanodine from high-affinity sites. Occupancy of the low-affinity ryanodine binding sites of ryanodine-sensitive Ca²⁺ channels results in a slowing of the dissociation of ryanodine from high-affinity sites.²⁷ An impairment involving low-affinity binding would therefore be expected to manifest as a reduction in the ability of ryanodine at high concentrations to slow the dissociation of ryanodine from high-affinity binding sites. Microsomes from four nonfailing hearts (in which the stimulation of Ca²⁺ transport rate by ryanodine was 23.4±9.4%) and four failing hearts (in which the percent stimulation of Ca²⁺ transport rate by ryanodine was -3.9±7.8%) were incubated in the presence of 20 nmol/L [³H]ryanodine, at which concentration high-affinity sites are saturated, and then diluted into KCl solutions in the absence or presence of 0.1 mmol/L ryanodine. Fig 5 shows the results from a single preparation. The percentage of bound [³H]ryanodine released on dilution into KCl solution in the presence and absence of 0.1 mmol/L ryanodine was determined at 100 and 600 minutes in microsomes from nonfailing and failing hearts. At both times, the dissociation of [³H]ryanodine from high-affinity binding sites was similar in microsomes from nonfailing and failing hearts in the absence and presence of 0.1 mmol/L ryanodine (P=.000, absence versus presence of ryanodine; P=.847, nonfailing versus failing; Fig 6). These results, which demonstrate comparable inhibition of the dissociation of [³H]ryanodine from high-affinity sites by 0.1 mmol/L ryanodine in preparations from nonfailing and failing myocardium, indicate that the diminished stimulation of Ca²⁺ accumulation by 0.5 mmol/L ryanodine in microsomes from failing myocardium cannot be attributed to impaired binding of ryanodine to low-affinity binding sites.

View larger version (18K): [in this window] [in a new window]   Figure 5. Plot showing the dissociation of [3H]ryanodine from high-affinity binding sites in myocardial sarcoplasmic reticulum. Microsomes from a single preparation from failing myocardium were incubated in the presence of 20 nmol/L [3H]ryanodine as described in "Methods" and diluted into KCl buffer in the absence () or presence () of 0.1 mmol/L ryanodine. Protein-bound [3H]ryanodine was determined as described in "Methods."

View larger version (21K): [in this window] [in a new window]   Figure 6. Bar graph showing the inhibition of [3H]ryanodine dissociation by 0.1 mmol/L ryanodine in microsomes prepared from nonfailing and failing human left ventricular myocardium. Bound [3H]ryanodine was measured at 100 and 600 minutes after dilution into KCl solution and expressed as a percentage of [3H]ryanodine bound before dilution. Values are the mean±SEM of four preparations from nonfailing hearts and four preparations from failing hearts. Results were compared using a repeated-measures ANOVA, with time (minutes) as the repeated measure and the source of myocardium (nonfailing vs failing) and the absence/presence of 0.1 mmol/L ryanodine in the diluting solution as the independent variables.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The experimental results demonstrate that the stimulation of oxalate-supported, ATP-dependent Ca²⁺ accumulation by high concentrations of ryanodine is diminished in sarcoplasmic reticulum–enriched microsomes prepared from failing human left ventricular myocardium and that this diminution cannot be attributed to a reduction in the density of functional ryanodine-sensitive Ca²⁺ channels in these preparations or an impairment of low-affinity ryanodine binding to these channels.

The stimulation of oxalate-supported, ATP-dependent Ca²⁺ accumulation in myocardial sarcoplasmic reticulum–enriched microsomes by high concentrations of ryanodine results from the inhibition of concurrent Ca²⁺ efflux through ryanodine-sensitive Ca²⁺ channels by the occupancy of low-affinity ryanodine binding sites on these channels. Our experimental results strongly suggest therefore that the ability of 0.5 mmol/L ryanodine to block concurrent Ca²⁺ efflux at high extravesicular [Ca²⁺] is for some reason impaired in microsomes from failing myocardium despite the occupancy of low-affinity ryanodine binding sites. Several possible explanations merit consideration. Concurrent Ca²⁺ efflux during active Ca²⁺ transport could occur through ryanodine-sensitive Ca²⁺ channels despite the presence of 0.5 mmol/L ryanodine if the occupancy of low-affinity ryanodine binding sites on these channels in failing myocardium were somehow uncoupled from the reduction in the open probability of these channels. There is at least one paradigm for the uncoupling of the ryanodine binding and Ca²⁺ channel properties of ryanodine-sensitive Ca²⁺ channels in cardiac muscle: the open probability of ryanodine-sensitive Ca²⁺ channels can be increased by proteolysis by calpain II without affecting high-affinity ryanodine binding.¹⁶ In previous studies, however, no evidence of increased proteolysis of ryanodine-sensitive Ca²⁺ channels was detected in immunochemical studies of sarcoplasmic reticulum–enriched microsomes from failing human myocardium.⁸ Furthermore, in the example cited, proteolysis with calpain II resulted in the uncoupling of changes in Ca²⁺ channel activity and high-affinity ryanodine binding: low-affinity ryanodine binding reduced the open probability of ryanodine-sensitive Ca²⁺ channels appropriately after proteolysis by calpain II, indicating that low-affinity ryanodine binding and Ca²⁺ channel properties remained coupled. The applicability of the results of the calpain II experiments to our observations is therefore questionable. Other investigators have reported that the amount of Ca²⁺ released from sarcoplasmic reticulum–enriched microsomes by doxorubicin and caffeine is higher in preparations from the myocardium of rats with pressure overload–induced hypertrophy than in preparations from nonhypertrophic rat myocardium despite the similar densities of ryanodine-sensitive Ca²⁺ channels in the two groups.¹⁸ These observations, which are evidence of a qualitative alteration in Ca²⁺ release in the sarcoplasmic reticulum of the hypertrophic hearts, are at least superficially similar to our findings in failing human myocardium. The fact that the ability of ryanodine to inhibit Ca²⁺ release was not examined in the experiments involving microsomes from rat heart prevents a more direct comparison of the results in hypertrophic rat and failing human myocardium. Finally, it should be noted that other proteins in the junctional sarcoplasmic reticulum can modulate the function of the ryanodine-sensitive Ca²⁺ channel, so changes in ryanodine-sensitive Ca²⁺ efflux in microsomes from failing myocardium could result from alterations involving these regulatory proteins.^{37 38}

Alternatively, the diminished stimulation of Ca²⁺ accumulation in microsomes from failing human myocardium in the presence of 0.5 mmol/L ryanodine could be explained by concurrent Ca²⁺ efflux during active Ca²⁺ transport through a mechanism that does not involve ryanodine-sensitive Ca²⁺ channels. One such mechanism that functions in diverse cell types is the release of Ca²⁺ from endoplasmic reticulum stores by inositol trisphosphate (for review, see Reference 39). This mechanism does not appear to be operative in myocardium, however, in which inositol trisphosphate receptors have been shown to be localized to intercalated disks rather than the sarcoplasmic reticulum.^{40 41} More recently, release of Ca²⁺ from skeletal muscle sarcoplasmic reticulum vesicles by cADP-ribose was reported to be unaffected by inhibitors of ryanodine-sensitive Ca²⁺ channels, suggesting the involvement of an independent mechanism.⁴² In myocardium, however, cADP-ribose seems to induce Ca²⁺ release through a direct interaction with ryanodine-sensitive Ca²⁺ channels, and the physiological relevance of this effect is uncertain.^{43 44} At this point, therefore, the possibility of Ca²⁺ efflux from myocardial sarcoplasmic reticulum vesicles through a mechanism that does not involve ryanodine-sensitive Ca²⁺ channels remains a matter of speculation.

Finally, the reduced stimulation of Ca²⁺ accumulation in microsomes from failing myocardium could potentially have resulted from an impairment in sarcoplasmic reticulum Ca²⁺ channel function that reduced the open probability of these channels in the presence of high extravesicular [Ca²⁺]. Stimulation of microsomal Ca²⁺ accumulation by ryanodine results from a reduction in concurrent Ca²⁺ efflux through ryanodine-sensitive channels. If the ability of channels to open were impaired in failing myocardium, concurrent efflux in the absence of ryanodine would be diminished, and the stimulation of Ca²⁺ accumulation in the presence of high concentrations of ryanodine would be reduced. In our experiments, however, levels of functional Ca²⁺-transporting ATPase were comparable in preparations from nonfailing and failing human myocardium, and the ratio of functional ryanodine-sensitive Ca²⁺ channels to functional Ca²⁺-transporting ATPase molecules was similar in the two groups. If the ability of sarcoplasmic reticulum Ca²⁺ channels to open in the presence of high extravesicular [Ca²⁺] were reduced in microsomes from failing myocardium, Ca²⁺ accumulation in these microsomes should have been higher than in microsomes from nonfailing hearts when measured in the absence of ryanodine and comparable when measured in its presence. Our data showed instead that Ca²⁺ accumulation was comparable in microsomes from nonfailing and failing hearts when measured in the absence of ryanodine and higher in microsomes from nonfailing hearts when measured in its presence. For this reason, the possibility that our results could be explained by a reduction of the open probability of ryanodine-sensitive Ca²⁺ channels in failing myocardium seems unlikely.

Whatever the molecular mechanism, an increased efflux of Ca²⁺ from the sarcoplasmic reticulum of failing myocardium could contribute to the abnormal [Ca²⁺]_i homeostasis that has been described in dilated cardiomyopathy in humans. Gwathmey et al² observed two components of the decline of the Ca²⁺ transient in trabeculae isolated from human myocardium. The second component was prolonged in failing myocardium, and its amplitude increased with increases in [Ca²⁺]_o. Interestingly, this prolonged elevation of [Ca²⁺]_i was only partially responsive to ryanodine, so the possibility of ryanodine-insensitive Ca²⁺ efflux raised earlier offers a plausible explanation for their findings. In addition, Beuckelmann et al³ observed a decrease in peak systolic [Ca²⁺], an increase in basal diastolic [Ca²⁺], and a slowing of the rate of decline of [Ca²⁺]_i in myocytes isolated from failing human myocardium. All of these observations could be explained by increased Ca²⁺ efflux from the sarcoplasmic reticulum in these myocytes. Further experiments involving direct measurement of Ca²⁺ release from sarcoplasmic reticulum–derived microsomes from nonfailing and failing myocardium under conditions that mimic those that exist in vivo will be necessary to determine the relevance of our observations to the cellular pathophysiology of dilated cardiomyopathy.

	Acknowledgments

 
This work was supported by the Department of Veterans Affairs Medical Research funds and by grants from the American Heart Association, Utah Affiliate; the Muscular Dystrophy Association; NHLBI (5 T32 HL-07576, HL-08927); and the National Institute of Arthritis, Musculoskeletal and Skin Diseases (AR41802). Dr Movsesian is the recipient of a Department of Veterans Affairs Career Development Award.

Received December 19, 1994; revision received May 29, 1995; accepted June 4, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Movsesian MA. Calcium uptake and release by cardiac sarcoplasmic reticulum. In: Allen PD, Gwathmey JK, Briggs M, eds. Inotropic Drugs: Basic Mechanisms and Clinical Practice. New York, NY: Marcel Decker Inc; 1993:101-120.

2. Gwathmey JK, Copelas L, MacKinnon R, Schoen FJ, Feldman MD, Grossman W, Morgan JP. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ Res. 1987;61:70-76. [Abstract/Free Full Text]

3. Beuckelmann DJ, Nabauer M, Erdmann E. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation. 1992;85:1046-1055. [Abstract/Free Full Text]

4. Movsesian MA, Bristow MR, Krall J. Ca²⁺ uptake by cardiac sarcoplasmic reticulum from patients with idiopathic dilated cardiomyopathy. Circ Res. 1989;65:1141-1144. [Abstract/Free Full Text]

5. Movsesian MA, Colyer J, Wang JH, Krall J. Phospholamban-mediated stimulation of Ca²⁺ uptake in sarcoplasmic reticulum from normal and failing hearts. J Clin Invest. 1990;85:1698-1702.

6. Movsesian MA, Smith CJ, Krall J, Bristow MR, Manganiello VC. Sarcoplasmic reticulum-associated cyclic adenosine 5'-monophosphate phosphodiesterase activity in normal and failing human hearts. J Clin Invest. 1991;88:15-19.

7. Movsesian MA, Karimi M, Green K, Jones LR. Ca²⁺-transporting ATPase, phospholamban, and calsequestrin levels in nonfailing and failing human myocardium. Circulation. 1994;90:653-657. [Abstract/Free Full Text]

8. Movsesian MA, Leveille C, Krall J, Colyer J, Wang JH, Campbell KP. Identification and characterization of proteins in human cardiac sarcoplasmic reticulum. J Mol Cell Cardiol. 1990;22:1477-1485. [Medline] [Order article via Infotrieve]

9. Hasenfuss F, Reinecke H, Studer R, Meyer M, Pieske B, Holtz J, Holubarsch C, Posival H, Just H, Drexler H. Relation between myocardial function and expression of sarcoplasmic reticulum Ca²⁺-ATPase in failing and nonfailing human myocardium. Circ Res. 1994;75:434-442. [Abstract/Free Full Text]

10. Studer R, Reinecke H, Bilger J, Eschenhagen T, Bohm M, Hasenfuß G, Just H, Holtz J, Drexler H. Gene expression of the cardiac Na⁺-Ca²⁺ exchanger in end-stage human heart failure. Circ Res. 1994;75:443-453. [Abstract/Free Full Text]

11. Steenbergen C, Murphy E, Levy L, London RE. Elevation in cytosolic free calcium concentration in myocardial ischemia in perfused rat hearts. Circ Res. 1987;60:700-707. [Abstract/Free Full Text]

12. Feher JJ, Lebolt WR, Manson NH. Differential effect of global ischemia on the ryanodine-sensitive and ryanodine-insensitive Ca²⁺ uptake of cardiac sarcoplasmic reticulum. Circ Res. 1989;65:1400-1408. [Abstract/Free Full Text]

13. Davis MD, Lebolt W, Feher JJ. Reversibility of the effect of normothermic global ischemia on the ryanodine-sensitive and ryanodine-insensitive calcium uptake of cardiac sarcoplasmic reticulum. Circ Res. 1992;70:163-171. [Abstract/Free Full Text]

14. Abdelmeguid AE, Feher JJ. Effect of perfusate [Ca²⁺] on cardiac sarcoplasmic reticulum Ca²⁺ release channel in isolated rat hearts. Circ Res. 1992;71:1049-1058. [Abstract/Free Full Text]

15. Okabe E, Kasumi K, Tsukasa S, Naoaki S, Kazuyuki T, Haruo I. The effect of ryanodine on oxygen free radical-induced dysfunction of cardiac sarcoplasmic reticulum. J Pharmacol Exp Ther. 1991;256:868-875. [Abstract/Free Full Text]

16. Rardon DP, Dominic CC, Mitchell RD, Seiler SM, Hathaway DR, Jones LR. Digestion of cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles with calpain II. Circ Res. 1990;67:84-96. [Abstract/Free Full Text]

17. Fujii J, Otsu K, Zorzato F, de Leon S, Khana VK, Weiler JE, O'Brien PJ, MacLennan DH. Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia. Science. 1991;253:448-451.[Abstract/Free Full Text]

18. Kim DH, Mkparu F, Kim CR, Caroll RF. Alteration of Ca²⁺ release channel function in sarcoplasmic reticulum of pressure-overload-induced hypertrophic rat heart. J Mol Cell Cardiol. 1994;26:1505-1512. [Medline] [Order article via Infotrieve]

19. Naudin V, Oliviero P, Rannou F, Sainte-Beuve C, Charlemagne D. The density of ryanodine receptors decreases with pressure overload-induced rat cardiac hypertrophy. FEBS Lett. 1991;285:135-138. [Medline] [Order article via Infotrieve]

20. Dodd DA, Atkinson JB, Olson RD, Buck S, Cusack BJ, Fleischer S. Doxorubicin cardiomyopathy is associated with a decrease in calcium release channel of the sarcoplasmic reticulum in a chronic rabbit model. J Clin Invest. 1993;91:1697-1705.

21. Cory CR, McCutcheon LJ, O'Grady M, Pang AW, Geiger JD, O'Brien PJ. Compensatory downregulation of myocardial Ca channel in SR from dogs with heart failure. Am J Physiol. 1993;264:H926-H937. [Abstract/Free Full Text]

22. Vatner DE, Sato N, Kiuchi K, Shannon RP, Vatner SF. Decrease in myocardial ryanodine receptors and altered excitation-contraction coupling early in the development of heart failure. Circ Res. 1994;90:1423-1430.

23. Finkel MS, Shen L, Romeo RC, Oddis CV, Salama G. Radioligand binding and inotropic effects of ryanodine in the cardiomyopathic Syrian hamster. J Cardiovasc Pharmacol. 1992;19:610-617. [Medline] [Order article via Infotrieve]

24. Sapp JL, Howlett SE. Density of ryanodine receptors is increased in sarcoplasmic reticulum from prehypertrophic cardiomyopathic hamster heart. J Mol Cell Cardiol. 1994;26:325-334. [Medline] [Order article via Infotrieve]

25. Lachnit WG, Phillips M, Gayman KJ, Pessah IN. Ryanodine and dihydropyridine binding patterns and ryanodine receptor mRNA levels in myopathic hamster heart. Am J Physiol. 1994;267:H1205-H1213. [Abstract/Free Full Text]

26. Holmberg SRM, Williams AJ. Single channel recordings from human cardiac sarcoplasmic reticulum. Circ Res. 1989;65:1445-1449. [Abstract/Free Full Text]

27. Brillantes A-M, Allen P, Takahashi T, Izumo S, Marks AR. Differences in cardiac calcium release channel (ryanodine receptor) expression in myocardium from patients with end-stage heart failure caused by ischemic versus dilated cardiomyopathy. Circ Res. 1992;71:18-26. [Abstract/Free Full Text]

28. Bristow MR, Ginsburg R, Umans V, Fowler M, Minobe W, Rasmussen R, Zera P, Menlove R, Shah P, Jamieson S, Stinson EB. ß₁- and ß₂-adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective ß₁-receptor down-regulation in heart failure. Circ Res. 1986;59:297-309. [Abstract/Free Full Text]

29. Tada M, Kadoma M, Inui M, Fujii JI. Regulation of Ca²⁺-pump from cardiac sarcoplasmic reticulum. Methods Enzymol. 1988;157:107-154. [Medline] [Order article via Infotrieve]

30. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 1988;157:378-417. [Medline] [Order article via Infotrieve]

31. Chu A, Diaz-Munoz M, Hawkes MJ, Brush K, Hamilton SH. Ryanodine as a probe for the functional state of the skeletal muscle sarcoplasmic reticulum calcium release channel. Mol Pharmacol. 1990;37:735-741. [Abstract]

32. Cleland WW. Statistical analysis of enzyme kinetic data. Methods Enzymol. 1979;63:103-138. [Medline] [Order article via Infotrieve]

33. Wang JP, Needleman DH, Hamilton SL. Relationship of low affinity [³H]ryanodine binding sites to high affinity sites on the skeletal muscle Ca²⁺ release channel. J Biol Chem. 1993;268:20974-20982. [Abstract/Free Full Text]

34. Bradford MM. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. [Medline] [Order article via Infotrieve]

35. Anderson K, Lai FA, Liu QY, Rousseau E, Erickson HP, Meissner G. Structural and functional characterization of the purified cardiac ryanodine receptor-Ca²⁺ release channel complex. J Biol Chem. 1989;264:1329-1335. [Abstract/Free Full Text]

36. Jones LR, Cala SE. Biochemical evidence for the functional heterogeneity of cardiac sarcoplasmic reticulum vesicles. J Biol Chem. 1981;256:11809-11818. [Abstract/Free Full Text]

37. Ikemoto N, Ronjat M, Meszaros LG, Koshita M. Postulated role of calsequestrin in the regulation of calcium release from sarcoplasmic reticulum. Biochemistry. 1989;28:6764-6771. [Medline] [Order article via Infotrieve]

38. Brillantes A-M, Ondrias K, Scott A, Kobrinsky E, Ondriasova E, Moschella MD, Jayaraman T, Landers M, Ehrlich BE, Marks AR. Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell. 1994;77:513-523. [Medline] [Order article via Infotrieve]

39. Berridge MJ, Irvine RF. Inositol phosphates and cell signalling. Nature. 1989;341:197-205. [Medline] [Order article via Infotrieve]

40. Movsesian MA, Thomas AP, Selak M, Williamson JR. Inositol trisphosphate does not release Ca²⁺ from permeabilized cardiac myocytes and sarcoplasmic reticulum. FEBS Lett. 1985;185:328-332. [Medline] [Order article via Infotrieve]

41. Kijima Y, Saito A, Jetton TL, Magnuson MA, Fleischer S. Different intracellular localization of inositol 1,4,5-trisphosphate and ryanodine receptors in cardiomyocytes. J Biol Chem. 1993;268:3499-3506. [Abstract/Free Full Text]

42. Morrissette J, Heisermann G, Cleary J, Ruoho A, Coronado R. Cyclic ADP-ribose induced Ca²⁺ release in rabbit skeletal muscle sarcoplasmic reticulum. FEBS Lett. 1993;330:270-274. [Medline] [Order article via Infotrieve]

43. Meszaros LG, Bak J, Chu A. Cyclic ADP-ribose as an endogenous regulator of the non-skeletal type ryanodine receptor Ca²⁺ channel. Nature. 1993;364:76-79. [Medline] [Order article via Infotrieve]

44. Sitsapesan R, McGarry SJ, Williams AJ. Cyclic ADP-ribose competes with ATP for the adenine nucleotide binding site on the cardiac ryanodine receptor Ca²⁺-release channel. Circ Res. 1994;75:596-600.[Abstract/Free Full Text]

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