AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 278: H1429-H1438, 2000;
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Vol. 278, Issue 5, H1429-H1438, May 2000

Thyroid hormone-induced overexpression of functional ryanodine receptors in the rabbit heart

M. Jiang1, A. Xu1, S. Tokmakejian2, and N. Narayanan1

1 Department of Physiology and 2 London Health Sciences Center, The University of Western Ontario, London, Ontario, Canada N6A 5C1


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Modifications in the Ca2+-uptake and -release functions of the sarcoplasmic reticulum (SR) may be a major component of the mechanisms underlying thyroid state-dependent alterations in heart rate, myocardial contractility, and metabolism. We investigated the influence of hyperthyroid state on the expression and functional properties of the ryanodine receptor (RyR), a major protein in the junctional SR (JSR), which mediates Ca2+ release to trigger muscle contraction. Experiments were performed using homogenates and JSR vesicles derived from ventricular myocardium of euthyroid and hyperthyroid rabbits. Hyperthyroidism, with attendant cardiac hypertrophy, was induced by the injection of L-thyroxine (200 µg/kg body wt) daily for 7 days. Western blotting analysis using cardiac RyR-specific antibody revealed a significant increase (>50%) in the relative amount of RyR in the hyperthyroid compared with euthyroid rabbits. Ca2+-dependent, high-affinity [3H]ryanodine binding was also significantly greater (~40%) in JSR from hyperthyroid rabbits. The Ca2+ sensitivity of [3H]ryanodine binding and the dissociation constant for [3H]ryanodine did not differ significantly between euthyroid and hyperthyroid hearts. Measurement of Ca2+-release rates from passively Ca2+-preloaded JSR vesicles and assessment of the effect of RyR-Ca2+-release channel (CRC) blockade on active Ca2+-uptake rates revealed significantly enhanced (>2-fold) CRC activity in the hyperthyroid, compared with euthyroid, JSR. These results demonstrate overexpression of functional RyR in thyroid hormone-induced cardiac hypertrophy. Relative abundance of RyR may be responsible, in part, for the changes in SR Ca2+ release, cytosolic Ca2+ transient, and cardiac systolic function associated with thyroid hormone-induced cardiac hypertrophy.

sarcoplasmic reticulum; calcium-release channel; cardiac hypertrophy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HEART IS a major target for thyroid hormone action, and changes in thyroid status lead to striking alterations in cardiac contractile function and energy metabolism (9, 43). Increases in thyroid hormone levels have been found to enhance myocardial contractility, the speed of systolic contraction and diastolic relaxation, cardiac output, and heart rate (12, 28, 33). On the other hand, decreases in these parameters were observed in hypothyroid state. Evidence from a number of studies suggests that the mechanisms underlying these changes include direct transcriptional regulation of cardiac genes by thyroid hormone, which in turn impacts on myocyte Ca2+ cycling at the level of the myofilaments and the sarcoplasmic reticulum (SR) (9, 34). Thyroid hormone-responsive elements have been identified in the promoter region of cardiac alpha -myosin heavy chain (alpha -MHC) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) genes, and thyroid state-dependent alterations in the transcriptional activity of these genes have been documented (1, 4, 9, 14, 18, 31, 44). A shift from the beta -myosin heavy chain (beta -MHC) (slow) to alpha -MHC (fast) has been demonstrated during transition from hypothyroid to hyperthyroid state. Changes in MHC isoforms correlated with alterations in myosin ATPase activity and actomyosin cross-bridge cycle rate (23). Thyroid hormone-induced increases in the levels of myocardial SERCA2 mRNA and protein have been documented (1, 18, 21, 22, 35). It has also been reported that thyroid hormone-mediated changes in SERCA2 protein levels were inversely related to alterations in the levels of SERCA2 inhibitor protein phospholamban (20, 22). On the basis of these observations, it has been suggested that thyroid hormone-mediated changes in the relative ratio of phospholamban to Ca2+-ATPase regulate the Ca2+-uptake rates by SR and the relaxation properties of the myocardium (20, 22).

In addition to the Ca2+-cycling properties of the myofilaments and the rate of Ca2+ sequestration by the SR, the speed of Ca2+ delivery to the myofilaments and the intensity of Ca2+ signal are important determinants of cardiac systolic function, strength of contraction, and contraction duration. However, relatively less is known about the influence of thyroid hormone on the cellular events associated with the rise in cytoplasmic Ca2+ on myocyte excitation. In the cardiomyocyte, the major mechanism for excitation-induced elevation of intracellular Ca2+ involves Ca2+-induced Ca2+ release from the SR (3). Recent evidence suggests that sarcolemmal L-type Ca2+ channels are closely associated with a cluster of SR Ca2+-release channels (CRC), called ryanodine receptors (RyR), in the diadic junctions forming discrete Ca2+-release units (41). According to the "cluster bomb" model (39), voltage activation of Ca2+ influx via the L-type Ca2+ channel serves as the local Ca2+ signal to activate the coupled RyR, causing further increase in local Ca2+ and cross-activation of other RyR within the release unit. These local Ca2+-release events have been visualized directly as "Ca2+ sparks" (6), and the recruitment of these events, as a function of Ca2+-channel activation, underlies the whole cell Ca2+ transient induced by transsarcolemmal Ca2+ influx (5, 26). Evidence from a study using ferret ventricular muscle indicates that, in the hypothyroid state, decreased peak tension during isometric contraction is associated with a Ca2+ transient of decreased amplitude and prolonged duration, whereas opposite changes in the time course of Ca2+ transient and associated isometric tension occur in the hyperthyroid state (28). Thyroid hormone-induced increase in Ca2+ influx across the sarcolemma, apparently due to an increase in the number of L-type Ca2+ channels, has been reported in cultured ventricular myocytes (19). The steady-state level of mRNA encoding the RyR-CRC has been shown to be increased in the hyperthyroid and decreased in the hypothyroid rabbit heart (1). It is not known whether the thyroid state-dependent changes in mRNA levels are accompanied by parallel changes in the cardiac tissue levels of RyR protein and RyR function. The present study was undertaken to determine the influence of thyroid hormone on RyR protein expression and the functional properties of the RyR in the rabbit heart.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Twelve-week-old New Zealand White male rabbits were obtained from a local breeder and were maintained on ordinary rabbit chow in the Health Sciences Center animal care facility of this institution. Hyperthyroidism was induced by injecting L-thyroxine (T4) intramuscularly at 200 µg/kg body wt daily for 7 days (1). Age-matched untreated rabbits were used as controls (euthyroid group). The care of animals and the protocols used were in accordance with the guidelines established by the Canadian Council on Animal Care and were approved by the Animal Ethics Committee of the University of Western Ontario.

Chemicals

Reagents for electrophoresis were obtained from Bio-Rad Laboratories (Mississauga, Ontario, Canada). 45CaCl2 and [3H]ryanodine were from NEN (Mississauga, Ontario, Canada). Monoclonal antibody against cardiac RyR isoform (RyR2) was purchased from Affinity BioReagents (Golden, CO). Polyclonal antibody against the skeletal muscle RyR isoform (RyR1) was a generous gift from Drs. V. Sorentino and A. Conti (San Raffaele Scientific Institute, Milan, Italy). All other chemicals were from Sigma Chemical (St. Louis, MO) or BDH Chemicals (Toronto, Ontario, Canada).

Preparation of SR Membranes

SR membranes were prepared as described previously (17) and then subjected to sucrose-density gradient fractionation according to the procedure of Feher and Davis (11) to yield membrane vesicles enriched in junctional SR (JSR) and longitudinal SR (LSR). Briefly, the ventricular tissue was minced and homogenized in 6 vols (based on tissue weight) of ice-cold buffer (10 mM NaHCO3, pH 6.8), using a Brinkman Instruments Polytron homogenizer (three 15-s bursts with 30-s interval between bursts, speed setting 5.5). The homogenate was centrifuged at 1,000 g for 10 min at 4°C. The supernatant was decanted and kept in an ice slurry. The pellet was resuspended in 4 vols of ice-cold buffer and centrifuged as before. The supernatant was decanted and combined with the first supernatant, and the pellet was discarded. The combined supernatant was centrifuged at 8,000 g for 20 min at 4°C. The supernatant was collected and the pellet discarded. Solid KCl (44 mg/ml) was added to the supernatant (final concentration 0.6 M), swirled to dissolve, left on ice for 25 min, and then centrifuged at 40,000 g for 60 min at 4°C. The supernatant was discarded, and the pellet enriched in SR membranes was resuspended in ice-cold buffer (10 mM imidazole HCl and 0.6 M KCl, pH 7; 0.5 ml/g ventricular wt) and layered onto a discontinuous sucrose gradient, which consisted of 19% sucrose (5 ml) and 29% sucrose (5 ml) in 10 mM imidazole HCl (pH 7) and 1 M KCl. After centrifugation at 29,000 g for 2 h in a Beckman JA20 rotor, the light fraction at the interface between the 19% and 29% sucrose gradient was collected, diluted with an equal volume of buffer (10 mM imidazole-0.6 M KCl, pH 7), and sedimented at 29,000 g for 30 min. This fraction constituted the LSR used in this study and was enriched in membrane vesicles of LSR as judged from the abundance of Ca2+-ATPase (SERCA2) and minimal contamination by calsequestrin and RyR-CRC. A heavy fraction formed as a pellet at the bottom of the tube after the sucrose-density gradient centrifugation was collected, and this fraction constituted the JSR used in this study. This fraction was enriched in calsequestrin and RyR-CRC and also contained nearly similar amounts (~85%) of Ca2+-ATPase (SERCA2) as the LSR fraction. After isolation, the LSR and JSR fractions were resuspended in 10 mM Tris-maleate-100 mM KCl buffer (pH 6.8), divided into small aliquots (100-200 µl), quick-frozen in liquid N2, and stored at -80°C. Protein was determined by the method of Lowry et al. (27) using BSA as standard.

Preparation of Muscle Homogenates

In addition to SR membranes, cardiac muscle homogenates from euthyroid and hyperthyroid rabbits were used in some experiments. The homogenates were prepared by homogenizing the ventricular tissue in 10 vols (based on tissue weight) of 10 mM Tris-maleate-100 mM KCl buffer (pH 6.8) using a Polytron homogenizer (three 15-s bursts with 30-s interval between bursts; speed setting 5.5). The homogenates were filtered through four layers of cheese cloth and used for experiments.

SDS-PAGE and Immunoblotting of RyR

The protein composition of JSR and LSR vesicles isolated from euthyroid and hyperthyroid hearts was analyzed by SDS-PAGE using 4-18% gradient gels as described previously (16). Western immunoblotting procedure was used to localize and quantify RyR in SR membrane vesicles and cardiac muscle homogenates. For this, samples of homogenate and SR vesicles (25 µg protein/lane in each case) were first subjected to SDS-PAGE on 6% homogenous or 4-18% gradient gels. The fractionated proteins were then electroblotted to nitrocellulose membranes. The membranes were probed with cardiac RyR2-specific antibody (monoclonal, dilution 1:2,500) or skeletal muscle RyR1-specific antibody (polyclonal, dilution 1:3,000). A peroxidase-linked anti-mouse IgG (for RyR2) and anti-rabbit IgG (for RyR1) at a dilution of 1:5,000 was used as the secondary antibody. Protein bands reactive with antibodies were visualized using the enhanced chemiluminescence detection system from Amersham. The images of the protein bands were optimized, captured, and analyzed by ImageMaster VDS gel documentation system (Pharmacia Biotech, San Francisco, CA). The Western blotting detection system was determined to be linear with respect to the amount of SR/homogenate protein in the range 10-40 µg using this camera-based densitometry system.

Measurement of High-Affinity [3H]Ryanodine Binding

High-affinity ryanodine binding was measured as described by Timmerman et al. (42) with minor modifications. Briefly, JSR vesicles (25 µg) were incubated at 37°C for 60 min in a buffered medium (total volume 100 µl) containing 150 mM KCl, 200 mM HEPES (adjusted to pH 7 with KOH), 0.25-50 nM of [3H]ryanodine (~57,000 counts · min-1 · pmol-1), 0.2 mM EGTA, and variable amounts of CaCl2 to obtain free [Ca2+] ranging from 0.05 to 12.94 µM as calculated by the computer program of Fabiato (10). The binding reaction was terminated by filtration through 0.22-µm GS Millipore filters and washed sequentially with 4 ml of washing buffer [150 mM KCl, 200 mM HEPES (adjusted to pH 7 with KOH)] and then twice with 4 ml each of ice-cold 10% ethanol. Specific binding of ryanodine was determined as the difference between total counts and nonspecific counts (measured in the presence of 10 µM unlabeled ryanodine).

Measurement of Unidirectional Ca2+ Release

Ca2+ release was determined by measuring unidirectional 45Ca2+ efflux from passively 45Ca2+-loaded JSR vesicles as described previously (38). Passive 45Ca2+ loading was performed by incubating JSR vesicles at 4°C for 1 h in medium A [50 mM Tris-maleate (pH 6.8), 120 mM KCl, 1 mM 45CaCl2, and 2 mM potassium oxalate; total volume 150 µl] or for 16 h in medium B [50 mM Tris-maleate (pH 6.8), 120 mM KCl, 5 mM MgCl2, and 5 mM 45CaCl2; total volume 150 µl]. To initiate Ca2+ release, aliquots of 45Ca2+-loaded vesicles were diluted 40-fold into a Ca2+-release medium [50 mM Tris-maleate (pH 6.8) containing (in mM) 120 KCl, 0.1 CaCl2, and 1 EGTA] that was preincubated for 5 min at 37°C. Subsequently, aliquots of the incubation mixture were filtered through Millipore filters at 15-s intervals for a period of 5 min. The filters were washed with 3 ml of ice-cold 10 mM Tris-maleate buffer (pH 6.8) containing 10 mM MgCl2 and 10 µM ruthenium red and dried at 60°C, and the 45Ca2+ radioactivity was determined by liquid scintillation counting.

Determination of Ca2+ Uptake

ATP-dependent Ca2+ uptake by JSR vesicles was measured using a Millipore filtration technique as described previously (32). The standard Ca2+ uptake assay medium (total volume 1 ml) contained 50 mM Tris-maleate (pH 6.8), 5 mM MgCl2, 5 mM ATP, 120 mM KCl, 5 mM potassium oxalate, 5 mM sodium azide, 0.1 mM EGTA, 0.1 mM 45CaCl2 (12,000-15,000 counts · min-1 · nmol-1), and JSR membranes (30 µg of protein). The assays were performed at 37°C; the Ca2+-uptake reaction was initiated by the addition of membrane fraction after preincubation of the rest of the assay components for 3 min. After 0.5, 1.0, and 1.5 min, 0.2-ml aliquots of the reaction mixture were filtered through 0.45-µm filters (Millipore, Bedford, MA). The filters were washed with 3 ml of a solution containing 10 mM Tris-maleate (pH 6.8), 100 mM KCl, and 10 mM MgCl2 and dried at 60°C, and the filter-associated radioactivity was determined by liquid scintillation counting. The rate of Ca2+ uptake was calculated from the linear regression line derived from the uptake determined at the three time points. The Ca2+-uptake rates were also measured under conditions of CRC blockade by including 25 µM ruthenium red (45) or 625 µM ryanodine (8) in the incubation medium.

Measurement of Blood Hormone Levels

Blood samples were collected by cardiac puncture at the time when the rabbits were killed, and the samples were then centrifuged. The serum samples were treated with polyethylene glycol to precipitate any endogenous antibodies (24), and the hormones were assayed by a fully automated chemiluminescent immunoassay analyzer (Chiron ACS-180).

Data Analysis

Results are presented as means ± SE. Statistical significance was evaluated by the Student's t-test; P < 0.05 was taken as the level of significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Establishment of Hyperthyroid State and Cardiac Hypertrophy

Ventricular weight normalized for body weight was increased significantly in the thyroid hormone-treated rabbits (hyperthyroid group) compared with the untreated control (euthyroid group) animals (Fig. 1A). This difference in the ratio of ventricular weight to body weight was due to both an increase in ventricular weight (euthyroid: 5.43 ± 0.11 g, n = 10; hyperthyroid: 6.08 ± 0.08 g, n = 10) and a decrease in body weight (euthyroid: 3.06 ± 0.05 kg, n = 10; hyperthyroid: 2.70 ± 0.04 kg, n = 10) in the hyperthyroid group. The blood levels of T4 and triiodothyronine (T3) were significantly elevated (Fig. 1, B and C), whereas thyroid-stimulating hormone levels were significantly decreased (Fig. 1D) in the hyperthyroid compared with euthyroid. These data demonstrate that the thyroid hormone treatment protocol used in this study resulted in the development of hyperthyroid state with attendant cardiac hypertrophy in the rabbit.


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  		Fig. 1.   Comparison of ventricular weight-to-body weight (g/kg) ratios (A) and blood thyroid hormone levels [free T4 (B), triiodothyronine (T3) (C), and thyroid-stimulating hormone (TSH) (D)] in control (euthyroid, open bars) and L-thyroxine (T4)-treated (hyperthyroid, filled bars) rabbits. Values are means ± SE for 5 animals in each group. *P < 0.01 vs. euthyroid hearts.

Effects of Thyroid Hormone on RyR Protein Expression

The relative levels of RyR protein in euthyroid and hyperthyroid rabbit hearts were determined by quantitative immunoblotting using a monoclonal antibody specific for the cardiac isoform, RyR2. Experiments using unfractionated cardiac muscle homogenates revealed significantly higher expression levels (~43% increase) of RyR2 in the hyperthyroid compared with the euthyroid (Fig. 2) heart. In additional experiments, cardiac muscle homogenates were fractionated to yield membrane vesicles enriched in JSR and LSR, and Western blotting analysis of RyR2 was performed using these membrane vesicles. As shown in Fig. 3, the relative amount of RyR2 was markedly higher (~2-fold) in JSR vesicles from the hyperthyroid heart, compared with those from the euthyroid heart. LSR vesicles from both euthyroid and hyperthyroid hearts had much lower levels of RyR2 than JSR vesicles. No significant difference in the relative amount of RyR2 was evident between LSR vesicles from hyperthyroid and euthyroid hearts. The protein profile of JSR and LSR vesicles isolated from hyperthyroid hearts was essentially similar to the protein profile of corresponding membrane vesicle preparations from euthyroid hearts (Fig. 4). Therefore, differences in the relative purity of the membrane vesicles isolated from the hyperthyroid versus euthyroid hearts does not contribute to the observed increase in expression levels of RyR2 in the hyperthyroid JSR. Densitometric analysis of Coomassie blue-stained RyR2 band in SDS-PAGE gels showed a significantly higher content of RyR protein in JSR vesicles derived from the hyperthyroid compared with euthyroid hearts (Fig. 5). Also evident was a significant increase (~75%) in the Ca2+-ATPase content of both JSR and LSR vesicles derived from the hyperthyroid compared with euthyroid hearts (Fig. 4).


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  		Fig. 2.   Detection and estimation of relative amounts of ryanodine receptor (RyR) in cardiac muscle homogenates from euthyroid and hyperthyroid rabbits by Western blotting. Identical amounts of homogenate protein (25 µg) from euthyroid and hyperthyroid hearts were subjected to Western blotting analysis after fractionation by SDS-PAGE on 6% homogenous gels (see METHODS). Representative immunoblots obtained using 3 separate homogenate preparations each from euthyroid and hyperthyroid hearts (bottom). RyR content was quantified by computer-assisted analysis of Western blots using ImageMaster VDS software, and the results obtained using 5 separate homogenate preparations each from euthyroid and hyperthyroid hearts are presented as means ± SE in bar graph. *P < 0.05 vs. euthyroid hearts.



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  		Fig. 3.   Detection and estimation of relative amounts of RyR in cardiac junctional sarcoplasmic reticulum (JSR, A) and longitudinal SR (LSR, B) from euthyroid and hyperthyroid rabbits by Western blotting. Identical amounts (25 µg protein) of JSR and LSR from euthyroid and hyperthyroid hearts were subjected to Western blotting analysis after fractionation by SDS-PAGE on 4-18% gradient gels (see METHODS). Representative immunoblots obtained using 4 separate JSR preparations and 3 separate LSR preparations each from euthyroid and hyperthyroid hearts (bottom). RyR content was quantified by computer-assisted analysis of Western blots using ImageMaster VDS software, and the results obtained using 9 separate JSR preparations and 5 separate LSR preparations each from euthyroid and hyperthyroid hearts are presented as means ± SE in bar graph. *P < 0.01 vs. euthyroid hearts.



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  		Fig. 4.   Comparison of protein profiles of cardiac LSR and JSR isolated from euthyroid and hyperthyroid rabbits. Identical amounts (30 µg protein) of LSR (A) and JSR (B) derived from euthyroid and hyperthyroid hearts were fractionated by SDS-PAGE on 4-18% gradient gel. Coomassie blue-stained gels showing typical protein profiles of LSR and JSR preparations from euthyroid and hyperthyroid rabbit hearts. Identity of peptide bands designated RyR-Ca2+-release channel (CRC) and Ca2+-ATPase was confirmed by Western immunoblotting analysis using monoclonal antibodies specific for cardiac RyR (RyR2 isoform) and cardiac sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) isoform.



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  		Fig. 5.   Estimation of relative amounts of RyR in cardiac JSR from euthyroid and hyperthyroid rabbits by densitometric analysis of Coomassie blue-stained RyR protein band in SDS-PAGE gel. After SDS-PAGE and staining of gels (see legend, Fig. 4), RyR content was quantified by computer-assisted analysis of the peptide band representing RyR using ImageMaster VDS software. Results obtained using 4 separate JSR preparations each from euthyroid and hyperthyroid hearts are presented as means ± SE. *P < 0.01 vs. euthyroid hearts.

Although the heart normally expresses only the RyR2 isoform, it was of interest to examine whether other RyR isoforms are also expressed in the hyperthyroid state. Western immunoblotting analysis using a polyclonal antibody specific for the RyR1 isoform showed no evidence of RyR1 expression in cardiac muscle of hyperthyroid or euthyroid rabbits; under similar experimental conditions, RyR1 isoform was readily detected in rabbit fast-twitch skeletal muscle, which normally expresses this isoform (results not shown).

Functional Properties of RyR in Euthyroid and Hyperthyroid Hearts

Having established thyroid hormone-induced overexpression of RyR in the rabbit heart, the functional properties of these receptors in euthyroid versus hyperthyroid hearts were compared using various criteria as described below.

Ca2+-dependent, high-affinity [3H]ryanodine binding. The plant alkaloid ryanodine binds preferentially to SR CRC that are open, and changes in [3H]ryanodine binding are thought to reflect changes in gating properties of the ryanodine receptors (7, 30). Figure 6A shows the specific [3H]ryanodine binding to JSR vesicles derived from euthyroid and hyperthyroid hearts as a function of free Ca2+ concentration. For these experiments, the binding assay medium contained 40 nM [3H]ryanodine, which permitted saturation of high-affinity binding sites (see below). In both euthyroid and hyperthyroid groups, specific [3H]ryanodine binding had a threshold for detection at 0.1 µM free Ca2+ and increased to a maximal value at 1.3 µM free Ca2+. At a given subsaturating (0.38 µM) or saturating (3.25 µM) free Ca2+ in the assay, the levels of specific [3H]ryanodine binding were significantly higher (~50% increase) in the hyperthyroid, compared with the euthyroid, group (Fig. 6, B and C). In additional experiments, specific [3H]ryanodine binding to JSR vesicles from euthyroid and hyperthyroid hearts was determined at varying concentrations of the radioligand in the presence of a saturating concentration of free Ca2+ (3.25 µM). Results from a typical experiment are shown in Fig. 7. At the range of [3H]ryanodine concentrations used (0.25-50 nM), specific binding was saturable in both groups (Fig. 7A). Nonspecific binding was <15% under these assay conditions. Scatchard plots of the data indicated a single binding site (Fig. 7B). Average values for maximum binding sites (Bmax) and the dissociation constant (Kd) for [3H]ryanodine derived from experiments using six separate JSR preparations each from euthyroid and hyperthyroid hearts are summarized in Table 1. It can be seen that the value for Bmax was significantly greater for hyperthyroid, compared with euthyroid, hearts; the Kd for [3H]ryanodine did not differ between the two groups.


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  		Fig. 6.   Ca2+-dependent high-affinity [3H]ryanodine binding to JSR vesicles from euthyroid and hyperthyroid rabbit hearts. High-affinity specific [3H]ryanodine binding was determined (see METHODS). A: Ca2+ concentration dependence of [3H]ryanodine binding; results obtained using 2 separate JSR preparations each from euthyroid and hyperthyroid hearts are shown and each data point represents mean of duplicate determinations. B and C: specific [3H]ryanodine binding data generated at 2 selected free Ca2+ concentrations (0.38 and 3.25 µM, respectively) using 5 separate JSR preparations each from euthyroid and hyperthyroid hearts are presented as means ± SE in B (free Ca2+ = 0.38 µM) and C (free Ca2+ = 3.25 µM). *P < 0.05 vs. euthyroid hearts.



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  		Fig. 7.   Specific [3H]ryanodine binding to JSR vesicles from euthyroid and hyperthyroid rabbit hearts at varying concentrations of [3H]ryanodine. Specific [3H]ryanodine binding was determined (see METHODS); the free Ca2+ concentration in assay was 3.25 µM. A: saturation binding curves. B: Scatchard plot of data (correlation coefficient: euthyroid, 0.97; hyperthyroid, 0.93). Representative data from a typical experiment are shown. Average values for maximum binding sites (Bmax) and dissociation constant (Kd) for [3H]ryanodine derived from experiments using 6 separate JSR preparations each from euthyroid and hyperthyroid hearts are summarized in Table 1.


                              
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  		Table 1.   Comparison of high-affinity specific [3H]ryanodine binding sites in JSR vesicles from euthyroid and hyperthyroid rabbit hearts

Ca2+ release from Ca2+-preloaded JSR vesicles. To assess the Ca2+-release function of the ryanodine receptors, JSR vesicles isolated from euthyroid and hyperthyroid hearts were subjected to passive Ca2+ loading, and the rate of Ca2+ release from the Ca2+-preloaded vesicles was measured (38). For these experiments, the Ca2+ loading of JSR vesicles was performed for 1 h in the presence of oxalate in the incubation medium, or for 16 h in the absence of oxalate in the incubation medium. As shown in Fig. 8, A and C, irrespective of the Ca2+ loading conditions used, the rate of Ca2+ release was significantly greater in the hyperthyroid group compared with the euthyroid group. However, during passive Ca2+ loading (in the absence or presence of oxalate), JSR vesicles from the hyperthyroid hearts accumulated a greater amount of Ca2+ than did JSR vesicles from the euthyroid hearts. Therefore, the Ca2+-release rates expressed as a percentage of the initial Ca2+ load did not differ appreciably between the euthyroid and hyperthyroid groups (Fig. 8, B and D).


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  		Fig. 8.   Ca2+ release from passively Ca2+-preloaded JSR vesicles of euthyroid and hyperthyroid rabbit hearts. JSR vesicles were subjected to passive Ca2+ loading in presence of oxalate (A and B) or in the absence of oxalate (C and D) and then incubated in a Ca2+-release medium to initiate Ca2+ release (see METHODS). In A and C, amount of Ca2+ released from JSR vesicles is plotted as a function of incubation time in the Ca2+-release medium. In B and D, Ca2+-release rates are expressed as a percentage of the initial intravesicular Ca2+ load (i.e., Ca2+ content of JSR vesicles before initiating Ca2+ release). When Ca2+ loading was performed in the presence of oxalate, the initial intravesicular Ca2+ load amounted to 1,226 ± 168 and 2,014 ± 286 nmol of Ca2+/mg of protein, respectively, for euthyroid and hyperthyroid JSR. When Ca2+ loading was performed in absence of oxalate, the initial intravesicular Ca2+ load amounted to 8.08 ± 0.55 and 13.61 ± 1.61 nmol of Ca2+/mg of protein, respectively, for euthyroid and hyperthyroid JSR. Each data point represents mean ± SE of 3 (C and D) or 5 (A and B) experiments using separate JSR preparations. *P < 0.05 vs. euthyroid JSR.

Assessment of RyR function using CRC blockers. Difference in the rates of ATP-energized Ca2+ uptake measured in the presence and absence of CRC blockers is a commonly used parameter to assess RyR function in isolated SR vesicles (8). The results presented in Fig. 9 compare the rates of ATP-driven Ca2+ uptake in JSR vesicles from euthyroid and hyperthyroid hearts in the absence and presence of CRC blockers. At concentrations known to block Ca2+ release (8, 45), ruthenium red (25 µM) and ryanodine (625 µM) both stimulated the rates of Ca2+ uptake in JSR vesicles of euthyroid and hyperthyroid hearts (Fig. 9, A and B). JSR vesicles from hyperthyroid hearts exhibited significantly higher rates of Ca2+ uptake compared with those from euthyroid hearts both in the absence and presence of CRC blockers. The rate of Ca2+ release, defined as the difference in the rate of Ca2+ uptake observed in the absence and presence of CRC blockade, was significantly greater (2- to 5-fold) in the hyperthyroid compared with the euthyroid group (Fig. 9C).


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  		Fig. 9.   Effect of RyR-CRC blockers on ATP-energized Ca2+ uptake by JSR vesicles from euthyroid and hyperthyroid rabbit hearts. Rates of ATP-dependent Ca2+ uptake were determined in absence of RyR-CRC blockers and in presence of RyR-CRC blocker ruthenium red (RR, 25 µM) or ryanodine (Ryn, 625 µM), as described in METHODS. A and B: effects of RR and Ryn, respectively, on Ca2+-uptake rates. C: rate of Ca2+ release, defined as difference in rates of Ca2+ uptake observed in absence and presence of RyR-CRC blockade. Data represent means ± SE of 5 experiments using separate JSR preparations in each case. *P < 0.05 vs. euthyroid hearts.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results presented here demonstrate that induction of hyperthyroid state, with attendant cardiac hypertrophy, is accompanied by overexpression of RyR protein in the rabbit myocardium. The elevated expression of RyR protein in the hyperthyroid compared with euthyroid heart could be observed in Western blotting experiments using unfractionated cardiac muscle homogenates as well as isolated membrane vesicles enriched in JSR. The polypeptide composition of JSR vesicles isolated from euthyroid and hyperthyroid hearts was similar except for the selective abundance of RyR (and Ca2+-ATPase) in membranes from the hyperthyroid group. Therefore, differences in the relative purity of membrane vesicles derived from euthyroid versus hyperthyroid hearts do not contribute to the thyroid hormone-induced overexpression of RyR protein reported here. Membrane vesicles of LSR isolated from euthyroid and hyperthyroid hearts showed essentially similar protein profiles and had only small amounts of RyR protein when compared with corresponding JSR vesicles. The relative amount of RyR protein in LSR vesicles did not differ significantly between euthyroid and hyperthyroid groups. These findings suggest that in the hyperthyroid cardiomyocyte, the overexpressed RyR are specifically targeted to JSR, the membrane locus at which they are thought to function in concert with voltage activation of L-type Ca2+ channels in the sarcolemma (see below). A previous study has reported an increased steady-state level of RyR-mRNA in the hyperthyroid rabbit heart (1), which is in accordance with the thyroid hormone-induced upregulation of RyR protein expression described here. To our knowledge, the present study is the first to characterize thyroid state-dependent alterations in RyR protein expression and functional properties of the RyR in the myocardium.

We used various criteria to compare the functional properties of RyR in euthyroid and hyperthyroid hearts, and these included specific high-affinity [3H]ryanodine binding, unidirectional Ca2+ release, and ATP-driven Ca2+ uptake in the presence and absence of CRC blockers. At low nanomolar concentrations ryanodine opens the RyR-CRC or locks it in the open state, whereas at high micromolar concentrations it promotes the closed conformation of the channel (7, 30). Therefore, high-affinity [3H]ryanodine binding, which is dependent on free Ca2+concentraion (7, 30), can be correlated with the functional state of the RyR. Our results show that high-affinity [3H]ryanodine binding in JSR vesicles from euthyroid and hyperthyroid hearts was Ca2+ dependent and saturable in the physiological range of cytosolic free Ca2+. Bmax was significantly greater in the hyperthyroid compared with euthyroid group, which is in accordance with the data from Western blotting experiments demonstrating upregulation of RyR protein expression. The dissociation constants for [3H]ryanodine and the Ca2+ sensitivity of [3H]ryanodine binding did not differ significantly between euthyroid and hyperthyroid hearts. These findings imply that the gating properties of RyR and the affinity of their Ca2+ activation sites remain unaltered in the hyperthyroid heart. Measurement of the rates of Ca2+ release from passively Ca2+-preloaded JSR vesicles demonstrated significantly higher rates of release in the case of hyperthyroid compared with euthyroid group, which could be attributed to the relatively higher density of RyR in the hyperthyroid JSR. However, in these experiments, the initial Ca2+ load was invariably greater in JSR vesicles from the hyperthyroid compared with euthyroid group, and the Ca2+-release rates (expressed as a percentage of initial Ca2+ load) did not differ significantly between the two groups. Previous studies have demonstrated that the rate of Ca2+ release from the SR is also dependent on intraluminal Ca2+ load (13, 37). Thus the SR Ca2+ load dependence of Ca2+-release function is retained in the hyperthyroid heart. The higher level of Ca2+ accumulation in hyperthyroid JSR during passive Ca2+ loading reflects an enhanced Ca2+ storage capacity of the membranes in the hyperthyroid state and is presumably due to larger intravesicular volume and/or an increased level of the Ca2+ storage protein, calsequestrin. Cardiac mRNA level of calsequestrin, however, was found not to be influenced by thyroid state in the rabbit (1).

Assessment of the effect of RyR-CRC blockade on active Ca2+-uptake rates also revealed significantly higher CRC activity in the hyperthyroid, compared with euthyroid, JSR (Fig. 9C). Previous studies have demonstrated abbreviation of cytosolic Ca2+ transient (Ca2+i) in hyperthyroid, compared with euthyroid heart, which is due to an increase in both the rate of rise and rate of decline in Ca2+i (2, 29). Because the rising phase of Ca2+i transient predominantly reflects the release of Ca2+ from the SR (3), the thyroid hormone-induced overexpression of RyR and associated enhancement in Ca2+-channel activity reported here provide a mechanistic basis for the faster rate of rise of the Ca2+i transient in the hyperthyroid heart, at the molecular level. According to the current concept of excitation-contraction coupling, one or a small number of L-type Ca2+ channels in the sarcolemma and a cluster of directly proximal RyR in the adjacent JSR serve as discrete Ca2+-release units producing spatially localized transient elevations of Ca2+i (Ca2+ sparks) on myocyte excitation (5, 6, 26, 39, 41). In this scheme, the functional efficacy of RyR would be expected to depend on their spatial proximity to the L-type channels as well as the maintenance of optimal stoichiometry of coupling between the two molecular components of the Ca2+-release unit. Our findings indicate that the overexpressed RyR in the hyperthyroid heart belong to the RyR2 isoform normally expressed in the myocardium and are targeted to their membrane locus in the JSR; this would ensure their spatial proximity to the L-type channels and functional compatibility. However, it is unclear whether thyroid state-dependent changes occur in the number of L-type channels in the sarcolemma. Radioligand binding studies have reported conflicting findings in this regard. In the rat heart, the number of [3H]nitrendipine binding sites was found to be decreased in the hyperthyroid state (15, 36), whereas no change (36) or an increase (15) was observed in the hypothyroid state. In contrast, in cultured chick ventricular cells, thyroid hormone treatment was shown to result in an increase in the number of L-type Ca2+ channels ([3H]PN200-110 binding sites), which was also associated with augmented transsarcolemmal Ca2+ influx (19).

Our results also showed significantly higher rates of ATP-energized Ca2+ uptake by JSR vesicles from the hyperthyroid, compared with those from the euthyroid heart (Fig. 9, A and B). This observation is in conformity with previous studies demonstrating enhanced Ca2+-uptake activity of cardiac SR from hyperthyroid animals (25, 40) and cardiomyocytes cultured in the presence of thyroid hormone (20). This enhancement in SR Ca2+ sequestering activity likely results from the thyroid hormone-induced upregulation of SERCA2 and downregulation of phospholamban (20-22) and contributes to faster rate of decline of Ca2+i (2, 29) and acceleration of diastolic relaxation (12, 28, 33) in the hyperthyroid heart.

In conclusion, this study demonstrates overexpression of RyR protein in the hyperthyroid rabbit heart. The relative abundance of RyR may be responsible, in part, for the changes in SR Ca2+ release, Ca2+i transient, and cardiac systolic function associated with thyroid hormone-induced cardiac hypertrophy.


    ACKNOWLEDGEMENTS

We are grateful to Drs. V. Sorrentino and A. Conti, San Raffaele Scientific Institute, Milan, for the generous gift of RyR1 polyclonal antibody. We thank Lily Jiang for secretarial assistance and Bruce Arppe for preparing photographs of illustrations.


    FOOTNOTES

This work was supported by Medical Research Council of Canada Grant MT9553. M. Jiang is the recipient of a Graduate Student Scholarship award from the Medical Research Council of Canada.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: N. Narayanan, Dept. of Physiology, Medical Sciences Bldg., The Univ. of Western Ontario, London, Ontario, Canada N6A 5C1 (E-mail: nnarayan{at}physiology.uwo.ca).

Received 9 June 1999; accepted in final form 28 October 1999.


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