Alterations in cardiac SR Ca2-release channels during development ...

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The cardiomyopathic Syrian ... SYRIAN CARDIOMYOPATHIC HAMSTERS (BIO 14.6 strain or ... under identical conditions on a normal laboratory diet (MF,.
Alterations in cardiac SR Ca21-release channels during development of heart failure in cardiomyopathic hamsters TAKESHI UEYAMA, TOMOKO OHKUSA, YUJI HISAMATSU, YASUMA NAKAMURA, TAKESHI YAMAMOTO, MASAFUMI YANO, AND MASUNORI MATSUZAKI Second Department of Internal Medicine, Yamaguchi University School of Medicine, Ube, Yamaguchi 755, Japan Ueyama, Takeshi, Tomoko Ohkusa, Yuji Hisamatsu, Yasuma Nakamura, Takeshi Yamamoto, Masafumi Yano, and Masunori Matsuzaki. Alterations in cardiac SR Ca21release channels during development of heart failure in cardiomyopathic hamsters. Am. J. Physiol. 274 (Heart Circ. Physiol. 43): H1–H7, 1998.—The cardiomyopathic Syrian hamster develops a progressive cardiomyopathy characterized by cellular necrosis, hypertrophy, cardiac dilatation, and congestive heart failure. This study aimed to identify alterations in cardiac mechanical function and in the cellular content of sarcoplasmic reticulum (SR) Ca21-release channels (ryanodine receptors, RyR) in the heart of the UM-X7.1 cardiomyopathic hamster during the development of heart failure. Experimental and healthy control hamsters were examined at 8, 18, and 28 wk of age. The UM-X7.1 hamsters had developed left ventricular (LV) hypertrophy at 8 wk and a marked LV dilatation at 18–28 wk. During the latter stage, the UM-X7.1 hamster hearts showed global hypokinesis. Equilibrium binding assays of high-affinity sites for [3H]ryanodine were performed in ventricular homogenate preparations. There was no significant difference between the two groups in the maximum number of [3H]ryanodine binding sites (Bmax ) at either 8 or 18 wk of age, although the cardiac pump function was impaired in UM-X7.1 hamsters at 18 wk of age. By 28 wk, Bmax was significantly lower in the UM-X7.1 hamsters. Quantitative immunoblot assay revealed that the content of RyR protein in cardiomyopathic hearts, which was increased at the early stage, declined to below normal as heart failure advanced. These results suggest that the number of RyR in the UM-X7.1 cardiomyopathic hamsters was preserved at both the hypertrophic and early stages of heart failure with a possibly compensatory increase in the level of protein expression, although the cardiac function already showed a tendency to be impaired. [3H]ryanodine binding; immunoblot assay

SYRIAN CARDIOMYOPATHIC HAMSTERS (BIO 14.6 strain or UM-X7.1 strain) display hereditary abnormalities in both cardiac and skeletal muscle (8). These strains develop a hypertrophic form of cardiomyopathy resulting in a greatly thickened ventricular wall and septum. In the heart of these hamsters, progressive cardiac necrosis begins at 40–50 days of age, the lesions reach maximum severity at ,80–90 days, and a process of hypertrophy followed by dilatation ultimately results in premature death from congestive heart failure at 250–300 days (11). This hamster model provides an opportunity to study changes that might also occur in human primary muscle disorders, which frequently involve the myocardium. Changes in the failing myocardium of the myopathic hamster include a deficient production of adenosine 38,58-cyclic monophosphate

(30), a decrease in Na1-K1-adenosinetriphosphatase (18), a decline in the function of the sarcoplasmic reticulum (SR) (4, 21, 29), an inhibition of the Na1/Ca21 exchanger (22), excessive Ca21 accumulation (3, 12), fibrosis (23), high inorganic phosphate (11), and a depressed phosphorylation potential (31). An elevation in the level of free intracellular Ca21 (‘‘Ca21 overload’’) is a common finding in cardiac necrosis and is suspected to be of central importance in the cardiomyopathic hamster (2, 6, 19). A previous study showed that Ca21 overload is detectable in hearts from very young (24- to 45-day-old) cardiomyopathic hamsters (6). Moreover, some investigators (13, 24, 25) recently reported that an increase in the density of SR Ca21-release channels (ryanodine receptors, RyR) occurs in conjunction with the development of Ca21 overload. However, this finding could be interpreted in one of two ways. The increase in RyR density could be an important causative factor in the development of Ca21 overload in cardiomyopathy, but it is equally possible that it is an early compensatory reaction to the disease process. The Ca21-uptake function and Ca21-pumping activity of the SR have already been studied in cardiomyopathic hamster hearts suffering end-stage congestive heart failure (21, 29). However, many previous studies reported physiological and biochemical alterations at the end of decompensated heart failure, and one major deficiency in many previous studies of failing heart tissue is the absence of a serial physiological correlate for the alterations in biochemistry detected as heart failure develops. The goal of the present study was to investigate alterations in cardiac pump function and to determine the cellular content of cardiac RyR protein and ryanodine binding in the heart of the myopathic hamster during the development of heart failure. Our results demonstrate that 1) at an early (hypertrophic) stage of this disease, the cardiac pump function was normal, with a normal level of ryanodine binding, although the expression level of RyR protein was increased [the latter may be a compensatory reaction to some factor(s) in the disease process and may prevent a reduction in ryanodine binding]; and 2) as cardiac pump failure advanced, the expression level of RyR protein was reduced and the ryanodine binding gradually decreased. MATERIALS AND METHODS

Animals. Cardiomyopathic (UM-X7.1) hamsters (kindly provided by Dr. Lemanskie, State Univ. of New York Health Science Center, Syracuse, NY, and inbred in our laboratory) and sex- and age-matched normal golden hamsters (Japan SLC, Hamamatsu, Japan) were used. Both cardiomyopathic

0363-6135/98 $5.00 Copyright r 1998 the American Physiological Society

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and normal hamsters were housed at five hamsters per cage in an air-conditioned room with automatic 12:12-h day-night cycling in the Experimental Animal Facility of the Yamaguchi University School of Medicine. They were all maintained under identical conditions on a normal laboratory diet (MF, Oriental Yeast, Chiba, Japan) with ad libitum access to tap water. The care of the animals and the protocols used conformed with the American Physiological Society ‘‘Guiding Principles in the Care and Use of Animals’’ and were in accord with guidelines laid down by the Animal Ethics Committee of Yamaguchi University School of Medicine. General protocols and study objectives. To evaluate the left ventricular (LV) dimensions and the contractile state in vivo, transthoracic echocardiography (15) was performed at 8, 18, and 28 wk of age (model SSD-280, Aloka, Tokyo, Japan, with a 7.5-MHz sector scan probe). The animal was placed on its back under light anesthesia induced with pentobarbital sodium (15 mg/kg im) (26). This relatively small dose of pentobarbital sodium was shown not to affect the level of blood pressure, heart rate, or the state of respiration in either type of hamster at any stage (10). The probe was gently placed so that it was in contact with the middle of the thorax through an ultrasound transmission medium (Aquasonic 100, Parker Laboratories, Orange, NJ). M-mode echocardiograms were obtained at the papillary muscle level, guidance being obtained from two-dimensional long-axis images (5), and were recorded on an image printer (UP-500, Sony, Tokyo, Japan). We measured the LV end-diastolic diameter (LVEDD) as the widest and the end-systolic diameter (LVESD) as the narrowest dimension in the M-mode recording. From these measurements, LV fractional shortening (LVFS) was calculated according to previously reported formulas (10). After we had obtained the echocardiograms, each hamster was anesthetized with additional pentobarbital sodium (30 mg/kg im), and the chest was opened immediately. The hamsters were divided into two groups according to the study protocol. In the first group, the heart was excised from each hamster and immersed in ice-cold 0.9% NaCl solution. After atrial tissue, visible fat, and connective tissues had all been removed, the biventricular tissue was weighed and immediately used to prepare a crude homogenate for the assay of RyR binding. In the second group, hearts were quickly frozen in liquid nitrogen and stored at 280°C until used to produce a membrane preparation for the immunoblot assay. Whole ventricle crude homogenate preparation. A crude membrane fraction for [3H]ryanodine binding assay was prepared as previously described with some modifications (28, 33). The whole ventricular tissue was homogenized twice for 20 s each time, using a Brinkmann Polytron, in 20 mM tris(hydroxymethyl)aminomethane · maleate containing 0.3 M sucrose, 0.1 M KCl, 5 mg/l leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride, at pH 7.0 (solution A). The homogenate was filtered through two layers of cheesecloth. Protein concentration was determined by the method of Lowry et al. (16), using bovine serum albumin as standard. Aliquots of homogenate were frozen in liquid nitrogen and stored at 280°C until used. Membrane preparation. Each frozen ventricle was cut into pieces in 3–5 vols (wt/vol) of solution A (see Whole ventricle crude homogenate preparation). The ventricle was finely minced and subjected to two 20-s bursts with a Brinkmann Polytron. The homogenate was then centrifuged at 12,000 g for 15 min at 4°C. The supernatant was frozen in liquid nitrogen and stored at 280°C until used. Protein concentration was determined by the method of Lowry et al. (16), using bovine serum albumin as standard. In the experiments

described here, a single heart was used to generate each fraction; thus n refers to the number of hearts in each group. Assay of [3H]ryanodine binding. [3H]ryanodine binding assays were carried out according to previously described methods (20, 33). Briefly, crude homogenate (0.4 mg/ml) was incubated for 90 min at 37°C in 25 mM imidazole (pH 7.4), 1.0 M KCl, 1.103 mM CaCl2, and 0.95 mM ethylene glycol-bis(baminoethyl ether)-N,N,N8,N8-tetraacetic acid (EGTA; 20 µM free Ca21 ), in each case with a concentration of [3H]ryanodine in the range of 0.6–20 nM. The reaction was terminated by rapid filtration of 1 ml of the incubation mixture through a glass fiber filter (Whatman GF/C, Maidstone, UK) under reduced pressure. To minimize the nonspecific binding component, each filter was immediately washed with 5 ml of ice-cold buffer (25 mM imidazole, 1.0 M KCl, 1.103 mM CaCl2, 0.95 mM EGTA, pH 7.4) and removed while under vacuum. After 5 ml of scintillation fluid were added, radioactivity was counted in a scintillation counter (LSC-5100, Aloka). Nonspecific binding was determined in the presence of 2 µM unlabeled ryanodine. Immunologic quantification of RyR protein. Immunoblot analysis was performed as previously described with some modifications (25). Ventricle homogenates (400 µg protein/ lane) from UM-X7.1 (n 5 5) and normal hamster (n 5 5) hearts at each stage were electrophoresed on 4% sodium dodecyl sulfate-polyacrylamide gels using Laemmli’s buffer system (14). The proteins in the gel were transferred to a Protran nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany). The membranes were treated with 5% nonfat dry milk in phosphate buffer, incubated with a monoclonal antibody solution, and incubated further with peroxidase-conjugated secondary antibody (1:1,000 dilution). For this, we used the monoclonal antibody Ry-9 against the cardiac RyR (kindly provided by Dr. M. Shigekawa, National Cardiovascular Center Research Institute, Osaka, Japan; Refs. 9, 25). The amount of protein recognized with the aid of the antibodies was quantified by means of the ECL immunoblotting detection system (Amersham, Amersham, UK), the membrane being exposed to X-ray film. Quantitative densitometry of immunoblots was analyzed using a microcomputer imaging device (AE-6900M, ATTO, Tokyo, Japan). Statistical analysis. All data are presented as means 6 SD. Comparisons between data from UM-X7.1 and data from normal hamsters were performed by a two-way analysis of variance with Scheffe´’s test. Differences were taken to be significant at P , 0.05. RESULTS

Morphological characteristics. Table 1 shows the morphological characteristics of the hamsters used in Table 1. Pathological findings in hamsters Age

8 wk UM-X 7.1 Control 18 wk UM-X 7.1 Control 28 wk UM-X 7.1 Control

n

BW, g

BVW, mg

BVW/BW, mg/g

8 10

80 6 3* 103 6 5

221 6 19* 250 6 22

2.8 6 0.3* 2.4 6 0.2

8 10

121 6 6* 161 6 14

331 6 13* 364 6 31

2.7 6 0.1* 2.3 6 0.1

8 10

136 6 8* 164 6 12

404 6 64 396 6 19

3.0 6 0.6* 2.4 6 0.1

Values are means 6 SD; n, no. of hearts. BW, body weight; BVW, biventricular weight. * P , 0.05 vs. appropriate control.

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Fig. 1. M-mode echocardiography serially obtained from representative UM-X7.1 and control hamsters at 8, 18, and 28 wk. Echoes were recorded at level of papillary muscles (using guidance by 2-dimensional long-axis images). For group data, see Table 2. Assessments of inter- and intraobserver variability were performed according to method of Bland and Altman (Ref. 1). To determine interobserver variability, 36 M-mode tracings were photocopied, and end-diastolic diameter and end-systolic diameter were measured independently by 2 different observers (T. Ueyama and T. Ohkusa). Another set of copies was measured (by T. Ueyama) to give estimate of intraobserver variability.

this study. Although the body weight of normal golden hamsters was significantly greater than that of UMX7.1 hamsters at each stage, the value of the biventricular-to-body weight ratio (calculated using wet wt) was significantly higher in each UM-X7.1 group than that in the corresponding group of golden hamsters. On this basis, a mild cardiac hypertrophy can be considered to be detectable at 8 wk of age in UM-X7.1 hamsters. Transthoracic echocardiography. Figure 1 shows M-mode echocardiograms of the LV at the papillary muscle level at three different stages in both UM-X7.1 and control hamsters. At 8 wk, the echocardiograms from UM-X7.1 hamsters did not differ from those obtained from control hamsters. At 18 wk, LVEDD and LVESD were both greater in the UM-X7.1 hamsters than in the controls and, at 28 wk, LVEDD and LVESD were markedly increased, whereas LV motion was significantly reduced (i.e., failing stage) (Table 2). There was no significant difference between these two groups in terms of LV fractional shortening (LVFS) at the age of 8 wk. However, at the failing stage, LVFS was much smaller in UM-X7.1 than in control hamsters (Table 2). [3H]ryanodine binding assay. Figure 2 shows representative examples of [3H]ryanodine binding curves derived from the hearts of UM-X7.1 and control hamsters. Nonspecific binding was ,10% of total binding at 2.5 nM [3H]ryanodine. In each case, because the Scatchard plot showed a well-fitted linear regression for the relationship between bound ligand and bound/ free ligand, it indicated a single binding site. The mean values obtained for the number of binding sites (Bmax ) and the dissociation constant (Kd ) are shown for all the hearts in Table 3. At 8 and 18 wk, the Bmax for the crude Table 2. Echocardiographic data in hamsters Age

8 wk UM-X 7.1 Control 18 wk UM-X 7.1 Control 28 wk UM-X 7.1 Control

n

LVEDD, mm

LVESD, mm

LVFS, %

HR, beats/min

8 10

3.0 6 0.5 3.2 6 0.4

1.6 6 0.4 1.5 6 0.2

47 6 9 53 6 9

422 6 28 451 6 29

8 10

5.6 6 0.3* 4.0 6 0.3

4.6 6 0.4* 2.1 6 0.4

19 6 3* 50 6 11

434 6 17 442 6 41

8 10

7.2 6 0.8* 4.1 6 0.4

6.3 6 0.9* 2.3 6 0.5

13 6 5* 45 6 11

406 6 15 400 6 49

Values are means 6 SD; n, no. of hearts. LVEDD, left ventricular (LV) end-diastolic diameter; LVESD, LV end-systolic diameter; LVFS, LV fractional shortening; HR, heart rate. * P , 0.05 vs. appropriate control.

homogenates obtained from UM-X7.1 hamster showed no significant difference from their respective controls. However, by 28 wk, the Bmax values had become significantly lower in the UM-X7.1 hamsters than in the corresponding controls. On the other hand, the Kd of [3H]ryanodine binding was not significantly different between the two groups at any stage. Immunologic assay. We used a quantitative immunoblot assay to make an estimation of the content of RyR protein in total ventricular homogenates (Fig. 3). This procedure should permit estimation of all tissue proteins without proteolytic degradation. At 8 and 18 wk, the content of RyR protein in UM-X7.1 hamster hearts was larger than that in the control hearts by 2.6 6 0.4 times and by 1.2 6 0.4 times, respectively. However, by 28 wk, as heart failure continued to develop, the content of this protein had become lower than in the controls (0.6 6 0.3 times control). DISCUSSION

In this study, we have examined alterations occurring in cardiac function and in cardiac SR Ca21-release channels (ryanodine receptors, RyR) during the development of heart failure in cardiomyopathic hamsters. We studied UM-X7.1 cardiomyopathic hamsters (a derivative of the BIO 14.6 strain) from the hypertrophic to the failing stage, and we examined the in vivo LV size and contractile state using transthoracic echocardiography. For the RyR binding assay, we used crude ventricular homogenates, which contain all of the ligand binding sites. By so doing, we hoped to avoid technical artifacts and the damage that might be incurred during the purification of the microsome fraction. In addition, we used a sensitive immunoblot assay method to quantify the amounts of RyR protein in ventricular homogenates. By using these methods, we have shown that 1) at the hypertrophic stage and at the early stage of heart failure, although cardiac pump function was impaired, ryanodine binding did not differ significantly from control, although the level of RyR protein expression was increased; and 2) as cardiac pump failure advanced, ryanodine binding came to be markedly lower than control and the level of RyR protein expression was also reduced below that seen in normal hearts. Time course of development of heart failure in UMX7.1 hamsters. Several types of animal models have been developed for investigating heart failure. The hereditary polymyopathy discovered in an inbred strain of Syrian hamsters has generated considerable interest

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Fig. 2. Representative data for [3H]ryanodine binding in crude homogenates obtained from UM-X7.1 and control hamster hearts at 8 (A), 18 (B), and 28 wk (C). Nonspecific binding was ,10% of total binding at [3H]ryanodine concentration of 2.5 nM. Each data point is average of duplicate determinations. Left, saturation binding curve; right, Scatchard plots.

in the field of genetically determined myopathies. This unique strain (BIO 14.6 and UM-X7.1) develops progressive cardiac necrosis beginning at 40–50 days of age (11). Cardiac hypertrophy is detectable at ,80 days, and the progressive hypertrophy followed by dilatation ultimately results in death from congestive heart failTable 3. [3H]ryanodine binding assay data Age

8 wk UM-X 7.1 Control 18 wk UM-X 7.1 Control 28 wk UM-X 7.1 Control

Bmax , pmol/mg

Kd , nM

0.45 6 0.08 0.51 6 0.03

0.63 6 0.11 0.79 6 0.23

0.43 6 0.03 0.51 6 0.11

0.45 6 0.05 0.78 6 0.30

0.32 6 0.03* 0.44 6 0.03

0.78 6 0.16 0.87 6 0.44

Values are means 6 SD; n 5 6 hearts for each group. Bmax , maximal number of binding sites; Kd , dissociation constant. * P , 0.05 vs. appropriate control.

ure at 250–300 days (11). As seen in our data (Tables 1 and 2), mild cardiac hypertrophy could be detected at 8 wk (50–60 days) of age without impaired cardiac pump function in our cardiomyopathic hamsters. Jasmin and Proschek (11) reported that cardiac hypertrophy starts to develop at the age of 80 days and that congestive heart failure starts to develop at 180 days. In contrast, the UM-X7.1 hamsters in our laboratory 1) showed evidence of cardiac hypertrophy at a younger age (50–60 days), 2) had an LVEDD and an LVESD that were already somewhat increased and LV motion that was somewhat reduced at the age of 120–130 days, and 3) died from congestive heart failure at 200–250 days. Although we are unsure why our UM-X7.1 hamsters started to develop cardiac hypertrophy and heart failure at younger ages than those reported by Jasmin and Proschek (11), we suspect that the explanation lies in the animals being transferred from the original laboratory to our laboratory and inbred for more than the 15th generation. Strictly speaking, our UM-X7.1 hamsters

CARDIAC RYANODINE RECEPTORS IN CARDIOMYOPATHIC HAMSTERS

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Fig. 3. Representative immunoblot analysis of ventricular homogenates from control (lane 1) and UM-X7.1 (lane 2) hamster hearts at 8, 18, and 28 wk. MW, molecular weight; CBB, Coomassie brilliant blue staining. Arrow, 220 kDa.

were a substrain of the original UM-X7.1. Possibly, the expression of the relevant abnormalities was more concentrated and the effects became manifest earlier than in the original strain. Alterations in cardiac RyR during progress of cardiomyopathy. Recently, some investigators have reported that ryanodine binding is increased in SR obtained from prehypertrophic cardiomyopathic hamster hearts (13, 24, 25). Such results could suggest that an increase in the amount or velocity of Ca21 release from the SR may contribute to the development of a Ca21 overload in this model of cardiomyopathy. However, there has as yet been no direct demonstration of an increase in the amount or velocity of Ca21 release from the SR or of an increase in intracellular Ca21 concentration ([Ca21]i ) transients in the cardiomyopathic hamster heart. In fact, Wikman-Coffelt et al. (32) reported that in the cardiomyopathic hamster heart there is an inhibition of glycolysis, and that the corresponding increase in intracellular H1 concentration in the myopathic heart itself causes a disturbance in Ca21 buffering, a severe rise in diastolic [Ca21]i, a decrease in [Ca21]i transients, and depressed cardiac performance. Moreover, on the basis of the published evidence, the possibility could not be excluded that the reported increase in ryanodine binding might be an early compensatory reaction to some factor(s) in the disease process rather than a cause of the development of Ca21 overload. If the former is true, the increase in ryanodine binding might possibly occur as a compensation for depressed SR storage capacity or reduced Ca21 influx through voltage-sensitive channels. In our study, at the age of 8 wk (hypertrophic stage), ryanodine binding did not differ from normal, at a time when there was an increased expression level of RyR protein. This could suggest that at this stage in the cardiomyopathic hamster RyR had completely compensated in terms of protein expression, perhaps via an increase in protein synthesis and/or a suppression of protein degradation (17). Moreover, at the early stage of heart failure (18 wk), although cardiac function was showing signs of impairment, ryanodine binding was still preserved. Whitmer et al. (29) reported no differ-

ence from normal in the cardiac Ca21-transport properties of SR from the BIO 14.6 strain of hypertrophic cardiomyopathic hamsters, the strain from which our UM-X7.1 strain is derived, but found that the BIO 53.58 strain of dilated cardiomyopathic hamsters exhibited a progressive defect in the SR. Although they measured cardiac SR Ca21 uptake, a different parameter from ours, their results, taken together with ours, could suggest that the cardiac SR Ca21-transport function may be preserved in our hypertrophic cardiomyopathic hamster (UM-X7.1) as a result of the modifications in protein synthesis and/or degradation during the development of heart failure. In contrast to the results of the present study, we recently reported that volume-overload cardiac hypertrophy in rats, even at its hemodynamically compensatory stage, is accompanied by a decrease in ryanodine binding (7). Moreover, Vatner et al. (28) reported that RyR binding was significantly reduced after 1 day of rapid pacing in dogs, at a time when severe heart failure was not manifest. These reports, and the present study, seem to suggest that the changes occurring in the intracellular processes underlying cardiac excitation-contraction coupling, especially cardiac SR function, may well differ between the various disease states studied and may be caused by different factors in the development of the different types of heart failure. A further discrepancy is that our data, which indicate normal ryanodine binding in crude homogenates from the cardiomyopathic hamster at the hypertrophic and early stages of this disease, are not consistent with previous reports on cardiomyopathic hamsters (see above). The following may help to explain this inconsistency. 1) The UM-X7.1 hamsters used in our experiment were at a different stage from those used by others; we used UM-X7.1 hamsters at the hypertrophic stage rather than the prehypertrophic stage. 2) We used crude cardiac homogenates for our RyR binding assay because such homogenates contain all of the ligand binding sites. Lachnit et al. (13) reported an increase in ryanodine binding when using membrane fractions but a small decrease when using whole tissue

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CARDIAC RYANODINE RECEPTORS IN CARDIOMYOPATHIC HAMSTERS

homogenates from cardiomyopathic hamsters at a prehypertrophic stage. They explained this discrepancy as follows: a loss of structural integrity in the T tubules allows easier extraction of T-tubule material after mechanical disruption, and this may be responsible for the increase in ryanodine binding in purified membranes of the myopathic heart. For this reason, we decided to use whole tissue homogenates to avoid mechanical artifacts and to preserve any pathophysiological conditions. There has been previous controversy about ryanodine binding in crude homogenate preparations of the cardiomyopathic heart. Some investigators (13, 25) observed either no difference or a slight decrease in ryanodine binding when they compared the cardiomyopathic hamster heart with the normal heart. In contrast, Sapp and Howlett (24) reported an increase in ryanodine binding in their binding study. It seems likely that the differing results may be caused by the use of differing assay conditions and membrane preparations. Expression of RyR protein in UM-X7.1 hamsters. Biochemical data suggest that cardiac hypertrophy is primarily the result of increased RNA and protein synthesis. As shown in our data, at the hypertrophic stage in the myopathic heart, the level of RyR protein expression was increased, and its level was still higher than that of the controls at the age of 18 wk. However, ryanodine binding was normal at the early stage of heart failure. Finally, both the level of RyR protein expression and ryanodine binding were markedly reduced in the myopathic heart. As the disease advanced, the compensatory mechanism that acts to increase the level of RyR protein was disturbed and finally resulted in decompensated heart failure. These results suggest that a conformational abnormality of RyR protein, which may involve an alteration in the phosphorylation of RyR and/or a potentiation or inhibition of [3H]ryanodine binding by some modulator(s)(27), may exist in myopathic hearts. However, at this stage, these notions can be only speculative, and further studies will be required before any firm proposals can be made. In conclusion, we have studied alterations in cardiac function and cardiac RyR in UM-X7.1 myopathic hamsters. Although a complete understanding of the intracellular processes, including Ca21 handling, underlying cardiomyopathy has not yet been achieved, the results of this study indicate that ryanodine binding is preserved at the hypertrophic stage and at the early stage of heart failure in UM-X7.1 hamsters. The increase in cardiac RyR protein expression seen at those stages may, at least in part, represent an important adaptive mechanism during the development of this disease. We thank Dr. M. Shigekawa of the National Cardiovascular Center Research Institute for providing the antibody against the cardiac ryanodine receptor (Ry-9) used in this study. We are also grateful to Dr. T. Noma and Dr. S. Inouye of the Yamaguchi University School of Medicine for advice and Dr. R. J. Timms for editing the language of the manuscript. Address for reprint requests: M. Matsuzaki,, Second Dept. of Internal Medicine, Yamaguchi Univ. School of Medicine, 1144, Kogushi, Ube, Yamaguchi 755, Japan. Received 21 April 1997; accepted in final form 10 September 1997.

REFERENCES 1. Bland, J. M., and D. G. Altman. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1: 307–310, 1986. 2. Bond, M., A. R. Jaraki, C. H. Disch, and B. P. Healy. Subcellular calcium content in cardiomyopathic hamster hearts in vivo: an electron probe study. Circ. Res. 64: 1001–1012, 1989. 3. Camacho, S. A., J. Wikman-Coffelt, S. T. Wu, and W. W. Parmley. Improved myocardial performance and energetics in Syrian cardiomyopathic hamsters after isoproterenol treatment: a 31P-NMR study. Circulation 77: 712–719, 1988. 4. Dusko, J., J. H. K. Vogel, and C. A. Chidsey. Reduced calcium uptake and ATPase of the sarcoplasmic reticular fraction prepared from chronically failing calf hearts. Circ. Res. 27: 235– 247,1970. 5. Feigenbaum, H. The echocardiographic examination. In: Echocardiography, edited by H. Feigenbaum. Philadelphia, PA: Lea and Febiger, 1984, p. 50–104. 6. Hano, O., and E. G. Lakatta Diminished tolerance of prehypertrophic cardiomyopathic Syrian hamster hearts to Ca21 stresses. Circ. Res. 69: 123–133,1991. 7. Hiramatsu, Y., T. Ohkusa, Y. Kihara, M. Inoko, T. Ueyama, M. Yano, S. Sasayama, and M. Matsuzaki. Early changes in the function of cardiac sarcoplasmic reticulum in volumeoverloaded cardiac hypertrophy in rats. J. Mol. Cell. Cardiol. 29: 1097–1109, 1997. 8. Homburger, F. Myopathy of hamster dystrophy: history and morphologic aspects. Ann. NY Acad. Sci. 317: 2–17, 1979. 9. Imagawa, T., T. Takasato, and M. Shigekawa. Cardiac ryanodine receptor is absent in type I slow skeletal muscle fibers: immunochemical and binding studies. J. Biochem. (Tokyo) 106: 342–348, 1989. 10. Inoko, M., Y. Kihara, I. Morii, H. Fujiwara, and S. Sasayama. Transition from compensatory hypertrophy to dilated, failing left ventricles in Dahl salt-sensitive rats. Am. J. Physiol. 267 (Heart Circ. Physiol. 36): H2471–2482, 1994. 11. Jasmin, G., and L. Proschek. Hereditary polymyopathy and cardiomyopathy in the Syrian hamster. I. Progression of heart and skeletal muscle lesions in the UM-X7.1 line. Muscle Nerve 5: 20–25, 1982. 12. Jasmin, G., and L. Proschek. Calcium and myocardial cell injury: an appraisal in the cardiomyopathic hamster. Can. J. Physiol. Pharmacol. 62: 891–898, 1983. 13. Lachnit, W. G., M. Phillips, K. J. Gayman, and I. N. Pessah. Ryanodine and dihydropyridine binding patterns and ryanodine receptor mRNA levels in myopathic hamster heart. Am. J. Physiol. 267 (Heart Circ. Physiol. 36): H1205–H1213, 1994. 14. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970. 15. Litwin, S. E., S. E. Katz, J. P. Morgan, and P. S. Douglas. Transthoracic echocardiographic evaluation of post-infarction ventricular remodeling in rat (Abstract). J. Am. Coll. Cardiol. 19: 362A, 1992. 16. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265–275, 1951. 17. Magid, N. M., J. S. Borer, M. S. Young, D. C. Wallerson, and C. DeMonteiro. Suppression of protein degradation in progressive cardiac hypertrophy of chronic aortic regurgitation. Circulation 87: 1249–1257, 1993. 18. Norgaard, A., U. Baandrup, J. S. Larsen, and K. Kjeldsen. Heart Na/K-ATPase activity in cardiomyopathic hamster as estimated from K-dependent 3–0-MPFase activity in crude homogenates. J. Mol. Cell. Cardiol. 19: 589–594, 1987. 19. Nylen, E. G., and K. Wrogemann. Mitochondrial calcium content and oxidative phosphorylation in heart and skeletal muscle from dystrophic mice. Exp. Neurol. 80: 69- 80, 1983. 20. Ohkusa, T., Y. Hisamatsu, M. Yano, S. Kobayashi, H. Tatsuno, Y. Saiki, M. Kohno, and M. Matsuzaki. Altered cardiac mechanism and sarcoplasmic reticulum function in pressure overload-induced cardiac hypertrophy in rats. J. Mol. Cell. Cardiol. 29: 45–54, 1997.

CARDIAC RYANODINE RECEPTORS IN CARDIOMYOPATHIC HAMSTERS 21. Panagia, V., S. L. Lee, A. Singh, G. N. Pierce, G. Jasmin, and N. S. Dhalla. Impairment of mitochondrial and sarcoplasmic reticulum functions during the development of heart failure in cardiomyopathic hamsters. Can. J. Cardiol. 2: 234–247, 1986. 22. Panagia, V., J. N. Singh, M. B. Anand-Srivastava, G. N. Pierce, G. Jasmin, and N. S. Dhalla. Sarcolemmal alterations during the development of genetically determined cardiomyopathy. Cardiovasc. Res. 18: 567–572, 1984. 23. Proschek, L., and G. Jasmin. Hereditary polymyopathy and cardiomyopathy in the Syrian hamster. II. Development of heart necrotic change in relation to defective mitochondrial function. Muscle Nerve 5: 26–32, 1982. 24. Sapp, J. L., and S. E. Howlett. Density of ryanodine receptors is increased in sarcoplasmic reticulum from prehypertrophic cardiomyopathic hamster heart. J. Mol. Cell. Cardiol. 26: 325– 334, 1994. 25. Tawada-Iwata. Y., T. Imagawa, A. Yoshida, M. Takahashi, H. Nakamura, and M. Shigekawa. Increased mechanical extraction of T-tubule/junctional SR from cardiomyopathic hamster heart. Am. J. Physiol. 264 (Heart Circ. Physiol. 33): H1447– H1453, 1993. 26. Tong, J., P. K. Ganguly, and P. K. Signal. Myocardial adrenergic changes at two stages of heart failure due to adriamycin treatment in rats. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H909–H916, 1991. 27. Valdivia, H. H., J. H. Kaplan, G. C. R. Ellis-Davies, and J. W. Ledere. Rapid adaptation of cardiac ryanodine receptors: modu-

28.

29.

30.

31.

32. 33.

H7

lation by Mg21 and phosphorylation. Science 267: 1997–2000, 1995. Vatner, D. E., N. Sato, K. Kiuchi, R. P. Shannon, and S. F. Vatner. Decrease in myocardial ryanodine receptors and altered excitation-contraction coupling early in the development of heart failure. Circulation 90: 1423–1430, 1994. Whitmer, J. T., P. Kumar, and R. J. Solaro. Calcium transport properties of cardiac sarcoplasmic reticulum from cardiomyopathic Syrian hamsters (BIO 53.58 and BIO 14.6): evidence for a quantitative defect in dilated myopathic hearts not evident in hypertrophic hearts. Circ. Res. 62: 81–85, 1988. Wikman-Coffelt, J., R. Sievers, R. J. Coffelt, and W. W. Parmley. Biochemical and mechanical correlates at peak systole in myopathic Syrian hamster. In: Cardiac Adaptation to Hemodynamic Overload, Training and Stress, edited by R. J. Jacob. Tubingen, Germany: Steinkopff-Verlag, 1983, p. 197–203. Wikman-Coffelt, J., R. Sievers, W. W. Parmley, and G. Jasmin. Cardiomyopathic and healthy acidotic hamster hearts: mitochondrial activity may regulate cardiac performance. Cardiovasc. Res. 20: 471–481, 1986. Wikman-Coffelt, J., T. Stefenelli, S. T. Wu, W. W. Parmley, and G. Jasmin. [Ca21]i transients in the cardiomyopathic hamster heart. Circ. Res. 68: 45–51, 1991. Zucchi, R., S. Ronca-Testoni, G. Yu, P. Galbani, G. Ronca, and M. Mariani. Postischemic changes in cardiac sarcoplasmic reticulum Ca21 channels: a possible mechanism of ischemic preconditioning. Circ. Res. 76: 1049–1056, 1995.