JA Wagner, HF Weisman, AM Snowman, IJ Reynolds, ML Weisfeldt and SH Snydercardiomyopathic hamster tissues

Alterations in calcium antagonist receptors and sodium-calcium exchange in

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205

Alterations in Calcium Antagonist Receptorsand Sodium-Calcium Exchange inCardiomyopathic Hamster Tissues

John A. Wagner, Harlan F. Weisman, Adele M. Snowman, Ian J. Reynolds,

Myron L. Weisfeldt, and Solomon H. Snyder

The Syrian cardiomyopathic (CM) hamster (BIO 14.6) develops a progressive cardiomyopathycharacterized by cellular necrosis, hypertrophy, and, eventually, cardiac dilatation andcongestive heart failure. Several lines of evidence implicate cellular calcium overload as anImportant etiologic factor. We previously reported an increased number of receptors forcalcium antagonist drugs, which block voltage-dependent calcium channels, in heart, skeletalmuscle, and brain tissue of these hamsters in the early necrotic stage of the disease. To bettercharacterize the pathophysiological significance of this abnormality we evaluated calciumantagonist receptor binding and Na+-Ca2+ exchange in CM and control hamsters at differentstages of disease as documented by quantitative histopathologic assessment. In CM hamsters asyoung as 10 days, an age previously thought to be before the onset of disease, we identifiedcardiac myocyte hypertrophy, a twofold increase in calcium antagonist receptor binding inheart and brain, and a 50% increase in skeletal muscle. Overt histological lesions were presentin skeletal muscle at 25 days and in heart between 28-30 days. The size of cardiac lesionsincreased over time and changed from necrotic foci with cellular infiltration to fibrotic orcalcined lesions by 360 days. Myocardial cellular hypertrophy persisted through the late stagesof the disease (360 days), but increased calcium antagonist binding was present in heart only to6 months of age, in skeletal muscle to 90 days, and in brain to 30 days. Na+-Ca2+ exchange inheart was normal until 15 days and then increased by 400% at 30 days suggesting that thisaugmentation might be a secondary response to the earlier increase in calcium antagonistreceptors. At 360 days cardiac Na+-Ca2+ exchange was decreased by 50%, likely reflectingprogressive cardiac damage. The increase in calcium antagonist receptors in CM animals asyoung as 10 days supports the hypothesis that abnormalities in voltage-dependent calciumchannels play a role in the pathophysiology of CM hamsters. (Circulation Research 1989;65:205-214)

An inbred strain of hamsters, the Syrian car-diomyopathic (CM) hamster (BIO 14.6,Bio Breeders, Fitchburg, Massachusetts),

displays hereditary abnormalities in cardiac andskeletal muscle. Heart involvement results in myo-cardial hypertrophy followed by progressive car-diac dilatation and premature death from congestive

From the Departments of Neuroscience, Pharmacology andMolecular Sciences, Psychiatry and Behavioral Sciences andDivision of Cardiology, Department of Medicine, The JohnsHopkins Medical Institutions, Baltimore, Maryland.

Supported by US Public Health Service grants MH-18501,RSA award DA-00074 to S.H.S., and HL-17655 to M.L.W.H.F.W. is the recipient of a Oinician-Scientist Award from TheJohns Hopkins School of Medicine.

Address for correspondence: Dr. Solomon H. Snyder, TheJohns Hopkins University School of Medicine, Department ofNeuroscience, 725 North Wolfe Street, Baltimore, MD 21205.

Received August 23, 1988; accepted December 28, 1988.

heart failure.1-3 Calcium overload of myocytes hasbeen implicated in the etiology of abnormalities inCM hamsters; since calcium uptake in the myocar-dium is increased,4-5 cardiac action potentials dis-play prolongation compatible with augmentedvoltage-dependent calcium channels,6 and calciumantagonist drugs such as verapamil are uniquelyefficacious in ameliorating the manifestations of thedisease.4'7-9 CM hamsters may provide a usefulmodel of human cardiac diseases such as ischemiaand reperfusion and hypertrophic cardiomyopathythat share common pathophysiological mechanismsas well as a selective efficacy of verapamil.10

Previously, we reported enhanced numbers ofcalcium antagonist receptors in heart, skeletal mus-cle, and brain tissue of CM hamsters,11 observa-tions confirmed by others.12-16 In the present study,we characterize in greater detail the alterations in

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206 Circulation Research Vol 65, No 1, July 1989

calcium antagonist receptor binding in several tis-sues of CM hamsters at different stages of diseaseas determined by quantitative histopathology andalso describe alterations in Na+-Ca2+ exchange.

Materials and MethodsHistopathologic Studies

Male hamsters (BIO 14.6 and age-matched FIBcontrols; Bio Breeders) were killed at various ages todocument the presence and extent of disease. Atsacrifice, the animals received sodium heparin (100units i.p.) and were anesthetized with intraperitonealsodium methohexital (35 mg/kg). They were thenweighed, and their body lengths were measured fromnose tip to the beginning Of the tail. The hearts wereexcised, placed in iced 30 mM KC1 to achievediastolic arrest, blotted dry, weighed, and fixed byimmersion in a 4% buffered formaldehyde solutionfor 24 hours. The brains were also removed, weighedafter blot drying, and fixed whole by immersion inthe formaldehyde solution. Small samples of skeletalmuscle were obtained from the tongue, acromiotra-pezius, biceps femoris, rectus abdominis, and exter-nal oblique and also fixed in formaldehyde.

The fixed hearts were serially sliced into 2-mmrings parallel to the atrioventricular groove frombase to apex. Each of the slices was embedded inglycol methacrylate. The plastic-embedded blockswere then cut in 3 jxm thick serial sections with aglass knife and Sorvall JB-4 microtome. The serialsections were cut in groups of four with 200 fimbetween groups. The sections were mounted onglass slides, and the first section from each groupwas stained with toluidine blue, the second withhematoxylin-eosin, the third with Masson's tri-chrome for connective tissue, and the fourth withthe Von Kossa method for calcium deposits andKernechtrot counterstain.17

The brains were sliced serially into 2-mm coronalsections, which were embedded in paraffin. Theskeletal muscle specimens were also embedded inparaffin. The embedded brain slices were cut into5-^tm-thick sections, mounted on slides, and stainedwith hematoxylin-eosin. Three sections of 5-^rnthickness from each skeletal muscle block were cutand mounted and stained with hematoxyMn-eosin,Masson trichrome, or Von Kossa/Kernechtrotstains.

The image of each histological heart section wasprojected at a magnification of 7.5-10.5 dependingon the heart size and traced onto paper. Intramyo-cardial lesions were identified and also traced. Thelesions were classified into four subtypes: 1) cellu-lar: myocytolysis and contraction band necrosiswith cellular infiltration; 2) mixed: cellular infiltra-tion with fibrosis; 3) fibrotic: collagen fibers andfibroblasts without other cell types; and 4) calcified:lesions with calcification with or without cellularinfiltration or fibrosis. The presence, type, andextent of cardiac lesions were confirmed by micros-

copy. The areas encompassed by the epicardialcontour, the endocardial contour and lesions, weredigitized using a Videoplan image analysis micro-computer (Carl Zeiss, Thornwood, New York).Cross-sectional left ventricular cavity diameter wasmeasured along a line connecting the centers ofmass of the right ventricular and left ventricularchambers. The myocardial area of each section wascalculated by subtracting the endocardial area fromthe epicardial area. For each heart, total left ven-tricular myocardial volume, left ventricular cavityvolume, and lesion volume (total and by eachsubtype) were estimated by serial reconstruction ofintegrated cross-sectional areas of the histologicalheart rings. The amount of each type of myocardialinjury as well as total amount of tissue injury wereexpressed as a percentage of total calculated ven-tricular volume.

To assess the development and extent of myocytehypertrophy, maximal myocyte diameter was deter-mined from the serial sections. Cross-sectional pro-files of cells were outlined at a magnification ofx500 by projecting the cursor of the Videoplancomputer through a camera lucida attached to alight microscope. The maximal diameter of thedigitized profile was then automatically measuredand recorded. To assure that measurements weremade near the cell centers, only cell profiles con-taining nuclei were measured. Between 25 and 50cells per serial section were measurable with thesecriteria. Because the 3- .̂m sections from which themeasurements were made were taken at 200 -fimintervals—a distance two to three times the lengthof myocytes—no cell was measured more thanonce. At least 200 cells from each of the originalheart rings were measured, resulting in a minimumof 600 measured cells for the smallest hearts to over1,000 cells for the largest hearts. Differences in celldiameter between CM and control hamsters at eachage were tested for significance by Student's t tests.

Tissue Preparation for Binding AssaysLittermates of the CM and control hamsters used

in the pathological studies were killed at the sameages by cervical dislocation and decapitation, andtheir brains, hearts, and hindleg skeletal musclewere rapidly removed and placed on ice. All furthersteps took place at 4° C.

Brains were homogenized in 50 mM HEPES, pH7.4, 50 mg wet wt tissue/ml buffer, using a Brink-mann potytron (setting on half-maximal for 30 sec-onds; Brinkmann Instruments, Westbury, NewYork). Homogenates were centrifuged for 15 min-utes at 40,000g. The pellet was resuspended in 50mM HEPES, pH 7.4, and centrtfuged for 15 min-utes at 40,0G0#. This pellet was resuspended in 50mM HEPES, pH 7.4, 100 mg wet wt brain/mlbuffer, and stored at -20° C until use.

Hearts were homogenized in 260 mM sucrose, 50mM HEPES, pH 7.4, 50 mg wet wt tissue/ml bufferusing a Brinkmann polytron (setting on half-

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Wagner et al Ca2+ Antagonist Receptors and Na+-Ca2+ Exchange in Hamster Cardiomyopathy 207

maximal for 30 seconds). Homogenates were cen-trifuged at l.OOQg for 10 minutes, and the resultingsupematants were filtered through four layers ofgauze and centrifuged at 10,000g for 20 minutes.Supematants were diluted with enough 3 M KC1, 50mM HEPES, pH 7.4, to bring the final KC1 concen-tration to 1 M, and the suspensions were incubatedon ice for 30 minutes followed by centrifugation at150,000g for 60 minutes. Resulting pellets wereresuspended in 50 mM HEPES, pH 7.4, 5 mgprotein/ml buffer, and stored at -20° C until use.Skeletal muscle was prepared similarly. Calciumantagonist binding activity remains unaltered insamples frozen up to 6 months.

Tissue Preparation for Na+-Ca2+ Exchange AssayHamsters were killed, and brains and hearts were

rapidly removed and placed on ice. All further stepstook place at 4° C. Synaptosomes were preparedaccording to the method of Hajos18 with the follow-ing modifications. Brains were homogenized in 260mM sucrose and 50 mM HEPES, pH 7.4, using amotor-driven, loose-fitting Teflon-glass homoge-nizer (12 up-and-down strokes). The homogenatewas centrifuged at l,00Qg for 10 minutes, and thepellet was discarded. The supernatant was dilutedwith an equal volume of basal buffer (mM: NaCl160, Tris 20, pH 7.4) and centrifuged at 9,000g for15 minutes. The resulting pellet was gently resus-pended in 3 ml of 260 mM sucrose and 50 mMHEPES, pH 7.4, using the Teflon-glass homoge-nizer, and the suspension was layered over 20 ml of800 mM sucrose. This two-step gradient was cen-trifuged at 10,000g for 25 minutes. The interfacebetween the low-density supernatant and the pelletwas aspirated, diluted with basal buffer, and centri-fuged at ll,000g for 15 minutes. This pellet wasresuspended in 2 ml basal buffer. The synaptosomeswere loaded with Na by overnight incubation inbasal buffer at 4° C.

Hearts were prepared as described.19 Briefly,hearts were rinsed with ice-cold 300 mM sucrose, 5mM MgSO4,10 mM imidazole, pH 7.0, minced withscissors, and combined with 4 volumes of 300 mMsucrose, 5 mM MgSO4, 10 mM imidazole, pH 7.0.All further steps took place at 4° C. This mixturewas subjected to four 5-second bursts with a Brink-mann polytron at setting 8. The mixture was homog-enized using a motor-driven Teflon-glass homoge-nizer (two up-and-down strokes). The sucroseconcentration was adjusted to 600 mM with a 1 Msucrose solution and centrifuged at 28,00Qg for 30minutes. The supernatant was diluted 1.5-fold withbasal buffer and centrifuged at 43,6O0g for 30 min-utes. The resulting pellet was resuspended in basalbuffer and incubated overnight at 4° C.

Calcium Antagonist BindingDihydropyridine (DHP) binding using pHJnitrendi-

pine was assayed as described.11 Membranes, dilutedwith 50 mM HEPES, pH 7.4, to a concentration of

100-150 fig protein/1 ml assay, were incubated with0.1-0.3 nM [3H]nitrendipine and varying concentra-tions of cold nitrendipine. Assays were terminatedafter 1 hour at room temperature (22-24° C) byfiltration over glass fiber filters (Schleicher andSchuell, Keene, New Hampshire) using a Brandelcell Harvester (Brandel, Gaithersburg, Maryland).Filters were washed with three 4 ml aliquots ofice-cold 50 mM NaCl. Filters were placed in 4 ml ofFormula 963 liquid scintillation cocktail (NEN-DuPont, Boston, Massachusetts), and trapped radio-activity was counted in a Beckman LS2800 scintil-lation counter (Beckman Instruments, Fullerton,California) at an efficiency of 55%. Specific bindingwas defined as total binding less binding in thepresence of 1 yuM nitrendipine. Saturation plotswere analyzed with the UGAND computer program.20

Protein was measured with the BCA assay (Pierce,Rockford, Illinois) with BSA fraction V (SigmaChemical, St. Louis, Missouri) as protein standard.

45Ca2+ Flux Measurements45Ca2+ uptake through N-type Ca2+ channels into

hamster brain synaptosomes was measured asdescribed.21 Measurement of Na+-Ca2+ exchangewas essentially as described.19 Briefly, Na+-loadedsynaptosomes and cardiac membrane vesicles werebriefly preincubated at 30° C; 25 fil aliquots wereinjected into 975 (JL\ of basal buffer or Na+-freebuffer (160 mM choline chloride recrystallized fromethanol, 20 mM Tris HC1, pH 7.4) and incubated for1 second at 30° C. Reactions also contained appro-priate concentrations of 45CaCl2. Reactions wereterminated by the addition of 3 ml ice-cold stopbuffer (160 mM choline chloride, 1 mM CaCl2, 20mM Tris HC1, pH 7.7,1.8 mM LaCl3) timed with theuse of an electronic metronome. Vesicles werecollected on glass fiber filters, as above, using aBrandel cell harvester and rinsed twice with 4 mlice-cold stop buffer without LaCl3. Retained radio-activity was determined as above. Na+-Ca2+

exchange was defined as the difference betweenuptake in Na-free and basal buffers. Data wereanalyzed by Lineweaver-Burke transformation andsimple linear regression.

Materials[3H]Nitrendipine (specific activity 78 Ci/mmol)

and ^CaC^ were obtained from NEN-DuPont.Nitrendipine was obtained from sources describedpreviously.11 Choline chloride was obtained fromCalbiochem, La Jolla, California; choline used forNa+-Ca2+ exchange incubation buffers was recrys-tallized from ethanol and stored under high vacuumuntil use. All other chemicals were reagent gradefrom commercial sources.

ResultsGross Pathological Findings

Animal weight, body length, heart weight, andbrain weight increased progressively with age.

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208 Circulation Research Vol 65, No 1, July 1989

TABLE 1. Morphologic Features of Cardiomyopathic Hearts

A. Extent

Age(days)

10

15

30

180

240

360

B. PercentDays

1015

30

180

240

360

of hypertrophyHeart wt/bodyFIB

4.6±1.84.9±0.14.1±0.13.9±0.13.8±0.24.1±0.1

wt(x10~3)14.6

4.6±0.14.9±0.14.1±0.15.4±0.2*6.2±0.6*7.4±1.5*

of total myocardium occupied by lesions (14.6Cellular

0

0

0.1±0.050

0

0

Mixed

0

0

0.1 ±0.053.1±0.10.8±0.4

0

HeartFIB

0.07±0.030.14±0.020.24±0.020.52±0.020.56±0.010.48±0.02

only)

wt/brain wt14.6

0.08±0.010.14±0.020.25±0.010.61+0.05*0.79±0.0S*O.70±O.O3*

Fibrotic

0

0

0

3.6±0.211.2±1.0

13.2

MaximumF I B

10.0±0.111.5+0.114.5±0.117.3±0.218.7±0.321.2±0.6

Calcified

00

0.2±0.21.2±0.20.5±0.1

1.4

cell diameter (/un)14.6

11.4+0.2'12.5 ±0.1*16.3 ±0.2*24.5±0.4f27.4 ±0.4f28.8 ±0.4f

Total

0

0

0.4+0.38.0+0.01

12.6±0.514.7

Results are expressed as mean±SEM. Four to 11 animals for each group were studied except that the volume fraction of lesions wasmeasured in only one 360-day-old CM (BIO 14.6) hamster. Comparisons between cardiomyopathic hearts and control (FIB) hamsterswere made with Student's t tests.

•p<0.05.

weight, did not show significant differences betweenthe control and CM hamsters until 180 days of age.Although the ratio of heart weight to body weight ismore often used as an index of gross hypertrophy,the ratio of heart weight to brain weight may bepreferable, because the CM hamsters have slowerincreases in their body weight than controls, whichalters the usual relation between heart and bodyweight.

Heart lesions were not usually seen until theanimals were 30 days of age, but a few animalsshowed small myocytolytic necrotic lesions at 28days (Table IB). Although some CM animals at 30days showed myocytolysis without cellular infiltra-tion, these lesions comprised less than 0.1% of themyocardium and thus are not shown separately inthe table. The typical lesion at 30 days showednecrosis including the presence of contraction bandsand a dense cellular infiltrate composed of cellsresembling histiocytes, lymphocytes, and mono-cytes (Figure 1A, Table IB). There was evidence ofearly healing in some lesions with fibroblasts andcollagen deposition. A large proportion of the cel-lular and mixed lesions showed at least some calci-fication on the Von Kossa stained sections (FigureIB). Aside from a small dip in the amount ofcalcium deposition between 180 and 240 days, whichlikely was due to aberrant sampling, the amount ofcalcium deposition became more prevalent withtime (Table IB). Widespread fibrosis with no exclu-sively cellular lesions was evident at 180 days andmore pronounced at 240 and 360 days. These quan-titative morphometric findings are in agreementwith earlier qualitative descriptions of the histolog-ical features of the cardiomyopathy.'

Between 10 and 30 days of age, there were nosignificant differences in these values between con-trol and CM hamsters at each age (data not shown).At 180 days, control hamsters had significantlygreater body weight (141±1 g vs. 114±2g) andgreater body length (18.8±0.8 cm vs. 15.9±0.1 cm).By contrast, heart weight at 180 days of age wasless in the control hamsters (0.56±0.02 g vs.0.65 ±0.04 g), but there continued to be no differ-ence in brain weight (1.06+0.02 g vs. 1.02+0.02 g).This pattern of greater body weight and length, nearequal brain weight, but lower heart weight in con-trols compared with CM animals was also seen at240 and 360 days (data not shown). Left ventricularcavity size was not significantly different betweenthe younger control and CM hamsters (e.g., 30 daymean cavity diameter was 1.9±0.1 mm in the con-trols and 2.1 ±0.2 mm in the CM hamsters). At 180days, mean cavity diameter was 2.9 ±0.1 mm incontrols and 3.8±0.2 mm in CM animals (p<0.05).Thereafter, the CM hamsters exhibited progres-sively greater cavity dilatation.

Histological FindingsCardiac myocyte hypertrophy was apparent in

CM hamsters as early as 10 days, the youngest agewe examined (Table 1). This is striking because it isgenerally felt that the animals are free of disease atthis early age and by gross measurement there is nosignificant increase in heart mass.1-3 As in previousstudies, we did not identify any other histologicalabnormalities at this age. CM hamster myocyteswere significantly larger at all other ages, but grosserindexes of hypertrophy, such as the ratios of heartweight to body weight and heart weight to brain

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Wagner et al Ca2+ Antagonist Receptors and Na+-CaJ+ Exchange in Hamster CardJomyopathy 209

FIGURE 1. A: Cardiac histopathology ofa 30-day-oid cardiomyopathic hamster. Thecentral lesion is characterized by a densecellular infiltrate with myocyte necrosis.The upper margin of the lesion consists ofcontraction band necrosis, a finding typi-cal of injury related to cellular calciumoverload (hemataxylin-eosin, original mag-nification, x64). B: A more advancedlesion in a 90-day-old hamster is charac-terized by calcium deposition and fibrosiswith minimal cellular components(hemataxylin-eosin, original magnifica-tion, x64).

B

Focal myocytolysis was seen in skeletal muscleas early as 25 days (Figure 2). In general, theprogression of skeletal muscle lesions paralleledthose of the heart but was somewhat accelerated.No brain lesions were apparent.

Ontogeny of Altered Calcium AntagonistReceptor Binding Sites in CardiomyopathicHamsters

In our initial studies, we showed increases innumbers of dihydropyridine calcium antagonistreceptors as well as phenylalkylamine calcium antag-onist receptors at 30, 60, and 90 days in several CMhamster tissues.11 An important concern is whetheralterations in receptor binding sites are secondaryto cardiac pathology. Accordingly, in the presentstudy we have examined [3H]nitrendipine binding todihydropyridine receptors at ages ranging from 10to 360 days (Table 2; Figures 3 and 4). At 10 days,the earliest time examined, [3H]nitrendipine bindingwas enhanced about twofold in membranes of heartand brain and almost 50% in skeletal muscle. Sta-

tistically significant increases continued to be appar-ent for [3H]nitrendipine binding to 180 days inheart, 90 days in skeletal muscle, and 30 days inbrain. To ascertain whether alterations in [3H]nitrendipine binding derived from abnormalities inthe affinity of the ligand for binding sites or in thenumbers of binding sites, we conducted saturationanalysis monitoring binding of different concentra-tions of the [3Ff]nitrendipine. The [3H]nitrendipineincreases in binding can be attributed solely toaugmented B ^ values with no change in KD levelsin the three tissues (Figures 3 and 4; data forskeletal muscle not depicted).

Na+-Ca2+ Exchange in Heart and Brainof Cardiomyopathic Hamsters

Previously we reported augmented 45Ca2+ accu-mulation in nerve ending preparations (synapto-somes) of CM hamster brain.11 Calcium accumula-tion can be mediated either by voltage-dependentcalcium channels or by Na+-Ca2+ exchange.Recently, we21-22 and others23-25 have observed

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210 Circulation Research Vol 65, No 1, July 1989

FrGURE 2. A: Skeletal muscle from a 28-day-old control (FIB) animal demonstrating normal histological findings(hematoxylin-eosin, original magnification, x64). B: Skeletal muscle from a 28-day-old cardiomyopathic (BIO 14.6)hamster showing myocytofysis and cellular infiltration (hematoxylin-eosin, original magnification, x64).

that, when substantial levels of sodium are includedin the incubation buffer, synaptosomal 45Ca2+ accu-mulation is attributable primarily to Na+-Ca2+

exchange rather than to voltage-dependent calciumchannels.21 Since sodium was included in incuba-tion buffers employed for our studies of synapto-somal calcium uptake in CM hamsters,11 the result-ant calcium accumulation presumably involvedNa+-Ca2+ exchange. To examine this question directly,we compared synaptosomal 45Ca2+ accumulation withsodium in the buffer as in earlier experiments11 andwith choline in the buffer under conditions in whichwe have shown that calcium uptake involves OnlyN-type voltage-dependent calcium channels.21 Asobserved previously, when sodium was included inthe incubation buffer, synaptosomes from CM ham-sters displayed augmented Ca2+ accumulation (con-trol = 1.22±0.23 and CM- = 1.78±0.19 nmol/mg;p<0.05). Nitrendipine (1 /*M) did not alter control orCM 45Ca2+ accumulation in the presence of sodium.However, with choline in the buffer, no abnormalityin 45Ca2J|" uptake was detected (control = 3.60±0.41and CM = 3.73±0.62 nmol/mg). Therefore, theincrease in Ca2+ uptake by synaptosomes is not medi-ated through N-type Ca channels.

To directly examine Na+-Ca2+ exchange, we mea-sured ^Ca2* uptake into membrane vesicles pre-pared from brain and heart (Figures 5 and 6).

Vesicles were loaded for 16 hours with 160 mMNaCl and 45Ca2+ uptake then monitored in a 1-second incubation in buffers containing either 160mM sodium or 160 mM choline. In the presence ofcholine, the large inside-outside gradient for sodiumwill result in sodium efflux associated with 45Ca2+

influx via the Na+-Ca2+ exchange mechanismwhereas the Na+-Ca2+ exchange mechanism shouldnot be operative with identical concentrations ofsodium on the inside and outside of the vesicles.Accordingly, Na+-Ca2+ exchange can be monitoredas the difference between 45Ca uptake in bufferscontaining choline in the medium minus uptake inbuffers containing sodium in the medium. In theseexperiments, we evaluated Na+-Ca2+ exchange inthe presence of different concentrations of calciumto ascertain whether any alterations would be deter-mined by changes in affinity (Km) or maximal veloc-ity of uptake (V,^).

Na+-Ca2+ exchange was increased in both brainand heart of CM hamsters. However, unlike calciumantagonist receptor binding, which was most ele-vated in young hamsters, no changes were evident inNa+-Ca2+ exchange in 10- and 15-day-old hamsters,while V ^ (nmoles per milligram protein ±SEM)was doubled at 30 days in CM brain (control 25±3;CM 53±6) and increased fivefold at 30 days in CMheart (control 22±3; CM 110±15). At 360 days

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Wagner et al Ca1+ Antagonist Receptors and Na+-Ca2+ Exchange in Hamster Cardiomyopathy 211

TABLE 2. [3H]NItiTndiplne Binding In the Heart, Brain, andSkeletal Muscle of Cardiomyopathic Hamsters

(fmol/mg protein)

(days)

Heart10

15

30

180

360

Brain10

15

30

90

180

240

360

Skeletal Muscle10

15

30

90

180

240

360

FIB

120±35140±35210±22220±322O0±25

150±35134+32110±10120±22125±15138±21135 ±27

1,210±100l,260±801,410±1401,350+1801,430±210l,4OO±2301,490+200

14.6

234±38*410±120370±37*320±28*26O±35

31O±61*250+40*210±llf

150±17126+1113O±29147±32

l,780±190'1,7^160*

l,920±130t

l,870±190'l,880±210l,840±2401,620±230

Results are expressed as mean±SD for groups of six to 10animals of each strain for each age. KD values did not differsignificantly between groups and were similar to previouslyreported values.11 In brain, KD values ranged from 0.22 to 0.42nM. In heart, KD values varied from 0.15 to 0.33 DM. In skeletalmuscle, KD values ranged from 2.9 to 4.3 nM. There was a small,but not statistically significant, tendency for KD values to increasein all three tissues in older animals (180-360 days). Comparisonsbetween CM (BIO 14.6) and control (FIB) hamsters were madewith Student's t tests.

*p<0.05.V<0.01.

Na+-Ca2+ exchange was not altered in brains andreduced 50% in hearts of CM hamsters (control 18 ±4;CM 9±3). The alterations in Na+-Ca2+ exchangeinvolved V,^ with no change in the Km values.

DiscussionIn the present study we have confirmed and

extended our previous observations that calciumantagonist receptor binding levels are augmented intissues of CM hamsters. Using the same BIO 14.6strain of CM hamsters, Finkel et al12-13 and Koba-yashi et al14 have confirmed the increase in cardiac[3H]nitrendipine binding. Some investigators havebeen unable to demonstrate this increase in dihy-dropyridine binding sites in related cardiomyo-pathic strains. Howlett and Gordon26 did not find anincrease in the number of dihydropyridine bindingsites in cardiac muscle homogenates from 50- to60-day-old CHF 146 CM hamsters compared withmatched controls (CHF 148). However, this CMstrain is not as well characterized as the BIO 14.6

EgaoaO

<MMn

SOLOS-

0.2 0.4

[•«] NITRENDIPINE SOUND (»mol/ag)

B

0.2 CL4

[•«] N1TMNDIPINE SOUND (»«ol/m«>

•aoi-

0.1 0.4

[*H] NITRCNOIPINE I0UND (»Ml/*g)

FIGURE 3. Saturation analysis of [3H]nitrendipine bind-ing in heart of 10- (A), 30- (B), and 360-day-old (C) control(FIB) and cardiomyopathic (CM) (BIO 14.6) hamsters.The concentration of nitrendipine varied between 0.05 and100 nM. Data shown are from typical experiments per-formed in duplicate. Experiments were repeated six to 10times. The total number of binding sites (B^ is increasedin CM hamsters, with no change in binding affinity.

strain, and it is possible that the progression ofdisease differs between the two strains. Because wefound that the presence and extent of the abnormal-ity in dihydropyridine binding sites changes as thedisease progresses, differences in disease progres-sion between the two strains may explain the dif-ferences in our results. In another study, no increasein [3H]PN 200-110 binding was observed in crudeheart homogenates from 35- to 41-day-old 53.58 CMhamsters.27 However, when Kuo et al15 examined

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212 Circulation Research Vol 65, No 1, July 1989

A

o CONTROL

O.I 0.2 0.5

[>H] H|T*CNDIPINE BOUND IpwM/n«)

B

ai ai

[>H] NITTCNDIPINE BOUNP (pro

o CONTROL• CU

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FIGURE 4. Saturation analysis of [3H]nitrendipine bind-ing in brain of 10- (A), 30- (B), and 360-day-old (C)control (FIB) and cardiomyopathic (CM) (BIO 14.6)hamsters using the same techniques as described forheart (Figure 3). The total number of binding sites (B^Jis significantly increased in CM hamsters at 10 and 30days of age but similar to controls at 360 days. As withheart tissue the increase in B^a is seen with no change inbinding affinity.

[3H]PN 200-110 binding to a purified cardiac sar-colemmal preparation from 2-month-old hamstersof the same 53.58 strain, they found an augmenta-tion in calcium antagonist receptor number com-pared with matched controls. In addition, in anotherstudy, Kuo et al16 observed an increase in dihydro-pyridine calcium antagonist receptors in cardiacsarcolemma from 30-day-old BIO 14.6 CM ham-sters using the reversible ligand [JH]PN 200-110 aswell as photoaffinity techniques with the dihydro-

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FIGURE 5. Lineweaver-Burke plots of brain Na+-Ca2+

exchange in 10- (A), 30- (B), and 360-day-old (C) controland cardiomyopathic (CM) hamsters. Typical plots aredepicted. Experiments were repeated seven or eight times.

pyridine [3H]aziodopine. The two studies that failedto demonstrate an increase in calcium antagonistreceptor sites used crude muscle homogenates ratherthan purified membrane preparations employed instudies showing an increase in dihydropyridine bind-ing. Other tissue components in the homogenatesmight have masked increases in dihydropyridinebinding.

One striking finding of the present study was thatthe augmented number of calcium antagonist bind-ing sites was most apparent in younger hamstersand was no longer detectable in older animals withmore extensive cardiac hypertrophy and congestiveheart failure. The fact that the largest increasesoccur in the very young hamsters before the devel-opment of necrotic lesions or heart failure suggests

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Wagner et al Ca1+ Antagonist Receptors and Na+-Ca2+ Exchange in Hamster Cardiomyopathy 213

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FIGURE 6. Lineweaver-Burke plots of heart Na+-Ca2+

exchange in 10- (A), 30- (B), and 360-day-old (C) conWand cardiomyopathic (CM) hamsters. Typical plots aredepicted of experiments repeated on seven or eightoccasions.

that the abnormalities are not secondary to cardiacpathology. Moreover, the increased numbers ofreceptors in brain and skeletal muscle support thisconclusion.

We found evidence of myocardial cell hypertro-phy in CM animals at the same early age that theincrease in calcium antagonist receptors was iden-tified. Other investigators have suggested that myo-cardial hypertrophy is a secondary phenomenon inthe CM hamster, serving as a compensatory responseto myocardial tissue injury and resultant loss ofventricular function.28 However, in the presentstudy, myocyte hypertrophy was present at a timebefore either structural or functional impairmenthad been documented.2-4 The discrepancy betweenthe quantitative morphometric data indicating anearly increase in cardiac myocyte diameter and the

grosser estimates of cardiac hypertrophy (heartweight-to-body weight and heart weight-to-brainweight ratios) is probably seen because these lattertechniques have less sensitivity in detecting modestdegrees of hypertrophy, especially in the extremelysmall hearts of the younger hamsters. For example,the approximate 10% greater cell diameter seen inthe CM animals 30 days old or younger wouldaccount for an approximate 20% increase in cellmass, using a cylindrical model for myocytes. Thisdifference was detected by several thousand celldiameter determinations in each group of animals.Assuming a<0.05 and jS<0.2, we would have had toweigh about 100 hearts in each group to be certainthat we could detect the same degree of hypertro-phy, given the variance present in our heart weightdeterminations. Nevertheless, we cannot excludethe possibility that the CM hamsters have fewer,but larger, left ventricular myocytes than the con-trol hamsters, accounting for the increased cellmass with little or no change in overall heart mass.

In the present study, we provide evidence thatthe increased synaptosomal accumulation of cal-cium in brains of CM hamsters is attributable toaugmented Na+-Ca2+ exchange rather than to anabnormality in voltage-dependent calcium channelsin nerve terminals. The calcium antagonist receptorbinding sites we have monitored are associated withvoltage-dependent calcium channels that mediatecalcium influx. At least three subtypes of voltage-dependent, calcium channels have been differenti-ated,29-30 and the calcium antagonist receptors areonly associated with one of these, the L subtype.N-type calcium channel activity is readily observedin buffers containing choline in place of sodium.21

L-type calcium channel activity is not reliablydetected in synaptosomal preparations. We moni-tored voltage-dependent ^Ca accumulation intobrain synaptosomes mediated by the N-type calciumchannel and detected no abnormality in CM ham-sters. Accordingly, it appears that the geneticallydetermined defect in CM hamsters is restricted tothe L-type calcium channel.

The increased Na+-Ca2+ exchange occurs subse-quent to the alteration in calcium antagonist recep-tors and so may reflect a secondary response toincreased calcium influx following the increased num-bers of L-type voltage-dependent channels. Thenotion that the increased Na+-Ca2+ exchange is asecondary compensatory phenomenon may alsoexplain why more pronounced increases in Na+-Ca2+

exchange are seen in heart, which displays patholog-ical alterations, than in the brain, where no alter-ations are apparent. Increases in the Na+-Ca2+

exchange system, which physiologically extrudesCa2+ from cells, may occur in response to the intra-cellular Ca2+ overload characteristic of CM hearts.

In summary, the present study supports conclu-sions from our earlier work11 suggesting that abnor-malities in calcium antagonist receptors associatedwith voltage-dependent calcium channels play a

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214 Circulation Research Vol 65, No 1, July 1989

major role in the pathophysiology of CM hamsters.The CM hamsters may provide a model for humanhypertrophic cardiomyopathy, which shares somepathophysiological features as well as the selectivetherapeutic response to calcium antagonist drugs.Supporting this possibility, Ferry and Kaumann31 aswell as ourselves32 have observed increased cal-cium antagonist receptor binding in cardiac tissuefrom these patients comparable in degree to thealterations in CM hamsters.

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KEY WORDS • hamster cardiomyopathy • calcium antagonistreceptors • sodium-calcium exchange • hypertrophy

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